key: cord- -d szap authors: permyakova, n. v.; uvarova, e. a.; deineko, e. v. title: state of research in the field of the creation of plant vaccines for veterinary use date: - - journal: russ j plant physiol doi: . /s sha: doc_id: cord_uid: d szap transgenic plants as an alternative of costly systems of recombinant immunogenic protein expression are the source for the production of cheap and highly efficient biotherapeuticals of new generation, including plant vaccines. in the present review, possibilities of plant system application for the production of recombinant proteins for veterinary use are considered, the history of the “edible vaccine” concept is briefly summarized, advantages and disadvantages of various plant systems for the expression of recombinant immunogenic proteins are discussed. the list of recombinant plant vaccines for veterinary use, which are at different stages of clinical trials, is presented. for many thousands of years plants have served humanity as a source of medicinal substances. how ever, only at the turn of the century xxi with the appearance of dna technologies, it became possible to modify plant genomes and to create new types of plants (transgenic plants), which are capable of syn thesizing and accumulating in their tissues recombi nant proteins from various heterologous systems. to date transgenic plants in which nuclear and chloro plast genomes have been transformed with genes encoding heterologous proteins that are important in the treatment of various diseases -antigens of infec tious agents, antibodies, immunomodulators, etc. have been created [ , ] . of principal importance of this work development was the creation of the "edible vaccine" concept, the essence of which is the use of genetically modified plants containing protein anti gens of infectious agents for oral delivery of relevant antigens to the mucosa of the gastrointestinal tract of warm blooded animals. vaccination based on the programming of the spe cific mechanisms of warm blooded animal protection against pathogens is the most efficient method for the struggle against infectious diseases, which often result in a mass mortality. in agriculture, there is no alterna tive to livestock vaccination, because there are no anti viral drugs that are suitable for a wide use in animal husbandry. the importance of animal vaccination indirectly affects human health, because the use of vaccines significantly reduces the amount of pharma ceuticals in the food chain. as a rule, animal immune mechanisms are acti vated by the direct introduction of infectious agents or their components. at present, most of used vaccines are preparations on the basis of inactivated agents. although these vaccines manifest the high immunoge nicity, they are not without serious shortcomings. among such disadvantages are the increased sensitiv ity of the organism to them, the large load on the immune system, the reactogenicity of vaccines (side effects), their toxicity. etc. the application of molecular biology and genetic engineering methods opened wide prospects for the manufacturing of vaccines of new generation, which immunogenic components can be biological mole cules or their fragments. dna fragments or proteins of the infectious agent cell envelopes can serve as immu nogenic components. when the gene encoding the envelope protein of the infectious agent is transferred into the genome of another organism, for example, plant, then the cells of such plant will synthesize the protein antigen capable of formation of resistance to this agent. thus, the introduction into the organism not the whole pathogen but only its part, which is not capable of inducing infection development, will pro vide for the effect of vaccination. one fourth of the total pharmaceutical market of drugs for veterinary use, and it is constantly expanding. [ ] . preparation of medicinal substances for the pro duction of veterinary products is based on various approaches, including biotechnology using genetically modified (transgenic) organisms for these purposes; such expression systems as bacteria, yeast, cells of insects and mammals are used. the application of genetically modified plants with genes encoding phar maceutic proteins inserted in the genome opens new prospects for obtaining recombinant proteins, includ ing plant vaccines [ ] . this review is devoted to the analysis of possibilities of producing recombinant immunogenic proteins for veterinary use on the basis of plant expression systems, and the history of the concept of "edible vaccines" for animal immunization. of plant vaccines the idea of the usage of plant cells for the synthesis and accumulation of recombinant protein antigens was for the first time successfully realized in by c. arntzen and his colleagues [ ] . just this team of researchers not only demonstrated a possibility of the accumulation of the surface hbsag antigen of hepati tis b virus but also its capability for self assembling in the virus like particles in transgenic tobacco plants. the virus like particles isolated from plant tissues were identical to the particles of hbsag antigen of indus trial recombinant vaccine obtained in the yeast expres sion system and also to virus like particles from the blood plasma of patients infected with hepatitis b virus. thus, it became obvious that genetically modi fied plants producing and accumulating protein anti gens of various infectious agents can be used for oral delivery of corresponding agents to the mucosa of the gastrointestinal tract of warm blooded animals, i.e., as "edible vaccines." the next important step in the development of the "edible vaccine" concept on the basis of genetically modified plants was the creation of transgenic plants producing the heat labile enterotoxin of escherichia coli [ , ] and b subunit of the cholera toxin [ ] . heat labile toxin of e. coli consists of two parts: lt a (enzyme) and lt b (pentamer of receptor binding polypeptides). lt b binds to the receptors on the sur face of membranes of epithelial cells of the mammal small intestine and transports lt a in the intestinal cells, where it induces changes in the cell metabolism and cell dehydration. when the two parts of the heat labile enterotoxin are separated, the appearance of the lt b protein complex on the surface of epitheliocytes will stimulate a strong immune response of intestine mucosa without the appearance of any disease signs. just this feature was the basis for the research of c. arntzen [ ] team on the creation of plant vaccine providing for the resistance to enterotoxigenic e. coli toxins. the authors established that lt b synthesized in transgenic tobacco and potato plants and also lt b isolated from e. coli delivered orally to mice induced similar immune responses. later, lt b sequence was optimized for expression in plant cells and transferred into the potato genome [ ] . in potato tubers, the protein assembled correctly in oligomers and was accumulated in amounts suffi cient for the induction of the immune response at oral delivery to the organism. on the basis of clinical trials of the "candidate" plant lt b vaccine, it was estab lished that the consumption of raw potato tubers con taining . - mg lt b by volunteers resulted in the formation of serum and mucosal immune responses with high titers of antibodies [ ] . the initial concept of "edible vaccine" was heavily criticized by researchers who believed that in the aggressive medium of the gastrointestinal tract the recombinant protein should be destroyed. however, later it was experimentally established that the recom binant b subunit of the cholera toxin fused with green fluorescent protein (gfp) protected by the plant cellu lose cell wall at oral delivery is capable of passing through the gastrointestinal tract and reaching the antigen containing cells of the mouse intestine [ ] . the results obtained confirmed the possibility of using plants synthesizing protein antigens of various infec tious agents for oral delivery of antigens to the mucosa of the gastrointestinal tract and experimentally con firmed a consistency of the "edible vaccine" concept. it became evident that genetically modified plants can be used for the creation of plant vaccines as a raw mate rial, and separate plant parts (fruits, roots, berries, leaves, etc.) can be used directly in food without pre liminary heat treatment. response formation in the process of evolution, mammals developed secondary lymphoid mucosal tissue capable of antigen absorption, processing them, and using for the induc tion of the mucosal response. it was established that in this case both cellular and humoral immunity were formed. it is of importance that adaptive mucosal immunity can distinguish between usual food and symbiotic antigens and infectious agents [ ] . the scheme of the mechanism of mucosal immune response formation is presented in fig. . in the digestive tract, which is one of the pathways for the penetration of a variety of pathogens into organism, the main associated lymphoid tissue is the peyer's patches. peyer's patches, inductive sites of the intestine, which contain the dome, underlying the fol licle (b zones with germinal center) and interfollicu lar region containing t cells. the surface of the dome is covered by a specialized follicle associated epithe lium containing the folded cells (m cells), which are able to absorb and transport antigens from the intesti nal lumen. after successful capture, the antigen is par tially cleaved and enters into dendritic cells (see fig. , step ). dendritic cells are especially important in the initiation of adaptive immune responses, since they migrate to the lymph nodes (see fig. , step ), and the mediators act in the development of various subpopu lations of t helper cells from naive t lymphocytes and also can interact with b lymphocytes (see fig. , step ). the activated b and t cells leave the peyer's patches and penetrate into the circulatory system. mature t helper cells then return to the mucosa sur pathogens (antigen) epithelium t helper cells face to function as effectors (see fig. , step ). t helper expressing interleukin (il ) increases the expression of the receptors of polymeric immunoglobulin (pig) and secretion of antigen spe cific iga (fig. , step ) . subsequent generation of mature plasma cells producing iga leads to the induction of antigen specific protection of local and distal mucosal surfaces. since the mucosal immune response has a general ized nature, oral (i.e., mucosal) vaccination is not only the immune response of the mucous membranes, but also the overall immune response of the organism [ ] [ ] [ ] [ ] . it is proved that vaccination via the surface of mucosa membrane can specifically activate the immune response to infection without the develop ment of such processes related to the disease as inflammation or toxicity [ , ] . of plant vaccines in comparison with traditional expression systems, plant systems are attractive to researchers in many ways, primarily due to the absence of the risk of plant cell infection with animal pathogens, viruses, prions, etc. plants are capable of the synthesis of most recom binant antigens with the same posttranslational modi fications as in animal cells [ ] . plant vaccines can play especially important role in the protection of ani mals against diarrheal diseases and diseases, which infectious agents penetrate into the organism through the mucosal tissues. modern techniques of genetic engineering allow to selectively direct the recombinant proteins expressed in plant cells to various plant organs (seeds, tubers, fruits, etc.) [ ] . this possibility greatly simplifies the large scale production of plant vaccines and reduces their cost. according to the experts, the final price of the product (recombinant protein) produced in a plant expression system will be much less than the price of a similar protein produced, for example, in mammalian cell culture [ ] . since recombinant proteins can be accumulated in the storage organs or seeds, they are able to be main tained without any changes and the loss of biological activity for a long time (months and years). it was established that the recombinant protein of cholera toxin b subunit remained stable in transgenic rice grains for at least months, when grains were stored under room temperature conditions [ ] . recombi nant protein antigens remained stable in rice grains during three years and provided for the formation of the protective immunity in mice against cholera agent or against enterotoxigenic e. coli [ ] ; in soybean seeds and soy milk, the preservation of antigen stabil ity was observed for four years [ ] .thus, grains of transgenic plants can be transported to the site of final destination without additional freezing and treatment, and this ensures the retention of activity of the recom binant protein activity, its stability, and the constancy of dosage. a somewhat different picture is observed when lyo philization is used as a method of the conservation of protein antigens synthesized by plant cells. it was established that the recombinant protein of the norovirus envelope retained its immunogenicity in tis sues of both lyophilized and air dried tomato fruits [ ] , but the tested samples differed in immunogenic ity. the immunogenicity of this recombinant protein in air dried tomato fruits was somewhat higher in comparison with lyophilized fruits. similar results were obtained in experiments on the lyophilization of potato tubers synthesizing the protein of norovirus envelope, e.g., the immunogenicity of lyophilized tubers was lower than that of fresh tubers [ ] . how ever, additional studies are required for the final solu tion of this question. despite these advantages, plant vaccines are not without disadvantages. one of them is differences in protein posttranslational modifications in plants and animals, e.g., in glycosylation of the recombinant pro tein [ ] . it is known that more than a half of proteins synthesized by eukaryotic cells are glycosylated and more than a third of currently applied biopharmaceu tics are glycoproteins [ ] . although the activity of most proteins does not depend on glycosylation, in some cases it may be critical. specific features of pro tein glycoforms can affect their folding, stability, trans portation, and changes in their functional activity and immunogenicity. examples of biopharmaceuticals, which functional activity depends on the specific gly coform, are erythropoietin, antibodies, blood anti gens, some interferons and hormones [ ] . scheme of the n glycan complex formation in plants and animals (humans, for example) is shown in fig. . the most significant difference in glycosylation is that the plant β , xylose is attached to the core mannose residue and α , fucose -to n acetylglu cosamine residue of the core glycan. in human cells xylose is not used at all in glycosylation and a proximal fucose residue is attached to glycans through the α , bond. it was established that sugar residues attached at posttranslational modifications of recom binant proteins in plant cells themselves were capable of exhibiting the immunogenicity. approximately in one quarter of patients with allergy symptoms, ige antibodies specific for complex glycans, which include xylose or fucose, were revealed [ ] . differ ences in glycosylation during posttranslational modi fications of recombinant proteins in plant and mam mal cells can be eliminated by genetic engineering techniques, which was successfully demonstrated on the moss physcomitrella patens, in which the genes encoding the enzymes β , xylosyltransferase and α , fucosyltransferase responsible, respectively, for xylosylation and fucosylation of proteins were switched off by the "knock down" method [ ] . it is known that when the foreign gene is inserted in the genome of the transgenic plant, the level of its expression depends on the site of its insertion, which determines the level of protein (antigen in particular) accumulation in plant tissues. in this connection, one of disadvantages of plant vaccines is the difficulty in the standardization of their dosage. it is at this stage the development of the "edible vaccine" concept has undergone substantial revision, since the possibility of oral delivery of the recombinant antigen with the raw plant material was untenable because of the variability of the recombinant protein content. according to the researchers involved in the development of "candi date" plant vaccines, the antigen dosage problem in plant tissues can be successfully solved by the intro duction of additional treatment of the plant material: its refinement (to equalize the concentration of the antigen), drying or lyophilization. it is also necessary to introduce an additional stage associated with the development of rapid methods for determining and monitoring the dosage of the recombinant antigen. after appropriate preparation, plant vaccines can be encapsulated, tableted, and used in practice under corresponding medical supervision [ , ] . thus, performed studies allow a suggestion that the plant vaccine based on the genetically modified plants is capable of inducing protective immunity and open new opportunities for the creation of low cost and easy handling vaccines against infectious diseases of animals. it is of importance that developed to date, highly effective methods of cultivation of agro eco nomically important plant species, as well as the seed production system for a particular culture make the plants attractive to be used as "biofactories" for man ufacturing cheap recombinant proteins for medical purposes. roplasts). among plant expression systems with stable integration of the transgene in the nuclear genome, a separate group includes the cultivated duckweed, microalgae, and cell culture systems in vitro: cell sus pensions, cultures of "hairy roots", moss protonemas, which cultivation conditions are a completely closed environment (bioreactors). promising is the transient expression system, in which the target gene is intro duced into plant cells and is expressed for a short period of time (several days), but is not integrated into the genome. each of these expression systems has its advantages and disadvantages; the main details of these systems are considered below. the most widely used system of heterologous gene expression, in particular for plant vaccine production, are transgenic plants with the stable integration of the transgene into the nuclear genome. the creation of these plants involves the transfer of foreign genes into the genome of plant cells using agrobacterium tumefa ciens or bioballistics and subsequent regeneration of transformed plants from these cells. being inserted into the nuclear genome, transgene becomes its resi dent part, is stably expressed, and is maintained in subsequent generations. using this expression system for producing recom binant proteins, including plant vaccines, is still ham pered by relatively low levels of transgene expression, which is, as a rule, less than % of total soluble protein (tsp) and also by its variability in different plant organs and tissues within a single plant and in different plants. most often, the variability in expression of the transgene is due to the random nature of its insertion into the nuclear genome (effect of position) and may be associ ated with partial or complete transgene inactivation [ , ] . experts believe that the use of plant expression sys tems for obtaining plant vaccines is economically bene ficial at the level of expression of a target gene, which allows the accumulation of recombinant protein in an amount not less than % of tsp [ ] . a lot of ways to increase the level of foreign gene expression in transgenic plants is developed; they are very fully discussed in the reviews [ , , ] . among them are the optimization of the codon composition of the target sequence, the usage of strong promoters, the addition of introns or regions of binding with the nuclear matrix (sar), and others. the search for tis sue and organ specific promoters, i.e., promoters providing for target gene expression in definite plant tissues or organs, is of a special interest. for example, the usage of promoters directing transcription of the target gene predominantly in the seeds can increase the yield of the target protein by an order or several fold: up to - % of tsp in rice grains and up to % of tsp in tobacco seeds [ ] . it is of importance that as compared with leaves, seeds contain less pro teases and much less water; therefore, recombinant protein is saved better. despite the fact that transgenic plants with stable transgene expression in the nuclear genome are used most widely, it is just this expression system that induces a cautious attitude of human society. one of the fears is associated with the possibility of transgene transfer from the cross pollinated plants into the genomes of wild relatives at growing biotechnological crops in open field. to solve this problem, researchers developed various agricultural technologies as well as fixing male sterility in transgenic plants to prevent unwanted cross pollination of cultivated and wild spe cies. examples of production of various immunogenic proteins using for this purpose transgenic plants with stable transgene integration into the nuclear genome are presented in table . chloroplasts are most attractive among plant expression systems. genetically modified plants with the stable transgene integration into the chloroplast genome were called transplastome plants. the specific organization of chloroplasts allows achieving a high dose of foreign gene in transplastome plants, which provides for the efficient production of the target pro tein. the record of the recombinant protein yield was achieved by transplastomic tobacco plants with the bacteriophage lysin gene plygbs, encoding a hydro lase of the bacterial cell wall, the level of which accu mulation in the leaves amounted to % of tsp [ ] . although in some cases, negative physiological changes were observed in plants with such high level of foreign gene expression, usually there were no devia tions in the development of such plants. problems of transplastome plant adaptation to the high level of for eign gene expression are discussed in the review of bally et al. [ ] . transgene delivery to chloroplasts is performed using bioballistics (gene gun); its integration into the chloroplast genome occurs via homological recombi nation [ ] . the advantage of chloroplast expression system is in the absence of the effect of position observed in the case of random pattern of transgene distribution in the nuclear genome. in plastids, there is no transgene splicing (inactivation); therefore, its expression is stably preserved in subsequent genera tions. due to the prokaryotic organization of chloro plast genome, there is a possibility of co expression of several genes within a single operon [ ] . an impor tant specificity of plastids is that they are inherited through the maternal line and usually are absent from pollen. therefore, as distinct from usual transgenic plants, transplastome plants are safe for environment because the uncontrolled spread of the transgene into other plants is prevented [ , ] . it should be noted that protein posttranslational modifications, e.g., assembling multimer proteins, the tmv-tobacco mosaic virus; camv-cauliflower mosaic virus; tsp-total soluble protein; (+) immune response is revealed at oral immunization; (++) immune response is revealed at oral immunization, animals did not die after virus infection. species of immunized animals are indicated, the way of antigen delivery is indicated in the cases when it was not oral; the amount of survived infected animals is indicated in the cases when the survival was less than %. no. formation of disulfide bridges, lipid modifications, etc., occur successfully in chloroplasts. it is estab lished that recombinant proteins synthesized in chlo roplasts do not differ from native ones in their func tional activities [ , ] . chloroplast attractiveness as the system of recombinant protein expression is in the fact that they are closed structures and it preserves the metabolites, which when released into the cytosol are toxic to plant cells, such as b subunit of cholera toxin [ ] and trihalose in tobacco cells [ ] . on the basis of the above works, it becomes appar ent that transplastome plants can be regarded as the most promising system for efficient production of pro tein antigens. however, the main disadvantage of such expression system at plant vaccine manufacturing is that chloroplasts cannot glycosylate proteins. at present, the creation of transplastome plants is also associated with some technological problems related to the absence of the efficient system of regeneration for most plant species and also with the absence of the efficient system of transgen delivery to the chloroplast genome providing for the high percent of transformed plant yield. the suspension cell cultures on the basis of geneti cally modified plants attract the attention of research ers as promising potential systems for biopharmaceu tics production. such cultures can be obtained from loose callus tissues induced from genetically modified explants or on the basis of co culturing of the cell sus pension and a. tumefasciens. after the assessment of growth characteristics and cell line screening to obtain promising lines capable of the accumulation of great amounts of recombinant proteins, such lines can be cultivated in bioreactors for target protein obtaining. an attractive feature of the cell cultures as expres sion systems for recombinant protein production, as compared to the use of whole plants for this purpose, is that cell culture can be unified in growth character istics, cell dimensions and types. moreover, cells are grown under strictly controlled conditions, when the product accumulation does not depend on the sea sonal weather changes and allows the permanent product obtaining in bioreactors. the additional insertion of signal peptide nucleotide sequences into the construct permits a protein secretion into the intercellular space, which allows target protein isola tion directly from the culture liquid. the addition of recombinant protein stabilizers to the suspension cul ture increases the yield of the target protein [ ] . by their capabilities, plant cell cultures are compa rable in production of therapeutic recombinant pro teins with the conventionally used mammalian cell cultures, such as chinese hamster ovary cells. how ever, as distinct from the mammal suspension cultures, they are not infected by any animal pathogens. to data, there are many examples of plant cell cultures with the yield of recombinant protein in the amount more than mg/l, which is a threshold value for starting the commercial manufacture of the product [ ] . an example of a commercially successful produc tion of veterinary vaccine products is developed by dow agro sciences company (united states) system concert™, patented as an effective and safe system for the production of vaccine proteins in cultured plant cells, cultured in a bioreactor. the first plant vaccine against newcastle disease virus of birds obtained in the tobacco cell culture was approved for use by the min istry of agriculture of the united states in . the disadvantages of this expression system are still insufficiently high yield of the target recombinant pro tein and the instability of foreign genes in cultured plant cells due to the epigenetic silencing of the trans gene transcription [ ] . advantages, disadvantages, and specific features of recombinant protein produc tion in the plant cell cultures are discussed in reviews [ , ] . there are many examples of successful use of aqueous plants, such as duckweed, unicellular algae, and mosses, which are cultivated similarly as plant cell suspensions in bioreactors, for recombinant pro tein expression. duckweed attracts the attention of researchers as a potential highly efficient system for recombinant pro tein expression due to its capability of a rapid biomass accumulation: it can be doubled for - h. geneti cally modified duckweed as a potential producer of biopharmaceutic proteins can be used by animals as a raw or dried food. duckweed is a monocotyledonous angiosperm; for foreign gene transfer into its genome, the methods of agrobacterial transformation and bioballistics are used. examples are known when genetically modified duckweed accumulated recombi nant protein in the amounts up to % of tsp, as assessed after the accumulation of gfp [ ] . sequenc ing the duckweed chloroplast genome is close to com pleting, which opens up some prospects for a signifi cant increase in the yield of recombinant proteins. the systems of biopharmaceuticals production using genetically modified microalgae are actively developed. algae combine advantages of both bacteria (rapid growth and simplicity of cultivation) and higher plants (a capability of posttranslational modifications and photosynthesis). chlamydomonas reinhardtii is most promising among algae: it has a short time of bio mass doubling (about h), it is easily subjected to nuclear and chloroplast transformation, it can be grown under photoautotrophic conditions or with the addition of acetate as the source of carbon. nuclear transformants usually give rather low yield of protein product; therefore, recombinant protein production by this alga is based on the transformation of chloro no. plast, which occupies about % of the cell volume [ ] . c. reinhardtii nuclear and chloroplast genomes are sequenced, and this simplifies substantially any genetic engineering manipulations. known examples of protein antigen production in chloroplasts of c. reinhardtii are b subunit of cholera toxin fused with the coat protein of foot and mouth disease virus [ ] or with d fibronectin binding domain of staphylococ cus aureus [ ] , as well as protein virus cryptokary osis (shrimp disease) [ ] and e protein of swine fever [ ] . the green moss physcomitrella patens is the only representative of bryophytes, the genome of which is currently completely sequenced and approaches to its transformation are developed. the peculiarity of this moss is that at the stage of the haploid juvenile game tophyte (protonema) this moss is morphologically similar to filamentous algae and easily enough culti vated in a bioreactor [ , ] . under certain culture conditions the moss can be in the stage of protonema indefinitely long. the attractiveness of this moss spe cies as the system for recombinant protein expression is that, as distinct from plants, fragments of foreign dna can be integrated in its genome through homo logical recombination, which reduces substantially a possibility of transferred gene inactivation. the p. pat ens cells are capable of postranslational modification of proteins of eukaryotic origin. since at the step of gametophyte the moss has the haploid number of chromosomes, it becomes possible to modify the func tioning of individual genes, in particular the moss lines conducting glycosylation of recombinant proteins as in mammalian cell type were obtained [ ] . the firm grenovation (germany) is developing the technology of biopharmaceutical protein production on the basis of p. patens in bioreactors. expression system for pro ducing biopharmaceuticals based on the green moss is not the part of the food chain and is characterized by a high degree of biosafety. as distinct from above described systems based on the stable expression of foreign genes integrated into nuclear or chloroplast genomes, during transient expression target proteins are synthesized in the plant cell during relatively short time (several days) without insertion into the plant genome. at present, the fol lowing approaches are used for transient gene expres sion in plants: gene delivery with the help of agrobac terium, the use of plant virus vectors, and magnifec tion [ ] [ ] [ ] . tobacco mosaic virus (tmv), potato x virus, alfalfa mosaic virus, and cowpea mosaic virus are used as virus vectors [ ] . the availability of infec tious cdna clones, the small size of the viral genomes, the short time required for the expression of a target gene, and a high level of expression provides for a high attraction of this system. the rapid development of this expression system led to substantial modifications of the first gene inser tion vectors or full virus vectors, which is a recombi nant virus that behaves as wild type virus but is capable of expressing additional genes. the next step was the creation of "disarmed vectors" (deconstructed vec tors) lacking a number of original virus genes, and gene replacement vectors, in which a portion of the viral genes is replaced by alien genes [ ] . viral vectors have several substantial disadvantages: a tendency to the loss of foreign insertion in the process of virus spreading over the plant and a potential risk for envi ronment related to the presence of infectious recombi nant viral particles. launch vectors represent an alternative to recom binant plant viruses; cdna of these viruses is deliv ered to plants within t dna region of agrobacterial ti plasmid. firstly, primary transcription of t dna occurs in the nucleus; then viral rna is released into the cytoplasm, where its further amplification, trans lation, and protein synthesis occur [ ] . by , the system of agroinfiltration based on the use of plant viruses and agrobacterial binary plasmids was upgraded and named as magnifection [ ] . at magnifection, multiple agrobacterial lines carrying different parts of the tmv genome are used simulta neously. after agrobacterial transfer into the plant cell nucleus, separate parts of viral genome are assembled in plants in the completely functional viral replicon [ ] . the substantial modification of the viral genome, including numerous point mutations for the removal of potential sites of splicing, intron insertion, and the removal of the gene encoding envelope proteins, pro vided for the highly efficient system capable of recom binant protein synthesis (up to g/kg of fresh tissue), which is more than % of tsp [ ] . among disadvantages of transient system is a necessity for recombinant protein isolation and purifi cation immediately after its accumulation in the plant, because, as distinct from seeds and fruits, plant leaves and stems cannot be stored for a long time. the systems of transient expression of recombinant proteins are rather promising in the cases when a rapid production of a small amount of proteins is required. experiments with transient expression in plants are held indoors, which reduces the risks associated with biosafety to almost zero. examples of manufacturing immunogenic proteins for veterinary using transient expression systems are presented in table . the sys tems of transient expression for the production of recombinant proteins are described in more details in reviews [ , , , , ] . "candidate" plant vaccines for veterinary use table presents examples of using various expres sion systems for the production of "candidate" plant vaccines for veterinary use. main specific immunogenic no. proteins synthesized at respective diseases (structural proteins, hemagglutinins, glycoproteins) are usually used as antigens. the most commonly used method of transgene construct delivering into plant cells is still the agrobacterial transformation. in some cases, the level of target protein expression was rather high [ ] [ ] [ ] [ ] , espe cially in the systems of transient expression [ ] [ ] [ ] , and suitable for product commercialization. in all experiments using the "candidate" plant vac cines, the formation of a specific immune response was demonstrated in vivo, and in most experiments immunogenic proteins were delivered to animals just orally. "candidate" plant vaccines were usually tested on mice, but in approximately a quarter works the ani mals subjected to the disease were tested. protein s of the transmissible swine gastroenteritis virus synthe sized in the cells of transgenic maize [ , , ] or tobacco [ ] and delivered into the body of pigs as a food supplement, provided for % survival of ani mals after infection [ ] . rabbit protein vp virus synthesized in potato [ ] and other plants (tobacco, pea, rape) [ ] and delivered orally enhanced protective immunity: after infecting rabbits with this virus all ani mals survived [ ] . the effect of plant recombinant antigen was comparable with commercially used vac cines. the use of plant vaccines for the vaccination of wild animals using edible baits (e.g., vaccine against rabies) will lead to an increase in the proportion of wild animal populations having immunity to the rabies virus. a potential possibility to reduce the cost of produc tion of biopharmaceutics using genetically modified plants served at the end of the xx century as an impe tus for more than twenty biotechnological companies to initiate commercial programs. as seen from the table , many biological products of plant origin are developed, expressed in different types of plants and plant cell cultures. for a variety of reasons, including the still skeptical attitude of the human community to the biosafety of genetically modified plants, many of these works remained in the framework of laboratory tests. at the moment three companies function on the biotechnology market of veterinary preparations, two from the united states and one from canada (table ). in the united states the dow agro sciences company presented a recombinant plant viral hn protein of the newcastle disease virus (approved by usda) and a mixture of antiviral vaccines at the first stage of clinical trials. the second american company at thomas jef ferson university has developed a plant anti rabies vaccine (completion of phase ). the canadian guardian biosciences company presented plant vac cine against chicken coccidiosis at the second phase of clinical trials. production and wide distribution of biopharmaceu ticals is hampered by a number of circumstances. the first of them is related to the problem of biosafetythe cultivation of genetically modified plants in the field can lead to the accidental introduction of foreign genes into crops grown for human consumption. therefore, most companies producing biopharma ceutics focused on plant species, which are absent from the food chain of humans and animals and also on growing of genetically modified plants preventing their cross pollination with other crops. the second difficulty is related to the necessity of plant material treatment for the removal of various undesired com pounds, such as lignin, proteases, phenolic com pounds, and pigments, especially in the case of plant species, which are not consumed. all these facts result in the requirement of additional studies. the third cir cumstance is due to the fact that until now all aspects of maintaining and growing of plants producing biop harmaceuticals are not settled at the legislative level. ambiguity and vagueness of the existing legislation in this area lead to the fact that large biopharmaceutical and biotechnology companies do not tend to invest in the development of technological lines and research programs in this area, which significantly inhibits the development of the industry. a significant problem for the development of vac cines for veterinary use, especially those used in agri culture, is the need to minimize the price of the final product. the vaccine should be inexpensive for entre preneurs engaged in commercial animal breeding and fully subsidized, if you intend to use the vaccine for mass immunization and the prevention of the disease spread in underdeveloped regions. as a result, the potential income of manufacturers of vaccines for ani mals is much less than that for vaccines intended for humans. for example, in , the volume of the mar ket for the vaccine against human papilloma virus was estimated at more than billion dollars, but the mar ket for the most popular animal vaccines (against foot and mouth disease of cattle and against mycoplasma hyopneumoniae in pigs) together amounted to only - % of this amount [ ] . animal vaccines are cheaper and the volume of market for them is less; therefore, the investments in their development are substantially less as compared with investments in the production of vaccines for humans, whereas the com plexity and diversity of both hosts and pathogens in the case of vaccines for animals is much higher. expression systems based on the use of plant cells still have a limited application or are used primarily in some laboratories. nevertheless, biopharming (the biotechnological production of various substances for medicine) in plants has attracted the attention of researchers and manufacturers in developed countries. first biopharmaceuticals of plant origin, such as anti bodies (anti hbsag required to purify the hepatitis b vaccine), therapeutic and dietary proteins ("intrinsic factor" required at vitamin b deficiency, gastric lipase), have entered the market, that is an excellent illustration of this progress [ ] . despite the fact that today the number of biophar maceutical proteins expressed in plant cells is enor mous, many questions still remain unresolved. the methods for target recombinant protein quantification and purification are still not developed for most of products. the problems of transgene silencing and increased expression of target protein genes are still at the stage of research. the important task that has yet to be solved is to achieve a stable level of expression in different batches of plant raw material. not much work appeared for judging about maintaining the sta bility of recombinant proteins after the harvest, pro cessing, and storage. all of these problems require additional expenses for further research. one of the main obstacles for the leading research groups working on the development and production of plant vaccines, given the financial constraints, is the fulfillment of the relevant official regulations govern ing the use of oral medications. to date, the purified vaccines and therapeutic proteins of plant origin must meet the same standards relating to the production, biosafety, purification, storage, dosage, etc. as any other recombinant proteins for medical purposes. nevertheless, despite these difficulties, there were first biopharmaceuticals of plant origin that passed all the necessary tests and were approved for use by the relevant authorities. some new products having spe cific advantages over similar products obtained in mammalian cell cultures were developed. such com panies as sembiosys genetics inc. (calgary, can ada), medicago inc. (quebec, canada), protalix bio therapeutics (karmiel, israel), and orf genetics (iceland) proved the possibility of quick establishing of the production of purified plant proteins, which are quite competitive in today's market. progress has been made in the formation of the legal framework related to the cultivation of transgenic plants, testing and use of plant biopharmaceuticals. several production pro cesses based on transgenic plants have already received a brand gmp (good manufacturing practice), the interest of manufacturers to this field of biotechnology began to increase again. this work was performed within the framework of the project vi. . . (no. ) development and improvement of genetic constructs to optimize the expression of target genes and the production of recombi nant proteins for medical purposes in transgenic plants and animals. plant produced vaccines: promise and reality evolution of plant made pharmaceu ticals current status of veterinary vac cines clinical trials fuel the promise of plant derived vaccines expression of hepatitis b surface antigen in transgenic plants oral immunization with a recombinant bacterial anti gen produced in transgenic plants edible vaccine protects mice against escher ichia coli heat labile enterotoxin (lt): potatoes express ing a synthetic lt b gene effi cacy of food plant based oral cholera toxin b subunit vaccine immunogenicity in humans of a recombinant bacte rial antigen delivered in a transgenic potato receptor mediated oral delivery of a bioencapsulated green fluorescent protein expressed in transgenic chlo roplasts into the mouse circulatory system delivery of plant made vaccines and therapeutics defending the mucosa: adjuvant and carrier formula tions for mucosal immunity induction of secretory immunity and memory at mucosal surfaces, vaccine oral delivery of human biopharmaceuti cals, autoantigens and vaccine antigens bioencapsu lated in plant cells the mucosal immune response to plant derived vaccines posttranslational modifica tion of therapeutic proteins in plants seed based expression systems for plant molecular farming the economic potential of plant made pharmaceuticals in the manu facture of biologic pharmaceuticals rice based mucosal vac cine as a global strategy for cold chain and needle free vaccination secretory iga mediated protection against v. cholerae and heat labile enterotoxin producing enterotoxigenic escherichia coli by rice based vaccine stability of a soybean seed derived vaccine antigen following long term storage, processing and transport in the absence of a cold chain tomato is a highly effective vehicle for expression and oral immunization with norwalk virus capsid protein production of plant made pharma ceuticals: from plant host to functional protein post translational modifica tions in the context of therapeutic proteins current achievements in the production of complex biopharmaceuticals with moss bioreactors low dose oral immunization with lyophilized tissue of herbicide resistant lettuce expressing hepatitis b surface antigen for prototype plant derived vaccine tablet formulation rna mediated chromatin based silencing in plants transcriptional gene silencing in plants plant based production of biopharma ceuticals production of heterologous proteins in plants: strategies for optimal expression expression of heterologous genes in plant systems: new possibilities exhaustion of the chloroplast protein synthesis capac ity by massive expression of a highly stable protein anti biotic metabolic adaptation in transplastomic plants massively accumu lating recombinant proteins transplastomic plants overexpression of the bt cry aa operon a ¸n´l in chloroplasts leads to formation of insecticidal crys tals chloroplast vector systems for biotechnology applications determining the transgene containment level provided by chloroplast transformation expression of the native cholera toxin b subunit gene and assembly as functional oligomers in transgenic tobacco chloroplasts chloroplast expression of his tagged gus fusions: a general strategy to over produce and purify foreign proteins using transplas tomic plants as bioreactors accumulation of trehalose within transgenic chloro plasts confers drought tolerance plants as bioreactors for the production of vaccine anti gens towards high yield production of pharmaceutical proteins with plant cell suspension cultures position effects and epigenetic silencing of plant transgenes bioreactor systems for in vitro production of foreign proteins using plant cell cultures high expression of transgene protein in spirodela micro algae come of age as a platform for recombinant protein production foot and mouth disease virus vp pro tein fused with cholera toxin b subunit expressed in chlamydomonas reinhardtii chloroplast heat stable oral alga based vaccine pro tects mice from staphylococcus aureus infection factors effecting expression of vaccines in microalgae recombination and expression of classical swine fever virus (csfv) structural protein e gene in chlamydomonas rein hardtii chroloplasts viral vec tors for the expression of proteins in plants agrobacterium mediated transient expression as an approach to production of recombi nant proteins in plants a novel two component tobacco mosaic virus based vector system for high level expression of multiple ther apeutic proteins including a human monoclonal anti body in plants magnifec tion -a new platform for expressing recombinant vac cines in plants plant based vaccines: unique advan tages expression of the newcastle disease virus fusion protein in transgenic maize and immunological studies multimerization of peptide antigens for pro duction of stable immunogens in transgenic plants generation and immunogenicity of japanese encepha litis virus envelope protein expressed in transgenic rice induction of protective immunity in swine by recombinant bamboo mosaic virus express ing foot and mouth disease virus epitopes in planta production of two peptides of the classical swine fever virus (csfv) e glycoprotein fused to the coat protein of potato virus x expression in plants and immunogenicity of plant virus based exper imental rabies vaccine delivery of subunit vaccines in maize seed a corn based deliv ery system for animal vaccines: an oral transmissible gastroenteritis virus vaccine boosts lactogenic immu nity in swine immunogenicity of porcine transmissible gastroenteritis virus spike protein expressed in plants oral immunization using tuber extracts from transgenic potato plants expressing rabbit hemorrhagic disease virus capsid protein pea derived vaccines demonstrate high immunoge nicity and protection in rabbits against rabbit haemor rhagic disease virus plant made pharmaceuti cals: leading products and production platforms pro duction of immunogenic vp protein of bovine group a rotavirus in transgenic potato plants rotavirus vp expressed by pvx vectors in nicotiana benthamiana coats pvx rods and also assembles into viruslike parti cles expression of rotavirus capsid protein vp in transgenic potato and its oral immuno genicity in mice protective lactogenic immunity conferred by an edible peptide vaccine to bovine rotavirus produced in transgenic plants bovine herpes virus gd protein pro duced in plants using a recombinant tobacco mosaic virus (tmv) vector possesses authentic antigenicity induction of a protective antibody response to foot and mouth disease virus in mice fol lowing oral or parenteral immunization with alfalfa transgenic plants expressing the viral structural protein vp induction of a protective antibody response to fmdv in mice following oral immunization with transgenic stylosanthes spp. as a feedstuff additive expression of hemagglutinin protein of rinderpest virus in transgenic tobacco and immunogenicity of plant derived protein in a mouse model systemic and oral immunogenicity of hemagglutinin protein of rinderpest virus expressed by transgenic peanut plants in a mouse model expression of hemagglutinin protein of rinderpest virus in trans genic pigeon pea oral immunogenicity of the plant derived spike protein from swine transmissible gastroenteritis coronavirus cloning and sequence analysis of the korean strain of spike gene of porcine epidemic diarrhea virus and expression of its neutraliz ing epitope in plants successful oral prime immunization with vp from rabbit haemorrhagic disease virus pro duced in transgenic plants using different fusion strate gies mucosal and sys temic immunization elicited by newcastle disease virus (ndv) transgenic plants as antigens expres sion of the fusion glycoprotein of newcastle disease virus in transgenic rice and its immunogenicity in mice expression of immunogenic s glycoprotein of infectious bronchitis virus in trans genic potatoes transient expression of the ectodomain of matrix protein (m e) of avian russian immunization with plant expressed hemagglutinin protects chickens from lethal highly pathogenic avian influenza virus h n challenge infection immunogenicity study of plant made oral subunit vaccine against porcine reproductive and res piratory syndrome virus (prrsv) expression of the rabies virus glycoprotein in transgenic tomatoes immunization against rabies with plant derived antigen development of an edible rabies vaccine in maize using the vnukovo strain induction of a protective immune response to rabies virus in sheep after oral immunization with transgenic maize, expressing the rabies virus glycoprotein expression of the rabies virus nucleoprotein in plants at high levels and evaluation of immune responses in mice expression of rabies virus g pro tein in carrots (daucus carota) key: cord- -ss x g b authors: stobbe, anthony; roossinck, marilyn j. title: plant virus diversity and evolution date: - - journal: current research topics in plant virology doi: . / - - - - _ sha: doc_id: cord_uid: ss x g b historically, the majority of plant virology focused on agricultural systems. recent efforts have expanded our knowledge of the true diversity of plant viruses by studying those viruses that infect wild, undomesticated plants. those efforts have provided answers to basic ecological questions regarding viruses in the wild, and insights into evolutionary questions, regarding the origins of viruses. while much work has been done, we have merely scratched the surface of the diversity that is estimated to exist. in this chapter we discuss the state of our knowledge of virus diversity, both in agricultural systems as well as in native wild systems, the border between these two systems and how viruses adapt and move across this border into an artificial, domesticated environment. we look at how this diversity has affected our outlook on viruses as a whole, shifting our past view of viruses as purely antagonistic entities of destruction to one where viruses are in a mutually beneficial relationship with their hosts. additionally, we discuss the current work that plant virology has put forth regarding the evolutionary mechanisms, the life histories, and the deep evolution of viruses. until recent years, our knowledge of the breadth of plant virus diversity was limited. the field of plant virology traditionally has focused on agricultural systems, with little study of viruses found in wild plants. in the past decade, several efforts have begun to fill these gaps in the form of biodiversity surveys. these surveys have given us a new view into the true diversity of plant viruses, as well as their distribution. one of the most powerful advances in microbe discovery has been massively parallel sequencing, or next generation sequencing (ngs). previously the most common methods for virus detection were protein based immunological tests such as elisa, or nucleotide specific pcr assays. neither of these are sensitive enough to detect low titers of virus in wild plants or general enough to detect novel viruses, or even related strains or species. ngs technology has boosted our ability to fully sequence whole genomes, and advanced the field of metagenomics, the study of all the genetic information from a given environment. when the requirement for culture is removed, the ability to sequence and identify fastidious or unculturable microbes becomes possible. ngs has become the gold standard for metagenomics. metagenomics can be used to identify novel virus species, using various techniques to enrich viral nucleic acids such as isolating specific forms of rna (dsrna, sirna, ssrna) or virus particle isolation. each method has positive and negative aspects (stobbe and roossinck ; . while ngs can be used for virus discovery, it also has been applied to plant virus diagnostics (stobbe et al. ; massart et al. ) . ngs also has been used to look further into the population diversity of individual virus strains. using this deep sequencing, one is able to determine all of the minor variants found in a given infection (simmons et al. ) . ngs has applications in many evolutionary questions regarding systemic movement, vectoring and epidemiology. in this chapter we look into the recent work looking into the diversity of plant viruses, not only in species diversity but also diversity within the species or quasispecies. this variation comes from many sources, including high mutation rates of rna viruses, recombination and reassortment. the variation we see within a single plant host has profound effects on the how the virus responds to selective pressures associated with new hosts, and factors such as the bottleneck events associated with cell-to-cell movement or vectoring. additionally, with our ever increasing knowledge of the breadth of virus diversity, as well as advances in technology, questions of the deep evolutionary history of viruses and their relationship to their hosts are beginning to be answered. while there has been a large body of work on algae-infecting viruses (vanetten and dunigan ), here we only consider the viruses of vascular plants. agriculture has been an important aspect of virology from the beginning of the field (beijerinck ) , and has been the focus of most work in plant virology throughout its year history. much of the early work characterizing and describing viruses was done with viruses of crop plants. while it is understandable that so much work has been put into a few specific plant species, this has left out a lot of information about viruses in natural settings. in modern agriculture the use of vast areas of monoculture, extended growing seasons, irrigation, and artificial soil amendments have each impacted plant pathogen prevalence, including viruses. agriculture is a human invention, and the cultivation of crops has propelled the human race to increasing cultural and technological advances, but with this advancement we have disturbed many natural systems throughout our history. domestication of the earliest crops probably began about , to , years ago (balter ) , presumably with their viruses experiencing a shift in selective pressure as well (stukenbrock and mcdonald ) . densely spaced, monoculture crops have been increasingly favored due to the ease of production, but these conditions also are excellent for the spread and infection of viruses and other deleterious microbes within the crop (thresh ; power and mitchell ) . to combat the yield loss associated with virus-induced disease, breeders have focused efforts on engineering disease resistant cultivars of crops. however, several forms of virus variation, such as the high mutation rates of rna and some dna viruses, recombination, and reassortment lead to resistance breaking (duffy and holmes ; mcdonald and linde ; harrison ) . although breeding of resistant cultivars has had some success, other methods such as increasing the plant species diversity in a given area, breaking the spatial and temporal components of the disease cycle, have been suggested (ratnadass et al. ) . for example, genetic diversity (heterosis) induced tolerance to turnip mosaic virus in wild cress (lepidium sp.) hybrids, while plants that were selfed were more susceptable to disease, suggesting that small populations with low genetic diversity could lead to increased disease symptoms, and infection rates (houliston et al. ) . intercropping cowpea with cassava or plantains has reduced the incidence of viruses in central america (valverde et al. ) . these practices suggest that increases in plant diversity, either within a species or with diverse species, could lower the incidence or pathology of viruses (see sect. . . ) . with the globalization of today's society, it is not surprising to find that humans are playing a role in the movement of plant viruses. human movement of both the plants and vectors associated with pathogens has facilitated the spread of viruses. the effects of climate change in the form of co and ozone may change the impacts of viruses on their plant hosts (trebicki et al. ) . while many pathogens move closer to the poles as climate change occurs, there is some evidence that viruses and nematodes are moving closer to the equator (bebber et al. ) . this may be an analytical artifact as viral symptoms are often misdiagnosed. increases in the range of insect vectors of viruses due to numerous factors, including climate change, predicts increased virus spread (fereres ) . a majority of crop viruses are insect vectored, and relationships between plants, viruses and insects are complex (roossinck b ). using insect vectors as targets for virus discovery is an attractive opportunity. vector-enabled metagenomics is a recent method for virus discovery that allows one to discover and characterize viruses that are in the area that the vector occupies, including both cultivated and wild plants. in a recent study using vector-enabled metagenomics % of the sequences obtained were related to known viruses, suggesting that many vector transmitted viruses are known (ng et al. ). this number was much higher than the number of identifiable virus sequences found in wild plants, where as many as % of sequences from virusenriched pools have no similarity to sequences in genbank . the "viral manipulation hypothesis" states that by modifying the production of certain volatiles, the plant host will be more attractive to the virus' vectors (gutiérrez et al. ) . vector transmission mechanisms of plant viruses influence the effects the virus has on the plant host, with persistently transmitted viruses tending to either improve the host quality for the vector or mimic high quality, and nonpersistantly transmitted viruses lowering quality to facilitate the rapid dispersal of the viruliforous insect to neighboring plants (mauck et al. ) . host manipulation is seen in unrelated families of plant viruses, implying convergent evolution (wu et al. ) . barley yellow mosaic virus (bydv) is persistently transmitted by the aphid rhopalosiphum padi. virus-free aphids have a feeding preference for bydv-infected plants, while the reverse is true for bydv-carrying aphids (ingwell et al. ). cucumber mosaic virus (cmv), a member of the bromoviridae family, increases the release of volatiles that mimic healthy plants, attracting vectors despite the low quality of the plant for the aphids (mauck et al. b) . additionally, cmv effects other non-vectoring insects, repelling some while attracting others, in the absence of aphids (mauck et al. a ). in mixed infections, competition can favor one microbe over another, as seen with the potyvirus, zucchini yellow mosaic virus (zymv) out-competing another potyvirus, watermelon mosaic virus (wmv). zymv and wmv are very similar viruses, in terms of genetics, hosts, and vectors. these similarities places them in direct competition with each other. one important difference is in vector manipulation; zymv manipulates the host-aphid relationship, while wmv does not. when co-infecting a plant, zymv will maintain its typical level of replication, while the replication of wmv is reduced. despite this, wmv is still transmitted from a mixed infection, taking advantage of zymv host manipulation that attracts the aphid vectors (salvaudon et al. ) . in an analysis of genetic turnover during transmission, several clones containing the same mutation leading to a premature stop codon was found within a plant. further transmissions using this experimental isolate lost this mutation, but this suggests that zymv has the ability to complement defective zymv genomes in the aphid vector (simmons et al. ) . viruses not only manipulate their hosts, they also respond to the presence of a vector feeding. cauliflower mosaic virus (camv), a double-stranded dna virus in the caulimoviridae family, is acquired up by its aphid vector packaged into transmission bodies. the transmission bodies change their morphology in different contexts, such as in response to c levels or host wounding. in addition, the transmission bodies change to a morphology that favors transmission when in proximity to the saliva from aphid feeding (martinière et al. ). obviously there are many differences between agricultural systems, and ecosystems of wild undisturbed plants. we have already touched on the use of monoculture, and the effect of plant biodiversity on the diversity of viruses, in this section we look into how nearby systems can influence viruses. the intersection of wild and agricultural systems has been described as the agro-eco border (roossinck and garcía-arenal ) . this border may be the source of new pathogenic plant viruses that can impact crops in spillover events ( fig. . ). in most cases these infections will be dead-end: either the virus is not competent for further transmission in the new host, it may not establish sufficient virus titer to allow transmission, or it may rapidly kill the host. rarely, a spillover virus may develop the ability to be further transmitted to more similar hosts, resulting in an emerging virus infection due to the relatively recent introduction of modern agriculture to australia (in the past years), considerable work has been done on this continent to look at the effect of agriculture across this border. many of the native viruses in australia have not been influence by agriculture. three different new encounter events of legume-infecting potyviruses have been described in australia. the interspecies genetic diversity of each virus differed, with the native viruses having greater diversity than the exotic viruses (webster et al. ) . viruses that infect crop plants often find reservoirs within nearby wild plants or in volunteer plants from the previous crop. these viruses, that cause disease in crops, may be asymptomatic in other hosts. for example, peanut stunt virus causes disease in peanuts, but is asymptomatic in clover (sherwood ) . the presence of highly susceptible hosts in a plant community can increase the incidence of the virus across all susceptible species in the community (power ) . several scenarios can be seen where the spread of virus moves between an asymptomatic native host to cultivated plants (jones ) . emergent viral diseases may come from "silent" infections within a nearby wild population, and are driven by anthropogenic factors, such as food production or the introduction of vectors (anderson et al. ). in africa, there have been several emergent viruses in agricultural systems. while many changes in the pathogens themselves have promoted the emergence of disease, changes in agricultural practices have also promoted the introductions. rice yellow mottle virus (rymv, in the genus sobemovirus) infects oryza sp. in both wild and agricultural systems. rymv was first described in in kenya and is currently an economically important plant virus. the rise of rice production in kenya is thought to be the main driver of rymv spread, as epidemics of the virus were not seen until after the intensification of rice production in the s (fargette et al. (thresh ) . the level of biodiversity on the wild side of the agro-eco border effects the emergence of viral movement across the border. a lowered incidence of two begomoviruses was seen in wild peppers with decreasing levels of cultivation or management, suggesting a dilution effect with higher levels of biodiversity ). this correlation with biodiversity appears to hold true with cmv in wild plants, but not with cmv in crops (sacristán et al. ). the loss of biodiversity appears to increase the movement of a virus across the agro-eco border, but a high degree of biodiversity can lead to a large number of viral species, which may serve as a reservoir for new infections (keesing et al. ) . opportunistic viruses quickly move into susceptible crops, decrease, then recover in the susceptible population, causing a rapid cycles of epidemics and decline (harrison ; thresh ) . in france, where zymv and wmv are both present, only wmv has significant natural reserviours, which explains the fragmented nature of zymv incidence across france (lecoq et al. ) . in spain, two strains of the potexvirus pepino mosaic virus (pepmv-eu and pepmv-ch ) co-circulate among tomato crops, with the ch strain being the dominate strain. pepmv-eu primarily exists in coinfections with pepmv-ch , and these coinfections allow for an extended host range of pepmv, thus extending the potential number of reservoirs. this has implications for coinfection effecting the emergence of pepmv in tomato plants (gómez et al. ). early attempts to explore virus prevalence within wild plants was hampered by a lack of sensitive detection methods (cooper and jones ) . in recent years, there have been a number of plant virus diversity surveys, in which plant tissue was sampled without targeting symptomatic plants (wren et al. ; roossinck ) . these tissue samples were then enriched for viral nucleic acid, and sequenced using ngs. the prevalence and distribution of viruses in these studies varies, but inevitably evidence for many novel virus is found, and the variation of viruses in wild systems is much greater than what is seen in crops ). the enemy-release hypothesis states that plants invading a new territory may have an advantage because they have left behind their pathogens (power ; rúa et al. ) . however, in the invasive grasses of the pacific coastal region of north america, a non-native plant uses its own adapted barley/cereal yellow dwarf virus (b/cydv) to gain an advantage over the native grasses (malmstrom et al. ) . the reverse was seen in another related system; venetanata dubai (african wiregrass), an invasive non-native grass that is not adapted to b/cydv was slowed in its movement across the northwest grasslands of america (ingwell and bosque-pérez ) . the extended phenotype of viruses can change based on many contexts, including the genotype of the host (vanm€ olken and stuefer ), and biotic and abiotic conditions. these phenotypes vary from the classic disease symptoms to host benefitting-qualities such as drought or cold tolerance (roossinck b) . the context of the plant hosts can effect the spread and diversity of plant viruses more than the composition of the plant host species. competition between bydv and cydv was altered by changing the nutrition resources in the form of nitrogen and phosphorous for their hosts (lacroix et al. ) . in a b/cydv survey in north american pacific coast grasslands targeting three different host species (avena fatua, elymus glaucus, and bromus hordeaceus) virus prevalence was determined not only by host species identity, with a. fatua having the highest prevalence and b. hordeaceus having the lowest, but also by biotic and abiotic factors, including an increase of virus prevalence with a decrease in precipitation and increase in soil nitrogen (seabloom et al. ) . many viruses that are found in wild plants have either mild symptoms or are completely asymptomatic (prendeville et al. ; jones ; davis et al. ) . many wild plants host multiple viruses, in some cases up to seven different viruses were found co-existing in a single plant . in two studies in the united states and costa rica, over % of the virus sequences found in wild plants belonged to three virus families: partitiviridae, chrysoviridae, and totiviridae (roossinck b). these virus families, along with the endornavirdae, have been described as persistent plant viruses (roossinck (roossinck , a . most of the persistent plant viruses are double stranded rna viruses, although the endornaviridae are likely single-stranded rna viruses that are isolated as replicative intermediates (roossinck et al. ) . most interestingly, the persistent viruses are wholly transmitted vertically, with no known form of horizontal transmission. in fact, there is no cell-to-cell movement of persistent viruses, spreading throughout the host via cell division (roossinck (roossinck , a . the partitiviridae, endornaviridae and chrysoviridae infect both plants and fungi, while the totiviridae also infect fungi and protozoa. while there is no observable effect of these viruses on their hosts, there have been multiple instances of integration of some persistent viral genomes into plant and fungal genomes (liu et al. ; chiba et al. ). this should not be surprising given the intimate symbiotic nature of the relationship. currently the persistent viruses are understudied and many aspects of their nature are unknown (roossinck a) . in a study looking at the effect of both cmv and zymv in wild populations of cucurbita pepo, the context of the host population, either adjacent to a road, within a managed peanut field, or within an unmanaged pasture, seemed to be the dominate factor in whether zymv was detrimental, beneficial or neutral, respectively (prendeville et al. ) . while latent viruses are common in wild plants, there are of course pathogenic viruses found in the wild as well (cooper and jones it is not surprising that with an in depth look into the viral biodiversity of plants one would find novel viruses related to known viruses, nor is it surprising that sequences with little or no similarity to anything in a curated database would be found. often, even when there are related viruses within a database, the curation is not in a state to be useful. there are few centralized databanks to store metadata collected during large biodiversity surveys, though attempts have been made. metavir, a website service offering basic analysis of viromes, allows for viromes to be made public, and at the time of writing houses different viromes from different projects (roux et al. ) . it is unclear how these potential viruses should be treated. in a recent virus survey in costa rica, % of the sequence reads received no hit when searched against the genbank database ). for viruses with no known relative, a cluster analysis can give structure to a population of unknown microbes, including viruses (labonté and suttle ) . while having a sequence identity for these viruses can offer some answers, the viruses ultimately need to be characterized experimentally. evidence for genetic variation of plant viruses was reported as early as (kunkel ) . numerous studies have looked at variation both within individual virus isolates and among isolates of the same virus species. variation provides the basis for evolution of traits through natural selection, and has resulted in adaptation of plant viruses to new hosts, to new vectors, and to overcoming host resistance, including natural resistance, resistance introgressed through breeding, or genetically engineered resistance. the high levels of mutation generated by viral polymerases leads to high levels of variation within a single infection, known as a quasispecies. the term quasispecies refers to a single replicating population, and is an "individual" that selection acts upon (holland and domingo ) . do to the many different selective pressures an rna virus experiences (different hosts, cell tropisms, vectors, etc.), a population that is more genetically robust, having a high degree of evolvability may have a selective advantage. this means in a fitness landscape, quasispecies that have a narrow fitness peak (less robust) experience a sharp decrease in fitness due to a single mutation, and are less likely to adapt rapidly to a new environment. conversely, those with a wide fitness peak (more robust) will experience a small change in fitness, allowing for multiple mutations to accumulate for selection to act upon. this is known as the survival of the flattest (wilke ) . it was been thought for some time that the lack of error correction within the rna dependent rna polymerase (rdrp) of rna viruses is responsible for the size of the quasispecies (steinhauer et al. ) . however, in coronaviruses it is clear that error correction can occur (denison et al. ). the quasispecies is effected by not only the mutation rate of the rdrp, but also by the mode of replication, logrithmic or stamping machine (safari and roossinck ) . while double-stranded rna viruses replicate predominantly by the stamping machine method, the mode of replication of other rna viruses is not clear. although the mutation rate of rna viruses are high, the level of variation within the quasispecies may be lower than expected (garcía-arenal et al. ) . this is due to both positive and negative selection; however, defective genomes are often carried in the population and can provide extended function in some cases. while there are significant genetic bottleneck during systemic infection (li and roossinck ) , as well as vector transmission (ali and roossinck ) , viruses probably recover their diversity rapidly. the size of the quasispecies, or level of variation of a virus within a host, is dependent on factors in both the virus and the host. when comparing three related sindbis-like viruses, cmv, tobacco mosaic virus (tmv), and cowpea chlorotic mottle virus each had significantly different levels of variation within the same host background (schneider and roossinck ) . in both tmv and cmv, the level of variation changed based on the host background (schneider and roossinck ) . different strains of cmv, fny and ls, display different levels of diversity in tobacco and pepper plants, which maps to both the a and a proteins (pita and roossinck b) . by using a non-coding satellite rna the indel fidelity of the cmv rdrp was analyzed in planta. while insertion mutations were rare, deletion mutations were more abundant and their rates differed based on the host background and the sequence context (pita et al. ) . ngs is being used to identify minor variants within quasispecies. with a level of coverage of x, the full range of variation can be uncovered (simmons et al. ) . this type of analysis can lead to answers to previously difficult questions of quasispecies dynamics in nature. mutations within the zymv quasispecies were maintained through the aphid vector transmission, as well as seen throughout the plant, suggesting that the bottleneck of vector transmission and movement throughout the plant may be lower than previously thought (simmons et al. ) . there is evidence that some variants within a quasispecies are necessary for specific functions. several zymv variants were found in different seed transmitted lines, suggesting that these variants have a role to play in seed transmission (simmons et al. ) . randomly generated point mutations in tobacco etch virus (tev) were used to determine the effect the mutations had on the virulence and fitness of tev. the majority of the mutations were lethal, with the majority of non-lethal mutations leading to a significant reduction of fitness. (carrasco et al. ). the lab strain of tev is adapted to tobacco, but when tev was adapted to pepper, virulence increased, but was found to decrease in the tobacco host, suggesting a tradeoff in becoming more specialized. no tradeoff was found for becoming more of a generalist (bedhomme et al. ; elena et al. ) . furthermore, pepper-adapted tev acquires mutations that have a wide range of effects both positive and negative, implying pleiotropic effects. interestingly, the fitness of mutants in the tobacco host does not predict the fitness in other non-native hosts (lalic et al. ) . by passaging plum pox virus (ppv; m strain) though several different host species for six years and analyzing the fixed mutations after host adaptation, it was found that peach yielded the lowest number of fixed mutations (two fold lower than other hosts). this suggests that ppv-m is highly adapted to peach (vozárová et al. ) . passaging pepino mosaic virus (alphaflexiviridae) through several tomato cultivars with varying degrees of tolerance, convergently leads to isolates with higher pathogenicity. these passages also have an increase in the genetic diversity, with genetic diversity being a good indicator of pathogenicity (minicka et al. ) . previously it was thought that the high levels of variation within the begomoviruses (circular ssdna) was due to high levels of recombination (lima et al. ), but begomovirues have substitution rates much higher than other dna viruses, on the order of À substitutions/site/year, in line with rates seen in rna viruses (duffy and holmes ) . macroptilium yellow spot virus (maysv) and tomato severe rugose virus (tosrv), both begomoviruses, were analyzed for their variability. interestingly, maysv, which primarily infects wild weeds, but does occasionally infect phaseolus vulgaris (the common bean), had a greater diversity than tosrv, which primarily infects tomato, and has a low incidence in wild plants. several recombination events were detected for maysv, which drove the majority of the variability in the species. recombination is not only an important part of population variation, but also can be used as a repair mechanism, balancing the high mutation rate of rna viruses (nagy and simon ) . recombination is a frequent occurrence in camv, with over % of isolates being recombinants after only days of infection (froissart et al. ) . rna of bromoviruses may contain recombination hotspots (bruyere et al. ) . while recombination is an important part of increasing the variation of a species, recombination events that lead to hybrid proteins are most likely less fit than recombinants between whole genes or protein domains (bonnet et al. ) . recombination offers a path for the adaptation of viruses to a new environment as seen with the introduction of tylcv into spain (garcía-andrés et al. ). interestingly, it appears the eukaryotic hosts may have adapted a method for modulating or regulating the degree of recombination of their infecting viruses. in a yeast model system modified to allow infection by tomato bushy stunt virus, xrn , a host exoribonuclease, was found to degrade truncated viral rna. these truncated rnas are substrates for recombination (cheng et al. ) . recombination is commonly found in the ssdna geminiviruses, both within and between different species (padidam et al. ; pagán and holmes ) . citrus tristeza virus (ctv, closteroviridae) is interesting in that many strains of the virus are commonly found within a single host plant. these strains have been phylogenetically analyzed to elucidate their evolutionary history, and it can be inferred that the current diversity of ctv was influenced by the original ancestral diversification, selection pressure of genes between and within strains, and significant recombination among strains (harper ) . a number of studies have looked at recombination frequencies in experimental systems (bujarski ; sztuba-solinska et al. ) , especially in the bromoviridae. in general, recombination frequencies are high in rna viruses, and hot spots for recombination have been identified that result in exchanges between related rna molecules, or in deletions leading to defective rnas. in the cucumoviruses experimental infections with interspecific reassortants have frequently led to recombinants in rna , where the end is exchanged with that of rna , presumably to establish a minus strand promoter that is cognizant with the replicase (aaziz and tepfer ; dewispelaer et al. ; pita and roossinck a) . in a recent study different strains of cmv had very different frequencies of recombination. interestingly, the high recombination strain was the same strain that had low mutation frequency, and the a protein that encodes the rdrp was responsible for both phenotypes (pita et al. ) . the deep evolutionary history of viruses is a matter for considerable speculation. since no virus fossils are available, studies have relied on comparisons of extant sequences; however, the recent explosion of complete genome information for plants and many other hosts has led to the development of a new field of virology known as paleovirology, which considers the viral sequences integrated into genomes as molecular fossils (katzourakis ). the majority of known plant viruses are rna viruses. the origins of rna viruses are thought come directly from the ancient rna world, a time before cellular life, where rna replicated itself without a dna phase (bernhardt ) . it was proposed three decades ago that animal and plant viruses have a common ancestor, likely an insect virus (goldbach ) . specific motifs in virus hallmark genes (genes which are unique to viruses and are found across many families of viruses) such as the rdrp were analyzed for similarity across a wide range of animal and plant rna viruses, with positive, negative, and double-stranded genomes. motifs in both the positive and double-stranded rna viruses suggests that these groups are monophyletic, with the negative strand rna viruses less likely to be monophyletic . virus hallmark genes are shared with other selfish genetic elements, such as plasmids and transposons, suggesting that viruses have a deep lineage, which some suggest dates to a pre-cellular time (koonin and dolja ) . one can think of viruses as both an organism and a mobile genetic element, much as light can be thought of as both a particle and a wave. these entities evolve within their environment, and then move as genetic elements through higher organisms (forterre and prangishvili ) . indeed examples of viral elements being incorporated into their host's genome can be found across all of the tree of life (katzourakis ) . a high degree of recombination and/or reassortment of genetic material allows for modular evolution, where genes, protein motifs, or separate rna molecules will evolve independently from each other. within the luteoviruses, recombination is often seen near the gene borders of the rdrp and the coat protein (cp) genes but rarely within genes. this suggests that the genetic histories of these genes are independent of each other (pagán and holmes ) . phylogenetic analysis of cmv genes implies that each of the viruses three rna segments have unique histories (roossinck ) . the extent of movement of genetic material across viruses can be seen with the recently named amalgaviridae, a double-stranded rna monopartite virus with open reading frames, an rdrp and another gene. the rdrp most closely resembles that of another double-stranded rna virus family, the partitiviridae, while the other gene resembles that of the nucleoprotein of negative-stranded rna viruses of the bunyaviridae family . the iconic movement protein common and unique to plant viruses, may have been originally derived from the structural proteins used in the formation of the plasmodesmata (lucas and wolf ) . while there is a lot of movement of genetic material between viruses and their eukaryotic hosts, this movement is vastly biased towards viral genes being moved to their hosts; hence viruses have a major role in the evolutionary history of higher organisms (forterre and prangishvili ) . it is extremely difficult to extract preserved viral nucleic acid from more than a few decades ago, though it is possible (roossinck, unpublished results) . because of this difficulty, these rare integration events can be used to elucidate the life histories of the virus. long before so many virus sequences were found in genomes, geminivirus sequences were found within the nicotiana genome (bejarano et al. ) . begomoviruses have been described as being old world or new world, with several distinct qualities associated with each group. most notably, the new world begomoviruses are monopartite while the old world are bipartite. using the intergration events within nicotiana, an estimate of - mya was found for the old/new world split, suggesting that this virus crossed the beringian land bridge (lefeuvre et al. ) . cooperation of viruses in mixed infections may be the initial step towards multipartiate viruses. two different monopartite viruses are known to cooperate, using each other's proteins for their own function. if the relationship of the two viruses becomes too dependent, essential gene loss can occur in one or both viruses that may remain viable due to the complementary gene of the other virus. this will eventually lead to merging the two species into a single species (shirogane et al. ). a recent phylogenetic analysis of the potyvirus genus suggests that the genus diverged approximately years ago, in monocots from the southern eurasia or northern african regions (gibbs et al. ) . some zymv lineages have been shown to be no older than years old, suggesting that humans had a role to play in there movement and diversification (simmons et al. ) . lueteoviridae diversification happened in three stages; the luteo/polerovirus split estimated at years ago, diversification of each genus estimated at - years ago, and the diversification of the species within the past years (pagán and holmes ) . the knowledge gained from extensive sampling including wild samples give us more insight into the life histories of viruses (wylie et al. ) . potyviruses have a large amount of diversity within and between their species. yam mosaic virus (ymv) is thought to have been originated in africa. high levels of recombination are found within natural populations of ymv (bousalem et al. ) . the endogenization of caulimoviruses distantly related to rice tungro bacciliform virus into the rice genome have given us insight into the family's evolutionary history. the edongenization events occured before the divergence of the domestic rice progenitor oryza rufipogon, placing this event at about , years ago (chen et al. ). the diversity of plant viruses is still largely unknown, but what we have learned is that the virus diversity of agriculture systems is vastly different than that of natural ecosystems. while the majority of viruses infecting crops cause disease, it appears that viruses in natural areas are neutral or may provide some small benefit to their hosts. this paradigm shift opens the door to many future applications, as well as exciting implications to the field of virology as a whole. understanding the mechanisms and consequences of movement across the agro-eco boundary, as well as an increased understanding of the mechanisms underlying virus evolution, may provide us with methods of predicting future epidemics, or attenuating the outbreak of new crop pathogens. the modern era of genomics is revealing new and exciting areas of research into virus evolution, and studies on the origins of viruses will likely lead to an understanding of the very origins of life on earth. recombination between genomic rnas of two cucumoviruses under conditions of minimal selection pressure genetic bottlenecks during systemic movement of cucumber mosaic virus vary in different host plants emerging infectious diseases of plants: pathogen pollution, climate change and agrotechnology seeking agricultures ancient roots crop pests and pathogens move polewards in a warming world multihost experimental evolution of a plant rna virus reveals local adaptation and host-specific mutations concerning a contagium vivum fluidum as cause of the spot disease of tobacco leaves integration of multiple repeats of geminiviral dna into nuclear genome of tobacco during evolution the rna world hypothesis: the worst hypothesis of early evolution of life (except for all the others) role of recombination in the evolution of natural populations of cucumber mosaic virus, a tripartite rna plant virus high genetic diversity, distant phylogenetic relationships and intraspecies recombination events among natural populations of yam mosaic virus: a contribution to understanding potyvirus evolution frequent homologous recombination events between molecules of one rna component in a multipartite rna virus genetic recombination in plant-infecting messenger-sense rna viruses: overview and research perspectives a real-time rt-pcr assay for quantifying the fitness of tobacco etch virus in competition experiments rice genomes recorded ancient pararetrovirus activities: virus genealogy and multiple origins of endogenization during rice speciation suppression of viral rna recombination by a host exoribonuclease widespread endogenization of genome sequences of non-retroviral rna viruses into plant genomes wild plants and viruses: underinvestigated ecosystems environmentally dependent host-pathogen and vector-pathogen interactions in the barley yellow dwarf virus pathosystem an rna proofreading machine regulates replication fidelity and diversity a map of the diversity of rna recombinants appearing in plants infected with cucumber mosaic virus and tomato aspermy virus phylogenetic evidence for rapid rates of molecular evolution in the single-stranded dna begomovirus tomato yellow leaf curl virus experimental evolution of plant rna viruses molecular ecology and emergence of tropical plant viruses insect vectors as drivers of plant virus emergence the major role of viruses in cellular evolution: facts and hypotheses recombination every day: abundant recombination in a virus during a single multi-cellular host infection founder effect, plant host, and recombination shape the emergence populations of begomoviruses that cause tomato yellow leaf curl disease in the mediterranean basis variation and evolution of plant virus populations time--the emerging dimension of plant virus studies molecular evolution of plant rna viruses mixed infections of pepino mosaic virus strains modulate the evolutionary dynamics of this emergent virus plant feeding by insect vectors can affect ife cycle, population genetics and evolution of plant viruses citrus tristeza virus: evolution of complex and varied genotypic groups plant virus ecology: ingredients, interactions and environmental influences virus variation in relation to resistance-breaking in plants origin and evolution of viruses consequences of interspecies hybridization and virus infection on the growth and fecundity of three threatened coastal lepidium (brassicaceae) species from new zealand plant viruses alter insect behavior to enhance their spread plant virus ecology and epidemiology: historical perspectives, recent progress and future prospects paleovirology: inferring viral evolution from host genome sequence data impacts of biodiversity on the emergence and transmission of infectious diseases virus world as an evolutionary network of viruses and capsidless selfish elements origins and evolution of viruses of eukaryotes: the ultimate modularity plant viruses of the almagaviridae family evolved via recombination between virusese with double-stranded and negative-strand rna genomes variation in phytopathogenic viruses previously unknown and highly divergent viruses populate the oceans environmental nutrient supply alters prevalence and weakens competitive interactions among coinfecting viruses effect of host species on the distribution of mutational fitness effects for an rna virus comparative molecular epidemiology provides new insights into zucchini yellow mosaic virus occurrence in france evolutionary time-scale of the begomoviruses: evidence from integrated sequences in the nicotiana genome genetic bottlenecks reduce population variation in an experimental rna virus population synonymous site variation due to recombination explains higher genetic variability in begomovirus populations infecting non-cultivated hosts widespread horizontal gene transfer from double-stranded rna viruses to eukaryotic nuclear genomes plasmodesmata: the intercellular organelles of green plants virus infection in remnant native bunchgrasses from invaded california grasslands a virus responds instantly to the presence of the vector on the host and forms transmission morphs current impact and future directions of high throughput sequencing in plant virus diagnostics transmission mechanisms shape pathogen effects on host-vector interactions: evidence from plant viruses infection of host plants by cucumber mosaic virus increases the susceptibility of myzus persicae aphis to the parasitoid virus infection influences host plant interactions with non-vector herbivores and predators pathogen population genetics, evolutionary potential, and durable resistance molecular evolution of pepino mosaic virus during long-term passaging in different hosts and its impact on virus evolution new insights into the mechanisms of rna recombination exploring the diversity of plant dna viruses and their satellites using vector-enabled metagenomics on whiteflies possible emergence of new geminiviruses by frequent recombination long-term evolution of the luteoviridae: time scale and mode of virus speciation effect of biodiversity changes in disease risk: exploring disease emergence in a plant-virus system fixation of emerging interviral recombinants in cucumber mosaic virus populations mapping viral functional domains for genetic diversity in plants environment determines fidelity for an rna virus replicase mutation and recombination frequencies reveal a biological contrast within strains of cucumber mosaic virus community ecology of plant viruses. in: roossinck mj (ed) plant virus evolution pathogen spillover in disease epidemics virus infections in wild plant populations are both frequent and often unapparent effects of virus on plant fecundity and populations dynamics plant species diversity for sustainable management of pests and diseases in agroecosystems: a review evolutionary history of cucumber mosaic virus deduced by phylogenetic analyses lifestyles of plant viruses persistent plant viruses: molecular hitchhikers or epigenetic elements? in: witzany g (ed) viruses: essential agents of life plant virus metagenomics: biodiversity and ecology plant virus ecology evolution of persistent viruses in plants plants, viruses and the environment: ecology and mutualism ecosystem simplification, biodiversity loss and plant virus emergence ecogenomics: using massively parallel pyrosequencing to understand virus ecology the remarkable evolutionary history of endornaviruses plant virus metagenomics: advances in virus discovery metavir : new tools for viral metagnome comparison and assembled virome analysis the role of viruses in biological invasions: friend or foe? population dynamics of cucumber mosaic virus in melon crops and in weeds in central spain how does the genome structure and lifestyle of a virus affect its population variation? outcomes of co-infection by two potyviruses: implications for the evolution of manipulative strategies evolutionarily related sindbis-like plant viruses maintain different levels of population diversity in a common host genetic diversity in rna viral quasispecies is controlled by host-virus interactions viral diversity and prevalence gradients in north american pacific coast grasslands viruses of white clover in pastures of pennsylvania cooperation: another mechanism of viral evolution rapid evolutionary dynamics of zucchini yellow mosaic virus rapid turnover of intra-host genetic diversity in zucchini yellow mosaic virus deep sequencing reveals persistence of intra-and inter-host genetic diversity in natural and greenhouse populations of zucchini yello mosaic virus transgenic virus-resistance in crop-wild cucurbita pepo does not prevent transmission of zucchini yellow mosaic virus lack of evidence for proofreading mechanisms associated with an rna virus polymerase plant virus metagenomics: what we know and why we need to know more e-probe diagnostic nucleic acid analysis (edna): a theoretical approach for handling of next generation sequencing data for diagnostics the origins of plant pathogens in agro-ecosystems rna-rna recombination in plant virus replication and evolution the role of weeds and wild plants in the epidemiology of plant virus diseases cropping practices and virus spread plant virus epidemiology: the concept of host genetic vulnerability virus disease in wheat is predicted to increase with changing climate incidence and some ecological aspects of cowpea severe mosaic virus in two cropping systems in costa rica chloroviruses: not your everyday plant virus the potential of plant viruses to promote genotypic diversity via genotype x environment interactions plum pox virus accumulates mutations in different genome parts during a long-term maintenance in prunus host plants and passage in nicotiana benthamiana virus impact at the interface of an ancient ecosystem and a recent agroecosystem: studies on three legume-infecting potyviruses in the southwest australian floristic region quasispecies theory in the context of populations genetics plant virus biodiversity and ecology aphid behavioral responses to virus-infected plants are similar despite divergent fitness effects phylogenetic analysis of bean yellow mosaic virus isolates from four continents: relationship between the seven groups found and their hosts and origins key: cord- -biop gyd authors: ali, muhammad; khan, tariq; fatima, kaneez; ali, qurat ul ain; ovais, muhammad; khalil, ali talha; ullah, ikram; raza, abida; shinwari, zabta khan; idrees, muhammad title: selected hepatoprotective herbal medicines: evidence from ethnomedicinal applications, animal models, and possible mechanism of actions date: - - journal: phytother res doi: . /ptr. sha: doc_id: cord_uid: biop gyd insight into the hepatoprotective effects of medicinally important plants is important, both for physicians and researchers. main reasons for the use of herbal medicine include their lesser cost compared with conventional drugs, lesser undesirable drug reactions and thus high safety, and reduced side effects. the present review focuses on the composition, pharmacology, and results of experimental trials of selected medicinal plants: silybum marianum (l.) gaertn., glycyrrhiza glabra, phyllanthus amarus schumach. & thonn., salvia miltiorrhiza bunge., astragalus membranaceus (fisch.) bunge, capparis spinosa (l.), cichorium intybus (l.), solanum nigrum (l.), sapindus mukorossi gaertn., ginkgo biloba (l.), woodfordia fruticosa (l.) kurz, vitex trifolia (l.), schisandra chinensis (turcz.) baill., cuscuta chinensis (lam.), lycium barbarum, angelica sinensis (oliv.) diels, and litsea coreana (h. lev.). the probable modes of action of these plants include immunomodulation, stimulation of hepatic dna synthesis, simulation of superoxide dismutase and glutathione reductase to inhibit oxidation in hepatocytes, reduction of intracellular reactive oxygen species by enhancing levels of antioxidants, suppression of ethanol‐induced lipid accumulation, inhibition of nucleic acid polymerases to downregulate viral mrna transcription and translation, free radical scavenging and reduction of hepatic fibrosis by decreasing the levels of transforming growth factor beta‐ , and collagen synthesis in hepatic cells. however, further research is needed to identify, characterize, and standardize the active ingredients, useful compounds, and their preparations for the treatment of liver diseases. liver disorders have been classified in the high priority areas of health care. according to an estimate by the world health organization, approximately million people of the world are suffering from a severe form of liver disorders, that is, chronic hepatitis (al-asmari et al., ) . medicine of herbal origin may serve as a feasible therapy for the prevailing liver problems because of their safety, easier availability, cost effectiveness, and environment friendliness (izzo, hoon-kim, radhakrishnan, & williamson, ) . medicinal plants have acquired importance in healthcare system throughout the world for their proven and effective therapeutic properties (helmstädter & staiger, ). an estimated % of the world's population is relying on medicines that contain compounds of herbal origin (ekor, ) . the international union for conservation of nature has suggested that approximately , to , flowering plants are used for medicinal purposes (chen, li, ren, & hu, ) . many factors regarding these medicines are important. herbal medicines are claimed to both treat and prevent diseases, which adds to a deep belief that these abbreviations: alt, alanine aminotransaminase; asp, angelica sinensis polysaccharides; ast, aspartate transaminase; egf, epidermal growth factor; hbv, hepatitis b virus; lbps, lycium barbarum polysaccharides; wf , woodfordia fruticosa flower extract. treatments are safe because they are "natural and gentle" and therefore, a harmless alternative to the conventional medicine. moreover, the latter may sometimes cause disappointing results and undesirable side effects in patients (izzo et al., ) . in addition, the less expensive herbal products are often not subject to strict regulations and medication prescribed by a physician or other qualified practitioners (hunter & hegele, ) . although medicinal plants have been used globally, their wider usage is limited to a few countries like japan, india, china, pakistan, thailand, iran, and some african countries (bahmani et al., ; iwu, ; li, ; sivasankari, anandharaj, & gunasekaran, ) . other countries are also encouraging the use of plant-based medicinal products in their healthcare systems. for example, natural health product regulations of canada for the plant-based product in healthcare encourages usage of modern technology and evidencebased scientific support towards promoting medicinal plants and the associated products (tomlinson & akerele, ) . a major concern of scientists investigating herbal treatments is that the chemical composition of the plants contributing to their biological effects is mostly undetermined (ling et al., ). herbs and herbal medicines have been used for the treatment of liver diseases for a long time (dhiman & chawla, ) . there are many herbs having ingredients that are potential sources of medicine for the treatment of liver diseases having various modes of actions and bioactivities (babu, bhuvaneswar, sandeep, ramaiah, & rajendra, ; gnanadesigan, ravikumar, & anand, ; pereira, barros, & ferreira, ) . however, several of them are well-studied for their bioactive components and the mechanism of hepatoprotective activity. in the current review, we have selected some of these compounds for which elaborate detail about hepatoxicity is available in literature in the form of either in vivo studies, study into biochemical parameters and bioactive compounds. this article highlights the possible ways of inducing hepatoxicity in mice models and encompasses the mechanisms in which certain medicinally important plants perform their hepatoprotective activity. the article further aims to summarize studies conducted on the composition, pharmacology, and nature of the selected plants in the light of possible mechanism deduced from experimental trials. a thorough search was conducted on the electronic literature databases, google scholar, pubmed, scopus, and web of science. literature was retrieved using the key words and phrases "hepatitis c", "hepatoprotective activity", "mechanism of action", "medicinal plants", "herbal", and "treatment". about relevant articles were extracted after a narrow search for a combination of the keywords and subsequent analysis per the inclusion criteria. there were two sets of criteria applied to articles for inclusion in this manuscript. according to the first, "general criteria", articles selected for this manuscript were those which (a) reported plants and their parts that were traditionally applied to hepatitis and liver disorders and any other type of hepatoprotective activity; (b) reported extract or pure compounds important for their hepatoprotective role; and (c) attempted to explain the mechanism of hepatoprotective action of these plants. the nd criteria were used for selecting those plants that are discussed in detail (shown in figure ). for this purpose, seventeen plants were selected for which recent articles were available that (a) studied in vitro and in vivo hepatoprotective activities of herbal products, (b) reported active compounds from the plant, and (c) described the mechanism of action herbal hepatoprotective products. the plants described in detail were selected if literature available for them fulfilled at least two of the above -point criteria. each of the selected plants was discussed; mainly focusing its hepatoprotective activities, active compounds, and possible mechanism of action. in addition, featured hepatoprotective herbal combinations have been deliberated. toxicity and quality control issues associated with these herbs/herbal products have been debated. two of the authors independently reviewed all the full-text articles obtained during the electronic search. data from the eligible articles were extracted; all the disagreements were discussed and were referred to a third reviewer (one of the author) for a final decision. all the data were extracted in two tables (tables and ) , and the mechanisms of action were explained in respective subheadings and demonstrated through four different figures (figures - , and ). the figures were constructed in chembiodraw ultra (version . ) software package. furthermore, the plant database "plant list" was used for the taxonomic categorization of all the documented plant species (theplantlist, ) . the prerequisite to screen/study any medicinal compound for its hepatoprotective activity is to develop a model (animal model or cell culture model) in which hepatic injury is induced (salehi, karegar-borzi, karimi, & rahimi, ) . several studies have manipulated mice models to induce hepatotoxicity and then treat those induced liver diseases using herbs and herbal products (figure ). this approach provides insight into how hepatitis and other liver diseases are caused. however, for many plants, their mechanism of action against hepatotoxic agents is not well-documented. the common prototype applied for hepatoprotective drug screening is the carbon tetrachloride (ccl ) induced hepatic injury (rodrigues et al., ) . as ccl have been reported for its damaging effects on the liver because on metabolism by p , it produces free radicals (johnston & kroening, ) . these free radicals cause lipid peroxidation by binding to dna, proteins, or lipids (yasuda, izumi, shimada, kobayakawa, & nakanishi, ) . the degree of hepatic injury is evaluated by the higher level of biochemical parameters that is ascribed to the production of trichloromethyl free radicals which eventually causes lipids peroxidation present in cellular (dusheiko, ) calotropis procera (aiton) dryand. crude hydro-ethanol solution extract prevents of the depletion of gsh levels. c. procera contains flavonoids thus it also performs the antioxidant activity (mcomish et al., ) clerodendrum abilioi r. fern. ethanol extract decreased the serum enzyme alt, ast, alp, tgl, and total cholesterol and considerably increased the glutathione level (chamberlain, adams, saeed, simmonds, & elliott, ) ficus carica l. leaves crude petroleum ether extract reduction in the levels of alt and ast. the petroleum ether extract of ficus leaves repair the damaged liver cell (gond & khadabadi, ) glycyrrhiza uralensis -glycyrrhizin glycyrrhizin administered in plc/prf/ cells suppressed the secretion of hbsag into the culture medium and concluded that glycyrrhizin modifies the intracellular transport and the surface nature of the hepatocytes (sato et al., ) momordica dioica roxb. ex willd. alkaloids, phenolic compounds, glycosides, flavonoids oral administration of the extract significantly normalized and restored the elevated serum enzymatic levels of ast, alt, salp, and total bilirubin. its hepatoprotective activity is due to the antioxidant and free radical scavenging activity. (surai, ) nelumbo nucifera gaertn. leaves catechin glycoside, myricitrin- -o-glucoside, hyperin, isoquercitrin, quercetin- -o-rhamnoside, astragalin lotus leaf extract possess significant hepatoprotective and antioxidant activity in ccl -induced toxicity rat model. free radicalscavenging and antioxidant activity due to the presence of some flavonoids and phenolic compounds results in the hepatoprotective activity. (theplantlist, ) paeonia lactiflora pall. and a. membranaceus (fisch.) bunge. -progression of ccl -induced hepatic fibrosis was inhibited in rates by decreasing the level of tumor growth factor-β and inhibit collagen synthesis (sun et al., ) s. miltiorrhiza bunge. roots -s. miltiorrhiza could reverse the ccl -induced fibrosis treatment by decreasing the levels of transforming growth factor-β , procollagens i and iii, and metalloproteinase- and decreasing the levels of metalloproteinase- in liver of the affected rates (wasser et al., ) s. miltiorrhiza bunge. roots s. miltiorrhiza polysaccharides protects liver against immunological injury by adjusting the levels of alanine aminotransferase, aspartate aminotransferase, nitric oxide, tumor necrosis factor and interleukin- (zein et al., ) solanum nigrum l. total decoction crude aqueous extract inhibited thioacetamide-induced collagen (α ) and transforming growth factor-β mrna levels in the liver of mice with thioacetamide-induced liver fibrosis (hsieh et al., ) s. nigrum l. and cichorium intybus l. crude plant extract protect dna against oxidative damage in the reaction mixture containing calf thymus dna and free radical generating system (sultana et al., ) (continues) membrane (chen, yu et al., ) . figure shows the different strategies applied for studying the in vivo effects of induced hepatotoxicity in mice models. the leaves are marked by distinct white "milky" veins that give the plant its common name (theplantlist, ) . historically, s. marianum was used medicinally to treat disorders of the gallbladder, spleen, and liver, but the most important medicinal application of s. marianum is its use as a hepatoprotective herbal treatment and as supportive treatment for chronic inflammatory liver disorders such as hepatitis, cirrhosis, fatty infiltration, and some other forms of liver damages due to toxic chemicals, poisonous mushrooms, and alcohol (freitag et al., ) . the most important component extracted from s. marianum is silymarin (lu, lu, chen, zhang, & wu, ; wu, wang, & que, ) , which is used to treat a variety of liver disorders, including chronic and acute viral or drug/toxin-induced hepatitis, alcoholic liver disease, and liver cirrhosis (lu et al., ) . silymarin is a combination of different ingredients with silibinin as the most active among them (surai, ) . silymarin has been approved for clinical studies in treating the hepatitis c virus infection (ferenci et al., ) . there are many studies on the mechanism of hepatoprotective effects of silymarin. recently, tunca et al. ( ) showed that silymarin has a protective action on pyridine-induced hepatic injury in syrian hamsters. the study concluded that it decreases the metabolic activation of pyridine (by decreasing the cytochromes p a protein concentration) and control the elevation of inducible nitric oxide synthase expression. all these factors play a protective role in liver injury. in another study, farghali, kamenikova, hynie, and kmonickova ( ) concluded that in addition to inhibition of lipid peroxidation, the hepatoprotective activity against thioacetamide-induced hepatotoxicity (khatri, garg, & agrawal, ) tephrosia purpurea (l.) pers. decreased serum aspartate aminotransaminase ( % and %), alanine aminotransaminase ( % and %), gamma glutamyl transpeptidase ( % and %), alkaline phosphatase ( % and %), total bilirubin ( % and %), and liver mda levels ( % and %), and significant improvement in liver glutathione ( % and %) when compared with thioacetamide-damaged rats. (hosseinzadeh & nassiri-asl, ) vitex negundo l. administration of ethanol solution extract of vitex leaf caused a significant decrease in tb, ast, alt, and alp levels in rats. (abdulkarim et al., ) zanthoxylum armatum dc. bark berberine elevated serum enzymatic levels of serum transaminases, alkaline phosphatase. total bilirubin was considerably restored to a normal level. (cha et al., ) note. gpx = glutathione peroxidase; mda = malondialdehyde; ast = aspartate transaminase; alt = alanine aminotransaminase; ccl = carbon tetrachloride; sod = superoxide dismutase; gsh = glutathione; tb = total bilirubin; alp = alkaline phosphatase hbsag = hepatitis b surface antigen; tgl = triglyceride lipase. inhibition of the increased intracellular ca i plays a critical role in the hepatoprotective effect of silymarin. although, upadhyay, kumar, and singh ( ) showed that silymarin restores the changes in the expression and activity of cytochrome p (cyp) enzymes (cyp a , cyp a , and cyp e ), glutathione-s-transferase, glutathione reductase and glutathione peroxidase, and lipid peroxidation in male swiss albino mice. glycyrrhizin administered in plc/prf/ cells suppressed the secretion of hbsag into the culture medium and concluded that glycyrrhizin modifies the intracellular transport and the surface nature of the hepatocytes glycyrrhizin administered intraperitoneally inhibits the lipopolysaccharide-and d-galactosamine-induced liver injury by preventing inflammatory responses and il- production in mice glycyrrhizin inhibited anti-fas antibody-induced hepatitis in mice by acting upstream of cpp -like protease administration of glycyrrhizin or glycyrrhetinic acid, significantly suppressed α (i) collagen gene promoter activation and progression of liver fibrosis induced by repeated ccl injections in transgenic mice (liew, erali, page, hillyard, & wittwer, ; martell et al., ; ogata, alter, miller, & purcell, ; sato et al., ) phyllanthin phyllanthus amarus schum. et thonn. phyllanthin help in restoration of antioxidant potential of rat hepatocytes, level of gsh, and sod and gr activities reduced by ethanol (chirdchupunseree & pramyothin, ) p-methoxy benzoic acid capparis spinosa l. the compound alleviated the enzyme levels increased as result of administration of ccl , and pcl (gadgoli & mishra, ) silymarin silybum marianum (l.) gaertn. silymarin attenuated the rifampicinand/or pyrogallol-induced hepatotoxicity by restoring the alterations in the expression and activity of cyp a and cyp e , glutathione-s-transferase, glutathione reductase and glutathione peroxidase, and lipid peroxidation in male swiss albino mice. silymarin suppresses n-nitrosodiethylamine induced hepatocarcinogenesis by modulating the antioxidant defense status of the animals (farghali et al., ; upadhyay et al., ) note. hbsag = hepatitis b surface antigen; cpp = -kda putative cysteine protease; ccl = carbon tetrachloride; gsh = glutathione, sod = superoxide dismutase; gr = glutathione reductase; cyp = cytochrome p ; pcl = paracetamol. g. glabra is a member of the glycyrrhiza genus (isbrucker & burdock, ) , an ancient genus that contains the most commonly used herbs in chinese traditional medicine (hosseinzadeh & nassiri-asl, ) . glycyrrhiza species are considered among the most important herbaceous plants for a diverse array of pharmacological activities (hosseinzadeh & nassiri-asl, ) . chemical structures of ( ) cryptotanshinone, ( ) phyllanthin, ( ) quercetin, ( ) glycyrrhizin, ( ) silymarin, and ( ) p-methoxybenzoic acid, also known as p-anisic acid. all the images were adopted from ncbi-pubchem with the compound ids; , , , , , and , respectively (pubchem, ) (mao et al., ) . one of the bioactive compounds from p. amarus is phyllanthin, which is a lignan compound and is traditionally applied in the treatment of many liver diseases (hanh, sinchaipanid, & mitrevej, ) . it was shown to have hepatoprotective effects on ethanol-induced oxidative damage in primary culture of rat hepatocytes through its antioxidant activity especially the activities of superoxide dismutase (sod) and glutathione reductase (chirdchupunseree & pramyothin, ) . previously, naaz, javed, and abdin ( ) . | c. intybus l. c. intybus l., commonly known as chicory, belongs to the lactuceae family and is typical mediterranean plant indigenous to western asia, europe, north america, and egypt, which varies in perianth color from white, red to blue (norbaek, nielsen, & kondo, . | s. nigrum l. s. nigrum l., commonly known as "black nightshade", is a species in the family solanaceae ( ccl -induced hepatic necrosis. this hepatoprotective effect might be due to its adjustment of antioxidant activity, detoxification enzymes, and its free radical scavenger effects. in another study, hsieh, fang, and lina ( ) induced liver fibrosis by administering thioacetamide in mice and treated them with distilled water and s. nigrum extract via oral administration for weeks. this treatment alleviated the hepatic hydroxyproline and α-smooth muscle actin protein levels in mice and inhibited thioacetamideinduced collagen and transforming growth factor-β mrna levels in the liver. histological examination of liver also confirmed that this extract reduced the degree of fibrosis caused by thioacetamide treatment which is the probable reason for the reduction of hepatic fibrosis. . | s. mukorossi gaertn. s. mukorossi gaertn., commonly known as ritha or aritha, is abundantly found in india. its fruit is reported to have expectorant, purgative, antidotal, and abortifacient effects. additionally, it is used in epilepsy, extreme salivation, and chlorosis (suhagia, rathod, & sindhu, ) . the saponins extracted from this plant are spermicidal (in vitro) and due to this property, it has been used in contraceptive cream (rastogi & mb, ) . pharmacological studies of s. mukorossi have shown their potential effect as hepatoprotective agents (upadhyay & singh, ) . to assess the hepatoprotective activity of the s. mukorossi, wistar male rats were treated with ccl . administration of ccl to normal rats increased the serum levels of alt, ast, alp, and bilirubin. these enzymes eventually cause damage to the hepatic cells. the ccl -treated liver cells cultured on petri plates were treated with the extracts of s. mukorossi and were reported to alleviate the levels of these enzymes. when histopathological studies of the ccl -treated rats were performed, they showed that it also causes the demolition of architectural configuration of target cells. however, rats that were treated with s. mukorossi presented normal lobular structural design, which shows its reparative properties and thus its hepatoprotective effects. . | g. biloba l. g. biloba l. belongs to family ginkgoaceae (theplantlist, ) . it is one of the significant herbs of the chinese traditional medicine. g. biloba leaf extract has been reported to have therapeutic activities against age-related memory deficit problems, including alzheimer's and dementia; cardioprotective, antiasthmatic, antidiabetic, hepatoprotective, photoprotective effects, dna repair mechanism, antioxidant, and antiinflammatory activities (mohanta, tamboli, & zubaidha, ) . g. biloba has been associated with a strong hepatoprotective activity through numerous studies (parimoo et al., ) . g. biloba amplifies cellular antioxidant protection system consisting of glutathione peroxidase, glutathione s-transferase, glutathione reductase, nonprotein thiols, catalase, and antioxidant enzymes (sod). the binding of an individual part of herbal tracks to that of phosphatidylcholine produces phytosome having better efficacy compared with traditional herbal extracts (naik, pilgaonkar, & panda, ) . rifampicin is an antibiotic widely used in tuberculosis chemotherapy. it has been reported to cause hepatoxicity, the reason of which is unknown as it is always given in combined form with other antibiotics such as isoniazid and ethambutol. wistar albino rats were treated with rifampicin that caused hepatoxicity in them. their blood samples were taken, and assays of their blood samples were performed to know the levels of sgpt, sgot, and alp. the elevated levels of sgot, sgpt, and alp show liver damage as these enzymes escape from the liver into the blood in case of liver damage. with parallel treatment through ginkoselect phytosome® and the standard drug silymarin, the markers enzymes levels in serum were nearly at a normal level or marginally elevated. this suggests the hepatoprotective quality of g. biloba plant. it also elevates total protein levels and albumin, which shows its hepa- with activities against chronic hepatic fibrosis (nitha, prabha, ansil, & latha, ) . it also shows antiinflammatory, antibiotic, antileprosy, and antihelminthic properties (arya et al., ; shoaib et al., ; syed & khan, ) . in an experiment designed to check the hepatoprotective effect of w. fruticosa flower extract (wf ), albino wistar rats were administered with ccl which resulted in the increased level of alp, ast, alt, and lactate dehydrogenase. these enzymes leak from serum into the blood. thus, ccl damage causes loss of enzymes which are responsible for drug metabolism (chandan et al., ) . these rats were administered with wf . the extract reversed the elevated lipid peroxidation and regulated the liver glucose- -phosphate and gsh levels. these results are in line with former information for other hepatoprotective agents . . | v. trifolia l. v. trifolia l., known generally as chaste tree, is a high-value medicinal plant that belongs to the family verbenaceae. its leaves are effective as plaster against pains, infections, and fever. its fruits are used in curing amenorrhea, and the flowers are effective against fever (chan, baba, chan, kainuma, & tangah, ) . the active constituents of this plant are essential oil (kvasnicka, biba, sevcik, voldrich, & kratka, ) , viterifolins, and diterpenes. it also possesses some important pharmacological qualities, that is, antipyretic (rani & sharma, ) , antibacterial (lawitz et al., ) , antiallergic, and antiasthmatic properties (lawitz & gane, ) . medical practitioners use this plant in the treatment of acute jaundice. however, literature study suggested that this plant is not well screened for its hepatoprotective activity. nonetheless, the tribal groups of western ghats use this plant leaf extracts in treating jaundice, and these results give some scientific evidence of hepatoprotective activity. . | s. chinensis (turcz.) baill s. chinensis (turcz.) baill is widely used in traditional and modern chinese medicine for the treatment of many disorders including insomnia, respiratory failure, and weakness. moreover, mental health improving ability along with fatigue reduction property is also validated for s. chinensis in russian medicine (szopa, ekiert, & ekiert, ) . in general, dibenzocyclooctadiene lignans found in s. chinensis are known to exhibit potent hepatoprotective activity (zheng et al., ) . in one of the study of individual lignin, gomisin a was found responsible for the acceleration of hepatocytes proliferation and increase hepatic flow (panossian & wikman, ) . furthermore, elevation of mitochondrial glutathione concentration was found to be linked with γ-schisandrin hepatoprotective mechanism. the increase in vitamin c concentration in the liver of test animals upon treatment with γ-schisandrin also validates its hepatoprotective ability. another individual lignin, schisandrin b was also found to counter oxidative harm to liver tissues (thandavarayan et al., ; xin et al., ) . in one scientific study, the hepatoprotective mechanism against acetaminophen-induced liver injury of six schisandra lignans (deoxyschisandrin, schisantherin a, schisandrin b, gomisin a, schisandrin c, and schisandrin) was elucidated. the hepatoprotective ability of these lignins was found to be associated with inhibition of cytochrome-mediated bioactivation (jiang et al., ) . furthermore, another mechanistic study investigated the hepatoprotective effect of schisandra polysaccharide in nonalcoholic fatty liver disease mice models. the results demonstrate potential down regulation of hepatic lipogenesis genes and lxrα/srebp- c/ fas/acc and srebp- /hmgcr signaling pathways in the liver (wang, song et al., ) . . | c. chinensis lam. c. chinensis lam. also known as chinese dodder is a parasitic plant having diverse traditional medicinal uses as a tonic, sex enhancer, and abortion preventer (zheng, dong, & she, ) . studies also have scientifically validated the hepatoprotective activity of c. chinensis (donnapee et al., ) . yen, wu, lin, and lin ( ) chinensis seeds ethanol solution extract was found to be more effective in rats with acetaminophen-induced hepatotoxicity (yen, wu, lin, cham, & lin, ) . the mechanism of hepatoprotective potential as demonstrated by ethanol solution extract of c. chinensis is proposed to be the elevated activities of antioxidant enzymes. . | l. barbarum l. l. barbarum l. berries are very famous in traditional chinese medicine for the treatment of inflammation, cancer, eye disorders, throat infection, and anemia. the use of these berries has been validated as food and also has gained great importance due to its significant antioxidant potential (cheng et al., ) . the major active components of l. barbarum berries are l. . | a. sinensis (oliv.) diels a. sinensis (oliv.) diels is reported in chinese herbal medicine for the treatment of cardiovascular disease, anemia, and hepatic disorders (bunel, antoine, nortier, duez, & stévigny, ) . the a. sinensis polysaccharides (asp) extracted from a. sinensis roots having the average molecular weight of , da is regarded as a potential active component of a. sinensis that exhibits a wide range of pharmacognostic properties (hsu, tsai, & tsai, ) . the hepatoprotective potential of asp in ccl -induced liver injury and via using ischemia/reperfusion rat is widely established (zhang et al., ) . wang, wen, li, zhang, & yang ( ) the number of hepatoprotective products from plants is ever increasing. in addition to the hepatoprotective role of a general class of phenolics and flavonoids, many studies have defined specific compounds for their preferable role in hepatitis and other liver disorders (shehab et al., ) . some of the important plants and their products are highlighted in figure . among the many different compounds, few are distinguished for their promising role in liver inflammation. we have, therefore, selected six important compounds for their hepatoprotective role (table ) . quercetin, for instance, a major flavonol commonly found most of the plants, is a potent hepatoprotective agent. the first basis of quercetin-based potency has been attributed to the antioxidant activity of this compound. one of the specific mechanisms of quercetin supplementation was established through ethanol-induced cytotoxicity which affects the activity of superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase. for instance, quercetin supplementation restored the glutathione reductase activity which was affected by ethanol that in turn reduced the glutathione content of liver. quercetin supplementation has also been attributed to hepatoprotection against metals, pesticides, drugs, toxins, and viruses (miltonprabu et al., ) . rhein ( , -dihydroxyanthraquinone- -carboxylic acid) is another important hepatoprotective compound extensively found in medicinal herbs, such as rheum palmatum l., cassia tora l., polygonum multiflorum thunb., and aloe barbadensis miller. the mechanism of action through which rhein acts has been described as modulation of cyp enzymes in rat liver, attenuation of total cholesterol and triglyceride levels in serum, and amelioration of glucose and lipid metabolism. similarly, rhein downregulated the levels of serum alt, hyaluronic acid, procollagen type iii, and liver malondialdehyde and inhibited the expression of transforming growth factor beta and alpha-smooth muscle actin in tetrachloride/ethanol-induced liver fibrosis rats . similarly, flavonoids, lignans, terpenoids, and steroids from vitex negundo l. have also been shown to demonstrate hepatoprotective activities (zheng et al., ) . extracts containing these compounds have been shown to improve biochemical and functional parameters and thus alleviate ccl -induced damage in liver rats. negundoside, for instance, which is a glycoside, has been demonstrated to reduce calcium-mediated toxicity and ccl -induced oxidative stress through regulation of calcium homeostasis and decreasing the production of ros and lipid peroxidation (zheng et al., ) . table with their structures given in figure . the present literature is not sufficient to assess the safety of most of the hepatoprotective and liver regenerative herbs and herbal products, as most of the studies focus on their antihepatotoxic effects only. however, some previous experiments on rats show that no adverse effects were observed by administering intraperitoneal injection of the a. membranaceus extracts at . g/kg for days, whereas large doses ( g/kg) of a. membranaceus root extracts resulted in mutagenicity in mice when injected directly into the stomach lining ). in another study, sato et al. ( ) observed no significant toxicity of various concentrations of glycyrrhizin on plc/prf/ cells in vitro. gadgoli and mishra ( ) reported that p-methoxy benzoic acid extracted from c. spinosa was nontoxic at mg/ml when applied to rat hepatocytes in vitro. this supports the claims made in the traditional system of medicine. similarly, silymarin is shown to have a lack of toxicity and side effects even at high doses (upadhyay et al., ) . isbrucker and burdock ( ) reported that no-observed-effect levels for purified glycyrrhizin are in the range of - mg/ kg/day and concluded that current levels of consumption of licorice extract products and glycyrrhizinate are safe. although, several herbals show potential activity for the treatment of acute and chronic liver diseases premarketing drug-testing, and pharmacovigilance is needed as with any other drug. so far, herbals to treat chronic liver diseases should not be recommended outside clinical trials as the evidence supporting its use is insufficient (stickel, patsenker, & schuppan, ) , and publications relevant to the cytotoxicity of medicinal plants should be encouraged (mukhtar et al., ) . moreover, there are issues like approval of the plant products/extracts as a drug from regulatory agencies such as the food and drug administration or any other equivalent agencies. extensive literature survey of hepatoprotective plants clearly indicates that herbal drugs have an enormous potential for the treatment of liver diseases. in this article, we reviewed the scientific merit of selected plants studied for their hepatoprotective mechanism of action. the major hepatoprotective mechanism identified by the majority of the studies is through combating the oxidative stress that damages the liver. we have summarized the effect of extracts and compounds from different herbs on liver injury considering changes in their biochemical parameters. we also presented the possible data available in the literature for different plants regarding their phytochemical constituents. we, therefore, conclude that herbs and herbal preparations are among the most important sources of hepatoprotective and liver regeneration medicines. however, further research is needed to identify, characterize, and standardize the active ingredients, useful compounds, and their preparations for the treatment of liver diseases. moreover, a combination of the traditional herbal medicines with the modern and conventional medicine may be one of the best options for the treatment of liver disorders and other diseases and infections, soon. the importance of medicinal plants can be determined from world health organization's estimates, which states that up to % of the world's population fulfill their healthcare needs from medicinal plants (mukhtar et al., ) . there has been a significant rise in using overthe-counter medicinal plant products containing powerful medicinal drugs and are believed to have to produce progressive effects with reduced side effects. however, therapeutic failures or adverse effects have been observed in many cases as pharmacological mechanisms of the herbal mixtures/preparations are not well-studied. the most important concern involving the use of medicinal plants is to identify and standardize the exact method of preparation of an extract, identification of active ingredients and details of administration (yip & kwan, ) . in this relationship, the screening and characterization of other undiscovered herbal products in traditional medicine is needed. the integration of the therapeutic use of traditional chinese medicinal knowledge with the synthetic and traditional oriental medicinal knowledge is a key area of research (cho & leung, ) . however, medicinal plants cultivated in different geographical regions are believed to differ in therapeutic effects in different diseases and infections. for example, a. membranaceus used in chinese traditional medicine, from certain locality contains more favorable trace elements and fewer harmful trace elements than those from other localities. in this context, the use of new therapeutic strategies based on natural plants may be useful to provide minimal toxicity, higher effectiveness, and a wider therapeutic background for effective manipulation than existing pharmaceutical products (panico et al., ) . authors declare that they have no competing interests. animals not applicable. the current article is a review article and does not contain any studies with human participants performed by any of the authors. hepatitis c virus genotypes and hepatitis g virus in hemodialysis patients from syria: identification of two novel hepatitis c virus subtypes hepatoprotective activity of two plants belonging to the apiaceae and the euphorbiaceae family a review of hepatoprotective plants used in saudi traditional medicine. evidence-based complementary and alternative medicine: ecam a systematic review of treatment response rates in pakistani hepatitis c virus patients; current prospects and future challenges in vivo hepatoprotective activity of the aqueous extract of artemisia absinthium l. against chemically and immunologically induced liver injuries in mice effect of ethanol extract of flowers of vitex trifolia linn. on ccl induced hepatic injury in rats extract of woodfordia fruticosa flowers ameliorates hyperglycemia, oxidative stress and improves beta-cell function in streptozotocin-nicotinamide induced diabetic rats hepatoprotective role of ricinus communis leaf extract against d-galactosamine induced acute hepatitis in albino rats a review of the health effects and uses of drugs of plant licorice (glycyrrhiza glabra l.) in iran nephroprotective effects of ferulic acid, z-ligustilide and e-ligustilide isolated from angelica sinensis against cisplatin toxicity in vitro use of a signature nucleotide sequence of hepatitis c virus for detection of viral rna in human serum and plasma complete nucleotide sequence of a type hepatitis c virus variant, the predominant genotype in the middle east medicinal plants of sandy shores: a short review on vitex trifolia l. and ipomoea pes-caprae hepatoprotective activity of woodfordia fruticosa kurz flowers against carbon tetrachloride induced hepatotoxicity conservation and sustainable use of medicinal plants: problems, progress, and prospects efficiency of transcellular transport and efflux of flavonoids with different glycosidic units from flavonoids of litsea coreana l. in a mdck epithelial cell monolayer model global prevalence of preexisting hcv variants resistant to direct-acting antiviral agents (daas): mining the genbank hcv genome data the effect of lycium barbarum polysaccharide on alcohol-induced oxidative stress in rats an evidence-based update on the pharmacological activities and possible molecular targets of lycium barbarum polysaccharides. drug design protective activity of phyllanthin in ethanol-treated primary culture of rat hepatocytes in vitro and in vivo immunomodulating and immunorestorative effects of astragalus membranaceus herbal products: benefits, limits, and applications in chronic liver disease herbal medicines for liver diseases cuscuta chinensis lam.: a systematic review on ethnopharmacology, phytochemistry and pharmacology of an important traditional herbal medicine summary: antiviral treatment of hepatitis c virus the growing use of herbal medicines: issues relating to adverse reactions and challenges in monitoring safety hepatoprotective efficacy of cichorium intybus l. extract against carbon tetrachloride-induced liver damage in rats silymarin effects on intracellular calcuim and cytotoxicity: a study in perfused rat hepatocytes after oxidative stress injury silibinin is a potent antiviral agent in patients with chronic hepatitis c not responding to pegylated interferon/ribavirin therapy hepatoprotective effect of silymarin (silybum marianum) on hepatotoxicity induced by acetaminophen in spontaneously hypertensive rats antihepatotoxic activity of p-methoxy benzoic acid from capparis spinosa hepatoprotective activity of ceriops decandra (griff.) ding hou mangrove plant against ccl induced liver damage hepatoprotective activity of ficus carica leaf extract on rifampicin-induced hepatic damage in rats ameliorating effects of compounds derived from salvia miltiorrhiza root extract on microcirculatory disturbance and target organ injury by ischemia and reperfusion physicochemical characterization of phyllanthin from traditional use of medicinal agents: a valid source of evidence a biomedical investigation of the hepatoprotective effect of radix salviae miltiorrhizae and network pharmacology-based prediction of the active compounds and molecular targets pharmacological effects of glycyrrhiza spp. and its bioactive constituents: update and review inhibitory effect of solanum nigrum on thioacetamide-induced liver fibrosis in mice inhibitory effect of angelica sinensis extract in the presence of -hydroxypropyl-β-cyclodextrin functional foods and dietary supplements for the management of dyslipidaemia the efficacy of liv- on liver cirrhotic patients: a randomized, double-blind, placebo-controlled first approach. phytomedicin: international journal of phytotherapy and phytopharmacology a review on hepatoprotective and immunomodulatory herbal plants risk and safety assessment on the consumption of licorice root (glycyrrhiza sp.), its extract and powder as a food ingredient, with emphasis on the pharmacology and toxicology of glycyrrhizin handbook of african medicinal plants a critical approach to evaluating clinical efficacy, adverse events and drug interactions of herbal remedies a review on phytochemical and pharmacological properties of litsea coreana hepato-protective effects of six schisandra lignans on acetaminopheninduced liver injury are partially associated with the inhibition of cyp-mediated bioactivation comparison of the nutritional value and biological activities of the acetone, methanol and water extracts of the leaves of solanum nigrum and leonotis leonorus mechanism of early carbon tetrachloride toxicity in cultured rat hepatocytes evaluation of hepatoprotective activity of aerial parts of tephrosia purpurea l. and stem bark of tecomella undulata in vivo glycyrrhizin accelerates liver regeneration and rapidly lowers serum transaminase activities in % partially hepatectomized rats in vitro antioxidant activities of methanol extracts of five phyllanthus species from india. lwt -food science and technology analysis of the active components of silymarin hepatoprotective effects of chinese medicinal herbs: a focus on antiinflammatory and anti-oxidative activities a protein with antiproliferative, antifungal and hiv- reverse transcriptase inhibitory activities from caper (capparis spinosa) seeds sofosbuvir for previously untreated chronic hepatitis c infection simeprevir plus sofosbuvir, with or without ribavirin, to treat chronic infection with hepatitis c virus genotype in non-responders to pegylated interferon and ribavirin and treatment-naive patients: the cosmos randomised study aqueous extract of solanum nigrum inhibit growth of cervical carcinoma (u ) via modulating immune response of tumor bearing mice and inducing apoptosis of tumor cells chinese & related north american herbs: phytopharmacology & therapeutic values a review of recent research progress on the astragalus genus hepatitis c genotyping by denaturing high-performance liquid chromatography hepatoprotective effects of solanum nigrum linn extract against ccl( )-induced oxidative damage in rats a pharmaceutical preparation of salvia miltiorrhiza protects cardiac myocytes from tumor necrosis factor-induced apoptosis and reduces angiotensin ii-stimulated collagen synthesis in fibroblasts hepatoprotective effects of solanum nigrum against ethanol-induced injury in primary hepatocytes and mice with analysis of glutathione s-transferase a synchronized and sustained release of multiple components in silymarin from erodible glyceryl monostearate matrix system hepatoprotective activity of vitex trifolia against carbon tetrachloride-induced hepatic damage dual effects of lipophilic extract of salvia miltiorrhiza (danshen) on catecholamine secretion in cultured bovine adrenal medullary cells the genus phyllanthus: an ethnopharmacological, phytochemical, and pharmacological review hepatitis c virus (hcv) circulates as a population of different but closely related genomes: quasispecies nature of hcv genome distribution geographical distribution of hepatitis c virus genotypes in blood donors: an international collaborative survey hepatoprotective effect of quercetin: from chemistry to medicine phytochemical and medicinal importance of ginkgo biloba l antiviral potentials of medicinal plants hepatoprotective effect of ethanolic extract of phyllanthus amarus schum. et thonn. on aflatoxin b -induced liver damage in mice pharmacological effects of capparis spinosa l neuropharmacological evaluation of ginkgo biloba phytosomes in rodents methanolic extract of woodfordia fruticosa kurz flowers ameliorates carbon tetrachlorideinduced chronic hepatic fibrosis in rats anthocyanins from flowers of cichorium intybus nucleotide sequence and mutation rate of the h strain of hepatitis c virus. proceedings of the national academy of sciences of the united states of america protective effect of capparis spinosa on chondrocytes pharmacology of schisandra chinensis bail.: an overview of russian research and uses in medicine hepatoprotective effect of ginkgo biloba leaf extract on lantadenes-induced hepatotoxicity in guinea pigs preventive effects of a purified extract isolated from salvia miltiorrhiza enriched with tanshinone i, tanshinone iia and cryptotanshinone on hepatocyte injury in vitro and in vivo phyllanthus amarus: ethnomedicinal uses, phytochemistry and pharmacology: a review a review: the pharmacology of isoliquiritigenin extraction, identification, fractionation and isolation of phenolic compounds in plants with hepatoprotective effects neuropharmacological activity of solanum nigrum fruit hepatitis c virus (hcv) genotypes distribution: an epidemiological up-date in europe cytoprotective role of solanum nigrum against gentamicin-induced kidney cell (vero cells) damage in vitro national center for biotechnology information. pubchem compound database glycyrrhizin alleviates experimental allergic asthma in mice the genus vitex: a review compendium of indian medicinal plants potential anti-inflammatory effects of artemisia gorgonum on rat liver injury induced by ccl -erratum. microscopy and microanalysis: the official journal of microscopy society of america flavour profile of capers (capparis spinosa l.) from the eolian archipelago by hs-spme/gc-ms medicinal plants for management of gastroesophageal reflux disease: a review of animal and human studies therapeutic basis of glycyrrhizin on chronic hepatitis b impact of phenolic composition on hepatoprotective and antioxidant effects of four desert medicinal plants scientific investigation of crude alkaloids from medicinal plants for the management of pain an ethnobotanical study of indigenous knowledge on medicinal plants used by the village peoples of thoppampatti ripe fruit of solanum nigrum l. inhibits cell growth and induces apoptosis in mcf- cells herbal hepatotoxicity cichorium intybus: traditional uses, phytochemistry, pharmacology, and toxicology sapindus mukorossi (areetha): an overview evaluation of hepatoprotective effects of helminthostachys zeylanica (l.) hook against carbon tetrachloride-induced liver damage in wistar rats crude extracts of hepatoprotective plants, solanum nigrum and cichorium intybus inhibit free radical-mediated dna damage effects of purified herbal extract of salvia miltiorrhiza on ischemic rat myocardium after acute myocardial infarction effects and mechanisms of extract from paeonia lactiflora and astragalus membranaceus on liver fibrosis induced by carbon tetrachloride in rats silymarin as a natural antioxidant: an overview of the current evidence and perspectives chromatographic profiling of ellagic acid in woodfordia fruticosa flowers and their gastroprotective potential in ethanol-induced ulcers in rats current knowledge of schisandra chinensis (turcz.) baill.(chinese magnolia vine) as a medicinal plant species: a review on the bioactive components, pharmacological properties, analytical and biotechnological studies differences in the hepatitis c virus genotypes in different countries schisandrin b prevents doxorubicin induced cardiac dysfunction by modulation of dna damage, oxidative stress and inflammation through inhibition of mapk/p signaling the plant list version . . published on the internet medicinal plants: their role in health and biodiversity pyridine induction of cytochrome p a , inos and metallothionein in syrian hamsters and protective effects of silymarin pharmacological effects of sapindus mukorossi effect of silymarin on pyrogallol-and rifampicin-induced hepatotoxicity in mouse effects of an extract from phyllanthus niruri on hepatitis b and woodchuck hepatitis viruses: in vitro and in vivo studies in vitro plantlets from alginate-encapsulated shoot tips of solanum nigrum l protection of lethal toxicity of endotoxin by salvia miltiorrhiza bunge is via reduction in tumor necrosis factor alpha release and liver injury angelica sinensis polysaccharide attenuates concanavalin a-induced liver injury in mice ethnobotany, phytochemistry, and pharmacology of the genus litsea: an update schisandra polysaccharide inhibits hepatic lipid accumulation by downregulating expression of srebps in nafld mice salvia miltiorrhiza reduces experimentally-induced hepatic fibrosis in rats enhanced bioavailability of silymarin by self-microemulsifying drug delivery system effects of salvianolic acid a on oxidative stress and liver injury induced by carbon tetrachloride in rats lycium barbarum polysaccharide attenuates alcoholic cellular injury through txnip-nlrp inflammasome pathway schisandrin b attenuates the inflammatory response, oxidative stress and apoptosis induced by traumatic spinal cord injury via inhibition of p signaling in adult rats the protective effect of tinoridine against carbon tetrachloride hepatotoxicity nanoparticles formulation of cuscuta chinensis prevents acetaminopheninduced hepatotoxicity in rats hepatoprotective and antioxidant effects of cuscuta chinensis against acetaminopheninduced hepatotoxicity in rats salvia miltiorrhiza bunge and its active component cryptotanshinone protects primary cultured rat hepatocytes from acute ethanol-induced cytotoxicity and fatty infiltration molecular identification of astragalus membranaceus at the species and locality levels hepatitis c virus genotypes in the united states: epidemiology, pathogenicity, and response to interferon therapy. collaborative study group systematic review of the renal protective effect of astragalus membranaceus (root) on diabetic nephropathy in animal models extraction, chemical analysis of angelica sinensis polysaccharides and antioxidant activity of the polysaccharides in ischemia-reperfusion rats levistilide a inhibits angiogenesis in liver fibrosis via vascular endothelial growth factor signaling pathway phytochemical and pharmacological profile of vitex negundo modern study of traditional chinese medicine schisantherin a protects against liver ischemia-reperfusion injury via inhibition of mitogen-activated protein kinase pathway selected hepatoprotective herbal medicines: evidence from ethnomedicinal applications, animal models, and possible mechanism of actions key: cord- -n sjixg authors: sahoo, sabuj; sarangi, sarmistha; kerry, rout george title: bioprospecting of endophytes for agricultural and environmental sustainability date: - - journal: microbial biotechnology doi: . / - - - - _ sha: doc_id: cord_uid: n sjixg the term endophytes refers to a group of endosymbionts usually bacterium, fungus or interactive bacterium-fungal species residing asymptomatically and grows within plants for at least a part of their life cycle intra- and intercelullarly in the tissues of higher plants without causing any visible manifestation of disease. the endophytes represent a potential source of novel natural and ecofriendly products for medicinal, agricultural and industrial uses with least adverse effect on the environment. the enormous biological diversity coupled with their capability to biosynthesize bioactive secondary metabolites has provided the momentum for the researchers working on endophytes. the present review was undertaken to highlight the biotechnological processes and bioprospection of endophytes as potential antimicrobial agents, secondary metabolites, antibiotics, antagonists against disease causing phytopathogens, cytotoxic, anticancer, insecticidal, antioxidant antiviral compounds andisolation and production of bioactive compounds with potent enzymatic activities. endophyte enhances biodegradation and hydrolysis processes significantly important against pathogenic infection, biotransformation studies and production of compounds with immense industrial applications. the interaction of the endophytic microbiota with the plants are more protected and can withstand the adverse environmental conditions and contribute to plant growth, productivity, carbon sequestration, enhanced phytoremediation efficiencies and amelioration of metal induced toxicity. the strategies governed by the endophytes for efficient production of novel bioactive phytocompounds was comprehensively discussed. the review envisaged the biodiversity, transmission of endophytes, plant endophyte interactions for the production of bioactive compounds for therapeutic, environmental and agricultural sustainability. , which infect some grasses confined to cool regions and the non-clavicipitaceous endophytes (nce), which are widely distributed and found in asymptomatic tissues of non vascular plants, conifers, ferns and angiosperms. however, nce are reported to be restricted to ascomycota and basidicomycota groups (jalgaonwala et al. ; bhardwaj and agrawal ) . symbiotic criteria used to characterize fungal endophytic classes are shown in table . . typically ce occurs on plant shoots where they form systemic intercellular infections. ce are fastidious in culture and are restricted to some grasses that grow in warm and cool season (bischoff and white ) . nce are primarily ascomycetes fungi recovered from land plants, terrestrial eco systems ranging from agro biosystems to biomes, that are widespread from tropic to tundra (arnold ) . the examples of fungal and bacterial endophytes with their host range is shown in tables . and . respectively. bacterial endophytes are the second most studied endophytes after fungi. more than phyla of bacterial endophytes belonging to about genera were reported, most of which belong to the phyla actinobacteria, proteobacteria and firmicutes drugs (jia et al. ) . due the development of sophisticated new versatile pseudo branches of life-sciences such as proteomics, genomics and their subdivided branches such as metabolomics, trancriptomics, it became much easier to determine molecular interaction at the most nano level (rout and sahoo ; pervez et al. ; abdurakhmonov ) . on the basis of these advanced modes of evaluations it is found that the endophytic population may greatly be affected by the age of the plant, genetic variations in the plant, or environmental background of the host plant that lies in it (jia et al. ) . taking the benefits into count, further bioengineer the endophyte with desired efficiency can be designed and incorporated into the host plant to produce the required quantity of the primary, secondary phytocompounds or drugs. apart from the growth of the host plant, proper uptake of the nutrients by the host plant the disease resistance of the host plant against pathogen can also be improved (mei and flinn ) . endophytes which lives inside plants for a part or whole of their life cycle can be transmitted from parents to offspring or between two individual plants in a community. there are basically two types of transmission. vertical transmission is a method of direct transfer from parents to offspring. vertically transmitted fungal endophytes are typically considered clonal and transmit via fungal hyphae penetrating the embryo within the host's seeds (e.g., seed transmitting forms of epichloe) (white et al. ; david and manish ). horizontal transmission is among individual plants in a community. reproduction through asexual or sexual spores leads to horizontal transmission, where endophytes may spread between plants in a population or community (mariusz et al. ) . most fungi are terrestial, growing as hyphae and producing thick walled non motile spores. spores are single-celled propagules which separate from the organism and can get dispersed. in both sexual and asexual transmission fungi produce spores that disperse among plants via air or through animals. a fungal hyphae is a long branching filamentous structure required for its vegetative growth. in vertical transmission endophyte is found in the embryo of infected seed. as the seed germinates, the endophyte grows into emerging leaf and finally grows up the stem and into the seed head of the reproductive plant. mostly bacterial endophytes are originated from rhizosphere or phyllosphere, however some are transmitted through seeds rhizosphere is the narrow region of the soil that is directly influenced by root secretions and associated soil microorganisms. endophytic bacteria have been isolated from monocotyledaneous [miscanthus giganteus, iris pseudacorus, phragmites australis, lythrum salicaria and cladium mariscus], dicotyledonous [gosspium hirsutum, cucumis sativis and beta vulgaris] to herbaceous plants. the root system secrets chemical exudates (flavinoids, amino acids, carbohydrates, organic acids). bacterial endophytes attracted towards these chemicals and by chemotactic movement it gets attached to the root. various molecules like cellulose, pectinase, superoxide dismutase etc. secreted by bacteria to colonize plants. finally bacteria enter through roots and spread to other parts. plant roots exude many organic compounds that stimulates microbial growth and can have major impact on the composition of rhizosphere microbiome (grayston et al. ; miethling et al. ) . endophyte distribution within plants depends on a combination of ability to colonize and allocation of plant resources. root endophytes often colonize and penetrate the epidermis at sites of lateral root emergence, below the root hair zone, and in root cracks (dong et al. ; compant et al. ; zakria et al. ). these colonizers are capable of establishing populations both inter-and intracellularly (hurek et al. ; zakria et al. ) . after initial colonization, some endophytes can move to other areas of the plant by entering the vascular tissues and spreading systemically (compant et al. ; zakria et al. ; david and manish ) . using endophytes labeled with green-fluorescent-protein (gfp), david and manish ( ) demonstrated the transport of the endophytes from seeds into plant roots and tissues, and endophytes injected into stems moved into the roots and rhizosphere, suggesting that there may be a continuing movement of organisms throughout the root microbiome. endopytes which live a part or full of their life cycle inside the host plant and secrete wide variety of compounds essential for the growth of plants and protection from environmental conditions. bioactive compounds are of enormous importance to plants as well as human are produced by endophytes. the phytocompounds secreted by the endophytes acts as biocontrol agent by exerting protective action of the plants from repeated grazing of herbivores on same plant. the compounds produced by endophytes are of immense importance as antibiotics, drugs or medicinal compounds useful for food industry and the compounds of high relevance in research. endohytes are involved in phytoremediation, biodegradation, and nutrient cycling and thus reduces use of pesticides in agriculture and protect our environment from hazardioaus chemicals. summarising the profound applications of endophytes and its impact on plants, human and environment, they have been proved to be a boon and not a ban and have potential to sustain the agriculture in a better way. various application of endophytes by means of which it promote plant growth as well as sustain the environment and agriculture are given below. the efficiency of endophytes to produce novel bioactive compounds with unique structures and bioactivities have proven to be helpful in agricultural sustainability, environmental conservation and ecotoxicological importance is currently being explored to their maximum extent. in lieu of a huge reservoir, these vast potentialities of secondary products, as well as their exploitation for agricultural and environmental benefaction, are scanty. the current development in endophytic research is mainly focused on evaluating endophytic microbial populations inhabiting plants, which enhances plant growth, disease resistance and the ability to tolerate or withstand the external environment. it can be assumed that these humble researches will emerge as a boon and would certainly leave a wide impact on agricultural science, environmental sustainability as well as for the welfare of mankind through their personification in technological advancement in phytological technologies. the impact of endophytes that enhances plant growth, disease resistance, agricultural and environmental sustainability as well as its ecotoxicological importance is shown in table . . nutrient cycling is an important process of balancing of the nutrients and make it available for each ecosystem components. the nutrients are made available by the degradation of biomass by saprophytes and recycled into the environment thus become accessible to living system. endophytes have been reported to have important role in biodegradation of the litter of the inhabitating host plant (muller et al. ; kumaresan and suryanarayanan ; osono osono , korkama-rajala et al. ; fukasawa et al. ; osono and hirose ; promputtha et al. ) . the endophytes colonize with in the plants initially, during biodegradation of litter, and is facilitated through antagonistic interaction of the saprophytes (thormann et al. ; fryar et al. ; terekhova and semenova ) . endophytes orchestrate many ubiquitous roles in the plant growth by colonizing the internal tissue in almost every part of the plant (santoyo et al. ) . moreover, these endophytes could efficiently execute this spectacular attribution of plant growth enhancement via certain important interrelated mechanisms like phytostimulation, phytoimmobilization, phytostabilization, phytotransformation, phytovolatilization, phytofilterastion, biofertilization and biocontrol (conesa et al. ) . phytostimulation is a direct manifestation of plant growth promotion through the up-regulation of phytohormones directly or indirectly (bloemberg and lugtenberg ) . likewise, phytoimmobilization is another remediation technology where contaminants are effectually removed by the means of plants, the intake and sequential release into the soil is further followed by immobilization in either a mineralemended soil or a geomate commonly known as mineral-containingmat (arthur et al. ) . removal of the contaminants from the location is quite sophisticated therefore stabilization provides another important and effective mode of technical amelioration strategy adapted by nature for self-sustenance (perez-de-mora et al. ). together phytoimmobilization and phytostabilization leads to phytotransformation or otherwise known as phytodegradation (sequestration and/or compartmentalization) phytocompounds involve the transformation or deterioration of intricate organic molecules into unadorned or simple molecular form that could be further absorbed into plant tissue in necessary (etim ) . the efficient degradation of contaminants or the xenobiotics, the subsequent release is sometimes regarded as phytoextraction/phytovolatilization where they are efficiently removed out of the plant system into the atmosphere (limmer and burken ) . phytofiltration is a fascinating scheme tailored by the plants where, with the help of certain surface anchorage phytocompounds, contaminants or nutrients are either absorbed/bound to the nonliving part of the plant (biosorptation), or to the root (rhizofiltration), or to the seedlings (blastofiltration) (conesa et al. ) . though there is tremendous advantage in the usage of this phenomenon for the agricultural remediation but till date the advancement of research in the field is scanty. biofertilazation on the other hand is one of the most growing, demanding, advanced and widely researched area of agriculture and environmental science. it is a technique where living microorganisms effectively aggrandize the nutrients and mineral uptake of the plants by means of establishing a symbiotic relationship with the plant and simultaneously sustains the dynamic nature of the soil (roychowdhury et al. ) . and finally, the protection of plant from pathogens before or after the harvest by the help of endophytic microbes is another growing sector of agricultural research where the endophytes produce a bioactive compound or induces the host plant to produce a specific and/ or a group of phytoactive compound that inhibits the growth and survivability of the pathogens (eljounaidi et al. ; shahzad et al. ). the method of removal of wastes and hazardous pollutants from the environment employing micro-organisms is known as bioremediation. endophytes have the potential to breakdown complex compounds. the role of endophytes in bioremediation (mastretta et al. ) resulted in improved biomass production under conditions of stress due to cadmium, thus can withstand higher cadmium concentration when compared to uninoculated plants. the impact of endophytes that enhances plant growth by bioremediation is shown in table . . up-regulation or down-regulation of phytocompounds specifically growth hormones and other bioactive compounds plays an indispensable role in plant growth and development. these phytohormones and bioactive compounds are generally self-induced within the plants in coordination with the time and certain abiotic factors which influences plant sustenance within that particular biosphere. but in some case the modulation of these phytohormones or other bioactive phytocompounds is induced by biotic factors living within the plant in a symbiotic relation as endophytes. these endophytes or endo-rhizosphers are currently been exploited for their capacity to produce exopolysaccharides, growth hormones (indole- -acetic acid), phyto-enzymes ( -aminocyclopropane- -carboxylic acid [acc] deaminase), volatile compounds, osmoregulatory hormone and antioxidants (table . ) (vurukonda et al. ) . phytohormones such as auxins, cytokinins and gibberellic acids are produced by bacterial endophytes (bloemberg and lugtenberg ) . the most highly studied example of phytostimulation involves lowering plant hormone ethylene levels by -aminocyclopropane- -carboxylate deaminase (acc). several endophytesthat release acc deaminase have been shown to increase plant growth including arthrobacter spp. and bacillus spp. in pepper plants (capsicum annuum) as well as pseudomonas putida and rhodococcus spp. in peas (pisum sativum) (sziderics et al. ; belimov et al. ) . higher amounts of bioactive ga . ga and ga that induced maximum plant growth in rice and soyabean varieties by cladosporium sphaerosperrmum, a fungal endophyte isolated from the roots of glycine max (l) merr (humayun et al. ). endophytes also facilitate uptake of important nutrients from soil, water and organic matter for growth and development of plants. phyllobacterium brassicacearum and bacillus subtilis have been evaluated for enhancing the abscisic acid (aba) in arabidopsis thaliana and platycladus orientalis respectively. the up-regulation aba hormone resulted in decrement of leaf transpiration in a. thaliana while increment stomatal conductance in p. orientalis (bresson et al. ; liu et al. ) . p. putida embellished the growth of glycine max by inducing the secretion of the hormone gibberellins. in lavandula dentate the hormone indole- -acetic acid (iaa) was enhanced by b. thuringiensis, which further improved k-contents and proline with simultaneously decrease glutathione reductase and ascorbate peroxidase (armada et al. ). the production of iaa was also enhanced in triticum by a group of microbes namely rhizobium leguminosarum, r. phaseoli and mesorhizobium ciceri (hussain et al. ). in a different study a total of bacterial endophytes were isolated form vitis vinifera l cv. and were tested for plant growth promoting (pgp) abilities along with their effect of a. thaliana was also evaluated. it was found that the endophytes could able to promote ammonia production, phosphate solubilization, iaa and iaa-like molecules biosynthesis, siderophores and lytic enzyme secretion. further twelve effective endophytes mainly belonging to bacillus, mirococcus and pantoa genera were specifically selected for further studies (baldan et al. ) . again root endophytes from different strains of solanum lycopersicum mostly belonging to species of pseudomonas, rhizobium, rhodococcus and agrobacterium were isolated and analyzed for their pgp properties. improvement in the production of organic acids, iaa, acc deaminase and siderophores was observed. the impact was further verified on a. thaliana root growth using vertical agar plate assay (abbamondi et al. ) . in a most recent study, endophytic bacteria were isolated from simmondsia chinensis root of which endophytic bacteria belonging to bacillus sp., streptomyces sp., methylobacterium aminovorans, rhodococcus pyridinivorans and oceanobacillus kimchi were the partial sequencing of s rdna gene. later it was found that of these endophytic bacterial species only two endophytes namely r. pyridinivorans and o. kimchi showed efficient pgp properties (perez-rosales et al. ) . endophytic fungus also imparts phytostimulation by modulating the production of bioactive phytocompounds within their host plant species. this phenomenon was shown in a study where fungal endophytes were isolated from a halophyte suaeda japonica and were identified by internal transcribed spacer (its). their pgp ability was verified by their treatment with waito-c rice seedling and moreover, their secondary metabolites such as bioactive gibberellins (gas) and other inactive gas were detected by hplc and gc-ms sim analysis (you et al. ). in panax ginseng strains of endophitic fungus belong to aspergillus, cladosporium, engyodontium, fusarium, penicillium, plectosphaerella, verticillium and ascomycete species were isolated and investigated for their capacity to produce saponins and ginsenosides which were detected by hplc (wu et al. ) . phoma species isolated from two medicinal plants namely tinospora cordifolia and calotropis procera was evaluated for its pgp activity on zea mays, where it was observed that the fungus indeed showed the ability to promote the plant growth (kedar et al. ) . trichoderma endophytes specifically t. atroviridae, t. polysporum and t. harzianum isolated from the roots of phaseolus vulgaris could able to synthesize and release proteolytic enzymes and phosphate solubilization factors. furthermore most of the active volatile and non-volatile metabolites that had certain stimulatory or inhibitory impact on p. vulgaris seed germination were perticularly released by t. polysporum and t. harzianum (pierre et al. ) . a recent study proves that iaa, acc deaminase and solubilize phosphate secretion enhanced in the plant brassica campestris by mucor species which was identified by s and s rrna its and sequence homology (zahoor et al. ) . apart from endophytic bacteria and fungus other microorganisms such as algae, acticomycetes, protozoa and cyanobacteria also mediate phytostimulation and contribute in host plant growth and development (table . ) (vejan et al. ) . a study on nostoc in crop plants like oryza sativa and triticum under axenic conditions improves the growth and development by modulating the hormones such as iaa (hussain et al. ) . overall it can be stated that endophytic microbes are an effective means of phytostimulants. in the remediation process where in the use of phytocomponents are at the verge of being highly exploited and could be further accelerated in conjugation with the symbiotic endophytes like bacteria, fungus and many other related microorganisms (ma et al. ) . previously substantiation convey, bacterial endophytes as an effective tool that could be implemented in regulation of physiological changes including immobilization of osmolytes, certain micronutrients along with osmotic acclimatization, stabilization of membrane ion conductivity which is directly or indirectly linked with changes in the membrane phospholipid composition (compant et al. ) . phytoimmobilization and transformation ultimately leads to increased ability to sustain or tolerate the stress induced by the abiotic component or factor. plants like elsholtzia splendens and commelina communis which could tolerate or withstand copper concentration, have been reported to encounter an increase in dry weight of the root as well as the shoot when inoculated with endophytic bacteria in comparison to the control (sun et al. ). on the other hand endophytes that are genetically modified convey an additional channel for phyto-associated neutralization of the contaminants and there by ameliorating the stress induced by these contaminants in the site (divya and kumar ) . on the basis s rrna gene sequencing phylogenic analysis reveled of about isolates comprising of proteobacterial genera in the chromium treated plant sample of albizia lebbeck. the proteobacterial genera commonly comprised of bacillus, rhizobium, pseudomonas, xanthomonas, salinococcus and marinomonas (manikandan et al. ) . there are certain multi-metal resistance endophytes, one of such endophyte is achromobacter piechaudii a bacterium isolated from sedum plumbizincicola and characterized by morphological features, biochemical and s rdna sequencing and phylogenic analysis. the bacterial strain portrayed increased level of resistance to cadmium, zinc and lead along with other salutary properties such as solubilization of phosphors and production of iaa and lastly presence of the strain significantly increased the availability of cadmium, zinc and lead in soil (ma et al. ) . effective bioremediation can be generally achieved by constructed wetland vegetated with leptochloa fusca. it was found that, when a combination of three endophytic bacteria namely pantoea stewartii, microbacterium arborescens and enterobacter species used for bioaugmentation, the consortium of the bacteria could enhance the growth of l. fusca and simultaneously contributed to the removal of both organic and inorganic pollutants and also ameliorated the toxicity in the constructed wetland (ashraf et al. ) . fungus on the other side also plays an important role in phytoimmobilization, which is a direct representation of the capacity to tolerate and immobilize pollutants by concurrently increasing the amount of biomass (sudha et al. ) . currently studies are focused on evaluating the capacity of endophytic fungus to induce metal tolerance and partial immobilization in plants. one such example can be cited for the endophytic fungus neotyphodium that enhanced cadmium tolerance and its partial immobilization in infected plants named festuca arundinacea and f. pratensis. additionally it was observed that photochemical efficacy of photosystem ii increased, indicating the reduction of cadmium stress (soleimani et al. a, b) . species of exophiala, metarhizium, promicromonospora and pencillium also showed increased tolerance to metal stress of copper and cadmium. but it was pencillium funiculosum that highly ameliorated biomass yield, chlorophyll and total protein contents in its host plant glycine max l. under cu stress (khan and lee ) . arbuscular mycorrhizal fungus (amf) is a group of mycorrhizal fungus that could penetrate roots through cortical cells of the vascular plants. in triticum the grain and shoot yield was increased under different concentration of zn, cu, fe and mn when inoculated with amf compared to un-inoculated triticum (khan et al. ) . dark septate endophytes (dses) are asexual chlamydospores ascomycetous fungi that inhabits within the living plant root in a symbiotic relationship. one of such endophyte is exophiala pisciphila, which regulates physiological response in z. mays under soil cadmium stress. the mechanism is not clear how such tolerance is achieved by z. mays by the help dse (wang et al. ) . the immobilization of soluble arsenic is another interesting feature displayed by these endophytic fungus. piriformospora indica is one of such fungus the colony of which was isolated from oryza sativa root and was evaluated for its impact on the plant. primarily it was found that hyper-colonization of the fungus that ultimately alleviated biomass density, root amelioration, number of chlorophyll and stabilization of oxidoredox status by the modulation of the antioxidative enzyme system which finally protects the plants photo-system under stress induced due to hyperconcentration of arsenic (mohd et al. ) . the impact of endophytes that enhances plant growth by phytoimmobilization is shown in table . . in nature, plants commonly perform this phenomenon to neutralize soil pollutants and furthermore this property could be enhanced by the help of endophytes. mostly endophytic bacteria, fungus, actinomycites and up to some extent algae are widely being explored for their contribution to plant life, in phytotransformation (shakoor et al. ) . presently the persistent organic pollutants like pesticides, explosives, industrial byproducts and other xenobiotics are the major source of abiotic stress. the role of bacterial endophytes in the metabolism of toxic xenobiotics has already been described successfully in the phytotransformation of toluene and other organic pollutants into intermediate metabolites that could efficiently be used both by the plant and associated interacting micro-organisms (aken et al. ) . burkholderia fungorum, a bacterial strain generally present in poplar root tissue, was isolated from oil refinery discharge that had the capability to transform dibenzothiophene, phenanthrene, naphthalene, fluorine and their removal (andreolli et al. ) . prosopis juliflora was found to harborcertain endophytic bacterial species such as aerococcus, bacillus and staphylococcus which was confirmed by s rrna gene sequencing phylogenic analysis. the ability to ionize toxic heavy metals such as chromium, cadmium, copper, lead and zinc were also evaluated along with the capacity to promote plant growth was confirmed when inoculated in lolium multiflorum l. (khan et al. ) . four different plants, achillea millefolium, dactylis glomerata, solidag ocanadensis and trifolium aureum could able to grow bounteously in the soil contaminated with petroleum. it was later found that there were about endophytic bacterial species that support these plants in phytotransformation of hydrocarbons. s rdna sequencing showed the presence of microbacterium foliorum and plantibacter flavus in all the plants (lumactud et al. ) . endophytic fungus also contributes in phytotransformation of contaminants like bacterial endophytes and improves plant's fitness to withstand environmental stress. alleviation of stress induced due to salt accumulation was achieved by a gas producing basidiomycetous endophytic fungus porostereum spadiceum (hamayun et al. ). there are many other endophytic fungus that performs similar functions like penicillium minioluteum, p. funiculosum, metarhizium anisopliae, beauveria bassiana, mucor sp. etc. (khan et al. a, b; greenfield et al. ; zahoor et al. ) . the impact of endophytes that enhances plant growth by phytotransformation is shown in table . . the phenomenon through which a plant can completely remove contaminants from the site and release them into atmosphere in a volatile form can be termed as phytovolatilization. this phenomenon is highly exploited in phytotechnology programs in standardization plant growth and survivability (schiavon and pilon-smits ) . plants simultaneously interacts with diversified classes of chemical compounds including both organic and inorganic through either direct or indirect phytovolatilization (limmer and burken ; schiavon and pilon-smits ) . volatilization of metals such as as, se, hg and organic compounds such as petroleum hydrocarbons are now archived by certain endophytic bacterium. some of such endophytic bacterium includes pseudomonas aeruginosa, p. putida, p. stutzeri, rhodococcus wratislaviensis, acinetobacter sp., burkholderia sp., gordonia sp., dietzia sp., gordonia sp., mycobacterium sp., nocardioides sp., novosphingobium sp., ochrobactrum sp., polaromonas sp., rhodococcus sp., sphingomonas sp. etc. which are currently being explored for their ability to degrade organic compounds (gkorezis et al. ) . certain endophytic fungus are also involved in phytovolatilization of organic and inorganic compounds, the most common of them are alternaria alternate, cladosporium cladosporioides, cochliobolus sativus, fusarium oxysporum, muscodor yucatanensis, talaromyces wortmannii, trichoderma viride etc (ningxiao et al. ) . the impact of endophytes that enhances plant growth environmental sustainability by phytovolatilization is shown in table . . as previously mentioned biofertilization is a technique where living microorganisms in co-ordination with nutrients and minerals present in the surrounding, enhances the absorption properties of the plant without altering dynamic nature of the soil (roychowdhury et al. ) . the promotion of plant growth by increasing the accessibility or supply of major nutrients is termed biofertilization (bashan ) . the biofertilizers basically supply nitrogen, phosphorous, potassium along with certain ion scavenging molecules like siderophores and exopolysaccharides (saha et al. ; sanlibaba and Çakmak ) . a well-studied form of biofertilization is nitrogen fixation, which is the conversion of atmospheric nitrogen to ammonia. several plant growth promoting bacterial endophytes have been extensively evaluated for their efficiency to fix nitrogen including azospirillumsp (hill and crossman ) , pantoea agglomerans (verma et al. ) and azoarcus sp. (hurek et al. ) . in the present scenario much significance is given to biofertilizers in comparison to the conventional fertilizers due to its ecofriendly nature. generally these biofertilizers can be differentiated into azotobacter, phosphate solubilizers or rhyzobium on the basis of the major type of microorganisms they harbor and/or their solubilization property. almost all the microorganisms in these ecofriendly fertilizers are symbiotic in nature moreover these organisms also act as endophytes in some cases. biofertilizers that harbors azotobacter that is a genus of motile oval bacteria belongs to a family of azotobacteriaceae which are aerobic and heterotrophic in nature includes bacterial species like a. beijerinckii, a. chroococcum, a. insignis, a. macrocytogenes and a. vinelandii. all of these bacterial endophytes are commonly found in crop plants like rice, wheat, maize etc. and are already have been proven to be efficient in crop improvement as well as sustenance of fertility of soil (dursun et al. ; roychowdhury et al. ) . inorganic and organic compounds solubilization is a distinct property bestowed by these biofertilizers is remarkable. most common of these solubilization properties is phosphate solubilization where the bacterial species such as azospirillum sp., pseudomonas sp., bacillus sp., proteous sp. along with certain fungal species like aspergillus flavus, a. niger. a. ochraceus, a. sydawi, a. terreus, a. versicolor, chaetomium globosum, fusarium sp., mucor sp., penicillium sp. etc . are involved in solubilization of insoluble inorganic phosphate such as hydroxyapatite, rock phospahate tricalcium phosphate and dicalcium phosphate, into ions (selvi et al. ; roychowdhury et al. ) . lastly, biofertilizers, composed rhizobacterial species that could fix atmospheric nitrogen to the soil or to the root nodules are under vigorous study. common rhizobacterial species includes bacillus megaterium, bradyrhizobium japonicum, rhizobium, bradyrhizobium, stenotrophomonas rhizophila etc. (rajeswari et al. ; tarekegn and kibret ) . the promotion of plant growth through protection from phytopathogens is known as biocontrol. the use of synthetic chemicals for controlling plant diseases is the major risk factor raised against ecological and environmental niches. the search for an ecofriendly way to fight against these diseases is the major public and research concern. this concern for a sustainable means has paved the way for an alternative approach that is, the potential use of endophytes, as biocontrol (eljounaidi et al. (eljounaidi et al. ; shahzad et al. ; egamberdieva et al. ). the main and common mechanism exploited by thes endophytes is the elevation of certain growth hormones, induced systemic resistance, signal interference, production of antimicrobial proteins, siderophores, antibiotics and inhibitory compounds (eljounaidi et al. ) . siderophore produced by a microorganism can bind iron with high specificity and affinity making iron unavilable for other microorganism; thereby limiting their growth. it may stimulate plant growth directly by increasing availability of iron in the soil sorrounding the roots or indirectly by competitively inhibiting the growth of plant pathogens with less efficient iron uptake system.. siderophores, such as pyochelin and salicylic acid, chelate iron and can indirectly contribute to disease control by competing with phytopathogens for trace metals (duffy and defago ) . antimicrobial metabolites produced by plant growth promoting bacterial endophytes such as , diacetylphloroglucinol (dapg) can enhance disease suppression in plants. endophytic microorganisms are regarded as an effective biocontrol agent, alternative to chemical control. beauveria bassiana an endophytic fungi, was reported to control the borer insects in coffee seedlings, thus acts as an entomopathogen (posada and vega ) and sorghum (tefera and vidal ). the fungal pathogen botrytis cinerea causes severe rotting in tomatoes during storage and shelf life can be well antogonised by endophytic bacteria bacillus subtilis isolated from spaeranskiatuberculata (bge.) baill (wang et al. ) . a new strain of burkholderia pyrrocinia and b. cepacia, were identified as potential biocontrol agent against poplar canker (ren et al. ) . bioactive compounds from endophytes and their use against pathogenic micro-organisms is shown in table . . other endophytic microorganisms like fungus and actinomycites have also been identified to play an indispensible role as a biocontrol in various cases. some such endophytic fungus with biocontrol activity against woolly aphid, fusarium wilt, against various soil born bacterial pathogen and pests are gibberella fujikuroi, aspergillus tubingensis, a. flavus, trichoderma koningiopsis, galactomyces geotrichum, p. simplicissimum, p. ochrochloron, eupenicillium javanicum (potshangbam et al. ) . some endophytes and their use against pathogenic microorganisms are shown in table . . advancement proteomics, genomics studies have revolutionized the present prospective of evaluation biomolecules and their regulation, modulation as well as their impact on or within the host, both in active and in-active state. these advanced studies includes certain sophisticated instrumentations and certain gel-based approach such as d (ahsan et al. ; hu et al. ) . the bioactive compounds of the host plants such as enzymes are one of the most significant phytocompounds that regulates almost every aspect of plant life. much work has been done on the evaluation of enzymatic activity and their up or down-regulation based on the impact of the endophytic microbes the host plant harbor (castro et al. ) . form these evaluation it can be speculated that all phytoenzymes that have a direct or indirect heterogeneous impact on plants growth and survivable under biotic and/or abiotic stress possesses four basic enzymatic activity that is proteolytic, amilolytic/endoglucanase, lipolytic and esterasic (table . ) (carrim et al. ; castro et al. ). the mechanism, interaction and beneficial effect, of endophytes is summarized in fig. . . endophytes exhibit a complex network interact in association with the host plants were widely studied as inexhaustible sources of new bioactive natural products. enzymes of the endophytes degrade the polysaccharides available in the host plants. fungal strains, isolated from various plants of medicinal importance viz., alpinia calcarata, bixa orellana, calophyiium inophyllum and catharanthus roseus have been reported to produce enzymes such as amylase, cellulose, laccase, lipase, pectinase, xylanase, − , -glucan, phosphotases and proteinase e, − , -glucan, without lyase, phosphotases and proteinase and protease extracellularly. the hydrolytic enzymes produced through endophytes differs from species to species and depends on the interactions with host and their ecological factors (sunitha et al. ). endophytes are capable of synthesizing number of bioactive metabolites which are mostly used as effective drugs against various diseases and are having profound impact on agricultural and environmental sustainability. these secondary metabolites were categorised into various functional groups, alkaloids, benzopyranones, flavonoids, phenolicsacids, quinones, steroids, saponins, tannins, tertaralones, swine meat and meat products, milk and dairy products joseph and priya ( ) xanthones and many others (schulz et al. ; strobel and daisy ; jalgaonwala et al. ; pimentel et al. ; godstime et al. ) . source of bioactive compounds from endophytes is presented in table . . plant growth-promoting effects of rhizospheric and endophytic bacteria associated with different tomato cultivars and new tomato hybrids genomics era for plants and crop species -advances made and needed tasks ahead recent developments in the application of proteomics to the analysis of plant responses to heavy metals phytoremediation of polychlorinated biphenyls: new trends and promises exploring the potential of endophytes from medicinal plants as sources of antimycobacterial compounds bacterial l-forms endophytic burkholderia fungorum dbt can improve phytoremediation efficiency of polycyclic aromatic hydrocarbons macroalgal endophytes from the atlantic coast of canada: a potential source of antibiotic natural products? microorganisms expression of alkane monooxygenase (alkb) genes by plant-associated bacteria in the rhizosphere and endosphere of italian ryegrass (lolium multiflorum l.) grown in diesel contaminated soil screening of rhizospheric actinomycetes for various in-vitro and in-vivo plant growth promoting (pgp) traits and for agroactive compounds differential activity of autochthonousbacteria in controlling drought stress in native lavandula and salvia plants species under drought conditions in natural arid soil understanding the diversity of foliar fungal endophytes: progress, challenges, and frontiers phytoremediation-an overview endophytic bacteria enhance remediation of tannery effluent in constructed wetlands vegetated with leptochloa fusca beneficial bacteria isolated from grapevine inner tissues shape arabidopsis thaliana roots inoculants of plant growth-promoting bacteria for use in agriculture characterization of plant growth promoting rhizobacteria isolated from polluted soils and containing -aminocyclopropane- -carboxylate deaminase a review fungal endophytes: as a store house of bioactive compound. world the fungal community: its organization and role in the ecosystem molecular basis of plant growth promotion and biocontrol by rhizobacteria strain phyllobacterium brassicacearum stm induces a reproductive delay and physiological changes that result in improved drought tolerance in arabidopsis enzymatic activity of endophytic bacterial isolates of jacaranda decurrens cham. (carobinha-do-campo) isolation and enzyme bioprospection of endophytic bacteria associated with plants of brazilian mangrove ecosystem diversity and versatility of actinomycites and its role in antibiotic production distribution of culturable endophytic bacteria in aquatic plants and their potential for bioremediation in polluted waters endophytic colonization of vitis vinifera l. by plant growth-promoting bacterium burkholderia sp. strain psjn a critical view of current state of phytotechnologies to remediate soils: still a promising tool? conservation and diversity of seed associated endophytes in zea across boundaries of evolution, ethnography and ecology isolation of endophytic actinomycetes from roots and leaves of maize (zea mays l plant-microbe interaction with enhanced bioremediation quantitative assessments of the host range and strain specifi city of endophytic colonization by klebsiella pneumonia environmental factors modulating antibiotic and siderophore biosynthesis by pseudomonas fluorescens biocontrol strains effects of foliar application of plant growth promoting bacterium on chemical contents, yield and growth of tomato (lycopersicon esculentum l.) and cucumber (cucumis sativus l antimicrobial activity of medicinal plants correlates with the proportion of antagonistic endophytes bacterial endophytes as potential biocontrol agents of vascular wilt diseases -review and future prospects phytoremediation and its mechanisms: a review the influence of competition between tropical fungi on wood colonization in streams effects of attack of saprobic fungi on twig litter decomposition by endophytic fungi diversity and biopotential of endophytic actinomycetes from three medicinal plants in india the interaction between plants and bacteria in the remediation of petroleum hydrocarbons: an environmental perspective mechanisms of antimicrobial actions of phytochemicals against enteric pathogens -a review endophytic actinobacteria of medicinal plants: diversity and bioactivity selective influence of plant species on microbial diversity in the rhizosphere beauveria bassiana and metarhizium anisopliae endophytically colonize cassava roots following soil drench inoculation gibberellins producing endophytic fungus porostereum spadiceum agh rescues growth of salt affected soybean characterization of n -fixing bacteria associated with sweet potato roots advances in plant proteomics toward improvement of crop productivity and stress resistance cladosporium sphaerospermum as a new plant growth-promoting endophyte from the roots of glycine max l root colonization and systemic spreading of azoarcusstrain bh in grasses azoarcusgrass endophytes contribute fixed nitrogen to the plant in an un-culturable state exopolysaccharides producing rhizobia ameliorate drought stress in wheat effect of iaa on in vitro growth and colonization of nostoc in plant roots natural products from plant associated endophytic fungi a friendly relationship between endophytic fungi and medicinal plants: a systematic review bioactive compounds from endophytes and their potential in pharmaceutical effect: a review endophytic phoma sp. isolated from medicinal plants promote the growth of zea mays endophytic penicillium funiculosum lhl secretes gibberellin that reprograms glycine max l. growth during copper stress salinity stress resistance offered by endophytic fungal interaction between penicillium minioluteum lhl and glycine max l ameliorative symbiosis of endophyte (penicillium funiculosum lhl ) under salt stress elevated plant growth of glycine max l potential of am fungi in phytoremediation of heavy metals and effect on yield of wheat crop cr-resistant rhizo and endophytic bacteria associated with prosopis juliflora and their potential as phytoremediation enhancing agents in metal degraded soils decomposition and fungi of needle litter from slow-and fast-growing norway spruce (picea abies) clones endophyte assemblages in young, mature and senescent leaves of rhizophora apiculata: evidence for the role of endophytes in mangrove litter degradation phytovolatilization of organic contaminants cytokinin producing, plant growth promoting rhizobacteria that confer resistance to drought stress in platycladusorientalis container seedlings bacterial endophytes isolated from plants in natural oil seep soils with chronic hydrocarbon contamination plant growth promoting rhizobacteria and endophytes accelerate phytoremediation of metalliferous soils bioaugmentation with endophytic bacterium e s homologous to achromobacter piechaudii enhances metal rhizoaccumulation in host sedum plumbizincicola the contribution of endophytic bacteria to albizia lebbeck-mediated phytoremediation of tannery effluent contaminated soil epichloo spp. associated with grasses: new insights on life cycles, dissemination and evolution endophyticbacteria from seeds of nicotiana tabacum can reduce cadmiumphytotoxicity the use of beneficial microbial endophytes for plant biomass and stress tolerance improvement variation of microbial rhizosphere communities in response to crop species, soil origin, and inoculation with sinorhizobium meliloti l endophytic fungi piriformospora indica mediated protection of host from arsenic toxicity diversity of endophytic fungi of single norway spruce needles and their role as pioneer decomposers stop and smell the fungi: fungal volatile metabolites are overlooked signals involved in fungal interaction with plants effects of prior decomposition of beech leaf litter by phyllosphere fungi on substrate utilization by fungal decomposers role of phyllosphere fungi of forest trees in the development of decomposer fungal communities and decomposition processes of leaf litter effects of prior decomposition of camellia japonica leaf litter by an endophytic fungus on the subsequent decomposition by fungal colonizers phytostabilization of semiarid soils residually contaminated with trace elements using by-products: sustainability and risks isolation and characterization of endophytic bacteria associated with roots of jojoba (simmondsia chinensis (link) schneid) first report of bacterial soft rot of aloe vera (aloe barbadensis ) caused by pectobacterium chrysanthemi in bangladesh fungal endophyte of bracken (pteridium aquilinum), withsome reflections on their use in biological control integrated assessment of phytostimulation and biocontrol potential of endophytic trichoderma spp against common bean (phaseolus vulgaris l.) root rot fungi complex in centre region, cameroon use of endophytes to obtain bioactive compounds and their application in biotransformation process inoculation and colonization of coffee seedlings (coffea arabica l.) with the fungal entomopathogen beauveria bassiana (ascomycota: hypocreales) functional characterization of endophytic fungal community associated with oryza sativa l. and zea mays l can leaf degrading enzymes provide evidence that endophytic fungi becoming saprobes? isolation, identification and screening of rhizobium for plant growth promotion endophytic aquatic hyphomycetes of roots ofplantation crops and ferns from india isolation and characterization of a new burkholderia pyrrocinia strain jk-sh as a potential biocontrol agent antioxidant enzyme gene expression in response to copper stress in withania somnifera l the effect of biofertilizers and the effect of vermicompost on the cultivation and productivity of maize -a review microbial siderophores and their potential applications: a review endophytic actinobacteria associated with dracaena cochinchinensis lour.: isolation, diversity, and their cytotoxic activities exopolysaccharides production by lactic acid bacteria plant growthpromoting bacterial endophytes selenium biofortification and phytoremediation phytotechnologies: a review endophytic fungi: a source of novel biologically active secondary metabolites analyzing the efficacy of phosphate solubilizing microorganisms by enrichment culture techniques plant growth-promoting endophytic bacteria versus pathogenic infections: an example of bacillus amyloliquefaciens rwl- and fusarium oxysporum f. sp. lycopersici in tomato a comprehensive review on phytoremediation of cadmium (cd) by mustard (brassica juncea l.) and sunflower (helianthus annuus l characteristics of an endophytic pyrene-degrading bacterium of enterobacter sp. j from allium macrostemon bunge potentiality of endophytic actinomycetes isolated from sugar cane colonization of a submersed aquatic plant, eurasian water milfoil (myriophyllum spicatum), by fungi under controlled conditions effect of endophytic fungi on cadmium tolerance and bioaccumulation by festuca arundinacea and festuca pratensis phytoremediation of an aged petroleum contaminated soil using endophyte infected and non infected grasses observations on the seasonal occurrence of marine endophytic and parasitic fungi bioprospecting of microbial endophytes and their natural products biological properties of endophytic fungi genetic diversity and characterization of heavy metalresistant-endophytic bacteria from two copper-tolerant plant species on copper mine wasteland isolation, characterization and antimicrobial activity of endophytic bacteria from polygonum cuspidatum extracellular enzymatic activity of endophytic fungalstrains isolated from medicinal plants bacterial endophytes contribute to abiotic stress adaptation in pepper plants (capsicum annuuml effects of rhizobium, nitrogen and phosphorus fertilizers on growth, nodulation, yield and yield attributes of soybean at pawe northwestern ethiopia effect of inoculation method and plant growth medium on endophytic colonization of sorghum by the entomopathogenic fungus beauveria bassiana the structure of micromycete communities and their synecologic interactions with basidiomycetes during plant debris decomposition succession of microfungal assemblages in decomposing peat land plants isolation and screening of endophytic fungi from three plants used in traditional medicine in nigeria for antimicrobial activity role of plant growth promoting rhizobacteria inagricultural sustainability -a review evaluationof plant growth promoting and colonization ability of endophytic diazotrophs from deep water rice enhancement of drought stress tolerance in crops by plant growth promoting rhizobacteria isolation and characterization of bacillus subtilis eb- , an endophytic bacterium strain displaying biocontrol activity against botrytis cinerea pers induction of toluene degradation and growth promotion in corn and wheat by horizontal gene transfer within endophytic bacteria unraveling the role of dark septate endophyte (dse) colonizing maize (zea mays) under cadmium stress: physiological, cytological and genic aspects the potential of the ni-resistant tce-degrading pseudomonas putida w -tce to reduce phytotoxicity and improve phytoremediation efficiency of poplar cuttings on a ni-tce co-contamination taxonomy, life cycle, reproduction and detection of acremonium endophytes a proteomic approach suggests unbalanced proteasome functioning induced by the growth-promoting bacterium kosakonia radicincitans in arabidopsis diversity of endophytic fungi from roots of panax ginseng and their saponin yield capacities characterization of culturable bacterial endophytes and their capacity to promote plant growth from plants grown using organic or conventional practices biosorpiton of cadmium by endophytic fungus (ef) microsphaeropsis sp fungal diversity and plant growth promotion of endophytic fungi from six halophytes in suncheon bay alleviation of heavy metal toxicity and phytostimulation of brassica campestris l. by endophytic mucor sp colonization and nitrogen-fixing ability of herbaspirillumsp. strain b gfp and assessment of its growth-promoting ability in cultivated rice key: cord- -ldfa a authors: joung, young hee; park, se hee; moon, ki-beom; jeon, jae-heung; cho, hye-sun; kim, hyun-soon title: the last ten years of advancements in plant-derived recombinant vaccines against hepatitis b date: - - journal: int j mol sci doi: . /ijms sha: doc_id: cord_uid: ldfa a disease prevention through vaccination is considered to be the greatest contribution to public health over the past century. every year more than million children are vaccinated with the standard world health organization (who)-recommended vaccines including hepatitis b (hepb). hepb is the most serious type of liver infection caused by the hepatitis b virus (hbv), however, it can be prevented by currently available recombinant vaccine, which has an excellent record of safety and effectiveness. to date, recombinant vaccines are produced in many systems of bacteria, yeast, insect, and mammalian and plant cells. among these platforms, the use of plant cells has received considerable attention in terms of intrinsic safety, scalability, and appropriate modification of target proteins. research groups worldwide have attempted to develop more efficacious plant-derived vaccines for over diseases, most frequently hepb and influenza. more inspiring, approximately plant-made antigens have already been tested in clinical trials, with successful outcomes. in this study, the latest information from the last years on plant-derived antigens, especially hepatitis b surface antigen, approaches are reviewed and breakthroughs regarding the weak points are also discussed. hepatitis b (hepb) is an infection with the hepatitis b virus (hbv), which attacks the liver and can cause both acute and chronic disease. the world health organization (who) estimates that million persons are chronically infected with hbv and that more than , people die every year due to complications of hepb, including cirrhosis and liver cancer [ ] . the point that needs the most attention is the high rates of chronic infection found in the sub-saharan africa and east asia, where between % and % of the adult population is infected, as well as in the amazon and the southern parts of eastern and central europe. otherwise, less than % of the population in western europe and north america is chronically infected [ ] . hbv is a hepatotropic dna virus that replicates by reverse transcription. the human hbv is a small circular dna molecule of . kb [ ] . its genome consists of four partially overlapping open reading frames (orfs), namely the envelope gene (pre-surface/surface (pre-s/s)), the core gene (pre-core/core (pre-c/c)), the polymerase gene (pol) and the transactivating protein x (x). the orf pre-s/s encodes pre-s , pre-s and surface (s) proteins that form the surface antigen (hbsag), and hbsag protein is the main antigen to elicit virus-neutralizing and protective antibodies by the immune system [ , ] . understanding the hbv genome and structure is an essential prerequisite for preventive or therapeutic vaccination against hepb. a vaccine against hepb has been available since . this first licensed anti-hbv vaccine containing subviral particles of hbv purified from the inactivated serum of carriers revealed very high efficacy [ ] , and a subsequent subunit vaccine made using the small surface antigen (s-hbsag) was developed in the early s [ ] . the yeast system for the recombinant antigen was to ensure safety and low cost. yeast-derived s-hbsag assembled into virus-like particles (vlps) were as immunogenic as natural subviral particles, and highly effective vaccines containing s-hbsag have been widely used as prophylactic vaccines against hbv infection [ ] . however, some groups of vaccines do not develop protective immunity against the virus and immunosenescence frequently occurs in adults [ ] . additionally, high cost limit and the necessity of accompanying infrastructure for the cold chain distribution and intravenous administration still constituted a barrier to vaccination approaches in developing countries. in order to successfully solve these problems, many research projects have been undertaken to develop more efficacious, easily administrated, and thermostable vaccines. a new recombinant hbv vaccine containing the pre-s/s has greater immunogenic potential than the conventional s antigen-based vaccines in terms of antibody induction and cellular immune response. middle (pre-s + s, m-hbsag) or large (pre-s + pre-s + s, l-hbsag) surface antigens [ ] have been used as components of specific immunotherapeutic vaccines for chronic hbv carriers [ , ] . additionally, chimeric protein created by fusing the hbv core antigen (hbcag) to the pre-s showed strong anti-hbc and moderate anti-pre-s immune responses [ ] . although vaccination is one of the most powerful and cost-competitive achievements, some vaccines may still have certain limitations related to maintenance of the cold chain, downstream processing costs, administration risk, and expensive scalability [ ] [ ] [ ] [ ] [ ] . from these reasons, the use of plant cells as alternative production platforms have received considerable attention in terms of intrinsic safety, scalability, and posttranslational modification of target proteins [ , ] . plant systems can be scaled up quickly to generate large quantities of the protein product, are not susceptible to contamination with known human or mammalian pathogens and are resistant to enzymatic digestion in the gastrointestinal tract. in addition, transgenic plants can be engineered to express and translate multiple proteins concurrently with appropriate folding and assembly into multimeric proteins, especially the posttranslational adjustments of antibodies. not all recombinant antigens will benefit from plant-based systems, but the best production system for each recombinant protein should be chosen using a case-by-case approach [ ] . merlin et al. [ ] proposed that plants are the most the beneficial for the production of four major categories of pharmaceutical proteins: ones that are required in large quantities, that need to be rapid-response, that require complex posttranslational modifications, or that are intended for oral delivery. within these categories, they suggested appropriate candidates to meet a spectrum of research, development, commercial needs, such as human glutamic acid decarboxylase, norwalk virus-like particles, monoclonal antibody g , and human interleukin- . those plant-made antigens have already been tested in clinical trials, with successful outcomes ( table ). the enzyme glucocerebrosidase for gaucher's disease, the first pmf-derived enzyme "elelyso™", has been approved and marketed by protalix in . elelyso™ is based on the use of carrot cells to produce recombinant taliglucerase alpha, which is used in enzyme replacement therapy to treat adult patients. this special food and drug administration (fda) approval case was fast tracked based on its applicability to a rare genetic disease and the bioreactor production under stringent conditions [ , ] . medicago has ongoing phase ii clinical trials for a plant-derived vlp quadrivalent seasonal influenza vaccine and an h pandemic influenza vaccine [ ] . they are focusing on vlp vaccine development. vlps are self-assembled structures derived from viral antigens that mimic the native architecture of viruses but lack the viral genome and thus are not infective. another important advantage as emerging vaccine is the more effective activation of key aspects of the immune response to achieve potent immune stimulation and to provide immunological memory for long-lasting protection [ , ] plant-based platforms including whole plant, organs or cell and expression technology to produce target antigens of interest are diverse [ ] [ ] [ ] . representative plant species expressing the oral vaccine are potato, tomato, and tobacco; additionally, maize, rice, carrot, and soybean are also applied in this field [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . those plants are mainly focused on traditional and usually eaten crops in human, because it is known that inexperienced plants sometimes have problems with certain plant allergies. target antigen proteins were expressed by a plant cell nuclear genome expression system in these plant species. edible plant vaccines are based on different parts of plants, such as fruits, seeds, and root vegetables. such food vaccines are prepared directly without expensive purification of the antigens, which is essentially required for parenteral administration of vaccines [ ] . therefore, the lyophilization of organs expressing stable antigens would facilitate their processing, purification and storage, reducing costs and allowing more practical vaccines. although stable transformation into transgenic plants is commonly accepted, the low production level of the resultant recombinant protein remains an issue of concern. an efficient alternative to nuclear transformation for vaccine antigens and other therapeutic proteins is plastid transformation [ ] . the highest expression of transgenes, up to more than % of total soluble protein, are reported in chloroplast transformation [ , ] otherwise, the universal expression level in most studies has been % of total soluble protein (tsp) or µg/g fresh leaf tissue [ , ] . chloroplast technology can also avoid the controversy related to transgene containment [ ] and express multigenes as single operon [ ] . waheed et al. [ ] reviewed recent vaccine antigens against human diseases expressed via plastid genome since . two plant species, tobacco ( different antigens) and lettuce (four different antigens), have mainly been used in plastid transformation. these results suggest that more industrial interest is needed to strengthen the research/academia-industry linkages in the chloroplast-based vaccine market. stable transformation has its own advantages such as reliable harvest of target proteins over multiple generations and optimized protocols for delivery of foreign genes into various plant species. although there are problems with the time required, seed resources can be grown anywhere with minimal cost and labor once the plant has been developed for the first time [ ] . most clinical trials, except for the three cases of eleyso, prx- , and recombinant human intrinsic factor, have used a tobacco-based transient expression system (table ). in recent years, interest in transient expression has increased due to the containment of the system and the possibility of rapid upscaling due to the short interval between transformation and expression, which are attractive features for the industrial scale production and approval of the expressed products, e.g., the mass production of tobacco by medicago and kentucky bioprocessing. pogue et al. [ ] reviewed plant-based transient expression systems for the production of pharmaceutical-grade recombinant aprotinin and a monoclonal antibody product. transient expression provides a safe and environmentally friendly system for both indoor and outdoor application with high speed and low cost of the genetic manipulation, rapid manufacturing cycles, and economical production. transient production using an agrobacterium tumefaciens-mediated transfer-dna delivery system (agro-infiltration) and/or virus-based replicating systems, the two dominant approaches, guarantees both the quality of the resulting purified products and the speed of development [ , ] . spiegel et al. [ ] demonstrate the application of the classical nicotiana benthamiana/a. tumefaciens transient expression system to accelerate the development of a malaria vaccine candidate, with screens for expression, solubility, and stability using fluorescent fusion proteins. in marin viegas et al. [ ], a transient expression system for the production of human tg in n. benthamiana leaves was optimized, and the reactivity of plant-produced tg in a cd screening test was evaluated. hence, transient expression performed in contained facilities satisfies good manufacturing practices, and quick expression can avoid the time-consuming stable transformation [ , ] . in a comparison of productivity in terms of biomass production, hiatt and pauly [ ] reported that grams of product may take only two weeks plus a few weeks more. in large-scale biomanufacturing systems, recombinant proteins can be produced at levels of - mg/kg fresh weight tissue in as little as three months [ ] . the transient expression of human interleukin- in n. benthamiana ( . % tsp) produces -fold more than stable expression in tobacco plants ( . mg/g fresh weight (fw)) [ ]. conversely, hgad mut is expressed at higher levels in stable tobacco plants ( . µg/g fw) than in n. benthamiana ( . µg/g fw) [ ] . this result suggests that expression potential or level varies case by case, depending on the target protein. in last decade, there has been a considerable increase in the use of transgenic plants to generate recombinant proteins for medical and veterinary use ( table ) . research groups worldwide have attempted to develop more efficacious plant-derived vaccines for over diseases, most frequently hepatitis b and influenza. in the case of hepatitis b, both stable and transient expression systems have been developed in various plants, including potato, lettuce, tobacco, tomato, carrot, and arabidopsis for stable expression and n. benthamiana for transient expression. a detailed review of hepatitis b will be presented in the next part of this article. influenza is also a main target of this field because it is a widely distributed viral infection of humans and animals, and a new epidemic strain appears every one to two years. this pattern requires the production of new vaccines at the same frequency, and a promising solution is to establish a rapid, flexible, and safe system. the production of various antigens such as hemagglutinin (ha) and the extracellular domain of matrix protein (m e) has mainly focused on transient expression systems using n. benthamiana leaves. using the medicago "proficia™" system, vaccine production can be initiated within less than three weeks from the identification of the genetic sequence of a pandemic or seasonal influenza strain [ ]. vamvaka et al. [ ] reported the development of transgenic rice plant expressing the hiv-neutralizing antibody g in the endosperm ( µg/g dry seed weight) to evaluate the potential of rice seeds as a vehicle for inexpensive microbicide production. production is higher than the initial achievement of maize-derived g ( µg/g) by rademacher et al. [ ] . rubio-infante et al. [ ] demonstrated the immunogenic potential of tobacco chloroplast-derived multi-hiv in an oral immunization scheme and proposed it as a vaccine prototype capable of inducing broad immune responses as it carries various b and t cell epitopes from several hiv strains. dengue has become a significant public health problem, and the threat of dengue fever is now increasing in temperate regions due to dramatic climate change. the rice codon-optimized consensus domain iii of dengue virus envelope glycoprotein (e) has been fused to the m cell-binding peptide via agroinfiltration with a plant virus-based expression system [ ] [ ] [ ] . carrying these results a step further, kim et al. [ ] generated an ebola ric-based denv vaccine in tobacco plants using a geminiviral vector expression system and reported its immunogenic properties as a self-adjuvanting dengue vaccine candidate. previously, phoolcharoen et al. [ ] reported that plant-expressed ebola ric protected mice against a lethal ebola virus challenge. the expression of subunit vaccines for animal viral diseases, such as avian influenza [ , ] , foot-and-mouth disease (fmd) [ ] [ ] [ ] , and diarrhea [ , , ] , which are considered to be the most important causes of economic losses in plants, has been frequently reported. the commercialization of veterinary vaccine is relatively easy compared with that of human vaccines. to date, four cases in clinical trials are ready to enter the market. [ ] suggest that the highly immunogenic vp epitopes produced in n. benthamiana are candidates for a subunit vaccine, specifically for the g p rotavirus strain. the greatest problem of plant-derived vaccine development is the extremely low expression level of the foreign protein in plants. for this reason, many researchers have studied how to improve protein expression levels in plants. in the case of plant-derived hbv vaccines, the first report was on the expression of the small hepatitis b surface antigen (s-hbsag) in transgenic tobacco plants. in this report, the hbsag produced in transgenic tobacco was antigenically and physically similar to the hbsag particles derived from human serum and recombinant yeast [ ] . afterward, many research groups attempted hbsag expression in different tissues and plant species, such as tobacco, potato, lettuce, soybean, lupine, maize, tomato, peanut and laminaria japonica (table ). in the transgenic tobacco plant transformed with the s-hbsag gene controlled by the s promoter, expression levels were very low: less than . % total soluble protein and less than ng/g fresh weight in leaf tissues. the expression levels of s-hbsag in other plant species were not significantly higher; in some species, expression levels were even lower than in tobacco. to improve vaccine production in plants, the most widely used strategies involve: ( ) suitable promoters, such as strong constitutive promoters, tissue-specific promoters and promoters that are inducible by environmental factors; ( ) targeting systems to specific organelles; ( ) optimized codon usage; ( ) alternative polyadenylation signals; ( ) increased translation efficiency using leader sequences; and ( ) different vector systems. many hbsag-overexpressing transgenic plants have been developed using strong constitutive promoters, such as the s promoter with enhancer [ ] [ ] [ ] . in addition to tissue-specific promoters, the patatin promoter for potato tuber [ , ] , globulin promoter for maize seed [ ] and fruit-specific promoters [ , ] were used. specific organelle-, endoplasmic reticulum (er)-, vacuole-and chloroplast-targeted strategies have also been tried [ , ] . the hbsag has been expressed in non-edible plants, such as tobacco, using four different expression cassettes: the hbsag gene without er retention signal (hbs), the hbsag gene with er retention signal (her), and each gene controlled by the ubiquitin promoter (ubq) or ethylene forming enzyme promoter (efe) [ ] . in this report, the maximum expression level ( . ng/g fw of leaves) was observed in efe-hbs transformed plant growth in vitro, but a higher proportion of the particulate form of the antigen was observed when it was expressed with an er retention signal. the efe promoter is more effective in in vitro-cultured plantlets, whereas the ubq promoter is more effective in greenhouse-grown plantlets. the maximum expression level was µg/g fw in the ubq-her transformed nt-l cell suspension culture [ ] . the expression level was increased up to µg/g fw using hbsag fused with the region from the soybean vegetative storage protein gene and was controlled by a chimeric ocs-mas promoter. upon transformation into a soybean cell culture using the same vector, the maximum expression level was µg/g fw [ ] . soybean cell/transgenic pmsi /eha ubq /er ng/g fw n.a. [ ] banana/transgenic pbi /eha efe er ng/g fw (leaf) n.a. [ ] lupin/transgenic prok/c s/n.a. ng/g fw (callus) oral, igg antibodies in serum (maximum miu/ml) [ ] lupin/transgenic prok/gv , eha , lba s/n.a. . - µg/g fw (callus) n.a. [ ] maize/transgenic n.a./eha xglb /cell wall . % of tsp (seed) n.a. [ ] maize/transgenic n.a./eha glob /cell wall . % of tsp (seed) oral (germ, bioencapsulated), iga and igg antibodies in serum (maximum miu/ml) [ ] maize/transgenic n.a./eha kbglb /cell wall . % of tsp (seed) oral (germ, wafer feeding), iga and igg antibodies in serum [ ] maize/transgenic n.a./eha glob /cell wall . % of tsp (seed) oral (germ, wafer feeding), iga and igg antibodies in serum [ ] cherry tomatillo/transgenic pcambia /eha s/n.a. ng/g fw (fruit) oral, igg antibodies in serum [ ] tomato/transient pbi /eha efe/er . µg/g dw (leaf) n.a. [ ] tomato/transgenic pbinplus-ars/lba s/er n.a. n.a. [ ] tomato/transgenic pbm/lba d s/n.a. . %- . % of tsp (leaf) n.a. [ ] tomato/transgenic pbinplus-ars/lba s/n.a. . µg/g dw (fruit) oral, iga and igg antibodies in serum (maximum miu/ml) [ ] carrot cell/transgenic ppcv /n.a. mas/n.a. ng/g fw n.a. [ ] laminaria japonica/transgenic pcat/n.a. sv /n.a. . %- . % of tsp n.a. [ ] peanut oral, igg antibodies in serum (maximum miu/ml), oral, iga and igg antibodies in serum (maximum miu/ml) [ , ] lettuce/transgenic pgptv-bar/eha s/n.a. - µg/g fw n.a. [ ] tomato/transgenic pbinplus-ars/lba s/n.a. . % of tsp (fruit) n.a. [ ] tomato/transgenic pbinplus-ars/aglo s/n.a. . %- . % of tsp (fruit) oral (freeze-dried material), igg antibodies in serum [ , ] carrot/transgenic pbinplus-ars/n.a. s/er ng/g fw (leaf) n.a. [ ] l-hbsag hbsag has been expressed in vegetative crops, such as potato, tomato, soybean and lettuce. the expression level of transgenic potato tubers was - µg/g fw. the highest expression in a tuber was developed using a construct driven by the camv s promoter with dual enhancers, the tobacco etch virus -utr, and the region from the soybean vegetative storage protein gene [ ] . expression level of hbv-protein in potato was little increased when controlled by the tuber specific promoter [ ] . target dna is inserted into a genomic dna as a random event when a using agrobacterium-mediated transformation. for this reason, it is difficult to conclude what is the best method for increase of hbv-protein expression level because differences in the expression level between the transgenic lines even with the same vector construction. expression in tomato fruit has been reported at . µg/g dry weight. to achieve a higher level of expression, several strong and inducible promoters, such as the enhanced dual s, ubq and efe promoters, were tested, as well as organelle targeting sequences. the greatest improvement resulted from the hbsag gene with an er retention signal controlled by efe promoter [ ] . sunil kumar et al. [ ] reported hbsag transformation in banana. the maximum expression in banana leaves has been reported at ng/g fw. the expression levels in banana fruits were not presented in this report, but the expression level was presumably lower than in the leaf tissue. as with leafy vegetables, a variety of expression technologies have not yet been applied. in lettuce, the maximum expression level was µg/g fw, which is the maximum anti-hbsag antibody titer of miu in immunized mice serum [ , , ] . upon transformation into a soybean cell culture using a construct of hbsag fused with the region from the soybean vegetative storage protein gene and as controlled by a chimeric ocs-mas promoter, the maximum expression level was µg/g fw [ ] . grains are a further option for the expression of candidate vaccine antigens. they have long stability of expressed recombinant proteins with low water content [ ] . in maize seed, the maximum expression has been reported at . % of total soluble protein (approximately µg/g fw). this level of expression was achieved using a barley alpha amylase signal sequence-fused s-hbsag gene with a × globulin promoter [ ] . all of the results suggest that the expression levels of hbsag are highly variable and depend on plant species, tissue types and culture conditions. the major recombinant hepatitis b vaccines contain s-hbsag; therefore, the expression of this protein has been the focus in plants. the proteins pres -s, m-hbsag, and pres -pres -s, l-hbsag, have been much less studied than s-hbsag. m-hbsag and l-hbsag have been transformed into potato, tomato, and tobacco (table ) . although the expression was optimized using suitable promoters, leader sequences and targeting signals, the expression levels of m-/l-hbsag were lower than for s-hbsag. however, hbcag induces a heightened immune response [ , ] and spontaneously assembles into capsid-like particles [ ] . for these reasons, efforts devoted to the production of an anti-hbv vaccine have focused on hbcag in the last few years. especially, hbcag has been abundantly produced using transient expression systems mediated by icon binary vectors [ ] or viral vector systems [ ] (table ). transgenic tobacco plants-derived hbsag was antigenically and physically similar to the human serum and recombinant yeast derived-hbsag particles [ ] . to analyze the immunological response in vivo, tobacco-expressed hbsag was purified and injected into balb/c mice. the anti-hepb response to the tobacco-derived hbsag was qualitatively similar to the response obtained by immunizing mice with commercialized yeast-derived hbsag vaccine [ ] . these results showed a possibility of developing injected vaccines using plant-expressed hbsag. due to differences of the manufacturing processes between companies, the amount of hbsag protein per dose differs among the various hbv vaccine products [ ] . for this reason, there is no international standard for the hbsag protein quantity in vaccines, but there is a standard based on protective efficacy of vaccination related to the anti-hepb antibodies induction. an anti-hbsag of ≥ miu/ml measured - months after the last dose of the vaccine are considered to be immune to hepb. although no international standard of antigen concentration is defined, considering the feasibility and cost-effectiveness of the injected vaccine, the concentration of antigen should be over µg/ml [ ] . despite many attempts to increase hbsag expression in transgenic plants, the expression level remains too low for use as an injected vaccine. the currently used hbv vaccine contains hbsag and is produced by yeast cells. the yeast-derived hbv vaccine can be supplied inexpensively ($ - per single dose [ ] ); therefore, it is difficult for plant-derived vaccines to have a competitive price. however, plant suspension cultures may be used as an alternative to yeast to produce antigens for purification. expression levels have approached µg/g fw ( mg/l culture medium) in transgenic soybean culture [ ] . although the expression level ( µg/g fw) was lower in transgenic tobacco cell suspension culture than in soybean [ ] , the former has been used to secrete hbsag into the culture medium, with a six-fold increase in secretion in response to jasmonic acid or salicylic acid treatment during cell culture, and the amount of antigen secreted was µg/l medium [ ] . another breakthrough regarding expression problems was achieved through the utilization of virus-based transient expression systems for the robust production of hbv antigens, such as s-hbsag and hbcag, with yields as high as mg/g fw [ , , ] . tobacco-derived proteins showing the maximum anti-hbsag antibody titer of miu in immunized mice serum [ ] are preferred the application of injection after purification process, rather than oral administration in order to remove many toxic alkaloids and phenolic substances which have a tobacco plant [ ] . however, improvements of several orders of magnitude are still needed for plant cell culture systems to be competitive, particularly given the slow growth rates of plant cells compared with yeast. in transgenic suspension cell culture, the formation of vlps by hbv antigens made it possible to exploit relatively inexpensive protein purification techniques, such as the sucrose gradient [ , , ] or cesium chloride gradient ultracentrifugation [ ] . the highest expressed soybean cell culture was used for antigen purification, and the antigen was suitable for injection [ ] , but the yield remained unsatisfactory and was not cost-effective. the biggest advantage of edible plant-derived vaccines is their easy application to oral delivery. the benefits of plant-derived edible vaccines are as follows: ( ) during oral delivery, plant-derived vaccines are protected in the stomach by plant cell wall and slow release in the gut; ( ) the plant tissue expressing antigen may be used as raw or dried food; ( ) capsules can also be made from partially or fully purified vaccine proteins; ( ) no need for cold chain systems for storage and delivery of the plant tissues or extracts; and ( ) the plant-derived vaccines are cost efficient compared with traditional vaccines. edible plant-derived hbv antigens have been administered by oral injection or feeding in mice with/without adjuvants [ , [ ] [ ] [ ] ]. an oral vaccine candidate has also been administered to human volunteers in small-scale clinical trials without adjuvants. the first trial was administered to three human volunteers in row lettuce leaves in two doses ( . - µg of s-hbsag/dose) without the use of an adjuvant. all volunteers responded, with two of them having serum responses in excess of the protective minimum level ( miu/ml of serum). however, the antibody levels declined rapidly [ , ] . in the second trial, previously vaccinated human volunteers were fed two or three doses of g of raw potato tubers (approximately mg of the s-hbsag/dose). more than half of the subjects showed increased antibody titers [ ] . the animal experiments and trials showed the potential for plant-derived hbv antigens to be used as an oral vaccine for the prevention of hbv, but there remain many problems to be solved for practical application, such as the administration of bulky plant material, declining long-term responses, individual differences in the immune response and the difficulty of defining the antigen dose [ ] . the expression level of plant-derived hbv antigen is only / - / of the expression of yeast-derived hbv antigen; however, the expression yield and plant production scale are still increasing [ , ] . tomato is possible intake without any processing or cooking. therefore, tomato fruit is a very attractive crop to develop an oral vaccine. according to the study to date, the expression level of hbv antigen was very low as ng/g fw ( table ). the maximum titers of anti-hbsag antibody in serum is miu using oral application. this antibody yield was high compared to the expression level of hbv antigen in tomato fruits [ ] . hbv antigen expression in maize produced much higher levels of antigen, and the palatability and digestibility were better than for potato. that is, cereal crops can easily transport or storage in dry state. in addition, the maize system induced a strong immune response with miu of maximum titer by both injection and oral administration [ , ] . this result suggests the possibility of providing a raw material for thermostable formulation at $ . per dose [ ] . plant components such as saponin, flavonoids, and plant oils also function as adjuvants [ ] [ ] [ ] and help maintain the immune response in the long term [ ] . the lyophilization method is an excellent way to increase the stability and shelf life of the plant-derived vaccines. in the previous study, the storage stability of lyophilized powder form was limited at • c [ ] . in a recent study, successful long-term storage at • c was achieved though improvements in the process [ ] . it is easier to control the concentration and standardize antigen doses and process the antigen into a tablet or capsule form using a powdered tissue instead of freeze-drying [ ] . despite over years of effort, no commercial plant-based anti-hbv vaccine has been developed. to commercialize a plant-derived hbv vaccine, several points should be considered. first, the greatest barrier is the low expression levels of hbv antigen in plants; however, expression yield and plant production scale can still be increased using plant expression vector optimization, which should be focused on the target plant. the process can also be more competitive by improving the plant-derived antigen to increase the immune response to the vaccine. second, an hbv antigen expressed in an edible plant has the advantage of being usable as an oral vaccine without processing. it is first necessary to analyze the characteristics of the target plants and the expressed protein for the development of an oral vaccine because plant components, secondary metabolites and foreign protein expression characteristics vary with plant species. to obtain feasible and cost-effective vaccines, the target plants for edible vaccines should have a long shelf life, be heat stable and be edible as a raw material. candidate grain crops are maize and rice; candidate vegetative crops are tomato and banana. third, consideration should be made of the public's acceptance of gm crops, especially plant-derived edible vaccines. for injected vaccine development, the most cost-effective method is a suspension culture in a closed environment, according to the regulations of good manufacturing practice. further safety of plant-derived vaccines can be obtained by following the same regulations established for traditional vaccines. in addition, for oral vaccines produced from gm crops, environmental risk assessment and human risk assessment should be performed. for these reasons, plant-derived oral vaccines cannot be called cost efficient compared with traditional vaccines, and the current concern over the use of gm plants is now affecting research in this field. world health organization (who) compact organization of the hepatitis b virus genome. hepatotlogy structural and functional analysis of full-length hepatitis b virus genomes in patients: implications in pathogenesis overview of hepatitis b virus mutations and their implications in the management of infection the newly licensed hepatitis b vaccine: characterics and indications for use human hepatitis b vaccine from recombinant yeast recombinant hepatitis b triple antigen vaccine: hepacare comparative immunogenicity of a pres/s hepatitis b vaccine in non-and low responders to conventional vaccine hepatitis b virus morphogenesis specific vaccine therapy in chronic hepatitis b: induction of t cell proliferative responses specific for envelope antigens induction of strong hepatitis b virus (hbv) specific t helper cell and cytotoxic t lymphocyte responses by therapeutic vaccination in the trimera mouse model of chronic hbv infection recombinant hepatitis b core antigen carrying pres epitopes induce immune response against chronic hbv infection medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants biological safety concepts of genetically modified live bacterial vaccines role of genetic factors and environmental conditions in recombinant protein production for molecular farming evolution of plant-made pharmaceuticals a perspective on the use of pleurotus for the development of convenient fungi-made oral subunit vaccines immunological aspects of using plant cells as delivery vehicles for oral vaccines comparative evaluation of recombinant protein production in different biofactories: the green perspective carrot cells: a pioneering platform for biopharmaceuticals production plants as factories for human pharmaceuticals: applications and challenges molecular pharming-vlps made in plants heat-precipitation allows the efficient purification of a functional plant-derived malaria transmission-blocking vaccine candidate fusion protein malaria vaccine candidate antigen targeting the pre-erythrocytic stage of plasmodium falciparum produced at high level in plants plant-based production of recombinant plasmodium surface protein pf and evaluation of its potential as a vaccine candidate a plant-produced pfs vlp malaria vaccine candidate induces persistent transmission blocking antibodies against plasmodium falciparum in immunized mice transgenic tomato plants expressing the antigen gene pfcp- . of plasmodium falciparum production, characterisation and immunogenicity of a plant-made plasmodium antigen the kda c-terminal fragment of plasmodium yoelii merozoite surface protein a plant-produced pfs vaccine candidate blocks transmission of plasmodium falciparum production and characterization of an orally immunogenic plasmodium antigen in plants using a virus-based expression system rice endosperm produces an underglycosylated and potent form of the hiv-neutralizing monoclonal antibody g a plant-derived multi-hiv antigen induces broad immune responses in orally immunized mice oral delivery of plant-derived hiv- p antigen in low doses shows a superior priming effect in mice compared to high doses chloroplast expression of an hiv envelop-derived multiepitope protein: towards a multivalent plant-based vaccine biochemical and immunological characterization of the plant-derived candidate hiv- mucosal vaccine ctb-mpr anchorage to the cytosolic face of the endoplasmic reticulum membrane: a new strategy to stabilize a cytosolic recombinant antigen in plants expression of hiv- antigens in plants as potential subunit vaccines rapid high-yield expression of a candidate influenza vaccine based on the ectodomain of m protein linked to flagellin in plants using viral vectors high-yield expression of m e peptide of avian influenza virus h n in transgenic duckweed plants oral immunization of haemaggulutinin h expressed in plant endoplasmic reticulum with adjuvant saponin protects mice against highly pathogenic avian influenza a virus infection intranasal vaccination with a plant-derived h ha vaccine protects mice and ferrets against highly pathogenic avian influenza virus challenge. hum. vaccines immunother biochemical composition of haemagglutinin-based influenza virus-like particle vaccine produced by transient expression in tobacco plants plant-derived h vlp vaccine elicits protective immune response against h n influenza virus in mice and ferrets safety and immunogenicity of a plant-produced recombinant monomer hemagglutinin-based influenza vaccine derived from influenza a (h n ) pdm virus: a phase i dose-escalation study in healthy adults human antibody response to n-glycans present on plant-made influenza virus-like particle (vlp) vaccines. vaccine a plant-based system for rapid production of influenza vaccine antigens. influenza other respir the human potential of a recombinant pandemic influenza vaccine produced in tobacco plants setting up a platform for plant-based influenza virus vaccine production in south africa formulation development of a plant-derived h n influenza vaccine containing purified recombinant hemagglutinin antigen. hum. vaccines immunother plant-produced recombinant influenza vaccine based on virus-like hbc particles carrying an extracellular domain of m protein preclinical and clinical development of plant-made virus-like particle vaccine against avian h n influenza a plant-produced influenza subunit vaccine protects ferrets against virus challenge. influenza other respir plant-expressed ha as a seasonal influenza vaccine candidate highly pathogenic avian influenza as a zoonotic agent transient expression of the ectodomain of matrix protein (m e) of avian influenza a virus in plants a transgenic plant cell-suspension system for expression of epitopes on chimeric bamboo mosaic virus particles development of a new vector using soybean yellow common mosaic virus for gene function study or heterologous protein expression in soybeans expression of vp protein of serotype a and o of foot-and-mouth disease virus in transgenic sunnhemp plants and its immunogenicity for guinea pigs immunogenicity of foot-and-mouth disease virus structural polyprotein p expressed in transgenic rice foliar extracts from transgenic tomato plants expressing the structural polyprotein, p - a, and protease, c, from foot-and-mouth disease virus elicit a protective response in guinea pigs induction of protective immunity in swine by recombinant bamboo mosaic virus expressing foot-and-mouth disease virus epitopes optimization of ammonium sulfate concentration for purification of colorectal cancer vaccine candidate recombinant protein ga -fck isolated from plants optimization of elisa conditions to quantify colorectal cancer antigen-antibody complex protein (ga -fck) expressed in transgenic plant expression of ga -fc fusion protein as a vaccine candidate for colorectal cancer in transgenic plants plant-derived epcam antigen induces protective anti-cancer response apple latent spherical virus vector as vaccine for the prevention and treatment of mosaic diseases in pea, broad bean, and eustoma plants by bean yellow mosaic virus generation of protective immune response against anthrax by oral immunization with protective antigen plant-based vaccine a plant based protective antigen [pa (div)] vaccine expressed in chloroplasts demonstrates protective immunity in mice against anthrax recombinant plants provide a new approach to the production of bacterial polysaccharide for vaccines production of a plant-derived immunogenic protein targeting apob and cetp: toward a plant-based atherosclerosis vaccine a plant-produced antigen elicits potent immune responses against west nile virus in mice suppression of inhibitor formation against fviii in a murine model of hemophilia a by oral delivery of antigens bioencapsulated in plant cells effects of plant cultivation density and light intensity on the production of a vaccine against swine edema disease in transgenic lettuce production of double repeated b subunit of shiga toxin e at high levels in transgenic lettuce plants as vaccine material for porcine edema disease immunogenicity of eit chimeric protein expressed in transplastomic tobacco plants towards development of an oral vaccine against escherichia coli o :h induction of toxin-specific neutralizing immunity by molecularly uniform rice-based oral cholera toxin b subunit vaccine without plant-associated sugar modification oral immunogenicity and protective efficacy in mice of transgenic rice plants producing a vaccine candidate antigen (as ) of ascaris suum fused with cholera toxin b subunit synthesis and assembly of an adjuvanted porphyromonas gingivalis fimbrial antigen fusion protein in plants transient expression of vp in nicotiana benthamiana and its use as a plant-based vaccine against infectious bursal disease virus vp antigen produced in tobacco transplastomic plants confers protection against bovine rotavirus infection in a suckling mouse model greenhouse and field cultivations of antigen-expressing potatoes focusing on the variability in plant constituents and antigen expression oral administration of plant-based rotavirus vp induces antigen-specific igas, iggs and passive protection in mice efficacy of a bvdv subunit vaccine produced in alfalfa transgenic plants immunocompetent truncated e glycoprotein of bovine viral diarrhea virus (bvdv) expressed in nicotiana tabacum plants: a candidate antigen for new generation of veterinary vaccines the effect of plant tissue and vaccine formulation on the oral immunogenicity of a model plant-made antigen in sheep use of the wound-inducible ntqpt promoter from nicotiana tabacum for production of a plant-made vaccine the release and induced immune responses of a plant-made and delivered antigen in the mouse gut plant expressed coccidial antigens as potential vaccine candidates in protecting chicken against coccidiosis immunogenicity study of plant-made oral subunit vaccine against porcine reproductive and respiratory syndrome virus (prrsv) extraction, purification and characterization of the plant-produced hpv subunit vaccine candidate e ggg plastid expression of a double-pentameric vaccine candidate containing human papillomavirus- l antigen fused with ltb as adjuvant: transplastomic plants show pleiotropic phenotypes anti-cancer activity of plant-produced hpv e vaccine production of human rotavirus and salmonella antigens in plants and elicitation of fljb-specific humoral responses in mice piri, i. isolation of ompa gene from salmonella typhimurium and transformation into alfalfa in order to develop an edible plant based vaccine expression of helicobacter pylori tonb protein in transgenic arabidopsis thaliana: toward production of vaccine antigens in plants expression of an immunogenic f -v fusion protein in lettuce as a plant-based vaccine against plague biopharmaceutical protein production under controlled environments: growth, development, and vaccine productivity of transgenic tomato plants grown hydroponically in a greenhouse plant-derived recombinant fl, v, and f -v fusion antigens of yersinia pestis activate human cells of the innate and adaptive immune system effective plague vaccination via oral delivery of plant cells expressing f -v antigens in chloroplasts a plant-produced plague vaccine candidate confers protection to monkeys plant-made subunit vaccine against pneumonic and bubonic plague is orally immunogenic in mice expression and immunogenicity of the mycobacterial ag b/esat- antigens produced in transgenic plants by elastin-like peptide fusion strategy superexpression of tuberculosis antigens in plant leaves oral immunogenicity of a plant-made, subunit, tuberculosis vaccine continued expression of plant-made vaccines following long-term cryopreservation of antigen-expressing tobacco cell cultures toward the development of a plant-based vaccine against reovirus generation of plant-derived recombinant dtp subunit vaccine analysis and stability of the respiratory syncytial virus antigen in a t generation of transgenic tomato plants immunogenic properties of plant-derived recombinant smallpox vaccine candidate pb accumulation of recombinant sars-cov spike protein in plant cytosol and chloroplasts indicate potential for development of plant-derived oral vaccines clinical safety and immunogenicity of tumor-targeted, plant-made id-klh conjugate vaccines for follicular lymphoma recombinant antibody g produced in maize endosperm efficiently neutralizes hiv- and contains predominantly single-glcnac n-glycans m cell-targeting ligand and consensus dengue virus envelope protein domain iii fusion protein production in transgenic rice calli expression of a cholera toxin b subunit and consensus dengue virus envelope protein domain iii fusion gene in transgenic rice callus novel vaccination approach for dengue infection based on recombinant immune complex universal platform a nonreplicating subunit vaccine protects mice against lethal ebola virus challenge immunization with plant expressed hemagglutinin protects chickens from lethal highly pathogenic avian influenza virus h n challenge infection engineering and expression of a human rotavirus candidate vaccine in nicotiana benthamiana expression of hepatitis b surface antigen in transgenic plants expression and characterization of hepatitis b surface antigen in transgenic potato plants production of hepatitis b surface antigen in transgenic plants for oral immunization oral immunization with hepatitis b surface antigen expressed in transgenic plants expression of the hepatitis b surface s and pres antigens in tubers of solanum tuberosum production of highly concentrated, heat-stable hepatitis b surface antigen in maize transient and stable expression of hepatitis b surface antigen in tomato (lycopersicon esculentum l.) expression of hepatitis b surface antigen in transgenic banana plants expression of the human hepatitis b virus large surface antigen gene in transgenic tomato plants hepatitis b surface expression in transgenic tobacco (nicotiana tabacum) plants using four different expression cassettes expression of hepatitis b surface antigen in transgenic banana plants and nt- cell line of tobacco hepatitis b surface antigen (hbsag) expression in plant cell culture: kinetics of antigen accumulation in batch culture and its intracellular form immunogenicity of transgenic plant-derived hepatitis b surface antigen analysis of transgenic tobacco plants carrying the gene for the surface antigen of the hepatitis b virus immunogenicity of parenterally delivered plant-derived small and medium surface antigens of hepatitis b virus oral administration of low doses of plant-based hbsag induced antigen-specific igas and iggs in mice, without increasing levels of regulatory t cells tissue specific expression of hepatitis b virus surface antigen in transgenic plant cells and tissue culture immunogenicity and tolerance following hiv- /hbv plant-based oral vaccine administration production of marker-free plants expressing the gene of the hepatitis b virus surface antigen virus-like particles expression and assembly in plants: hepatitis b and norwalk viruses secretion of hepatitis b surface antigen in transformed tobacco cell suspension cultures a plant signal peptide-hepatitis b surface antigen fusion protein with enhanced stability and immunogenicity expressed in plant cells process options in hepatitis b surface antigen extraction from transgenic potato study of the immunogenicity of hepatitis b surface antigen synthesized in transgenic potato plants with increased biosafety a plant-derived edible vaccine against hepatitis b virus low-dose oral immunization with lyophilized tissue of herbicide-resistant lettuce expressing hepatitis b surface antigen for prototype plant-derived vaccine tablet formulation freeze-drying of plant tissue containing hbv surface antigen for the oral vaccine against hepatitis b analysis of the limitations of hepatitis b surface antigen expression in soybean cell suspension cultures obtaining tomato plants transgenic for the pres -s-hdel gene, which synthesize the major hepatitis b surface antigen agrobacterium tumefaciens-mediated transformation of yellow lupin to generate callus tissue producing surface antigen of hbv in a long-term culture bioencapsulation of the hepatitis b surface antigen and its use as an effective oral immunogen supercritical fluid extraction provides an enhncement to the immune response for orally-delivered hepatitis b surface antigen oral delivery of wafers made from hbsag-expressing maize germ induces long-term immunological systemic and mucosal responses oral immunization of animals with transgenic cherry tomatillo expressing hbsag construction of a plant effective expression vector containing the gene of hepatitis b virus surface antigen the integration of a major hepatitis b virus gene into cell-cycle synchronized carrot cell suspension cultures and its expression in regenerated carrot plants expression of hepatitis b surface antigen gene (hbsag) in laminaria japonica (laminariales, phaeophyta) transforming hbsag into peanut and detection of its immunogenecity plant expression, lyophilisation and storage of hbv medium and large surface antigens for a prototype oral vaccine formulation oral immunogenicity of potato-derived hbsag middle protein in balb/c mice retention of the ability to synthesize hiv- and hbv antigens in generations of tomato plants transgenic for the tbi-hbs gene synthesis of hepatitis b virus surface antigen in tomato plants transgenic for the pres -s gene candidate mucosal vaccine against hepatitis b based on tomatoes transgenic for the pres -s gene comparative analysis of hbv m-antigen production in leaves of individual transgenic carrot plants application of the human hepatitis b virus core antigen from transgenic tobacco plants for serological diagnosis high-level production of hepatitis b core antigen in plant leaf and its immunogenicity in mice extremely high-level and rapid transient protein production in plants without the use of viral replication a dna replicon system for rapid high-level production of virus-like particles in plants tandem fusion of hepatitis b core antigen allows assembly of virus-like particles in bacteria and plants with enhanced capacity to accommodate foreign proteins seed-based expression systems for plant molecular farming the use of viral vectors to produce hepatitis b virus core particles in plants hbv core particles as a carrier for b cell/t cell epitopes envelopment of the hepatitis b virus nucleocapsid weekly epidemiological record; hepatitis b position paper world health organization (who) toward the global elimination of hepatitis b virus transmission optimization of elastin-like polypeptide fusions for expression and purification of recombinant proteins in plants plant-based edible vaccine against hbv oral immunization of human with transgenic lettuce expressing hepatitis b surface antigen the twenty-year story of a plant-based vaccine against hepatitis b: stagnation or promising prospects? recombinant hepatitis b vaccines: disease characterization and vaccine production. in production of recombinant proteins. novel microbial and eukaryotic systems plants as bioreactors for the production of vaccine antigens immunogenicity of a novel, bivalent, plant-based oral vaccine against hepatitis b and human immunodeficiency viruses saponin-adjuvanted particulate vaccines for clinical use immunomodulatory properties of vitamins, flavonoids and plant oils and their potential as vaccine adjuvants and delivery systems activation of antigen-presenting cells by immunostimulatory plant dna: a natural resource for potential adjuvant stability of s-hbsag in long-term stored lyophilised plant tissue key: cord- - gcpk x authors: koprowski, hilary title: vaccines and sera through plant biotechnology() date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: gcpk x nan during the last years, more vaccines and sera have become available for prophylaxis or treatment of disease than during the preceding century. yet at the same time, the number of obstacles preventing the use of those biomedical discoveries for the benefit of mankind has risen almost proportionately. stricter regulations for safety and efficacy have led to increased cost of production of biomedicals, but secondly the price to the consumer has risen to a degree that now precludes prophylactic vaccination against some diseases on a global scale. in addition to the extraordinarily high price of new vaccines and sera, the method of administration of these products through syringe and needle presents another burden in their worldwide use. in , i organized a mass vaccination trial with my oral polio vaccine in the then belgian congo. two hundred and fifty thousand individuals were vaccinated in weeks by oral spraying. this became a model for worldwide polio vaccination-a project requiring only one oral administration of a product-to eradicate polio in the world. the global use of vaccine requires that the vaccines be inexpensive and easily maintained and distributed. after considering various alternatives of fulfilling the criteria established for a global approach to immunization, it has become clear that our only choice is the production of vaccines or other materials of biomedical importance in plants. the advantages of plants are quite clear. plants are very inexpensive to grow in mass; some can grow in almost any soil or climate in the world. various parts of plants (leaves, seeds, fruits, and chloroplasts) can also be used as vehicles for the biomedical products. production of biomedicals in plants and their administration to humans and animals also provides an added margin of safety as compared to biologicals produced in animal tissues. early concerns that plants might not be capable of producing biomedicals of animal origin, and that plant-specific glycosylation patterns might functionally alter plant-produced antibodies have proven unwarranted. basic methods to transfer foreign genese into plants are well established. in the first approach, the plant is transfected with a foreign gene in combination with the plant parasite, agrobacterium tumefaciens. in this constitutive approach, future generations of the same transgenic plant species heritably retain the capacity to express the original product. the product is isolated from the extract of leaves, seeds, etc. (table ). in the second approach, the gene is inserted into a plant virus (for example, alfalfa or tobacco mosaic virus), which is used in combination with a foreign antigen to infect plants. here, the final product is harvested from the plant in tandem with the plant virus. in both cases, purification of the vaccine or antibody is a rather simple procedure. the achievements in production of plant-derived vaccines and sera, can be classified in two categories: ( ) vaccines and antibodies for infectious diseases; ( ) vaccines and antibodies for cancer. table shows the list of infectious disease agents against which plant-derived products were made in the biotechnology foundation laboratories, in chronological order, respiratory syncytial virus (rsv), hepatitis b, hiv, rabies, anthrax, diphtheria, sars, and smallpox virus. one of the first vaccines produced in this system was against rsv, which causes disease of newborn children. we produced the vaccine antigen as a fusion protein with the capsid protein of alfalfa mosaic virus in tobacco. immunogenicity was tested in mice, which were either injected with or fed the plant-produced vaccine ( as compared to controls; high-titer antibodies against rsv were also induced. plant-derived vaccines for hiv and anthrax will be presented by dr. alexander karasev. scientists under the leadership of professor andrzej legocki in poznan, poland, have produced a vaccine against hepatitis b in transgenic lettuce. these scientists have shown that vaccines expressed in lettuce leaves can be ground, frozen, dried and powdered without loss of immunogenicity. observation of immune response in mice fed the plant-derived vaccine indicates that two feedings at -month intervals produce optimal immunogenic responses. rabies is a worldwide disease of humans and warmblooded animals. except for the time-honored pasteur type vaccine, all modern vaccines are too expensive for use on a worldwide scale. rabies antibody is essential in treating severe bites but is unavailable because it is not sufficiently lucrative for production by the pharmaceutical industry. rabies in dogs is a major, but not unique, source of infection in southeast asia and in many african countries. an inexpensive bait vaccine for dogs could lead to the eradication of rabies in those parts of the world. to express rabies vaccine in plants, we have used a recombinant alfalfa mosaic virus in spinach leaves. this virus expressed a chimeric peptide containing antigenic determinants of rabies virus glycoprotein, which elicits immune responses; and of nucleoprotein, which increases glycoprotein immunogenicity. the chimeric gene antigen was pcr-amplified and cloned for fusion with the gene encoding the coat protein of alfalfa mosaic virus. tobacco and spinach were the plants used to express the antigen. the presence of rabies virus protein in spinach leaves was checked by western blot and immunogenicity was tested in mice. then volunteers were fed g of spinach extract recombinant virus twice at -week intervals. half of the subjects responded to the oral administration of spinach as indicated by a vigorous booster response after a single injection of commercial vaccine. there is a global crisis in post-exposure treatment of rabies because of worldwide unavailability of the antirabies serum required for severe bites. thus, it was decided on an emergency basis to produce rabies antibody in plants. research conducted by dr. kisung ko, led to the production of a transgenic tobacco plant containing the heavy and light chains of human rabies antibody. the two chains recombined in the plants to produce a complete antirabies antibody, which was as effective as the original antibody in animals, before and after exposure to rabies (table ). two very interesting results emerged from the research with this antibody. first, leaf extract of the transgenic tobacco was found to be virtually free of nicotine and other alkaloids (fig. ) . second, the glycosylation pattern of the plant-derived antibody was considerably different from that of the animal tissue-derived antibody (fig. ) . mannose is the only glycan prevalent in the plantibody, yet the antibody was at least as efficacious as the native antibody produced in animals. table in vivo efficacy of mab p for post-exposure prophylaxis of hamster injected with a lethal dose of coyote rabies street viruses (dr. c. rupprecht, cdc) post-exposure treatment antibody a (iu/animal) vaccine (hdcv) b − + mab p ( ) / c / mab p ( . ) / / mab p ( . ) / / hrig ( ) / / untreated control / / ko et al. [ ] , pnas. a iu: international unit. b hdcv: (imovax, lot mo ) human diploid cell culture rabies virus vaccine, "−" and "+" indicate treatments without or with hdcv, respectively. c number of surviving hamsters/number of hamsters tested. strict guidelines regarding the amount and potency of antirabies antibody for use in post-exposure treatment has enabled calculation-based on the yield of antibody per tobacco plant-of plant acreage needed to produce a given number of antibody international units. if, as expected, kg of tobacco produces - mg of antibody, ha or . acres is needed to produce enough antibody for vaccination of , people. i leave it to accountants to calculate the production cost of such an antibody as compared to antibody produced in animal tissue. one of the many advantages of plants for production of biological products is their ability to express multiple genes in the same plant. vaccines against diphtheria, tetanus, and oral pertussis are the triad injected into infants during their first - months of life. a plan to substitute oral products for injectable vaccines was initiated through production of transgenic tobacco (as a model) and transgenic carrots expressing all three vaccines (table ). plants expressing current diphtheria toxoid have already been produced (fig. ) . the spike protein of sars coronavirus is presumed to be immunogenic. transgenic tobacco and tomato expressing the spike protein were generated (fig. ) . interestingly, only months elapsed between the introduction of the spike construct into the plant and the first observation of sars coronavirus mrna expression by the plant (fig. ) . theoretically, the vaccine could be obtained by growing large numbers of transgenic plants. it is doubtful whether other methods currently available would produce vaccine material against a newly discovered disease in such a short time. after decades of neglect, we are now witnessing a revived interest in immunological approaches to cancer treatment. thus, we sought expression of colorectal cancer anti-gen ga and of the corresponding - a antibody in plants. attempts were also made to express antibodies recognizing epidermal growth factor (egf) which is present at the surface of epithelial tumors in plants. the tobacco mosaic virus-based gene vector (p b) was used for expressing the - ga antigen. nicotiana betthamiana was infected with the construct and checked expression by western blot analysis of the leaf extract. a group of mice were then immunized with the plant-produced - antigen and, for comparison, another group with the same antigen expressed in baculovirus systems. sera obtained from both groups of mice recognized human colorectal cell lines expressing this antigen (fig. ) . a cross reactivity test capable of discriminating the two antigens, that produced in plants and that produced in baculovirus vector, ruled out any "contaminant" basis for the immune response. complement-dependent cytotoxicity assays of sera obtained from mice immunized with the plant-derived - antigen showed lysis of breast cancer cells, but not of antigenically unrelated melanoma cells (fig. ) . the plantderived - antigen also induced antigen-specific proliferation of t cells. using the same technique as for antirabies antibodies, we expressed - -reactive antibody - a in tobacco plants. purified leaf extract of these plants specifically recognized receptors present in human colorectal cancer cells. perhaps the most interesting results obtained from the study of the two plantibodies against rabies and cancer relate to the glycosylation pattern. the plant-derived antirabies heavy chain was fused to kdel (lysineasperagine-glycosamine-leucine) endoplasmic reticulum retention signal. as a result, the anti-rabies plantibody contained abundant mannose, whereas the plant-derived anticancer antibody contained less mannose and more nacetylglucosamine (fig. ) . despite the different glycosylation patterns of the two plantibodies and the difference of both patterns from those of the native antibody, their biological activity was equivalent to each other and to that of the native antibody. antibodies reactive with the epidermal growth factor were recently licensed to treat certain types of epithelial cancers. such antibodies were first developed at the wistar institute many years ago. they have recently been adapted to production in tobacco plants and within the near future might provide an inexpensive therapeutic tool in human cancer. however, plant production of vaccines and sera is not a simple procedure with assured success in each undertaking. one of the most important remaining problems is the low yield of the bioproduct in plants. to date, no magical solution to this problem has been found. codon optimization, careful approaches to harvesting and purifying plant products, use of plant parts such as chloroplasts to increase uptake of the material are but a few potential avenues to help increase the yield of the final product. at present, it is still difficult to produce sizeable amounts of plant-derived products. great strides have been made in our knowledge and expertise in the use of plants as hosts for production of biomedicals. table there are four stages of adopting new ideas the first is "it's impossible" the second is "maybe it's possible, but it's weak and uninteresting" the third is, "it's true and i told you so" and the fourth is, "i thought of it first" as shown in the last table (table ), a segment of the population remains to be convinced that the future of biologicals is linked with production in plants. some people will consider the facts and ultimately opt for acceptance. others will remain "true believers" in harmful effects caused by transgenic plants. their voices may become subdued or even silenced by increasing evidence that the future of global health improvement rests strictly in the progress made on the worldwide use of plants to combat diseases. function and glycosylation of plant-derived antiviral monoclonal antibody key: cord- -zfh im authors: saxena, jyoti; rawat, shweta title: edible vaccines date: - - journal: advances in biotechnology doi: . / - - - - _ sha: doc_id: cord_uid: zfh im in recent years edible vaccine emerged as a new concept developed by biotechnologists. edible vaccines are subunit vaccines where the selected genes are introduced into the plants and the transgenic plant is then induced to manufacture the encoded protein. foods under such application include potato, banana, lettuce, corn, soybean, rice, and legumes. they are easy to administer, easy to store and readily acceptable delivery system for different age group patients yet cost effective. edible vaccines present exciting possibilities for significantly reducing various diseases such as measles, hepatitis b, cholera, diarrhea, etc., mainly in developing countries. however, various technical and regulatory challenges need to overcome in the path of this emerging vaccine technology to make edible vaccine more efficient and applicable. this chapter attempts to discuss key aspects of edible vaccines like host plants, production, mechanism of action, advantages and limitations, applications, and different regulatory issues concerned to edible vaccines. vaccine is a biological preparation intended to produce immunity to a disease by stimulating the production of antibodies. dead or attenuated organisms or purified products derived from them are generally used to produce various vaccines. over the past decade, scientific advances in genetics, molecular biology, and plant biotechnology have improved the understanding of many infectious diseases and led to the development of vaccination programs. the most common method of administering vaccines is by injection but some are given by mouth or nasal spray. though immunization is the safest method to combat the diseases worldwide but there are many constraints regarding its mode of production, distribution, delivery, cost, and lack of enough research. hence it is desirable to look for an effectual and powerful yet cost effective, easy for storage and distribution yet safe method of immunization. it should also be readily acceptable to all sociocultural groups around the globe. research underway is dedicated to solving these problems by finding ways to produce edible vaccines in the form of transgenic plants which have been investigated as an alternative means to produce and deliver vaccine. edible vaccines are called by several alternative names such as food vaccines, oral vaccines, subunit vaccines, and green vaccines. they seem to be a viable alternative especially for the poor and developing countries. they have come up as great boon in medicinal science for which biotechnologists should be given all credit. the concept of edible vaccines lies in converting the edible food into potential vaccines to prevent infectious diseases. it involves introduction of selected desired genes into plants and then inducing these altered plants to manufacture the encoded proteins. it has also found application in prevention of autoimmune diseases, birth control, cancer therapy, etc. edible vaccines are currently being developed for a number of human and animal diseases. this new technology hopefully will contribute positively toward the global vaccine programs and have a dramatic impact on health care in developing countries. many people in developing countries do not have access to the vaccines they need, as the traditional vaccines are costly and require skilled medical people for administration and are less effective in inducing mucosal immune response. it was these needs which inspired hiatt et al. ( ) who attempted to produce antibodies in plants which could serve the purpose of passive immunization. the first report of edible vaccine (a surface protein from streptococcus) in tobacco, at . % of total leaf protein level, appeared in in the form of a patent application published under the international patent cooperation treaty. by conceiving the idea of edible vaccine dr. charles arntzen tried to realize it (arntzen ) . in , arntzen and coworkers introduced the concept of transgenic plants as a production and delivery system for subunit vaccines in which edible tissues of transgenic crop plants were used (mor et al. ) . they found that this concept could overcome the limitations of traditional vaccines, thereby triggering the research on edible vaccine. in s, streptococcus mutans surface protein antigen a was expressed for the first time in tobacco. the same group also pioneered the field with work on hepatitis b and heatlabile toxin, b subunit in tobacco plants and potato tubers. in the same year, the successful expression of hepatitis b surface antigen (hbsag) in tobacco plants was also achieved (mason et al. ) . to prove that plant-derived hbsag could stimulate mucosal immune responses via oral route, potato tubers were used as an expression system and were optimized to increase the accumulation of the protein in plant tubers (richter et al. ) . parallel to the evaluation of plant-derived hbsag, mason and arntzen explored plant expression of other vaccine candidates including the labile toxin b subunit (lt-b) of enterotoxigenic escherichia coli (etec) and the capsid protein of norwalk virus. the plant-derived proteins correctly assembled into functional oligomers that could elicit the expected immune responses when given orally to animals (mason et al. ). in a new era was opened in vaccine delivery when researchers supported by the national institute of allergy and infectious diseases (niaid) have shown for the first time that an edible vaccine can safely generate significant immune responses in people. the report by collaborators from the university of maryland in baltimore, the boyce thompson institute for plant research in ithaca, n.y., and tulane university in new orleans appeared in the may issue of nature medicine. according to the then director of niaid ''edible vaccines offer exciting possibilities for significantly reducing the burden of diseases like hepatitis and diarrhea, particularly in the developing world where storing and administering vaccines are often major problems,'' mor et al. ( ) also discussed the rapid increase of research in the edible-vaccine field and pointed out that plants could be used to create multicomponent vaccines that can protect against several pathogens at once. this is an aspect of the edible-vaccine approach that further strengthens its impact. later, in sala and research group reported that proteins produced in these plants induced the mucosal immune response which was the main aim behind this concept. research into edible vaccine is still at a very early stage and scientists have a long way to go before it will become a major part of immunization program world wide. to date, many plant species have been used for vaccine production. the choice of the plant species is important. an edible, palatable plant is necessary if the vaccine is planned for raw consumption. in case of vaccine for animal use, the plant should preferentially be selected among those consumed as normal component of the animal's diet. some food vehicles are discussed below: the concept of edible vaccine got impetus after arntzen and coworkers expressed hbsag in tobacco. the first edible vaccine was produced in tobacco in in which . % recombinant protein (a surface protein from streptococcus) of the total soluble leaf proteins was found. it appeared in the form of a patent application published under the international patent cooperation treaty. transgenic tobacco is successfully engineered for the production of edible vaccines against hepatitis b antigen using 's' gene of hepatitis b virus (hbv). the optimum level of recombinant protein was obtained in leaves and seeds. since acute watery diarrhea is caused by enterotoxigenic e. coli and vibrio cholerae that colonize the small intestine and produce one or more enterotoxin, an attempt was made toward the production of edible vaccine by expressing heat-labile enterotoxin (lt-b) in tobacco. besides, antibodies against dental caries, expressed in tobacco, are already in preclinical human trials. italian researchers have now developed an immunologically active, cost-efficient vaccine against human papilloma viruses (hpv). hpv are the causative agents for cervical cancer, and are also involved in skin, head, and neck tumors. cervical cancer is one of the main causes of cancer-related deaths. genetically modified potatoes are also a viable option and seem to be the desired vector. many of the first edible vaccines were synthesized in potato plants. in serum antibodies at some point after immunization, and of the ( %) also showed fourfold mount in intestinal antibodies. the potatoes were well tolerated and no one experienced serious adverse side effects. vaccine development has successfully tested a potato-based vaccine to combat the norwalk virus, which is spread by contaminated food and water. the virus causes severe abdominal pain and diarrhea. a research team led by william langridge of the loma linda university in california has reported that transgenic potatoes engineered with a cholera antigen, ctb can effectively immunize mice. mice fed transgenic potatoes produce cholera-specific antibodies in their serum and intestine; iga and igg antibodies reach their highest levels after the fourth feeding. in yet another experiment genetically engineered potatoes containing a hepatitis b vaccine have successfully boosted immunity in their first human trials. attempts have also been made to boil the potatoes as raw potatoes are not very appetizing but unfortunately the cooking process breaks down about % of the proteins in the vaccine. while some proteins are more tolerant to heat, for most proteins it will be necessary to amplify the amount of protein in the engineered foods if they are to be cooked before consumption. tomatoes are an excellent candidate because they are easy to manipulate genetically and new crops can be grown quickly. moreover, they are palatable and can be eaten raw. while tomatoes do not grow well in the regions in which the edible vaccines are most needed, the engineered tomatoes can be dried or made into a paste to facilitate their delivery. the anti-malaria edible vaccines in different transgenic tomato plants expressing antigenic type(s) have been proposed by chowdhury and bagasara in . they hypothesized that immunizing individuals against - antigens and against each stage of the life cycle of the multistage parasites would be an efficient, inexpensive and safe way of vaccination. tomatoes with varying sizes, shapes, and colors carrying different antigens would make the vaccines easily identifiable by lay individuals. tomatoes serve as an ideal candidate for the hiv antigen because they unlike other transgenic plants that carry the protein, are edible and immune to any thermal process, which help to retain their healing capabilities. scientists have claimed that tomatoes could be used as a vaccine against alzheimer's disease. the work is in progress to genetically modify the fruit to create an edible vaccine that fires up the immune system to tackle the disease by attacking the toxic beta-amyloid protein that destroys vital connections between brain cells, causing alzheimer's. researchers have engineered tomato plants (lycopersicon esculentum mill var. uc b) to express a gene for the glycoprotein (g-protein), that coats the outer surface of the rabies virus. the recombinant constructs contained the gprotein gene from the era strain of rabies virus, including the signal peptide, under the control of the s promoter of cauliflower mosaic virus (camv). a common fruit-the banana-is currently being considered as a potential vehicle for vaccines against serious as well as too common diseases. the advantage of bananas is that they can be eaten raw as compared to potatoes or rice that need to be cooked and can also be consumed in a pure form. furthermore, children tend to like banana and the plants grow well in the tropical areas in which the vaccines are needed the most. hence, the research is leaning toward the use of banana as the vector since a large number of third-world countries, who would benefit the most from edible vaccines have tropical climates. on the negative side, a new crop of banana plants takes about months to bear fruit. after fruiting, the plants are cut down and a new crop of vaccine-bearing plants must be planted. researchers have also developed bananas that deliver a vaccine for hbv. the banana vaccine is expected to cost just cents a dose, as compared to the $ for the currently available injectable vaccine. maize has also been used as a vector for various edible vaccines. egyptian scientists have genetically engineered the maize plants to produce a protein known as hbsag which elicits an immune response against the hepatitis b virus and could be used as a vaccine. if human trials are successful more than billion people infected with hepatitis b, and about million of these at high risk of serious illness and death from liver damage and liver cancer would be benefited. researches are in offing at iowa state university with the aim to allow pigs and humans to get a flu vaccination simply by eating corn or corn products. it is quite likely that corn vaccine would work in humans when they eat corn or even corn flakes, corn chips, tortillas, or anything that contains corn. genetically modified maize could provide protection to chickens against a highly contagious and fatal viral disease affecting most species of birds. mexican researcher octavio guerrero-andrade and his colleagues at the centre for research and advanced studies in guanajuato, central mexico, genetically modified maize to create an edible vaccine against newcastle disease virus (ndv). they inserted a gene from the ndv, a major killer of poultry in developing countries, into the maize dna and found antibodies against the virus in chickens that ate the genetically modified maize. one pig vaccine has also been produced in corn successfully. efforts are being made by us company prodigene to genetically modify maize to contain a key protein found on the surface of the monkey form of hiv. according to us national institute of health this development brings an edible, more effective, hiv vaccine for people a step closer. transgenic maize expressing the rabies virus glycoprotein (g) of the vnukovo strain has also been produced using ubiquitin maize promoter fused to the whole coding region of the rabies virus g gene, and a constitutive promoter from camv. maize embryogenic callus were transformed with the above construct by biolistics. regenerated maize plants were recovered and grown in a greenhouse. the amount of g-protein detected in the grains was approximately % of the total soluble plant protein. rice is another potential crop which has been used for developing vaccines. it offers several advantages over traditional vaccines; it does not require refrigeration. in fact, the rice proved just as potent after months of storage at room temperature and the vaccine did not dissolve when exposed to stomach acids. in an attempt, predominant t cell epitope peptides, which were derived from japanese cedar pollen allergens, were specifically expressed in rice seeds and delivered to the mucosal immune system (mis); the development of an allergic immune response of the allergen-specific th cell was suppressed. furthermore, not only the specific ige production and release of histamine from mast cells were suppressed, but the inflammatory symptoms of pollinosis, such as sneezing, were also suppressed. these results suggest the feasibility of using an oral immunotherapy agent derived from transgenic plants that accumulate t cell epitope peptides of allergens for allergy treatment. the transfer of genetic material from the microbe responsible for producing cholera toxin into a rice plant has been achieved. the plants produced the toxin and when the rice grains were fed to mice they provoked immunity from the diarrhea-causing bacterium. genetically modified spinach has also been considered for the development of edible vaccine. spinach is being investigated as a plantderived, edible vehicle for anthrax vaccine, as well as a vehicle for the hiv- tat protein (a prospective vaccine candidate). in an experiment a fragment of protective antigen (pa) that represents most of the receptor-binding domain was expressed as a translational fusion with a capsid protein on the outer surface of tobacco mosaic virus, and spinach was inoculated with the recombinant virus. the plant-expressed pa is highly immunogenic in laboratory animals. among other food crops with potential to be developed as edible vaccine; sweet potato, peanuts, lettuce, watermelon, and carrots are on the top priority. the development of plant-based vaccines to protect against many other diseases, such as hiv- , hepatitis b, rabies, and non-hodgkin's lymphoma are ongoing throughout the globe using one of these edible plants. the advantages and disadvantages of various plant host systems are given in conventional subunit vaccines are expensive and technology-intensive, need purification, require refrigeration, and produce poor mucosal response. in contrast, edible vaccines would enhance compliance, especially in children, and because of oral administration would eliminate the need for trained medical personnel. their production is highly efficient and can be easily scaled up. for example, hepatitis b antigen required to vaccinate whole of china annually, could be grown on a -acre plot and all babies in the world each year on just acres of land. they are cheaper, sidestepping demands for purification (single dose of hepatitis b vaccine would cost approximately . cents), grown locally using standard methods and do not require capitalintensive pharmaceutical manufacturing facilities. mass-indefinite production would also decrease dependence on foreign supply. fear of contamination with animal viruses-like the mad cow disease, which is a threat in vaccines manufactured from cultured mammalian cells, is eliminated as plant viruses do not infect humans. edible vaccines activate both mucosal and systemic immunity, as they come in contact with the digestive tract lining which is not possible with subunit vaccines which provide poor mucosal response. this dual effect of edible vaccines provides first-line defense against pathogens invading through mucosa, such as mycobacterium tuberculosis and agents causing diarrhea, pneumonia, stds, hiv, etc. the specific advantages are stated below: . edible means of administration. . no need of medical personnel and syringes. . sterile injection conditions are no more required. . economical in mass production by breeding compared to an animal system. . easy for administration and transportation. . effective maintenance of vaccine activity by controlling the temperature in plant cultivation. . therapeutic proteins are free of pathogens and toxins. . storage near the site of use. . heat stable, thus eliminating the need of refrigeration. potential for outcrossing in field; deep root system problematic for cleaning field (mason et al. ) . antigen protection through bioencapsulation. . subunit vaccine (not attenuated vaccine) means improved safety. . seroconversion in the presence of maternal antibodies. . generation of systemic and mucosal immunity. . enhanced compliance (especially in children). . delivery of multiple antigens. . integration with other vaccine approaches. . plant-derived antigens assemble spontaneously into oligomers and into virus like particles. . no serious side effect problems have been noticed until now. . reduced risk of anaphylactic side effects from edible vaccine over injection system is one benefit reported by the bio-medicine.org. they reported that the edible vaccine carries only part of the allergen compared to injection methods which reduce anaphylactic risk. . administration of edible vaccines to mothers to immunize the fetus-in utero by transplacental transfer of maternal antibodies or the infant through breast milk. edible vaccines have a potential role in protecting infants against diseases like group-b streptococcus, respiratory syncytial virus (rsv), etc., which is under investigation. . edible vaccines would also be suitable against neglected/less common diseases like dengue, hookworm, rabies, etc. they may be integrated with other vaccine approaches and multiple antigens may also be delivered. with advancement come many hurdles and problems, so is true for edible vaccines. like, one could develop immunotolerance to the vaccine peptide or protein, though a little research has been done on it. one of the key goals of the edible-vaccine pioneers was to reduce immunization costs but later many limitations were reported as given below: . consistency of dosage from fruit to fruit, plant to plant, lot to lot, and generation to generation is not similar. . stability of vaccine in fruit is not known. . evaluation of dosage requirement is tedious. . selection of best plant is difficult. . certain foods like potatoes are generally not eaten raw and cooking the food might weaken the medicine present in it. . not convenient for infants as they might spit it, eat a part or eat it all, and throw it up later. concentrating the vaccine into a teaspoon of baby food may be more practical than administering it in a whole fruit. . there is always possibility of sideeffects due to the interaction between the vaccine and the vehicle. . people could ingest too much of the vaccine, which could be toxic, or too little, which could lead to disease outbreaks among populations believed to be immune. . a concern with oral vaccines is the degradation of protein components in the stomach due to low ph and gastric enzymes. however, the degradation can be compensated by repeating the exposure of the antigen until immunological tolerance is accomplished (mason et al. ) . . potential risk of spreading the disease by edible vaccine delivery is a concern of many. potential contamination of the oral delivery system is a possible danger. foreign proteins in plants accumulate in low amounts ( . - % of total protein) and are less immunogenic, therefore the oral dose far exceeds the intranasal/parenteral dose. for example oral hepatitis b dose is - times the parenteral dose and g potato expressing b subunit of labile toxin of etec (lt-b) is required in three different doses to be immunogenic. attempts at boosting the amount of antigens often lead to stunted growth of plants and reduced tuber/fruit formation as too much mrna from the transgene causes gene silencing in plant genome. techniques to overcome these limitations are given below: . optimization of coding sequence of bacterial/ viral genes for expression as plant nuclear genes . expression in plasmids . plant viruses expressing foreign genes . coat-protein fusions . viral-assisted expression in transgenic plants . promoter elements of bean yellow dwarf virus with reporter genes gus (b -glucuronidase) and green fluorescent protein (gfp), substituted later with target antigen genes. . antigen genes may be linked with regulatory elements which switch on the genes more readily or do so only at selected times (after the plant is nearly fully grown) or only in its edible regions. exposure to some outside activator molecule may also be tried. development of edible vaccines is a possible high-volume, low-cost delivery system for thirdworld countries to fight against fatal maladies like aids, hepatitis, and diarrhea. researches by the niaid and the university of maryland showed no significant side effects in a small study using genetically engineered potatoes to make toxin of the e. coli, a diarrhea-causing bacterium. volunteers reported no serious adverse reactions to genetically altered potatoes used to deliver edible vaccine toxin, according to the national institutes of health (nih). the nih reported that - volunteers who ate the raw potato bites developed four times the antibodies against e. coli without obvious side effects. long-term reactions to edible vaccines are yet to be determined and possible delayed reactions have not yet been discovered. an organized large scale study is required before edible vaccines are put into large scale production. creating edible vaccines involves the genetic engineering approach for the introduction of selected desired genes into plants and then inducing these altered plants to manufacture the encoded proteins. this process is known as ''transformation'' and the plants altered with new characteristics are called ''transgenic plants''. like conventional subunit vaccines, edible vaccines are composed of antigenic proteins and are devoid of genes responsible for pathogenicity. thus, they have no way of establishing infection, assuring its safety, especially in immune-compromised patients. the gene which codes the active antigenic protein is first isolated from the pathogen and is incorporated in a suitable ''gene vehicle''. this gene vehicle is integrated into the genome of the plant and is allowed to express the corresponding antigen. then these plant parts are fed to animals and humans to run their course. two main strategies are used for the production of candidate vaccine antigen in plant tissues (fig. . a, b) . . stable genomic integration: this is the most popular method used for the published edible vaccine clinical trials to date. under this method the genetic line is produced that can be propagated either by vegetative (stem cuttings) or sexual (seeds) reproduction methods. the stable expression strategy provides an opportunity to introduce more than one gene for possible multicomponent vaccine production. furthermore, the choice of genetic regulatory elements allows organ and tissue-specific expression of foreign antigens. stable transformation causes the desired gene to be incorporated either in nucleus or chloroplast. agrobacterium mediated gene transfer is used for transforming the plants in which the gene is integrated in nucleus. besides, direct delivery of dna into the tissue can also be applied, biolistic being the most popular method. however, chloroplast engineering has also got impetus in last decade due to its various advantages over nuclear engineering. gram-negative soil bacterium, which infects the wound sites in dicot plants causing the formation of the crown gall tumor. this bacterium is capable of transferring a particular dna segment (t-dna) of the tumor-inducing (ti) plasmid into the nucleus of infected cells where it is subsequently integrated into the host genome and transcribed. the t-dna usually contains cancer-causing oncogenic genes and genes that synthesize opines which are excreted by infected crown gall cells and are a food source for bacterium. during the genetic manipulation, the ti plasmid is engineered to carry the desired gene for vaccine and the virulent genes that cause tumor growth in plants are deleted. the transgene is integrated, expressed, and inherited in mendelian fashion. the whole plant can be then regenerated from individual transformed plant cell. it has been studied that genes are successfully expressed in experimental model plants and when given orally to animals, the extract of transgenic plant containing the antigen induced serum antibodies, thus can be used to produce the edible vaccine. the application of agrobacterium mediated transformation is at present possible to most species of agronomic interest, including members of family graminae and leguminosae. this opens interesting new aspect for the development of edible vaccines for both human and veterinary uses. the second approach for nuclear transformation is based on the microprojectile bombardment method, also known as the gene gun or biolistic method. this method is especially beneficial for those plants which can not be transformed by a. tumefaciens mediated gene transfer method. selected dna sequences are precipitated onto metal microparticles and bombarded with a particle gun at an accelerated speed in a partial vacuum against the plant tissue placed within the acceleration path. microparticles penetrate the walls and release the exogenous dna inside the cell where it will be integrated in the nuclear genome. thus, this method effectively introduces dna. the cells that take up the desired dna, are identified through the use of a marker gene (in plants the use of gus is most common), and then cultured to replicate the gene and possibly cloned. this method has various advantages including ( ) thousands of particles are accelerated at the same time causing multiple hits resulting in transferring of genes into many cells simultaneously, ( ) since intact cells can be used, the difficulties encountered with the use of protoplast are automatically circumvented, and ( ) the method is universal in its application so that cell type, size, and shape or the presence/absence of cell wall do not significantly alter its effectiveness. another important use of the gene gun involves the transformation of organelles such as chloroplasts, and yeast mitochondria. the biolistic particle delivery system ''shoots'' adequately processed dna particles, which penetrate into the chloroplast and integrate with its genome. the chloroplast's transformation is an interesting alternative to nuclear transformation which has come up in recent past. all plant cells have chloroplasts that capture light energy from the sun to produce free energy through a process called photosynthesis. in chloroplast genetic engineering, the recombinant dna plasmid is bound to small gold nanoparticles that are injected into the chloroplasts of the leaf using a gene gun as described above. this device uses high pressure to insert the plasmid coated particles into the cells. these plasmids contain multiple genes of importance such as the therapeutic gene, a marker gene (may or may not be for antibiotic resistance), a gene that enhances the translation of therapeutic gene and two targeting sequences that flank the foreign gene. the foreign genes are inserted through homologous recombination via flanking sequences at a precise and predetermined location in the organelle genome. the gene expression level in plastids is predominately determined by promoter and -untranslated regions ( -utr elements) (gruissem and tonkyn ) . therefore, suitable -utrs including a ribosomal binding site (rbs) are important elements of plastid expression vectors (eibl et al. ) . in order to obtain high-level protein accumulation from expression of the transgene, the first requirement is a strong promoter to ensure high levels of mrna. most laboratories use the strong plastid rrna operon (rrn) promoter (prrn). besides gene gun, peg mediated transformation and galistan expansion femto syringe microinjection techniques are also used for gene delivery in chloroplast. some of the advantages of chloroplast transformation technology are its low cost, natural gene containment, site specific insertion, very high level of stable expression, generation of production lines with a competitive timeline, elimination of gene silencing, and high accumulation of the recombinant protein. precise steps are given below: step : a heteroplasmic diploid plant cell (first round of selection) a homoplasmic diploid plant cell (second round of selection) step : multiple gene expression step : reproductive organs disintegration of paternal plastids step : maternal inheritance of transgenic traits. the most common entry point for pathogens is at mucosal epithelia lining the gastrointestinal, respiratory, and urino-reproductive tracts, which are collectively the largest immunologically active tissue in body. the mis is the first line of defense and the most effective site for vaccination against those pathogens; nasal, and oral vaccines being the most effective. the goal of oral vaccine is to stimulate both mucosal and humoral immunity against pathogens. edible vaccines have plant parts which are fed directly and the outer tough wall of plant cell acts to protect the antigens against attack by step : single gene step an antigen in a food vaccine is taken up by m cells in the intestine and passed to various immune system cells, which then start a defensive attack, as antigen is a true infectious agent, not just part of one. the response leaves longlasting ''memory cells'' able to promptly neutralize the real infectious agent. the whole procedure can be explained in two stages (fig. . a, b) antibodies and antibody fragments produced against specific antigens are given in table . . there are numerous therapeutic and diagnostic applications of edible vaccines which are summarized in table . . some of the diseases on which the work is going on are described below: malaria is a disease of humans transmitted by the bite of an infected mosquito. it remains one of the most significant causes of human morbidity and mortality worldwide. according to who's world malaria report there are more than million cases of malaria killing around , people. three antigens are currently being investigated for the development of a plant-based malaria vaccine, merozoite surface protein (msp) , msp from plasmodium falciparum and msp / from p. yoelli. wang et al. ( ) have demonstrated that oral immunization of mice with recombinant msp , msp / , and msp , co-administered with ctb as a mucosal adjuvant, induces antibody responses effective against blood stage parasite. measles is an infection of the respiratory system caused by a virus. in an experiment mice fed with tobacco expressing mv-h (measles virus haemagglutinin from edmonston strain) antibody titers five times the level considered protective for humans could be attained and secretory iga was found in their feces. prime boost strategy by combining parenteral and subsequent oral mv-h boosters could induce titers times the human protective levels. these titers were significantly greater than with either of the vaccine administered alone. mv-h edible vaccine does not cause atypical measles, which may be occasionally seen with the current vaccine. thus, it may prove better for achieving its eradication. the success in mice has prompted similar experiments in primates. transgenic rice, and lettuce, and baby food against measles are also being developed. when given with ctb (adjuvant), - g mv-h lettuce is enough; however, an increased dose would be required if given alone. rabies is a deadly viral infection that is transmitted to humans from animals. tomato plants expressing rabies antigens could induce antibodies in mice. alternatively, tmv may also be used. transformed tomato plants using camv with the glycoprotein (g-protein) gene of rabies virus (era strain) was shown to be immunogenic in animals. hepatitis b is a potentially life-threatening liver infection caused by the hepatitis b virus. it is estimated to have infected million people throughout the globe, making the virus one of the most common human pathogen. first human trials of a potato-based vaccine against hepatitis b have reported encouraging results. since immunization is the only known method to (das ) prevent the disease of the hepatitis b virus, any attempt to reduce its infection requires the availability of large quantities of vaccine hbsag. the amount of hbsag needed for one dose could be achieved in a single potato. levels of specific antibodies significantly exceeded the protective level of miu/ml in humans. when cloned into camv, the pcmv-s plasmid encoding the hbsag subtype ayw showed higher expression in roots as compared to leaf tissue of the transgenic potato. furthermore, expression of the antigen was found to be higher in roots of transgenic potato than in leaf tissues. however, the expression of hbsag in transgenic potatoes is not sufficient for using as oral vaccine. further studies are underway to increase the level of hbsag by using different promoters e.g., patatin promoter, and different transcription regulating elements. cholera is an infection of the small intestine that causes a large amount of watery diarrhea. it causes up to million deaths per year in the developing world, primarily among children. studies supported by who have demonstrated possibility of an effective vaccine for cholera, which provides cross protection against enterotoxic e. coli. to address this limitation, plants were transformed with the gene encoding b subunit of the e. coli heat liable enterotoxin (lt-b). transgenic potatoes expressing lt-b were found to induce both serum and secretory antibodies when fed to mice; these antibodies were protective in bacterial toxin assay in vitro. this is the first ''proof of concept'' for the edible vaccine. since people eat only cooked potatoes, the effect of boiling on the properties of ctb expressed in transgenic potatoes was examined. after boiling for min, over half of the vaccine protein survived in its biologically active form, providing evidence that cooking does not always inactivate edible vaccines. thus, the spectrum of plants for producing edible vaccines may be expanded beyond raw food plants such as fruits. co-expression of mutant cholera toxin subunit (mct-a) and lt-b in crop seeds has been shown to be effective by nasal administration and is extremely practical. the prevalence of diabetes is increasing globally and india is no exception. more than million people are affected with diabetes worldwide. type-i diabetes, also known as insulindependent diabetes mellitus (iddm) or juvenileonset diabetes, primarily affects children and young adults and accounts for - % of the diagnosed diabetes in north america. research by ma and hein ( ) at the university of western ontario showed that diabetes can be prevented in mice by feeding them with plants engineered to produce a diabetes related-protein. the idea is based on 'oral tolerance' where the autoimmune system is selectively turned off early by teaching the body to tolerate the ''antigenic proteins''. the pancreatic protein, glutamic acid decarboxylase (gad ) is linked to the onset of iddm, and when injected into mice it is known to prevent diabetes. the canadian group developed transgenic potato and tobacco plants with the gene for gad , fed them to nonobese diabetic mice, which developed insulin-dependent diabetes spontaneously. the results were intriguing, only % of the prediabetic mice fed with transgenic plants developed the diabetes, while % nontreated mice developed the disease. the treated mice also showed increased levels of ig , an antibody associated with cytokines, which suppresses harmful immune responses. thus, the antigen produced in plants appears to retain immunogenicity and prevent diabetes in an animal model. according to canadian scientists, this is the first proof of principle for the use of edible vaccines in the treatment of the autoimmune diseases. human immunodeficiency virus (hiv) is a retroviral that causes acquired immunodeficiency syndrome (aids), a condition in humans in which progressive failure of the immune system allows life-threatening opportunistic infections and cancer to thrive. in order to produce edible vaccine initial success in splicing hiv protein into cpmv has been achieved. two hiv protein genes and camv as promoter were successfully injected into tomatoes with a needle, and the expressed protein was demonstrable by polymerase chain reaction (pcr) in different parts of the plant, including the ripe fruit, as well as in the second generation plants. recently, spinach has been successfully inoculated for tat protein expression cloned into tmv. each gram of leaf tissue of spinach was shown to contain up to - lg of tat antigen. mice fed with this spinach followed by dna vaccinations resulted in higher antibody titers than the controls, with the levels peaking at weeks post-vaccination. it is still unclear whether the edible vaccines would be regulated under food, drugs. or agricultural products and what vaccine component would be licensed-antigen itself, genetically engineered fruit or transgenic seeds. they would be subjected to a very close scrutiny by the regulatory bodies in order to ensure that they never enter the food supply. this would include greenhouse segregation of medicinal plants from food crops to prevent outcrossing and would necessitate separate storage and processing facilities. although edible vaccines fall under ''genetically modified'' plants, it is hoped that these vaccines will avoid serious controversy, because they are intended to save lives. edible vaccines are future vaccines and some challenges are yet to be overcome before these can become a reality. like all products regulated by food and drug administration, edible vaccines undergo a rigorous review of laboratory, and clinical testing that are conducted to get information regarding safety, efficacy, purity, and potency of these products. these trials can take place only after satisfactory information has been collected on the quality of the nonclinical safety. successful expression of antigens in plants has been demonstrated in the past. the vaccines have also been checked for their efficacy in humans. results from the primary phase of the first-ever human clinical trial of an edible vaccine were published in the journal nature medicine in (blaine p. friedlander, boyce thomson institute of plant research), which indicated that consumption of servings of raw potatoes resulted in immunity to specific diseases. the human clinical study was conducted under the direction of dr. carol tacket at the center for vaccine development, university of maryland school of medicine in baltimore. in the first phase of human testing, the potatoes eaten by volunteers contained a vaccine against travelers' diarrhea, a common condition resulting from intestinal infection by the bacterium e. coli, which contaminates food or water supplies. the clinical trials were approved in advance by the food and drug administration. encouraged by the results of this study, scientists started exploring the use of this technique for administering other antigens. in thanavala's group has developed a potato vaccine booster for use in conjunction with injected hepatitis b vaccine. it is currently in phase ii clinical trial and phase i for patients who have previously been vaccinated. in , tacket and his team mates studied the human immune response to the norwalk virus capsid protein expressed in potatoes. overall, % ( out of volunteers) developed some kind of immune response, although the antibody increase in some cases was modest. in same year, pogrebnyak's lab developed an effective vaccine against the coronavirus which causes severe acute respiratory syndrome (sars). tomato and tobacco plants are used for high expression of the coronavirus spike protein (s ). first, lyophilized tomato fruit was fed to mice and then boosting occurred with s protein expressed in tobacco roots; high igg immune responses and significant igg a and igg b responses were observed in their sera. research is also going in the direction to engineer the plants to produce a variety of functional monoclonal antibody (ma et al. ) . in the first human study of transgenic plant vaccine designed to induce active immunity, adult volunteers were given either g of transgenic potato, g of transgenic potato or g of wild type potato, each transgenic potatoes containing from . to . lg/g of lt-b. the variable dose per gram of potato was due to the tissue specificity of the promoter, therefore, that lt-b was expressed to a different degree in the different tissues of the potatoes. the potatoes in this study were ingested raw; however, subsequent studies have shown that transgenic potatoes expressing the b subunit of cholera toxin could be boiled for min until the tissue becomes soft with loss of only about % of the ct-b pentameric gm binding form. serologic responses were also detected after vaccination. totally out of the volunteers ( %) who ingested transgenic potatoes developed igg anti-lt and in half of them responses occurred after the first dose. there are of the ( %) volunteers developed fourfold rise in serum iga anti-lt. researchers supported by the niaid have shown for the first time that an edible vaccine can safely trigger significant immune responses in people. the goal of the phase proof-ofconcept trial study was to demonstrate that an edible vaccine could stimulate an immune response in humans. volunteers ate bite-sized pieces of raw potato that had been genetically engineered to produce part of the toxin secreted by e. coli, which causes diarrhea. the trial enrolled healthy adults, were chosen at random to receive the genetically engineered potatoes and received pieces of ordinary potatoes. the investigator periodically collected blood and stool samples from the volunteers to evaluate the vaccine's ability to stimulate both systemic and intestinal immune responses. the potatoes were well tolerated and no one experienced serious adverse side effects. niaid supported scientists are exploring the use of this technique for administering other antigens. edible vaccines for other intestinal pathogens are already in the pipeline. potatoes and bananas that might protect against norwalk virus, a common cause of diarrhea, and potatoes and tomatoes that might protect against hepatitis b are being developed. thirty million children throughout the world do not receive even the most basic immunizations each year. as a result, at least three million of these children die from diseases that are fully vaccine-preventable. the solution to vaccinate these children might seem simple with the idea of large scale production of edible vaccines for various diseases. as a recent progress, the first human clinical trials for plant-based vaccine have been performed; it brings many challenges like optimization of expression levels, stabilization during post harvest storage, etc. long-term reactions to edible vaccines are yet to be determined. possible delayed reactions not yet discovered may be the point of consideration. in addition to that, edible vaccines can be further improved for their oral immunogenicity by the use of specific adjuvant which can be applied either as a fusion to the candidate gene or as an independent gene. some of the diseases to which edible vaccines have shown promising application may be elaborated in the veterinary as well as human spectrum. these studies conclude plant-derived vaccines as a new hope and promise for more immunogenic, more effective, and less expensive vaccination strategies against both respiratory as well as intestinal mucosal pathogens. research in the field of edible vaccines holds immense potential for the future and every advancement made in this direction is bringing the dream of edible vaccine one step closer. there is hope that in coming future edible vaccines will conquer all serious diseases and make the planet beautiful to live in. edible vaccines an edible vaccine for malaria using transgenic tomatoes of varying sizes, shapes and colors to carry different antigens plant derived edible vaccines in vivo analysis of plastid psba, rbcl and rpl utr elements by chloroplast transformation: tobacco plastid gene expression is controlled by modulation of transcript levels and translation efficiency control mechanisms of plastid gene expression production of antibodies in transgenic plants immunotherapeutic potential of antibodies produced in plants production of biologically active human interleukin- in transgenic tomato and potato transgenic plants as vaccine production systems expression of hepatitis b surface antigen in transgenic plants edible vaccine protects mice against escherichia coli heat-labile enterotoxin (lt): potatoes expressing a synthetic lt-b gene edible plant vaccines: applications for prophylactic and therapeutic molecular medicine edible vaccines: a concept comes of age plant biotechnology and in vitro biology in the st century production of hepatitis b surface antigen in transgenic plants for oral immunization human immune responses to a novel norwalk virus vaccine delivered in transgenic potatoes immunogenicity in humans of an edible vaccine for hepatitis b oral immunization with a combination of plasmodium yoelii merozoite surface protein and / enhances protection against lethal malarial challenge key: cord- - yd bkf authors: strobel, gary; daisy, bryn; castillo, uvidelio title: novel natural products from rainforest endophytes date: journal: natural products doi: . / - - - - _ sha: doc_id: cord_uid: yd bkf endophytic microorganisms are found in virtually every higher plant on earth. these organisms reside in the living tissues of the host plant and do so in a variety of relationships, ranging from symbiotic to pathogenic. endophytes may contribute to their host plant by producing a plethora of substances that provide protection and survival value to the plant. ultimately, these compounds, once isolated and characterized, may also have potential for use in modern medicine. novel antibiotics, antimycotics, immunosuppressants, and anticancer compounds are only a few examples of what has been found after the isolation and culturing of individual endophytes followed by purification and characterization of some of their natural products. the potential of finding new drugs that may be effective candidates for treating newly developing diseases in humans is great. the need for new and useful compounds to provide assistance and relief in all aspects of the human condition is ever growing. drug resistance in bacteria, the appearance of new life-threatening viruses, the recurrent problems of diseases in persons with organ transplants, and the tremendous increase in the incidence of fungal infections in the world's population all underscore our inadequacy to cope with these medical problems. environmental degradation, loss of biodiversity, and spoilage of land and water also add to problems facing humanity, and each of these in turn can have health-related consequences. endophytes, microorganisms that reside in the tissues of living plants, are relatively unstudied as potential sources of novel natural products for exploitation in medicine. however, some of the most extensive and comprehensive work on natural products produced by endophytes has been done on the neotyphodium sp. found on grasses ( ) . alkaloids synthesized by this fungus in its grass hosts have been implicated in fescue toxicosis in rangeland animals ( ) . the chemistry and biology of this and other grass endophytes are reviewed elsewhere ( ) . unfortunately, because this work is so comprehensive, one may be led to the conclusion that endophytes produce toxic compounds only in their respective hosts and hold no promise for any medicinal applications whatsoever ( ) . it turns out that this is simply not the case. as endophytes are examined from a plethora of sources, an overwhelming number have been found to produce natural products with promising potential for medicinal applications. of the approx , higher plant species that exist on the earth, each individual plant, of the billions that exist here, is host to one or more endophytes. only a handful of these plants (grass species) have ever been completely studied relative to their endophytic biology ( ) . consequently, the opportunity to find new and interesting endophytic microorganisms among myriads of plants in different settings and ecosystems is very great. the intent of this review is to provide insights into their occurrence in nature, the products that they make, and indicate how some of these organisms are beginning to show some potential for human use. the majority of the report discusses rationale for study, methods used, and examples of a number of endophytes isolated and studied in the authors' laboratories over the course of many years. this review, however, also includes some specific examples that illustrate the work of others in this emerging field of bioprospecting the microbes of the world's rainforests. there is a general call for new antibiotics, and for chemotherapeutic agents that are highly effective and possess low toxicity. this search is driven by the development of resistance in infectious microorganisms (e.g., staphylococcus, mycobacterium, streptococcus) to existing drugs and by the menacing presence of naturally resistant organisms. the ingress to the human population of new disease-causing agents such as acquired immunodeficiency syndrome (aids), ebola, and severe acute respiratory syndrome (sars) requires the discovery and development of new drugs to combat them. not only do diseases such as aids require drugs that target them specifically, but new therapies are needed for treating ancillary infections, which are a consequence of a weakened immune system. furthermore, others who are immunocompromised (e.g., cancer and organ transplant patients) are at risk of infection by opportunistic pathogens, such as aspergillus, cryptococcus, and candida, which normally are not major problems in the human population. in addition, more drugs are needed to efficiently treat parasitic protozoan and nematodal infections such as malaria, leishmaniasis, trypanomiasis, and filariasis. malaria by itself is more effective in claiming lives each year than any other single infectious agent with the exception of aids and tuberculosis (tb) ( ) . however, the enteric diseases claim the most lives each year of any disease complex, and unfortunately, the victims are mostly children ( ) . novel natural products and the organisms that make them offer opportunities for innovation in drug discovery. exciting possibilities exist for those who are willing to venture into the wild and unexplored territories of the world to experience the thrill of engaging in the discovery of endophytes, their biology, and potential usefulness. it may also be true that a reduction in interest in natural products for use in drug development has happened as a result of people growing weary of dealing with the traditional sources of bioactive compounds, including plants of the temperate zones and microbes from a plethora of soil samples gathered in different parts of the world by armies of collectors. in other words, why continue to do the same thing when robots, combinatorial chemistry, and molecular biology have arrived on the scene? furthermore, the logic and rationale for time and effort spent on drug discovery using a targetsite-directed approach has been overwhelming. while combinatorial synthesis produces compounds at random, secondary metabolites, defined as low-molecular-weight compounds not required for growth in pure culture, are produced as an adaptation for specific functions in nature ( ) . shutz notes that certain microbial metabolites seem to be characteristic of certain biotopes, both on an environmental as well as organismal level ( ) . accordingly, it appears that the search for novel secondary metabolites should center on organisms that inhabit unique biotopes. thus, it behooves the investigator to carefully study and select the biological source before proceeding, rather than to take a totally random approach in selecting the source material. careful study also indicates that organisms and their biotopes that are subjected to constant metabolic and environmental interactions should produce even more secondary metabolites ( ) . endophytes are microbes that inhabit such biotopes, namely higher plants, which is why they are currently considered as a wellspring of novel secondary metabolites offering the potential for exploitation of their medical benefits. in addition, it also is extremely helpful for the investigator interested in exploiting endophytes to have access to, or have some expertise in, microbial taxonomy, and this includes modern molecular techniques involving sequence analyses of s and s rdna. currently, endophytes are viewed as an outstanding source of bioactive natural products because there are so many of them occupying literally millions of unique biological niches (higher plants) growing in so many unusual environments. thus, it would appear that a myriad of biotypical factors associated with plants can be important in the selection of a plant for study. it may be the case that these factors may govern which microbes are present in the plant as well as the biological activity of the products associated with these organisms. since the discovery of endophytes in darnel, germany, in , various investigators have defined endophytes in different ways, which usually depended on the perspective from which the endophytes were being isolated and subsequently examined ( ) . bacon et al. give an inclusive and widely accepted definition of endophytes: "microbes that colonize living, internal tissues of plants without causing any immediate, overt negative effects" ( ) . while the symptomless nature of endophyte occupation in plant tissue has prompted focus on symbiotic or mutualistic relationships between endophytes and their hosts, the observed biodiversity of endophytes suggests they can also be aggressive saprophytes or opportunistic pathogens. both fungi and bacteria are the most common microbes existing as endophytes. it would seem that other microbial forms most certainly exist in plants as endophytes, such as mycoplasmas, rickettsia, and archebacteria; however, no evidence for them has yet been presented. the most frequently isolated endophytes are the fungi ( ) . it turns out that the vast majority of plants have not been studied for their endophytes. thus, enormous opportunities exist for the recovery of novel fungal forms, including genera, biotypes, as well as species in the myriad of plants yet to be studied. hawksworth and rossman estimated there may be as many as million different fungal species, yet only approx , have been described ( ) . as more evidence accumulates, estimates keep rising as to the actual number of fungal species. for instance, dreyfuss and chapela estimate there may be at least million species of endophytic fungi alone ( ) . it seems obvious that endophytes are a rich and reliable source of genetic diversity and may represent previously undescribed species. finally, in our experience, novel microbes (as defined at the morphological and/or molecular levels) often have novel natural products associated with them. this fact alone helps eliminate the problems of dereplication in compound discovery. it is important to understand the methods and rationale used seem to provide the best opportunities to isolate novel endophytic microorganisms at the genus, species, or biotype level. thus, since the number of plant species in the world is so great, creative and imaginative strategies must be used to quickly narrow the search for endophytes displaying bioactivity ( ) . a specific rationale for the collection of each plant for endophyte isolation and natural product discovery is used. several hypotheses govern this plant selection strategy, and these are as follows: . plants from unique environmental settings, especially those with an unusual biology, and possessing novel strategies for survival, are seriously considered for study. . plants that have an ethnobotanical history (use by indigenous peoples) that are related to the specific uses or applications of interest are selected for study. these plants are chosen either by direct contact with local peoples or via local literature. ultimately, it may be learned that the healing powers of the botanical source, in fact, may have nothing to do with the natural products of the plant, but of the endophyte inhabiting the plant. . plants that are endemic, having an unusual longevity, or that have occupied a certain ancient land mass, such as gondwanaland, are also more likely to lodge endophytes with active natural products than other plants. . plants growing in areas of great biodiversity, it follows, also have the prospect of housing endophytes with great biodiversity. just as plants from a distinct environmental setting are considered to be a promising source of novel endophytes and their compounds, so too are plants with an unconventional biology. for example, an aquatic plant, rhyncholacis penicillata, was collected from a river system in southwest venezuela where the harsh aquatic environment subjected the plant to constant beating by virtue of rushing waters, debris, and tumbling rocks and pebbles ( ) . these environmental insults created many portals through which common phytopathogenic oomycetes could enter the plant. still, the plant population appeared to be healthy, possibly owing to protection by an endophytic product. this was the environmental biological clue used to pick this plant for a comprehensive study of its endophytes. eventually, an unusual and potent antifungal strain of serratia marcescens, living both as an epiphyte and an endophyte, was recovered from r. penicillata. this bacterium was shown to produce oocydin a, a novel antioomycetous compound having the properties of a chlorinated macrocyclic lactone ( fig. ) ( ) . it is conceivable that the production of oocydin a by s. marcescens is directly related to the endophyte's relationship with its higher-plant host. currently, oocydin a is being considered for agricultural use to control the ever-threatening presence of oomyceteous fungi such as pythium spp. and phytophthora spp. oocydin a also has activity against a number of rapidly dividing cancer cell lines ( ) . plants with ethnobotanical history, as mentioned above, also are likely candidates for study, since the medical uses for which the plant was selected may relate more to its population of endophytes than to the plant biochemistry itself. for example, a sample of the snakevine, kennedia nigriscans, from the northern territory of australia, was selected for study since its sap has traditionally been used as bush medicine for many millenia. in fact, this area was selected for plant sampling because it has been home to the world's longest standing civilization-the australian aborigines. the snakevine is harvested, crushed, and heated in an aqueous brew by local aborigines in southwest arnhemland to treat cuts, wounds, and infections. as it turned out, the plant contained a streptomycete that possessed unique partial s rdna sequences when compared to those in genbank. the organism was designated streptomyces nrrl , and it produces broad-spectrum novel peptide antibiotics called munumbicins, which are discussed below ( ) . it seems likely that some of the healing properties in plants, as discovered by indigenous peoples, might be facilitated by compounds produced by one or more specific plant-associated endophytes as well as the plant products themselves. in addition, it is worthy to note that some plants generating bioactive natural products have associated endophytes that produce the same natural products. such is the case with taxol, a highly functionalized diterpenoid and famed anticancer agent that is found in each of the world's yew tree species (taxus spp.) ( , ) . in , a novel taxol-producing fungus, taxomyces andreanae, from the yew taxus brevifolia, was isolated and characterized ( ). of the myriad of ecosystems on earth, those having the greatest general biodiversity seem to be the ones also having the greatest number and most diverse endophytes. tropical and temperate rainforests are the most biologically diverse terrestrial ecosystems on earth. the most threatened of these spots cover only . % of the land's surface, yet they harbor over % of the world's terrestrial biodiversity ( ) . in addition, each of the - areas identified as supporting the world's greatest biodiversity support unusually high levels of plant endemism ( ) . as such, one would expect, with high plant endemism, there also should exist specific endophytes that may have evolved with the endemic plant species. biological diversity implies chemical diversity, because of the constant chemical innovation that is required to survive in ecosystems where the evolutionary race to survive is most active. tropical rainforests are a remarkable example of this type of environment. competition is great, resources are limited, and selection pressure is at its peak. this gives rise to a high probability that rainforests are a source of novel molecular structures and biologically active compounds ( ) . bills et al. describe a metabolic distinction between tropical and temperate endophytes through statistical data that compare the number of bioactive natural products isolated from endophytes of tropical regions to the number of those isolated from endophytes of temperate origin ( ) . not only did they find that tropical endophytes provide more active natural products than temperate endophytes, but they also noted that a significantly higher number of tropical endophytes produced a larger number of active secondary metabolites than did fungi from other substrata. this observation suggests the importance of the host plant as well as the ecosystem in influencing the general metabolism of endophytic microbes. tan and zou believe the reason why some endophytes produce certain phytochemicals, originally characteristic of the host, might be related to a genetic recombination of the endophyte with the host that occurred in evolutionary time ( ) . this is a concept that was originally proposed as a mechanism to explain why t. andreanae may be producing taxol ( ) . thus, if endophytes can produce the same rare and important bioactive compounds as their host plants, this would not only reduce the need to harvest slow-growing and possibly rare plants, but also help to preserve the world's everdiminishing biodiversity. furthermore, it is recognized that a microbial source of a high-value product may be easier and more economical to produce effectively, thereby reducing its market price. all aspects of the biology and interrelatedness of endophytes with their respective hosts is a vastly under-investigated and exciting field ( , ) . thus, more background information on a given plant species and its microorganismal biology would be exceedingly helpful in directing the search for bioactive products. presently, no one is quite certain of the role of endophytes in nature and their relationship to various host plant species. although some endophytic fungi appear to be ubiquitous (e.g., fusarium spp., pestalotiopsis spp., and xylaria spp.), one cannot definitively state that endophytes are truly host-specific or even systemic within plants, any more than one can assume that their associations are chance encounters. frequently, many endophytes of the same species are isolated from the same plant, and only one or a few biotypes of a given fungus will produce a highly biologically active compound in culture ( ) . a great deal of uncertainty also exists between what an endophyte produces in culture and what it may produce in nature. it does seem possible that the production of certain bioactive compounds by the endophyte in situ may facilitate the domination of its biological niche within the plant or even provide protection to the plant from harmful invading pathogens. furthermore, little information exists relative to the biochemistry and physiology of the interactions of the endophyte with its host plant. it would seem that many factors changing in the host, related to the season, age, environment, and location, may influence the biology of the endophyte. indeed, further research at the molecular level must be conducted in the field to study endophyte interactions and ecology. all of these interactions are probably chemically mediated for some purpose in nature. an ecological awareness of the role these organisms play in nature will provide the best clues for targeting particular types of endophytic bioactivity with the greatest potential for bioprospecting. after a plant is selected for study, it is identified, and its location is plotted using a global positioning device. small stem pieces are cut from the plant and placed in sealed plastic bags after excess moisture is removed. every attempt is made to store the materials at °c until isolation procedures can begin ( , ) . in the laboratory, the surfaces of plant materials are thoroughly treated with % ethanol, sometimes flamed, and ultimately they are air dried under a laminar-flow hood. this is done in order to eliminate surface-contaminating microbes ( ) . then, with a sterile knife blade, outer tissues are removed from the samples and the inner tissues carefully excised and placed on water agar plates. after several days of incubation, hyphal tips of the fungi are removed and transferred to potato dextrose or other suitable agar. bacterial forms also emerge from the plant tissues, including, on rare occasions, certain streptomyces spp. the endophytes are encouraged to sporulate on specific plant materials and are eventually identified via standard morphological and molecular biological techniques and methods. eventually, when an endophyte is acquired in pure culture, it is tested for its ability to be grown in shake or still culture using various media and growth conditions ( ) . it is also immediately placed in storage under various conditions including % glycerol at - °c. ultimately, once appropriate growth conditions are found, the microbe is subjected to fermentation, extraction, and the bioactive compounds are isolated and characterized. virtually all of the common and advanced procedures for product isolation and characterization are utilized in order to acquire the product(s) of interest. central to the processes of isolation is the establishment of one or more bioassays that will guide the compound purification processes. one cannot put too much emphasis on this point, since the ultimate success of any natural-product isolation activity is directly related to the development or selection of appropriate bioassay procedures. these can involve target organisms, enzymes, tissues, or model chemical systems that relate to the purpose for which the new compound is needed. the following section shows some examples of natural products obtained from endophytic microbes and their potential in the pharmaceutical and agrochemical arenas. many of the examples are taken from our work, and thus, this review is by no means inclusive of all natural-product work in endophytes. fungi are the most commonly isolated endophytic microbes. they usually appear as fine filaments growing from the plant material on the agar surface. generally, the most commonly isolated fungi are in the group fungi imperfecti or deuteromycetes. basi-cally, they produce asexual spores in or on various fruiting structures. also, it is quite common to isolate endophytes that are producing no fruiting structures whatsoever, such as mycelia sterilia. quite commonly endophytes do produce secondary metabolites when placed in culture. however, the temperature, the composition of the medium, and the degree of aeration will affect the amount and kind of compounds that are produced. sometimes endophytic fungi produce antibiotics. natural products from endophytic fungi have been observed to inhibit or kill a wide variety of harmful microorganisms including, but not limited to, phytopathogens, as well as bacteria, fungi, viruses, and protozoans that affect humans and animals. described below are some examples of bioactive products from endophytic fungi. cryptosporiopsis cf. quercina is the imperfect stage of pezicula cinnamomea, a fungus commonly associated with hardwood species in europe. it was isolated as an endophyte from tripterigeum wilfordii, a medicinal plant native to eurasia ( ) . on petri plates, c. quercina demonstrated excellent antifungal activity against some important human fungal pathogens, including candida albicans and trichophyton spp. a unique peptide antimycotic, termed "cryptocandin," was isolated and characterized ( ) . this compound contains a number of peculiar hydoxylated amino acids and a novel amino acid, -hydroxy- -hydroxy methyl proline (fig. ) . the bioactive compound is related to known antimycotics-the echinocandins and the pneumocandins ( ) . as is generally true, not one but several bioactive and related compounds are produced by an endophytic microbe. thus, other antifungal agents related to cryptocandin are also produced by c. quercina. cryptocandin is also active against a number of plant pathogenic fungi, including sclerotinia sclerotiorum and botrytis cinerea. cryptocandin and its related compounds are currently being considered for use against a number of fungi causing diseases of the skin and nails. cryptocin, a unique tetramic acid, is also produced by c. quercina (discussed previously) (fig. )( ) . this unusual compound possesses potent activity against pyricularia oryzae, the causal organism of one of the worst plant diseases in the world, as well as a number of other plant pathogenic fungi ( ) . the compound was generally ineffective against a general array of human pathogenic fungi. nevertheless, with minimum inhibitory concentrations against p. oryzae at . μg/ml, this compound is being examined as a natural chemical control agent for rice blast and is being used as a platform for the synthesis of other antifungal compounds. as mentioned earlier, p. microspora is a common rainforest endophyte ( ) ( ) ( ) ( ) . it turns out that enormous biochemical diversity does exist in this endophytic fungus, and many secondary metabolites are produced by various strains of this widely dispersed organism. one such secondary metabolite is ambuic acid, an antifungal agent, which has been recently described from several isolates of p. microspora found as representative isolates in many of the world's rainforests (fig. ) ( ) . this compound as well as another endophyte product, terrein, have been used as models to develop new solidstate nuclear magnetic resonance (nmr) tensor methods to assist in the characterization of the molecular stereochemistry of organic molecules. a strain of p. microspora was also isolated from the endangered tree torreya taxifolia and produced several compounds having antifungal activity, including pestaloside, an aromatic β-glucoside (fig. ) , and two pyrones-pestalopyrone and hydroxypestalopyrone ( ) . these products also possess phytotoxic properties. other fig. . ambuic acid, a highly functionalized cyclohexenone produced by a number of isolates of pestalotiopsis microspora found in rainforests around the world. this compound possesses antifungal activity and has been used as a model compound for the development of solid-state nuclear magnetic resonance methods for the structural determination of natural products. newly isolated secondary products obtained from p. microspora (endophytic on taxus brevifolia) include two new caryophyllene sesquiterpenes-pestalotiopsins a and b ( ) . additional new sesquiterpenes produced by this fungus are α-hydroxydimeninol and a highly functionalized humulane ( , ) . variation in the amount and kinds of products found with this fungus depends on both the cultural conditions as well as the original plant source from which it was isolated. pestalotiopsis jesteri is a newly described endophytic fungal species from the sepik river area of papua new guinea, and it produces jesterone and hydroxyjesterone, which exhibit antifungal activity against a variety of plant pathogenic fungi ( ) . these compounds are highly functionalized cyclohexenone epoxides. jesterone, subsequently, has been prepared by organic synthesis with complete retention of biological activity (fig. ) ( ) . jesterone is one of only a few products from endophytic microbes in which total synthesis of a bioactive product has been successfully accomplished. phomopsichalasin, a metabolite from an endophytic phomopsis sp., represents the first cytochalasin-type compound with a three-ring system replacing the cytochalasin macrolide ring. this metabolite exhibits antibacterial activity in disk diffusion assays (at a concentration of μg/disk) against bacillus subtilis, salmonella gallinarum, and staphylococcus aureus. it also displays moderate activity against the yeast candida tropicalis ( ) . an endophytic fusarium sp. from the plant selaginella pallescens, collected in the guanacaste conservation area of costa rica, was screened for antifungal activity. a new pentaketide antifungal agent, cr , was isolated from the culture broth of the fungus and showed potent activity against c. albicans in agar diffusion assays ( ) . colletotric acid, a metabolite of colletotrichum gloeosporioides, an endophytic fungus isolated from artemisia mongolica, displays activity against bacteria as well as against the fungus helminthsporium sativum ( ) . another colletotrichum sp., isolated from artemisia annua, produces antimicrobial metabolites as well. a. annua is a traditional chinese herb that is well recognized for its synthesis of artemisinin (an antimalarial drug) and its ability to inhabit many geographically different areas. not only did the colletotrichum sp. found in a. annua produce metabolites with activity against human pathogenic fungi and bacteria, but also metabolites that were fungistatic to plant pathogenic fungi ( ). there are only a limited number of bacterial species known to be associated with plants, and one of the most common is pseudomonas spp. pseudomonas has representative biotypes and species that are epiphytic, endophytic, and pathogenic. they have been reported from every continent including the antarctic. some of these species produce phytotoxic compounds as well as antibiotics. the ecomycins are produced by pseudomonas viridiflava ( ) . this bacterium is generally associated with the leaves of many grass species and is located on and within the tissues ( ) . the ecomycins represent a family of novel lipopeptides and have masses of and . besides common amino acids such as alanine, serine, threonine, and glycine, some nonprotein amino acids are incorporated into the structure of the ecomycins, including homoserine and β-hydroxyaspartic acid ( ) . the ecomycins are active against such human pathogenic fungi as cryptococcus neoformans and c. albicans. the pseudomycins produced by a plant-associated pseudomonad are another group of antifungal peptides ( , ) . they are active against a variety of plant and human pathogenic fungi, including candida albicans, cryptococcus neoformans, and a variety of plant pathogenic fungi, including ceratocystis ulmi (the dutch elm disease pathogen) and mycosphaerella fijiensis (causal agent of black sigatoka disease in bananas). the pseudomycins are cyclic depsipeptides formed by acylation of the oh group of the n-terminal serine with the terminal carboxyl group of l-chlorothreonine. variety in this family of compounds is imparted via n-acetylation by one of a series of fatty acids, including , -dihydroxydecanoate, -hydroxy-tetradecanoate ( ) . the pseudomycins contain several nontraditional amino acids, including l-chlorothreonine, l-hydroxyaspartic acid, and both d-and l-diaminobutryic acid. the molecules are candidates for use in human medicine, especially after structural modification by chemical synthesis has successfully eliminated mammalian toxicity ( ) the pseudomycins are also effective against a number of ascomycetous fungi, and are being considered for agricultural use for the control of the black sigatoka disease in bananas (strobel, unpublished). streptomyces spp. are filamentous bacteria, belonging to the order actinomycetales, that live in widely diverse ecological settings. generally, this group is gram positive, has a high g+c content, and does not have an organized nucleus. to date, actinomycetes have been the world's greatest source of natural antibiotics ( ) . in fact, just one genus, streptomyces, is the source of % of these compounds. the majority of the antibiotic producers are from soil sources, and until recently it was not realized that these organisms can exist as endophytes. one of the first endophytic streptomyces spp. isolated was that from lolium perenne, a grass species ( ) . this isolate produces a diketopiperazine that is a weak antibiotic and has been designated "methylalbonoursin" ( ) . using the ethnobotanical approach to plant selection, the snakevine plant, k. nigriscans, was chosen as a possible source of endophytic microbes because of its long-held traditional use by australian aborigines to treat cuts and open wounds, resulting in reduced infection and rapid healing. this plant was collected near the aboriginal community of manyallaluk in northern territory, australia, and consistently yielded an endophytic actinomycete designated streptomyces nrrl ( ) . the organism was not found in several tree species supporting the vine, suggesting a host-selective or -specific association of the endophyte with a specific plant genus. this streptomycete produces a family of extremely potent peptide antibiotics, and these compounds may not only protect the plant from fungal and bacterial infections, but also have unknowingly served the aborigines as a source of bush medicine. the antibiotics produced by streptomyces nrrl , called "munumbicins," possess widely differing biological activities, depending on the target organism. in general, the munumbicins demonstrate activity against gram-positive bacteria such as bacillus anthracis and multidrug-resistant mycobacterium tuberculosis, as well as a number of other drug-resistant bacteria. however, the most impressive biological activity of any of the munumbicins is that of munumbicin d against the malarial parasite plasmodium falciparum, having an ic of . ± . ng/ml ( ) . the munumbicins are highly functionalized peptides, each containing threonine, aspartic acid (or asparagine), and glutamic acid (or glutamine). since the peptides are yellowish orange, they also contain one or more chromophoric groups, whose structures have not been determined. their masses range from to da. the isolation of this endophytic streptomycete represents an important finding, providing one of the first examples of plants serving as reservoirs of actinomycetes. more than of these endophytic streptomycetes, now in hand in our laboratory, possess antibiotic activity (castillo, u., strobel, g.a., unpublished data). endophytic actinomycetes are now being tested and considered for use in controlling plant diseases ( ) . another endophytic streptomyces sp. (nrrl ), from a fern-leaved grevillea (grevillea pteridifolia) tree growing in the northern territory of australia, produces novel antibiotics called "kakadumycins," which are related to the echinomycins ( ) . each of these antibiotics contains alanine, serine, and an unknown amino acid. kakadumycin a has wide-spectrum antibiotic activity similar to that of munumbicin d, especially against gram-positive bacteria, and it generally displays better bioactivity than echinomycin. for instance, against b. anthracis strains, kakadumycin a has mics of . - . μg/ ml, in contrast to echinomycin at . - . μg/ml. both echinomycin and kakadumycin a have impressive activity against p. falciparum, with ld s in the range of - ng/ml ( ) . kakadumycin a and echinomycin are related by virtue of their very similar structures (amino acid content and quinoxoline rings), but differ slightly with respect to their elemental compositions, aspects of their spectral qualities, chromatographic retention times, and biological activities ( ) . echinomycin and kakadymycin a were studied as inhibitors of macromolecular synthesis, with control substances such as ciprofloxacin, rifampin, chloramphenicol, and vancomycin used as standards with well-established modes of action. tests were done for dna, rna, protein, and cell-wall synthesis inhibition activities, respectively. kakadumycin a significantly inhibited rna synthesis in b. subtilis ( ) . kakadumycin a also inhibited protein synthesis and cell-wall synthesis substantially, but had a lower effect on dna synthesis. kakadumycin a shares a very similar inhibitory profile with echinomycin in four macromolecular synthesis assays. kakadumycin a preferentially inhibits rna synthesis, and may have the same mode of action as echinomycin, which inhibits rna synthesis by binding to a dna template ( ) . more recently, endophytic streptomycetes have been discovered in an area of the world claimed to be one of the most biologically diverse-the upper amazon of peru. the inner tissues of the follow me vine, monstera sp., commonly yielded a verticillated streptomycete with outstanding inhibitory activities against pythiaceous fungi as well as the malarial parasite plasmodium falciparum. the bioactive component is a mixture of lipopeptides named "coronamycins" ( ). another fascinating use of products from endophytic fungi is the inhibition of viruses. two novel human cytomegalovirus (hcmv) protease inhibitors, cytonic acids a and b, have been isolated from solid-state fermentation of the endophytic fungus cytonaema sp. their structures were elucidated as p-tridepside isomers by ms and nmr methods ( ) . it is apparent that the potential for the discovery of compounds having antiviral activity from endophytes is in its infancy. the main limitation to com-pound discovery to date is probably related to the absence of common antiviral screening systems in most compound-discovery programs. muscodor albus is a newly described endophytic fungus obtained from small limbs of cinnamomum zeylanicum (cinnamon tree) ( ) . this xylariaceaous (non-spore producing) fungus effectively inhibits and kills certain fungi and bacteria by producing a mixture of volatile compounds ( ) . the majority of these compounds have been identified by gas chromatography (gc)/ms, synthesized or acquired, and then formulated into an artificial mixture. this mixture not only mimicked the antibiotic effects of the volatile compounds produced by the fungus, but also was used to confirm the identity of the majority of the volatiles emitted by this organism ( ) . each of the five classes of volatile compounds produced by the fungus had some microbial effects against the test fungi and bacteria, but none was lethal. however, they acted synergistically to cause death in a broad range of plant and human pathogenic fungi and bacteria. the most effective class of inhibitory compounds was the esters, of which isoamyl acetate was the most biologically active. the composition of the medium on which m. albus grows dramatically influences the kind of volatile compounds that are produced ( ) . the ecological implications and potential practical benefits of the "mycofumigation" effects of m. albus are very promising, given the fact that soil fumigation utilizing methyl bromide will soon be illegal in the united states. methyl bromide is not only a hazard to human health, but it has been implicated in causing destruction of the ozone layer. the potential use of mycofumigation to treat soil, seeds, and plants may soon be a reality. the artificial mixture of volatile compounds may also have usefulness in treating seeds, fruits, and plant parts in storage and while being transported. muscodor albus already has a limited market for the treatment of human wastes. its gases have both inhibitory and lethal effects on such fecal-inhabiting organisms as escherichia coli and vibrio cholera. using m. albus as a screening tool, it has now been possible to isolate other endophytic fungi producing volatile antibiotics. the newly described m. roseus was obtained twice from tree species growing in the northern territory of australia. this fungus is just as effective in causing inhibition and death of test microbes in the laboratory as m. albus ( ) . in addition, for the first time, a nonmuscodor species (gliocladium sp.) was discovered as a producer of volatile antibiotics. the volatile components of this organism are totally different from those of either m. albus or m. roseus. in fact, the most abundant volatile inhibitor is [ ] -annulene, formerly used as a rocket fuel and discovered for the first time as a natural product. however, the bioactivity of the volatiles of this gliocladium sp. is not as good or comprehensive as those from muscodor spp. ( , ) . taxol and some of its derivatives represent the first major group of anticancer agents that are produced by endophytes (fig. ) . taxol (fig. ) , a highly functionalized diterpenoid, is found in each of the world's yew (taxus) species, but was originally isolated from taxus brevifolia ( ) . the original targets for this compound were ovarian and breast cancers, but now it is used to treat a number of other human tissue proliferating diseases as well. the presence of taxol in yew species prompted the study of their endophytes. by the early s, however, no endophytic fungi had been isolated from any of the world's representative yew species. after several years of effort, a novel taxol-producing endophytic fungus, taxomyces andreanae, was discovered in taxus brevifolia ( ) . the most critical line of evidence for the presence of taxol in the culture fluids of this fungus was the electrospray mass spectrum of the putative taxol isolated from t. andreanae. in electrospray mass spectroscopy, taxol usually gives two peaks-one at mass , which is m+h + , and the other at , which is m+na + . fungal taxol had a mass spectrum identical to authentic taxol ( ) . then, c labeling studies showed the presence of fungal-derived taxol in the culture medium ( ) . this early work set the stage for a more comprehensive examination of the ability of other taxus species and many other plants to yield endophytes producing taxol. some of the most commonly found endophytes of the world's yews and many other plants are pestalotiopsis spp. ( ) ( ) ( ) ( ) . one of the most frequently isolated endophytic species is pestalotiopsis microspora ( ) . an examination of the endophytes of taxus wallichiana yielded p. microspora, and a preliminary monoclonal antibody test indicated that it might produce taxol ( ) . after preparative tlc, a compound was isolated and shown by spectroscopic techniques to be taxol. labeled ( c) taxol was produced by this organism from several c precursors ( ) . furthermore, several other p. microspora isolates that produce taxol were obtained from a bald cypress tree in south carolina ( ) . this was the first indication that endophytes, residing in plants other than taxus spp., produce taxol. therefore, a specific search was conducted for taxolproducing endophytes on continents not known for any indigenous taxus spp., e.g., south america and australia. from the extremely rare, and previously thought to be fig. . taxol, the world's first billion-dollar anticancer drug, is produced by many endophytic fungi. it too, possesses outstanding anti-oomycete activity. extinct, wollemi pine (wollemia nobilis), pestalotiopsis guepini was isolated, which was shown to produce taxol ( ) . also, quite surprisingly, a rubiaceous plant, maguireothamnus speciosus, yielded a novel fungus, seimatoantlerium tepuiense, that produces taxol. this endemic plant grows on the top of the tepuis in the venzuelan-guyana border in southwest venezuela ( ) . furthermore, fungal taxol production has also been noted in periconia sp. ( ) and seimatoantlerium nepalense, another novel endophytic fungal species ( ) . simply, it appears that the distribution of taxol-making fungi is worldwide and is not confined to endophytes of yews. the ecological and physiological explanation for fungi making taxol seems to be related to the fact that taxol is a fungicide, and the organisms most sensitive to it are plant pathogens such as pythium spp. and phytophthora spp. ( ) . these pythiaceous organisms are some of the world's most important plant pathogens and are strong competitors with endophytic fungi for niches within plants. in fact, their sensitivity to taxol is based on their interaction with tubulin, in an identical manner as in rapidly dividing human cancer cells ( ) . thus, bona fide endophytes may be producing taxol and related taxanes to protect their respective host plant from degradation and disease caused by these pathogens. other investigators have also made observations on taxol production by endophytes, including the discovery of taxol production by tubercularia sp. isolated from the chinese yew (taxus mairei) in the fujian province of southeastern mainland china ( ) . at least three endophytes of taxus wallichiana produce taxol, including sporormia minima and trichothecium sp. ( ) . using hplc and esims, taxol has been discovered in corylus avellana cv. gasaway ( ) . several fungal endophytes of this plant (filbert) produce taxol in culture ( ) . it is important to note, however, that taxol production by all endophytes in culture is in the range of sub-micrograms to micrograms per liter. also, commonly, the fungi will attenuate taxol production in culture, with some possibility for recovery, if certain activator compounds are added to the medium ( ) . efforts are being made to determine the feasibility of making microbial taxol a commercial possibility, e.g., the discovery of endophytes that make large quantities of one or more taxanes that could then be used as intermediates for the organic synthesis of taxol or one of its anticancer relatives. torreyanic acid, a selectively cytotoxic quinone dimer and potential anticancer agent, was isolated from a p. microspora strain (fig. ) . this strain was originally obtained as an endophyte associated with the endangered tree torreya taxifolia (florida torreya) ( ) . torreyanic acid was tested in several cancer cell lines, and it demonstrated to times more potent cytotoxicity in lines that are sensitive to protein kinase c agonists; it causes cell death by apoptosis. recently, torreyanic acid has been successfully synthesized by a biomimetic oxidation/dimerization cascade ( ) . alkaloids are also commonly found in endophytic fungi. fungal genera such as xylaria, phoma, hypoxylon, and chalara are representative producers of a relatively large group of substances known as the cytochalasins, of which more than are now known. many of these compounds possess antitumor and antibiotic activities, but because of their cellular toxicity they have not been developed into pharmaceuticals. three novel cytochalasins have recently been reported from rhinocladiella sp., as an endophyte on tripterygium wilfordii. these compounds have antitumor activity and have been identified as -oxa- [ ] -cytochalasins ( ) . thus, it is not uncommon to find one or more cytochalasins in endophytic fungi, and this provides an example of the fact that redundancy in discovery does occur, making dereplication an issue even for these under-investigated sources. two compounds, pestacin and isopestacin, have been obtained from culture fluids of pestalotiopsis microspora, an endophyte isolated from a combretaceaous plant, terminalia morobensis, growing in the sepik river drainage system of papua new guinea ( , ) . both pestacin and isopestacin display antimicrobial as well as antioxidant activity. isopestacin was attributed with antioxidant activity based on its structural similarity to the flavonoids (fig. ) . electron spin resonance spectroscopy confirmed this antioxidant activity; the compound is able to scavenge superoxide and hydroxyl free radicals in solution ( ) . pestacin was later described from the same culture fluid, occurring naturally as a racemic mixture and also possessing potent antioxidant activity (fig. ) ( ) . the proposed antioxidant activity of pestacin arises primarily via cleavage of an unusually reactive c-h bond and, to a lesser extent, through o-h abstraction ( ) . the antioxidant activity of pestacin is at least one order of magnitude more potent than that of trolox, a vitamin e derivative ( ). a nonpeptidal fungal metabolite (l- , ) was isolated from an endophytic fungus (pseudomassaria sp.) collected from an african rainforest near kinshasa in the democratic republic of the congo ( ). this compound acts as an insulin mimetic but, unlike insulin, is not destroyed in the digestive tract and may be given orally. oral administration of l- , in two mouse models of diabetes resulted in significant lowering of blood glucose levels. these results may lead to new therapies for diabetes. immunosuppressive drugs are used today to prevent allograft rejection in transplant patients, and in the future they could be used to treat autoimmune diseases such as rheumatoid arthritis and insulin-dependent diabetes. the endophytic fungus fusarium subglutinans, isolated from t. wilfordii, produces the immunosuppressive but noncytotoxic diterpene pyrones subglutinols a and b (fig. ) ( ) . subglutinol a and b are equipotent in the mixed lymphocyte reaction (mlr) assay and thymocyte proliferation (tp) assay, with an ic f f of . μm. in the same assay systems, the famed immunosuppressant drug cyclosporin a, also a fungal metabolite, was roughly as potent in the mlr assay and more potent in the tp assay. still, the lack of toxicity associated with subglutinols a and b suggests that they should be explored in greater detail as potential immunosuppressants ( ). of some compelling interest is an explanation as to how the genes for taxol production may have been acquired by p. microspora ( ) . although the complete answer to this question is not at hand, relevant genetic studies have been performed on this organism. p. microspora ne is one of the most easily genetically transformable fungi that have been studied to date. in vivo addition of telomeric repeats to foreign dna generates extrachromosomal dnas in this fungus ( ) . repeats of the telomeric sequence '-ttaggg- ' were appended to nontelomeric transforming dna termini. the new dnas, carrying foreign genes and the telomeric repeats, replicated independently of the chromosome and expressed the information carried by the foreign genes. the addition of telomeric repeats to foreign dna is unusual among fungi. this finding may have important implications in the biology of p. microspora ne , because it explains at least one mechanism as to how new dna can be captured by this organism and eventually expressed and replicated. such a mechanism may begin to explain how the enormous biochemical variation may have arisen in this fungus ( ) . also, this initial work represents a framework to aid in the understanding of how this fungus may adapt itself to the environment of its plant hosts and suggests that the uptake of plant dna into its own genome may occur. in addition, the telomeric repeats have the same sequence as human telomeres, and this points to the possibility that p. microspora may serve as a means to make artificial human chromosomes, a totally unexpected result. endophytes are a poorly investigated group of microorganisms that represent an abundant and dependable source of bioactive and chemically novel compounds with potential for exploitation in a wide variety of medical applications. the mechanisms through which endophytes exist and respond to their surroundings must be better understood in order to be more predictive about which higher plants to seek, study, and employ in isolating microfloral components. this may facilitate the natural-product discovery process. although work on the utilization of this vast resource of poorly understood microorganisms has just begun, it has already become obvious that an enormous potential for organism, product, and utilitarian discovery in this field holds exciting promise. this is evidenced by the discovery of a wide range of products, and microorganisms that present potential. it is important for all involved in this work to realize the importance of acquiring the necessary permits from governmental, local, and other sources to pick and transport plant materials (especially from abroad) from which endophytes are to be eventually isolated. in addition to this aspect of the work is the added activity of producing the necessary agreements and financial sharing arrangements with indigenous peoples or governments in case a product does develop an income stream. certainly, one of the major problems facing the future of endophyte biology and natural-product discovery is the rapidly diminishing rainforests, which hold the greatest possible resource for acquiring novel microorganisms and their products. the total land mass of the world that currently supports rainforests is about equal to the area of the united states ( ) . each year, an area the size of vermont or greater is lost to clearing, harvesting, fire, agricultural development, mining, or other human-oriented activities ( ) . presently, it is estimated that only a small fraction ( - %) of what were the original rainforests existing - yr ago, are currently present on the earth ( ). the advent of major negative pressures on them from these human-related activities appears to be eliminating entire mega-life forms at an alarming rate. few have ever expressed information or opinions about what is happening to the potential loss of microbial diversity as entire plant species disappear. it can only be guessed that this loss is also happening, perhaps at the same frequency as the loss of mega-life forms, especially because certain microorganisms may have developed unique specific symbiotic relationships with their plant hosts. thus, when a plant species disappears, so too does its entire suite of associated endophytes and consequently all of the capabilities that they might possess to make natural products with medicinal potential. multistep processes are needed now to secure information and life forms before they are lost. areas of the planet that represent unique places housing biodiversity need immediate preservation. countries need to establish information bases of their biodiversity and at the same time begin to make national collections of microorganisms that live in these areas. endophytes are only one example of a life-form source that holds enormous promise to impact many aspects of human existence. the problem of the loss of biodiversity should be one of concern to the entire world. coevolution of fungal endophytes with grasses: the significance of secondary metabolites microbial endophytes niaid global health research plan for hiv/aids, malaria and tuberculosis industrial microbiology bioactive fungal metabolites-impact and exploitation. british mycological society endophytes: a rich source of functional metabolites endophytic fungi in grasses and woody plants where are the undescribed fungi? potential of fungi in the discovery of novel, low-molecular weight pharmaceuticals hotspots: earth's biologically richest and most endangered ecoregions. washington dc. cemex conservation international oocydin a, a chlorinated macrocyclic lactone with potent anti-oomycete activity from serratia marcescens munumbicins, wide-spectrum antibiotics produced by streptomyces nrrl , endophytic on kennedia nigriscans taxomyces andreanae a proposed new taxon for a bulbilliferous hyphomycete associated with pacific yew lessons from nature: can ecology provide new leads in the search for novel bioactive chemicals from rainforests? recent and future discoveries of pharmacologically active metabolites from tropical fungi taxol and taxane production by taxomyces andreanae microbial gifts from rain forests rainforest endophytes and bioactive products endophytic taxol producing fungi from bald cypress taxodium distichum taxol from pestalotiopsis microspora, an endophytic fungus of taxus wallichiana an endophytic gliocladium sp. of eucryphia cordifolia producing selective volatile antimicrobial compounds cryptocandin, a potent antimycotic from the endophytic fungus cryptosporiopsis cf. quercina inhibitors of β-glucan synthesis cryptocin, a potent tetramic acid antimycotic from the endophytic fungus cryptosporiopsis cf. quercina ambuic acid, a highly functionalized cyclohexenone with antifungal activity from pestalotiopsis spp. and monochaetia sp the relationship between an endangered north americn tree and an endophytic fungus pestalotiopsin-a and pestalotiopsin-b-new caryophyllenes from an endophytic fungus of taxus brevifolia a new isodrimeninol from pestalotiopsis sp metabolites of endophytic fungi of taxus brevifoliathe first highly functionalized humulane of fungal origin jesterone and hydroxy-jesterone antioomycete cyclohexenenone epoxides from the endophytic fungus pestalotiopsis jesteri exploring chemical diversity of epoxyquinoid natural products: synthesis and biological activity of jesterone and related molecules phomopsichalasin, a novel antimicrobial agent from an endophytic phomopsis sp cr , a new pentaketide antifungal agent isolated from an endophytic fungus metabolites of colletotrichum gloeosporioides, an endophytic fungus in artemisia mongolica new bioactive metabolites produced by colletotrichum sp., an endophytic fungus in artemisia annua ecomycins, unique antimycotics from pseudomonas viridiflava pseudomycins, a family of novel peptides from pseudomonas syringae, possessing broad spectrum antifungal activity structure of the pseudomycins, new lipodepsipeptides produced by pseudomonas syringae msu h synthesis and antifungal activities of novel -amido bearing pseudomycin analogs practical streptomycetes genetics. the john innes foundation biosynthesis of -n-methylalbonoursin by an endophytic streptomyces sp endophytic actinomycetes: attractive biocontrol agents kakadumycins, novel antibiotics from streptomyces sp. nrrl , an endophyte of grevillea pteridifolia coronamycins, peptide antibiotics produced by a verticillated streptomyces sp. (msu- ) endophytic on monstera sp cytonic acids a & b: novel tridepside inhibitors of hcmv protease from the endophytic fungus cytonaema species muscodor albus gen. et sp. nov., an endophyte from cinnamomum zeylanicum volatile antimicrobials from a novel endophytic fungus effect of substrate on the bioactivity of volatile antimicrobials produced by muscodor albus muscodor roseus anna. nov. an endophyte from grevillea pteridifolia plant antitumor agents,v . the isolation of taxol, a novel antitumor agent from taxus brevifolia pestalotiopsis guepinii, a taxol producing endophyte of the wollemi pine, wollemia nobilis seimatoantlerium tepuiense gen. nov. a unique epiphytic fungus producing taxol from the venezuelan guyana the induction of taxol production in the endophytic fungus periconia sp. from torreya grandifolia seimatoantlerium nepalense, an endophytic taxol producing coelomycete from himalayan yew (taxus wallichiana) antifungal properties of taxol and various analogues taxol from tubercularia sp. strain tf , an endophytic fungus of taxus mairei evidence for paclitaxel from three new endophytic fungi of himalayan yew of nepal bioprospecting for taxol in angiosperm plant extracts torreyanic acid: a selectively cytotoxic quinone dimer from the endophytic fungus pestalotiopsis microspora total synthesis of the quinine epoxide dimer (+) torreyanic acid: application of a biomimetic oxidation/ electrocyclization/diels-alder dimerization cascade three new chytochalasins produced by an endophytic fungus in the genus rhinocladiella ispoestacin, an isobenzofuranone from pestalotiopsis microspora, possessing antifungal and antioxidant activities pestacin: a , -dihydro isobenzofuran from pestalotiopsis microspora possessing antioxidant and antimycotic activities discovery of small molecule insulin mimetic with antidiabetic activity in mice subglutinols a & b: immunosuppressive compounds from the endophytic fungus fusarium subglutinans in vivo addition of telomeric repeats to foreign dna generates chromosomal dnas in the taxol-producing fungus pestalotiopsis microspora we thank dr. gene ford and dr. david ezra for helpful discussions. the authors express appreciation to the nsf, usda, novozymes biotech, nih, the bard foundation of israel, the r&c board of the state of montana, and the montana agricultural experiment station for providing financial support for some of the work reviewed in this chapter. key: cord- -xcx c da authors: sahai, aastha; shahzad, anwar; shahid, mohd. title: plant edible vaccines: a revolution in vaccination date: - - journal: recent trends in biotechnology and therapeutic applications of medicinal plants doi: . / - - - - _ sha: doc_id: cord_uid: xcx c da plants have been used as a source for many pharmaceutical since long. however, utilization of plant systems for production of edible vaccines has been a comparatively recent phenomenon. there are several potential advantages of plant derived vaccines over other conventional systems of vaccine production such as mammalian or avian cell culture. the cost of vaccines is one factor preventing further use of vaccination, leaving hundreds of thousands of children susceptible to preventable diseases. especially for developing world this novel technique proved to be a boon for its low cost of production, convenient administration, easy storage and negligible chances of infection whereas the conventional system of vaccine production limits the applicability of vaccines in many parts of the world. these vaccines are prepared by introducing selected desired genes into plants and inducing these genetically modified plants to manufacture the encoded proteins. transgenic plants may provide an ideal expression system, in which transgenic plant material can be fed directly as oral dose of recombinant vaccines. expression of vaccines in plant tissue eliminates the risk of contamination with animal pathogen, provides a heat stable environment and enables oral delivery thus eliminating infection related hazards. identification of transgenic material, containment of the transgenes and control of recombinant protein may be potential problems for large scale production of vaccines in plants. factors like scaling up production as well as distribution and handling of transgenic plant material must comprise the future consideration in this field. since time immemorial, plants besides providing food and shelter have also been used for basic preventive and curative health care by human beings. plants were the fi rst and only source for every kind of medication in primitive times. gradually, animal derived products were also started being used as medicines for some ailments like snake venom has been used for medical purposes for thousands of years. in chinese medicine snake skin was used to treat superfi cial diseases, including skin eruptions, eye infections or opacities, sore throat, and hemorrhoids. however, plants still remained to be the primary source of medication till now for their ease of availability and safe nature. with the advent of modern science and technology, pharmacology has shown remarkable advances with the artifi cial production of plant and animal based medicines in laboratories. apart from this some novel medicinal compounds were also synthesized to combat life threatening diseases. all these advancements were aimed to increase average human life span and to decrease the child mortality rate. a signifi cant step taken in this regard was the universal immunization programme (uip) launched by who in s in which children all over the world are vaccinated against six deadly diseases (bcg, opv, diphtheria, tetanus, pertussis, and measles) with the aim of immunizing % of all children by . however, even after all these efforts % of infants are still left unimmunized; responsible for approximately two million unnecessary deaths every year, especially in remote and impoverished parts of the globe ( langridge ) . moreover, immunization remains an unfi nished agenda with an estimated . million children were not reached with three doses of dtp vaccine in . those who are not immunized -about every fifth child -are mostly among the poorest and the most vulnerable. vaccination is an important tool for prevention of many diseases. it is the administration of vaccine to produce immunity to a disease. vaccination involves the stimulation of the immune system to prepare it for the event of an invasion from a particular pathogen for which the immune system has been primed (arntzen ) . on recognizing the foreign substance (live/ weakened microbes, antigen or proteins) which enters the body through vaccine, the immune system activates, producing antibodies to destroy the invader. not only this but this attack leaves behind the 'memory' t and b cells which guards body in case of any future attack by that pathogen. some vaccines provide lifelong protection; others (such as those for cholera and tetanus) must be re-administered periodically (langridge ) . classically, this has been achieved by presenting the immune system with whole viruses or bacteria that have been killed or made too weak to proliferate much. however, these conventional vaccines poses threat of actually acquiring the infection from weakened or attenuated pathogenic strains. thus newer approach uses sub unit vaccines and recombinant vaccines. these vaccines consist of specifi c macromolecules that induce a protective immune response against a pathogen rather than administering the whole pathogen in the body. a "subunit vaccine" refers to a pathogen-derived protein (or even just an immunogenic domain of a protein, i.e. "an epitope") that cannot cause disease but can elicit a protective immune response against the pathogen. very often the subunit-vaccine candidate is a recombinant protein made in transgenic production-hosts (such as cultured yeast cells), then purifi ed, and injected as vaccines to immunize against a specifi c disease (mor and arntzen ) . these sub-unit vaccines increases vaccine safety by circumventing the need to use live viruses or microbes and has thus made them the preferred approach for vaccine manufacturers (national institute of allergy and infectious diseases ). unfortunately, traditional subunit vaccines are expensive to produce and not heat stable which limits their availability and use in developing countries. although these vaccines have an undue advantage over traditional conventional vaccines but they are expensive and their storage and transportation pose a problem as many of them require refrigeration. this is a disadvantage in many of the developing countries where the vaccines were needed most. thus, to ensure a successful global immunization a whole new idea of plant edible vaccines was presented by arntzen and co-workers in , by introducing the concept of transgenic plants as a production and delivery system for subunit vaccines (mason et al. ; mason and arntzen ) . they envisaged that production would be as cheap as agriculture, that distribution would be as convenient as marketing fresh produce and that administration would be as simple and safe as eating (mor et al. ). plants have long been considered an ideal expression system for many of the animal derived proteins, antibodies and pharmacologically important compounds. factors in favor of plant systems as sources of animal derived proteins include: the potential for large-scale, low-cost biomass production using agriculture; the low risk of product contamination by mammalian viruses, blood borne pathogens, oncogenes and bacterial toxins; the capacity of plant cells to correctly fold and assemble multimeric proteins; low downstream processing requirements for proteins administered orally in plant food or feed; the ability to introduce new or multiple transgenes by sexual crossing of plants; and the avoidance of ethical problems associated with transgenic animals and the use of animal materials (doran ) . the fi rst pharmaceutically relevant protein made in plants was human growth hormone, which was expressed in transgenic tobacco in (barta et al. ). since then, many other human proteins have been produced in an increasingly diverse range of crops. in , the fi rst antibody was expressed in tobacco (hiatt et al. ) . the idea of suitability of plants as vaccine production and delivery system was presented by research group of charles arntzen in s. this idea was quite promising in terms of providing a successful tool for mass immunization in developing countries where till now vaccination is hampered by high cost of production and delivery of vaccines. basic concept of edible vaccines involves the production of plants containing antigens required to stimulate the immune response in human body. thus by simply eating the plant product people will get vaccination against diseases. however the concept sounds quite simple but its development was equally complex. to produce edible vaccine, plants are engineered to contain desired gene of interest which codes for the antigen. this process is accomplished by transformation and the altered plant containing foreign gene is called transgenic plant. thus, the process remains the same as with subunit vaccine preparation because it contains only desired antigen and not the whole pathogen. these vaccines basically work in the same way as the injected dna vaccine, since a peptide sequence similar to an infectious part of a pathogen is synthesized, by itself, and is used to prime t and b cells in the body. the big difference in this case is that the protein sequences are encoded in a plant to form the desired protein. this protein is then ingested, as the plant or its fruit is eaten. one becomes immune against the ingested protein, as t and b cells become stimulated to proliferate and differentiate (mor et al. ). expression of vaccines in plant tissues eliminates the risk of contamination with animal pathogens, provides a heat-stable environment, and enables oral delivery, thus eliminating injection-related hazards (walmsley and arntzen ) . for production of edible vaccines, it is desirable to select a plant whose products are consumed raw to avoid degradation during cooking. thus, plants like tomato, banana and cucumbers are generally the plants of choice. vaccines made from plant material have enormous potential for use, in the developing world. also it may be much easier to persuade people to eat protective vegetables than to accept injections or take pills. vaccines made from mashed potato, designed to protect against travellers' diarrhoea, have already been tested in humans. vaccines made from dried tomatoes have also been developed. both have been developed by the cambridge-uk-based biotechnology company axis genetics, who pioneered the techniques involved. edible vaccines are mucosal-targeted vaccines that stimulate both the systematic and mucosal immune network, activating the fi rst line of defense of human body through mucosa. the mucosal surfaces are found lining the digestive tract, respiratory tract and urinoreproductive tract. mucosal immune system (mis) is the fi rst line of defense and the most effective site of vaccination, nasal and oral vaccines being the most effective (mor et al. ; korban et al. ) . before understanding the mechanism of action of edible vaccines it is important to understand the functioning of mis. induction of a mucosal immune response starts with the recognition of an antigen by specialized cells called m-cells. these cells are localized in the mucosal membranes of lymphoid tissues such as peyer's patches within the small intestines. the m-cells channel the antigen to underlying tissues where antigenpresenting cells internalize and process the antigen. the resulting antigenic epitopes are presented on the apc surface, and with the assistance of helper t cells activate b cells. the activated b cells migrate to the mesenteric lymph nodes where they mature into plasma cells and migrate to mucosal membranes to secrete immunoglobulin (ig) a. on passing through the mucosal epithelial layer towards the lumen, the iga molecules complex with membrane-bound secretary components to form secretary iga (siga). transported into the lumen, the siga interacts with specifi c antigenic epitopes and neutralize the invading pathogen (walmsley and arntzen ) . when edible vaccines are eaten they degrade majority of the plant cells in the intestine as a result of the action of digestive and bacterial enzymes. this degradation results in the release of antigens present in the plant product. the whole process occurs near the peyer's patches (rudzik et al. ) where m cells recognize the released antigen and lead to the production of iga antibodies in mucosal lymphoid tissues through the above mentioned mechanism. these iga antibodies get transported across the epithelial cells into the lumen where they interact with released antigens depicting immunogenic response. plant edible vaccines are basically prepared using two strategies. either through the introduction of antigen producing gene in plant genome via agrobacterium species; the process known as transformation. this can also be done by directly inserting the target gene in plant genome without the help of any vector. in this case gene gun eliminates the requirement of vector by directly bombarding the target gene in plant tissue, through the technique known as bolistic method. second strategy involves the use of plant viruses as vectors which results in transient expression of the foreign gene. the effect of the added genes is to make the plant or plant virus produce antigens which are normally found only on the surfaces of the pathogen. however, both the techniques require introduction of genes taken from the pathogenic microbe that infect humans but transformation technique offers certain advantages. there are three types of plant transformation methods: . agrobacterium mediated transformation . micro projectile bombardment/bolistics method . electroporation first method involves the use of agrobacterium tumefaciens as a carrier or vector system for introduction of antigen producing gene in plant genome. agrobacterium tumefaciens is a soil bacterium responsible for producing crown gall tumor in plants via its ti (tumor inducing) plasmid. a. tumefaciens and a. rhizogenes has the property to integrate their plasmid dna with nuclear genome of host (plants) cells. this property is exploited for introducing foreign gene in plants and the process is called 'transformation' while the plants produced through this process are 'transgenic plants'. during transformation the bacterial plasmid is fi rst disarmed by removing tumor inducing gene and the target gene for a selected immunogen is introduced. along with the target gene, antibiotic resistance genes were also added in the plasmid which acts as marker genes to identify the transformed plant tissues containing bacterial plasmid. the bacterium is then co cultivated with the wounded plant tissues so that the bacterial dna penetrate the plant cells. following this the target gene randomly incorporates in the plant nuclear genome. after selection through antibiotic resistance successfully transformed plant tissues are identifi ed and regenerated into whole plants under in vitro conditions. these transformed plant lines show varying degree of expression for introduced gene thus producing varying amount of desired antigen (shah et al. ) . plant line showing highest expression is identifi ed and propagated on large scale to be used as edible vaccine. the drawback of this method is that it gives low yield and the process is slow. it requires about weeks to months for whole plants to be formed through this process depending on regeneration ability of various plant species under in vitro conditions. this method is more successful in dicotyledonous plants like potato, tomato and tobacco which are easy to transform through agrobacterium in comparison to monocotyledons. second technique is capable of directly introducing the gene in plant cells through gene gun. this method is called 'bolistic method'. in this method selected dna sequences are precipitated onto metal microparticles and bombarded against the plant tissue with a particle gun at an accelerated speed. microparticles penetrate the walls and release the exogenous dna inside the cell where it will be integrated in the nuclear genome through mechanisms that have yet to be clearly understood (mishra et al. ). this method is quite attractive because dna can be delivered into cells of plant which makes gene transfer independent of regeneration ability of the species. but the chief limitation is the need for costly device particle gun (shah et al. ) . in another technique called 'electroporation', dna is introduced into cells by exposing them for brief period to high voltage electrical pulse which is thought to induce transient pores in the plasma lemma (singh ) . the cell wall presents an effective barrier to dna, therefore, it has to be weakened by mild enzymatic treatment so as to allow the entry of dna into cell cytoplasm. transformation technique results in stable expression of the foreign gene allowing production of subsequent generations of large numbers of transgenic plants, either by vegetative or sexual means. the seeds collected from these transgenic lines could be stored, and used as and when necessary for the molecular farming (malabadi et al. ) . it also provides the opportunity to introduce more than one gene for possible multi-component vaccine production. in addition, judicious choice of genetic regulatory elements allows organ and tissue-specifi c expression of foreign antigens . furthermore, plant dna is not known to interact with the animal dna and plant viral recombinants do not invade mammalian cells (sala et al. ). another approach of plant edible vaccine production utilizes plant viral expression systems which addresses the low yield issue of transformation approach. the rationale of this approach is based on the notion that during viral replication, gene copy number becomes amplifi ed, resulting in a much higher level of transgene expression than observed with stable transformation. among the potential advantages of transient viral expression of transgenes over stably transformed transgenic plants are the shorter time for cloning of foreign genes in the viral genome as compared with the time required to transform plants, the ease at which antigen production can be scaled up, and the wide host range of plant viruses that allows the use of multiple plant species as biofactories (koprowski and yushibov ) . capsid proteins of many types of viruses can assemble into virus-like particles (vlps). devoid of the viral genetic material, vlps often resemble the native virions in their morphology, antigenic properties and stability. these vlps are genetically engineered to express the desired peptides/proteins. two techniques namely 'overcoat technology' and 'epicoat technology' are used to design vlps that contain antigen dna on their surface. some plant viruses like cpmv (cowpea mosaic virus), alfalfa mosaic virus, tmv (tobacco mosaic virus), camv (caulifl ower mosaic virus), potato virus x and tomato bushy stunt virus can be used effectively for this purpose. overcoat technology permits the plant to produce the entire protein, whereas epicoat technology involves the expression of only foreign protein (lal et al. ). the recombinant virus is then inoculated into the host plant which produces several antigenic proteins (mor et al. ). these particulate antigens generally elicit stronger mucosal immune responses than soluble antigens, which can often repress the immune response by inducing immunotolerance (garside and mowat ) . however, each single antigen expressed in plants must be tested for its proper assembly and can be verifi ed by animal studies, western blot; and quantifi ed by enzyme-linked immunosorbent assay (elisa) (haq et al. ) . this method offers rapid onset of expression, and the systemic spread of virus so that protein is produced in every cell (desai et al. ) . vlps are therefore predicted to make excellent mucosal vaccines (estes et al. ) . however, the method poses certain limitations such as transient expression is less easy to initiate. also the subsequent plant generations do not inherit the foreign gene as it is not incorporated into the plant genome. thus the production of a plant virus-derived vaccinogen requires an extra step of inoculation of the host plants with the chimeric virus. before administration, chimeric virus particles are often purifi ed from host-plant tissues that are unpalatable, containing toxins or are not practical for direct consumption. nevertheless, the high level of foreign protein expression (up to g/kg of plant tissue) within a short period ( - weeks after inoculation) makes this an attractive alternative for vaccine production (walmsley and arntzen ) . plants and plant viruses were not used until , however, when an epitope from the foot-and-mouth disease virus (fmdv) was expressed on the surface of cowpea mosaic virus (cpmv) (usha et al. ) . chimeric plant viruses were proven effective as carrier proteins for vaccinogens in after rabbits raised an immune response against purifi ed chimeric cpmv particles expressing epitopes derived from human rhinovirus (valenzuelz et al. ) . majorly tmv and cpmv were used initially as plant viral expression systems because of advantages of high yield (l- g of virus per kg of host tissue), thermostability and ease of virus purifi cation, but now a days other plant viruses like alfalfa mosaic virus (amv), camv (caulifl ower mosaic virus) etc. are also being used. an increased interest in using plant virus vectors was witnessed for development of vaccines against many animal and human diseases (adams et al. ; clarke et al. ; dedieu et al. ; mastico et al. ; lomonossoff and johnson ) . in , who estimated that three to fi ve million children's lives could be saved each year if new vaccines were available to control or prevent commonly occurring infectious diseases. cvi emphasized on the need to create technologies that would make vaccine cheap and more reliable, especially for the developing world. cvi (children's vaccine initiative) also presented idea of needle free immunization to save millions of children from the pain of needles and to avoid risk of death due to needle carried infections. the attention was laid on oral vaccines as they fulfi ll all the requirements and are an effective means of vaccination by activating the mucosal immunity where most of the pathogens attack fi rst. development of heat stable oral vaccines were demanded which could serve the purpose of mass immunization in developing countries. being heat stable they can eliminate the requirement of cold chains which are very costly and generally not found those parts where vaccination is needed most. subunit vaccines based upon recombinant cell culture expression systems are feasible but, for commercial-scale production, these systems require fermentation technology and stringent purifi cation protocols so that suffi cient amounts of recombinant protein can be obtained for oral delivery. even with technological improvements, fermentation-based subunit vaccine production may be a prohibitively expensive technology for developing countries where novel oral vaccines are urgently needed . plant derived edible vaccines are the answer to all these requirements. these vaccines offer all those advantages which make them an ideal candidate to create a disease free world. following are the advantages of plant edible vaccines: (a) plant derived edible vaccines are cheap as they eliminate the expenses associated with fermenters, purifi cation, adjuvant, cold chain logistics, storage/transportation, needle free-administration, and sterile delivery (davoodi-semiromi et al. , walmsley and arntzen ; pascual ; malabadi ; daniell et al. ; gomez et al. ). (b) plant derived vaccine offer higher safety because they are needle/syringe free that is why risk of infections occurring from syringes is eliminated. (c) these vaccines do not require trained medical personnels for its administration unlike conventional vaccines which uses syringes as delivery system (d) plant derived vaccines are safe and have less chances of contamination with human and animal pathogenic microorganisms, because plants are not hosts for human infectious agents (giddings et al. ; ma et al. ). (e) as edible vaccine can be administered orally, they elicit both mucosal and systemic immunity which is not observed in traditional vaccines. (f) these vaccines are heat stable and can be stored at room temperature, unlike traditional vaccine which need cold chain storage which increases the yearly cost to preserve vaccines and limits their availability to areas having cold storage facility (nochi et al. ) (g) a concern with oral vaccines is the degradation of protein components in the stomach (due to low ph and gastric enzymes) and gut before they can elicit immune responses (daniell et al. b ) but in plant edible vaccines bioencapsulation of antigen in the rigid plant cell walls could provide protection from intestinal degradation (webster et al. ) . (h) for production of edible vaccines, costly equipments and machines are not necessary as they could be easily grown in farms as compared to cell culture grown in fermenters. (i) plant derived vaccines can be designed to contain numerous antigens for various disease. these multicomponent vaccines are called second generation vaccines and provide immunization against many diseases in a single dose. (j) an edible vaccine provides higher safety of individual as compared to traditional vaccine as edible vaccines are subunit preparation and do not involve attenuated pathogens which sometimes causes disease when administered as vaccine. (k) they can be ingested by eating the plant/part of the plant. so, the need to process and purify does not arise. (l) if the local/native crop of a particular area is engineered to produce the vaccine, then the need for transportation and distribution can be eliminated. (m) plant cells are suitable for vaccine production due to their capability to correctly fold and assemble, not only antibody fragments and single chain peptides, but also full-length multimeric proteins. (n) low downstream processing requirements for proteins administered orally. (o) as the antigen is present in edible plant product and can be administered by directly eating the plant tissue, it surpasses the purifi cation requirement which otherwise is a costly process. (p) new or multiple transgenes can be introduced by sexual crossing of plants thus creating novel vaccines against multiple diseases. (q) as plants are a safe biological system and harbor no pathogen of human infections, there are no ethical problems associated with plant edible vaccines unlike the vaccines produced from animal cell cultures and transgenic animals. (r) expression of antigen in plant seeds provides a convenient system of vaccine storage for long time duration thus reducing storage and shipping costs under ambient conditions. (s) plant cells are able to perform complex posttranslational modifi cation of recombinant proteins, such as glycosylation and disulfi de bridging that are often essential for biological activity of many mammalian proteins, allowing for the retention of native biological activity (lienard et al. ; rybicki ; tremblay et al. ) the fi rst report of the concept of using a plant expression system for the production of an edible vaccine appeared in a patent application published under the international patent cooperation treaty (curtiss and cardineau (mason et al. ). in parallel with evaluation of plant-derived hepatitis b surface antigen, mason and arntzen explored plant expression of other vaccine candidates including the labile toxin b subunit (lt-b) of entertotoxigenic escherichia coli (etec) and the capsid protein of norwalk virus (nvcp). it was found that the plant derived proteins correctly assembled into functional oligomers that could elicit the expected immune responses when given orally to animals (haq et al. ; mason et al. mason et al. , . subsequently, a number of attempts were made to express various antigens in plants (table . ). a. sahai et al. hepatitis b virus (hbv) is one of the major causes of chronic viremia in humans (purcell ) . the hepatitis b virus is estimated to have infected million people throughout the globe, making it one of the most common human pathogens. since immunization is the only known method to prevent the disease of hepatitis b, any attempt to reduce its infection requires the availability of large quantities of vaccine, hepatitis b surface antigen (hbsag) (malik et al. ) . mason et al. demonstrated the expression of hbsag at levels equal to . % of total soluble protein in tobacco. however, the low level of expression of the hbsag in transgenic tobacco ( . % of soluble protein) and the alkaloids present in the crude plant extract prevent direct feeding studies. thus, mason et al conducted further studies with tobacco-derived recombinant hbsag (rhbsag) protein that was recovered from leaf extracts as a vlp with an average size of nm, which are similar to those found in the sera of infected humans and in the commercial vaccine (cabral et al. ) . these plant-derived vlps mimic the appearance of recombinant yeastderived hbsag particles (scolnick et al. ) , which is the material that is used in commercially available recombinant vaccine for hepatitis b (recombivaxm; distributed by merck, sharpe, and dohme) and can be injected as vaccinogen directly. in addition, the plant-derived material had similar buoyant density and antigenicity to human and yeast-derived hbsag, indicating faithful preservation of protein folding characteristics in the plant system'. thus a crude extract of rhbsag from plants was used in parenteral immunization studies with mice (thanavala et al. ) . the extract caused an immune response that was similar to the one achieved with recombivaxm, so the studies concluded that the rhbsag from plants demonstrate that b-and t-cell epitopes of hbsag are preserved when the antigen is expressed in transgenic plants, and that the recombinant antigen is produced as a vlp that mimics the currently available commercial vaccine. subsequently many papers were published characterizing the recombinant product which assembled into virus like particles (vlps), and could invoke specifi c immune responses in mice upon parenteral delivery. further studies were made by arntzen group to prove that plant-derived hbsag can stimulate mucosal immune responses via the oral route, for which they employed potato tubers as an expression system and optimized it to increase accumulation of the protein in the plant tubers (richter et al. ) . the resulting plant material proved superior to the yeast-derived antigen in both priming and boosting of immune responses to oral immunogen in mice (kong et al. ; richter et al. ) . the hbsag has also been expressed in banana (may et al. ) and lettuce (kapusta et al. ) . recently kumar et al. ( ) transformed embryogenic cells of banana with the 's' gene of hepatitis b surface antigen (hbsag) using agrobacterium mediated transformation. the expression levels of the antigen in the plants grown under in vitro conditions as well as the green house hardened plants were estimated by elisa. maximum expression level of ng/g f.w. of leaves was noted in plants transformed with pefehbs grown under in vitro conditions, whereas pher transformed plants grown in the green house showed the maximum expression level of . ng/g f.w. of leaves. hbsag obtained by them from transgenic banana plants was found to be similar to human serum derived one in buoyant density properties. kumar et al. ( ) advocated that although expression levels of the antigen are low in banana fruits, the expression levels of the vaccine antigens can be increased by the use of promoter of abundant pulp protein (clendennen et al. ) , or promoters of the proteins found in abundance in the ripe banana fruits (peumans et al. ) . mcgarvey et al. ( ) have reported the expression of rabies virus (rv) glycoprotein in transgenic tomatoes. although mcgarvey et al. did not reported on the immunogenicity of the tomato rv glycoprotein, yusibov et al. ( ) have reported the induction of neutralizing antibodies in mice that were parenterally vaccinated with a peptide of rv glycoprotein fused to a plant virus coat protein. antigen-capsid fusion is one of the alternative strategies of producing a plant-based vaccine where plants are infected with recombinant viruses carrying the desired antigen in viral coat protein. the infected plants have been reported to produce the desired fusion protein in large amounts in a short time. it should, however, be kept in view that recombinant viruses need to be highly purifi ed for parenteral administration or partially purifi ed for oral administration. modelska et al. ( ) reported that immunization of mice intraperitoneally or orally by gastric incubation or by feeding of plants infected with the recombinant alfalfa mosaic virus (aimv) carrying rabies peptide cpdrg showed local as well as systemic immune response. after immunization, % of the mice were protected against the challenge with a lethal dose of the virus. this report also demonstrated stronger immune responses in mice that consumed the chimeric viruses in planta rather than after purifi cation (modelska et al. ). recently, loza-rubio and coworkers developed transgenic maize expressing the rabies virus glycoprotein of the vnukovo strain and they evaluated its immunogenicity in mice by the oral route. animals were fed once with μg of protein and challenged -days later with a rabies virus isolated from vampire bats. the edible vaccine induced viral neutralizing antibodies which protected mice % against challenge (loza-rubio et al. ). the vp capsid protein of the foot-and-mouth disease virus (fmdv) was also successfully expressed in transgenic arabidopsis thaliana (carrillo et al. ). carrillo and co-workers have shown that mice injected intraperitoneally with the partially purifi ed vp protein are totally resistant to a challenge with a virulent strain of the virus. oral administration of the plant-derived vp subunit of fmdv has not been demonstrated. because fmdv virions are complex structures containing several subunits therefore it is unlikely that the vp peptide alone will assemble into stable vlps. another important study was regarding enterotoxigenic escherichia coli (etec) and vibrio cholera which are the primary pathogens responsible for acute watery diarrhea. cholera is a devastating diarrheal disease that has caused recurrent pandemics throughout the world since (yu and langridge ) . the heatlabile enterotoxin (lt) of etec is closely related to cholera toxin (ct). haq et al. ( ) proved that a bacterial antigen (lt-b) could be expressed in edible tissues of transgenic plants and could assemble into pentameric ring structures (as shown by its ability to bind gangliosides). in addition, and most importantly, lt-b expressed in plants was shown to induce the production of both serum and mucosal antibodies in mice fed with transgenic potato tubers. however, the low level of expression of lt-b in potato tubers, implied that people would have to eat an unreasonably large amount of tuber to receive the desired dose. mason et al. ( ) improved expression levels of lt-b in transgenic plants by the construction of a 'plant-friendly' synthetic lt-b gene. the protein product of this synthetic gene accumulates to ~ . % of the soluble protein, which is . -fold higher than the best levels obtained previously by haq et al. ( ) . these higher expression levels have allowed this 'edible vaccine' to be tested in humans, representing the fi rst human clinical trial of a plant-derived vaccine where g of raw potato tubers expressing lt-b of etec in three doses had to be consumed in order to overcome digestive losses of the antigen and to elicit a signifi cant immune response (tacket et al. ) . these results showed, for the fi rst time, that food plant-based vaccines are immunogenic in humans. cholera toxin, which is very similar to e. coli lt, has also been expressed in plants. hein et al. ( ) generated tobacco plants expressing ct-a or ct-b subunits of the toxin. ct-a produced in plant was not cleaved into a and a subunits, which happens in epithelial cells. while ct-b undergone similar processing in plants as the ct-b derived from v. cholerae , and was thus recognized by mouse anti-ct-b antibody. antigenically it was found to be similar to the bacterial protein. even after boiling transgenic potato tubers till they became soft, approximately % of the ct-b was present in the pentameric gm ganglioside-binding form (arakawa et al. (arakawa et al. , . higher expression levels (up to . % tsp) were obtained when ctb- l fusion protein was expressed in transgenic chloroplasts (molina et al. ). according to the who, hiv-induced acquired immunodefi ciency syndrome (aids) kills - million people annually (unaids ) . stable chimeric cpmv particles that express epitopes derived from human rhinovirus and hiv- were described by porta et al. in . the inserted epitopes were immunogenic in rabbits. initial success was also achieved in splicing hiv protein into cpmv by prakash ( ) . two hiv protein genes and camv as promoter were successfully injected into tomatoes with a needle and the expressed protein was demonstrable by polymerase chain reaction (pcr) in different parts of the plant, including the ripe fruit, as well as in the second generation plant. norwalk virus is known to cause acute gastroenteritis in humans. norwalk virus capsid protein (nvcp) from the diarrhea causing norwalk virus, expressed in transgenic tobacco and potato with . % of total soluble protein, also assembled vlps and stimulated serum igg and gut iga specifi c for nvcp when fed to mice (mason et al. ) . the clinical trial was conducted at the center for vaccine development with nvcp potatoes ( tacket et al. ) . twenty adults ingested either two or three doses each of g raw potato containing - lg nvcp. nineteen of twenty adults showed signifi cant increases in the numbers of specifi c anti-nvcp antibody-secreting cells of the iga subtype, and six developed increases in igg antibody secreting cells. this study proved that orally delivered plantexpressed vlps could stimulate immune responses and further that gm binding activities not required for oral immunization. these results suggest a new strategy for the development of plant vaccines. individual soluble protein antigens are relatively ineffective for oral immunization because of intestinal digestion and lack of antigen tropism for galt (gut associated lymphoid tissues). in contrast, vlps are stable in the acidic environment of the stomach and resistant to enzyme digestion in the small intestine. most important, vlps preserve conformational epitopes located on the surface of viral particles, which are recognized by the host immune system. compared with other viral antigens, the plant-produced vlp may be the more effective antigen for protection against infectious enteric viral diseases (yu and langridge ) . attempts are underway to engineer bananas and powdered tomatoes expressing norwalk virus. over two billion individuals reside in the malaria endemic areas and the disease affects - million people annually. as a result of malarial-infection, an estimated three million lives are lost annually, among them are over one million children (majority under years of age).the world malaria situation has become signifi cantly worse in recent years as the main forms of malaria control, spraying programmes and chemotherapy, becoming less effective in the development of vector and parasite resistance. in a study by beachy et al. ( ) a -amino-acid epitope of zona pellucida , zp , protein and another epitope from malarial sporozoites have been expressed as fusion proteins with tmv capsid protein with the idea of developing anti-fertility and anti-malarial vaccines. the antigenicity of the products has been found to be positive. currently, three malarial antigens are under investigation for the development of plant-based malaria vaccine, merozoite surface protein (msp) ,msp from plasmodium falciparum, and msp / from p. yoelli. wang et al. ( ) has demonstrated that oral immunization of mice with recombinant msp , msp / and msp , co-administered with ctb as a mucosal adjuvant, induced antibody responses effective against blood-stage parasite. the study however involved complexity as the protein was expressed in e. coli and protection was only evident when high dose antigen was administered. thus it is still uncertain if the oral delivery of a plant-derived malaria vaccine would induce signifi cant immune responses in humans. it has been suggested that antigen expression level in plants are so low that an unrealistic quantity of plant material would have to be consumed to achieve meaningful immunity. for this approach to become realistic improve antigenic expression has to be achieved. moreover, due to high levels of antigen anticipated to be necessary, it is likely that strong adjutants will also be required (wang et al. ). hence, appropriate adjuvants have to be identifi ed and tested. finally, in the face of reports showing induction of tolerance or immunity through comparable oral immunizations vaccination regimens must be rigorously tested in preclinical studies (arakawa et al. ). applications of edible vaccines are expanding to autoimmune diseases where the body's own proteins recognized as foreign by the immune system. autoimmune diseases include arthritis, myasthenia gravis, multiple sclerosis and type i diabetes. it was established by arakawa et al. ( ) that food plants are feasible production and delivery systems for immunotolerization against autoimmune diseases. they found cholera toxin b to be useful as a carrier molecule for specifi c targeting to the gut-associated lymphoid tissue (galt). a cholera toxin b-insulin fusion protein produced in transgenic potato plants successfully protected non obese diabetic mice from development of autoimmune diabetes mellitus type i. ma and jevnikar ( ) expressed glutamic acid dehydrogenase in potatoes and fed them to non-obese diabetic mice, in which the reduced pancreatic islet infl ammation suggested immuno-tolerization of cytotoxic t-cell-mediated autoimmune disease. an optimal oral dose of a plant-derived auto antigen can potentially inhibit development of the autoimmune disease (carter and langridge ; sala et al. ) . edible vaccine development for the prevention or treatment of cancer is diffi cult since tumor antigens are also auto-antigens (zhang et al. ). recently, a poly-epitope isolated from a human melanoma tumor was integrated into the nuclear and chloroplast dna of tobacco in an attempt to develop a plant-derived melanoma vaccine (sala et al. ) . mccormick et al. ( ) expressed a scfv antibody fragment of the immunoglobulin from a mouse b-cell lymphoma in tobacco with a viral vector and showed that mice injected with this vaccine were protected from challenge by a lethal dose of tumor. another scfv fused to the potato virus x coat protein generated protection against lymphoma and myeloma (savelyeva et al. ). there were many questions regarding plant edible vaccines when the concept was given by arntzen and group in s. some of them were answered in the course of successive development in this fi eld while some still remain unanswered. even after all the research and developments made in plant derived edible vaccines, there are some limitations and issues which are still to be addressed. a major impediment in the successful application of these vaccines is the low expression level of antigen and uncertain dosage. in all the studies mentioned above the expression level of antigen in plant tissue was not of practical utilization as to achieve the required immunity very large amount of plant product is need to be consumed. in addition, not all vaccine candidate proteins are highly immunogenic in plant tissues and secondary metabolites found in plants may compromise the ability of the vaccine candidate protein to induce immunity (teli and timko ) . two solutions to overcome this limitation are being explored. first, techniques to enhance antigen accumulation in plant tissues are being explored. a number of factors, including genes encoding vaccine antigens can be optimized for plant codon use; plant promoters can be engineered to increase transcription levels; rna splice sites and intron sequences can be removed codon usage, the type of ′-untranslated sequence incorporated, the presence of specifi c intra-and extracellular targeting or compartmentalization sequences present, the site of gene integration into the genome, etc. affect transgene expression and ultimately vaccine epitope accumulation in plants. optimization of coding sequences of bacterial or viral genes for transient expression, as well as defining the best subcellular compartment for product accumulation to obtain optimal quantity and quality, is also being studied (teli and timko ) . to enhance the immunogenicity of the orally delivered antigens, the use of carrier proteins may also be required, especially for small, non-particulate subunit vaccine antigens (walmsley and arntzen ) . another approach is to use bacterial enterotoxins such as ct or lt , mammalian and viral immunomodulator, or plant-derived secondary metabolites (yu and langridge ) . although the constitutive expression of foreign genes may lead to the accumulation of foreign proteins to levels toxic in the plant, inducible promoters, which stimulate gene expression at specifi c points in plant development, may not only prevent accumu lation of toxic levels of the foreign gene product but may conserve the plant's photosynthate for generation of maximum plant growth, resulting in higher yields of recombinant proteins (arakawa et al. (arakawa et al. , . thus, temporal and organ-and tissue-specifi c promoters activated in fruit, tubers, or seeds must be explored (fiedler and conrad ) . another problem is that the glycosylation of trans proteins in plants differs slightly from those produced in transgenic animals or animal cells in vitro (lerouge et al. ). the addition of xylose and change from a b ! to a b ! linkage of fucose are typical in plants. a signifi cant difference with transprotein production in plants is their inability to add sialic acid to glycoproteins (lerouge et al. ) . this sugar has been implicated in longer clearance times for proteins in the blood and therefore is a major factor for a select group of pharmaceutical proteins. however, plant glycan patterns may not represent a problem in terms of human health; they may affect conformational epitopes, or clearance of plant derived antibodies (bakker et al. ; ma et al. ) . this potential problem is likely to be overcome as we learn more about the requirements for these processes in both plant and animal cells (bakker et al. ) . another concern is about the potential contamination of plant-derived recombinant proteins with potentially toxic factors due to the fact that some plant species contain numerous toxic alkaloids and other secondary metabolites. careful selection of appropriate plant materials for heterologous expression can help alleviate this potential problem (teli and timko ) . another major concern is the possibility of development of immunotolerance to the vaccine protein or peptide. also there will be a challenge in controlling transgene escape as the identifi cation of "vaccine" fruit vs a normal fruit would be diffi cult. fruit vaccines should be easily identifi able to avoid the misadministration of the vaccine, which may lead to complications such as immunotolerance. also the oral vaccines are complicated by the need to protect the antigen from the effects of the acidic and proteolytic environment of the gut. hence, to be effective, oral subunit vaccines generally require higher doses than oral replicating vaccines (walker ; mestecky et al. ) allergic reactions to plant protein glycans and other plant antigens is a challenging issue. it has been suggested that plant derived recombinant proteins or antibodies may have increased immunogenicity or allergenicity as compared to mammalian counterparts (ma et al. ; penney et al. ) . this is well explained by the fact that a state of tolerance or energy has been gained by the daily consumption of plant glycolproteins in our food (ma et al. ) . some other potential problems related to plant edible vaccines include: (a) plant and product contamination by mycotoxins, pesticides, herbicides and endogenous metabolites. (b) regulatory uncertainty, particularly for proteins requiring approval for human drug use (doran ) . (c) consistency of dosage from fruit, plant to plant, generation to generation is not similar (d) stability of vaccine in fruit is not known and differs from plant to plant. (e) some food cannot be eaten raw (e.g. potato) and needs cooking which will denature or weaken the protein present in it (moss et al. ) (f) variable conditions for edible vaccine are also a major problem. potatoes containing vaccine to be stored at °c and could be stored for longer time while a tomato does not last long. thus these vaccines need to be properly stored to avoid infection through microbial spoilage. thus while the plant edible vaccines are a lucrative option in the fi eld of vaccination, there is much remains to be done in this fi eld. with many potential issues to be addressed this area of health care is open for exhaustive research and development. transgenic chloroplasts have become attractive systems for heterologous gene expressions because of unique advantages. chloroplast genetic engineering offers several advantages, including high levels of transgene expression, transgene containment via maternal inheritance, and multi-gene expression in a single transformation event (chebolu and daniell ). the highest level of protein expression through transgenic tobacco chloroplast was obtained for bacillus thuringiensis (bt) cry aa protein ( . % tsp) (de cosa et al. ) . as chloroplasts are genetically semi autonomous having their independent genome, many self replicating copies of transformed plastids can be produced in a single plant cell thus enhancing the antigen/ protein yield considerably. there are several other advantages of chloroplast transformation system over nuclear transformation. environmental concerns about mixing genetically modifi ed pollen with other crops or weeds have been continually raised (stokstad and vogel ) . this objection may be partially addressed by engineering the foreign gene into the chloroplast dna (ruf et al. ) . as chloroplast genome is maternally inherited it does not follow mendelian pattern of inheritance (zhang et al. ) so gene pollution caused by transgene escape through pollen can be controlled. even if the pollen from plants that exhibit maternal inheritance contains metabolically active plastids, the plastid dna is lost during pollen maturation and is not transmitted to the next generation . therefore, the chloroplast expression system is an environmentally friendly approach. chaperones present in chloroplasts facilitate correct folding and assembly of monoclonal antibody in transgenic chloroplasts ) and also result in fully functional human therapeutic proteins, as seen in interferon alpha and gamma (falconer ; leelavathi and reddy ) . chloroplasts have the ability to process eukaryotic proteins, including correct folding of subunits and formation of disulfi de bridges (daniell et al. b ). chloroplast-synthesized cholera toxin-b subunit binds to the intestinal membrane gm -ganglioside receptor, thereby confi rming the correct folding and disulfi de bond formation through functional assay (daniell et al. a ; molina et al. ) . also, chloroplasts have the ability to express multiple genes in a single transformation event. expression of polycistrons in transgenic chloroplasts is a unique feature, which facilitates the expression of entire pathways in a single transformation event (de cosa et al. ; daniell and dhingra ) . de cosa et al. ( ) for the fi rst time expressed a complete bacterial operon in transgenic chloroplasts, resulting in the formation of stable cry aa crystals. this should facilitate expression of polyvalent vaccines or multisubunit proteins in transgenic chloroplasts (chebolu and daniell ). problem of gene silencing is also eliminated in chloroplast expression systems. inspite of higher expression level gene silencing was not observed in transgenic chloroplast derived plants (de cosa et al. ) . chloroplasts can be a good place to store the biosynthetic products that could otherwise be harmful when accumulated in cytosol (bogorad ) . this was demonstrated when cholera toxin b subunit was accumulated in large quantities in transgenic chloroplasts and it had no toxic effect (daniell et al. a ) , whereas when accumulated in the cytosol in very small quantities, ctb was toxic (mason et al. ) . similarly, trehalose, was toxic when accumulated in cytosol but was nontoxic when compartmentalized within chloroplasts (lee et al. ) . however chloroplast transformation is also not untouched with certain limitations and issues. as with any fresh tissue molecular farming system, protein stability over time will change even with refrigeration. extraction and purifi cation must be performed at very specifi c times following harvest. tobacco is currently a highly regulated crop and is not edible. large volume products and edible vaccines would not appear to be feasible using this system (horn et al. ). tobacco appears to be the only species in which plastid transformation has been established as routine (svab and maliga ; . however, recently kanamoto et al. ( ) developed plastid transformation system for lettuce. currently the researches in plant edible vaccine production are emphasizing on good manufacturing practices (gmp) with increased antigen expression being the everlasting objective of this technology. recently a novel technique developed by icon genetics (bayer crop science, germany), based on the magnifection, has additional advantageous than the routine methods used for the production of subunit vaccines in plants. this system allows very fast production, high recombinant protein expression levels for example hepatitis b virus (hb core) with an accumulation level exceeding % of total soluble protein in tobacco (gomez et al. ; yusibov and rabindran ; gleba et al. ; huang et al. ) . recently alvarez et al. ( ) conducted an important study on enhancing recombinant protein expression in transgenic plants. they suggested that in maize, γ-zein is the major storage protein synthesized by the rough endoplasmic reticulum (er) and stored in specialized organelles called protein bodies (pb). zera® (γ-zein er-accumulating domain) is the n-terminal proline-rich domain of c-zein that is suffi cient to induce the assembly of pb formation. fusion of the zera® domain to proteins of interest results in assembly of dense pb-like, er-derived organelles, containing high concentration of recombinant protein. to confi rm this, they expressed f -v antigen protein from yersinia pestis (causal pathogen of plague) in three plant models ( ncotiana benthamiana , medicago sativa (alfalfa) and nicotiana tabacum nt cells) with and without a fused zera® domain. they found that f -v protein with fused zera® domain showed three times more accumulation in plant cells than f -v protein alone. the list of diseases that could potentially be prevented with plant-based, edible vaccines is keeping on increasing. clinical trials have already succeeded in increasing the production of antibodies against hiv in mice (karasev et al. ) on the other hand, soybean has been genetically modifi ed to produce monoclonal antibo dies that act as carriers of cancer-attacking compounds. the possibility of developing hiv and cancer vaccines through transgenic plants could be a major step in the fi ght against these devastating diseases. highly pathogenic h n avian infl uenza responsible for global pandemics and with mortality rates exceeding % encouraged global efforts to develop vaccines against this highly pathogenic avian infl uenza (hpai). shoji et al. ( ) recently described the production of ha from the a/indonesia/ / strain of h n infl uenza virus by transient expression in nicotiana benthamiana plants. they demonstrated that immunization of mice and ferrets with this plant-derived ha protected ferrets against challenge infection with a homologous virus. sexually transmitted diseases are also now included in the domain of plant based vaccination. maclean et al. ( ) have reported the expression of l capsid protein from hpv- . by a transient expression assay, the authors determined not only that l protein was capable of assemble into vlps, maintaining its immunological properties, but that a human instead of a plant codon usage and the protein-direction to the chloroplast, were the best conditions to achieve high yields of recombinant l protein ( % of tsp in transgenic tobacco). these results showed an effi cacious way of producing vlps onward hpv vaccine, maybe a cheaper one than the new hpv vaccine produced in insect cells. immunizing experiments are needed to better prove this hypothesis. obregon and coworkers showed a new strategy for increasing recombinant protein production in hiv by improved the stability of p core protein. this stable protein formed dimmers that were retained within the cell resulting in an enhanced expression, apparently related to protein folding processing and assembly, subcellular targeting and protein stability (obregon et al. ) . recently, saejung et al. ( ) reported, for the fi rst time, the production of a dengue vaccine in plants. takagi et al. ( ) reported gm rice expressing two t-cell epitope peptides of cry j i and cry j ii allergens of japanese cedar ( cryptomeria japonica ) pollen. multicomponent edible plant vaccines providing immunologic protection simultaneously against several infectious diseases are under construction. recently in this context (shchelkunov et al. ) demonstrated a bivalent vaccine synthesis through a synthetic chimeric gene, tbi-hbs, encoding the immunogenic env and gag epitopes of human immunodefi ciency virus (hiv- ) and the surface protein antigen (hbsag) of hepatitis b virus (hbv), was expressed in tomato plants. tomato fruits containing the tbi-hbs antigen were fed to experimental mice and, on days and post-feeding, high levels of hiv and hbv-specifi c antibodies were present in the serum and feces of the test animals. intraperitoneal injection of a dna vaccine directing synthesis of the same tbi-hbsag antigen boosted the antibody response to hiv in the blood serum; however, it had no effect on the high level of antibodies produced to hbv. although no plant-based edible vaccines are currently commercially available, a secretory antibody vaccine was approved in the eu, a poultry vaccine against newcastle disease was approved by the usda, and a hepatitis b virus vaccine using a tobacco plant has been approved in cuba (kim and yang ) . vaccines for diarrhea, hepatitis b and rabies, and antibodies for non-hodgkin's lymphoma, colorectal cancer and dental caries, have been submitted for phase i or phase ii clinical trials in humans (ma et al. ). korban and colleagues at the university of illinois (urbana-champaign, ill.) have reported a plant-based oral vaccine against respiratory syncytial virus (rsv) in tomato fruit (korban et al. ) with the ultimate aim of moving the product into apple. hsv is a viral pathogen that causes respiratory diseases and is a leading cause of viral lower respiratory tract illness in infants and children worldwide. aziz et al. ( ) paved way for developing edible vaccines against anthrax by successfully expressing the protective antigen against anthrax in potato tubers. plant edible vaccines have the potential to change the whole scenario of vaccination. upto few years ago vaccination was only limited to six deadly diseases in chil dren but now not only human but animal diseases of bacterial, viral even protozoan origin are being successfully studied for vaccination. not only this but the researches are ongoing to provide plant based vaccines for autoimmune diseases like diabetes and cancer which has risen the need of these vaccines for developed countries also which till now were emphasized to be important majorly for developing countries. apart from many advantages of these vaccines there are some very relevant social, environmental and ethical issues concerning them which are need to be addressed. besides future research is needed to overcome limitations like low expression, immunotolerance, glycosylation, immunogenicity, and stability of the transproteins if the practical application of these vaccines is to be realized. one very important point is the proper coordination between academia and industry to help these vaccines reach people. both technical and regulatory hurdles have to be overcome. it will be a challenge to create a positive public perception regarding safety and effi cacy of these vaccines after all the fuss created over the safety issues of transgenic crops during last few years. lastly timely funding for this research and participation of corporate giant will certainly help in making this dream a reality soon. the expression of hybrid hiv-ty virus-like particles in yeast higher accumulation of f -v fusion recombinant protein in plants after induction of protein body formation expression of cholera toxin b subunit ologomers in transgenic potato plants a plant-based cholera toxin b subunit-insulin fusion protein protects against the development of autoimmune diabetes edible vaccines expression of protective antigen in transgenic plants: a step towards edible vaccine against anthrax galactose extended glycans of antibodies produced by transgenic plants the expression of a nopaline synthase human growth hormones chimeric gene in transformed tobacco and sunfl ower callus tissue use of plant viruses for delivery of vaccine epitopes engineering chloroplasts: an alternative site for foreign genes, proteins, reactions and products neutralising immunogenicity of a polyepitope antigen expressed in a transgenic food plant: a novel antigen to protect against measles cellular and humoral immunity in guinea pigs to two major polypeptides derived from hepatitis b surface antigen in vitro selection and characterization of human immunodefi ciency virus type variants with increased resistance to abt- , a novel protease inhibitor plant-based vaccines for protection against infectious and autoimmune diseases chloroplast-derived vaccine antigens and biopharmaceuticals: expression, folding, assembly and functionality presentation and immunogenicity of viral epitopes on the surface of hybrid hepatitis b virus core particles produced in bacteria the abundant -kilodalton banana pulp protein is homologous to class iii acidic chitinases world intellectual property organization multigene engineering: dawn of an exciting new era in biotechnology expression of native cholera toxin b subunit gene and assembly as functional oligomers in transgenic tobacco chloroplasts medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants milestones in chloroplast genetic engineering: an environmentally friendly era in biotechnology chloroplast derived antibodies, biopharmaceuticals and edible vaccines plant-made vaccine antigens and biopharmaceuticals the green vaccine: a global strategy to combat infectious and autoimmune diseases chloroplast-derived vaccine antigens confer dual immunity against cholera and malaria by oral or injectable delivery over expression of the cry aa operon in chloroplasts leads to formation of insecticidal crystals poliovirus chimeras expressing sequences from the principle neuralising domain of human immunodeficiency virus type production of heterologous proteins in plants: strategies for optimal expression foreign protein production in plant tissue cultures virus-like particle vaccines for mucosal immunization expression of interferon alpha b in transgenic chloroplasts of a low-nicotine tobacco high-level production and long-term storage of engineered antibodies in transgenic tobacco seeds mechanisms of oral tolerance transgenic plants as factories for biopharmaceuticals magnifection-a new platform for expressing recombinant vaccines in plants oral immunogenicity of the plant derived spike protein from swine-transmissible gastroenteritis coronavirus developments in plant-based vaccines against diseases of concern in developing countries oral immunization with a recombinant bacterial antigen produced in transgenic plants expression of cholera toxin subunits in plants production of antibodies in transgenic plants plant molecular farming: systems and products rapid, high level production of hepatitis b core antigen in plant leaf and its immunogenicity in mice cholera toxin b protein in transgenic tomato fruit induces systemic immune response in mice effi cient and stable transformation of lactuca sativa l. cv. cisco (lettuce) plastids high-level expression of the neutralizing epitope of porcine epidemic diarrhea virus by a tobacco mosaic virus based vector. protein expression and purifi cation a plant-derived vaccine against hepatitis b virus plant based hiv- vaccine candidate: tat protein produced in spinach synthesis and assembly of anthrax lethal factor cholera toxin b-subunit fusion protein in transgenic potato current trends in edible vaccine development using transgenic plants oral immunization with hepatitis b surface antigen expressed in transgenic plants the green revolution: plants as heterologous expression vectors foods as production and delivery vehicles for human vaccines expression of hepatitis b surface antigen in transgenic banana plants edible vaccines: current status and future edible vaccines accumulation of trehalose within transgenic chloroplasts confers drought tolerance chloroplast expression of his-tagged gus-fusions: a general strategy to overproduce and purify foreign proteins using transplastomic plants as bioreactors n-glycoprotein biosynthesis in plants: recent developments and future trends pharming and transgenic plants eukaryotic viral expression systems for polypeptides development of an edible rabies vaccine in maize using the vnukovo strain auto antigens produced in plants for oral tolerance therapy of autoimmune diseases induction of oral tolerance to prevent diabetes with transgenic plants requires glutamic acid decarboxylase (gad) and il- antibody processing and engineering in plants, and new strategies for vaccine production optimization of human papillomavirus type (hpv- ) l expression in plants: comparison of the suitability of different hpv- l gene variants and different cell-compartment localization production of edible vaccines for oral immunization in transgenic plants, current and future prospective recent advances in plant derived vaccine antigens against human infectious diseases edible vaccine-vegetables as alternative to needles transgenic plants as vaccine production systems expression of hepatitis b surface antigen in transgenic plants expression of norwalk virus capsid protein in transgenic tobacco and protein and its oral immunogenicity in mice edible vaccine protects mice against e. coli heat-labile enterotoxin (lt): potatoes expressing a synthetic lt-b gene multiple presentation of foreign peptides on the surface of an rna-free bacteriophage capsid generation of transgenic banana ( musa acuminata ) plants via agrobacterium -mediated transformation rapid production of specifi c vaccines for lymphoma by expression of tumor-derived single-chain fv epitopes in tobacco plants expression of the rabies virus glycoprotein in transgenic tomatoes routes of immunization and antigen delivery systems for optimal mucosal immune responses in humans edible vaccines: a new approach to oral immunization immunization against rabies with plant-derived antigen high yield expression of a viral peptide animal vaccine in transgenic tobacco chloroplasts plants and human health: delivery of vaccines via transgenic plants edible vaccines: a concept come of age implications of human immunodefi ciency eradications of measles development of a plant derived subunit vaccine candidate against hepatitis c virus rice based mucosal vaccine as a global strategy for cold chain and needle free vaccination hiv- p -immunoglobulin fusion molecule: a new strategy for plant-based protein production vaccines are for dinner plant-made vaccines in support of the millennium development goals the abundant class iii chitinase homolog in young developing banana fruits behaves as a transient vegetative storage protein and most probably serves as an important supply of amino acids for the synthesis of ripening-associated proteins development of cowpea mosaic virus as a high yielding system for the presentation of foreign peptides edible vaccines and antibody producing plants hepatitis virus: changing pattern of human disease production of hepatitis b surface antigen in transgenic plants for oral immunization repopulation with iga-containing cells of bronchial and intestinal lamina propria after transfer of homologous peyer's patch and bronchial lymphocytes stable genetic transformation of tomato plastids and expression of a foreign protein in fruit plant-made vaccines for humans and animals production of dengue envelope domain iii in plant using tmv-based vector system vaccine antigen production in transgenic plants: strategies, gene constructs and perspectives oral immunization of mice with transgenic tomato fruit expressing respiratory syncytial virus-f protein induces a systemic immune response plant viral genes in dna idiotypic vaccines activate linked cd (+) t-cell mediated immunity against b-cell malignancies clinical evaluation in healthy adults of a hepatitis b vaccine made by recombinant dna edible vaccine: a better way of immunization immunogenicity of a novel, bivalent, plant-based oral vaccine against hepatitis b and human immunodefi ciency viruses plant-derived hemagglutinin protects ferrets against challenge infection with the a/indonesia/ / strain of avian infl uenza agrobiotechnology. mixed message could prove costly for gm crops high-frequency plastid transformation in tobacco by selection for a chimeric aaada gene immunogenicity in humans of a recombinant bacterial antigen delivered in a transgenic potato human immune responses to a novel norwalk virus vaccine delivered in transgenic potatoes a rice-based edible vaccine expressing multiple t cell epitopes induces oral tolerance for inhibition of th -mediated ige responses recent developments in the use of transgenic plants for the production of human therapeutics and biopharmaceuticals immunogenicity of transgenic plant-derived hepatitis b surface antigen new advances in the production of edible plant vaccines: chloroplast expression of a tetanus vaccine antigen tetc tobacco, a highly effi cient green bioreactor for production of therapeutic proteins report on the global hiv/aids epidemic expression of an animal virus antigenic site on the surface of a plant virus particle antigen engineering in yeast: synthesis and assembly of hybrid hepatitis b surface antigenherpes simplex gd particles new strategies for using mucosal vaccination to achieve more effective immunisation plants for delivery of edible vaccines plant cell factories and mucosal vaccines oral immunization with a combination of plasmodium yoelii merozoite surface proteins and / enhances protection against lethal malaria challenge oral immunogenicity of human papillomavirus-like particles expressed in potato successful boosting of a dna measles immunization with an oral plant-derived measles virus vaccine oral immunization with rotavirus vp expressed in transgenic potatoes induced high titers of mucosal neutralizing iga novel approaches to oral vaccines: delivery of antigens by edible plants a plant-based multicomponent vaccine protects mice from enteric diseases recent progress in the development of plant derived vaccine antigens produced in plants by infection with chimeric plant viruses immunize against rabies virus and hiv- antibodies can eradicate cancer micrometastasis examination of the cytoplasmic dna in male reproductive cells to determine the potential for cytoplasmic inheritance in angiosperm species acknowledgements dr. anwar shahzad gratefully acknowledges the fi nancial support provided by ugc and up-cst in the form of research projects (vide no. - / sr and vide no. cst/ d respectively). dr. aastha sahai is also thankful to csir, new delhi, for the award of senior research fellowship for providing research assistance. key: cord- - ggaqf authors: nan title: abstracts of the papers presented in the xix national conference of indian virological society, “recent trends in viral disease problems and management”, on – march, , at s.v. university, tirupati, andhra pradesh date: - - journal: indian j virol doi: . /s - - - sha: doc_id: cord_uid: ggaqf nan patients showed rashes on face, hand and foot. ev detection carried out in vesicular fluid, stool, serum and throat swab specimens by rt-pcr of ncr gene. serotyping was carried out by using rt-pcr of viral protein of vp / a junction region followed by sequencing and phylogenetic analysis using neighbor-joining-algorithm and kimura- parameter model of mega- software. overall ev positivity detected in hfmd patients from kerala, tamil nadu, west bengal and orissa states was found to be . %, . %, . % and . % respectively. typing of vp gene sequences indicated presence of ca- , ev- , echo- strains in kerala and ca- in west bengal, orissa and tamil nadu. phylogenetic analysis indicated ca- , ev- , echo- strains showed . - . % and - . % homology with japanese, australian and french strains. however, ca- strains were closer to malaysian strains with . - . % nucleotide homology. the present study documents the association of multiple types of ev's i.e., ca- , ev- , echo- and ca- strains contributing as prime viral pathogens in hfmd epidemics in the reported regions with new emergence of ca- circulating strain in kerala, india. tasgaon september . sera were collected from suspected hepatitis cases and there contacts and tested for anti hev igm/igg antibodies (elisa) and liver enzymes like alanine aminotransferase (alt). anti hev igm antibodies were detected in . % ( / ) of the suspected cases. the overall attack rate was . %. male to female ratio was : . majority ( . %) of the cases were in the age group - years and recovered without any clinical complications. weekly distribution of cases showed that the majority ( . %, / ) cases occurred between nd and rd week of june. dark urine ( . %), jaundice ( . %), fatigue ( . %), abdominal pain ( . %), anorexia ( . %), vomiting ( . %), fever ( . %), giddiness ( . %), diarrhoea ( . %) and arthalgia ( . %) were the prominent symptoms. sera collected from antenatal cases (ancs) showed anti hev igm antibody in . affected pregnant women had a normal outcome. a death of year, male hepatitis e case was reported during the outbreak period that had cirrhosis of liver with oesophageal varices. sanitary survey revealed that water pipelines were laid down in close proximity of sewerage system, and water posts were without tap. these are the likely sources of faecal contamination of water supplies. among water samples collected from various places, were found to be unfit for drinking based on the routine bacteriological tests conducted at state public health laboratory, pune. no case occurred after the pipelines were repaired. this typical outbreak of hepatitis e re-emphasizes need for proper water supply/sewage disposal pipelines and adequate maintenance measures. jayanthi shastri, nilima vaidya, sandhya sawant, umesh aigal department of molecular biology, kasturba hospital for infectious diseases, mumbai, india dengue and dengue haemorrhagic fever are amongst the most important challenges in tropical diseases due to their expanding geographic distribution, increasing outbreak frequency, hyperendemicity and evolution of virulence. the gobal prevalence of dengue has grown dramatically in recent decades. who estimates - million cases of dengue virus infections worldwide every year resulting in , to , cases of dhf and , deaths each year. public health laboratories require rapid diagnosis of dengue outbreaks for application of measures such as vector control. laboratory diagnosis of dengue virus infection can be made by the detection of specific virus, viral antigen, genomic sequence and/or antibodies. currently basic methods used by laboratories for diagnosis of dengue virus infection are virus isolation and characterisation, detection of genomic sequence by nucleic acid amplification technology assay and detection of dengue virus specific antibodies/antigen. molecular diagnosis based on reverse transcription (rt)-pcr s.a. one step or nested pcr, nucleic acid sequence based amplification (nasba), or real time rt-pcr, has gradually replaced the virus isolation method as the new standard for the detection of dengue virus in acute phase serum samples. several pcr protocols for detection have been described that vary in the extraction method, genomic location of primers, specificity, sensitivity and the methods to determine the products and the serotype. pcr-based dengue tests, due to the specificity of amplification, enable a definitive diagnosis and serotyping of the virus. in addition dna sequencing of the amplification product enables the virus to be genotyped, providing important information on the sources of infection. more recently tests have incorporated flurogenic probe, so called taq man technology for the specific real time detection of dengue - amplicons. product is detected by a specific oligodeoxy nucleotide probe that is labelled with carboxy-fluorescein (fam). this technology offers the advantage of being both rapid and potentially quantitative. second, the detection of product by hybridisation of flurochrome labelled probes increases specificity. third, as the product is detected without the need to open the reaction tube, the risk of contamination by product carry over is minimised. the advantages of speed, contamination minimisation and reduced turn around time justify application of this assay over the currently used nested pcr assay. during the period january to october , molecular laboratory received samples from patients presenting with acute onset fever for dengue . %) samples were tested positive by this method. the disease peaks in the monsoon season with a percentage of . %. rapid tests, igm and igg capture elisa are popularly used tests for diagnosis of dengue infection. its utility is limited for diagnosing dengue in convalescecce ( - days) . specificity is also compromised due to infections with flaviviruses: japanese encephalitis and chikungunya. dengue ns ag elisa with its cost effectiveness, specificity and sensitivity should be considered as the test of choice for diagnosing dengue in the acute phase of illness in the developing countries. molecular diagnosis enables confirmatory diagnosis of dengue in the acute phase of the illness and is suitable for further typing methods. assistant general manager and r&d coordinator, division of quality control and r&d, bharat immunologicals and biologicals corporation ltd., village chola, bulandshahr, up vaccine development in india, though slow to start, has progressed by leaps and bounds in the past years. it was dependent on imported vaccines but now it is not only self-sufficient in the production of vaccines conforming to international standards with major supplier of the same to unicef. the role of drug authorities is to enhance the public health by assuring the availability of safe and effective a indian j. virol. (september ) (suppl. ):a -a vaccines, allergenic extracts, and other related products. vaccine development is tightly regulated by a hierarchy of regulatory bodies. guidelines provided by the indian council of medical research (icmr) set the rules of conduct for clinical trials from phase i to iv studies as well as studies on combination vaccines. these guidelines address ethical issues that arise during a vaccine study. a network of adverse drug reaction (adr) monitoring centers along with the adverse events following immunization (aefi) monitoring program provide the machinery for vaccine pharmacovigilance. genetic modifications have been developed to develop effective and cheaper vaccines by the use of recombinant technology. to ensure safety of consumers, producers, experimental animals and environment, governments all over the world are following regulatory mechanisms and guidelines for genetically modified products. as with other industrializing countries undergoing rapid shifts, india clearly recognizes the need to restructure its regulatory system so that its biopharmaceutical industry can compete in international markets. genetic engineering approval council (geac), recombinant dna advisory committee (rdac), review committee on genetic manipulation (rcgm), institutional biosafety committees (ibsc) are responsible for development, commitment for parameters and commercialization of recombinant vaccines. to centralize and coordinate the whole system, government has taken to form two agencies to regulate the regulation laws to develop recombinant pharmaceuticals products including vaccines. the first is the creation of the national biotechnology regulatory authority (nbra), under the department of biotechnology (dbt), as part of india's long-term biotech sector development strategy. the second major initiative will affect the entire indian pharmaceutical industry. this is the replacement of most state, district, and central drug regulatory agencies with a single, central, fda-style agency, the central drug authority (cda). the cda is expected to have separate, semi-autonomous departments for regulation, enforcement, legal, and consumer affairs; biotechnology products; pharmacovigilance and drugs safety; medical devices and diagnostics; imports; quality control; and traditional indian medicines. it will set up offices throughout india and will be paid for inspection, registration, and license fees. its enforcement powers will be strengthened by a new law increasing the criminal penalties for illegal clinical trials. in the manufacturing area, though, the country has been tightening the rules and enforcement. an amendment to the regulations, ''schedule m'' of the drug and cosmetics act, now specifies the good manufacturing practice (gmp) requirements for factory premises and materials. these requirements were modeled after us fda regulations, to improve regulatory coordination between indian and us regulators. india has realized the importance of regulations in pharmaceutical specially in vaccine field but it will take several years to implementation of these. india has coordinated some of its regulatory functions with western organizations. the us pharmacopoeia established an office in hyderabad in . a representative of the indian pharmaceutical lobby also recently has expressed openness to an expansion of the fda's oversight of indian manufacturing. as india expands its global drug and biologicals production, us and europe, as the world's largest drug importers, will likely expand their regulatory support in the development of the country's regulatory systems. rapid diagnosis of japanese encephalitis virus (jev) infections is important for timely clinical management and epidemiological control in areas where multiple flaviviruses are endemic. however, the speed and accuracy of diagnosis must be balanced against test cost and availability, especially in developing countries. an antigen capture enzyme-linked immunosorbent assay (elisa) for detection of circulating jev specific nonstructural protein (ns ) was developed by using monoclonal antibodies (mabs) specific to recombinant (ns ). the applicability of this jev ns antigen capture elisa for early clinical diagnosis was evaluated with acute phase serum/ cerebrospinal fluid (csf) specimens collected from different epidemics during [ ] [ ] [ ] . jev ns antigen was detected in circulation from day to . the sensitivity and specificity of jev ns detection in serum/csf specimens with reference to reverse transcriptase pcr was %, and . % respectively. no crossreactions with any of the other closely related members of the genus flaviviruses (dengue, westnile, yellow fever and saint louis encephalitis (sle) viruses) were observed when tested with either clinical specimens or virus cultures. these findings suggested that the reported jev specific mab-based ns antigen capture elisa will be a rapid and reliable tool for early confirmatory diagnosis as well as surveillance of je infections in developing countries. manmohan parida the recent emergence of a novel human influenza a virus (h n ) poses a serious global health threat. the h n virus has caused a considerable number of deaths within a short duration since its emergence. a two-step single tube accelerated rapid real-time and quantitative swine flu virus specific h rtlamp assay is reported by targeting the h gene of the novel h n hybrid virus. the feasibility of swine flu h rtlamp for clinical diagnosis was validated with a panel of suspected throat wash samples comprising confirmed positive and confirmed negative cases of ongoing epidemic. the comparative evaluation of h specific rtlamp assay with real-time rt-pcr demonstrated exceptionally higher sensitivity by picking up all the h n positive and additional positive cases amongst the negatives that were sequence confirmed as h n . none of the real-time rtpcr positive samples were missed by rtlamp system. the comparative study revealed that rtlamp was -fold more sensitive than rtpcr with a detection limit of copy number. these findings suggested that rtlamp assay is a valuable tool for rapid, real-time detection as well as quantification of h n virus in acute phase throat swab samples without requiring any sophisticated equipments. because of its recurrent nature. despite considerable progress in understanding of the virus at cellular and molecular levels, the proper management of the disease in its different stages is still a dilemma particularly whether to use antiviral or steroids or both. the risk of using steroids with its attendant complications has to be weighed against the risk of progression of the disease if avoiding the use of steroids. this dilemma can be reduced to a considerable extent if basic principles of virology and pathogenesis are kept in mind. this article reviews current concepts of virological and clinical aspects of hsv keratitis to enable a broad understanding of the disease process. it is recognized several influential host factors including the fact that hsk is more common in men than women. it is observed that the ability of hsv to establish latent infection in sensory neurons and possibly cornea, but have as yet been unable to use this knowledge to prevent the disease limitations. acknowledging limitations may further stimulate application of laboratory knowledge in coping with hsk which constitutes to present major challenge in terms of management. mvo- study on effect of human bhsp in immunity of hcv core protein and hbv hbsag there are more than million individuals with hepatitis b and c in the world. in spite of vaccination in the different areas there are several reports about patients who got vaccine before. also there is not efficient vaccine against of hepatitis c and one of the important problems in vaccine project is development of effective and suitable adjuvant in human vaccines. at present research we applied human bhsp protein as adjuvant and chaperon. this protein injected to balbc mice as adjuvant together with recombinant proteins of hcv core and hbv hbsag. then humoral and cellular immune systems of the mice were studied. core and hbsag genes were cloned into petduet- vector and thermal vector of pgp - was used for human heat shock protein expressions. the different combination of these three proteins was injected to mice and we evaluated the total igg and igg a of mice serums after a week. two weeks after booster injection, we studied the proliferation and cytokine secretion of spleen, inguinal and popliteal lymph nodes lymphocytes in vitro and ex vivo conditions. so the core/hbsag + hsp and core + hbsag + hsp complexes induced total igg and igg a secretion. the spleen lymphocytes proliferation were increased equal to serum igg a level that was constant in second time bleeding with significant different to complexes with freund's adjuvant. at first il- and il- cytokines were increased and then decrease of il- meaned no hypersensitivity. the chaperon effect of hsp on structure of core and hbsag proteins was studied by cd and flourometer. it could fold the proteins after heating and unfolding. hepatitis b virus (hbv) infection is vaccine preventable global public health problem. all commercially available vaccines contain one or more of the recombinant hepatitis b envelope protein or surface antigen (hbsag). measurement of antigen responsible for immunogenicity of vaccine is central to quality assessment. the problems associated with the use of a polyclonal antibody in an assay with regard to its poorly defined nature and batch-to-batch variation has been mitigated by the use of mabs as described in this paper. the initial capture of hbsag by the mab could orientate it such that the same antibody could bind to it as a detection antibody after labeling with out steric hindrance. the development of an immuno-capture elisa (ic-elisa) to measure the hbsag content using a monoclonal antibody (mab) specific to determinant ''a'' of hbsag in the experimental vaccine formulations is being discussed. murine mabs developed against hbsag, subtype adw were found to cross-react with the other subtypes viz. ad and ay too. the mabs have been characterized following which, one mab hbs was chosen for developing ic-elisa format for the quantification of the hbsag in the final algel adsorbed vaccines. the unadsorbed hbsag was used to establish the standard curve of hbsag/a. the elisa had a sensitivity of ng/ml of hbsag. the recovery rate of hbsag/a was found to be around % in the vaccines treated to desorb the antigen from algel. twenty seven experimental batches of monovalent hepatitis b vaccines were analyzed for the hbsag content, both by ic-elisa and a commercial kit (axsym kit, abbott laboratories, usa). the statistical analysis of ic-elisa results indicated that an experimental equation f(x) = . (x) + . , could precisely estimate the amount of hbsag in the adsorbed vaccines. the amounts of hbsag recovered from the adsorbed vaccines as estimated by the ic-elisa format had a good correlation with the estimates derived from a commercial kit, which is being used by several vaccine manufacturers in india for the quality control of vaccine antigen. the varying amounts of vaccine antigens that could be recovered seemed to depend on the quality of the hbsag and the methods of hbsag adsorption to the alum gel during vaccine manufacture. epidemiology of the spread of h n virus. children of school going age have become victim of this deadly virus as evident from the reporting data generated in the past few weeks. the mortality rate has also been slightly increased. the disease spread in wave pattern and presently the world is passing through the second wave of pandemic with more severity in young and otherwise health people with a predilection for lungs leading to viral pneumonia and respiratory failure. now the pandemic gained hold in the developing world affecting more severely as millions of people live under deprived conditions having multiple health problems, with little access to basic health care. current data about the pandemic from developed counties need to be very closely watched in relation to shift in virus sub type, shift of the highest death rate to younger populations, successive pandemic waves, higher transmissibility than seasonal influenza, and demographic differences etc. presently the world appears to be better prepared. vaccine is available in market in many countries. even vaccine trials are actively going on in indian population. effective antivirals are available. although till now h n diagnostic centers worked with cdc/who recommended h n specific primer, probes with taqman chemistry by real time pcr, efforts on the development of indigenous diagnostics, vaccines and chemoprophylaxis is going on to have a better combat against this highly infectious virus. were positive for rotavirus infection by either page or elisa methods. the available data highlights the importance of rotavirus as a cause of diarrhea in children, which is severe enough to deserve specialized care. the observed proportion of . % of all diarrhea cases being associated with rotavirus falls within the range of values reported by other workers. the reported positivity varies from . to . %. in our study a complete concordance of elisa and page results were observed in ( %) of the tested specimens. this finding closely correlates with the findings of other authors who found a . - . % concordance results between elisa and page methods. some authors found rna-page method that is as sensitive and rapid as elisa for detecting rotavirus in stool samples of cases of diarrhea and some others proposed elisa is more sensitive than page method fond to be % specific. the remaining ( %) samples showed conflicting results. in a lone sample in which the od value of elisa test was . , this value was almost at the cutoff level, the possibility of this sample being positive by elisa test is doubtful. negative result of the same sample in page method is difficult to explain, the possibility of presence of lot of empty virus particles or due to low concentration of viral rna in the fecal specimen and insufficient extraction of viral rna could be possible. on the other hand, of the samples which gave positive results by page method were negative by elisa test. these samples had a typical - - - rna pattern. the reason for their being elisa negative thus remains unexplained, however blocking factors or the presence of inhibitory substance in stools might have been responsible. the samples containing predominantly complete particles can also give false negative results. since, the group antigen is not exposed. earlier studies have also reported page to be the most sensitive technique although some are of view that it is laborious procedure. how ever, the page system used in this study was very simple to perform and the results were available on the same day. the main requirement was of trained personnel and proper standardization of the technique. most reports states that the greatest advantage of page and silver stain method are its lack of ambiguity and the fact that it provides information about viral electropherotypes. the modified page system was thus found to be reliable, simple and rapid, no expensive reagents were required. locally available reagents from hi media were used. the cost of the chemical for page per specimen was rs. approximately as compared to rs. per test by confirmatory elisa. a locally produced slab gel electrophoresis system with power pack was the only equipment required. this method could be used for the routine diagnosis of rotavirus infection in the laboratory. vaccine, rapid diagnosis plays an important role in early management of patients. in this study a qc-rt-pcr assay was developed to quantify chikungunya virus rna by targeting the conserved region of e gene. a competitor molecule containing an internal insertion was generated, that provided a stringent control of the quantification process. the introduction of -fold serially diluted competitor in each reaction was further used to determine sensitivity. the applicability of this assay for quantification of chikungunya virus rna was evaluated with human clinical samples and the results were compared with real-time quantitative rt-pcr. the sensitivity of this assay was estimated to be rna copies per reaction with a dynamic detection range of to copies. specificity was confirmed using closely related alpha and flaviviruses. the comparison of qc-rt-pcr result with real-time rt-pcr revealed % concordance. these findings demonstrated that the reported assay is convenient, sensitive and accurate method and has the potential usefulness for clinical diagnosis due to simultaneous detection and quantification of chikungunya virus in acute-phase serum samples. in india, measles vaccine was introduced as part of expanded programme of immunization in . measles, mumps and rubella (mmr) vaccine is still not part of the national immunization schedule of india. the indian association of paediatrics (iap) recommends measles vaccine at months of age and mmr vaccine at - months. however, in a recent policy update, iap committee on immunisation opined that there is a need for a second dose of mmr vaccine for providing adequate immunity against mmr. the aim of the present study was to assess the extent of sero-protection against mmr at - years of age in children who have received one dose of mmr between and months of age. an attempt has also been made to assess the sero-response to the second dose of mmr vaccine in - years old children. a total of consecutive children between the ages of - years who had received mmr vaccine between and months of age and attending the immunization clinic of gtb hospital, delhi were enrolled. the vaccination status, anthropometry and physical examination findings were recorded. three ml of venous sample was again withdrawn for estimation of post vaccination antibody titre. it was observed that . %, . % and . % children were seroprotected for mmr respectively after . - . year of receiving first dose of mmr vaccine. seroprotection rose to . %, % and % for mmr respectively after - weeks of receiving second dose of mmr vaccine. geometric mean concentration of antibody also rose significantly in all three diseases. in view of low seroprevalence of mmr and hence high susceptibility to infection at - years of age, who have already received mmr vaccine, there is need to boost the immune responses against these three diseases by giving a second dose of mmr vaccine. baseline information on the epidemiology of viral agents causing stis and types of risk behaviour of affected persons are essential for any meaningful targeted intervention. the present study documents the pattern of viral stis in patients attending a tertiary care hospital, correlating the syndromic approach and the laboratory investigations to determine the aetiology. three hundred consecutive patients attending the sti clinic were diagnosed and categorized according to the syndromic approach of the who along with detailed history and demographic data. majority of the patients were men ( . %) with a mean age of years. men received education up to middle school. half of the female subjects were illiterate. sixty percent of the patients were married and among these, % were regular condom users. first sexual contact at or before years of age was more in men ( % vs. . % in women). promiscuity was more among male patients who had contact with csw. genital herpes was the commonest viral sti ( / ) followed by genital wart ( / ). concomitant infection with more than one virus was seen in % of patients. hiv was prevalent in . % of sti patients. hepatitis b, hepatitis c, herpes simplex type and molluscum contagiosum were the other viral agents seen in sti clinic attendees at our centre. this disease currently prevalent in more than countries world wide and annually - million people are infected with dengue virus among which . - lakhs cases were dengue hemorrhagic fever (dhf) and dengue shock syndrome (dss) which are serious forms of dengue virus infection and due to this condition , deaths might occur annually world wide and approximately million children were hospitalized for the fast decades. this disease is characterized by sudden onset of high fever with sever headache, pain in the back and limbs, lymphadenopathy macuolo-papulur rash over the skin and retro-bulbar pain. early diagnosis can be established with simple and rapid lgg/ gm antibodies detection in the blood samples of the patients based on the bi-directional immunoassay system for its management and control to reduce morbidity and mortality. details will be presented. myocarditis and dilated cardiomyopathy (dcm) are common causes of morbidity and mortality both in children and adults. the most common viruses involved in myocarditis are coxsackievirus b or adenovirus. recently, the coxsackievirus and adenovirus receptor (car), a common receptor for coxsackieviruses b , b and adenoviruses , has been identified. increased expression of car has been reported in patients with dcm suggesting utilization of car by these viruses for cell entry. the present study was designed to study the expression of car in myocardial tissue of patients with dcm. formalin fixed myocardial tissues were obtained from autopsy cases. a total of cases of dcm and cases of controls which included non-cardiac (group-a) and cardiac disease other than dcm (group-b) were included in the study. expression of car was studied by immunohistochemical staining of myocardial tissue using car specific rabbit polyclonal antibody and biotin conjugated secondary antibody. the tissue sections were considered positive when[ % of the cell showed brown color staining by immunohistochemistry (ihc). the car positivity in dcm cases was found to be % ( / ) as compared to % in control group a and % in control group b respectively. the car positivity was significantly higher in the test group as compared to both the control groups. further car positivity in all the cellular types (myocytes, endothelial cells and interstitial cells) was found significantly higher in test group as compared to both the control groups. the expression of car was significantly higher in myocytes as compared to both endothelial and interstitial cells in all the groups. however, no significant difference was observed in car positivity between endothelial and interstitial cells. the present study highlights the increased expression of car in dcm cases with further significance of car expression in myocytes and endothelial cells. this may help further in understanding the tropism of viruses or cellular susceptibility, which in turn will help in appropriate diagnostic and therapeutic approach in management of viral myocarditis and dcm cases. food security and safety vary widely around the world, and reaching these goals is one of the major challenges, raising public concern for the wellbeing of mankind, in particular. industrialized production and processing as well as improper environmental protection have clearly shown severe limitations such as worldwide contamination of the food chain and water. contaminated water and food during the processes of production, processing and handling are essentially responsible for food and water borne viral infections/diseases. the cases of viral food borne outbreaks are on the rise, creating a threat to human health. recent researches indicate that epidemiological studies are meager to focus the frequently contaminated foods and food borne viral diseases. current paper projects the etiology of select food borne viral diseases, probable reasons for non availability of appropriate methods to detect the viruses responsible for the diseases, routes of water and food borne transmission of enteric viral infections, currently available methods of detection of select viruses and bio safety measures to prevent food borne viral infections. dietary/nutritional management in food borne viral diseases is crucial to control weakness and gastro enteric intolerance due to disease condition and antibiotic therapy. it will principally improve food intake, resulting in better nutritional status leading to optimum immune response. food borne viruses are mainly belong to rotaviruses, enteropathogenic viruses, astroviruses, adenoviruses and caliciviruses, causes acute gastroenteritis (ag) which is an important health problem. the frequency of rotavirus as a cause of sporadic cases of ag ranges between . % and . %. astroviruses cause ag, with a frequency ranging between and %: outbreaks have been described in schools and kindergartens, but also in adults and the elderly. the frequency of identification of adenoviruses and as causes of sporadic ag in non-immuno suppressed children ranges between . % and . %, although there is probably underreporting because the sensitivity of conventional techniques is low. caliciviruses are separated phylogenetically into two genera: norovirus and sapovirus. norovirus is frequently associated with food-and water-borne outbreaks of ag. it is estimated that % of cases of ag due to norovirus are food borne. in sweden and some regions of the united states, norovirus is the first cause of outbreaks of food borne diseases. sapovirus outbreaks due to person-to-person and food borne transmission affecting both children and adults have recently been reported in countries such as canada and japan. it has been predicted that the importance of diarrhoeal disease, mainly due to contaminated food and water, as a cause of death will decline worldwide. evidence for such a downward trend is limited. this prediction presumes that improvements in the production and retail of microbiologically safe food will be sustained in the developed world and, moreover, will be rolled out to those countries of the developing world increasingly producing food for a global market. sustaining food safety standards will depend on constant vigilance maintained by monitoring and surveillance but, with the rising importance of other food-related issues, such as food security, obesity and climate change, competition for resources in the future to enable this may be fierce. in addition the pathogen populations relevant to food safety are not static. food is an excellent vehicle by which many pathogens (bacteria, viruses/prions and parasites) can reach an appropriate colonization site in a new host. although food production practices change, the well-recognized food-borne pathogens, such as salmonella spp. and escherichia coli, seem able to evolve to exploit novel opportunities, for example fresh produce and even generate new public health challenges, for example antimicrobial resistance. in addition, previously unknown food-borne pathogens, many of which are zoonotic, are constantly emerging. awareness and surveillance of viral food-borne pathogens is generally poor but emphasis is placed on norovirus, hepatitis a, rotaviruses and newly emerging viruses such as sars. it is clear that one overall challenge is the generation and maintenance of constructive dialogue and collaboration between public health, veterinary and food safety experts, bringing together multidisciplinary skills and multi-pathogen expertise. such collaboration is essential to monitor changing trends in the well-recognized diseases and detect emerging pathogens. it is also necessary to understand the multiple interactions between these pathogens and their environments during transmission along the food chain in order to develop effective prevention and control strategies. to analyse the effectiveness of these sirnas targeting rabies virus l gene, the bhk- cells expressing sirnas in shrna form were produced by transduction of cells with radv-l. the transduced bhk- cells expressing sirna were infected with rabies virus pv- strain. there was reduction in rabies virus multiplication as analysed by reduction in fluorescent foci forming unit (ffu) count by . % ( ffu in bhk- cells expressing sirna-l compared to ffu in bhk- cells expressing negative sirna). the expression of l gene mrna was reduced by . fold in rabies virus infected radv-l transduced cells compared to radv-neg transduced cells (negative control) as detected using real-time pcr. after analyzing the effectiveness of radv-l in vitro, its effectiveness was also evaluated in vivo in mice after virulent rabies challenge. the mice were inoculated with plaque forming units (pfu) of radv-l in masseter muscle (i/m route) and challenged with ld rabies virus challenge virus standard (cvs) strain. the results indicated % protection with improved median survival from to days compared with group of mice treated with radv-neg. the results of this study indicated that sirnas targeting rabies virus polymerase (l) gene delivered through adenoviral vector inhibited rabies virus multiplication in vitro and in vivo. and were successfully produced and purified from the infected spodoptera frugiperda (sf- ) cells using these recombinant baculovirus. the morphology of the vlps was validated by electron microscopy in comparison to the authentic bt virions. the vlps produced here were stable and were highly immunogenic with intact outer layer which is rapidly lost during normal infection of btv. these btv-vlps elicited long lasting protective immunity in vaccinated sheep against virulent virus challenge. with the use of btv-vlps it was also possible to differentiate the infected and vaccinated animals (diva). vlp-based btv vaccine has potential advantages with regard to controlling the spread of btv with multiple serotypes. it is possible to produce milligram quantities of correctly folded and processed protein complexes using this baculovirus expression system and hence it is a more promising system for producing new generation vaccines like vlp subunit vaccine against any viral diseases in large scale. peste des petits ruminants (ppr), goatpox and orf are oie notifiable diseases of small ruminants especially goat and sheep. these diseases are economically important, in enzootic countries like india and cause significant loss and are major constraints in the productivity. considering the geographical distribution of ppr, goat pox and orf infections and prevalence of mixed infection, in the present study, safety and potency of the experimental triple vaccine comprising attenuated strains of thermostable-ppr virus (pprv jhansi, p- ) grown at °c, high passaged goat poxvirus (gtpv uttarkashi, p ) and attenuated orf virus (orfv mukteswar, p ) was evaluated in sub-himalayan local hill goats. goats simultaneously immunized with ml of vaccine consisting of either tcid or tcid of each of pprv, gtpv and orfv were monitored for clinical and serological responses for a period of - weeks post-immunization (pi) and post challenge (pc). specific immune responses i.e., antibodies directed to pprv, gtpv and orfv could be demonstrated by ppr competitive elisa kit and capripox indirect elisa, snt, respectively following immunization. all the immunized animals resisted infections when challenged with virulent strains of either gtpv or pprv or orfv on day dpi, while in contact control animals developed characteristic signs of respective disease. further, ppr viral antigen could be detected by using ppr sandwich elisa kit in the excretions (nasal, ocular and oral swab materials) of unvaccinated control animals after challenge but not from any of the immunized goats. triple vaccine was found safe at dose as higher as tcid and induced protective immune response even at lower dose ( tcid ) in goats, which was evident from sero-conversion as well as challenge studies. the study indicated that these viruses are compatible and did not interfere with each other in eliciting immune response, paving the feasibility of use of this triple vaccine in combating these infections simultaneously. toll like receptors (tlrs), primary sensors of microbial origin, plays a crucial role in the innate immunity. till now mammalian tlrs have been identified, while there is no information available on tlrs of yak. this study is part of world bank funded-icar project. yak, named bos grunniens for its distinctive vocalization and relationship with cattle, is natural habitant of extremely cold environment. when these animals comes to a lower altitude grazing land, adjacent to villages, become susceptible to the diseases of cattle, buffalo etc. thus, present study was undertaken to with genetic characterization and evolutionary lineage analysis of yak tlrs. we worked on tlr gene, which plays an important role in recognition of ssrna viruses. total rna was extracted from mitogen stimulated pbmcs of yak. the rt-pcr conditions were standardized for full length amplification of tlr gene using specific self designed primers. the expected amplicon of bps was obtained. it was cloned in pgemt-easy vector followed by transformation in e. coli top strain. the recombinant clones were screened, picked up for plasmid isolation and release of tlr was confirmed by restriction digestion. the cloned tlr product was sequenced and analyzed for the nucleotide and deduced amino acid sequences, and d structure analysis. the results revealed that yak shows more than % sequence homology with other bos indicus breeds and bos taurus breeds. however, identity was less than % with other animal species (equine, murine, feline, canine etc.). the evolutionary lineage findings cluster yak more closely with bovine species. point mutations revealed changes at nucleotide positions with corresponding amino acid change at positions. smart analysis of yak protein domain architecture revealed toll-interleukin i receptor (tir), leucine rich repeats (lrr) and signal peptide region. the variations in yak mainly lie in the lrr region. homology modeling revealed horse shoe shaped structure with alpha helix. the additional alpha helix present in bos indicus was not detected in yak. the present study shows existence of genetic variability in tlr gene of yak, in particular the lrr region, which plays an important role in the pathogen recognition and the evolutionary lineage analyses shows its closeness with other bovine species. a.p. aquaculture and fisheries, tirupati in this new millennium, aquatic animal health management strategies in asia expanded and adjusted to the current disease problems faced by the aquaculture sector. this presentation will briefly discuss some of the most serious trans-boundary pathogens affecting asian aquaculture including a newly emerging disease and highlight recent regional and national efforts on responsible health management for mitigating the risks associated with aquatic animal movement. a regional approach is fundamental since many countries share common social, economic, industrial, environmental, biological and geographical characteristics. capacity and awareness building on aquatic animal epidemiology, science-based risk analysis for aquatic animal transfers, surveillance and disease reporting, disease zoning and establishment of aquatic animal health information systems to support development of national disease control programs and emergency response to disease outbreaks are needed. molecular diagnostics with emphasis towards standardization and harmonization, inter-calibration exercises and quality assurance in laboratories, accreditation program and utilization of regional resource centres on aquatic animal health will also be needed. whilst most of these strategies are directed in support of government policies, implementation will require pro-active involvement, effective cooperation and strategic networking between governments, farmers, researchers, scientists, development and aid agencies, and relevant private sector stakeholders at all levels. their contributions are essential to the health management process. generally, aquaculture plays an important role in economy as harvests from natural waters have declined or, at best, remained static in most countries. fish and shrimp, the main aquaculture product sources, have gained the most attention. many factors can cause losses in yields of fish products and infectious disease in fish and shrimp is the biggest threat to the fishery industry. shrimp and fish aquaculture has grown rapidly over several decades to become a major global industry that serves the increasing consumer demand for seafood and has contributed significantly to socio-economic development in many poor coastal communities. however, the ecological disturbances and changes in patterns of trade associated with the development of shrimp and fish farming have presented many of the pre-conditions for the emergence and spread of disease. shrimp and fish are displaced from their natural environments, provided artificial or alternative feeds, stocked in high density, exposed to stress through changes in water quality and are transported nationally and internationally, either live or as frozen product. these practices have provided opportunities for increased pathogenicity of existing infections, exposure to new pathogens, and the rapid transmission and trans boundary spread of disease. not surprisingly, a succession of new viral diseases has devastated the production and livelihoods of farmers and their sustaining communities. this review examines the major viral pathogens of farmed shrimp and fish, the likely reasons for their emergence and spread, and the consequences for the structure and operation of the shrimp farming industry. in addition, this review discusses the health management strategies that have been introduced to combat the major pathogens and the reasons that disease continues to have an impact, particularly on poor, smallholder farmers in asia. btv isolates from the same geographic region have been termed as 'topotypes' and initial observation on segment nucleotide sequences identified a correlation between topotypes and genetic information. later topotyping was proposed based on segment , on the premise that the encoding protein ns , which is involved in virus egress from insect cells, would lead to evolutionary fitness in parallel with the geographic distribution of the different culicoides species. further studies attempted to extend this to nucleotide sequence homology in segments and , but failed to identify clear cut correlations or any evidence for positive selection. for example, south african isolates were found not to cluster into separate african lineage. in this study, we carried out a more extensive analysis of segment sequences. our analysis showed no segregation of isolates into topographically distinct groups. instead we observed topological clustering of the clades, and we attribute this to genetic bottleneck resulting in genetic drift and founder effect leading to homogenous gene pool in a geographical area. we hypothesize that when a new virus enters a geographical area where local btv strains are already circulating, the new genes/segments would enter into a bigger gene pool. consequently, the newer incursions into a heavily endemic area tend to get diluted and disappear from the population because the rate of drift is inversely proportional to the population size, unless they are positively selected. use of live attenuated vaccine in israel, europe, south africa and usa also led to more homogenous population similar to the vaccine strains due to continuous infusion of the vaccine type genes into the gene pool. we conclude that restriction of specific strains to certain geographical areas could generate uniquely imprinted genotypes which would not only indicate origin but also predict movement of viral strains to new areas. vvo- viral diseases of zoonotic importance: indian context k. prabhudas pd-admas, ivri, campus, hebbal, bangalore zoonoses are generally defined as animal diseases that are transmissible to humans. they continue to represent an important health hazard in most parts of the world, where they cause considerable expenditure and losses for the health and agricultural sectors. the emergence of these zoonotic diseases are very distinct, hence their prevention and control will require unique strategies, apart from traditional approaches. such strategies require rebuilding a cadre of trained professionals of several medical and biologic sciences. the article discusses virus infections that have significant zoonotic implications for india. buffalopox is a contagious viral disease affecting milch buffaloes and rarely, cows, with a morbidity rate up to % in the affected herd. although the disease is not responsible for high mortality, it adversely affects the productivity of the animals, resulting in large economic losses. furthermore, the disease has zoonotic implications, as outbreaks are frequently associated with human infections, particularly in the milkers. the causative agent, buffalopox virus (bpxv), is closely related to vaccinia virus. the outbreaks of febrile rash illness among humans and buffaloes were investigated in the villages of districts solapur and kolhapur of western maharashtra. clinico-epidemiological investigations of humans and buffaloes were carried out and representative clinical samples were collected respectively. the samples include vesicular fluid, scab, and blood. laboratory investigations for buffalo-pox virus (bpxv) was done by pcr on blood samples, scabs and vesicular fluid. in vitro virus isolation attempts were carried out by using vero e- cells. negative staining electron microscopy was also employed for detection of virus particles. a total of human cases with pox lesions on hand and other body parts from village kasegaon, district-solapur and cases from different villages of kolhapur district were reported. besides pox lesions patients were having fever, malaise, pain at site of lesion and axillary and inguinal lymphadenopathy. in kasegaon village, attack rate in human cases was . % and in buffaloes . % ( / ). whereas in kolhapur area attack rate in buffaloes was . % ( / ). bpxv was confirmed in blood, vesicular fluid and scab specimens from human cases and scab specimen from buffalo by polymerase chain reaction (pcr) method. the bpxv was also isolated from different clinical specimens and further identified by pcr and electron microscopy. clinical manifestation of the disease in buffaloes from solapur district was as reported earlier like common pox lesions on teats and udders whereas the buffaloes from kolhapur district had lesions on hairless parts of ears and on the eyelids with purulent discharge. bpxv from human and buffalo cases showed similarity. vaccines have been made against several diseases and used for controlling the afflictions. however a few of them were not effective for successfully controlling the disease. the reasons for the failure are many, the major being, either the pathogen is not completely cleared from the vaccinated animal or it reemerges after changing its antigenic structure, thus making the vaccination programme less effective. in addition to this, emergences of newer diseases such as hiv the development of suitable vaccines have become a challenging task. this is especially true in the case of viral diseases. these challenges have warned the researchers ''that protection by vaccination is not that simple and strait forward approach'', and lot need to be understood in terms of host virus interaction and role of environment in perpetuating the disease. so the immediate step that was considered was the environmental safety by way using non infectious materials as vaccines. with the understanding that has been developed in molecular immunology and molecular biology and with the availability of molecular tools that have been developed through recombinant dna technology the field of vaccinology has changed dramatically to emerge as modern vaccinology. this presentation deals with the modern approaches that are being used to produce effective vaccines in the case of foot and mouth disease of cloven footed animals. the similar approach may be worked out for other viral diseases also. despite the availability of an inactivated vaccine that is noted to provide solid immunity against the disease over a short period of time, the search for an ideal vaccine, the criteria for which are; safety of the vaccine for environment, easy in its preparation, does not require a cold chain for its storage, provides longer lasting immunity, economically viable and may be able to clear the virus in case of persistent infection is on. the advent of recombinant dna technology together with the information available on the molecular biology of viruses has enabled to design the development of newer vaccines that can induce strong cellular and humoral responses. the underlying principal in the present vaccine development strategy world over is the virus antigen gene has to be expressed in the tissue and the vaccine backbone has to trigger the immune system for eliciting desired immune response. bangalore campus of ivri has been vigorously pursuing research to develop ideal vaccines for foot and mouth disease keeping above principal in mind to achieve the previously mentioned criteria. the approaches selected are to see that the virus antigen/s replicate transiently in the host. the self replicating vaccines that have been developed are pox virus vectored vaccines, alpha virus replicase based vaccines and fmdv vectored vaccines. the approach and the result obtained so far will be discussed. silkworm, bombyx mori is affected with various diseases caused by viruses viz., nuclearpolyhedrosis (bmnpv), densosnucleosis (bmdnv) and infectious flacherie (bmifv). silkworm viral diseases form major constraints for the silk cocoon production in all the sericultural countries. the losses due to silkworm diseases is estimated about - % and among them viral diseases are most common. in sericulture, prophylactic measures play a vital role in the management of silkworm diseases. these include disinfection of silkworm rearing house and appliances, rearing area, rearing surroundings, silkworm egg and body, and rearing bed disinfection associated with maintenance of general hygiene and personnel hygiene. all these activities are generally carried out as rituals by using general disinfectants often with partial success. recent trends in complete management of silkworm diseases include development of silkworm hybrids evolved from disease resistant/tolerant breeds, effective eco-and user-friendly disinfectants, anti-microbial feed-supplements and use of transgenic silkworms. biotechnological breakthrough in this regard is through rna interference (rnai) approach involving dsrna mediated nuclear polyhedrosis management and this is presently pursued by apssrdi, hindupur in collaboration with centre for dna fingerprinting and diagnostics (cdfd), hyderabad. nadu and karnataka. the disease appears to be more severe in rural flocks than organized farms. our investigations revealed the morbidity, mortality and case fatality rates among rural and organised farms as . %, . %, . % and . %, . %, . % respectively. higher morbidity and mortality in rural areas may be due to stress factors like poor nutrition, parasitic burden, fatigue due to long walks and non availability of veterinary aid. kulkarni et al. also reported the severe bt outbreaks in rural areas of maharashtra with overall morbidity, mortality and case fatality of %, % and % respectively. all the south indian sheep breeds were found to be susceptible and clinical farm of the disease is evident in all of them though saravanabava ( ) reported variations in susceptibility among the indigenous sheep. trichy black and ramnad white sheep were found to be more susceptible than the vambur and mecheri sheep of tamil nadu. prevalence of bluetongue in sheep, goat and cattle appears to be high in the region. serological surveys conducted in andhra pradesh during revealed the prevalence of btv antibodies in sheep ( . %) goats ( . %) cattle ( %) and buffaloe ( %). similar high prevalence of btv antibodies in sheep and goats were also reported from the other states in the region. clinical disease has not been recorded in kerala though btv antibodies were recorded in sheep ( . %) and goats ( . %) (ravi sankar ) . culicoides are the known biological vectors of btv. all the culicoides species are not capable of transmitting the btv. the occurrence of the disease is related to the presence of the competent vectors in the area. jain et al. ( ) established the involvement of the culicoides in transmitting the btv by isolating the virus from culicoides at haryana, the north indian state. c. imicola and c. oxystoma were found to be prevalent in andhra pradesh and tamil nadu. narladakar et al.( ) reported the presence of c. schultzei, c. perigrinus and c. octoni in marathwada region of maharastra. culicoid vectors are significantly affected by the climate and annual variations in the climate reflects the outcome of the disease. the monsoon season (june to dec) with the temperature ranging from . to . °c appears to be favourable period for the multiplication of culicoides. the maximum no of outbreaks were recorded during the north east monsoon period (oct-dec) followed by south west monsoon period (june to sep) in the region. however, details on the distribution of the competent vectors, feeding habits and their dynamics in the region is lacking multiple btv serotypes were found to be circulating in the region. (kulkarni and kulkarni ; janakiraman etal. ; mehrotra et al. ) a total of serotypes viz. - , , , , , and were identified based on the virus isolations. sreenivasulu et al. isolated btv serotype from an outbreak of bt in native sheep of andhra pradesh. btv serotype , and were also isolated from the outbreaks occurred in andhra pradesh. some of the isolates need to be serotyped. deshmukh and gujar ( ) isolated btv type from maharashtra. following is the summary of the distribution of btv serotypes in this region. clinical picture of bt in native sheep appears to be slightly different, the major difference being that swelling of lips and face was less conspicuous. mucocutaneous borders appeared to be very sensitive to touch and bleed easily upon handling. the classical signs of cyanosis of tongue and reddening of coronary band are not the common features of the disease in native sheep. the disease was also confirmed by the virus isolation and identification. clinical disease has not been reported in cattle, buffaloes and goats in spite of high seroprevalence. in conclusion bt is established in native sheep and causes severe economic losses to the farmers. the disease is concentrated in the southern peninsula of the country. the disease is seasonal and is associated with the rain fall. multiple serotypes appear to be circulating in this region. the btv serotypes were of virulent in nature as evident by severe outbreaks. s. janardana reddy*, d. c. reddy department of fishery science and aquaculture, sri venkateswara university, tirupati in less than three decades, the penaeid shrimp culture industries of the world developed from their experimental beginnings into major industries providing hundreds of thousands of jobs, billions of u.s. dollars in revenue, and augmentation of the world's food supply with a high value crop. concomitant with the growth of the shrimp culture industry has been the recognition of the ever increasing importance of disease, especially those caused by infectious agents. in india viral diseases have become an important limiting factor for growth of shrimp aquaculture industry. although more than different viral pathogens have been identified in different species of shrimp world wide, only a few viruses have identified which are causing disease problems in cultured tiger shrimps in india, east coast of andhra pradesh, in particular. diagnostic methods for these pathogens include the traditional methods of morphological pathology (direct light microscopy, histopathology, and transmission electron microscopy), enhancement and bioassay methods, traditional microbiology, and the application of serological methods. while tissue culture is considered to be a standard tool in medical and veterinary diagnostic labs, it has never been developed as a useable, routine diagnostic tool for shrimp pathogens. the need for rapid, sensitive diagnostic methods led to the application of modern biotechnology to penaeid shrimp disease. the industry now has modern diagnostic genomic probes with nonradioactive labels for viral pathogens like infectious hypodermal and hematopoietic necrosis (ihhnv), hepatopancreatic virus (hpv), taura syndrome virus (tsv), white spot syndrome virus (wssv), monodon baculo virus (mbv), and bp. highly sensitive detection methods for some pathogens that employ dna amplification methods based on the polymerase chain reaction (pcr) now exist, and more pcr methods are being developed for additional agents. these advanced molecular methods promise to provide badly needed diagnostic and research tools to an industry reeling from catastrophic epizootics and which must become poised to go on with the next phase of its development as an industry that must be better able to understand and manage disease. within this field, shrimp immunology is a key element in establishing strategies for the control of diseases in shrimp aquaculture. research needs to be directed towards the development of assays to evaluate and monitor the immune state of shrimp. the establishment of regular immune checkups will permit the detection of shrimp immunodeficiencies but also to help monitor and improve environment quality. for this, immune effectors must be first identified and characterised. in the end, however, the assumption may be made that the sustainability of aquaculture will depend on the selection of disease-resistant shrimp, i.e. to develop research in immunology and genetics at the same time. the development of strategies for prophylaxis and control of shrimp diseases could be aided by the establishment of a collaborative network to contribute to progress in basic knowledge of penaeid immunity. however, to improve efficiency, it appears essential also to open this network to complementary research areas related to shrimp pathology, physiology, genetics and environment. bluetongue is an important viral disease of sheep causing severe economic losses to the farmers. lack of effective vaccine is the major impediments in controlling the disease. multiple serotypes were found to be circulating in the state. attempts are being made to develop the vaccine employing the available serotypes to control the disease. hence, it is essential to identify the antigenic relationship among the serotypes to identify the candidate vaccine strains to be incorporated in the preparation of vaccine. reciprocal cross neutralization test was employed to find out the r% values between btv- , - and - which indicated the extent of antigenic relationship between the serotypes. r% value between btv- and btv- was recorded as . r% value of . and . were observed between btv- and - and btv- and - respectively. the r% values recorded in the present study revealed a weak antigenic relationship between the btv serotypes. the extent of antigenic relationship between the btv serotypes was also determined by multiple sequence alignment of the nucleotide and amino acid sequences of the reference btv serotypes , and . the sequence analysis of the vp gene revealed a homology of - % and - % at the nucleotide and amino acid levels respectively. r% values obtained using reciprocal cross neutralization test with the btv- , and serotypes isolated in native sheep of andhra pradesh and the genomic analysis of the reference serotypes of btv- , and revealed very weak antigenic relationship and were highly divergent. diseases especially those by viral pathogens cause greater economic losses in most horticultural crop species throughout the world as compared to agricultural crops. non-genetic methods of management of these diseases include quarantine measures, eradication of infected plants and weed hosts, crop rotation, use of certified virus-free seed or planting stock and use of pesticides to control insect vector populations implicated in transmission of viruses. however, none of these measures is likely to provide an enduring solution against these diseases especially those caused by viruses due sometimes to the huge expenditure involved, but mostly to the questionable effectiveness and reliability of those methods. as key control pesticides are getting increasingly abandoned, development of alternative methods to control diseases has been a felt-need in the recent past. though breeding for disease resistance generally provides a reliable security in a long run, introgression of host plant resistance did not materialise in most important crops. non-availability of an appropriate source of resistance in inter-fertile relatives, linkage to undesirable traits, or often times polygenic nature of such sources of resistance are the stumbling blocks in breeding programs. the limitations of conventional breeding and routine cultural practices prompted the need for the development of other approaches of virus control that could be fully incorporated into traditional methods. in this perspective, the concept of pathogen-derived resistance offers an attractive strategy to evolve newer methods of virus management, by transforming crop plants with nucleotide sequences derived from the pathogen's genome. an increasing number of molecular characterisation of plant virus genomes and the stable transformation of a number of horticultural crop species have in fact opened an avenue for molecular breeding against virus pathogens. successful field-testing of genetically modified crop cultivars renders proof of their supremacy over existing cultivars. it also contributes to demonstrate their capability with regard to environmental safety with a view to winning over public concern and scepticism. in general, the eventual commercialisation transgenic lines expressing virus resistance will rely upon a host of factors including their field performance, genetic stability, public acceptance and the resolution of environmental concerns and patent related issues. as such, elaborate field trials and allied studies are now required to adapt genetically engineered horticultural crops expressing virus resistance for their implementation into practical agriculture. a few examples from current research at tnau, in india or elsewhere will be discussed in this presentation. virology unit, division of plant pathology, iari, new delhi in recent times there has been greater emphasis on vegetatively propagated crops in india to help diversify the indian agriculture. fruit, flower, spice and plantation crops are important vegetatively propagated horticultural crops, which have become a driving force for economic development in several parts of india. however, most of the vegetatively propagated crops are threatened by biotic stress caused by plant pathogens in general and plant viruses in particular. plant viruses produce specific and non specific symptoms and in some cases no symptoms are produced. correct identification and diagnosis of viral diseases is first step in the management of any disease including viral diseases. there have been two major breakthroughs in virus diagnostics during last four decades. the first one was serological assay using monoclonal or polyclonal antibodies in enzyme linked immunosorbent assay (elisa) and the other one was the use of in vitro amplification of dna in polymerase chain reaction (pcr). a significant development in serological assays has been its simplification in form of user's friendly quick strip/dip stick method. the one-step lateral-flow (lf) tests have been developed for the on-site detection and identification of several plant viruses. rapid advancement in virus genome characterization has led to the development of novel approaches of nucleic acid based diagnostics which include conventional pcr, real time pcr, multiplex pcr, micro/macro arrays and biochips. pcr protocols already exist for many plant viruses of citrus, banana, apple, papaya, vegetables, ornamental and spice crops. a further advancement has led to development of realtime pcr assay which is relatively easy but requires training for diagnosticians. in real-time pcr assays, results can be available within min. the nucleic acid template preparation in pcr has been simplified. membrane based dna template protocol and co-isolation of nucleic acid template preparation are novel approaches in pcr detection of virus and virus like pathogens. since many of the horticultural crops are often infected by more than one virus, their individual detection by pcr is not only expensive but also time consuming. therefore, multiplex pcr has been developed where in genome of more than one virus could be amplified and detected in the same reaction mixture. development of nucleic acid based chip is now one of the fastest and recent growing areas in the field of pathogen detection. these nucleic acid based chips have been named as dna/rna chips, biochips, genechips, biosensors or dna arrays. when it comes to applications of microarray technology for plant viruses, it is not too difficult to see the value of a method that could potentially detect a whole range of viruses using a single test. however, microarrays are unlikely to become the only method in use in a diagnostic laboratory. processing of germplasm including transgenic planting material imported for research purposes into the country. during the last two decades, a total of , samples of wheat including transgenics were imported from cimmyt (mexico), icarda (syria) and many other countries. these were grown in post-entry quarantine nursery each year at nbpgr, new delhi and the transgenic samples were grown in national containment facility of level- (cl- ) since its inception to ensure that no viable biological material/pollen/pathogen enters or leaves the facility during quarantine processing of transgenics. in addition, post-entry quarantine inspections of the transgenic wheat grown by indenters are also undertaken by nbpgr quarantine scientists. virus-induced gene silencing (vigs) is a technique in which viral genomes are used, usually after appropriate modifications, for transient gene silencing in plants. the mechanism behind vigs is the phenomenon called rna-interference (rnai), which is widespread in many organisms and is believed to be form of inherent defence system against intracellular pathogens, such as viruses and transposons. double-stranded rna or rna containing strong secondary structures, commonly produced during viral infections, are believed to cause triggering of rnai, which employs a battery of proteins and nucleoprotein complexes to identify and degrade specific viral transcripts. in vigs, viral genomes not causing severe symptoms, but which can accumulate and spread efficiently in the host plant are used as vectors in which a host gene is cloned and introduced into the plant. upon replication, the viral vector triggers rnai response in the host plant, which also targets the host gene, leading to its silencing and subsequently, the silenced phenotype revealing gene function in vivo. vigs has been used extensively to study gene functions in dicot plants, such as tobacco, tomato, pea, soybean, etc., using vectors derived from reference genes are commonly used as an/the endogenous normalisation measure for the relative quantification of target genes. the expression (characteristics) of seven potential reference genes was evaluated in tissues of healthy, physiologically stressed and barley yellow dwarf virus (bydv) infected cereal plants. these genes were tested by rt-qpcr and ranked according to the stability of their expression (characteristics) using three different methods (two-way anova, genorm and normfinder tools). in most cases, the expression (characteristics) of all genes did not depend on the abiotic stress conditions or on the virus infections. all the genes showed significant differences in expression (characteristics) among plant species. glyceraldehyde- -phosphate dehydrogenase (gapdh), beta-tubulin (tubb) and s ribosomal rna ( s rrna) always ranked as the three most stable genes. on the other hand, elongation factor- alpha (ef a), eukaryotic initiation factor a (eif a), and s ribosomal rna ( s rrna) for barley and oat samples; and beta-tubulin (tubb) for wheat samples were consistently ranked as the less reliable controls. the bydv titre was determined in two oat varieties by rt-qpcr by three different quantification approaches. statistically, there were no significant differences between the absolute and the relative quantification, or between quantification using gapdh + tubb + tuba + s rrna and ef a + eif a + s rrna. the geometric average of gapdh, s rrna, tuba and tubb is suitable for normalisation of bydv quantification in barley and oat tissues. for wheat samples, a combination of gapdh, s rrna, tubb, eif a and e fa is recommended. department of microbiology, yogi vemana university, vemanapuram, kadapa large scale production and import of propagative material poses potential risk of introducing several destructive pathogens particularly viruses and mycoplasma like organisms in our country. this demands adequate quarantine safe guards such as growing them under approved post entry quarantine facility for specific period so as to facilitate virus detection, thereby curtailing risk. when such facilities are coupled with propagation by tissue culture will ensure virus free propagative plant material. the requirement of nationwide network of post entry quarantine facility working in close collaboration with crop institutions are very much emphasized for considering import of high risk plant genera for agriculture development. present paper discusses about virus disease of quarantine importance affecting ornamental and fruit plants such as chrysanthimum, dahlia, dianthus, rosabengalensis, cattleya, cymbidium, dendrobium, lilium, citrus, vitis etc. the paper also discusses on immunodiagnostic methods of detection and methods of obtaining virus free propagative material. rice tungro occurs as epidemics in regular cycles and has been reported in the last years from all the major rice growing regions of india, especially prevalent in the southern and eastern states. development of the durable resistant varieties to tungro is crucial for the management of the disease. molecular breeding, involving the use of dna markers linked to the resistant gene(s) for selection, can overcome the difficulties encountered in conventional resistant breeding programs. for successful marker-assisted selection (mas), the identification of closely linked markers through the process of gene tagging and mapping is a prerequisite. attempts have been initiated for identification of tungro resistance genes through molecular mapping and their introgression into the target varieties using marker-assisted selection at drr, hyderabad. the inheritance of resistance to rice tungro virus disease was studied in seven resistant rice cultivars with field evaluation at hot spot locations. the microsatellite markers linked to rice tungro resistance in utri merah was studied and found that resistance genes were linked to rm on chromosome . through molecular mapping two qtl were identified controlling rtv resistance on chromosomes and in 'utri rajapan' explaining . % and . % of the phenotypic variance. in variety 'vikramarya', another two qtl for rtv resistance were detected on chromosomes and explaining . % and . % of the phenotypic variance. the closely linked markers identified in this study flanking the gene of interest through mapping will improve the efficiency and precision of introgression programs in marker assisted breeding for rtv resistance. functional characterization of these qtl for rtv resistance is under progress. there is only a limited pool of natural virus resistance in cassava against cassava mosaic geminiviruses and cassava brown streak ipomovirus hence the development of transgenic resistance in this significant crop might present an option. rna mediated resistance through the expression of inverted-repeat dsrna sequences derived from the virus genome and the modification of plant microrna to produce antiviral artificial microrna are strategies that have recently been proven very effective for induction of virus resistance (immunity) against a number of rna viruses. results from rna interference strategies against geminiviruses never resulted in immunity of transgenes. however, it suggest that viral mrna are targets of rna silencing and that the success of the strategy depends on the relevance of the target gene in the systemic spread of the virus. we have generated a number of rna silencing constructs to induce resistance against cbsv and the indian cassava mosaic viruses icmv and slcmv. due to the serious problems inherent with transformation of cassava and subsequent resistance screening, these constructs were tested for efficiency either by transient-or by transgenic expression in n. benthamiana. complete immunity was reached in transgenic n. benthamiana against cbsv using inverted repeat or amirna constructs. using different species of cbsv for resistance screening, immunity was broken, to show the minimum context for broad spectrum resistance. similarly, highly specific resistance was reached in expression of amirna. in contrast, virus resistance against icmv/ slcmv using single amirna constructs was not successful. results from the experiments to generate virus resistance against cbsv and icmv/slcmv will be shown; methods to evaluate efficiency of rnai gene constructs by transient gene expression in n. benthamiana and strategies to develop efficient resistance against rna and dna viruses in cassava will be discussed. bitter gourd (momordica charantia l.) which is also called bitter melon, balsam apple and balsam pear belongs to family cucurbitaceae. it is an important traditional vegetable of nutritive and medicinal value that is cultivated in tropical and sub-tropical asia, but is considered as a weed host reservoir for viruses in jamaica. viral disease-like symptoms were observed occurring naturally on the crops of bitter gourd grown in the fields of northern india during - . an incidence of . % of diseased plants was recorded which showed chlorotic spots and mosaic ranging from mild mottling to green blisters along with leaf smalling, leaf and fruit deformations, bud necrosis and stunted growth whereas . % plants exhibited leaf curling alone or in combination with mosaic-type disease. a reduction of . % in fruit yield was recorded in mosaic-like disease which could be attributed to lesser fruit setting due to bud necrosis, smaller fruit size and stunted plant growth. such plants produced deformed, notched, irregularly shaped fruits wherein pre-mature yellowing and necrosis on the anterior and posteriors ends made . % fruits unfit for marketability. the dwindling yield and production of unmarketable fruits posed a major constraint for profitable cultivation of this economically important crop, thus warranting for studies on etiology and management of these diseases. the mosaic-like disease was transmitted to healthy seedlings of bitter gourd at -leaves stage by sap inoculation as well as by aphid viz., myzus persicae sulz. and aphis gossypii glov. initially studies were carried out to optimize protocols for efficient plant regeneration and agrobacterium-mediated transformation for nagpur sweet orange, which is a popular and elite citrus cultivar in india. organogenesis was induced in etiolated epicotyl explants of one-month-old axenically raised polyembryonic seedlings by culturing them in mt medium supplemented with g/l sucrose with varying concentrations of plant hormones. it was found that bap at mg/l without auxin was best for efficient shoot regeneration in citrus using epicotyl explants. a % regeneration frequency was obtained and multiple shoot formation was obtained from both the cut ends of all the explants. an average of . well-differentiated shoots per explant were obtained, all of which rooted normally under the influence of mg/l iba. this improved regeneration protocol was utilized in standardizing agrobacterium-mediated transformation of citrus using a. tumefaciens strain eha , containing binary plasmid pcambia that harbors gus reporter gene and npt-ii plant selection marker gene. one-month-old epicotyl explants infected with over-night grown agrobacterium (a . - . ) for min and co-cultured for days were found to be optimum for transformation as assessed on the basis of pcr analysis and gus activity displayed by the stem and leaf sections of putative transgenics. overall transformation frequency ranged from to %. current study focuses on the generation of citrus transgenics for ctv resistance using a. tumefaciens strain eha containing binary plasmid pbinar harboring portion of coat protein gene of ctv and npt-ii gene employing the standardized protocols. several putative transgenic shoots were recovered on selection medium and they are being utilized for molecular analyses and resistance against ctv. work is also in progress on the generation of citrus transformants using rnai construct harboring ctv cp and p genes, singly and in conjunction. our lab was also involved in developing rice transgenics for resistance against rice tungro disease, which is one of the most important and widespread virus diseases of rice in south and southeast asia, causing an annual estimated loss in crop yield of economic losses worth millions of rupees are caused due to these diseases annually. virus diseases are frequently less conspicuous than those caused by other plant pathogens and last for much longer. this is especially true for perennial crops and those that are vegetatively propagated. one further problem with attending to assess losses due to various diseases on a global basis is that what most of the data are from small comparative trials rather than wide scale comprehensive surveys, even the small trials do not necessarily give data that can be used for more global estimates of losses. this is for several reasons, including: ( ) variation in losses by a particular crop from year to year; ( ) variation from region to region and climatic zone to climatic zone: ( ) differences in loss assessment methodologies; ( ) identification of the viral etiology of the disease; variation in the definition of the term 'losses' and ( ) chilli is the major vegetable and spice crop grown in thar desert areas of rajasthan. leaf curl disease (chlcd) is one of the major constrains in chilli cultivation faced by farmers and cause yield loss up to %. a survey was conducted in major chilli growing areas of thar desert; bikaner, nagur, jodhpur and jalore districts of rajasthan during november, to understand the present status of leaf curl disease in chilli. among the four district surveyed for chlcd, the disease incidence was recorded maximum (up to %) in jodhpur district followed by jolore district (up to %). no relation was found between the disease incidence and varieties. the major varieties grown in these area are; mehsana, rch (mandoria), haripur raipur, mathania and local cultivars. the number of whitefly was also counted in top, middle and bottom leaf of chilli grown in these areas. the average number of whitefly per plant ranged from . to . . more number of whitefly ( . ) was recorded in jodhpur district and lowest ( . ) in jalore district. total dna was extracted from three leaf curl infected samples from each district and tested for the presence of begomovirus using coat protein (cp) and dna-b specific primers. all the samples were positive for cp and dna-b amplifications by pcr. the cloning and sequencing of selected cp gene and dna-b fragments are in progress. the preliminary investigations shows that the leaf curl disease of chilli is widespread in the arid region of rajasthan and may be caused by begomovirus associated with satellite dna-b. bittergourd (momordica charantia) is an important vegetable crop of kerala. the crop is affected by several diseases of which mosaic is a prominent one. a field experiment was conducted to evaluate the efficacy of potentised resistance inducing substances (ris) viz., mosaic affected bittergourd plant tissue, ash of mosaic affected bittergourd plant tissue, plumbago and salicylic acid for control of bittergourd mosaic in march . ris were applied as drench and foliar spray at three potency levels twice, before flowering of the crop. the experimental crop was grown as per the package of practice recommendations in split plot design with five replications per treatment. the disease incidence, disease severity and yield of the crop were recorded. the result of the experiment shows that spraying was more effective than drenching of treatments for reducing mosaic incidence and severity. among treatments, infected plant extract at potency was the most effective one for reducing mosaic incidence and it showed the maximum incubation period and minimum disease severity. the spray application of treatments produced significantly higher yield than drenching. among the treatments, ash of infected plant at and potency and infected plant extract at potency were on par and produced comparatively higher yield. elephant foot yam (amoprhophallus paeoniifolius), colocasia (colocasia esculenta) and tannia (xanthosoma sagittifolium) are the major edible aroids cultivated in india. the elephant foot yam cultivation is gaining importance due to its high production potential, nutritional and medicinal values and good economic returns. all these aroids are vegetatively propagated and viral diseases are spreading through planting materials. ctcri has the mandate of producing healthy planting materials of these edible aroids. accurate diagnosis and identification of the virus is essential for production of healthy planting material and effective management of the disease. though occurrences of viral diseases on edible aroids in india were known in s, not much attention was given for detection and identification of the virus involved. in case of elephant foot yam - % mosaic incidence was observed with varying symptoms of mosaic, puckering, filiformy etc. in colocasia and tannia, - % incidence was noticed. rt-pcr amplification with potyvirus group specific primers and subsequent cloning and sequencing of the amplified product has confirmed the association of dasheen mosaic virus (dsmv) with all the three edible aroids cultivated in india. the complete full length coat protein gene of dsmv infecting elephant foot yam was cloned in pgem-t vector and sequenced. further sequence analysis revealed that the cp of dsmv consisted of nucleotides and the utr comprised of nucleotides. blast and phylogenetic analysis showed highest similarity of % with that of dsmv isolate af , reported from usa. the deduced amino acid sequence of cp had . - . % identity with other dsmv isolates. blast analysis of the partial cp gene sequences of colocasia and tannia also confirmed that the virus involved is dsmv. rt-pcr analysis of large number of samples from all the three crops confirmed that the potyvirus group specific primers (mj and mj ) are good for rapid detection of dsmv in these crops. dsmv specific biotinylated cdna and digoxigenin labelled crna probes were also prepared and dsmv in elephant foot yam was detected through nucleic acid spot hybridization. yellow leaf disease (yld) caused by sugarcane yellow leaf virus (scylv) is a recently recorded disease in india and is found wide spread throughout country. in popular varieties, the disease incidence varied from to . % and attained epidemic levels under field conditions. detailed studies on the impact of yld on sugarcane revealed that the virus infection significantly reduces various cane growth parameters, cane yield and juice quality. sequence comparisons of the coat protein (cp) and movement protein (mp) of scylv isolates from india and database sequences showed a significant variation between indian isolates and the database sequences both at nt and aa level in the cp/mp coding regions. the significant variation in our isolates with the database isolates, even in the least variable region of the scylv genome showed that the population existing in india is different from rest of the world. further, comparison of partial sequences encoding for orf and revealed that yld in sugarcane in india is caused at least by three genotypes viz., cub, ind and bra-per, of which a majority of the samples were found infected with cuban genotype (cub). the genotype ind was identified as a new genotype and this was found to have significant variation with the reported genotypes. we have identified specific primers from cp region of the virus and optimized rt-pcr conditions to diagnose the virus. this assay has been found efficient in detecting the virus in asymptomatic plants and tissue culture derived seedlings. elimination of the virus through meristem culture has been demonstrated to purify the virus from the infected planting materials and this technique needs to be adopted to supply disease-free planting materials for effective management of the disease. studies are also in progress to identify the yld-resistant sources in sugarcane germplasm to initiate breeding for yld-resistance in sugarcane. mycoviruses are viruses that infect fungi. they have been identified in all major fungal families. in the present scenario, mycoviruses are the important means of biocontrol of plant fungal pathogens. most identified fungal viruses have double stranded rna genomes, often with more than one dsrna present per virus particle, and have been spherical in shape. these viruses are mostly vesicle bound, as other viruses have protein coatings. to be a true mycovirus, they must demonstrate an ability to be transmitted-in other words be able to infect other healthy fungi through anastomosis and spores. mycoviruses lead 'secret lives', reduce the ability of their fungal hosts to cause disease in plants. this property, known as hypovirulence (hypovirulence is a term used to describe reduced virulence found in strains of pathogens), this phenomenon was first observed in cryphonectria (endothia) parasitica (chestnut blight fungus) on european castanea sativa in italy, where naturally occuring hypovirulent strains were able to reduce the effect of virulent ones. these slower growing hypovirulent strains of c. parasitica contain a single cytoplasmic element of double-stranded rna (ds rna) similar to that found in mycoviruses that was transmitted by anastomosis in compatible strains through natural virulent populations of c. parasitica. hypovirulence has also been reported in many other fungal plant pathogens, including rhizoctonia solani, gaeumannomyces gramini var. tritici, ophiostoma ulmi, sclerotinia homoeocarpa, diaporthe ambigua alternaria alternata, and fusarium sp. etc. hypovirulence has attracted attention owing to the importance of fungal diseases in agriculture and the limited strategies that are available for the control of these diseases. it reduces the use of toxic fungicides which also affect the plant growth. the symptoms resulted by the mycoviruses are reduction in growth, reduction in pigmentation and sporulation, excessive sectoring and aerial mycelial collapse. these are the consequences of alteration in complex physiological and biochemical processes involving interaction between host and virus. cassava (manihot esculenta crantz.) is the major tuber crop in peninsular india, it is grown in an area of . lakh hectares with the annual production of . million tonnes both for direct consumption and the starch grain (sago) producing industries, mainly in the southern states of tamil nadu, kerala and andhra pradesh (fao ) . in tamil nadu, cassava primarily produced for sago producing industries where it is considered as an industrial crop rather than food crop, so the resource rich farmers are cultivating the cassava as irrigated crop in their fertile land and the poor farmers are raising the crop under rainfed conditions. in south india in addition to cassava there is a practice of intercropping important vegetable crops like, tomato, brinjal, legumes and gourds in cassava fields since all the above mentioned crops are short duration and are money spinners for the farmers. unfortunately, the major production constraint in these vegetable crops including cassava is the geminiviruses belonging to the family of in recent years there has been growing concern regarding the standard of scientific researches in india. the strengths, weaknesses, opportunities and threats (swot) analysis on indian scientific research reviewed the progress of science during the last six decades. although the 'strengths' were highlighted in good measure, it was the list of 'weaknesses' that called for attention to upgrade the standard of research and 'opportunities' that provide scope for overall scientific growth. a comparison between india and other countries in terms of research papers published revealed that india's contribution to science has come down enormously. what ails indian science? should we compare the growth of indian science with other developed countries? what criteria should be adopted to judge the quality and standard of scientific research? how to motivate the scientists to improve their scientific output? how do motivate the scientists to improve their scientific output? how do indian journals perform in maintaining quality? this paper analyses critically the scientific journals around the world, based on the scores allotted by the national academy of agriculture sciences (naas) in and for and journals respectively. in general, the indian journals performed poorly irrespective of the disciplines with only - % in the high standard. the paper dealt with the reasons for low impact factor, the anomalies in the allotment of scores to wide spectrum of the journals and the disadvantages the scientists face with the scoring system. a case study was presented of an institute with over scientists whose publications were analyzed to discuss the merits and demerits of the system. the performance of the journals published by prestigious academics, societies and councils was also projected. the paper concluded with the need for enhancing the image of the country through research publications in high standard journals and the role of various scientific bodies with shore and long term measures. poster session herpes simplex virus (hsv) keratitis is a leading cause of corneal blindness throughout the world. the infection can be diagnosed by clinical manifestations but in case of atypical ocular cases, laboratory diagnosis is more helpful in timely management of disease. collection of corneal scrapings in all cases of stromal and epithelial keratitis may not be possible, but collecting tear fluid is a convenient procedure causing less discomfort to the patients. therefore, the present study was intended to evaluate the suitability of tear specimens for detecting hsv by polymerase chain reaction (pcr) and immunofluorescence (ifa). tear fluid and corneal scrapings were collected from patients of suspected herpetic keratitis. hsv- antigen was detected by ifa using rabbit anti-hsv antibodies. pcr was performed to amplify bp region of thymidine kinase (tk) coding gene and bp region from dna polymerase coding gene of hsv. out of patients hsv antigen was detected in ( . %) of corneal scrapings and ( . %) of tear specimens and in ( . %) patients from both the specimens. hsv gene could be amplified in ( . %) of corneal scrapings and ( . %) of tear fluids and in ( . %) patients from both the specimens. although, corneal scraping seemed to be marginally superior material for detection of hsv, tear fluid may also serve as an appropriate alternative clinical specimen, due to ease of collection and least discomfort to the patients. in either cases pcr detected higher number of hsv cases than ifa. therefore if and when feasible, both ifa and pcr should be used simultaneously on each specimen to obtain best results. cytokines play a key role in the regulation of immune responses. in hepatitis c virus infection (hcv), the production of inappropriate cytokine levels appears to contribute to viral persistence and to affect response to therapy. il- is produced by a variety of cells including t cells, phagocytes and fibroblast. cytokine genes are polymorphic at specific sites, and certain mutations located within coding/regulatory regions have been shown to affect the overall expression and secretion of cytokines in patients with hcv infection. to correlate the serum levels and polymorphism of il- gene in chronic hepatitis c patients and healthy controls. forty patients positive for hcv rna attending the medicine out patient department and wards of lok nayak hospital, new delhi as well as forty healthy controls were enrolled for the study. the serum level of il- was detected by using elisa. genomic dna was extracted from whole blood of hcv infected patients and healthy controls by using accuprep genomic dna extraction kit according to manufacture's instruction. the genotyping of il- promoter (- variant) was carried out by pcr and direct sequencing using the method of patricia woo et al. . the serum level of il- was significantly down regulated in hcv infected chronic patients as compared to the healthy controls. genotyping of - promoter variant of il- was performed by pcr and direct sequencing. il- polymorphism in the g/g, g/c and c/c allele was non significant when compared to hcv patients and healthy controls. the il- serum levels were significant among hcv infected patients when compared to healthy controls. the polymorphism in the promoter region of il- (- ) was found nonsignificantly associated in hcv patients compared to healthy controls. in conclusion, the present study suggests that the host il- polymorphism alone may not play a significant role in the outcome of hcv infection. acute gastroenteritis (age) is a global health problem and has been associated with multiple etiological agents, which include bacteria, protozoa and viruses. viral gastroenteritis is considered as the second most common illness in children after upper respiratory tract infection. among enteric viruses, rota, noro, enteric adeno, astro and enterovirus are found to be associated with gastroenteritis. although, association of enteric viruses has been established in children hospitalized for age no such data is available from hospitalized children other than enteric infections. to determine the prevalence of enteric viruses circulating in hospitalized children. fecal samples, n = ( symptomatic and asymptomatic for age) were collected from children \ year of age from three different hospitals across the city of pune from june to feb. . detection of group a rotavirus was carried out by using antigen captured elisa. rt-pcr and pcr was carried out for the detection of norovirus, enterovirus, astrovirus and enteric adenovirus detection by using primers targeted to rdrp gene, ncr gene and consevered gene for serine protease and hexon gene respectively. out of fecal samples tested for enteric viruses in age cases, the prevalence of rota, entero, noro, enteric adeno and astrovirus were . % ( ), . % ( ), . % ( ), . % ( ) and . % ( ) respectively. however, the presence of these viruses in the asymptomatic cases (n = ) was detected at . % ( ), . % ( ), . % ( ), . % ( ) and . % ( ) levels respectively. mixed infections of enterovirus and rotavirus were found in both symptomatic . % ( ) and asymptomatic cases . % ( ). however, mixed infection of enterovirus with adenovirus were found only in asymptomatic cases . % ( ). no marked difference was observed in the seasonal pattern of all viruses in the patients with or without gastroenteritis. the findings of this study document highest circulation of rotaviruses in patients symptomatic and asymptomatic for age. the entero and noroviruses remain second most important enteric viruses in these patients. influenza in humans is a major public health concern and the understanding of its evolution in the light of its ''antigenic drift'' helps prediction of epidemics and update of yearly influenza vaccine. to antigenically characterize influenza a (h n ) isolates and study antigenic drift during to in pune city. patients with influenza like illness were identified using a strict case definition from dispensaries located in different areas in pune and clinical samples (ns/ts) were collected after obtaining informed consent. these clinical samples were processed in vivo (in fertile eggs) and in vitro ( overall, an additional ( . %) positive cases of dengue could be detected when ns antigen assay was also used in the study. highest ns antigen positivity was encountered among the samples collected on the rd day of fever whereas mac elisa for anti igm antibody was positive after th day and gradually there was an increase in the positivity towards the convalescent phase of the disease. the results of this study indicate that ns antigen based elisa test can be an useful tool to detect the dengue virus infection in patients during the early acute phase of disease since appearance of igm antibodies usually occur after fifth day of the infection. concurrent use of both diagnostic assays namely ns antigen as well as mac elisa will improve the overall detection of dengue infection. early detection of acute dengue virus infection is crucial to provide timely information for the management of patients. human parvovirus b , a member of the parvoviridae family, is a pathogen associated with a wide variety of diseases. most commonly, it causes childhood rash erythema infectiosum, but in some cases more serious symptoms such as persistent arthropathy, critical failures of red cell production causing transient aplastic crisis, this infection in pregnancy causes hydrops fetalis and myocarditis. traditional immunosuppressive therapy being unsuccessful, anti-viral therapy might be worthy of consideration. functional annotation would provide role of viral proteome in its survival and pathogenic mechanisms. svmprot functional family annotations of vp protein had deciphered its zincbinding, coat protein, outer membrane, chlorophyll biosynthesis, dna repair and calcium-binding nature. vp protein is having a key role in viral assembly of b virus and being non-homologous to human proteome, it was identified as an attractive molecular target for structure based drug discovery. the vp protein crystal structure was energy minimized using charmm. a structure based virtual screening method was applied using ligandfit to identify potential inhibitors of vp protein from chembank database and ten potential human parvovirus b vp inhibitors were proposed. prism genetic analyzer. the drafting of the sequences was performed using bioedit software and were submitted in genbank. for phylogenetic interpretation denv representing the full extent of genetic diversity in denv- , denv- and denv- were collected from genbank. neighbor joining algorithm was implemented with bootstrap value of , replicates for phylogenetic inference using mega . . . the genomic region to (c-prm gene junction) of denv were amplified directly from patient serum. twelve of samples were positive for dengue viral rna. of these were dengue type , was dengue type and were dengue type . for molecular epidemiological survey and genotyping of the sequences more than sequences from different geographical areas including sequences form previously reported north indian isolates were compared with our present data set. the critical analysis of the sequences revealed: dengue type sequences were clustered within sub-type of genotype iii and all the sequences of den- clustered along with genotype iii. thus, among the dengue types , and currently circulating in north india, dengue type , genotype iii, being the predominant one followed by, genotype iii of dengue type . although there is no specific treatment or vaccine available currently, the confirmative rapid diagnosis based on detection of viral nucleic acid or igm antibodies in serum, an indication of recent infection, helps in epidemiological monitoring, symptomatic treatment of patients and determining prognosis. serological detection of anti-cgv igm antibodies was performed using rapid immuno-chromatographic assay (rica) and igm-antibody capture enzyme linked immunosorbant assay (mac-elisa). eighty convalescent sera were tested by rica and of them were found positive for anti-cgv igm antibodies. twenty-five anti-cgv igm antibody rica positive sera were further assayed using mac-elisa. more sera from the patients are currently being tested to compare the sensitivity of these two serological assays in anti-cgv igm antibody based early serological diagnosis of cgv infection and the findings will be presented. thus the present study was designed to evaluate the utility of multiplex pcr (mpcr) for simultaneous and rapid detection of dengue and chikungunya viral infections. seventy-two acute phase blood samples from clinically suspected dengue cases were subjected for dengue and chikungunya uniplex pcr using dengue genus specific primers and e gene specific primers for chikungunya virus as well as multiplex pcr was developed for simultaneous detection of dengue and chikungunya infection. standard strains of dengue and chikungunya virus were used as controls. of the clinically suspected dengue samples were found to be positive for dengue viral rna by dengue uniplex pcr as well as dengue chikungunya mpcr whereas none of the samples were positive for chikungunya virus infection by both uniplex chikungunya pcr and dengue chikungunya mpcr. the result of dengue and chikungunya uniplex pcr was found to be % concordant with dengue chikungunya multiplex pcr. dengue chikungunya multiplex pcr was found to be a potential rapid test to detect dengue and chikungunya viral infections simultaneously in clinical samples. sheetal malhotra, neelam marwaha, karan saluja, ratti ram sharma department of transfusion medicine, pgimer, chandigarh transmission through blood and blood products can be reduced to a great extent by efficient and reliable testing of the blood. the newer fourth generation elisa assays simultaneously detect antibodies against hiv- and and the presence of p antigen and thus shorten the window period to about days, as compared to days with third generation elisa. to compare the hiv seroprevalance among blood donors using fourth generation elisa (antigen-antibody) versus third generation elisa (antibody) assay. this was a prospective study involving blood donors of which were voluntary donors ( being students and being non students) and were replacement donors. sex workers are one of the core group for transmission of sti/hiv and as a ''bridge group'' to the general population. accordingly, highest priority is given to this group in targeted intervention for prevention of hiv/aids. here we are describing one such female sex worker who was harbouring concomitant sti including viral sti. a year old female sex worker was brought to the sti clinic of a tertiary care hospital by ngo with complaint of genital discharge for days. on per speculum examination, cervix was slightly erythematous, tender with mucopurulent discharge. there was no vaginal discharge or ulcer in anogenital area. however, there was a wart at lateral wall of vagina. as per naco syndromic management guideline, treatment was given for n. gonorrhoeae, c. trachomatis and hpv. cervical swab was taken and subjected to various microbiological investigation for the detection of sti viz n. gonorrhoeae, c. trachomatis, t. pallidum, candida spp., t.vaginalis, hsv- , hsv- , hiv, hbv, hcv, hpv and m. contagiosum. saline wet mount showed pus cells, but no yeast cells or trophozoite of trichomonas vaginalis. gram stained smear showed more than four polymorphonuclear leucocytes in the absence of gramnegative intracellular diplococci and a presumptive diagnosis of non gonococcal urethritis was made. no organism was isolated on any culture media after appropriate incubation. cervical swab was negative for antigen of c. trachomatis. serum was tested positive for hbv, hcv, hsv- and t. pallidum though it was seronegative for hiv. in the present case, the female sex worker was harbouring four viral sti viz hsv- , hbv, hcv and hpv alongwith t. pallidum. however clinically she was diagnosed and treated accurately only for genital wart while cervical discharge due to hsv- was misdiagnosed. it is necessary to try to test alternative approaches such as periodic presumptive therapy of viral sti, because this will not only boost up the efforts of sti control in the target group but also help in hiv control. alternatively, regular clinical and laboratory screening for viral sti may be tried. densonucleosis viruses (dnv) belong to parvoviridae family. they are the etiological agents of insect's disease known as densonucleosis, which leads to death or loss of vital functions of the infected insect. densonucleosis virus of mosquitoes has generated lot of scientific interests because of its tremendous potential in biological control and its application as a transducing vector. earlier, we have reported the isolation and characterization of a dnv from aedes aegypti mosquitoes and its prevalence among different ae. aegypti populations from india. there are reports suggesting that when aedes albopictus mosquitoes co-infected with dengue- and dnv, the multiplication of den- is suppressed. the present study focus on the effect of coinfection of ae. aegypti mosquitoes with dnv and chikungunya virus (chik). the first instar mosquito larvae were infected with dnv and the emerging dnv infected females were then infected with chikv by oral feeding. thus obtained chik infected female mosquitoes were analyzed by real time pcr for both dnv and chikv on alternate days post-infection, up to the th day. the data showed no significant difference in the multiplication of either of the viruses after co-infection. results suggest that chikv neither stimulates the replication of dnv nor is its own replication suppressed due to co-infection. this study forms an initial step in understanding the role played by such endogenous viruses on the vector dynamics. chandipura virus pathogenesis is manifested as encephalitis in young children with a very high mortality rate. this damage could be due to direct replication of the virus in brain parenchymal tissue or immune system mediated. this study aims at elucidating the role of brain infiltrating lymphocytes in pathogenesis using mice as the model system. mice were inoculated intracerebrally with the virus and the perfused brain tissue was used to isolate the lymphocytes. control mice were inoculated with an equal amount of media. in order to standardize the procedure for isolation of lymphocytes from brain tissue, splenocytes were processed to isolate the lymphocytes using histopaque density gradient method. methods to isolate lymphocytes from brain tissue as described by earlier workers were tested for the ease and efficiency of procedure using known suspension of lymphocytes from spleen. percoll density gradient method provided optimum yield of lymphocytes with an ease of handling. in this, brain cell suspension used to prepare % percoll is layered over % percoll prepared using media in : ratio. density gradient centrifugation is carried out at g for min at °c to obtain lymphocyte layer at the interface. leishman staining was performed to analyze the morphological characteristics of isolated lymphocytes. normal lymphocytes showed dark blue stained nucleus. some bigger sized cells with diffused nucleus characteristic of atypical lymphocytes were observed and some of the cells were surrounded by hair like structures. phenotypic characterization was carried out using flow cytometry. the presence of cd + , cd + and cd + cells was observed. the percentages of cd + , cd + and cd + cells was found to be . %, . % and . % respectively in the lymphocytes isolated from infected animal and . %, . % and . % respectively from control animal. hence, cd + cells showed maximum infiltration after infection. (santosh et al. ; pradeep et al. ). in the present study chikv suspected blood samples were collected and the acute phase samples were subjected to rt-pcr for the presence of virus specific rna by using the primer pair dvrchk-f/dvrchk-r as described by us earlier (naresh kumar et al. ). the convalescent phase samples were screened for chikv specific antibodies by using sd bioline chikungunya igm rapid test. six sets of primers were designed to amplify the complete nsp and complete structural genes of chikungunya virus. the products were further gel purified, cloned in ptz r/t vector and the recombinant clones were sequenced and submitted to the genbank. the complete ns gene and structural genes were compared with other available sequences in the genbank. sequence analysis results will be presented. the present study discusses these aspects in detail. . some of these phages (viz. v , v ) showed plaques at °c but not at °c. thus they seem to be lysogenic. for propagating and increasing the titre of all the above isolates, various previously described methods were attempted, but none of these methods were satisfactory. but when siliconized glassware and plastic-ware were used, propagation was successful. we showed that siliconization of glassware and plastic-ware was essential for the propagation of our mycobacteriophage isolates v , v , v , v and v . also, phage dilution medium (pdm) as described by chaterjee et al. ( ) was found to be effective for picking out of the plaques made by the phages. in this way, the phage isolates were propagated up to p . the various passages of the phage isolates v , v , v , v and v (i.e. original, p , p and p ) were stored at - °c. the four major routes of transmission are unsafe sex, contaminated needles, transmission from an infected mother to her baby at birth (vertical transmission) and breast milk. screening of blood products for hiv has largely eliminated transmission through blood transfusions or infected blood products in the developed world. in , globally, about million people died of aids, . million were living with hiv and . million people were newly infected with the virus. hiv infections and aids deaths are unevenly distributed geographically and the nature of the epidemics vary by region. more than % of people with hiv are living in the developing world. there is growing recognition that the virus does not discriminate by age, race, gender, ethnicity, socioeconomic status-everyone is susceptible. however, certain groups are at particular risk of hiv, including men who have sex with men (msm), injecting drug users (idus), and commercial sex workers (csws). the present study indicates the prevalence of hiv infection among the people residing in the northern region of india predominantly among the foothills of the himalayas. the study was carried out on the patients visiting herbertpur christian hospital (a unit of emmanuel hospital association) under the integrated counselling and testing centre scheme at the respective hospital during the - . the study indicates the screening of people groups residing in the respective area through community health schemes. the diagnosis of the hiv infection is done by three types of assays namely. the tridot method which is the rapid method of diagnosis followed by the. hiv coombs test which involves the dot immunoassay principle. the third assay is the enzyme linked immunosorbent assay (elisa). the number of patients screened during the period of september to march is which include patients coming from four different states namely haryana uttarakhand uttarpradesh and himachal pradesh. the number of people who were tested positive are and the number of people who were tested negative are . the people tested positive are sent to the higher centre for other confirmatory tests such as pcr and western blot analysis. these patients are sent for treatment and prophylaxis at a respective recognised centre in dehradun. the present study determines a consistent community hiv screening and treatment approach through diagnostics counselling and awareness programmes. classical swine fever (csf) also known as hog cholera is a highly contagious and fatal disease of swine. csf became rapidly a major issue of pig industries. it still causes important economical losses worldwide. it is considered as a major health problem of swines in india. during the month of august to october there was an outbreak of classical swine fever in bihar. from three districts darbhanga, patna and supol, total numbers of different infected tissue samples like kidney, spleen and lymphnode were collected from the dead morbid/pigs. total rna was isolated from % homogenate of infected tissues in sterile pbs by tri-reagent (sigma, usa) according to the manufacturer's instructions and cdna was prepared by using commercial available kit. the cdna was stored frozen at - °c until used. for the molecular detection of classical swine fever virus specific nested pcr amplification of e and ntr was done along with ns b and e rns amplification. primarily these samples were found positive with these primers. further confirmation by sequencing was done by cloning of these pcr products in pgem-t easy vector. e and ntr sequences were considered for phylogentic analysis along with complete available sequences of csfv. nucleotide sequence alignments were carried out using the clustalw program (dnastar) and phylogenetic tree analysis (dnastar) showed that ntr have close proximity with taiwan strain (accession no. ay ) and e shows close proximity with chinese isolate csfv- (accession no. af ). peste des petits ruminants (ppr) and sheeppox are oie notifiable diseases of small ruminants especially sheep and goat. both the diseases are economically important, in enzootic countries like india and are major constraints in the productivity of animals. considering the geographical distribution of both ppr and sheep pox infections and prevalence of mixed infection, in the present study, safety and potency of the experimental duel vaccine comprising attenuated strains of thermostable-ppr virus (pprv-revati, p- ) grown at °c and attenuated sheep poxvirus (sppv-srinagar, p ) was evaluated in local non-descript sheep. experimental animals were grouped into four groups and each group was comprising six animals, received doses ( tcid ), dose ( tcid ) and / th dose of vaccines and normal saline as control in ml volume subcutaneously, respectively. serum samples were collected on , , , and th day post vaccination. sheep simultaneously immunized with ml of vaccine consisting of either or doses of each of pprv and sppv were monitored for clinical and serological responses for a period of - weeks post-immunization (pi) and post challenge (pc). specific immune responses i.e., antibodies directed to both pprv and sppv could be demonstrated by ppr competitive elisa kit and capripox indirect elisa, respectively following immunization. all the immunized animals' resisted infection when challenged with virulent strain of sppv (srinagar isolate at p- ) on day dpi, while in contact control animals developed characteristic signs of sheeppox. the challenge of the sheep against ppr was not carried out, however, the antibody titre after immunization determined by snt and elisa, indicated that protective titre, as per earlier report on the goats. dual vaccine was found safe at higher dose and induced protective immune response even at lower dose ( tcid ) in sheep, which was evident from sero-conversion as well as challenge study with sppv. the study indicated that both the viruses are compatible and did not interfere with each other in eliciting immune response, paving the feasibility of use of this dual vaccine in combating both infections simultaneously. goatpox is one of the highly contagious, oie notifiable and economically important viral diseases of goats. the disease is caused by goatpox virus (gtpv) is classified of the genus capripoxvirus in the family poxviridae. the disease incurs severe economic losses in terms of high morbidity in adults and heavy mortality in young kids and is a major constraint in goat farming in india. considering the enzotic nature and economic impact of the disease, it is all important to control the infection by developing an effective vaccine. recently, vero cell based a live attenuated goat pox vaccine; using gtpv uttarkashi isolate (p ) has been developed in authors' laboratory and evaluated in goats. the vaccine was found safe, potent and immunogenic experimentally and even at field trials. the vaccine has been evaluated at large-scale at different regions of the country and found suitable for mass vaccination. however, the longevity of potency was not evaluated. therefore, a long term potency trials were studied for a period of years with annual challenge by using virulent goatpox virus and sero-monitoring. a sufficient number of hill goats has been vaccinated with dose of vaccine ( . tcid /ml) and monitored for clinical and serological response. every year, significant number of vaccinated (n = ) and control animals (n = ) were used for challenge with virulent strain ( . srd /ml, gtpv mukteswar). sera of pre-and post-challenged ( dpc) animals including controls have been collected and monitored for serological response in the form of specific antibody production by snt and indirect elisa. all the vaccinated animals were protected on challenge, whereas, all unvaccinated controls developed infections. the same has been reflected in sero monitoring of collected sera. so the developed live attenuated goat pox vaccine was found safe, immunogenic and potent for a period of years of immunization and suitable for mass scale vaccination in control and eradication of goat pox along with a/are suitable diagnostic tool/s in goatpox enzootic country like india. rotavirus infection in avian species varies from subclinical infections to outbreaks of diarrhea. the economic significance of rotaviral enteritis to the poultry industry has not yet been defined, but by analogy to the situation in mammals, it is likely to be significant. unlike the extensive studies performed on rotavirus infection in humans and animals, limited studies have been carried out to determine the extent of exposure of poultry birds to rotaviruses. to determine the prevalence of avian rotavirus antibodies in commercial broiler chickens. a total of chicken serum samples were collected from the lairage of a poultry slaughter house where birds from four different broiler farms in and around pune city were supplied to. the serum samples were tested by an igg antibody capture elisa wherein purified chicken rotavirus ch was used as coating antigen. sera from specific pathogen free (spf) chick (n = ) served as negative control in the test. cut off was calculated as mean negative control ? sd (standard deviation). s/co (mean sample od /cut off) values above ( . - . ) in % ( / ) serum samples were indicating positivity to rotavirus antibodies. the result of the study indicates exposure of the birds to avian rotavirus or similar agent that is circulating in pune city. bluetongue has become established in south india causing regular outbreaks in sheep. btv serotypes , , and were isolated from native sheep of andhra pradesh. the other serotypes circulating in the state need to be identified. however the major constraint is the serotype identification. to overcome the difficulties of traditional serotyping methods (neutralization tests), nucleic acid based tests are being tried. rt-pcr for serotyping was standardized using primers specific to vp gene of btv- , and serotypes. rt-pcr resulted in bp product of btv- , bp product of btv- which was defined by specific primers. however non specific amplification at two different sites i.e. bp and bp was noticed for btv- . specificity of rt-pcr was evaluated. btv- and btv- specific primers could amplify only btv- and btv- respectively where as btv- type specific primers amplified not only btv- but also btv- and btv- . nucleic acid sequence data obtained from btv- pcr product and btv- cloned products were specific to vp gene of btv- and btv- respectively. however, and bp products of btv- were identical to vp gene of btv- , , , , and and vp gene of btv- , and respectively, indicating the non specific amplification of btv- . foot and mouth disease is the most contagions and highly economically impotent disease of cloven footed animals. the disease is controlled by regular vaccination using the vaccine produced from the virus grown in the cell culture. the vaccine strain used for vaccine production is selected from the field isolates based on the adaptability and growth kinetics in bhk cells and antigen coverage. however the field viruses need to be passaged several times to adapt in tissue culture. passage of field viruses in tissue culture may results in development of mutants whose genetic makeup may differ from the field samples also some of the field strains may fail to adapt or may grow poorly in the tissue culture, thus the efficiency of the vaccine gets affected. structural proteins of fmdv carry the sequences which determine the serotype specificity and immunogenicity. thus one may replace the gene coding for structural proteins from the full length cdna copy of the vaccine strain that has been adapted to the tissue culture with the poly-structural protein gene (pi) so that the chimeric virus gets the serotype specificity of the field strain besides retaining the other characteristics that are needed for a vaccine virus. we have made replication competent fmdv asia i full length genome and cloned under t and cmv promoter separately in plasmid vectors. bam h sites were created for inserting pi- a gene of other field strains. the p - a of type 'o' vaccine strain was amplified directly from the cattle tongue material, cloned in plasmid vector and studied the specificity by sequence analysis and gene expression. we have introduced 'o' p - a gene into the full length construct devoid of asia structural protein gene, p - a. the in vitro transcribed rna in case of t promotered construct and plasmid dna in case of cmv promotered construct were transfected into the bhk cells. after the passaging the virus obtained was studied for the speciality. this approach may be used not only for rapid selection of vaccine strain and also as a repository of the cdna copy of the virus. the p is composed of a, b, c and d (vp , vp , vp , and vp ) respectively of which the vp is the most immunogenic and subunit vaccine produced with vp alone was able to induce high level of neutralising antibodies. thus to control the disease in india polyvalent vaccine consisting of the inactivated virus of all the three serotypes are in use. however the conventional vaccines have several drawbacks which include safety and temperature sensitivity. hence alternatively sub-unit vaccines consisting of vp protein has been tried. however this showed limited success due to the antigenic variations occurring in the field viruses thus escaping the neutralization from the antibodies generated from single cloned protein. hence the present study was undertaken with an objective to include all the neutralizing epitopes present in the three serotypes by linking vp ( d) genes and produce a poly valent protein for using as poly subunit vaccine. in this study we have constructed a cassette by linking the genes of three serotypes 'o' ( bp), 'a' ( bp) and 'asia ' ( bp). these genes were cloned individually in commercially pbsk vector and confirmed by sequence analysis before linking in pc dna vector. the linked gene construct was sub-cloned in pet expression vector. the expression of the protein gene from the pet vector was induced with iptg and analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (sds-page). a fusion protein of size kda was observed in page gels. since the protein contains his residues from the vector at the n-terminal end, affinity purification was carried out using nickel nitrilo-tri-acetic-acid (ni-nta) agarose matrix. the immunoreactivity of the purified protein was assayed by western blot with the anti fmdv type 'o' and 'asia ' specific sera. the may be used as a subunit vaccine. silkworm diseases caused by viruses, bacteria, fungi and protozoans form major constraints for the silk cocoon production in all the sericultural countries and among these silkworm viral diseases viz., nuclear polyhedrosis and infectious flacherie caused by bmnpv and bmifv cause severe crop loss. the traditional disease management strategies include prophylactic measures and use of disease free silkworm eggs. the prophylactic measures such as disinfection of silkworm rearing house and appliances, egg surface, silkworm bed disinfection and rearing surroundings. the disinfectants used presently in sericulture are either formaldehyde or chlorine based products, but these chemicals are neither eco-nor user-friendly. the awareness about health hazards caused by formaldehyde and environmental pollution caused by cl necessitated the development of eco-and user-friendly disinfectant products for use in sericulture. these include alternative disinfectant products developed using biodegradable chemicals and plant based ingredients by apssrdi, hindupur and central silk board for the management of silkworm diseases in india. the ideal disinfectant for sericulture would be the one which can inactivate silkworm pathogens of diverse origin and economical for sericulture. the paper discusses on the disadvantages of hcho and cl based disinfectants and advantages of eco-and user-friendly disinfectant for the management of silkworm diseases especially the ones caused by viruses. the baculovirus expression vector system (bevs) is widely used for the production of high levels of properly post-translationally modified, biologically active and functional recombinant proteins and has facilitated basic biomedical research on protein structure, function, drug discovery and the roles of various proteins in disease. bevs is based on the introduction of a foreign gene into nonessential for viral replication genome region via of homologous recombination with a transfer vector containing target gene. the resulting recombinant baculovirus lacks one of nonessential gene (polh, v-cath, chia etc.) replaced with foreign gene encoding heterologous protein which can be expressed in cultured insect cells and insect larvae. insect cell-bev system is widely used to produce recombinant proteins. bevs also eliminates concerns regarding pathogens that could potentially be transmitted to humans as it is non-infectious to vertebral animals. these features make silkworm system an ideal expression and delivery package for producing proteins of medicinal importance. the efficiency, low cost and large-scale production of proteins using bevs represents breakthrough technology that is facilitating highthroughput proteomic studies. the bevs has become a core technology for cloning and expression of genes for study of protein structure, processing and function; production of biochemical reagents; study of regulation of gene expression; commercial exploration, development and production of vaccines, therapeutics and diagnostics; drug discovery research; exploration and development of safer, more selective and environmentally compatible biopesticides. utilization of silkworm larvae and pupae as bioreactor with recombinant bmnpv producing foreign proteins extends the usages of silkworms. due to its large-size and high protein synthesis ability as well as the expediency in mass culture, silkworm is considered as good candidate for producing recombinant proteins. wssbv is the causative agent of a disease, which has recently caused high shrimp mortalities and severe damage to shrimp culture. wssbv has been found across different penaeid shrimp species. in order to develop a effective diagnostic tool, a wssbv genomic library was constructed by cloning wssbv genomic dna extracted from purified virions. in the present study wssv disease free (confirmed by pcr analysis) were collected from hatcheries from different areas of guntur and prakasam districts and analysed to study the effect of various physical parameters like temperature, p h , salinity and turbidity on the prevalence of above disease. the studies on the surface water temperature revealed fluctuations in the ponds ranging between to . °c in diseased ponds and . to . °c in healthy ponds. these results show definite influence of temperature on the prevalence of wssv. present day strategy in vaccine development is to include marker facility that helps in distinguishing antibody response due to vaccination vis-à-vis infection in vaccinated animals. such information becomes relevant for effective disease control programmes especially when using inactivated virus vaccines like foot and mouth disease (fmd). the antibodies generated in the animals, only through vaccination, is the measure of vaccine efficacy and safety. presently inactivated fmd virus (fmdv) vaccines are used to control the disease in the endemic countries like india. the quality assurance of the vaccine depends on the efficacy of the vaccine in generating protective antibody without causing subclinical disease due to improper inactivation. since protective antibody response in vaccinated animals can not be distinguished from that of infected animals one needs to assay the antibody response against non structural proteins (nsps) and the vaccine must be free of contaminated nsps. production of vaccine free of nsps requires the cumbersome method of virus purification which adds to the cost of the vaccine. alternatively one may develop a positive marker vaccine by including a foreign protein or epitope which is not expected to be present in the vaccine and the antibodies generated against which helps in detecting the vaccine related response. here we report a molecular approach by which we introduced a immuno-dominant epitope of green fluorescent protein (gfp) into the structural protein gene of foot and mouth disease virus vaccine strain asia ( / ). our laboratory has produced a mini-genome of fmdv asia that lacks structural protein gene (p - a) coding for all the structural proteins (vp - ) of fmdv asia as a vector (pcfl dasia ). the p - a of the asia vaccine strain was cloned separately into a plasmid vector and by successive pcr mutagenesis and cloning we have introduced nucleotide sequence corresponding to amino acid epitope of gfp into p - a gene. gfp epitope was inserted by replacement at n-terminal region of vp- which is not immunogenic. the modified p - a was expressed in e. coli and studied. the modified p - a gene with gfp epitope was inserted into the pcfl dasia to get full length replication competent cdna cloned under cmv promoter in pcdna (pcflasiagfp). this can be used to produce synthetic virus with gfp epitope that can generate antibodies not only against neutralizing epitopes but also against gfp epitope. presence of antibody against gfp epitope in the vaccinated animal will reveal vaccine efficacy. elisa against gfp can be used as a companion test not only for safety evaluation but also for quick evaluation of efficacy. further absence of nsp antibodies in the serum may reveal the quality of the vaccine in respect of safety. self replicating dna vaccines are developed to achieve robust immune response through enhanced antigen production and gamma interferon expression in vaccinated animals. since self replicating dna vaccines induce gamma interferon expression which helps in viral clearance such vaccines are expected to be useful to cure even the carrier and persistently infected animals. understanding the events that help in the elicitation of both the arms of immune response in vaccinated animals is necessary to understand the effectiveness of the vaccine. the work presented here deals with the immunological evaluation of a sindbis virus replicase based dna vaccine carrying linked fmdv vp genes in vaccinated guinea pigs. we have constructed self replicating dna vaccine vector and to the down stream of a sub genomic promoter we have inserted secretory signal followed by linked-vp genes of fmdv serotypes (o-a-asia ) with glycine and proline bridge in between. guinea pigs were vaccinated with the construct and the sera at days post vaccination were evaluated both for cellular response by studying the cd levels and by mtt and cytokine profiles by real time assays. the humoral response was evaluated by studying cd levels in the whole blood by facs analysis and serum antibody levels by snt and elisa. the animals were challenged with gp infective dose of fmdv type 'o' virus lesions were scored. further the replicative efficiency of the challenge virus was studied by ab elisa. the results showed that all the assays except antibodies against ab protein have positive correlation with the protection. as expected the titre of the antibodies against ab protein was lower indicating that the challenge virus replication was inhibited in the vaccinated animals. the limited studies conducted by us showed that self replicating vaccine has a potentiality to emerge as potent vaccine for fmd. ganjam virus (ganjv) belongs to the genus nairoviruses (family bunyaviridae). these viruses cause diseases in livestock. it has been isolated from different animal hosts and tick vectors from india. genus nairoviruses includes a total of tick-borne viruses, classified into serogroups. the important serogroups are crimean congo hemorrhagic fever (cchf) and the nairobi sheep disease (nsd). the main members of the nsd group are nsd and dubge viruses. their genome consists of three segments of single stranded rna, viz. s, m and l that encodes viral nucleocapsid protein, viral glycoprotein g and g and the viral polymerase respectively. ganjv is very closely related to (nsdv). nsdv is found in east and central africa, causes very high morbidity and mortality in livestock. the present study involves phylogenetic comparison of ganjv isolates from india with other nairoviruses based on complete n gene. it will help to understand the kind of nucleotide (nt) and amino acid (aa) changes that have occurred in ganjv strains from different geographical areas. eight strains of ganjv isolated at niv during - from different parts of india were used in this study. virus stocks were prepared in vero e cell line these were used as the source of viral rna. the n gene was amplified either as a complete gene in one reaction or in fragments whenever necessary. thus obtained sequences were analyzed; annotated to get a consensus sequence, aligned against the sequence of prototype strain of ganjv and other representative nairoviruses. the nt sequences were converted to aa sequences and analysis was done at both nucleotide and amino acid levels. based on what nt or aa phylogenetic tree was constructed and compared with other nairoviruses (cchf, dugv, hazv, kupv and nsdv) where complete s segment sequences were available gen-bank database (ncbi). the phylogenetic data at both the nt and aa levels showed that all the strains of ganjv form monophyletic lineage with the nsdv. cchfv and hazv together formed another clade, whereas dugv and kupv made a separate branch in the tree. the different ganjv strains showed - % difference with nsdv at the nucleotide level and - % difference at the amino acid level. hazv showed - % difference at the nt level and % difference at the aa level with ganjv as well as nsdv. the present data obtained suggests that ganjv and nsdv are minor variants of the same virus. diarrhoeal syndrome is one of the major concerns of the livestock industry. most of the diarrheic cases in animals go unnoticed and limited attention is paid on viral etiology. presence of large amount of fecal matter in animal shed acts as a source of infection for calves via drinking water, feed, or contaminated soil. keeping this in view, investigation was planned to detect the association of rotaviruses with diarrhea in dairy calves and to observe the genomic diversity among the circulating viruses in tarai area of uttarakhand. a total of diarrheic fecal samples collected from instructional dairy farm, nagla, pantnagar, uttarakhand were screened during the study. samples were collected from both cow calves and buffalo calves in - months of age. for the diagnosis of rotavirus, all the fecal samples were subjected to rna-electrophoresis after nucleic acid extraction. viral genome segments were visualized by silver staining. out of the total samples tested, seven were found positive in rna-page showing typical genome segments migration pattern of bovine rotavirus. in the given samples prevalence of bovine rotavirus was . % and % in cow and buffalo calves, respectively. on the basis of migration patterns of rotavirus in rna-page, group a were identified with typical : : : pattern. variation within movement of various genome segments among isolates of bovine rotaviruses was observed during the study that may be indicative of emergence of mutants in the circulating isolates. the vp gene based group a specific rt-pcr was standardized and all the isolates in this area were confirmed to be of group a type. work is in progress to genotype the bovine rotaviruses of this region based on vp and vp genes. this study emphasizes the need to explore the prevalence of bovine group a rotaviruses in different places of uttarakhand and their genetic characterization which could help in selection of control strategies for rotavirus infections. foot-and-mouth disease (fmd) is endemic in india causing enormous economic loss to the animal keepers and trade embargo with fmd free countries in livestock and animal products. rapid diagnosis of fmd is of immense importance in prevention and control of the disease. fmd is initially diagnosed clinically and confirmed by laboratory tests. virus isolation in cell culture and sandwich elisa for antigen detection are commonly practiced in laboratories. the virus isolation though is very sensitive but it can be slow and analytical sensitivity of the elisa is lower and can not be used with certain sample types. the use of molecular techniques in the diagnostic laboratory has greatly increased the speed, specificity and sensitivity of fmd diagnostic tests. molecular techniques like rt-pcr, pcr-elsa and dot hybridization can be used with more success for detecting carrier animals and animals harboring sub-clinical infection and can be applied in a wide range of clinical sample types. these techniques can be used as genus and serotype specific test including detection of particular lineage/genotypes with in the serotype. multiplex pcr has been used to differentiate serotypes of fmdv and the technique is sensitive, experimentally simpler, cost effective and less time consuming. the assay can be used for serotyping on elisa negative samples. the molecular techniques not only help in diagnosis but also useful for epidemiological studies. lineage differentiating rt-pcr has been useful in identifying different lineages of serotype asia (lineage b, c and d) before proceeding with sequencing of d region. similarly genotype differentiating rt-pcr has been developed and used in differentiating two different genotypes of serotype a (genotype vi and vii). these assays have the potential to be applied on clinical samples directly, thereby saving much time needed for sample processing and nucleotide sequencing. recent development of real time rt-pcr methodology has allowed the diagnostic potential of molecular assays to be realised. advancement in real time pcr technology made it possible to combine several assays within a single tube which is in the progress in our laboratory. integration of these assays onto automated high throughput platforms provides diagnostic laboratories with the capability to test large numbers of samples. microarray technology was provided greater screening capabilities for pathogen detection. the microarray allows the addition of large number of oligonucleotide probes for identification of mutant pathogen and also for subtype determination. the combined properties of high sensitivity and specificity, low contamination risk, and speed has made realtime pcr and microarray technology a highly attractive alternative to conventional methods in increasing percentage of outbreaks confirmed and analyzed and for tracing the origin of fmd virus responsible for outbreaks. dna vaccines are expected to elicit both humoral and cellular responses, cellular response being long lasting. however the approach has several limitations like poor stability of dna, poor expression and risk of integration. poor expression becomes the major limitation in the case of fmd as fmdv proteins are poor immunogens. also dna vaccine vectors carrying only eukaryotic promoters elicit strong cmi response and weak humoral response. the methodology to achieve humoral response involves the expression and secretion of the expressed protein so that the antigen presenting cells will be able to process the antigen and produce humoral response. in case of fmd humoral response is as important as cellular response. the present project aims at addressing these issues; achieving higher expression and getting the protein secreted out by constructing self replicating gene vaccines for fmd and studying their efficacy. the vector for humoral immune response contains eef promoter, sindbis virus polymerase gene and secretory and anchoring signals. the integrity of the vectors was confirmed by sequence analysis. the linked polyvalent protein genes of fmdv serotype a, o and asia were cloned into the vectors and the presence of the insert was confirmed by restriction enzyme digestion. the functionality of the constructed dna vaccine vector (pvac self rep ) was assayed by transfecting the dna into bhk cell monolayer and studying the s labeled proteins in immuno-precipitation assays. the studies showed high level of expression in case of constructed vector as compared to infected virus for the specific protein. the secretion of the expressed protein was assayed by immuno-fluorescence assay and found to be positive. encouraged with these studies the preliminary studies were conducted on vaccine efficacy studies in guinea pig model. the immunized guinea pigs showed high antibody titres by snt and elisa, as compared to conventional dna vaccines (pup cd) even at / th of the dose. this approach of constructing self replicating dna vaccine for humoral response is the first report. genetically engineered microorganisms are important sources of industrial and medicinal proteins. over the past decade, plant host system has been investigated as potential host system for expressing proteins of therapeutic and diagnostic use. however concerns regarding the stability and environmental safety need to be addressed. chloroplast engineering is expected to resolve some of these issues since, plastids/chloroplasts are inherited maternally and are not disseminated through pollen. this makes plastid transformation a valuable tool for transgenic creation besides offering biological containment. since foot and mouth disease (fmd) of cloven footed animals is a major concern in the world over. foot and mouth disease (fmd) is the most feared, viral disease of the cloven footed animals causing heavy losses to the live stock industry. the disease is enzootic in many parts of the world including asia. the conventional vaccines for fmd have several limitations which include safety, temperature sensitivity and duration of immunity. attempts have been made to overcome these limitations using recombinant dna technology. amongst the newer vaccines, edible vaccines are cost effective and easy to administer. since the stability of the gene of interest is the major concern in the case of plant transgenics, marker genes are used for regular selection. the detection methods based on the available marker proteins like b glucoronidase (gus) protein/antibiotic selection are cumbersome and cost intensive. however selection based on herbicide resistance is much simpler and easy. hence in the present study, the -enolpyruvylshikimate- -phosphate synthase (epsp) gene was used as a marker along with the immunogen gene of fmdv. epsp is the key enzyme in the shikimate biosynthesis pathway necessary for the aromatic amino acids production. in order to investigate the mechanism of long term immunity and the effect of protective immunity induced by cationic plg micro particle coated dna vaccination. we constructed the expression plasmid containing a foot-and-mouth disease virus (fmdv) id gene sero type a. intramuscular vaccination of guinea pigs with the micro particles coated plasmid dna induced a strong antibody response and neutralization antibodies, cellular mediated immune response which lasted year. we further analyzed the persistence and expression of id gene by polymerase chain reaction and reverse transcriptase polymerase chain reaction and quantitative pcr. the results showed that id gene was present and expressed in the muscle cells up to year after days post vaccination. furthermore, guinea pigs vaccinated with micro particles coated plasmid dna were protected against a challenge with fmdv virus. therefore the micro particles coated plasmid dna vaccination dose induce a protective immunity and long term humoral, cellular immuno responses against fmdv, which could be maintained by persistent expression of id gene in muscle cells. foot and mouth disease virus (fmdv) causes a highly contagious viral disease of cloven hoofed animals, which has a considerable socioeconomic impact on the countries affected. interleukin- (il- ) enhances the il- driven th immune response that is important in immunity against intracellular pathogens. the multiple roles of il- in many physiological and pathological processes have generated a great deal of interest in recent years. antiviral effects of il- have been reported. we evaluated the effects interleukin- (il- ) on the replication of fmdv in vitro in bhk- cells. bovine il- mature protein coding sequence was amplified from the bovine pbmc cells and cloned into prokaryotic expression vector pet a. protein expressed was purified and specificity was confirmed by immunoblotting. bhk- cells were treated with purified expressed il- protein with ( lg/ml) h prior to fmd infection. cell culture supernatants were collected at h post infection were subjected for elisa and virus titration assay. rna extracted from the cells was subjected to real time pcr for viral rna quantification. log titer reduction was observed in the fmd virus titer in il- treated cells compared to the untreated cells where as virus antigen quantified by elisa has shown a reduction of -folds. -fold reduction in the fmd viral rna copy number was observed in the il- treated cell compared to the untreated measured by qpcr. current study demonstrated the potent anti viral activity of il- on fmdv by inhibiting the viral replication. these results further suggests that il- has the potential role of il- as molecular adjuvant in fmd vaccine development and development of therapeutic for fmd. foot and mouth disease is the most contagious viral disease of farm animals. control of the disease in animals is by vaccination and slaughtering of infected animals. conventional oil adjuvant vaccine has its own limitation. alternate to this genetic vaccines where the dna encoding viral antigen may be a promising approach. naked dna vaccine has limitations like poor uptake of dna by cells and more importantly by nucleus. as a result delivery of naked dna through calcium phosphate nanoparticle was attempted. calcium phosphate nanoparticle is a potential delivery agent which proved to enhance the immune response. fmdv p - cd ''o'' vaccine gene constructs in pcdna . ? entrapped by the nanoparticles was prepared by using different molarity of calcium chloride and disodium hydrogen orthophosphate. the nanoparticles entrapping fmdv p - cd ''o'' and naked dna were presented to the guinea pigs through intramuscular injection to study the mrna expression of antigen by rt-pcr. animals were sacrificed at defined time to collect different organs and total rna was extracted. each time blood was collected to analyse the fmdv specific serum antibodies. dna vaccines presented through calcium phosphate produced transcripts in the injected muscle up to days whereas naked dna up to days. serum antibody levels of naked dna vaccine showed antibody titre till days. whereas nanoparticle injected animals showed serum antibody till days. serum neutralization titres of . were observed in calcium phosphate dna vaccines at about - days, where as naked dna sn titers were observed for short period of - days. the study clearly showed calcium phosphate nanoparticle entrapping fmdv vaccine dna may be a better delivery system for dna vaccines as it confirms availability of the antigen and persistence of antibody for longer duration than naked dna. capripox is highly infectious, contagious, and oie notifiable disease of small ruminants, caused by sheeppox and goatpox viruses which are members of capripoxvirus genus of the family poxviridae. in the present study, we analyzed the partial gene sequences of p protein, an immunogenic envelope protein of capripox viruses (capv) to assess the genetic relationship among different sheep pox and goat pox virus isolates from different geographical areas of the country. product of this gene has been shown to be important in attachment of capv to host cell surface receptors during viral entry and host immune response. the following virus isolates have been used in the analysis: gtpv-uttarkashi, p , vaccine virus; gtpv mukteswar, p , challenge virus; gtpv (akola), gtpv bareilly/ , gtpv ladakh/ and gtpv sambalpur/ , field isolates and sppv srinagar, p ; sppv ranipet, p ; sppv-rf, p , vaccine viruses and sppv makdhoom/ , sppv cirg/ , sppv pune/ , sppv bareilly, sppv / and sppv / , field isolates. in this study, all virus isolates were confirmed by pcr amplification and analysed in pcr-restriction fragment length polymorphism (pcr-rflp) using ecori enzyme to confirm their specificity. further, the amplicons were cloned and sequenced commercially. nucleotide and the deduced amino acid (aa) sequences were compared with published sequences of the members of the genus capripox virus. sequence analysis of partial bp sequence has shown high sequence identity among all indian sppv and gtpv isolates at both nt and aa levels. it revealed a . - % and . for gtpv field isolates where as, % for sppv field isolates at both the nt and aa levels. in general, capv isolates in this study shown . - . and . % homology between gtpv and sppv at nt and aa levels as reported earlier. further, it revealed a unique change of g a in all gtpv isolates resulting in formation of drai site in place of ecori and possible development of restriction enzyme specific pcr-rflp for differentiation of sppv and gtpv from field isolates. orf or contagious ecthyma is considered as non-contagious, proliferative disease and is caused by orf virus of the genus parapox virus of the family poxviridae. it is reported most commonly in sheep and goats and also a zoonotic agent. camels are also infected by orf virus and reported in camel rearing countries as a mixed infection with camel pox, the later is caused by an orthopox virus. in india, there are few reports of the orf virus infection in camels and identified by clinical signs and pcr. in this study, we identified the presence of orf virus from clinical samples of suspected case of sporadic infection in camels by serological and molecular techniques. viral dna isolated from processed scabs used initially in nested polymerase chain reaction as diagnostic pcr which successfully amplified bp fragments and also sequenced to check the fidelity of the product. after confirming the infection by pcr, some of the structural and non-structural genes were amplified for sequence analysis. out of the five genes characterized, the major important one selected for sequence and phylogenetic analysis is b l gene which is homologous to a major envelope protein p k of vaccinia virus. full open reading frame of bp from orf b l was amplified by pcr, cloned and sequenced commercially. nucleotide and deduced amino acid sequences of b l were compared with other published sequences of the members of the genus papapox virus. sequence analysis shows a maximum percent identity of . and (indian orf virus isolates); . and . (other orf isolates); . and . (orf-camel/jodhpur/ ); and . (bovine popular stomatitis virus) and finally . and . (pseudo cowpox virus) respectively at nt and aa levels. phylogenetic analysis of the isolate was also performed using the neighbour joining method in mega program to know the phylogeny relatedness of the virus, which revealed that the isolate is well grouped with the jodhpur isolate and closely related to pseudo cowpox virus. it warrants further analysis of other potential genes to confirm the causative agent of the contagious ecthyma in camels as pseudo cowpox virus. chikungunya an arboviral disease is transmitted through the bite of an infected aedes mosquito. it causes a self limited febrile illness along with arthralgia and myalgia. in some cases neurological and severe hemorrhagic manifestations has been observed. chikv epidemic has been reported in africa, india, south east asian countries and during the current out break imported cases of chikv has been encountered in most of the european countries. the causative agent belongs to the genus alphavirus family togaviridae. human beings serve as the chikungunya virus reservoir host during epidemic periods. outside these periods the main reservoirs are monkeys, rodents, birds, and other unidentified vertebrates. antibodies to chikv have been detected in domestic animals. in the present study we surveyed madanapalli, palamaner, b. kotta kota and tirupati and collected a total of rodent samples, bovine samples; sheep samples and canine samples. total rna was isolated from all these samples and subjected to rt-pcr using a primer pair dvrchk-f/dvrchk-r which could amplify a bp e gene product specific to chikungunya virus (naresh kumar et al. ). all the serum samples were further screened for chikv specific igm antibodies using commercially available ctk biotech strips. none of the samples were found positive either for chikv specific rna or chikv specific igm antibodies. more number of samples from domestic animals as well as rodents are being screened to study their possible role if any in the maintenance of chikv in nature and during the inter epidemic periods. the present study discusses these aspects in detail. petunia hybrida is widely used as experimental host plant for begomovirus identification and its characterization. hitherto, natural infection of begomovirus on petunia has not been reported in india. recently, petunia hybrida grown in and around ludhiana were found to be depicting typical symptoms caused by begomovirus. the symptoms include severe reduction in leaf size, downward curling and distorted leaves. severely infected plant became bushy, stunted and produces no flower. total genomic dna was extracted from the plants showing symptoms of begomovirus, by ctab method. the presence of virus was confirmed by using degenerated primers, designed to identify all the begomovirus prevailing in the world. to identify the strain associated with the disease, the positive samples along with healthy control were tested against different strain specific primers of tomato leaf curl virus, so far reported in india i.e. tomato leaf curl new delhi virus, tomato leaf curl palampur virus, tomato leaf curl banglore virus, tomato leaf curl karnataka virus and tomato leaf curl gujarat virus. among these, only tomato leaf curl new delhi virus specific primer was able to give the desired amplicon of * bp. hence, it is confirmed that the leaf curl disease of petunia hybrida is associated with tomato leaf curl new delhi virus. this disease of petunia can become a sever production constraint in coming years. from last years ( and ) it was observed that some varieties of brinjal grown in rainy season, showed typical leaf curl type of symptoms. the symptoms include upward curling of the leaves, cupping, vein thickening, reduction in leaf size and distortion of leaves. the severely infected plant remains stunted and bushy, became unproductive or produces only few fruits. the disease was experimentally transmitted from naturally infected brinjal to healthy seedlings by whiteflies (bemisia tabaci) and grafting, but not by mechanical or aphid transmission. to detect the begomovirus associated, total genomic dna was extracted from the plants showing disease symptoms. the presence of virus was confirmed by using pcr based begomovirus geneus-specific primers designed by deng et al., wyatt and brown and rojas et al. these degenerated primers give the expected product size of * , * and * bp, respectively. core coat protein (cp) gene and dna-b was also amplified in the samples using specific primers. to identify the strain associated with leaf curl virus, dna was subjected against primers of different indian tomato leaf curl virus strain i.e. tomato leaf curl new delhi virus, tomato leaf curl palampur virus, tomato leaf curl banglore virus, tomato leaf curl karnataka virus and tomato leaf curl gujarat virus, using pcr. among these, only tomato leaf curl new delhi virus primer was able to show the desired product size of * bp. therefore, it was confirmed that leaf curl disease of brinjal is caused by tomato leaf curl new delhi virus in association with satellite b-dna. to identify the strain associated with the disease, all samples were further subjected to the specific primers, designed to amplify all the tomato leaf curl virus strains, so far reported from india i.e. tomato leaf curl new delhi virus, tomato leaf curl palampur virus, tomato leaf curl banglore virus, tomato leaf curl karnataka virus and tomato leaf curl gujarat virus, using pcr. among these, only tomato leaf curl palampur virus specific primer was able to give the expected product size of * bp. this shows the association of tomato leaf curl palampur virus with leaf curl disease of calendula and marigold. thus, calendula and marigold can act as a reservoir for the tomato leaf curl palampur virus and may cause severe constrain in the production of these important ornamental plants. groundnut bud necrosis virus (gbnv) belongs to serogroup iv of the genus tospovirus in bynayaviridae family and infects several economically important crops all over india. the nucleocapsid protein (np) encoded by the small rna of gbnv encapsidates the viral rnas. apart from this structural role, the np has also been implicated in the replication, transcription, maturation and cell to cell movement. with a view to study the structure and function, the np of gbnvtomato isolate from karnataka was over expressed in e. coli and purified by ni-nta chromatography. the purified np was present as ribonucleoprotein complex and as heterogeneous mixture containing monomers, tetramers and higher order multimers. in order to determine the regions involved in oligomerization and nucleic acid binding, mutational approach was taken. n-and c-terminal deletion clones were generated (n np, n np, c np and c np), over expressed in e. coli, and were purified by a procedure identical to that used for the wild type protein. initial studies on oligomeric status suggested that in addition to n-and c-terminal regions there may be additional regions or residues which contribute to multimerization of np. the amount of rna bound to the truncated proteins was reduced in case of n np, n np and c np. interestingly removal of amino acid residues (natively unfolded region) from the c terminus resulted in complete loss of nucleic acid binding suggesting that the rna binding domain was located in c-terminal region of np. further np was observed to get phosphorylated in in vitro kinase assays by a kinase present in the soluble fraction of tobacco plant sap. both atp and gtp were utilized as phosporyl donors and mn ? was the preferred metal ion which suggests that np might be phosphorylated by a ck -like protein kinase. phosphorylation studies with n-and c-terminal truncated proteins revealed that the site of phosphorylation lies within the amino acid residues - . by mass spectrometric analysis of the protein threonine- and serine- were identified as possible phosphorylation sites. a naturally occurring isolate of virus infecting gherkin (cucumis anguira l.) showing mosaic symptoms of mosaic, leaf distortion and dark green islands in the lamina was identified in the export cultivars of gherkin grown in commercial fields of kuppam rural, chittoor district, andhra pradesh. the virus infection was deadly prevalent among the field that caused a lot of economic damage to the crop that resulted in yield losses and reduced quality of fruits meant for export. symptoms of the infected fruit included blistering and malformation of the fruit. the virus infected leaf samples were collected and initial host range tests were conducted with different cucurbit species showed that the host range include propagation hosts like cucumis anguira (gherkin), cucumis sativus, cucurbita pepo, cucumis melo, langeneria vulgaris, momordica charantia and local assay host like chenopodium amaranticolor. the virus host range was only restricted to cucurbit species and chenopodium. the virus was maintained for further studies on cucurbita pepo by sap or mechanical inoculation. the virus induced mosaic, vein clearing symptoms on pumpkin. electron microscopy of the leaf dip preparations stained with % uranyl acetate from the pumpkin leaves showing symptoms revealed the presence of a long flexuous filamentous particle measuring nm. the virus positively reacted to the polyclonal antisera of papaya ringspot virus-w, potato virus y, tobacco etch virus and also strongly reacted with the polyclonal antiserum of zucchini yellow mosaic virus in direct antigen coated-enzyme linked immunosorbent assay (dac-elisa). because of very strong reaction to polyclonal antisera of zucchini yellow mosaic virus, we tried to amplify the partial nib and cp genes of the virus along with the utr by using two primers zy gctccatacatagctgag acagc and zy taggctttttgcaaacggagtcta at c . total rna from gherkin infected leaves was isolated using trizol ls reagent (sigma). rt-pcr was performed to obtain an amplicon of * . kbp, cloned into fermentas ptz r/t vector and sequenced at mwg biotech, bangalore. sequence analysis revealed that the virus was isolate of zucchini yellow mosaic virus and was showing % of homology to that of the zucchini yellow mosaic virus strain b genome ay and zucchini yellow mosaic virus nat genome ef which were strains reported from israel. the sequence of the present study was submitted to the genbank gq . the results state a suspicion that the virus could have been mobilized by some infected source brought by the commercial israeli based companies into india due to poor quarantine regulations as the gherkin cultivation in these regions is chiefly supported, purchased, exported and marketed by these private companies that are based from israel. this is the first report on molecular characterisation of zucchini yellow mosaic virus infecting cucumis anguira (gherkin) from india. they also exhibited synergism with other virus which was region specific. fifty percent of the total symptomatic plant population was found be positive only for carla while remaining showed mixed infection of carla with tospo in some regions while in others carla virus was found to be associated with cmv. presence of only carlavirus was up to - % incidence, without association of tospo, cmv, poty or tobamo viruses was also observed in some fields. avijit tarafdar, raju ghosh, k. k. biswas plant virology unit, division of plant pathology, indian agricultural research institute, new delhi citrus tristeza virus (ctv), a brown citrus aphid (toxoptera citricidus) transmitted closterovirus under family closteroviridae, is one of the major limiting factors in cultivation of citrus worldwide. ctv is a longest known plant virus having flexuous particle of nm in size. ctv genome is a positive sense ssrna of about kb nucleotide containing open reading frames (orfs) encoding proteins. several biological as well as genetic variants of ctv are reported in all the citrus growing countries in the world. ctv causes decline and death of millions of citrus trees in the world. in india, ctv is a century old problem, and has killed an estimated one million citrus trees till today. in molecular and genetic level, ctv isolates from india were not fully characterized. genetic diversity and sequence divergence in ctv isolates of india are not fully established. further, evidence of recombination and causes of evolution of ctv variants in india have not been studied till date. therefore, in the present study, effort has been made to characterize several indian ctv isolates in genetic level, examine their genetic diversity, identify recombination events and analyze evolution of divergent ctv. a total number of ctv isolates from different regions of india ( from darjeeling hills, five from bangalore, from delhi and from vidarbha) were under taken for genetic study. two genomic regions of ctv, i.e., entire cp gene (cpg) ( nt) and a gene fragment of orf a (orf a) ( nt) were amplified, cloned, sequenced and nucleotides were analyzed. based on cpg, indian isolates shared - % nucleotide identity, and based on orf a they shared - % identity, among them. incongruence of phylogenetic relationship was observed as on sequence analysis five phylogenetic clades based on cpg, and eight clades based on orf a, were generated suggesting the recombination events have been occurred between the sequences of indian ctv isolates. thus, to identify the potential recombination events, and determine the parental sequences in ctv isolates, six recombination detecting algorithms, namely, rdp, genconv, bootscan, maxchi, chimera and siscan were used. out of indian ctv, cpg of and orf a of isolates of ctv showed recombination events suggesting orf a was more prone and fragile to rna recombination as compared to cpg. this findings indicated that high degrees of genetic diversity and incongruent relationships of indian ctv isolates are due to genetic recombination occurred, which may be the important factors in driving evolution ctv variants in india, that was also supported by a splittree decomposition analysis. b. v. bhaskara reddy, y. sivaprasad, k. rekha rani, k. raja reddy department of plant pathology, regional agricultural research station, acharya n.g. ranga agricultural university, tirupati, andhra pradesh sunflower (helianthus annus l.) is one of the most important oil seed crops in the world which ranks third in area after soyabean and groundnut. the sunflower necrosis disease (snd) is characterized by necrosis of leaves, necrosis streaks on petioles, stem, floral parts and stunted growth. the causal agent of the disease has been identified as tobacco streak virus (tsv) which belongs to genus ilarvirus of the family bromoviridae. the suspected tsv infected sunflower samples collected from chittoor district in andhra pradesh were found positive for tsv-dac elisa. total rna was extracted from sunflower using rnaeasy isolation kit (qiagen). the tsv coat protein (cp) gene, movement protein (mp) gene and replicase (rep) gene were amplified by rt-pcr with specific primers, cloned in ptz r/t vector, sequenced and deposited in genbank (gu , gu and gu ). the size of cloned cp gene was bp and codes for amino acids. the cp gene sequence analysis revealed that the tsv-tpt infecting sunflower has - % homology at nucleotide level with soybean, tagietus-tpt and okra-tn isolates and - % homology at amino acid level. the movement protein gene was bp and codes for amino acids. the mp gene sequence analysis showed that it has - % homology at nucleotide level and - % at aminoacid level. chilli (capsicum annuum), the important commercial vegetable/spice of himachal pradesh, is affected by several viral diseases; of them cucumo, tospo, poty and gemini viruses are the most common genera. however, these viruses are not identified clearly and characterized fully, which are foremost needed to formulate the management strategy. therefore, in the present study, effort has been made to identify and characterize the important viruses causing diseases in chilli. in this study, several farms in major chilli growing areas of bilaspur and kangra districts in himachal pradesh were surveyed and infected plant samples were collected randomly. virus infection in these samples were detected by das-elisa using antisera to cucumber mosaic virus (cmv) and potyvirus (group specific) and through slot-blot hybridization (sbh) using cmv, iris severe mosaic poty virus (ismv), tomato spotted wilt tospo virus (tswv) and chilli leaf curl gemini virus (clcuv). based on das-elisa and sbh, the incidence of disease was estimated and ranged from . to . % by cmv and . to . % by potyvirus. to detect tospo and geminivirus in the infected chilli, sbh test was carried out. infected samples showed maximum virus titer in both das-elisa and sbh test were further confirmed by pcr using specific primers. desired sizes of amplicons; * bp, * bp, * bp and * bp of cmv, poty, gemini and tospo viruses, respectively, were obtained. as the present study clearly indicated that cmv appeared as a major one among the viruses infecting chilli in the hilly region of himachal pradesh, two isolates of cmv were characterized in genetic level. thus the amplified products (* bp) of cmv, palampur and palampur were cloned in pgemt cloning vector, sequenced and the sequences were submitted to ncbi database (palampur : acc-fm and palampur : acc-fm ). the sequences were then analyzed and compared with other sequences available in the data base. based on sequence analysis, it was found that present cmv isolates shared % nucleotide identity between them, are closely related with australian cmv isolate cmv-ly (acc-af ) by % nucleotide identity. in phylogenetic tree analysis, it was observed that indian cmv isolates formed same cluster along with cmv-ly. as it is known that cmv subgroup ii comprises cmv-ly, it is concluded that the cmvs of this hilly region of himachal pradesh belong to subgroup ii. chilli is essentially a crop of the tropics and grows better in hotter regions. chlii (capsicum annuum), a member of family solanaceae is an important vegetable and spice crop of immense commercial importance. the pungency in pepper is due to an alkaloid known as capsaicine and peppers are characterized as sweet, hot or mild depending on capsaicine content. the present investigation were conducted to find out the highly resistant cultivars of capsicum annuum against cmv and tylcv among ten cultivars of chilli in agroclimatic condition of aligarh. the highest ( and ) percentage of infection was observed in hc- and kalyanpur type- by showing the positive reaction to cmv by elisa test. no symptoms was recorded in case of bc- , lca- and jca- and showed negative reaction to cmv by elisa. bc- and lca- also showed negative reaction to tylcv by elisa and these were symptomless. maximum infection ( and ) was registered in hc- and c , cultivar. so, the bc- , lca- and jca- has proved highly resistant varieties against cmv and tylcv and these may be used in breeding programmes against viruses. cotton leaf curl virus belongs to the family geminiviridae, genus begomovirus. the members of this family contain circular single stranded dna molecules as their genomes. there are two kinds of begomoviruses-bipartite viruses with genomes consisting of two dna molecules designated dna-a and dna-b and the monopartite viruses which contain only dna-a but not dna-b. in monopartite viruses, the dna-a is accompanied by a small circular dna molecule called dna-b which is essential for the development of typical disease symptoms. cotton leaf curl virus is a monopartite virus and causes the cotton leaf curl disease which has emerged as a major disease of cotton in the indian subcontinent. the non-structural protein ac of cotton leaf curl kokhran virus-dabawali isolate (clcukv-dab) was cloned into pgex x vector and overexpressed in bl (de )plyss e. coli cells. the overexpressed gst-ac protein was purified by glutathione sepharose chromatography. the purified gst-ac protein was found to possess atpase activity. the optimum temperature and ph for the activity were °c and . respectively. the atpase activity was inhibited in presence of edta, showing that it is dependent on divalent metal ions. the activity was supported by magnesium, manganese and zinc ions but inhibited in presence of calcium ions. it was also inhibited by the non-hydrolyzable atp analogue adenosine-b, c-imido triphosphate and in the presence of other nucleotides like ctp and gtp. the k m and the v max of the reaction for atp as the substrate are . mm and . nmol/min/ mg of the protein respectively. the enzyme could also utilize gtp as the substrate. the fact that ac is specifically an ntpase and not a general phosphatase is revealed by the finding that it does not hydrolyze p-nitrophenyl phosphate to yield yellow colour while a similar reaction carried out in parallel with alkaline phosphatase readily yields the colour. it has been suggested earlier that ac may be involved in cell to cell movement of the virus (rojas et al. ) . it is possible that by its ability to hydrolyze atp, ac serves to power viral movement in the plant. thirteen sugarcane yellow leaf virus isolates causing yellow midrib and irregular yellow spot pattern from six states of india were characterized by rt-pcr assays. scylv- f and scylv- r primers were used as forward and reverse primer pairs and the amplified products were cloned and sequenced. comparative coat protein sequence analysis confirmed that all the scylv-indian isolates were clustered into two major groups confirming the existence of two strains of scylv affecting sugarcane crops of india. in a separate experiment, the member of both of the phylogenetic groups were found to be transmitted by the sugarcane aphid, melanaphis sacchari from infected to healthy sugarcane suggesting its secondary spread in nature. the symptoms produced by the virus causing cotton mosaic disease were little bit different in both sap inoculation and under natural field condition. in natural field condition it has shown clear chlorosis type of symptoms on major leaves of plants but in sap inoculated plants veinal chlorosis and mosaic type of symptoms are found to be common. in field conditions infected plants grows erect and have less boll formation. there is no effect found on seed shape or seed size. the initial symptoms produced on cotton leaves after inoculation were wonderful. local lesions observed in second week from inoculation and then they changes to chlorotic type of symptoms and some are necrotic symptoms also. the plants at early stage are found to be affected, has less lateral branch development and hence reduction in yield production. the naturally field infected plants showing good symptoms are also difficult to identify in lateral stage of plant. because they disappear with time. the virus is very easily sap transmissible. the virus is found to be transmitted by thrips palmi and thrips tobacci in persistent manner. no seed transmission is observed. virus showed same physical properties as it shows in stem necrosis of peanut or sunflower necrosis disease. the physical properties are found to be thermal inactivation point (tip) - °c, dilution end point (dep) - to - and longevity in vitro (liv) h, virus infecting nineteen different host plants are identified belonging to five different types of families viz. malvaceae, chenopodiaceae, compositae, leguminaceae and solanaceae. however they found to produce same types of symptoms as in most of the host that have been tested before. in elisa test report it is found that the virus showing positive test only with anti serum of tsv of a cowpea and cotton but negative reaction with pbnv of cowpea and cotton which clearly denied possibility of presence of pbnv in cotton producing these kinds of symptoms elisa report clearly shows that tsv antiserum of cowpea is showing positive results with clear chlorotic types of symptoms. a powerful approach to functional genomics, and an alternative to the massive generation of transgenic plants, is the use of the recently described virus induced gene silencing (vigs) process, which allows viral vectors to knock out the function of a gene-of-interest. vigs is based on a silencing mechanism that regulates gene expression by the specific degradation of rna. as a tool for reverse genetics, vigs has many advantages over other common ways to study gene function because of the ability of viruses to replicate and move systemically within a plant. vigs can generate a phenocopy of a mutant without all the troubles of traditional methods of mutagenesis. geminiviruses with their small dna genomes and ease of inoculation through agrobacterium, are excellent candidates for vigs vector development. as a first step, the geminivirus bhendi yellow vein mosaic virus, characterized in our lab (jose and usha, virology : [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ) has been chosen. the satellite b dna associated with this virus has a single open reading frame (bc ). bc is essential for symptom development but not for replication. therefore, bc has been replaced by a multiple cloning site harbouring sali, xbai, bamhi, bsrgi and xhoi, initially in a cloning vector and then in the binary vector containing the partial tandem repeat of the b dna. in the place of the bc orf, the plant phytoene desaturase gene has been cloned and the resulting construct was used for agroinfiltration along with the partial tandem repeat clone of the begomovirus (dna a component chilli (capsicum annuum l.) plants exhibiting prominent symptoms of begomovirus like: leaf curl, vein swelling, shortening of petioles, crowding of leaves and stunting of plants were collected from rorkee, uttarakhand and dhaulpur, rajasthan, india. total genomic dna was isolated from naturally infected chilli samples and pcr was carried out with coat protein (located in dna-a) gene specific primers. as expected to the primers, * bp dna fragments were amplified from the infected chilli samples. to know the bipartite nature of the virus isolates, nuclear shuttle protein (located in dna-b) gene specific primers were employed which also resulted in positive amplification of * bp dna bands with all the coat protein tested positive samples. to ascertain the association of dnab component with the virus isolates, a set of dna-b specific primers were used which resulted in positive amplification of full length (* . kb) dna bands in the chilli samples collected from rorkee, uttarakhand, however, multiple sizes bands were resulted with the samples collected from dhaulpur, rajasthan. these findings confirmed that both the virus isolates under study are bipartite begomovirus associated with dna-b satellite. the sequencing of the pcr products is under progress which analysis will be discussed. groundnut bud necrosis virus (gbnv) belonging to the genus tospovirus, which is a unique member of the family bunyaviridae, infects several economically important crops. the virus has three genomic ssrna segments namely s (ambisense), m (ambisense) and l (negative sense). the s rna codes for nucleoprotein (np) and non-structural protein (nss) from viral complimentary and viral strands respectively. many viral nonstructural proteins such as ns of hepatitis c virus, yellow fever virus, dengue virus, sv large t antigen and cytoplasmic inclusion protein of tamarillo mosaic potyvirus are known to exhibit rna/dna stimulated ntpase, dntpase and helicase activity. nss of gbnv does not have any sequence similarity with any of the above mentioned viral rna/dna helicases but has a ntp binding domain. however, it has been implicated as suppressor of gene silencing in vivo. with a view to elucidate the mechanism by which nss could act as a suppressor of gene silencing and examine the other potential roles of nss in the life cycle of the virus, the gbnv (to) nss was over-expressed in e. coli and purified by ni-nta chromatography. in vitro studies with the purified rnss suggest that it exhibits an rna stimulated ntpase activity. many of the proteins that possess the rna/ dna stimulated ntpase and datpase activity, are also shown to have atp dependent nucleic acid unwinding activity. it was therefore of interest to examine whether nss has the nucleic acid unwinding activity. the helicase assays revealed that nss has dna/rna helicase activity. helicase activity of nss was absolutely dependent on atp and mg ? ion. nss could unwind dsdna substrate with overhang, or overhang. mutation of the crucial lysine in walker motif a (k ) severely affected the unwinding activity where as mutation of aspartate residue in walker motif b (d ) resulted in only % loss of activity. in this regard, rnss is a unique enzyme which does not have the canonical helicase motifs but can catalyze dsdna/dsrna unwinding in an atp and mg ? dependent manner. the rnss might act as a suppressor of by unwinding the dsrna, the substrate for dicer. in addition to being a suppressor of ptgs, nss may also regulate the viral replication and transcription by modulating the secondary structure of the viral genome. this new research finding on nss might pave way for further studies on its role in viral replication and transcription. yellow vein mosaic disease of pumpkin (cucurbita moschata) poses a serious threat to the cultivation of this crop in india. the disease was found to be associated with whitefly-transmitted bipartite begomoviruses were detected in varanasi field using polymerase chain reaction (pcr) with primer design through coat protein conserved region of begomoviruses from ncbi database. all plant samples showing symptoms were infected with begomovirus. the virus species were provisionally identified by sequencing * bp of the viral coat protein gene (av ageratum conyzoides is commonly known as billygoat-weed, chick weed, goatweed and whiteweed. in india it is popularly known as bill goat weed. it is an annual herbaceous plant with a long history of traditional medicinal uses in several countries of the world and also reputed to possess varied medicinal properties including the treatment of wounds and burns. in cameroon and congo, it is used traditionally to treat fever, rheumatism, headache, and colic. during survey in and around gorakhpur in , ageratum plants were found affected with the symptoms of leaf curling, mosaic mottling and leaf yellows. the infected leaf samples were processed for virus identification and association with pcr assays. total dna was extracted and pcr were performed with begomovirus specific primers (tlcv-cp). a * bp band was consistently amplified on % agarose. the pcr products were directly sequenced and sequence was submitted in genbank with the accession no. gq . the blast search analysis showed highest similarity of % with the ageratum enation virus. vernonia cinerea leaves with yellow vein symptoms were collected around crop fields in madurai. a bp product amplified from total dna extracted from symptomatic leaves with degenerate primers designed to amplify a part of the av gene from begomoviral dna a component was cloned and sequenced. based on the above sequences, specific primers were designed and the full length dna a of nucleotides with typical genome organization of begomoviral dna a was obtained and was submitted to embl data base (acc no: am ). the sequence comparison with other begomoviruses revealed the closest identity ( %) with emilia yellow vein virus from china and less than % with all known begomoviruses. the international committee on taxonomy of viruses (ictv) has therefore recognized vernonia yellow vein virus (vyvv) as a distinct begomovirus species. conventional pcr could not amplify the dna b or dna b from the infected tissue. however, the b dna ( bp) associated with the disease was obtained (acc no: fn ) by the rolling circle amplification-restriction fragment length polymorphism method (rca-rflp) using phi dna polymerase. sequence analysis shows that dna b of vyvv has the highest identity ( %) with dna b of ageratum leaf curl disease and - % with the b dna associated with other begomoviruses. infectious clones of vyvv dna a and dna b as dimers were made using the products of rca-rflp. these infectious clones will be used for agroinfection of vernonia and the results will be discussed. this is the first report of the molecular characterization of vernonia yellow vein virus (vyvv) from vernonia cinerea in india. production of bulb and seed crop of onion (allium cepa l.) is hampered by onion yellow dwarf virus (oydv) and iris yellow spot virus (iysv) with an incidence of . % and . % in bulb crop and . % and . % in seed crop, respectively in the popularly grown cv. hisar- . four symptom-based variants of oydv designated as grade a, b, c and d produced varied types of symptoms in onion crop incurring heavy losses in bulb and seed production. iysv caused tiny hay coloured spots of different shapes and sizes on leaves and scapes which later coalesced and led to drying and lodging of scapes. the plant height, bulb weight and bulb size were . cm, . g and . cm in plants infected with oydv, . cm, . g and . cm in iysv infection, . cm, . g and . cm due to their combined infection, as compared to . cm, . g and . cm respectively, in healthy plants of bulb crop. in plants infected with oydv grade a the plant height was minimum ( . cm) whereas the number of umbels was maximum ( . umbels/pl.) but other yield parameters viz., weight/umbel ( . g), number of seeds/umbel ( ), seed weight/umbel ( . g) and seed yield/plant ( . g) were recorded to be the lowest. the minimum reduction in plant height ( . cm), weight/umbel ( . g), number of seeds/umbel ( ), seed weight/umbel ( . g) and seed yield/plant ( . g) were recorded in oydv grade d. the plant height was . cm with . umbels per plant, . g weight/umbel, seeds/umbel, . g seed weight/umbel and . g seed yield/plant in iysv infected plants. the plant height ( . cm), umbels/plant ( . ), weight/umbel ( . g), number of seeds/umbel ( ), seed weight/umbel ( . g) and seed yield/plant ( . g) were found to be the lowest in combined infection of oydv and iysv diseases in comparison to higher values in healthy controls ( . cm, . , . g, , . g, . g, respectively). a minimum reduction in the test weight, germination and seed vigour index were found ( . g, . % and ) due to oydv grade a infection, whereas these were . g, . % and in iysv disease infected plants and . g, . % and in combined infection of oydv and iysv diseases in comparison to . g, . % and in healthy plants. the maximum hampering of seed vigour parameters was recorded due to iysv infection. lodging of scapes caused by this disease was responsible for heavy losses in seed production and seed quality. cotton leaf curl disease is one of the major threats to cotton cultivation from northern india. survey conducted during , observed the disease incidence ranged from to % from bhatinda, abohar, fazilka, sri ganganagar, hanumanghar. in order to study genetic variability in the virus, twelve clcuv isolates were partially characterized ( bp common region, full length av gene and partial sequences of ac and av gene). full length characterization of representative isolates from bhatinda, abohar, fazilka, sri ganganagar, hanumanghar is under progress. partial sequence analysis of clcuv isolates revealed that, the virus isolates collected during cropping season are closely related to cotton leaf curl burewala virus from pakistan and results were discussed. pratibha singh, h. s. savithri department of biochemistry, indian institute of science, bangalore tospoviruses, belonging to the family bunyavirideae, infect economically important plants such as groundnut, tomato, watermelon etc. they have a tripartite genome, with l, m and s segments of rna, in pseudo circular (panhandle) form. the viral genomes encode four structural proteins (l, n, g and g ) in the antisense orientation, and two non structural proteins nss and nsm in the sense orientation. the nsm is the only protein unique to tospoviruseses that infect plants in the bunyaviridae family and hence is proposed to be important for cell to cell movement. ground nut bud necrosis virus (gbnv), a member of the tospovirus genus, is the most prevalent virus infecting several species of leguminosae and solanaceae plants in india. total rna was isolated from gbnv infected tomato leaves and rt-pcr was performed using appropriate primers to amplify the nsm gene. the pcr product was cloned in pgex x vector. the recombinant nsm clone was transformed into bl (de ) e. coli cells and over-expressed by induction with . mm iptg. sds-page analysis of induced and uninduced fraction revealed the presence of overexpressed protein of expected size. the soluble gst-nsm was purified by gsh sepharose affinity chromatography. purified gst-nsm was shown to interact with in vitro transcribed rna transcript by electrophoretic mobility shift assay. further nsm was shown to interact with viral encoded proteins np and nss using elisa and yeast two hybrid system. nsm was also shown to be phosphorylated in vitro by pellet fraction of plant sap. thus the recombinant gbnv nsm possesses the characteristic features of a movement protein such as nucleic acid binding, interaction with nucleocapsid protein, and ability to undergo posttranslational modification. solanum melongena, commonly called as egg plant is one of the most important vegetable crop in the world. it is cultivated widely in the tropical and sub tropical regions. several viruses such as cucumber mosaic cucumo virus (cmv), potato virus-y (pvy), potato virus-x (pvx) and tobacco ring spot virus (trsv) infect egg plant under natural conditions. in india major crop losses due to cmv infection in brinjal is % (fao stat- ) . in the present study the infected leaf samples were collected from local fields of ramapuram, chandamama palli, chandragiri, madanapalli, yadhamari, durgasamudram villages in and around tirupati, were tested for cmv infection by dac-elisa with cmv antisera. the resulting positive samples were further inoculated to the raised brinjal seedlings of selected varieties through mechanical sap inoculation. different varieties of brinjal like mullabadhine, ankhur, ravya, mattigulla, casper and easter egg were used for monitoring the susceptibility to cmv infection. the mosaic symptoms were observed after weeks of inoculation in all varities of brinjal except mullabadhina. among all these susceptible varities ankhur variety is selected to study induced biochemical changes such as chlorophylls, carbohydrates, proteins, nucleic acids and polyphenol oxidases in cmv infected brinjal leaves. in the infected leaves considerable reduction in chlorophyll and starch and increase in total proteins, sugars, rna and polyphenol oxidases was observed when compared to healthy leaves. the amount of total starch, protein and dna decreased to about , and lg/g respectively in infected leaves, where as sugars ( lg/g), rna content ( lg/g) and polyphenol oxidase activity was increased as compared to healthy leaves. the above results suggests that there is an altered concentrations of chlorophyll, proteins, nucleic acids, carbohydrates and polyphenol oxidase activity in the brinjal leaves due to the effect of cucumber mosaic cucumo virus infection. leaf analysis was found to be used as widely accepted diagnostic tool to assess the nutritional status of the vegetables. the present study deals with these aspects in detail. the total rna and dna was isolated from infected leaf samples. rt-pcr assays were performed using sugarcane yellow leaf virus (scylv) specific primers (scylv- f and scylv- r). the infection of scylv was detected in all the collected samples, which showed the expected size (* bp) amplicon during rt-pcr. in another experiment with nested pcr analysis, a phytoplasma characteristic . kb rdna pcr product were amplified from dnas of all infected samples but not in healthy sugarcane plants tested using phytoplasma universal primer pairs p /p and fu /ru . dna extracts from plants with yellow mid rib and leaf yellows produced products of bp, which gave typical phytoplasma profiles when digested with hae iii and hha i. no pcr amplifications were produced using dna from symptomless plants. our results suggest that the yellow mid rib and leaf yellows symptoms on sugarcane varieties in uttar pradesh and uttarakhand states of india exhibiting midrib yellowing and leaf yellows symptoms is mainly caused by mixed infection of scylv and scylp. the affected clumps showed reduction in stalk height as compared to healthy fields. thirty-one sugarcane mosaic isolates belonged to sugarcane mosaic virus (scmv) and sugarcane streak mosaic virus (scsmv were collected from china and india), confirmed in indirect elisa and rt-pcr amplification with scmv and scsmv-specific primers. the amplicons ( . kb) from the coding region of coat protein (cp) were cloned, sequenced and compared to each other as well as to the sequences of scmv isolates from sugarcane (australia, usa, china, brazil, mexico and south africa), maize (australia, china, iranian) and one scsmv isolate from sugarcane (india) in genbank. maximum likelihood and maximum parsimony analyses robustly supported two major monophyletic groups that were correlated with the host of origin: the scmv subgroup that included isolates from china and only isolates from india, and the scsmv subgroup that contained all isolates from india. maize dwarf mosaic virus (mdmv) and johnsongrass mosaic virus (jgmv) were not detected in any of the samples tested. a strong correlation was observed between the sugarcane groups and the geographical origin of the scmv isolates. the millable sugarcane samples from china contained a virus tentatively described as sorghum mosaic virus (srmv). three isolates from nine chewing canes in fujian, yunnan and guizhou provinces of china also contained srmv, and the other samples including five isolates from india was found infected with scmv. no srmv infection has been detected in sugarcane mosaic samples from india. sequence comparisons and phylogenetic analysis indicated that srmv can be considered as the most common and prevalent potyvirus infecting sugarcane in china, however in india sugarcane streak mosaic virus is dominant in causing mosaic symptoms on sugarcane. dig-labeled dna probe complementary to coat protein (cp) region of tobacco streak virus (tsv) sunflower isolate was designed for the sensitive and broad-spectrum detection of tsv isolates, the most devastating virus in india. dot-blot and tissue print hybridizations with the digoxigenin labeled probe were performed for the tsv detection at field levels. here, dot-blot hybridization was used to check a wide number of tsv isolates with a single probe and sensitivity with different sample extraction methods. the probe with cp conserved region prepared from sunflower pcr amplicon was hybridized with the tsv field isolates of gherkin, pumpkin, sunflower, marigold and globe amaranth samples because of highly conserved with little variability in cp region. the sensitivity limits were decreased from total nucleic acid to partially purified and crude extract preparations. in particular, tissue blot hybridization offers a simple, reliable procedure as dot-blot, but requires no sample processing. because there is minimal sample preparation, tissue-print hybridization could be an important component of tsv management programs. thus, the above non-radioactive labeled probe techniques can facilitate in screening the samples during tsv outbreaks and in quarantine services. savita patil, rupali sawant*, k. banerjee virology group, agharkar research institute, macs, g.g. agarkar road, pune two mycobacterium smegmatis strains (ari lab nos. v and v ) were employed for the isolation of mycobacteriophages from soil and sewage samples. mycobacteriophages were isolated from soil samples collected from an area surrounding the tuberculosis (tb) ward, naidu hospital, pune, against m. smegmatis strain v . these were numbered as v , v and v and were isolated by using washed-cell preparation method. the bacteriophages against the other m. smegmatis strain, i.e. v , were isolated from soil samples (collected from around tb ward, sassoon hospital, pune). some of these phages (viz.v , v ) showed plaques at °c but not at °c. thus they seem to be lysogenic. for propagating and increasing the titre of all the above isolates, various previously described methods were attempted, but none of these methods were satisfactory. but when siliconized glassware and plastic-ware were used, propagation was successful. we showed that siliconization of glassware and plastic-ware was essential for the propagation of our mycobacteriophage isolates v , v , v , v and v . also, phage dilution medium (pdm) as described by chaterjee et al. ( ) was found to be effective for picking out of the plaques made by the phages. in this way, the phage isolates were propagated up to p . the various passages of the phage isolates v , v , v , v and v (i.e. original, p , p and p ) were stored at - °c. pvp- effect on pigments due to geminivirus infection on cowpea (vigna unguiculata) shail pande*, naveen pandey, k. shukla mahatma gandhi p. g. college gorakhpur, d.d.u. gorakhpur university, gorakhpur geminiviruses are one of the most important group of viruses causing economic losses in tropics. the symptom produced are yellowing of leaves which directly affect the pigments of diseased plants it in turn affects productivity and yield of diseased plant. cowpea vigna unguiculata is one of the important crop cultivated throughout india for its green pods which are used as vegetables and seeds are used as pulse. cowpea is affected by many viruses amongst them geminiviruses are one of the important virus on the cowpea plant. in the present study total chlorophyll content was studied in leaf of cowpea of diseased and healthy plants using arnon's method. carotenoids were also studied using ikan's method. it was found that chlorophyll content in diseased plants were lower compared to healthy plant similar results were found with carotenoids so the geminivirruses infection lowers the chlorophyll and carotenoid content in diseased plants which reduces yield of diseased cowpea plant. shweta sharma , amrita banerjee , j. tarafdar , r. rabindran , indranil dasgupta * department of plant molecular biology, university of delhi, south campus, new delhi; bidhan chandra krishi vishwavidayalaya, kalyani, nadia, west bengal ; tamil nadu agricultural university, coimbatore, tamil nadu rice tungro disease is an important disease of rice, caused by a joint infection by two viruses: rice tungro spherical virus (rtsv) and rice tungro bacilliform virus (rtbv) in south and southeast asia. the complex of rtbv and rtsv is transmitted by an insect vector green leaf hopper (glh). previously we reported complete genomic sequences of two geographically distinct isolates of rtbv; rtbv-wb (west bengal) and rtbv-ap (andhra pradesh) collected from the field in mid- s. both the sequences showed high homology all along the genome but showed divergence from previously reported southeast asian isolate i.e. rtbv-phil (philippines). to check whether a time period of a decade has resulted into variability in the genomic sequence of different isolates of rtbv in india, we cloned and sequenced the complete genome of rtbv from two geographically distinct regions of india i.e. west bengal and kanyakumari collected from the field in . the complete nucleotide sequence of the dna fragments covering the whole genome of rtbv was determined using universal primers m f and m r and by primer walking, without any ambiguities remaining. the nucleotide sequences of overlapping clones were assembled and analyzed using the dna analysis software generunner and blastn program of ncbi. homology search at the nucleotide and amino acid level were performed using the blastn and blastp (respectively) programs of ncbi. multiple sequence alignments were performed using clus-tal-w software. sequence analysis results thus obtained showed that both the recently obtained complete genomic sequences of rtbv from two geographically distinct regions of india i.e. west bengal and kanyakumari showed very high homology (both at the nucleotide and amino acid levels) with the two previously reported rtbv isolates from india i.e. rtbv-wb (west bengal) and rtbv-ap (andhra pradesh) all along the genome. as observed earlier both the sequences diverged significantly from the southeast asian isolates. this suggests that even after the spatial and temporal difference (a time gap of approx years) between the two previously reported rtbv isolates and the recently reported one, there is very little sequence variability between them. this further strengthens the earlier reports that the rtbv genomes in india are highly conserved. homology search at the nucleotide level using blastn program with the previously existing rtbv isolates revealed a very high percentage identity of % with the rtbv west bengal isolate and % with the rtbv andhra pradesh isolate. this further strengthens the earlier reports that there is not much genetic variability in the rtbv genomes in indian subcontinent. complete genomic rna sequences of two geographically distinct isolates of rice tungro spherical virus (rtsv), a member of the genus waikavirus, family sequiviridae, were determined from india. out of the two previously reported sequences, the indian isolates were closer to the resistance breaking strain rtsv-[vt ] than rtsv- [phila] . between them, the indian sequences showed nucleotide as well as amino acid identities of %. a moderate homology was observed between the leader peptide and a putative helper component protein involved in insect transmission of the maize chlorotic dwarf virus, a closely related waikavirus, indicating its possible transmission-related function. unlike rice tungro bacilliform virus, which causes rice tungro disease jointly with rtsv, and is significantly different between isolates from india and philippines, rtsv genomes were observed to be much more conserved between isolates from the two countries. rice tungro bacilliform virus (rtbv) are believed to be the joint causative agents for the devastating tungro disease of rice prevalent in south and southeast asia [ ] . rice tungro disease has become the major cause of production losses in rice during last three decades in several rice growing states of india. here, we report, for the first time the complete sequence analysis of two geographically distinct indian isolates of rtsv. we analyze the deduced protein sequences and their phylogenetic relationship with the two complete rtsv sequences from philippines as well as with other members of sequiviridae family. we provide molecular evidence that the indian isolates of rtsv are closely related to those from the philippines. we had earlier reported that rtbv isolates between india and philippines differ significantly from each other [ ] . this study was undertaken in order to see whether rtsv isolates from india also show similar difference from those reported from the philippines. frequent outbreaks of tungro were reported near kanyakumari in the last - years. the present work was undertaken to clone and sequence the full-length rtbv and rtsv genomes from the infected rice plants collected from above region and to analyze the similarity of its genetic material with the existing indian isolates of rtbv and rtsv. a . kb dna fragment encoding the reverse transcriptase gene of rtbv genome was amplified and cloned in t/a vector and was sequenced commercially. homology search at the nucleotide level using blastn program with the previously existing rtbv isolates revealed a very high percentage identity of % with the rtbv west bengal isolate and % with the rtbv andhra pradesh isolate. this further strengthens the earlier reports that there is not much genetic variability in the rtbv genomes in indian subcontinent. similarly, the cp region of rtsv was amplified by rt-pcr and was cloned in t/a vector. recently, rice tungro disease has been reported from kanyakumari district of tamil nadu. it is important to determine the genetic nature of this isolate in order to develop resistance strategies. it is thus necessary to clone and characterize the viruses from kanyakumari and to determine the mechanism of virus resistance in transgenic lines. rice tungro disease is an important viral disease of rice. rice tungro is caused by infection by two viruses: rice tungro bacilliform virus (rtbv) and rice tungro spherical virus (rtsv). rtsv is a plant picornavirus with a kb single stranded rna genome. it belongs to genus waikavirus in the family sequiviridae and is necessary for transmission of the two viruses by the leafhopper vector nephotellix virescens. rtsv rna is translated to form a large polyprotein, which is then self cleaved to form the viral proteins, including the three coat proteins, replicase, protease. studies have been conducted on rtsv from philippines. correct information of sequence variability of viral isolates to check whether different geographical conditions like those present in india select for genotypically variable strain and to design for transgenic resistance strategy, information on rtsv from india is absolutely essential. the objective of this study was to clone rtsv isolates from india and compare the genetic diversity of indian isolates from other southeast asian isolates and amongst each other. also develop strategy to impair the attack of virus-complex on rice. the achieve this, complete genomes of two isolates from india were cloned by amplifying different genes by rt-pcr and subsequently cloned in ta vectors, followed by sequencing. subsequently constructs containing cp - , antisense replicase, sense replicase and double stranded replicase were cloned in plant transformation vector. these constructs were used to transform aromatic rice variety from indian-pusa basmati (pb ). pcr analysis of the above plants was done to check the stable insertion of insert in the transgenics. jatropha (jatropha curcas) of the family euphorbiaceae is being grown in india as a major commercial fuel (bio-diesel) crop. jatropha is cultivated in districts of potential states of india. unfortunately, the cultivation of jatropha is limited by the severe mosaic disease. recently, a severe mosaic disease with significant disease incidence was observed in - on j. curcas grown in experimental plots of nbri and j. gossypifolia, a weed growing road side around lucknow and kathaupahadi, madhya pradesh. the disease consisted of the symptoms of severe mosaic, blistering, leaf distortion and stunting of whole plant and no fruit/seed production in severely affected plants. symptomatology and whitefly population observed on them suggested the occurrence of begomovirus infection. to detect the begomovirus infection, the total dna from leaf samples of infected jatropha plants was extracted and polymerase chain reaction (pcr) were performed using three sets of begomovirus genus specific (cpit-i/cpit-t, paliv /paric and paliv /palic ) primers and the expected size * bp, . kb and . kb amplicons were obtained which confirmed the begomovirus infection. further to identify the begomovirus/es and investigate the genetic diversity among them exists if any, the * . kb amplicons were cloned and sequenced. the sequence data were deposited in the genbank database under accession nos.: gq and fj (from j. curcas) and eu and fj (from j. gossypifolia). during blast analysis gq and fj shared highest % sequence identity with each other and - %% with sri lankan cassava mosaic virus (aj , aj , aj , aj and aj ) and indian cassava mosaic virus from india (ay ) therefore, designated as two strains of jatropha mosaic india virus-lucknow. blast analysis of eu showed maximum % similarities with croton yellow vein mosaic virus (aj ), % with tomato leaf curl new delhi virus (dq ) and - % with papaya leaf curl virus (aj and y ), therefore, identified as strain of croton yellow vein mosaic virus. blast analysis of the virus isolate (fj ) showed highest % identities with tomato leaf curl virus-bangalore ii (tolcv-b ii-u ) and - % with tomato leaf curl karnataka virus (tol-ckv, ay , fj ), therefore, considered as new begomovirus species ''jatropha yellow mosaic india virus''. the phylogenetic analysis of gq and fj (from j. curcas) and eu and fj (from j. gossypifolia) was performed along with some selected isolates of begomovirus which showed [ % sequence identities during blast analysis. the isolate eu showed closest relationship with croton yellow vein mosaic virus while fj showed separate clustering of all the four begomovirus from jatropha species. during phylogenetic analysis these isolates formed three separate clusters, therefore, they were considered as three distinct begomoviruses. the above data clearly show that some genetic diversity exists among the begomoviruses infecting jatropha species in india. bitter gourd (momordica charantia l.) of the family cucurbitaceae, also known as bitter melon is extensively cultivated in north eastern region of uttar pradesh, india. it is regarded as one of the world's major vegetable crops and has great economic importance. a severe yellow mosaic disease on bitter gourd (momordica charantia) with a significant disease incidence was observed during the survey of different locations of eastern up, india in the year . the whitefly (bemisia tabaci) population was also observed in the vicinity. the characteristic disease symptoms and whitefly population indicated the possibility of begomovirus infection. total dna were isolated from infected as well as healthy leaf samples. two primer pair (tlcv-cp and roja's primer) were used to study, which resulted * bp with tlcv-cp in / samples and * . kb amplicons with roja's primer in / samples. for further identification of the begomovirus, the pcr amplicons were cloned and sequenced (genbank accession no. eu and eu , respectively). the blastn search analysis of eu indicated - % identity with several isolates of tomato leaf curl new delhi virus (tolcndv). the phylogenetic analysis also showed closest relationships of the isolate (eu ) with tolcndv isolates. based on highest sequence identity and closed relationships with tolcndv the virus isolated from bitter gourd was considered as an isolate of tomato leaf curl new delhi virus. while, blastn search analysis of eu isolate, shared highest - % identites with pepper leaf curl bangladesh virus (peplcbv) isolates. the phylogenetic analysis of the virus isolate with selected begomovirus isolates revealed a closest relationship with peplcbv. these results confirmed the association of peplcbv on bitter gourd. study revealed the variability of viruses on bitter gourd in eastern up, india. tobacco streak virus groundnut isolate was characterized biologically by taking six cultivars (jl , tmv , k , k , k ) and one pre-release culture (k ) using seedlings of - days old under glasshouse conditions. there were clear differences were observed among cultivars tested regarding incubation period, percent seedling wilt and time taken to death of seedlings. k- was least susceptible among all the cultivars tested and it supported least virus titer (a nm: . - . ). both localized (necrotic lesions on leaf, veinal necrosis, leaf yellowing, wilting) and systemic (petiole necrosis, necrotic lesions on young leaves, death of top growing buds not only on main stem but also on all primaries (side shoots), followed by stem necrosis, stunted growth, axillary shoot proliferation with small leaves having general chlorosis, peg necrosis, pod necrosis, pod size reduction, wilt of plants) symptom were observed in all cultivars tested. biological differentiation of tsv and gbnv was made by sap inoculation of both viruses separately using susceptible groundnut cultivar jl under glasshouse conditions. there were certain similarities and differences were observed between these viruses infecting groundnut. seed infection of tsv ranged from . to . % in seeds collected from naturally infected and sap inoculated groundnut cultivars/pre-releases (jl , tmv , k- , k- , k- and k- ) belonging to spanish and virginia types. tsv was detected both in pod shell and seed testa from pod samples produced by sap inoculation under glasshouse conditions. however, seed transmission of tsv was not observed in groundnut. coat protein (cp) gene of three groundnut tsv isolates (gn-ap- - ; gn-ap - ; gn-ap - ) were sequenced and all the three isolates contained a single open reading frame (orf) of bp nucleotide and could potentially code for amino acids (aa). cp gene of tsv isolates originating from different hosts shared high degree of sequence identity both at nucleotide ( . - %) and amino acid ( . - %) levels respectively. tones grown in an area of . . ha (fao stat ). in india papaya is grown in nearly , ha with an annual production of , , tones (fao stat ) and occupies fourth place in the world. the crop is severely affected by a number of viruses. papaya ring spot virus (prsv-p) is the most important virus. the detection of virus infection in plants has traditionally involved either bioassay on indexing plants and or immunological methods (hill , torrence and jones ) . use of nucleic acid probes has improved the detection and sensitivity of viruses. the most common non-radioactive probes are biotynilated probes, which are very specific and sensitive. papaya ring spot virus (prsv-p) is a positive sense ssrna virus belonging to the genus potyvirus family potyviridae and transmitted by aphids. prsv-p coat protein gene region was used as template cdna for probe preparation. dot-blot hybridization with the biotin labeled probe were performed for prsv-p detection. the clarified sap of healthy and infected plants were serially diluted and spotted onto the nitrocellulose membrane, hybridized to biotin labeled probe. biotin labeled rna's are employed as probes, with a subsequent detection based on streptavidin-alkaline phosphatase conjugates. the sensitivity for viral detection of the biotin labeled probe was found to be sensitive than enzyme linked immunosorbent assay (elisa). in recent years tospovirus is causing devastating damage to the yield of vegetables in india. it infects economically important crops viz., tomato, chilli, peppers, groundnut, watermelon and various legumes. now it is emerging as severe disease in brinjal also. in order to monitor the natural occurrence and distribution of tospovirus in vegetable, surveys were conducted in the predominant brinjal growing areas of gujarat, karnataka, maharashtra and andhra pradesh during - incidence ranging from to %, to %, to %, and to . % respectively. samples collected from different places of india were found positive to pbnv in direct antigen coating-enzyme linked immunosorbent assay (dac-elisa). pbnv infected brinjal plants showed mosaic mottling of leaves with leaf distortion, longitudinal streaks on the stem and necrotic rings on leaves and fruits. early infection led to severe stunting and abnormal fruiting. biological and molecular characterization of pbnv-brinjal isolates were compared with other isolates and results are discussed. for identification of virus causing mosaic symptoms on soybean various host plants were tested. plants species belonging to the different families viz. caricaceae, graminae, leguminosae, malvaceae and solanaceae were tested. the virus produced symptoms on diagnostic plant species like chenopodium album, c. quinoa, helianthus anus, phaseolus vulgaris and vigna ungiculata. among tested families the leguminosae that were the host of virus included arachis hypogea, the virus causing mosaic symptoms in soybean is inactivated between and °c and between dilution of - to - . all the inoculated plants of assay host showed the symptoms at °c but not at °c. similarly local lesions produced at - but not at - . the virus in crude sap was infectious up to h but not at h at room temperature. however, the percentage infectivity decreased progressively as the aging of the sap was increased at room temperature. on the basis of reactions on diagnostic hosts pvp- identification and characterization of potyvirus infected chilli (capsicul annum l the virus under study caused mild mosaic and severe mottling symptom in leaves of infected plants. the dilution end point (dep) of the virus was found to be - to - , longevity in vitro (liv) - days at room temperature ( °c), thermal inactivation point (tip) - °c. electron microscopy of purified virus preparation revealed the presence of flexuous particle of size nm long and nm in width with characteristic cytoplasmic inclusions: pinwheels and scrolls. the virus was transmitted by sap and by aphid myzus persicae. the host range study revealed that the host species were restricted to family chenopodiaceae and solanaceae. on the basis of above characteristic, the virus under study was identified as potyvirus associated with mild mosaic and severe mottling symptom in capsicum. phytoplasma causing grassy shoot disease and sugarcane yellow leaf viruses are important pathogens of sugarcane. these pathogens are causing severe losses in sugarcane productivity. with a view to producing virus and phytoplasma free planting material of sugarcane, experiments were undertaken using infected varieties of sugarcane growing at the farms of sugarcane research institute. apical meristems measuring about mm in length, were dissected out, surface sterilized and cultured on agar gelled murashige and skoog's (ms) medium containing growth regulators for shoot induction. the established shoot cultures were multiplied through repeated subcultures on fresh media at - days interval. elimination of gsd and scylv was confirmed through molecular analysis of regenerated plants using specific primers of scylv and gsd. results revealed that apical meristem culture technique is effective in eliminating the pathogens like scylv and phytoplasma (gsd) from the infected clones. this is probably the first report on elimination of grassy shoot disease in sugarcane through meristem culture. papaya ringspot virus (prsv), which causes the most widespread and devastating disease in papaya, isolates originating from different geographical regions in south india were collected and maintained on natural host papaya. the entire coat protein (cp) gene of papaya ringspot virus-p biotype (prsv-p) was amplified by reverse transcription-polymerase chain reaction (rt-pcr). the amplicon was inserted into pgem-t vector by t-a cloning method, sequenced and sub cloned into a bacterial expression vector prset-a using directional cloning strategy. the prsv coat protein was over expressed as fusion protein in e. coli. sds-page gel revealed that cp expressed as a * kda protein. the recombinant coat protein (rcp) fused with his-tag was purified from e. coli using ni-nta resin. the antigenicity of the fusion protein was determined by western blot analysis using antibodies raised against purified prsv. the purified rcp was used as an antigen to produce high titer prsv specific polyclonal antiserum. the resulting antiserum was used to develop an immunocapture reverse transcription-polymerase chain reaction (ic-rt-pcr) assay and compared its sensitivity levels with elisa based assays for detection of prsv isolates. ic-rt-pcr was shown to be the most sensitive test followed by dot-blot immunobinding assay (dbia) and plate trapped elisa. key: cord- -qnclsjym authors: gupta, ankit; gupta, rasna; singh, ram lakhan title: microbes and environment date: - - journal: principles and applications of environmental biotechnology for a sustainable future doi: . / - - - - _ sha: doc_id: cord_uid: qnclsjym microbes are omnipresent in the biosphere, and their presence invariably affects the environment in which they grow. the effects of microbes on their environment can be beneficial or harmful or inapparent with regard to human measure or observation. the most significant effect of the microbes on earth is their ability to recycle the primary elements that make up all living systems, especially carbon, oxygen, and nitrogen (n). primary production involves photosynthetic organisms which take up co( ) from the atmosphere and convert it to organic (cellular) material. the process is also called co( ) fixation, and it accounts for a very large portion of organic carbon available for synthesis of cell material. decomposition or biodegradation results in the breakdown of complex organic materials to other forms of carbon that can be used by other organisms. there is no naturally occurring organic compound that cannot be degraded by some microbe, although some synthetic compounds such as teflon, plastics, insecticides, and pesticides are broken down very slowly or not at all. through the microbial metabolic processes of fermentation and respiration, organic molecules are eventually broken down to co( ) which is returned to the atmosphere for continuous process of primary production. biological nitrogen fixation is a process found only in some bacteria which remove n( ) from the atmosphere and converts it to ammonia (nh( )), for use by the plants and animals. nitrogen fixation also results in replenishment of soil nitrogen removed by agricultural processes. thus along with all these benefits, microbes greatly contribute in maintaining sustainability of environment. this chapter mainly focuses on beneficial and harmful impacts of microbes on environment and their role to maintain quality, health, and sustainability of environment. the earth is known as a "closed system" where materials cycle between lithosphere (rocks), atmosphere (air), hydrosphere (water), and biosphere (organism) (fig. . ) . together, they make up all the components of our planet, both living and nonliving. earth produces everything it needs to ensure the survival and growth of its residents. environment is defined as the circumstances or conditions that surround an organism or group of organisms. environment is the complex of social or cultural conditions that affects an individual or community. since humans inhabit the natural world as well as the built or technological, social, and cultural world, all constitute an important part of our environment. environmental studies need to understand the life processes at the microscopic level and ecologist levels from species to ecosystem. species refer to organisms of the same kind that are genetically similar enough to breed in nature and produce live, fertile offspring. population consists of all members of the same species living in a given area in the same time. all the populations of organism living and interacting in a particular area make up a biological community. an ecological system or ecosystem is composed of a biological community and its physical environment. the environment includes abiotic factor such as climate, water, minerals, and sunlight as well as biotic factors such as organisms, their products, and effect in a given area. photosynthesis is the basis of energy economy of all, but a few specific ecosystem and ecosystem dynamics are based in how organisms share food resources. in fact one of the major properties of an ecosystem is its productivity, the amount of biomass produced in a given area during a given period of time. the rate of production of food creates a linked series of feeding known as food chain, whereas when individual food chains become interconnected, they form food web. an organism's feeding status in an ecosystem can be expressed as trophic level shown in fig. . . the groups of similar species create population which results in a community. microbial community means all microbial populations in a habitat. the activities of complex communities of microbes affect biogeochemical transformations in natural, managed, and engineered ecosystem. microbial communities are very important for the rigorous progress in the field of agriculture which increases the rate of crop production. microbial community may be terrestrial or aquatic. a community of microbes and their environment that occurs on the landmasses of continents and islands form a terrestrial microenvironment. terrestrial microenvironment is distinguished from the aquatic microenvironment ecosystems by the lower availability of water and the consequent importance of water as a limiting factor. rain forests are the most diverse and productive terrestrial microenvironment, but their soil is nutrient deficient due to extensive leaching by rainwater. soil formation is a slow process that involves physicochemical weathering and biological processes over millions of years. microbes play an important role in soil aggregate formation and soil stability that confer fertility and productivity to soil. the soil microbes participate in these processes through many ways, e.g., filamentous microbes assemble clay particles using extensive network of hyphae resulting into soil aggregates. additionally, some microbes secrete exopolysaccharides or cause compaction of clay particles that promote soil aggregation. the surface soil is always rich in indigenous population of bacteria (including actinomycetes), fungi, algae, and protozoans. additionally, human and animal activities also introduce specific microbes in the soil by several ways. human activity directly adds bacteria as biodegradative agents or applying sewage sludge to agricultural fields. animals introduce microbes through bird dropping or excretion. the atmosphere is an inhospitable climate for microbes because of stress due to dehydration. this results in a limited time frame for microbes to be active; however, some microbes get resistance to these stresses through specific mechanisms promoting loss of their biological activity. spore-forming bacteria, molds, fungi, and cystforming protozoans all have specific mechanisms through which they are protected from these harsh gaseous environments. therefore, viability is highly dependent on the environment, time they spend in the environment, and type of microbes. however, many other factors also influence the viability of microbes such as humidity, temperature, oxygen content, specific ions, uv radiation, various pollutants, and other air-associated factors (aofs). the relative humidity or relative water content of the air is critical for survival of airborne microbes. most of the gram-negative bacteria associated with aerosols are able to survive for longer period at low relative humidity, whereas in contrast grampositive bacteria remain viable longer in association with high relative humidity. the ability of microbes to survive in aerosol is related to the organism's surface biochemistry. one possible explanation of this fact could be a structural change in lipid bilayers of the cell membrane in response to very low humidity. during loss of water, the cell membrane bilayer changes from the typical crystalline structure to a gel phase and affects the surface protein configuration resulting into inactivation of the cell. the viruses with enveloped nucleocapsids (e.g., influenza virus) have longer airborne survival in low relative humidity below %, whereas viruses without nucleocapsids (e.g., enteric viruses) are able to survive in high relative humidity above %. temperature is a critical factor influencing the activity of microbes. in general, high temperature leads to inactivation due to desiccation and protein denaturation, whereas lower temperature promotes longer survival rates. at very low temperature, some microbes lose viability because of ice crystal formation on their surface due to freezing. mostly radiation at low wavelengths, e.g., uv radiation and ionic radiation (x-rays), is harmful for microbes causing dna damage. these radiations target dna by producing single or double strand breaks and changing the structure of nucleic acid bases. uv radiation causes damage by forming intra-strand thymidine dimers causing inhibition of biological activity such as replication of genome, transcription, and translation. several mechanisms including association of microbes with large airborne particles, pigments or carotenoids, high relative humidity, cloud cover, etc. protect microbes from these harmful radiations. however, many microbes (e.g., deinococcus radiodurans) have evolved mechanisms to repair dna damage caused by uv radiation. oxygen, open-air factors (oaf), and ions combine to inactivate many species of airborne microbes. some reactive forms of oxygen including superoxide radicals, hydrogen peroxide, and hydroxide radicals are produced due to lighting, uv radiation, or pollution and cause dna damage by producing mutations. similarly, oafs (mixture of ozone and hydrocarbons) also cause inactivation of microbes by damaging nucleic acids and enzymes. in addition to these factors, positive ions cause only physical decay, e.g., inactivation of cell surface proteins, whereas negative ions confer both physical and biological damages such as dna damage. aquatic microenvironments occupy more than % of the earth's surface including mostly ocean but also others such as estuaries, harbors, river, lakes, wetlands, streams, springs, aquifers, etc. the microbiota, living in aquatic environment, are the primary producers (responsible for approximately half of all primary production on earth) and primary consumers as well. a large variety of microbial communities live in aquatic environments such as the planktonic, sediment, microbial mat, biofilm communities, etc. planktons refer to photoautotrophic microbial community including both eukaryotes (algae) and prokaryotes (cyanobacteria) and heterotrophic community including bacteria (bacterioplankton) and protozoans (zooplankton). phytoplanktons are the primary producers in the food web using their ability to fix co into organic matter through photosynthesis. aquatic microenvironment is further classified into three microenvironments, occupied by microbes living in freshwater, brackish water, and marine water. the study of freshwater microenvironment is known as micro-limnology. there are two types of freshwater environment: standing water or lentic habitats (e.g., lakes, ponds, bogs) and running water or lotic habitats including springs, rivers, and streams. lentic habitats are dominated by phytoplankton, forming distinct community gradients based upon the wavelength and the amount of light that penetrates to a depth, e.g., chlorobium. chlorobium can utilize longer wavelength than other phototrophs and survive with little or no oxygen by consuming h s instead of h o for photosynthesis. in freshwater environment, two types of lakes are present: eutrophic and oligotrophic lakes. oligotrophic lakes have higher rate ( - mg carbon/m /day) than eutrophic lakes ( - mg carbon/m /day) because eutrophic lakes have much higher levels of organic matter causing turbidity and interfering with light penetration. however, in terms of secondary productivity, eutrophic lakes have much higher rates ( - mg carbon/m /day) as compared to oligotrophic lakes ( - mg carbon/m /day). brackish water environment is more saline than freshwater but less saline than marine water environment. an estuary, a part of river that meets with sea, is the best example of brackish water environment. estuaries are highly variable environments because salinity changes drastically over a relatively short distance. despite this, estuaries are highly productive environments, e.g., mangrove swamps in the everglades of florida, usa. estuaries are generally turbid due to the large amount of organic matter brought by rivers and the mixing action of tides; therefore, light penetration is poor. primary producers vary from to organisms/ml and in relation to depth and proximity to littoral zones. despite the low primary productivity, substrate availability is not limited, and heterotrophic activity is high ranging from to mg carbon/m /day. marine water environments are highly diverse and contain - % salinity. the ocean is divided into two zones on the basis of light availability: photic zone, where light can penetrate, and aphotic zone with lower light. marine microenvironment is further divided into four habitats: neuston, pelagic, epibiotic, and endobiotic. habitat at the surface of sea (air-water interface) is termed as neuston. on the basis of the precise depth, pelagic habitat is subdivided into epipelagic and benthopelagic zones. epipelagic zone is found in upper m of the water column, and a large proportion of organisms living in it are photosynthetic, whereas benthopelagic zone is sea-sediment interface. the third major habitat is the epibiotic habitats referring to surfaces on which attachment of communities occur, while the fourth is the endobiotic habitat with organisms (e.g., epulopiscium) found within the tissues of other larger organisms such as fish. the organisms living in physically or geochemically extreme conditions that are detrimental to most life on earth are termed as extremophiles. most of the extremophiles are microbes and belong to the domain archaea. here are some extreme environmental conditions where extremophiles survive. environments with high temperature (> °c ) including terrestrial and submarine springs with a temperature of °c , hydrothermal vents with a temperature more than °c are inhospitable for most forms of life except some bacteria and archaebacteria, e.g., thermus, methanobacterium, sulfolobus, pyrodictium, and pyrococcus. pyrodictium and pyrococcus are capable of surviving at temperature > °c . another example of such renowned extremophiles is thermus aquaticus having thermotolerant dna polymerase, which is widely used in the polymerase chain reaction (pcr). these thermophiles have developed such characteristic mechanisms facilitating proteins in folded state even at high temperatures due to increased salt bridges (cations that bridge charges between amino acid residues). some organisms require salt concentrations substantially higher than that found in seawater for their growth, and they are known as halotolerant. halobacterium and halanaerobium are two examples of halotolerant bacteria; however, some algae and fungi also exhibit halotolerance feature. the main mechanism of salt tolerance operates by internal sequestration of high balancing solute (k + in bacteria and glycerol in halotolerant eukaryotes) equal to external salt concentration. a second mechanism of salt tolerance involves proteins with acidic and low proportion of nonpolar amino acids. these proteins require high salt concentrations to balance their charge for their optimal activity. therefore, some obligate halophiles are unable to survive in the environment lacking high salt concentration due to these macromolecular modifications. there is a wide range of microbes present in our biosphere depending on their physical and other characteristics. microbes fall into two groups, prokaryotes and eukaryotes, depending upon whether they have nucleus or not. prokaryotes lack this membrane around their genetic material, and this group includes viruses, bacteria, and related archaea. the other category of microbes includes algae, fungi, protists, and other microscopic animals, having cell nucleus. bacteria and archaea are the smallest free-living, unicellular organisms present on the earth. their cell sizes typically range from . to . μm in diameter. both exist in various cell shapes, e.g., cocci, rods, or spirals, and some soil bacteria form branching filaments, e.g., actinomycetes. their dna is found free in the cell cytoplasm and lack a true nuclear envelope, and the genome is mainly composed of single double-stranded dna molecule with smaller dna elements known as plasmids. the size of bacterial genome typically ranges from four to six million nucleotides in length and enable to code , - , genes. a bacterial cell envelope is composed of two layers, the inner layer is cell membrane made of phospholipids and the outer layer is cell wall made of proteins, carbohydrates, and lipids, but its composition varies based on the type of organism. most of the microbes move through flagella (whiplike extensions from the cell) and file filaments, e.g., pili. the pili enable them to attach with each other or to soil particles. additionally these pili are also involved in transfer of genetic material between bacterial cells, known as conjugation. these microbes usually reproduce asexually, e.g., binary fission, resulting in the formation of two genetically identical bacterial cells. on the basis of gram staining, bacteria are of two types: gram positive and gram negative; both vary in cell structure and physiology ( fig. . ). bacteria and archaea both require carbon as building blocks of their cellular materials and energy to drive the reactions involved in cell biosynthesis and metabolism. most of the bacteria utilize oxygen, whereas some bacteria and archaea grow anaerobically by using alternative electron acceptors, e.g., nitrate and sulfate. basically microbes are classified into autotrophs and heterotrophs. autotrophs utilize sunlight or inorganic compounds such as fe + , nitrate, or nitrite as energy source to fix atmospheric carbon dioxide to produce carbohydrates, fats, and proteins. however, heterotrophic bacteria use organic compounds as a source of carbon and energy. archaea were originally known to be found in extreme environments and termed as "extremophiles," but now they are widely distributed and are found in many environments including soil. it is hard to distinguish both archaea and bacteria on the basis of their morphology. but most recently their classification using molecular phylogenetic tools based on a comparison of s ribosomal rrna sequences has revealed three separate domains of life: eukaryotes, bacteria, and archaea. archaea are closely related to eukaryotes (all multicellular organisms) than the bacteria (woese et al. ). fungi belong to eukaryotes and, therefore, are more closely related to plants and animals than bacteria or archaea. fungal cell consists of membrane-bound nucleus with chromosomes containing genetic material, e.g., dna, membrane-bound organelles, e.g., mitochondria, and a cell wall composed of glucans and chitin. fungi are basically heterotroph organisms meaning thereby that they derive their food from nonliving organic sources, e.g., saprophytic fungi, which feed on dead or decaying organic materials. few fungi also exist as unicellular organisms, e.g., yeast, which grow through cylindrical threadlike structures ( - cm in diameter) known as hyphae. these hyphae may be either septate, e.g., compartmentalized through cross walls, or nonseptate. the hypha is a main part of fungus and constitutes a mycelium. finely branched mycelium occupies a large surface area in the soil and produces a range of enzymes acting on soil organic matter to produce nutrients and energy required for fungal growth. fungi can reproduce by both sexually, e.g., through spores, and asexually, e.g., budding or binary fission. fungi are highly diverse and play a wide range of role in their surrounding environment such as decomposers, mutualists, endophytes of plants, pathogens, and predators. fungal hyphae are the basic components of soil food webs since they constitute a food source for soil biota, whereas fungal sporocarps provide food for larger animals. protists are unicellular eukaryotes having characteristic organelles but lacking cell wall. protists may be free living, parasitic, or opportunistic based on environmental changes. the size of protists varies from μm to several centimeters. these are classified into four groups: flagellates (mastigophora), amoebae (sarcodina), sporozoans (sporozoa), and ciliates (ciliophora). flagellates are characterized by the presence of flagella as a locomotive tool, multiplication by binary fission, and having both heterotrophic and autotrophic feeding mechanisms. flagellates are further classified into two major divisions: photosynthesizing (phytomastigophora) and non-photosynthesizing (zoomastigophora). autotrophic flagellates possess chloroplast and synthesize their own food or nutrients by photosynthesis such as euglena, whereas heterotrophic flagellates are parasitic and take their nutrients from their host, for example, leishmania and giardia. amoebae have pseudopodia as their locomotive tool. pseudopodia help in ingestion of food materials and provide an extended basis of further classification. the rhizopoda move by a fluid endoplasm, whereas the actinopoda have a spikelike pseudopodium for their movement and feeding. these organisms are vacuolated and covered in a shell-like outer layer known as "test." these shells may be composed of proteinaceous, siliceous, or calcareous substances and have either a single or multiple chambers. some testate amoebae are also found in soil, building their shells by excreting substances capable of aggregating soil particles. some important free-living soil amoebae are grouped in the family vahikampfia. entamoeba is well-known parasitic amoeboid causing dysentery in humans. the ciliates have hairlike structures in an ordered array surrounding the cell known as cilia that divides by transverse fission. cilia help in their locomotion and feeding. ciliates are generally free living such as paramecium, but some species are adapted to parasitic life cycles. sporozoans are mostly parasitic and form spores. few members are adapted in a symbiotic relationship and have no locomotive organ; therefore, they rely on vectors or direct contact with susceptible host to continue their growth and replication. for example, some species have evolved to enable digestion in the gut of domestic livestock. however, parasitic members are totally dependent on host for their nutrition, for example, toxoplasma, isospora, and plasmodium. most of parasitic protists are of obvious public health concern causing deadly diseases such as malaria, sleeping sickness, chagas disease, leishmaniasis, giardiasis, cryptosporidiosis, etc. these parasites have adapted themselves for surviving and reproducing in their hosts by evading host immune responses. many flagellated protists are capable of forming cysts that are known to survive conventional methods of disinfection and can be transmitted to their host via a water route. entamoeba histolytica is a parasitic amoeba and causes diarrhea and dysentery. naegleria is a free-living amoeba in freshwater, causing infections in nasal passage of humans and capable of invading brain tissues. toxoplasma is an invasive protist that causes blindness and serious illness or death in unborn fetuses. cryptosporidium is responsible for a number of epidemics including the largest us waterborne outbreak in milwaukee. plasmodium is a main causative agent of mosquito-borne disease, malaria. domestic animals are also at risk of serious illness and death from parasitic protozoans, for example, histomonas, trichomonas, etc. viruses are small, obligate, intracellular pathogens that require a host cell for their growth and replication. they can survive outside the host cell but not multiply without a host. viruses are generally species specific and can infect all types of life forms: bacteria, plants, and animals. public focus is most often on ( ) plant viruses affecting important crops such as tobacco, potatoes, and tomatoes or ( ) animal viruses causing deadly diseases: herpes, smallpox, rabies, mumps, measles, meningitis, hepatitis, encephalitis, influenza, diarrhea, yellow and dengue fever, etc. the basic virus structure includes a capsid protein coat and an internal nucleic acid (rna or dna), but some viruses may also have protein and lipid envelopes, glycoprotein spikes, or more complex tail and sheath structures. a number of protein capsomers held together by non-covalent bonds form a capsid coast surrounding the nucleic acid molecule. the size of capsids ranges from μm, as in small parvovirus of animals, to several hundred nanometers, as in some filamentous plant viruses. this outer coat protects and shields the viral nucleic acid and harbors specific receptor sites for attachment on hosts. the viral capsids have two types of symmetrical organization: helical and icosahedral. the helical viruses look like a spiral or a helix with a cylindrical shape, whereas icosahedral viruses adopt a -triangular-sided spherical shape when viewed with an electron microscope. viruses have either rna or dna in the double-stranded or single-stranded form. viral nucleic acid length varies from . to over kb, and it encodes - genes. early studies on diverse soil bacteria and fungi are mainly focused on what could be easily cultured from soils, but the fact is that less than % of the soil bacteria could be cultured, suggesting the requirement of other approaches. norman pace and colleagues in found that microorganisms could be identified in naturally occurring microbial populations without culturing them (hugenholtz et al. ), but this process requires the pcr amplification of the rrna genes using rrna specific primers and rna extracted directly from the cells present in soil. these specific primers may differentiate among various microbial communities at level of different domains such as bacteria, eukarya, and archaea or phylum (e.g., actinobacteria or bacteroidetes) ( fig. . ). however, a range of approaches could be adopted in order to separate and sequence the rrna genes. the advanced high-throughput dna sequencing now allows the identification of each individual in thousands of samples within a short duration (caporaso et al. ). once we compare these rrna gene sequences from cultivated species using various online databases, e.g., genbank, allowing identification of evolutionary (phylogenetic) relationships among various unknown and known organisms, it displays an estimate of the genetic diversity of organisms in a particular community. it is easy to speculate about the organism's characteristics and its closest cultivated relative on the basis of sequencing details. phylogenetic information sometimes also provides details about the physiology, e.g., all cyanobacteria constitute a monophyletic group in a similar way as sulfate-reducing bacteria, halophiles, and methanogenic archaea do. the composition of in situ environmental bacterial communities has been investigated using various molecular tools. the relative abundance of the major phyla varies among diverse soils and environmental conditions such as some members of the phyla, i.e., proteobacteria, acidobacteria, and actinobacteria, are abundant and widely distributed; however, members belonging to verrucomicrobia, bacteroidetes, firmicutes, chloroflexi, planctomycetes, and gemmatimonadetes are comparatively less prevalent. the proteobacteria is a diverse group of organisms among various subphyla out of which, α-, β-, γ-, and δ-proteobacteria are most commonly found in soil. the members belonging to α-, β-, and γ-subphyla are copiotrophs (an organism able to grow in nutrient-rich environments particularly carbon in contrast to oligotrophs, those found in environments with much lower carbon concentration). the proteobacteria are more prevalent in those area rich in resource availability, e.g., rhizosphere soils. the α-proteobacteria consist of various metabolically diverse heterotrophic and autotrophic bacteria, e.g., heterotrophic bacteria such as sphingomonas, that are able to degrade various toxic compounds including pentachlorophenol and polyaromatic hydrocarbons and also involved in weathering of minerals. some heterotrophs that are also able to fix atmospheric nitrogen by forming symbiotic relationships with legumes belong to family rhizobiaceae such as rhizobium, mesorhizobium, and bradyrhizobium. the autotrophic α-proteobacteria also include soil methane oxidizers, e.g., methylobacter and methylophilus, nitrite oxidizers, e.g., nitrospira and nitrobacter, and phototrophs, e.g., rhodospirillum and rhodobacter. the β-proteobacteria is also found into three groups: heterotrophs, autotrophs, and methanotrophs. some of the known heterotrophs found in soil belong to the genera burkholderia, alcaligenes, and acidovorax. out of these, burkholderia spp. is metabolically diverse and uses simple amino acids, sugars, and recalcitrant aromatic and phenolic compounds as a source of carbon and therefore plays a major role in carbon turnover. burkholderia spp. is also known to promote plant growth by fixing atmospheric nitrogen. some examples of heterotrophic β-proteobacteria are collimonas, able to degrade live hyphae by producing chitinase, and autotrophic β-proteobacteria are nitrosospira (ammonia oxidizer), thiobacillus (iron oxidizer), methylomonas (methane producer), rhodocyclus (phototroph), etc. the γ-proteobacteria are also categorized into heterotrophs, lithotrophs, and phototrophs. pseudomonas and xanthomonas are well-known heterotrophs. pseudomonas spp. has a remarkable nutritional versatility since most of them are able to grow on more than or different substrates including sugars, amino acids, fatty acids, alcohols, and hydrocarbons. the γ-proteobacteria also include various photolithotrophs, e.g., thiocapsa and chromatium, that utilize sulfide or elemental s as an electron donor and co as a source of carbon under anaerobic conditions in light. the δ-proteobacteria consist mainly of so − -and fe-reducing bacteria. desulfovibrio, a sulfate-reducing bacteria, grow anaerobically by utilizing lactate or ethanol as carbon sources, found in oxygen-depleted soil. this group also includes a parasitic bacteria named as bdellovibrio. acidobacteria are widely distributed and found in abundance in the soil with low ph. since they are poorly present in soil culture collections, a little knowledge is available about their metabolic capabilities. the complete genome sequencing of cultured soil acidobacteria, e.g., acidobacterium capsulatum, implies that they may be oligotrophs and able to metabolize a range of simple and complex carbon sources. these bacteria are also found in low nutrient conditions, tolerant to changes in soil moisture. they also play a role in nitrate and nitrite reduction but not in denitrification or nitrogen fixation. verrucomicrobia, also found commonly in soil, are oligotrophic in nature; however, the ecology of verrucomicrobia is poorly understood. the majority of bacteria of this group are chthoniobacter flavus and opitutus terrae. genome sequencing of a free-living heterotroph bacteria found in aerobic soil, e.g., chthoniobacter flavus, suggests that it is able to metabolize plant polysaccharides but not amino acids except pyruvate. however, genome sequencing of a verrucomicrobium from rice paddy soil, e.g., opitutus terrae, implies that it is an anaerobic bacterium, capable of producing propionate through fermentation of polysaccharides from plants. gram-positive bacteria are abundant in soil culture collections and categorized into two groups: actinobacteria and firmicutes. the actinobacteria are commonly found in soil and further classified into three subphyla: actinobacteridae, acidimicrobidae, and rubrobacteridae. the abundance of actinobacteridae in soil relatively increases with addition of labile carbon sources. the actinobacteridae includes metabolically diverse aerobic heterotrophs, e.g., arthrobacter, rhodococcus, streptomyces, and mycobacterium. streptomyces is a well-known bacterium producing antimicrobial compounds. the rubrobacteridae includes two genera not represent in soil culture collections, e.g., rubrobacter and solirubrobacter. acidimicrobium ferrooxidans is an acid-tolerant ferrous iron oxidizer bacterium and has been detected in soil culture collections among few members of acidimicrobidae. the firmicutes include bacteria that are able to form endospores such as bacillus and clostridium, and because of endospore production, they are able to survive longer in the soil during dry periods. bacillus is capable of degrading various carbon sources, including polysaccharides from plants, whereas clostridium may ferment sugars, starch, pectin, and cellulose. the addition of recalcitrant c compounds in soil favors the growth of clostridiales. planctomycetes multiply through budding and lack peptidoglycan in their cell walls. these bacteria are also involved in ammonium oxidation (anammox) in soil under anaerobic conditions. planctomycetes, verrucomicrobia, and chlamydia are important from evolutionary point of view since they share numerous features, e.g., presence of membrane-coat-like proteins and condensed dna, rarely found in bacteria but more common in archaea and eukaryotes. sphingobacteria are known to be involved in aerobic degradation of plant materials present in soil and complex organic molecules, e.g., starch, proteins, cellulose, and chitin. members belonging to genus chitinophaga are filamentous and chitinolytic and exhibit gliding movement. archaea are known to be widely distributed in the soil on the basis of s rrna gene sequences (bates et al. ) , and additionally these gene sequences suggest that members belonging to the phylum crenarchaeota are found in abundance in the marine environment. all cultured crenarchaeota are thermophilic or hyperthermophilic organisms: having the ability to survive at a temperature up to °c. crenarchaeota is sulfur-dependent extremophile; one of the best-characterized members is sulfolobus solfataricus. this organism was originally isolated from geothermally heated sulfuric springs in italy and grows at °c and ph of - . these organism stains are gram negative and are morphologically diverse having rod-, cocci-, filamentous-, and oddly shaped cells. the crenarchaea play a role in ammonia oxidation in the soil. mesophilic ammonia-oxidizing archaea (aoa) is abundant in a diverse range of marine environments, including the deep ocean, as revealed by the quantification of the archaeal amoa gene encoding the alpha-subunit of the ammonia monooxygenase. nitrososphaera viennensis is a most recent ammonia-oxidizer crenarchaea that was extracted from garden soil, and subsequent phylogenetic analysis confirmed its taxonomic affiliation. methanogens are strict soil anaerobes and grow in association with bacteria that participate in the anaerobic food chain and convert complex organic molecules to methane (ch ) and co . methanogens generate methane through various pathways. they display various activities such as reduction of carbon dioxide and methanol, cleavage of acetate, and methane production from methylated compounds. members belonging to genera methanosarcina, methanosaeta, and methanocella are widely distributed in the environment, and both methanosarcina and methanosaeta produce methane through acetate reduction. seven fungal phyla are currently recognized: chytridiomycota, blastocladiomycota, neocallimastigomycota, glomeromycota, ascomycota, basidiomycota, and parasitic endobionts, e.g., microsporidia. among these fungal phyla, the first three have flagellated cells at least during one stage of their life cycle and thereby turned into terrestrial organisms in contrast to higher fungi that lack motile cells. among these phyla, chytridiomycota is a basal group and may degrade chitin and keratin. batrachochytrium dendrobatidis is a known pathogenic unicellular chytrid of many amphibian species resulting into decline of worldwide amphibian population. blastocladiomycota differ from the chytridiomycota in reproduction since they exhibit different form of meiosis. glomeromycota exhibit some features identical to "lower" fungi, e.g., they have multinucleate aseptate mycelia and most of them have no known sexual stages. they reproduce through large thick-walled asexual spores, commonly found in soils, and may germinate in the presence of a plant root. this phylum includes arbuscular mycorrhizal (am) fungi that form obligate biotrophic symbioses with mosses, approximately % of all land plants, and cyanobacterium nostoc forming cyano-lichens. these higher fungal phyla have a characteristic feature having two compatible nuclei in a hyphal cell also known as dikaryon. ascomycota is one of the largest fungal phyla with , or more number of species. the members of this phylum have a characteristic spore-bearing saclike structure also known as asci, produced in large numbers during sexual reproduction. ascomycetes mostly reproduce asexually and are rarely found to reproduce through sexual mating. ascomycetes have a typical haploid mycelium with septate hyphae and cell wall made up of chitin and β-glucans. few macroscopic ascomycetes exhibit well-known reproductive structures such as morels, truffles, etc. however, many ascomycetes are microscopic unicellular organisms, e.g., yeast or saccharomyces, and filamentous fungi, e.g., aspergillus. most of the ascomycetes are saprobes and have various enzymes to degrade complex substrates such as cellulose, keratin, and collagen. therefore, ascomycetes are known to play a critical role in decomposing and nutrient recycling. approximately , species of ascomycetes form lichens through symbiotic relationship with green algae and cyanobacteria. however, other ascomycota constitute ectomycorrhizal and/or ectendomycorrhizal associations through symbiosis with woody plants. some ascomycetes are known plant parasites and predators. the family orbiliaceae includes carnivorous fungi having specialized hyphae to trap prey including a range of soil mesofauna, protists, nematodes, and arthropods. members belonging to basidiomycota are also known as club fungi and produce spores during sexual reproduction on club-like stalks known as basidia. these microscopic basidia are basically clustered on specialized structures also known as sporocarps. numerous haploid spores are produced after meiosis and released in the environment resulting into a new haploid mycelium after germination. microbes play a critical role in carbon cycle on the global scale that is a key constituent of all living organisms (fig. . ). microorganisms avail carbon for living organisms and for themselves as well through extracting it from nonliving sources. in aquatic habitats, microbes convert carbon anaerobically, present at oxygen-free zones such as deep mud of lakes and ponds. carbon dioxide (co ) is the most common form of carbon that enters into a carbon cycle. co is a water-soluble gas present in the atmosphere. plants and photosynthetic alga use co during photosynthesis to synthesize carbohydrates. additionally chemoautotrophs such as archaea and bacteria also utilize co to synthesize sugars. this carbon, present in the form of sugar, is further processed through a chain of reactions during respiration known as tricarboxylic acid cycle resulting into energy. microbes may also use carbon under anaerobic conditions to produce energy through a process called as fermentation. plants are the primary producers in a terrestrial ecosystem; however, free-living planktons, cyanobacteria, and symbionts such as lichens also contribute in fixing carbon in some ecosystems. nonliving organic material is recycled by heterotrophic bacteria and fungi, whereas saprobes utilize organic material and produce co during respiration, thereby contributing to carbon cycle. however, higher animals, e.g., herbivores and carnivores, also digest organic materials to obtain energy using gut microbiota residing in their intestinal tracts; the process is known as decomposition, resulting into inorganic products such as co , ammonia, and water. actinobacteria and proteobacteria are capable of degrading soluble organic compounds, e.g., organic acids, amino acids, and sugars (eilers et al. ) . similarly, bacteroidetes are also involved in degradation of more recalcitrant carbon compounds, e.g., cellulose, lignin, and chitin, and utilize higher level of available nitrogen that help in production of extracellular and transport enzymes (treseder et al. ). on the contrary, bacteria living in low-nitrogen environments are more able to metabolize nitrogen-rich organic compounds, e.g., amino acids. the abundance of α-proteobacteria and bacteroidetes favors carbon mineralization, whereas acidobacteria oppose it (fierer et al. ). some microbes execute anaerobic or fermentative degradation of organic compounds into organic acids and some gases, e.g., hydrogen and co . methanogens are able to use that hydrogen to reduce co into methane under strict anaerobic conditions ( fig. . ). in order to complete cycle, methane-oxidizing bacteria, e.g., methanotrophs, transform methane to co , water, and energy under aerobic conditions. other microbes such as green and purple sulfur bacteria participate in carbon cycle by degrading hydrogen sulfide (h s) into compounds having carbon during energy production (see in reaction). some bacteria, e.g., thiobacillus ferrooxidans, derive energy from oxidation of ferrous iron to ferric iron and thereby contribute to carbon cycle. few microbes such as bacteroides succinogenes, clostridium butyricum, and syntrophomonas spp. make a collaborative effort (also known as interspecies hydrogen transfer) for anaerobic degradation of carbon to produce co and methane in bulk. the following reaction shows anaerobic photoautotrophism in purple sulfur bacteria: nitrogen is an essential element present in protein and nucleic acid structure. microorganisms play a critical role in nitrogen cycle through various processes such as nitrogen fixation, nitrate reduction, nitrification, denitrification, etc. (fig. . ) . the microbial processes limit the productivity of an ecosystem because nitrogen availability is a limiting factor for plant biomass production. ammonification involves decomposition of organic nitrogen into ammonia. both bacteria and archaea are capable of fixing atmospheric nitrogen through reduction into ammonium ( fig. . ) . nitrogenase is an oxygen-sensitive enzyme that catalyzes nitrogen fixation under low oxygen environment. n-fixation requires energy in form of atp ( mol) per mole of fixed nitrogen. nitrification involves two steps: first, ammonia is oxidized to nitrite and then to nitrate. the oxidation of ammonia to nitrite is carried out by few soil bacteria, e.g., nitrosospira, nitrosomonas, crenarchaeum, or nitrososphaera, and thereafter nitrite is oxidized to nitrate by some bacteria, e.g., nitrobacter and nitrospira (fig. . ) . nitrification also changes the ionic state of soil from positive to negative through oxidation of ammonia to nitrite and release of energy, which is used by nitrifying microbes to assimilate co . denitrification involves sequential reduction of nitrate (no − ), nitrite (no − ), and nitric oxide (no) to the greenhouse gas nitrous oxide (n o) or benign nitrogen gas (n ). since this process requires limiting oxygen, therefore, it occurs mostly in waterlogged areas that provide anaerobic environment. nitrogen cycle involves denitrification process through which fixed nitrogen returns back to the atmosphere from soil and water in order to complete the nitrogen cycle. denitrification involves a range of soil microbiota belonging to proteobacteria, actinobacteria, and firmicutes and other soil eukaryotes. most of the bacteria lack single or multiple enzymes involved in denitrification and known to be incomplete denitrifier, for example, most of the fungi and bacteria lack nitrous oxide reductase and thereby produce n o as a final product. therefore, incomplete denitrification results into emission of greenhouse gases. sulfur is an important component of a couple of vitamins and essential metabolites, and it is found in two amino acids, cysteine and methionine. in spite of its paucity in cells, it is an absolutely essential element for living systems. like nitrogen and carbon, the microbes can transform sulfur from its most oxidized form (sulfate or so ) to its most reduced state (sulfide or h s) ( fig. . ) . the sulfur cycle, in fig. . role of microbes in sulfur cycle particular, involves some unique groups of prokaryotes. two unrelated groups of prokaryotes oxidize h s to s and s to so . the first is the anoxygenic photosynthetic purple and green sulfur bacteria that oxidize h s as a source of electrons for cyclic photophosphorylation. the second is the "colorless sulfur bacteria" (now a misnomer because the group contains many archaea) which oxidize h s and s as sources of energy. in either case, the organisms can usually mediate the complete oxidation of h s to so . → → sulfur-oxidizing prokaryotes are frequently thermophiles found in hot (volcanic) springs and near deep-sea thermal vents that are rich in h s. they may be acidophiles as well, because they acidify their own environment by the production of sulfuric acid. since so and s may be used as electron acceptors for respiration, sulfate-reducing bacteria produce h s during a process of anaerobic respiration analogous to denitrification. the use of so as an electron acceptor is an obligatory process that takes place only in anaerobic environments. the process results in the distinctive odor of h s in anaerobic bogs, soils, and sediments where it occurs. sulfur is assimilated by bacteria and plants as so for use and reduction to sulfide. animals and bacteria can remove the sulfide group from proteins as a source of s during decomposition. these processes complete the sulfur cycle. phosphorus is a critical element of various building blocks such as nucleic acids, e.g., dna and rna, adp, atp, and phospholipids. phosphorus is a rare element in the environment because of its tendency to precipitate in the presence of divalent and trivalent cations at neutral and alkaline ph. microorganisms (bacteria and fungi) mineralize organic phosphate in the form of phosphate esters into inorganic phosphate through a process driven by phosphatase enzymes (fig. . ) . additionally, they also convert insoluble phosphorus into soluble form by a reaction with resulting byproducts such as organic acids. mycorrhizal fungi help plants to overcome phosphorus limitation through its mobilization from insoluble mineral form by producing oxalate, e.g., various ectomycorrhizal basidiomycetous fungi express phosphate transporters in their extraradical hyphae during phosphorus deficiency in surrounding environments. bacteria and fungi are able to biodegrade or detoxify substances through various ways; thereby, microbial processes are extensively used for bioremediation. bioremediation/biotransformation is a waste management tool that involves naturally occurring organisms to remove or neutralize hazardous waste into less toxic or nontoxic substances. the most commonly used microorganisms are flavobacterium, arthrobacterium, and azotobacter. bioremediation focuses on different sources and hence is called with different names: fungi mycoremediation → → biotechnological treatment of waste management involves use of microorganisms to detoxify air, water, and soil pollutants and carried out at lower temperature and pressure; therefore, it requires less energy than the conventional physicochemical treatment method. depending upon the types of contaminants' site of monitoring and favorable environmental conditions, bioremediation may be carried out either in situ or ex situ (table . ). biostimulation and bioaugmentation processes promote the rate of degradation of organic and inorganic pollutants (fig. . ) . these treatment technologies have been found eco-friendly and cost-effective means of pollution control leading to increased public acceptance and compliance with environmental legislation. heterotrophic microbes such as pseudomonas, sphingomonas, and mycobacterium are known to be involved in oil degradation. pseudomonas is one of well-studied bacteria capable of degrading alkanes, monoaromatics, naphthalene, and phenanthrene under aerobic conditions. the hydrocarbon-degrading bacteria are dominant in soil contaminated with oil; however, higher concentration of hydrocarbons may deplete available nitrogen and phosphorus in that area since these elements are assimilated during biodegradation. microbes (bacteria and fungi) are able to degrade a range of biodegradable pesticides such as atrazine, which is degraded by a bacterium, e.g., arthrobacter nicotinovorans, and related derivatives such as simazine, propazine, and cyanazine (aislabie et al. ) . non-biodegradable pesticides, e.g., ddt (dichlorodiphenyltrichloroethane), are not readily degraded and still persist in the soil. some fungi having ability to degrade lignin, such as phanerochaete chrysosporium, are able to degrade various contaminants such as pentachlorophenol and dioxin, and the best example are zygomycetes that degraded various contaminants during wood-treating operation in whakatane (thwaites et al. ) . biodegradation of a contaminant depends upon its chemical structure and physical state since various contaminants, e.g., oil are readily degradable, but synthetic contaminants, e.g., ddt and aldrin, are nondegradable and persist in the environment. the ability for degradation also depends upon rare and novel structures and water solubility since less soluble compounds are difficult to degrade. additionally, poorly water-soluble or hydrophobic contaminants also readily bind to clay particles and, therefore, are easily available to microbes present in soil. these soil microbes utilize these contaminants as energy source, present at higher concentration, and these could be toxic for them, resulting into slow biodegradation. biodegradation also involves a contact between contaminants and microbes. some microbes, e.g., chemotactic bacteria and fungi, have an ability to sense and move toward them. the microbial ability to withstand metal toxicity and their physiological adaptation to metal stress has an important significance. indeed, the expression and activity of proteins involved in metal uptake are crucial for metal resistance, and different bacteria adapt distinct complements of these systems. bacteria have evolved some regulatory mechanisms to control membrane transporter activity that take up metals, and some of these activities are determined by regulators that bind to metal ions with femtomolar activities. in response to metal exposure, some microorganisms upregulate the expression of extracellular polymers or siderophores containing functional groups that are capable of coordinating metal ions and may be subject to reduced uptake or increased efflux by membrane transporters upon binding to toxic metals. many microbes have ability to precipitate metals as metal oxides, metal sulfides, metal protein aggregates, or metal crystals forming particulates in close association with cytoplasmic membranes. in addition, some microbes can use cytoplasmic proteins, e.g., bacterioferritin and metallothioneins, to bind, sequester, or store metals (carrondo ) . some microbes use metals in specific redox and covalent reactions that convert toxic metal species into less toxic forms either oxidative or reductive metabolism (fig. . ) . few microbes have evolved detoxification mechanisms during their exposure to heavy metals, e.g., copper, mercury, lead, zinc, cadmium, etc. one of the known examples is cadmium accumulation in agricultural soils in new zealand due to extensive use of superphosphate fertilizer (loganathan et al. ) . due to metal toxicity, microbes have evolved few defense mechanisms such as metal sequestration, detoxification, and efflux of ions. bacteria sequester heavy metals through their binding with cell membrane, cell wall, and extracellular polysaccharides (harrison et al. ) . microbes may also detoxify toxic metals through reduction using various cellular enzymes, e.g., mercury oxidase reduces hg + to hg, which has a low evaporation point and, therefore, diffuses from cell (nies ) . few gram-negative bacteria, e.g., alcaligenes eutrophus, have evolved a mechanism to fight with metal toxicity by expelling them from cytoplasm to external environment through cation/proton antiporter, present at cell membrane (silver and phung ) . nowadays, the microbial ability to transform heavy metals is being extensively used as a tool for bioremediation. microbes are ultimate garbage disposal of nature that clean up or transform contaminants into non-hazardous or less hazardous substances. various microorganisms such as bacteria and fungi detoxify hazardous substances by secreting various enzymes (table . ), also involved in various industrial applications. microbial oxidoreductases detoxify toxic xenobiotics such as phenolic compounds, produced from the decomposition of lignin through polymerization, copolymerization with other substances, or binding to humic substances. most of the metalreducing bacteria reduce the radioactive metals into insoluble forms that appear as a precipitant with the help of an intermediate electron donor (leung ) . the paper and pulp industry produces chlorinated phenolic compounds upon partial degradation of lignin during pulp bleaching process. these recalcitrant wastes are removed by the action of various fungal extracellular oxidoreductase enzymes such as laccase, manganese peroxidase, and lignin peroxidase that are released from fungal mycelium into their neighborhood environment. the plants belonging to families such as fabaceae, gramineae, and solanaceae release oxidoreductases, which are recruited in the oxidative degradation of certain soil constituents. oxygenases recruit oxidation of reduced substrates through oxygen transfer from molecular oxygen (o ) using various co-substrates (fad, nadh, nadph). oxygenases fall into two major categories on the basis of number of oxygen atoms used during oxygenation: monooxygenases and dioxygenases. oxygenases mediate dehalogenation of halogenated pollutants, methanes, ethanes, and ethylenes through association with multifunctional enzymes (fetzner and lingens ) . monooxygenases play an important role in bioremediation process as a biocatalyst due to their high selectivity and stereoselectivity on the wide range of substrates. most of the monooxygenases are known to have cofactors but few act without them. monooxygenases incorporate single atom of oxygen molecule into the substrate and are further classified into two subgroups based on the presence of cofactor. p monooxygenases are heme-containing oxygenases, while flavindependent monooxygenases consist of flavin as a prosthetic group and require nadp or nadph as a coenzyme. monooxygenases catalyze desulfurization, dehalogenation, denitrification, ammonification, hydroxylation, and biotransformation of various aromatic and aliphatic compounds. methane monooxygenase is the bestcharacterized monooxygenase involved in the degradation of various hydrocarbons. monooxygenases exhibit differential activity in the presence or absence of oxygen. monooxygenases catalyze oxidative dehalogenation under oxygen-rich conditions, whereas under low oxygen conditions, they catalyze reductive chlorination. microbial dioxygenases primarily oxidize aromatic compounds and are involved in bioremediation process. aromatic hydrocarbon dioxygenases belong to rieske non-heme iron oxygenase family and are involved in oxygenation of various substrates. an example is naphthalene dioxygenase having rieske ( fe- s) cluster and mononuclear iron molecule in each alpha-subunit (dua et al. ) . one of the best nature's strategies for bioremediation is the catechol dioxygenase found in soil bacteria which is involved in transformation and degradation of aromatic molecules into aliphatic products. laccases are the members of multicopper oxidase family, produced by certain plants, fungi, insects, and bacteria and catalyze oxidation of a wide range of phenolic and aromatic substrates. most of the microbes produce intra-and extracellular laccases, catalyzing the oxidation of aminophenols, polyphenols, ortho-or paradiphenols, lignins, aryl diamines, etc. these enzymes act not only by oxidizing phenolic and methoxy-phenoic acids but also through their decarboxylation and demethylation. laccases are also involved in depolymerization of lignin resulting into various phenols that are utilized by microorganisms. microbial peroxidases catalyze oxidation of lignin and other phenolic compounds in the presence of hydrogen peroxide (h o ) and a mediator. among all microbial peroxidases, lignin peroxidase (lip), manganese-dependent peroxidase (mnp), and versatile peroxidase (vp) have shown potent activity to degrade toxic substances. lignin peroxidases are heme-containing proteins secreted by white rot fungi during secondary metabolism and play an important role in degradation of lignin from plant cell wall. manganese-dependent peroxidase is an extracellular hemecontaining enzyme secreted by basidiomycete fungi. mn + stimulates mnp production and itself oxidizes to mn + by mnp. this results into mn + chelateoxalate, which in turn oxidizes various phenolic substances. versatile peroxidases directly oxidize mn + , methoxy benzenes, phenolic aromatic substrates similar to mnp and lip. vp exhibits broad substrate specificity and is able to oxidize substrates even in the absence of manganese as compared to other peroxidases. hence, bioremediation and biotechnological applications for industrial processing need efficient vp production. microbial hydrolases play an important role in bioremediation process and act by disrupting chemical bonds in toxic compounds and thereby reduce their toxicity up to some extent. these enzymes are readily available and do not need any cofactor for stereoselectivity. some extracellular hydrolases such as amylases, proteases, lipases, dnases, and xylanases exhibit potential role in various sectors, e.g., food industry, biomedical sciences, and chemical industries. other hydrolases, e.g., hemicellulases, cellulases, and glycosidases, have shown more importance because they are involved in biomass degradation. lipases are capable of degrading lipids (e.g., triglycerides) derived from a range of microbes, plants, and animals, into glycerol and free-fatty acids. recent reports suggest a close association of lipase with organic pollutants present in the soil, and its activity results into reduced hydrocarbon content in the contaminated soil. microbial lipases are extensively used in industries since these enzymes catalyze various chemical reactions such as hydrolysis, esterification, alcoholysis, aminolysis, etc. lipase activity is an important indicator or parameter for testing hydrocarbon content present in the soil. lipases are widely used in pharmaceutical, food, chemical, cosmetic, and paper industries, but the cost of their production limits their potent application in the industries. cellulases convert cellulosic waste materials to glucose and have been implicated in intense research for bioremediation processes. some bacteria and fungi express extracellular cellulases, hemicellulases, and pectinases at very low levels, e.g., bacillus strains produce alkaline cellulases and trichoderma and humicola fungi produce neutral and acidic cellulases. cellulases are widely used in paper and pulp industry for ink removal during paper recycling, in ethanol production from cellulosic biomass, and in brewing industry to enhance juice release from fruit pulp. proteases hydrolyze the proteinaceous substances in the atmosphere resulting from animal death, shedding, and molting of appendages, as a by-product of poultry, fishery, and leather industries. proteases are divided into two groups: exopeptidases and endopeptidases. exopeptidases are further classified into aminopeptidases and carboxypeptidases depending on their site of cleavage either at n-or c-terminus of a peptide chain. endopeptidases are also grouped based on the position of active site such as serine endopeptidases, cysteine endopeptidases, aspartic endopeptidases, and metallopeptidases. microbial proteases have been employed in cheese and detergent manufacturing industries since many years. some proteases have been used in production of noncalorific artificial sweetener, e.g., dipeptide aspartame. alkaline proteases are extensively used in leather industry for removal of hairs and parts on animal skin. some proteases are also used in combination with broad-spectrum antibiotics in the treatment of wounds, cuts, and burns. food chains show the relationships between producers, consumers, and decomposers, showing who eats whom with arrows. the arrows show the movement of energy through the food chain. for example, in the food chain shown below, the small fish (silverside) gets its energy by eating the plankton and the large fish (bluefish) gets its energy by eating the small fish. finally, the bacterium eats the fish after it dies, getting its energy from the large fish. the bacterium also returns nutrients back to the environment for use by the phytoplankton. zooplankton silverside b luefish phytoplankton thus the food chain becomes a complete circle. animals may eat more than one type of food. they may eat many different types of plants or many different animals. this makes everything more complicated, and the food chain becomes a food web. a food web is made up of interconnected food chains. most communities include various populations of producer organisms that are eaten by a number of consumer populations (fig. . ) . the green crab, for example, is a consumer as well as a decomposer. the crab will eat dead things or living things if it can catch them. a secondary consumer may also eat a number of primary consumers or producers. this nonlinear set of interactions which shows the complex flow of energy in nature is more easily visualized. in a food web, nutrients are recycled by decomposers in the end. animals like shrimp and crabs can break the materials down to detritus. then bacteria reduce the detritus to nutrients. decomposers work at every level, setting free nutrients that form an essential part of food chain. large number of primary producers such as bacteria and algae can maintain the base of pyramid to balance the biomass in trophic levels. in a food chain, energy is lost in each step of the chain in two forms, first by the organism producing heat and doing work and second by the food that is not completely digested or absorbed. therefore, the food web depends on the constant supply of energy from producer and nutrients that are recycled by the decomposition of organism. as food is passed along the food chain, only about % of the energy is transferred to the next level. for example, % of the energy phytoplankton received from the sun can be used by zooplankton at the next level. from one level to the next, about % of the energy used by the previous level is lost. this means that there has to be a lot more organisms at the lower levels than at the upper levels. the number of organisms at each level makes a pyramid shape and is called a food pyramid. to better understand this energy loss, it is helpful to look at a food pyramid. thus food web may create the capacity of coexistence which was responsible for species evolution and maintenance of microbial diversity. host-microbe symbiosis exists in almost all animals, and the symbiotic bacteria can be profitable, harmful, or of no effect to the host. for example, the harmless escherichia coli strains commonly found in intestine are a normal part of the gut flora and can advantage their hosts by producing vitamin k and by keeping pathogenic bacteria from colonizing the intestine. the interactions between host and microbe form complicated networks. by contrast, some others like e. coli strain o can cause disease in its host. host and microbe interaction can be beneficial or harmful but have an important impact on the environment. when the organism is able to produce disease even in an apparently healthy host, it is referred as primary pathogen, but when it causes disease only when host's defenses are impaired, it is called secondary pathogen. the microbes consistently associated with a host are called flora. these microbes have a full range of symbiotic interactions with their hosts. some host-microbe interactions are given below (table . ). symbiosis a relationship in which two dissimilar organisms (symbionts) live in close association with one another. commensalism a relationship between two species in which one is benefited and the other is not affected, neither negatively nor positively. mutually beneficial relationship between two species. parasitism a relationship between two species in which one benefits (parasite) from the other (host); it usually involves some detriment to the host. beneficial plant-microbial interactions are categorized into three parts. first, microbes either through direct interaction with the plants or indirectly through influencing biotic or abiotic parameters of soil supply minerals to support plant growth. second, some microbes inhibit the growth and activity of plant pathogens to promote plant growth. third, few microbes produce phytohormones that stimulate the growth of plants (table . ). additionally, some saprophytic microbes establish neutral interactions with plants without directly benefiting or harming them. these microbes enrich soil nutrient levels by decomposing organic components and thereby influence their productivity and growth. the plant rhizosphere is the major soil ecological environment for plant-microbe interactions. in rhizosphere different microbes colonize around growing roots, which may either result in symbiotic, neutralistic, or parasitic interactions depending upon nutritional status of soil, soil environment, plant defense mechanism, and the type of microbial proliferation in the rhizosphere zone. the microbial community living in the rhizosphere zone benefits plant by promoting their growth and are also known as plant growth-promoting rhizobacteria (pgpr). these pgprs include various bacteria, e.g., azospirillum, bacillus, pseudomonas, rhizobium, serratia, stenotrophomonas, and streptomyces and fungi, e.g., ampelomyces, coniothyrium, trichoderma, etc. these pgprs support plant growth by increasing soil fertility, secreting phytohormones, and protecting them from various diseases by producing antibiotics and inducing plant defense system. pseudomonas and bacillus are well-studied pgprs and dominating bacteria, present in rhizosphere. pgpr bacteria have following roles to play: . pgpr bacteria suppress the growth of pathogenic microbes by lowering iron availability through secretion of low molecular weight siderophores. . pgpr can reduce the activity of pathogenic microorganisms by activating the plant to induced systemic resistance (isr) or systemic acquired resistance (sar). these plant resistance systems are induced by signaling molecules, e.g., jasmonic acid, ethylene, and salicylic acid. . pgpr bacteria enhance the production of phytohormones (e.g., auxin, cytokinin, gibberellins), besides having nitrogen-fixing ability. these phytohormones play a critical role in root initiation, cell division, and cell growth. auxin is most prominently secreted by azospirillum spp. . several commercial pgprs support plant growth by several means such as bioprotectants, biostimulants, and biofertilizers. . pgpr bacteria, e.g., azospirillum, also provide nutrition to plants by liberating phosphorous from organic compounds and thereby support plant growth. root-colonizing microbes are guided by chemical plant signal overlap. for example, plant flavonoids act as chemoattractants for nitrogen-fixing bacteria, mobile zoospores, and symbiotic fungi. during interaction of microbes with plant epidermis, plants secrete signal molecules in the form of flavonoids and flavones in the rhizosphere that drive the differentiation between pathogenic, associative, symbiotic, or neutralistic adaptation of microbes with the plants. in legume rhizobium symbiosis, the rod-shaped soil bacterium rhizobium induces nitrogen-fixing nodules on the roots of leguminous plants that convert approximately % of chemically inert nitrogen present in the atmosphere into ammonia through reduction process using bacterial enzyme nitrogenase in nitrogendeficient condition (zahran ) . during this symbiotic relationship, plant root releases elicitors of nod gene expression, bacteria releases nod factor, and plant root demonstrates ion flux, expresses nodulin proteins, and undergoes nodule morphogenesis. the plant supports metabolism of bacterial endosymbionts by providing a micro-aerobic environment for effective functioning of the oxygen-sensitive nitrogenase, encoded by bacterial nif genes and carbohydrates. in return, bacteria fix atmospheric nitrogen for plants to meet their biological needs. the other diazotrophs such as azotobacter, azospirillum, as well as rhizosphere fungi and bacteria especially pseudomonas and bacillus also interact with rhizobium affecting nodulation and nitrogen fixation and help in creating a beneficiary region where interacting microbes get benefit from additional nutrient resources. therefore, a mutualistic relationship exists between azotobacter and azospirillum where both interact with rhizobium to improve plant growth, and these beneficiary effects are mainly attributed to improvements in root development, increase in water and mineral uptake by roots, the displacement of fungi and pathogenic bacteria, and, to lesser extent, biological nitrogen fixation (heath and tiffin ) . nodule formation involves expression of rhizobia specific genes: bacterial genes (nod genes) and plant genes (nodulin genes). the component, enzymes, and their function are leghemoglobin (protection against oxygen), nitrogenase (n fixation), glutamine synthetase (n-detoxification), and uricase (n-detoxification). a mycorrhiza is a symbiotic association of a fungus and roots with vesicular plant. in a mycorrhizal association, the fungus colonizes the host plant roots either intracellularly as in arbuscular mycorrhizal fungi (amf or am) or extracellularly as in ectomycorrhizal fungi (sikes ) . in ectomycorrhiza, a fungus does not enter into plant cell, whereas it colonizes the outer cell layers and forms a hartig net. hartig net is a soil network that connects several organisms and protects against pathogenic fungi and soil bacteria. in this association, fungi form a net around the roots (hairs) to extend their access to soil nutrients. ectomycorrhiza promotes growth of tree seedlings and germination of seeds. this mutualistic association provides the fungus with relatively constant and direct access to carbohydrates such as glucose and sucrose. the carbohydrates are translocated from their source (usually leaves) to root tissue and on to the plant's fungal partners. in return, the plant gains the benefits of the mycelium's higher absorptive capacity for water and mineral nutrients due to the comparatively large surface area of the mycelium/root ratio, thus improving the plant's mineral absorption capabilities. additionally, mycorrhizal plants are often more resistant to disease caused by soilborne pathogens and metal toxicity. the mycorrhizal fungi, especially the vesicular arbuscular mycorrhizae (vam) belonging to the zygomycetes class, play an important role in phosphorous mobilization in soils having a relatively low level of available phosphorous for the better growth of cereals as well as legumes. the fundamental characteristics of fungal species that form vam are: • they all belong to glomales (zygomycetes). • initiation of interaction through germinating spores on plant plasma membrane. • hyphae form appressorium (attachment site). • formation of an extracellular hyphal system in the apoplast. • formation of haustorium: penetration into plant cell (intracellular arbuscules). • enlargement of interaction surface. • lifetime of arbuscle: a few days. extracellular hyphae of the fungal species collect nutrients and transfer them to the fungus. the association of mycorrhizal fungi with legumes has a great impact on root and shoot development and phosphorous uptake resulting in the enhancement of nodulation and nitrogen fixation. benefited fungi activate the defense genes that encode defensin proteins and may produce the reactive oxygen species through nadh oxidase to protect crops against pathogenic microbes. the yield of crop plants may increase four times higher with mycorrhizal fungi. infectious diseases are caused by pathogenic microbes that attack and obtain their nutrition from the host they infect. a pathogen is a microorganism that has the potential to cause disease. an infection is the invasion and multiplication of pathogenic microbes in an individual or population. an infection does not always result in a disease. when an infection causes damage to the individual's vital functions in system, it leads to a disease. ability to cause disease is pathogenicity, whereas the degree of pathogenicity is known as virulence. to cause disease, a pathogen must gain an access to the host, adhere to host tissues, penetrate or evade host defense system, and damage the host either directly or indirectly by accumulation of microbial wastes. numerous fungi, bacteria, viruses, and nematodes are pathogenic in nature and caused many plant and animal diseases (tables . and . ). disease triangle is one of the first concepts published by stevens in and recognized the interaction among the host, pathogen, and environment ( fig. . ). this triangular relationship is unique to phytopathology in comparison to veterinary and medical sciences because terrestrial plants possess little thermal storage capacity, and their immobility prevents escape from inhospitable environment. the sophisticated immune system found in mammals is absent in plant, and this places an emphasis on the earth's genetic constitution. finally the predominance in the phytopathology of fungi, which are also highly dependent on environment, may have contributed to the development of this paradigm. any disease caused by a pathogen is a chain of events involved in the development of pathogen and the effects of disease on the host. all infectious disease-causing agents go through a disease cycle. a generalized disease cycle is illustrated (fig. . ) . mechanisms of pathogenesis determine the relationship between virulence and components of parasite fitness, such as transmission to new hosts and survival within the host. by making explicit how the biochemical mechanisms of pathogenesis set the relations between parasite fitness and virulence, we expand the conceptual framework of parasite virulence to encompass many cases that are not addressed by the prior theories of virulence. human pathogens may enter into the host by different routes such as the mucous membranes, skin, and parental route and cause many diseases (table . ). most microbes must enter through their preferred portal of entry in order to cause disease, whereas some can cause disease from many routes of entry. the likelihood of disease increases as the number of invading pathogens increases. infectious dose (id ) and lethal dose (ld ) are used to determine fig. . disease triangle, illustrating the factors required for disease development the number of microbes. id is the number of microbes required to produce infection in % of the population, whereas ld is the amount of toxin or pathogen necessary to kill % of the population necessary in a particular time frame. the role of microbes in plant diseases has been recorded as far back bc (table . ). whereas some bacteria cause hormone-based distortion of leaves and shoots known as fasciations or crown gall, a proliferation of plant cells leading to swelling at the intersection of stem and soil and on roots happen as well. however, symptoms may vary with photoperiod, variety of plants, temperature, humidity, and infective dose. some plant pathogenic microbes cause severe economically damaging disease such as spots, mosaic patterns, or pustules on leaves and fruits, smelly tuber rots, etc. (table . ). most of the plant pathogens induce a hypersensitive reaction (hr) in nonhosts or indicator plants, and this hr acts as a plant defense mechanism elicited by the presence of a pathogen in nonhost tissue. although most plant disease is caused by fungi ( %), only a small fraction of fungi in the environment cause disease. plant pathogenic fungi have played a powerful role in human and natural history. fungal pathogens produce toxins causing allergies, e.g., mushrooms produce hallucinogenic mycotoxins (black molds). protozoa can grow inside host cells causing lysis. they may use host cells, food source, and microbial wastes (table . ). in sum, it may be said that microbes play a significant role to maintain our environmental sustainability by maintaining biogeochemical and nutrient cycles. microbes protect our environment from hazardous compounds by using a technique known as bioremediation and keeping our environment healthy. this chapter also provides evidences to explore pgprs in sustainable agriculture to improve productivity and other environmental prospects. therefore, current agricultural practices need to be improved through use of biopesticides and biofertilizers in order to minimize environmental and health problems. characterization of arthrobacter nicotinovorans him, an atrazine-degrading bacterium, from agricultural soil new zealand examining the global distribution of dominant archaeal populations in soils ultra-high throughput microbial community analysis on the illumina hiseq and miseq platforms ferritins, iron uptake and storage from the bacterioferritin viewpoint biotechnology and bioremediation: success and limitations shifts in bacterial community structure associated with inputs of low molecular weight carbon compounds to soil bacterial dehalogenases: biochemistry, genetics and biotechnological applications towards an ecological classification of soil bacteria multiresistance and tolerance in microbial biofilms stabilizing mechanisms in legume-rhizobium mutualism impact of culture-independent studies on the emerging phylogenetic view of bacterial diversity bioremediation: techniques for cleaning up a mess fertiliser contaminants in new zealand grazed pasture with special reference to cadmium and fluorine: a review microbial heavy metal resistance when do arbuscular mycorrhizal fungi protect plant roots from pathogens? bacterial heavy metal resistance: new surprises plant pathology, an advanced treatise fungalbased remediation: treatment of pcp contaminated soil in new zealand evolutionary trade-offs among decomposers determine responses to nitrogen enrichment towards a natural system of organisms: proposal for the domains archaea, bacteria, and eucarya rhizobium-legume symbiosis and nitrogen fixation under severe conditions and in an arid climate suggested further readings beneficial plant-microbial interactions: ecology and applications environmental microbiology: fundamentals and applications applications of microbial engineering adaption of microbial life to environmental extremes environmental and microbial relationships anil prakash (eds) ( ) microorganisms in environmental management thermophilic microbes in environmental and industrial biotechnology environmental bioremediation technologies microbial ecology: organisms, habitats, activities key: cord- -z bx authors: herbers, karin; mönke, gudrun; badur, ralf; sonnewald, uwe title: a simplified procedure for the subtractive cdna cloning of photoassimilate-responding genes: isolation of cdnas encoding a new class of pathogenesis-related proteins date: journal: plant mol biol doi: . /bf sha: doc_id: cord_uid: z bx transgenic tobacco plants (ppa- ) constitutively expressing escherichia coli pyrophosphatase behind the s camv promoter accumulate high levels of soluble sugars in their leaves [ ]. these plants were considered a tool to study adaptation of leaves to photoassimilate accumulation at the molecular level. by differential hybridization of a subtractive library enriched for transcripts present in the transgenic plants different cdnas were isolated. by sequence analysis four cdnas could be identified as -aminocyclopropane- -carboxylate-oxidase and as three different pathogenesis-related proteins (pr- b, pr-q and sar . ). two cdnas were homologous to a calmodulin-like protein from arabidopsis and a human ribosomal protein l while six cdna clones remained unknown. one of these clones (termed par- for photoassimilate-responsive) displayed features similar to pathogenesis-related proteins: hybridizing transcripts, . and . kb in length, were strongly inducible by salicylate and accumulated in tobacco plants after infection with potato virus y (pvy) both in infected and uninfected systemic leaves. par- transcripts also accumulated in wildtype leaves upon floating on glucose and sucrose whereas sorbitol and polyethylene glycol had no effect. rescreening of the ppa- cdna library with the par- cdna as probe resulted in hybridizing cdnas which by homology were found to fall into three classes (par- a, b, c). the cdnas coding for par- a and b were . % homologous on the dna level while both were less related to the par- c cdna ( . % and . % homologous, respectively). one open reading frame was identified in all three par- cdna classes. translation would result in proteins with a theoretical molecular mass of about kda. the n-terminal amino acid sequences resemble a signal peptide which would direct the proteins to the secretory pathway. using selective ′ hybridization probes of the three par- cdnas it was possible to discriminate the different transcripts. both par- a and par- c mrnas are induced in plants treated with pvy. plants are confronted with sugar accumulation in their leaf mesophyll cells if their photoassimilate production exceeds their utilization in sink tissues. this situation may arise when they are exposed to high light, elevated levels of atmospheric co and/or when they suffer from sink limitations as under nitrogen deficiency or cold stress [ ] . as a result of sugar accumulation the rate of sucrose synthesis decreases, starch accumulates and photosynthesis is inhibited. in contrast to the well studied biochemical mechanisms in response to sugar accumulation the underlying molecular mechanisms are largely unknown. systems used to cause an increase of assimilates in photosynthetically active plant cells are girdling (cold or hot wax collar, respectively) of source leaves, the removal of sink tissues and sugar feeding experiments using detached leaves, cell suspension cultures or protoplasts [ , , , ] . all these experimental systems, however, are restricted to investigations of the short-term response to sugar accumulation. an alternative to study long-term adaptation of plant cells to elevated levels of sugars would be the use of transgenic plants. transgenic tobacco plants expressing e. coli pyrophosphatase behind the constitutive camv s promoter (ppa- plants) accumulate high levels of carbohydrates in their source leaves [ , ] . these biochemical changes have been explained to be due to increased sucrose synthesis and inhibited loading of sucrose into the phloem [ , , ] . in addition to the expected biochemical alterations in plant metabolism caused by the e. coli transgene a number of other phenotypical changes were observed in the transgenic tobacco plants. the high sugar levels in the source leaves lead to an increase in turgor pressure by a factor of about in the transgenic as compared to wild-type plants while the water potential was essentially not changed [ ] . presumably as a consequence of the elevated turgor, the mesophyll cell enlarged two-fold as compared to wild-type cells and cell walls were found to contain ~o and ~o more pectin and uronic acids, respectively [ ] . to study the long-term adaptation of gene expression of plants that are continuously confronted with high levels of endogenous soluble sugars we have chosen the transgenic ppa- plants as a tool. here we describe the cloning and description of cdnas from a subtractive library enriched for transcripts present in the sugaraccumulating ppa- plants. an efficient and simpined procedure for the establishment of subtractive cdna libraries will be presented. one of the cdnas that could not be identified by sequence homology will be characterized in more detail and is suggested to code for a new class of pathogenesis-related proteins (pr proteins). standard procedures were used for recombinant dna work [ ] . bacterial transformation were done into e. coli xll-blue (stratagene, la jolla, ca). total rna was extracted from frozen plant material [ ] . poly(a) rna was isolated by means of oligo(dt) cellulose type (pharmacia) according to the manufacturer's instructions. poly(a) rna was prepared from source leaves of wildtype and ppa- plants. /zg were transcribed into double-stranded cdna using the cdna synthesis kit from pharmacia. eco ri-not i adaptors (pharmacia) were ligated to the cdnas which were then cloned into eco ri-digested zap ii vector arms (stratagene). after in vitro packaging using the gigapack ii packaging extract (stratagene) recombinant lambda dna was transfected into e. coli xl -blue cells (strategene) and the titer of the cdna libraries determined by counting the plaques. thereafter the cdna libraries were amplified according to the amplification protocol of stratagene. the preparation of the subtractive library is schematically drawn in fig. . the wild-type and the ppa- cdna libraries prepared as described above were in vivo excised according to the in vivo excision protocol of stratagene. obtained bacterial colonies were ampfified and their recombinant plasmid dna isolated using standard procedures. ktg of the wild-type plasmid cdna library was digested with not i while plasmids of the ppa- cdna library were cut with eco ri. cdna fragments were then separated in agarose gels and fragments between . kb and . kb eluted. wild-type cdna was photobiotinylated (clontech) according to the protocol by strauss and ausubel [ ] . photobiotinylated wild-type cdna and ppa- cdna fragments were mixed in a ratio : , denatured at °c and renatured by slowly decreasing the temperature to °c. avidin (vectrex avidin) was added to the mixture and wild-type and hybrid cdna molecules removed by centrifugation. ppa- enriched cdna fragments were cloned into ecori-digested zap ii vector arms, packaged, transfected into xll-blue cells, the titer determined and the subtractive cdna library amplified as described above. screening of cdna libraries followed the dna screening protocol by stratagene. dna of x plaque-forming units of the subtractive library were transferred onto nylon filters (hybond n, amersham buchler) and hybridized to radioactively labelled wild-type cdna. hybridizing phage dnas were visualized by autoradiography before the filters were hybridized to radioactively labelled ppa- cdna. after autoradiographic exposure the two autoradiographies were compared to identify phage dnas specific to ppa- . purified clones were in vivo excised and characterized by sequencing. nicotiana tabacum l. cv. samsun nn was obtained from vereinigte saatzuchten ag (ebstorf, germany). the transgenic ppa- tobacco plants have been described [ ] . total rna was denatured in % formamide, subjected to agarose gel electrophoresis ( . % agarose, % formaldehyde) and blotted onto nylon membranes (hybond n; amersham buchler, braunschweig, germany). if not otherwise stated, membranes were hybridized at °c in a buffer containing polyethylene glycol and ~o formamide [ ] . radioactive labelling of dna probes was performed using a random-primed dna labeling kit (boehringer, mannheim, germany). filters were washed three times for min at °c w i t h x s s c ( i × ssc is . m nac , . m sodium citrate). radioactive probes for screening the subtractive library were prepared as follows: . #g mrna of wildtype and ppa- plants, respectively, were transcribed into double-stranded cdna (cdna synthesis kit, pharmacia). this cdna was radioactively labelled using the random primed dna labeling kit as described above. pvy n was obtained from the bundesanstalt far ztichtungsforschung an kulturpflanzen (aschersleben, germany). leaves of infected tobacco plants were homogenized in mm potassium phosphate buffer, ph . (ca. g leaf material in ml buffer) to obtain viral extract. leaves to be infection were dusted with carborundum (sic) and the viral extract applied by gently rubbing the upper face of the respective leaves with a pistil. a few minutes later the treated leaves were rinsed with water. mature source leaves were used to apply the viral extract. to days after infection symptoms appeared on the plants. plant material was homogenized with pb s buffer [ ] containing . % tween- , % polyvinylpyrollidone , . ~o bovine serum albumin ( g plant material/ ml buffer). serial dilutions of homogenized extract were analysed by means of the 'double antibody sandwich' test using monoclonal antibodies against pvy (bioreba, reinach, schweiz). the elisa procedure was performed accordir/g to the protocol by bioreba. in order to get ppa-l-specific cdna clones a subtractive library enriched for transcripts present in ppa- plants was prepared ( fig. ) . rna was isolated from ppa-l- plants which is the best characterized ppa- plant line [ ] . to avoid multiple cycles of subtraction with subsequent pcr amplification as developed by straus and ausubel for genomic subtraction [ ] , the subtractive edna library was made by only one cycle of subtraction with subsequent selective cloning of transcripts from ppa- plants (fig. ). to this end the recombinant plasmids of the original cdna libraries made from mrna of wild-type and ppa- plants, respectively, were digested with different restriction enzymes, i.e. wild-type cdna with not i and ppa- cdna with eco ri. the respective cdnas were eluted from agarose gels and the wild-type cdna fragments were then photobiotinylated and hybridized to the ppa- cdna. after addition of avidin wild-type as well as hybrid cdnas were removed by centrifugation. the remaining cdna fragments -already enriched for ppa- clones -were inserted into the eco ri restriction site of the zap ii vector thereby excluding the cloning of not i-digested wild-type cdnas. by hybridizing the same number of plaque-forming units of the subtractive library and the original ppa- cdna library with the e. coli pyrophosphatase dna (present only in the transgenic ppa- plants) it was found that this procedure had resulted in a ten-fold increase of ppa- -specific clones in the subtractive library. the main advantage of this method is that amplification of the library in vivo largely excludes artifacts which are commonly found in pcramplified cdna libraries. one step of subtraction was performed in order not to lose cdna clones which are only moderately induced in ppa- plants. homologous to the ribosomal protein l from man [ ] while the remaining six could not be identified by sequence homologies in the databanks. because of their responsiveness to sugar accumulation in the transgenic ppa- plants these cdnas were designated par ( to ) which stands for photoassimilate-responsive genes. to get a clue about possible functions we started to analyse the expression pattern of one of the isolated unknown cdna clones (par-l). in ppa-l- plants the steady-state rna levels hybridizing to this unidentified cdna increased in the order from sink to source leaves (fig. b , lanes to ) which are characterized by increasing sugar levels in the same order [ ] . much weaker hybridization signals were detected in source leaves of wild-type plants (fig. a, lane ) . signal strengths in leaves of ppa- plants were approximately four times more intense than in the corresponding leaves of wild-type plants as deter-isolation of ppa-l-specific cdna from the subtractive library by differential screening the subtractive library was successively hybridized to radioactively labelled cdna prepared from mrna of wild-type and ppa- plants. differentially hybridizing clones were purified and sequenced. by homology to known sequences four cdnas could be identified as coding for -aminocyclopropane-l-carboxylic acid oxidase [ ] and for three different pathogenesis-related proteins (pr proteins), pr-lb [ ] , pr-q [ ] and sars. [ ] . the dna sequence of one cdna clone shares homology with a calmodulinlike protein from arabidopsis [ ] and one is mined using a phosphoimager. the results were confirmed by analysing rna from two other independent ppa- lines (ppa-l- and ppa-l- ) [ ] . the unknown cdna which was bp in length hybridized to two mrna transcripts being ca. . kb and . kb in length. because the accumulation of transcript levels appeared to be correlated with corresponding steady-state levels of soluble sugar (fig. ) we wondered whether the m r n a transcripts would accumulate in wild-type leaves upon incubation with soluble sugars. leaf discs from mature leaves of wild-type tobacco plants were therefore floated on water, glucose and sucrose ( , , , , , mm) for h and the rna was analysed (fig. ) . the par- m r n a transcripts accumulated in response to sucrose at a concentration of mm and levels increased with higher concentrations of sucrose (fig. , lanes - ) . floating on glucose at concentrations of mm to mm also lead to accumulating levels of par- mrna although less strongly (fig. , lanes - ) . at a glucose concentration of mm levels of par-i transcripts had increased -fold while floating on sucrose resulted in an increase of about -fold compared to the water control as quantified by phosphoimaging. there are multiple ways by which sugars may possibly exert their effect on gene expression. they might mediate the inducing response by being the effector molecules themselves. alternatively, the water household may be perturbed (osmotic potential, water potential and resulting turgor pressure) or metabolic changes such as alterations in the c/n ratio may occur in response to sugar accumulation. alternatively, sugars might induce a state of general stress resulting in changed levels of phytohormones which would regulate gene expression. to study whether the accumulation of par- transcripts was due to osmotic effects caused by an increase in the levels of soluble sugars, leaf discs were incubated in different osmolytes and the rna was analysed. sorbitol at concentrations of and mm (measured to correspond to and mmol/ kg), did not cause par- transcript accumulation in floating experiments (fig. , lanes and ) . these concentrations were used as they were about equal in osmolality to mm and mm glucose (measured to be and mmol/kg, respectively). as expected, floating on mm glucose resulted in the accumulation of par- transcripts (fig. , lane ) . no induction was observed after floating leaf discs on ~o polyethylene glycol (fig. , lane ) . altogether the data suggest that the induction of the par- transcripts by glucose and sucrose cannot be caused by osmotic changes alone. glutamine used in floating experiments to perturb the cellular c/n ratio by increasing the level of amino acids did not lead to enhanced levels of par- transcripts either (fig. , lane ) . as cdnas coding for pr-proteins had been isolated from the ppa-l-specific library supporting the hypothesis that sugar accumulation in leaves would result in a general stress response we wondered whether par- also possessed any features of stress-related genes. to this end the expression of the par- transcripts was studied in response to hormones associated with stress in plants by means of floating experiments (fig. ) . no substantial differences in transcript levels were observed after floating leaf discs on water, methyl jasmonate and abscisic acid (fig. , lanes to )whereas levels of par- transcripts rose significantly as a result of floating leaf discs on mm salicylate for h (fig. , lane ) . floating leaves on gibberellic acid and indole acetic acid as well as spraying plants with ethephon did not induce par- transcripts (data not shown). because of their induction by salicylate the levels o f m r n a transcripts corresponding to the par- cdna were analysed in response to virus infection. to this end tobacco plants cv. samsun nn were infected with pvy which on this cultivar gives rise to systemic infections (unpublished data). first symptoms of infected leaves are the browning of veins which is followed by a crinkly and finally necrotic appearance of the leaves. in plants with to leaves pvy spreads systemically to all leaves above the directly infected one while in elder plants -depending on the number of leaves at the time of infection -the first to the fourth above the infected leaf does not get infected (unpublished observation). the expression of par- transcripts was studied after treatment of tobacco plants with pvy from to days after infection (dpi). samples were taken for rna analysis as well as for detection of pvy by the elisa technique from the directly infected as well as from the systemic noninfected and infected leaves. four days after mechanical treatment of leaves with pvy a weak induction of the par- transcripts could be observed (fig. a, lane ) . transcript levels increased up to dpi (fig. a, lanes - ) . a moderate accumulation of transcripts was also found in the systemically uninfected leaves (fig. b, lanes - ) . at no time virus could be detected in these leaves using monoclonal antibodies against pvy coat protein or by hybridizing northern blots with cdna encoding pvy coat protein (data not shown). systemic leaves which got infected accumulated high levels of par- transcripts starting dpi (fig. c, lanes - ) . pvy coat protein could be detected dpi after pvy treatment in locally infected leaves and dpi in systemically infected leaves. the data prove that the par- transcripts are inducible by virus both locally and systemically. the expression of the par- transcripts in response to pvy occurred co-ordinately with the expression of mrnas encoding pr-q and sar . (data not shown). to find out whether the two differently sized par- transcripts were due to the expression of homologous genes the original ppa- cdna library was screened with the par- cdna fragment. out of several hundred hybridizing plaques independent clones were purified and after in vivo excision analysed by restriction and sequencing. according to their homology the cdnas (between . and kb in length) could be grouped into three different classes (termed par-la, b, c). the first two classes (a and b) shared . ~o homology on the dna level, while both groups were less related to class c ( . ~o and . ~o homology, respectively). the three different cdnas contained one common open reading frame which would code for a protein with a theoretical molecular mass of ca. kda. comparisons between the putative coding regions of the three par- proteins essentially reflected the degree of similarity found on the dna level. par-la and par-ib proteins would be identical to . ~o while they would share only . ~o and ~o identical amino acids with the par-lc pro-tein, respectively. the amino acid sequences of the proteins deduced from the open reading frames of the three different par cdnas have been aligned in fig. . the dna homology in the putative coding region of the three par proteins is significantly higher than in the surrounding untranslated regions (data not shown) which additionally argues for this open reading frame as being the coding region. all three par proteins have the same hydrophilic profile with a hydrophobic stretch of amino acids at the nterminus which resembles a signal peptide. it seems likely that the three pars are targetted into the secretory pathway. to discriminate between the differently sized par- transcripts dna fragments were isolated from the putative '-untranslated regions of the three par- cdnas. these were used as probes in northern blots to analyse the accumulation of par- mrnas in infected and uninfected leaves of pvy-treated tobacco plants. as shown in fig. , par-la (lanes - ) and par-lc cdna fragments (lanes - ) differentially hybridized to the . kb and . kb mrna transcript, respectively. for comparison, the same blot was hybridized with the full-length par-la probe under low-stringent conditions (fig. , lanes - ) . the par-lb cdna fragment resulted in a very weak constitutive signal after days exposure. par-la and c transcripts were induced both in infected and uninfected leaves of pvy-treated plants (fig. ). plants are faced with photoassimilate accumulation in their cells in a number of natural conditions like elevated levels of atmospheric co , nitrogen deficiency, freezing, cold and osmotic stress. to study long-term adaptation ofgene expression to sugar accumulation we used transgenic photoassimilate-accumulating tobacco plants (ppa- ) [ ] as a model system. the strategy was to isolate cdna clones by differential hybridization of a subtractive library enriched for transcript present in the sugaraccumulating ppa- plants. in order to avoid cdna artifacts frequently resulting from pcr amplifications during the establishment of a subtractive library a novel strategy was employed (fig. ) . cdna libraries were prepared from mrnas of wild-type and ppa- plants allowing the in vivo amplification of the respective cdnas. the edna library prepared from mrna of ppa- plants was cut with the same restriction enzyme later used for establishment of the subtractive library. on the other hand, the edna of the library prepared from mrna of wild-type plants was excluded from cloning because a different restriction enzyme was used. the exclusion of wild-type edna cloning allowed for only one round of subtraction with an enrichment for ppa-l-specific cdnas by -fold. thus this efficient and simple procedure eliminated the need for multiple rounds of hybridizations/subtractions and subsequent pcr amplifications. by differential screening of the subtractive library different cdnas were isolated of which four could be identified as encoding -aminocyclopropane- -carboxylate-oxidase (acc oxidase) and three different pr-proteins (pr-lb, pr-q and sar . ). two cdnas were found to be homologous to a calmodulin-like protein from arabidopsis and the human ribosomal protein l [ ] . the remaining six did not show similarities to sequences in the databanks. • one of these unknown cdnas (termed par- for photoassimilate-responsive) hybridized to mrnas of about . and . kb. the steadystate levels of par- transcripts in leaves increased in the order from upper to lower leaves both in wild-type and ppa- plants thus being correlated with the levels of endogenous sugars (fig. ) [ , ] . in ppa- plants which accumulate much higher levels of soluble sugars as compared to wild-type plants [ , ] , par- transcripts also accumulated to higher levels. these correlations strongly indicate that the induction of par- mrna may be mediated by an increase in soluble sugars in the plant cells. this assumption has further been supported by the finding that wild-type leaves floated on different concentrations of glucose and sucrose also accumulated high levels of par- transcripts (fig. ) . thus the accumulation of par- edna by soluble sugars appears to be the cause for its isolation as a photoassimilate-responding gene. by rescreening the ppa- cdna library using par- as a probe independent cdnas were isolated and characterized. by comparison of their respective dna sequences the cdnas could be grouped into three different classes of which the first two (par-la and b) were . ~o homologous while they were less related to the third-group par-lc ( . ~o and . ~o homologous, respectively). par-la and par-lc transcripts were found to be strongly inducible by salicylate in floating experiments and were furthermore shown to accumulate in uninfected and infected leaves of tobacco plants treated with pvy ( fig. , , ). by these characteristics the par- cdnas may be classified as cdnas encoding a novel class of pathogenesis-related proteins. the par- cdnas would code for proteins with a theoretical molecular mass of kda which could be targetted into the secretory pathway due to an n-terminal signal peptide (fig. ) . different sets of pr proteins are known to be induced under a number of stress conditions like viral infection, pathogen invasion, injury, uv light, ozone, chemical treatment, salicylic acid, ethylene and other plant hormones ( [ ] ; reviewed in [ ] ). thus the accumulation of at least four different pr proteins under conditions where the plant cells permanently meet high levels of sugars and osmotic stress might have been expected. however, pr proteins have attracted attention mainly for their roles in host/pathogen relationships, particularly for their possible involvement in inducing resistance against further development of virus disease in tobacco leaves. less consideration has been attributed to pr protein function in response to other physiological conditions like osmotic stress. nevertheless, pierpoint et al. [ ] and ohashi and matsuoka [ ] reported on the induction of pr proteins under mannitolinduced osmotic stress. osmotin, a protein which belongs to the group of pr proteins, had first been studied in cultured tobacco cells osmotically adapted to low water potential [ , ] . under comparable osmotic conditions, namely floating leaves on sorbitol and peg, accumulation of par- mrnas was not induced (fig. ) . thus the induction of par- transcripts in wildtype leaves upon floating on osmolytes cannot be solely attributed to osmotic effects. penetrating and possibly metabolizable solutes such as sucrose and glucose (fig. ) may be required for the response. the question arises how the induction of the par- mrnas is mediated and whether there might be induction mechanisms common to other pr protein genes in physiological situations where plants accumulate photoassimilates in their cells. hypothetically, even the induction of pr proteins by pathogens might follow the same mechanism. viruses have been described to cause severe perturbations in carbohydrate metabolism in leaves leading to the accumulation of starch [ and references therein] and/or soluble sugars [ , ] . sturm and chrispeels [ ] found that bacterial infection caused rapid induction of extracellular invertase which hydrolyses extracellular sucrose. glucose and fructose may be taken up which might trigger the induction response. sugars have also been reported to induce other stress-related proteins, such as proteinase inhibitor ii [ , ] , cathepsin d inhibitor and leucine aminopeptidase from potato [unpublished data] and chalcone synthase [ ] . thus, as already suggested by j ang and sheen [ ] , there might be a common mechanism of sugar sensing in the repression of photosynthetic genes and the activation of stressrelated/pathogenesis-related genes. as the induction of par and pr protein-specific transcripts in the ppa- plants might be mediated by salicylate we have determined salicylate levels in these plants. there was no difference in the levels of salicylic acid or its conjugate between ppa- and wild-type plants (ph. meuwly, j.-p. m&raux, unpublished data). this indicates that the accumulation of pr protein-specific transcripts by soluble sugars follows a salicylate-independent pathway. other or additional mechanisms for the induction of pr proteins in the photoassimilateaccumulating plants cannot be ruled out. for instance, the accumulation of the mrna coding for acc oxidase might indicate a role of ethylene in the induction process. this is supported by the finding that carbohydrates stimulate ethylene production in tobacco leaf discs [ ] . current experiments are devoted to studies on the signal transduction mechanisms of stressrelated as well as photosynthetic genes in ppa- and other transgenic sugar-accumulating plants (manuscript in preparation). acceleration of nucleic acid hybridisation rate by polyethylene glykol embl data library, accession number x differential induction of acquired resistance and pr gene expression in tobacco by virus infection, ethephon treatment, uv light and wounding molecular characterization of messenger rnas for pathogenesis-related proteins la, lb and lc, induced by tmv infection of tobacco phloem-specific expression of pyrophosphatase inhibits long distance transport of carbohydrates and amino acids in tobacco plants regulation of photosynthesis by end-product accumulation in leaves of plants storing starch, sucrose, and hexose sugars structure and expression of an ethylene-related mrna from tomato sugar sensing in higher plants inorganic pyrophosphate content and metabolites in potato and tobacco plants expressing e. coli pyrophosphatase in their cytosol wound-inducible potato inhibitor ii genes: enhancement of expression by sucrose sugar response element enhances wound response of potato proteinase inhibitor ii promoter in transgenic tobacco regulation of the expression of rbc s and other photosynthetic genes by carbohydrates: a mechanism for the 'sink regulation' of photosynthesis? ribulose-l, -bisphosphate carboxylase-oxygenase, other photosynthetic enzymes and chlorophyll decrease when glucose is supplied to mature spinach leaves via the transpiration stream human cdnas encoding elongation factor lg and the ribosomal protein rl impaired photoassimilate partitioning caused by phloemspecific pyrophosphate removal can be complemented by a phloem-specific cytosolic yeast-derived invertase in transgenic plants improved method for the isolation of rna from plant tissues elevated mrna levels of the ribosomal protein l and a calmodulin-like protein in assimilate accumulating transgenic tobacco plants synthesis of stress proteins in tobacco leaves stress-induced expression of genes for pathogenesis-related proteins in plants isolation of complementary dna clones encoding pathogenesis-related proteins p and q, and acidic chitinases from tobacco carbohydrates stimulate ethylene production in tobacco leaf discs. ii. sites of stimulation in the ethylene biosynthesis pathway the pathogenesis-related proteins in tobacco: their induction by viruses in intact plants and their induction by chemicals in detached leaves molecular cloning: a laboratory manual metabolic repression of transcription in higher plants characterization of osmotin. thaumatinlike protein associated with osmotic adaptation in plant cells molecular cloning of osmotin and regulation of its expression by aba and adaptation to low water potential expression ofe. coli inorganic pyrophosphatase in transgenic plants alters photoassimilate partitioning plant responses to sugar accumulation in transgenic tobacco plants die vergilbungskrankheit der rtlbe. mitt biol zentralanst land-forstw berlin-dahlem genomic subtraction for cloning dna corresponding to deletion mutations cdna cloning of carrot extracellular fl-fructosidase and its expression in response to wounding and bacterial infection complex, localized changes in coz assimilation and starch content associated with the susceptible interaction between cucumber mosaic virus and a cucurbit host sugar-dependent expression of the ch s-a gene for chalcone synthase from petunia in transgenic arabidopsis regulation of the expression of photosynthetic nuclear genes by coz is mimicked by regulation by carbohydrates: a mechanism for the acclimation of photosynthesis to high co coordinate gene activity in response to agents that induce systemic aquired resistance the effect of infection with beet yellows and beet mosaic viruses on the carbohydrate content of sugar-beet leaves, and on translocation we are very grateful to susanne konig for sequencing the cdnas at the automated laser fluorescence dna sequencer. we would like to thank heike deppner and christiane prflgner for technical assistance and birgit schafer and heike ernst for the photographic work. we are also indebted to hellmuth fromme and his colleagues. for taking care of the greenhouse plants. the cdna encoding potato virus y coat protein was kindly provided by dr schlegel (bundesanstalt for ztlchtungsforschung an kulturpflanzen, aschersleben, germany). this work was supported by the deutsche forschungsgemeinschaft (grant so / - ). key: cord- - l fdmi authors: marquet-blouin, e.; bouche, f.b.; steinmetz, a.; muller, c.p. title: neutralizing immunogenicity of transgenic carrot (daucus carota l.)-derived measles virus hemagglutinin date: journal: plant mol biol doi: . /a: sha: doc_id: cord_uid: l fdmi although edible vaccines seem to be feasible, antigens of human pathogens have mostly been expressed in plants that are not attractive for human consumption (such as potatoes) unless they are cooked. boiling may reduce the immunogenicity of many antigens. more recently, the technology to transform fruit and vegetable plants have become perfected. we transformed carrot plants with agrobacterium tumefaciens to generate plants (which can be eaten raw) transgenic for an immunodominant antigen of the measles virus, a major pathogen in man. the hemagglutinin (h) glycoprotein is the principle target of neutralizing and protective antibodies against measles. copy numbers of the h transgene were verified by southern blot and specific transcription was confirmed by rt-pcr. the h protein was detected by western blot in the membrane fraction of transformed carrot plants. the recombinant protein seemed to have a % lower molecular weight than the viral protein. although this suggests a different glycosylation pattern, proper folding of the transgenic protein was confirmed by conformational-dependent monoclonal antibodies. immunization of mice with leaf or root extracts induced high titres of igg and igg a antibodies that cross-reacted strongly with the measles virus and neutralized the virus in vitro. these results demonstrate that transgenic carrot plants can be used as an efficient expression system to produce highly immunogenic viral antigens. our study may pave the way towards an edible vaccine against measles which could be complementary to the current live-attenuated vaccine. the development of genetic transformation technology has allowed the expression of foreign genes in an increasing number of plant species. the use of plants for the production of foreign antigen proteins that could serve as experimental immunogens was first reported in the early s (cardineau and curtis, ; mason et al., ) . since then, a number of viral and bacterial antigens have been expressed in a variety of plant species (mcgarvey et al., ; thanavala et al., ; carrillo et al., ; gomez et al., ; modelska et al., ; tacket et al., ; wigdorovitz et al., ) . despite differences in post-translational processing viral and bacterial antigens preserved their immunogenic properties when produced in plants and induced cross-reactive and sometimes neutralizing and protective antibodies. plants could therefore be an inexpensive source of antigens that could be easily purified for parenteral inoculation (thanavala et al., ; gomez et al., ) . moreover, oral ingestion of plants expressing high levels of antigens bear the potential of edible vaccines (kong et al., ) . strategies based on potent mucosal adjuvants such as cholera toxin and heat-labile enterotoxin of escherichia coli (haq et al., ; arakawa et al., a, b) may pave the road for oral immunization. research on edible plant vaccines has been carried out mostly in plant species largely inappropriate for human consumption (e.g. tobacco, nicotiana tabacum, thanavala et al., ; mason et al., ; huang et al., ; nicotiana benthamiana, modelska et al., ; arabidopsis thaliana, carrillo et al., ; gomez et al., ) . for most proteins of human pathogens expressed in edible plants, potatoes were used, which are normally boiled before consumption (mason et al., ; arakawa et al., a; richter et al., ; kong et al., ) . it can be anticipated that many antigens would not resist cooking without being denatured and that cooked plant material is less immunogenic than raw plants (kong et al., ) . therefore, there is a need to develop other and more appropriate transgenic plant species that can serve as edible vaccines. the technology for creating other transgenic edible plants, including fruits and vegetables has been further perfected (schenk et al., ; brodzik et al. ) . we chose to transform transgenic carrots, which can be grown in most parts of the world, which can be eaten both raw and cooked, and are part of the early diet of infants. current life-attenuated measles vaccines are given routinely at to months of age. after a single injection, seroconversion rates are high, complications are rare and protection is long-lasting. these advantages are difficult to match by other experimental measles vaccine. however, after years % of vaccinees are thought to have lost protective levels of antibodies (mossong et al., ) . revaccination with an oral vaccine which would boost the residual immunity would be a preferred strategy since it can be selfadministered, requires less training of health workers and avoids the risks associated with needle injections. the potential of a parenteral/oral prime-booster schedule has been demonstrated for a number of pathogens (kong et al., ; mantis et al., ) . such a schedule is probably less liable to problems of weak and variable responses after oral vaccination. a strategy based on oral vaccination could be a particularly useful for large-scale booster immunization in developing countries where the need to deliver parenteral vaccines may hamper eradication efforts. measles is caused by a paramyxovirus (mv) which projects two glycoproteins, the hemagglutinin (h) and the fusion protein, from the outer viral envelope. the h protein is responsible for the attachment of the virus to the host cell (naniche et al., ) , whereas the fusion protein is directly involved in the fusion of viral and target cell membranes required for the penetration of the virus (wild et al., ) . virus-neutralizing and protective antibodies are mainly directed against the hemagglutinin and, to a lesser extent, the fusion protein (mcfalin et al., ; giraudon and wild, ) . the aim of this study was ( ) to explore the potential of carrots as an expression system for antigens that is suitable for human consumption, and ( ) to test whether the measles virus hemagglutinin glycoprotein would preserve its neutralizing immunogenicity in this system. although some work has been done with transgenic carrot callus cells (brodzik et al., ) , this is one of the first reports of the expression of a transgenic antigen in mature carrots, showing that high levels of virus-neutralizing antibodies can be induced with a glycoprotein produced in this plant. the coding sequence corresponding to the measles virus hemagglutinin (mv-h) protein (bouche et al., a) was subcloned into the expression cassette of the prtl vector at the ncoi-bamhi sites (restrepo et al., ) . in this vector, the mv-h sequence was under the control of the constitutively expressed cauliflower mosaic virus (camv) s promoter fused to the tobacco etch virus (tev) -untranslated region, a translational enhancer, and the camv s terminator (odell et al., ; pietrzak et al., ) . these control sequences were flanked with hindiii restriction sites that allowed their transfer, together with the h sequence, into the t-dna region presents in binary vector pbin (bevan, ) , creating the recombinant plasmid pbin -mvh. the t-dna region, delimited by the right and left border sequences, also contains the neomycin phosphotransferase ii gene (nptii) that provides neomycin and kanamycin resistance to transformed plants ( figure ). after subcloning of the expression cassette in pbin and transformation of xl -blue cells kanamycin-resistant colonies were picked and checked for the presence of the expression cassette by pcr and automated sequencing ( a model, perkin elmer, netherlands). plasmid dna was then isolated from a positive clone and introduced in agrobacterium tumefaciens strain lba by electroporation ( µf, v, ). transformed bacteria were selected on yeb-agar solid medium containing µg/ml kanamycin ( • c, h) and were used for subsequent carrot transformation. the protocols of a. tumefaciens-mediated transformation of hypocotyls were modified for the production of transgenic carrot plants (hardegger and sturm, ; tokuji and fukuda, ; brodzik et al., ) . sterilized carrot seeds (herrera-estrella and simpson, ; daucus carota cv. senkou-gosun, kindly given by dr tokuji, japan) were sown in . % agar in the dark at • c. after days hypocotyl segments were harvested in gamborg b liquid medium (b ) supplemented with % sucrose and with the phytohormone , -dichlorophenoxyacetic acid ( mg/l) for h at • c. after washing, the segments were placed for days on phytohormone-free b solid medium ( . % agar) containing % sucrose. the hypocotyl fragments were then transformed by immersion ( h) in the bacterial suspension of a. tumefaciens containing the recombinant pbin -mvh binary plasmid. the segments were further co-cultured in the dark at • c with the bacteria on b solid medium with % sucrose. five days later, the explants were washed with b medium containing cefotaxime ( mg/l) to kill remaining agrobacteria. explants were grown in darkness at • c on b solid medium containing cefotaxime ( mg/l) and geneticin ( mg/l) for the selective growth of transgenic cells. after weeks of selection, somatic embryos resistant to geneticin appeared on the hypocotyl segments. each hypocotyl usually gave rise to a few embryos. embryos were subcultured and rooted on selective medium at • c under light. plantlets with adequate roots were transferred to potting soil and grown in a greenhouse under normal light and humidity conditions. several lines of transgenic carrots were obtained and further studied. genomic dna was isolated from both untransformed and transformed plants by macerating frozen leaves (ca. g) in liquid nitrogen. the resulting powder extract was re-suspended in the extraction buffer ( mm tris-hcl, mm edta, . m nacl, mm -mercaptoethanol and % n-cetyl-n,n,n,trimethylammonium bromide, ph . ) and incubated at • c for min. after a chloroform extraction, nucleic acids were precipitated with . volume of isopropanol and the pellet obtained after centrifugation was resuspended in tris-hcl ( mm, ph . )/ edta ( mm) buffer. after rnase treatment the dna was stored at − • c until being used. a fragment of the mv-h expression cassette (promotor-mvh-terminator) was specifically amplified with a forward primer ( -gcaagacccttcctctatat- ) from the s promoter region and a reverse primer ( -atctgggaactactcacac- ) from the s terminator region. the presence of the nptii gene was detected by pcr amplification with a pair of specific primers within the nptii gene ( -tgctcctgccgagaaagtatc- and -tcctgtatcgcaaccgatgggc- ). genomic dna of untransformed plants was used as negative control. southern blotting with genomic dna was performed following conventional protocols. briefly, µg of extracted dna was digested overnight at • c with ecori which cuts the recombinant t-dna at a single position (between the s promoter and the tev leader). after agarose gel ( . %) electrophoresis, digestion products were transferred overnight onto a nylon membrane (hybond+, amersham-pharmacia biotech, uk). the membrane was equilibrated with × sspe prior to immobilization of dna by uvcross-linking (uv stratalinker, stratagene, netherlands). for hybridization, a p-labelled h-specific cdna probe, generated by nick translation according to feinberg and volgelstein ( ) , was incubated with the membrane for h at • c. the membrane was then washed for min at • c successively with . % sds in × ssc, × ssc and finally in . × ssc. hybridized complexes were detected by autoradiography. total rna from leaves ( g) of transformed plants was isolated as described by hughes and galau ( ) . rna from untransformed plants was used as a negative control template. reverse transcription (rt) was carried out for h at • c in µl final reaction volume containing µg rna, ng random hexamers and a mixture of dntps ( mm each), mm dtt, units of rnasin (promega, netherlands) and units of m-mlv reverse transcriptase (superscript ii, gibco life sciences, belgium). after adding . volume of mm atp, ligation was carried out by incubation for min at • c in the presence of units of t dna ligase. pcr amplification was performed with two h-specific primers including the first and the last nucleotides of the coding sequence. the pbin -mvh vector was used as positive control template. a pcr reaction was also performed directly on the total rna to confirm the absence of specific dna in the extract. plant tissues were homogenized on ice in pbs containing mm edta and protease inhibitors ( µg/ml aprotinin, . mg/ml iodoacetamide, µg/ml leupeptin, mm pmsf). the mixture was centrifuged at × g ( min, • c) to remove insoluble debris. the membrane fraction was sedimented by ultracentrifugation at × g ( min, • c), and re-suspended in pbs containing . % np- . protein concentration was determined with the dc protein assay kit (biorad, belgium). one gram of wet weight of carrot plant gave about µg of membrane protein. proteins of the membrane fraction were separated by % sds-page under reducing and denaturing conditions ( mm dtt, m urea, % sds). proteins were blotted onto nitrocellulose membrane in tris-glycine buffer for h at ma. the membrane was blocked for h at room temperature with % non-fat dehydrated milk in pbs containing . % tween- . for detection of the mv-h protein two specific monoclonal antibodies (mabs; bh and bh , : dilution) and goat anti-mouse iggconjugated horseradish peroxidase secondary antibodies ( : dilution) were used. bound antibodies were detected by enhanced chemiluminescence (ecl kit, amersham-pharmacia biotech). to characterize recombinant h protein in leaf and root extracts microtiter plates (maxisorb, nunc, denmark) were coated with increasing concentrations of plant extract (in pbs) and revealed with h-specific mabs (dilution : ). bh recognizes the sequential helix-forming epitope h - (fournier et al., ; deroo et al., ) ; bh and bh are conformation-dependent antibodies (unpublished); bh binds only denatured protein (ziegler et al., ) ; bh binds to the hemagglutinin noose epitope (hne) h - (ziegler et al., ) . mouse sera ( : to : ) were titrated against immobilized purified h protein (bhk-h) produced in bhk- cells ( ng/well; bouche et al., b) . the coated antigens were washed and blocked with % bsa in tris-buffered saline ( mm, ph . ). after addition of mabs or mouse serum, alkaline phosphatase-conjugated goat anti-mouse igg ( : ; southern biotechnology association, usa) and pnitrophenylphosphate (sigma, usa) were used for detection. optical density (od) was measured after min at nm. data were expressed as net od values after subtracting either the absorbance of the negative control antigen (e.g. extract of wild-type roots or leaves, or bhk- antigen; figure ) or the absorbance of the conjugate without mouse serum (< . od). h-specific antibody isotypes and subclasses were determined in mouse sera ( : ) by elisa with specific conjugates and substrate of a commercial kit (biorad). data were expressed as od at nm after min as recommended. groups of four spf balb/c mice were primed by intraperitoneal injection of with µg of leaf or root extracts from transgenic or wild-type (wt) plants or µg of bhk-h or bhk- antigens emulsified ( : ) in freund's complete adjuvant (sigma). by comparison with the elisa signal of mammalianexpressed purified h protein, the plant extract and the bhk extract was estimated to contain about and µg of the specific protein, respectively. mice were boosted on days , and with the same antigen preparation emulsified in freund's incomplete adjuvant (sigma). sera were drawn days after boosting and were tested as pooled sera (after the second boost) or as individual sera (after the third boost). the reactivity of immune sera ( : ) with native h protein or mv was tested by flow cytometry as described before using permanently h-transfected mel-juso cells expressing the recombinant protein at their surface (mel-juso/h, gift of r. de swart, rotterdam, netherands; de swart et al., ) or mvsuperinfected ebv-transformed human b cell line (mv-wmpt; gift of b. chain, london, uk; muller et al., ) . for both assays wt or uninfected cells (mel-juso/wt or wmpt) were used as negative control cells. cells were incubated on ice ( min) with diluted serum of individual mice. after washing, fitc-conjugated goat anti-mouse fc-specific antibody ( : , sigma) was used for detection. fitcconjugate alone, naïve serum on positive and negative cells or test serum on negative cells served as negative controls. bh ( : ; ziegler et al., ) served as an antibody positive control. dead cells were excluded by propidium iodide staining ( µg/ml). to test mv-neutralizing activity, mouse sera were heated for min at • c to inactivate complement. duplicates of two-fold serial dilutions ( : to : ) of heat-inactivated serum were mixed with an equal volume of medium containing plaqueforming units of edmonston strain mv. after . h of pre-incubation at • c, the mixture was added to a subconfluent vero cell culture in -well plates ( . × cells per well). after one hour unbound virus was removed and cells were covered with a carboxymethylcellulose ( %) overlay. after four days of incubation under tissue culture conditions, a . % neutral red solution was added. on day , the overlay was removed and cells were fixed with a % formaline solution. the % neutralization titre, defined as the reciprocal of the dilution that reduces the number of plaques by %, was calculated accord-ing the spearman-kärber method. titres < were considered to be negative. the transformation of carrot plants was mediated by recombinant agrobacterium infection using the pbin -mvh plasmid (figure ). the regenerated transgenic plants showed no morphological changes in comparison to wt carrot plants (data not shown). about plants resulting from independent transformation events were selected and grown in the greenhouse. ten of them were analysed further. the presence of the mv-h expression cassette in transgenic plants was confirmed by pcr followed by gel electrophoresis of the amplified fragments (figure a) . from all transformed plants tested a product of the expected size ( . kb) was amplified (lanes - ). the same size was obtained with pbin -mvh vector as a positive control template (lane ). this product was absent in dna of untransformed plants (lane ). similarly, the presence of the nptii gene was confirmed as a specific product of bp in all geneticin-resistant plants ( figure b, lanes - ) . this product was absent from untransformed plants (lane ). copy numbers of the transgene and the integration pattern were determined by southern blot by digesting genomic dna of the transformed plants ( figure c ) with ecori (which has a unique restriction site in the t-dna, see figure ). hybridization with a mv-h-specific probe showed in every transgenic plant a unique restriction pattern (lanes - ) indicating that insertions occurred at random sites throughout the genome. all plants appeared to have integrated a single copy of the transgene, as illustrated by the presence of a single hybridization band. the insert corresponding to the radioactive probe was used as positive control template (lane ). no signal appeared when genomic dna of wt plants was used as a template (lane ). the transcription of the mv-h gene was analysed by rt-pcr on total rna extracted from transgenic plants ( figure a ). untransformed plants served as a negative control (lane ). all tested plants produced a specific major transcript of the expected size lanes - ) . the minor unspecific, smaller band was also found when other transgenes were tested. no amplified dna was detected when the pcr was directly performed on the rna preparations, confirming the rna-specificity of the reaction (lane ). crude membrane preparations of leaves ( ng protein) and of bhk-h cells ( ng protein) gave specific bands of similar intensities in western blots ( figure b ). based on elisa with purified h protein the content of specific protein was estimated at about % and % of specific protein in membrane fraction. the h-specific monoclonal antibodies bh and bh that bind to two non-overlapping epitopes revealed a strong under reducing conditions with an estimated molecular mass of about kda (lane ). under the same conditions, h protein produced in mammalian cells (bhk-h) migrated with an apparent size of ca. kda as reported earlier (lane ; bouche et al., b) . this difference in mass may correspond to different levels of glycosylation of the monomer, but this was not further explored (vialard et al., ) . negative controls included an irrele- vant mab (lanes - ) as well as a crude membrane preparation of wt leaves (lanes ). the western blot suggested that there may be differences in glycosylation. glycosylation is well known to be important for the proper folding of the h protein (hu et al., ) . several monoclonal antibodies were used to investigate the conformational integrity of the transgenic protein ( figure a ). microtitre plates were coated with increasing concentrations ( . - ng/well) of membrane fractions from both transgenic leaves and roots. high net signals were obtained with two conformational-dependent mabs (bh and bh ), and bh which recognizes the sequential epitope h - (fournier et al., ) with a putative helical conformation (deroo et al., ) . in contrast, bh , which binds denatured protein only, showed essentially no reactivity above the background of wt plants. bh binds to the hemagglutinin noose epitope (hne, h - ) with its oxidized cysteine bridge (ziegler et al., ) . the antibody binding pattern was essentially the same in root and leaf extracts and resembled that of purified h protein of mammalian origin ( figure b ). in general, signals tended to be higher in younger than in older tissues (data not shown). since the transgenic h protein seemed to be antigenically conserved, its immunogenicity was tested in mice. after or boosts with transgenic plant extracts all animals showed reactivity with purified h protein produced in mammalian cells ( figure a ). after the third boost, average antibody levels obtained with leaf extracts were about times higher than after immunization with root extracts. similar levels were found after boost for leaves, whereas antibodies still increased between boost and with root extracts. sera of control mice immunized with wt plant extract showed no cross-reactivity with the h protein (net od < . ; data not shown). interestingly, antibodies generated with the transgenic leaves were of both the igg and igg a subclass, whereas the immune response against the h protein produced in bhk- cells was essentially restricted to igg , suggesting a difference in the th /th balance ( figure b ). no h-specific igg b, igg , iga and igm were detected. to exclude that the reactivity may be due to partially denatured recombinant protein, the sera were further tested for antibodies against the intact native protein expressed on mv-superinfected wmpt cells ( figure a ) and h-transfected mel-juso cells (figure b ). the flow cytometry data showed that all mice vaccinated with transgenic leaf or root extracts produced high levels of antibodies cross-reacting with the native protein independently whether virus-infected or h-protein transfected cells were used. antibody levels were similar to those of mice immunized with h protein produced in mammalian cells, while wt plants induced no cross-reactive antibodies. in addition to a strong virus cross-reactivity, all sera showed high levels of neutralizing antibodies in a standard plaque reduction neutralization assay. in the group immunized with leaf extracts, mean neutralizing titres of (range - ) were observed, while root extracts induced significantly lower titres ( ; range - ). all sera from mice given extracts of untransformed plants had titres < and were considered negative. in general, both cross-reactive and neutralizing titres were higher in leaves than in roots. this could reflect a difference in expression or in the yield of the extraction procedure. although edible vaccines seem to be feasible, very few antigens have been expressed in plants fit for human consumption (modelska et al., ; kapusta et al., ; sandhu et al., ) . for instance, potatoes have been used to express antigens of human pathogens (mason et al., ; arakawa et al., a, b; richter et al., ; kong et al., ) . however, raw potatoes are not very appealing, and cooking can drastically reduce the immunogenicity of the vaccine (kong et al., ) . this is the first report of the expression of an antigenic protein as a transgene in mature carrots (which can be eaten raw by human beings) and of the antigenic and immunogenic properties of the heterologous protein in mice. genomic integration of the mv-h gene under the camv s double promoter was obtained using agrobacterium tumefaciens. southern blot analysis showed that all regenerated transgenic plants analysed integrated a single copy of the transgene and that integration occurred randomly in the carrot genome. in all clones a specific transcript of the expected size could be amplified by rt-pcr. under the fluorescence microscope, a fusion protein of mv-h with the green fluorescence protein (gfp) expressed in tobacco protoplasts showed a predominant association with the plasma membrane (data not shown). the membrane fraction of carrot cells produced a strong signal in western blot correspond-ing to an estimated µg of specific protein per gram of wet weight. interestingly, the transgenic protein migrated faster in sds-page than the same protein produced in mammalian cells, suggesting a size difference of about kda. a similar observation was made when the rabies virus glycoprotein g was expressed in plants (mcgarvey et al., ) . the expression of the c-terminally fused gfp demonstrates that the h protein is fully translated and that the difference in apparent size is most probably due to post-translational modifications. in the virus, the edmonston strain h protein undergoes glycosylation at of the predicted n-glycosylation sites (hu et al., ) . it has been shown that glycosylation of mammalian proteins is efficient in plants, but normally different carbohydrate side chains are utilized (bardor et al., ) . the complex glycans of plants are often smaller than those of animals, partially because they lack sialic acid (faye et al., ) . in insect cells, which also lack sialic acid, the reduced size of the recombinant h protein was explained by a difference in glycosylation (vialard et al., ) . authentic post-translational modifications such as glycosylation and cystine-bridge formation are thought to be important for intracellular trafficking (hu and norrby, ) as well as the antigenic conformation of the h protein (hu et al., ; hu and norrby, ) . recently, huang et al. ( ) reported the expression of the h protein in tobacco leaves. in this system, the native h protein was undetectable. after adding a retention signal for the endoplasmic reticulum low levels of protein became detectable by elisa with polyclonal sera from rabbits and man; the reactivity with mabs was even weaker or negative suggesting that the conformation may have been less than optimal. the immunogenic integrity of the h protein is critical for the induction of antibodies that protect against virus infection and disease. conformational dependent mabs confirmed the proper antigenic structure of the transgenic protein produced in carrots; while mab bh which recognizes only denatured protein showed no reactivity. after immunization with extracts of transgenic carrots high levels of virus-neutralizing antibodies were found, using the who recommended plaque neutralization assay (miller et al., ) . these titres were comparable to those obtained with the mammalian cell-derived h protein extract. the antibody isotype subclasses suggested that in contrast to the h protein of mammalian origin, which produced primarily a th (or antibody-dominated) response, the plant protein induced a th /th -balanced response, indicative of both a humoral and cellular response. this may be important to safeguard against atypical measles when such a vaccine is used in unprimed individuals. in many countries carrots are components of the diet of both adults and children and a stable antigenic transgene in an edible plant that can be consumed raw without further processing could bring a vaccine within reach of the most destitute. although plants can be used as efficient bioreactors for producing antigens, the full potential of plant-based vaccines becomes apparent when immunogenicity can be demonstrated after oral ingestion. a frequent problem of plant-based vaccines is that the response after oral delivery is inconsistent and variable. sometimes even low levels of antigen can give an effective response after oral administration in mice (mason et al., ; wigdorovitz et al., ) and man (kapusta et al., ) . in other cases mucosal immunity was improved by mucosal adjuvants (arakawa et al., b; kong et al., ) . in the study by huang et al. ( ) , the immune response after oral administration of transgenic tobacco was very weak even when exceedingly high levels ( times mg) of ctb where co-administered. however, when experimental oral vaccines were given after parenteral priming, results were more consistent than after oral immunization alone (kong et al., ; mantis et al., ) . therefore, a prime booster schedule with an oral vaccine could be an attractive alternative when immunity to the current (injectable) live-attenuated measles vaccine wanes in adults. further studies are required to confirm the efficacy of the carrot plant expressed measles antigen in such a scenario. efficacy of a food plant based cholera toxin b subunit vaccine a plant-based cholera toxin b subunit-insulin fusion protein protects against the development of autoimmune diabetes analysis of the n-glycosylation of recombinant glycoproteins produced in transgenic plants binary agrobacterium vectors for plant transformation immunosorbent assay based on recombinant hemagglutinin protein produced in a high-efficiency mammalian expression system for surveillance of measles immunity a simplified immunoassay based on measles virus recombinant hemagglutinin protein for testing the immune status of vaccinees transgenic plants as a potential source of an oral vaccine against helicobacter pylori oral immunization by transgenic plants protective immune response to foot-and-mouth disease virus with vp expressed in transgenic plants measles virus fusion protein-and hemagglutinin-transfected cell lines are sensitive tool for the detection of specific antibodies by facs-measured immunofluorescence assay enhanced antigenicity of a four-contact-residue epitope of the measles virus hemagglutinin protein by phage display libraries: evidence of a helical structure in the putative active site detection, biosynthesis and some functions of glycans n-linked to plant secreted proteins a technique for radiolabeling dna restriction endonuclease fragments to high specific activity antibodies to a new linear site at the topographical or functional interface between the haemagglutinin and fusion proteins protect against measles encephalitis correlation between epitopes on hemagglutinin of measles virus and biological activities: passive protection by monoclonal antibodies is related to their hemagglutination inhibiting activity expression of immunogenic glycoprotein s polypeptides from transmissible gastroenteritis coronavirus in transgenic plants oral immunization with a recombinant bacterial antigen produced in transgenic plants transformation and regeneration of carrot foreign gene expression in plants role of individual cysteine residues in the processing and antigenicity of measles virus hemagglutinin protein role of n-linked oligosaccharide chains in the processing and the antigenicity of the measles virus haemagglutinin protein plant-derived measles virus hemagglutinin protein induces neutralizing antibodies in mice preparation of rna from cotton leaves and pollen a plant-derived edible vaccine against hepatitis b virus oral immunization with hepatitis b surface antigen expressed in transgenic plants immunization of mice with recombinant gp in a systemic prime/mucosal boost protocol induces hiv- -specific serum igg and secretary iga antibodies expression of hepatitis b surface antigen in transgenic plants expression of norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice monospecific antibody to the haemagglutinin of measles virus expression of the rabies virus glycoprotein in transgenic tomatoes antibodies to measles, mumps, and rubella in uk children years after vaccination with different mmr vaccines immunization against rabies with plant-derived antigen modeling the impact of subclinical measles transmission in vaccinated populations with waning immunity cholera toxin b stimulates systemic neutralizing antibodies after intranasal co-immunization with measles virus human membrane cofactor protein (cd ) acts as a cellular receptor for measles virus identification of dna sequences required for activity of a plant promoter: the camv s promoter expression in plants of two bacterial antibiotic resistance genes after protoplast transformation with a new plant expression vector nuclear transport of plant potyviral proteins production of hepatitis b surface antigen in transgenic plants for oral immunization oral immunization in mice with transgenic tomato fruit expressing respiratory syncytial virus-f protein induces a systemic immune response promoters for pregenomic rna of banana streak badnavirus are active for transgene expression in monocot and dicot plants immunogenicity in humans of a recombinant bacterial antigen delivered in a transgenic potato human immune responses to a novel norwalk virus vaccine delivered in transgenic potatoes immunogenicity of transgenic plant-derived hepatitis b surface antigen a rapid method for transformation of carrot (daucus carota l.) by using direct somatic embryogenesis synthesis of the membrane fusion and hemagglutinin proteins of measles virus, using a novel baculovirus vector containing the β-galactosidase gene induction of a protective antibody response to foot and mouth disease virus in mice following oral or parental immunization with alfalfa transgenic plants expressing the viral structural protein vp measles virus: both hemagglutinin and fusion glycoproteins are requiered for fusion protection against measles virus encephalitis by monoclonal antibodies binding to a cystein loop domain of the h protein mimicked by peptides which are not recognized by maternal antibodies we are grateful to richard wagner and his team (ibmp) for taking care of the transgenic carrots. we also thank b. jérouville, s. willieme and w. ammerlaan for technical assistance. this research was supported by a grant from the ministère de l'education nationale et de la formation professionnelle du grand-duché de luxembourg, the european union th framework programme (project pl ) and the crp-santé, luxembourg. key: cord- - lc fcpe authors: rekha, kaliyaperumal; thiruvengadam, muthu title: secondary metabolite production in transgenic hairy root cultures of cucurbits date: - - journal: transgenesis and secondary metabolism doi: . / - - - - _ sha: doc_id: cord_uid: lc fcpe cucurbits are important group of vegetables due to their nutritional significance and are also used for valuable traditional medicine. the infection of plants by agrobacterium rhizogenes results in a hairy root (hr) phenotype characterized by rapid growth in hormone-free medium, an unusual ageotropism and extensive lateral branching. these genetically transformed root cultures (hairy roots) can produce levels of secondary metabolites comparable to that of intact plants. hairy root cultures offer promise for high production and productivity of valuable secondary metabolites in many plants. high stability and productivity features allow the exploitation of hrs as valuable biotechnological tool for the production of plant secondary metabolites. while these chemical compounds are employed by plants for interactions with their environment, humans have long since explored and exploited plant secondary metabolites for medicinal and practical uses. the main constraint for commercial exploitation of hairy root cultivations is the development and scaling up of appropriate reactor vessels (bioreactors) that permit the growth of interconnected tissues normally unevenly distributed throughout the vessel. emphasis has focused on designing appropriate bioreactors suitable to culture the delicate and sensitive plant hairy roots. to this end, hairy root culture presents an excellent platform for producing valuable secondary metabolites. for these reasons, this chapter describes the establishment of hairy roots and production of secondary metabolites from hairy roots of cucurbits and also phytochemicals uses for biological activity. cucurbits are the popular name to the plants of family cucurbitaceae, which include over genera and species [ ] , among the economically most important plant families [ , ] . it is a large group of plants which are medicinally valuable. cucurbitaceae members are primarily established in the tropical regions of the world. global production of cucumbers, including gherkins, was among the top ten vegetables produced globally (http://faostat.fao.org). cucurbits are a prominent source of secondary metabolites, and many genera of this family received a great level of scientific interest because of the extensive range of pharmacological and nutraceutical properties [ ] . it is reported that the bitter flavor of cucurbits is due to tetracyclic triterpenoids [ ] . various phytochemicals such as alkaloids and saponins are extracted from momordica, citrullus, cucurbita, and lagenaria [ ] . the family proved itself as a strong source of food and medicine. major species of importance include: citrullus lanatus (watermelon), cucumis sativus (cucumber), cucumis melo (musk melon), cucumis anguria (bur gherkin), cucurbita pepo (pumpkin), momordica charantia (bitter gourd), momordica dioica (spine gourd), coccinia grandis (ivy gourd), and praecitrullus fistulosus (tinda). in recent years, consumption of cucurbits in the average diet has been highlighted for its contribution towards lowering the risks of several life-threatening diseases such as coronary heart disease, stroke, pulmonary disease, and different types of cancer. plant species are capable of producing different types of secondary products which can be harnessed by humans for their beneficial properties in a large domain of industrial or medicinal applications [ ] . world health organization (who) estimates that up to % of people rely mainly on traditional herbs as remedies for their medicines [ ] . extracted from entire plants, secondary products are used by food and pharmaceutical industries, although most often numerous natural plantderived molecules remain undiscovered or unexplored for their pharmacological properties [ ] . roots play most important roles in plants and they anchor plants to the ground, take up minerals and water from the soil, store nutrients for perennial plants, and produce a diverse array of chemicals for symbiotic interactions or defensive with other plants or microbes in the rhizosphere. these plant-produced chemicals have traditionally been referred to as secondary metabolites and more recently tagged as specialized metabolites. bioactive compounds are extra nutritional constituents that naturally occur in small quantities in plant and food products [ ] . most common bioactive compounds include secondary metabolites such as antibiotics, mycotoxins, alkaloids, food grade pigments, plant growth factors, and phenolic compounds [ , ] . many secondary metabolites not only protect plants from pathogens, insects, and environmental stresses but also are valuable for human health. many plant species, including crop plants, are capable of producing and releasing biologically active compounds (allelochemicals). allelochemicals (e.g., phenolics, terpenoids, alkaloids, coumarins, tannins, steroids, and quinines) are released by the plant into the environment by root exudation, volatile emissions, leaching from the leaves and other aerial parts, and the decomposition of plant material [ , ] . plant roots release a range of compounds that are not directly involved in the growth and development of the plant but are very much important for plants during stress conditions (biotic/abiotic). these compounds include aliphatic acids, aromatic acids, fatty acids, sterols, phenolics, enzymes, and other secondary metabolites, including flavonoids [ , ] . many plant secondary metabolites of interest are accumulated in roots. however, plant cultivation is often time consuming and metabolite extraction from plant roots is destructive to plant growth. agrobacterium rhizogenes is a gram negative soil-borne bacterium of the family rhizobiaceae, which causes the hairy roots disease by infecting wounded higher plants. the transformed roots can be excised to establish axenic root cultures and indefinitely propagated in growth regulator free medium. the root exhibit fast, plagiotropic growth characterized by profuse lateral branching and rapid root tip elongation [ , ] . root loci (rol) genes harbored by the root-inducing (ri) plasmid of this bacterium are incorporated into the host plant genome, causing hairy root. rol genes are thought to affect growth and development of transformed roots and induce secondary metabolite synthesis by turning on the transcription defense genes [ , ] . the rolb and rolc genes are absolutely essential for induction of hairy roots [ ] . fast growing and genetically stable hairy roots can be efficiently cultured in large scale bioreactors [ ] . besides, hairy root cultures are usually capable of producing the same compound(s) of identical chemistry found in wild-type roots of the naturally occurring parent plant without loss of structural integrity and/or quantity or concentration of the product, which is frequently observed in callus or cell suspension cultures [ ] . a. rhizogenes to regulate the genes that were involved in the plant secondary metabolite production [ ] . hairy roots induced from different plant tissues generally grow fast, are genetically stable, and often, but not always, simulate the biochemical profiles of plant roots, which makes hairy roots an attractive system for producing valuable secondary metabolites. plant roots can synthesize, store, and secrete a vast array of compounds, and transformed root cultures have a wide range of biosynthetic capacities [ ] . various advantages of hairy root culture over cell suspension culture include genotypic and biochemical stability, cytodifferentiation, and growth in hormone free medium. these factors play a vital role during secondary metabolite production. fast growth, low doubling time, ease of maintenance of hairy roots, and their ability to synthesize a large range of chemical compounds offer an additional advantage as a continuous source for the production of valuable secondary metabolites [ ] . a number of secondary metabolites have been reported to be produced from hairy root cultures [ ] . progress has been made on commercialization of hairy root products. rootec bioactives ltd., founded in in switzerland, currently produces phytochemicals from hairy roots induced from plant species in their proprietary mist bioreactors. in the future, more investigations could be directed toward determining the efficacy of crude hairy root extracts or hairy root-produced chemicals. there have been few reviews in the literature on a wide variety of hairy root applications in secondary metabolites of medicinal plants. previously, very few studies of secondary metabolite production in hairy root cultures of cucurbits have been reported. first time, we focus this chapter on establishment of hairy roots and production of secondary metabolites in cucurbits. hairy roots were induced from various explants (leaf, cotyledon, hypocotyl, node, and root) after - weeks of culture. control explants failed to induce hairy root formation. high induction of hairy roots was observed in leaves compared to other explants in gynostemma pentaphyllum, cucumis anguria, momordica charantia, and m. dioica [ ] [ ] [ ] [ ] . cotyledon explants produced higher frequency of hairy root induction in cucumis melo [ ] [ ] [ ] and c. sativus [ , ] . transgenic frequency ( %) of the infected stems of luffa cylindrica formed vigorous hairy roots within weeks from the inoculation of the bacteria [ ] . table shows the different strains of agrobacterium rhizogenes (maff - , k , r , c c , a , , mtcc , atcc , r , kctc , and kctc ) that were examined for their ability to induce hairy roots of various cucurbits such as melon, pumpkin, cucumber, sponge gourd, chinese cucumber, southern ginseng, bitter melon, spine gourd, and bur gherkin [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . two strains of a. rhizogenes differed in their ability to induce hairy roots, with strain kctc being more effective than kctc [ ] [ ] [ ] . monocyclic phenolic compound, acetosyringone incorporated into the nutrient medium showed enhanced transformation frequency than the medium without it. acetosyringone was used for co-cultivation in c. sativus [ ] and m. charantia [ ] . acetosyringone is an amino acid derivative which served as a nutrient source for the invading agrobacterium and enhanced the transformation rate. it was reported that acetosyringone would induce the vir gene of agrobacterium cultures. the established hairy roots show typical morphological characteristics with rapid growth on phytohormone-free medium, lack of geotropism, and extensive lateral branching [ ] [ ] [ ] [ ] . the transformed root was confirmed by pcr to determine the presence of a t-dna sequence in their genomes in gynostemma pentaphyllum [ ] . the pcr products from the hairy roots for rolb regions but not from untransformed roots of g. pentaphyllum. this finding indicated that the rolb genes from the ri plasmid of a. rhizogenes were integrated into the genome of g. pentaphyllum hairy roots. the negative results of pcr amplification for the virc gene demonstrated that no bacterial dna was involved in rolb amplification leading to false positives [ ] . the transgenic nature of hairy roots was confirmed by pcr using rolc and aux gene specific primers, and transgenicity was also confirmed by polymerase chain reaction (pcr), reverse-transcriptase pcr (rt-pcr), and sequencing in c. anguria, m. charantia, and m. dioica [ ] [ ] [ ] . the integration of ri t-dna into the genome of plant cells caused the formation of hairy roots, in which rol and aux genes were harbored in m. dioica [ ] . pcr analysis targeted the a. rhizogenes rolc, aux , and vird genes in m. dioica. the rolc and aux genes, located on [ ] independent t-dnas (tl-dna and tr-dna, respectively) of the ri plasmid of a. rhizogenes strain, are diagnostic for t-dna integration into the host genome. the vird gene, located outside the t-dna, is diagnostic for the presence of any remaining agrobacteria in the root tissue [ ] . the rol and aux genes are essential for the induction of hairy roots, and they act as a potential activator of secondary metabolites in cucurbits [ ] [ ] [ ] . the vird gene was used to verify the complete absence of a. rhizogenes in the hairy roots lines of c. anguria and m. dioica. this result indicates that pri t-dna fragments of a. rhizogenes were successfully integrated into the genome of c. anguria and m. dioica without bacterial residues [ , ] . the obtained full length coding sequence of rolc gene of m. charantia and m. dioica [ , ] . the use of pcr combined with dna sequencing instead of southern blotting for the characterization of transgenic plants has the advantage that the newly inserted genes can be detected at an earlier stage with less dna and less plant material [ , ] . the presence of pri t-dna in pumpkin long-term hairy root cultures was determined by southern hybridization [ ] . integration of the t-dna region of ri-plasmids into the plant genome was confirmed by both opine assay on paper electrophoresis and pcr-based detection of rol genes in trichosanthes kirilowii var. japonica [ ] . successful integration of the t-dna into chromosomal dna of the kmh- was first examined by pcr amplifying the rolc gene located on the integrated t-dna in cucumis melo [ ] . an immunoblot analysis of the oriental melon transgenic hairy root extract revealed kda single bands coincident with the molecular weight of the gfp gus fusion proteins. elisa demonstrated that the highest level of gfp-gus fusion protein expression was . % of the total soluble protein in a transgenic hairy root of oriental melon [ ] . the integration of t-dna containing a gus reporter gene in hairy root lines was confirmed at low copy numbers ranging from to copies using quantitative real-time pcr, and histochemical staining of cucumber hairy roots showed overexpression of the gus gene when driven with the camv s promoter in c. sativus [ ] . the presence of gus activity and its localization were observed in all of the tissues of the root, especially in transgenic cucumber hairy root lines with the camv s and camv st/amv promoters. the transgenic cucumber hairy roots lines with the camv s promoter or the camv st promoter showed localized gus activity only in the vascular bundles in c. sativus [ ] . quantification of the copy number of the gus gene using absolute quantification in real-time pcr revealed a low copy number of the gus gene per genome [ ] . the transgenic plants looked normal and were positive for the neomycin phosphotransferase ii. southern blot analysis of the transgenic plants revealed that all plants contained vector dna, but only some of them contained dna from the ri plasmid [ ] . enzyme-linked immunosorbent assay (elisa) revealed the highest levels of the recombinant t-pa accumulation in transgenic hairy roots carrying the t-pa transgene under the control of single and dual rold promoters as compared to triple and quadruple rold promoters [ ] . previously, it was reported that changes in secondary metabolite production in hairy roots and ri plants correlate with changes in the phenotype induced by the insertion of rol genes and with the quantity of the polypeptide encoded by the rolc gene [ , ] . interestingly, both the capacity to grow and produce nicotine in hairy roots and ri plants of nicotiana tabacum cv. xanthi were higher after integration of the three rol genes (a, b, c) together than with rolc alone. in addition, the level of nicotine accumulation was positively correlated with the levels of the polypeptide encoded by the rolc gene, as detected by immunoassays [ ] [ ] [ ] . the rola gene appears to be an activator of growth and secondary metabolism. although the rolb gene has emerged as the most powerful stimulator, its use is presently disputed owing to its growth-suppressing effect. more positively, the self-activation of rolc gene seems to be promising [ ] [ ] [ ] . the time profile of the growth of hairy roots in liquid culture was reported in c. anguria, m. charantia, and m. dioica [ ] [ ] [ ] . the sixth day was the lag period of hairy root growth; then it began to increase gradually during the eleventh day. the exponential growth stage during the days was followed by the stationary phase during the - days. the higher fresh mass (fm) and dry mass (dm) was observed at , , and days of culture of c. anguria, m. charantia, and m. dioica [ ] [ ] [ ] . the culture duration of days of hairy roots increased about -fold compared to inoculum and the gypenoside content in g. pentaphyllum [ ] . hairy root cultures showed a sigmoidal growth curve, and crude extracts showed a progressively increasing translational inhibitory activity that reached the maximum value during the early stationary phase of l. cylindrica [ ] . sucrose is the most significant carbon source for plant tissue cultures and helps as the chief energy source and an important constituent in secondary metabolite biosynthesis in cucurbits [ ] . the amount of sucrose usually affects the accumulation of secondary metabolites in cultures. about % of sucrose produced the higher amount of biomass accumulation and metabolite production in c. anguria, m. charantia, and m. dioica [ ] [ ] [ ] ] . about % of sucrose induced hairy root induction in trichosanthes kirilowii var. japonica [ ] . many previous reports focus on the composition of medium nutrients to achieve maximum accumulation of metabolites in cultured cells [ ] . the different media, full and half strength ms, b , nn, and ls were employed in hairy root culture and the results shown that ms medium was superior for biomass accumulation in g. pentaphyllum, c. anguria, m. charantia, m. dioica, cucumis melo, c. sativus, and t. kirilowii [ - , , , ] . however, other media like b was also used to induce hairy roots in l. cylindrica [ ] . hairy root induction of cucurbits using carbohydrate source is by sucrose and nutrients media is by ms or b . increased secondary metabolite production in hairy roots cultured in vitro, over their wild-type counterparts, may be seen as one of the most exciting spin-offs of biotechnology. due to their great richness in secondary products, such as triterpenoids and phenolic compounds, plants represent an immense source of therapeutic and/or industrial compounds. for example, plant-derived biomolecules, such as saponins (g. pentaphyllum), triterpenoids of bryonolic acid, and chondrillasterol (trichosanthes kirilowii var. japonica), ribosome-inactivating protein (luffa cylindrica), charantin (momordica charantia), hydroxybenzoic acids, hydroxycinnamic acids, and flavonols (c. anguria, m. charantia and m. dioica), are efficient in the treatment of different pathology types relating to cancer, cardiovascular and metabolic disorders, and/or other infectious diseases (table and fig. ). many plant metabolites are commercially available as drugs, flavors, food additives, cosmetics, fragrances, and insecticides. here, several important phytochemicals from hairy roots of cucurbits are discussed (fig. ). phenolic compounds are secondary metabolites, ubiquitous in plants and plant derived foods. they show a large diversity of structures, including rather simple molecules (e.g., vanillin, gallic acid, caffeic acid) and polyphenols such as stilbenes, flavonoids, and polymers derived from these various groups [ ] . phenolic compounds are classified into three major groups based on the number and binding position of exchangeable hydroxyl groups on aromatic compounds: simple phenol and phenolic acid group, hydroxycinnamic acid derivative group, and flavonoid group. a majority of the plant phenolic metabolites are derived from the aromatic amino acids that are synthesized from the shikimate pathway. phenolics are collectively valued for their wide variety of health-promoting activities. flavonoids are phenylpropanoid metabolites, most of which are synthesized from p-coumaroyl-coa and malonyl-coa, and share their precursors with the biosynthetic pathway for lignin biosynthesis [ ] . flavonoids are low-molecular-weight compounds having approximately atoms of carbon, which are organized in a c Àc Àc configuration [ ] . more than flavonoids have thus far been identified in plants [ ] . phenolic acids are considered as simple phenolics, and they are categorized into two groups, i.e., the hydroxybenzoic and hydroxycinnamic acids. fig. (continued) antiallergenic, antimicrobial, cardioprotective, anti-inflammatory, antioxidant, artherogenic, and vasodilatory effects [ ] [ ] [ ] . phenolic compounds are synthesized via the phenylpropanoid pathway that begins with conversion of phenylalanine to cinnamic acid by phenylalanine ammonia lyase (pal). in the last few years, great attention has been paid to the bioactive compounds due to their ability to promote benefits for human health, such as the reduction in the incidence of some degenerative diseases like cancer and diabetes [ , ] , reduction in risk factors of cardiovascular diseases [ , ] , antioxidant, antimutagenic, antiallergenic, antiinflammatory, and antimicrobial effects [ , ] , among others. due to these countless beneficial characteristics for human health, researches have been intensified aiming to find fruits, vegetables, plants, agricultural, and agro-industrial residues as sources of bioactive phenolic compounds. the qualitative and quantitative analysis of phenolic compounds from hairy roots and untransformed (roots from in vitro seedling) root extracts of c. anguria, m. charantia, and m. dioica [ ] [ ] [ ] were studied using ultra-hplc. the phenolic compounds in the c. anguria, m. charantia, and m. dioica extracts were identified by comparisons of the retention time and uv spectra of authentic standards and the quantitative data were calculated from calibration curves [ ] [ ] [ ] . both transgenic and nontransgenic roots contained flavonols, hydroxycinnamic, and hydroxybenzoic acids. hairy roots contained higher amounts of flavonols compared to nontransgenic roots of c. anguria, m. charantia, and m. dioica [ ] [ ] [ ] . myricetin, quercetin, catechin, kaempferol, and rutin levels were higher in hairy roots compared to nontransgenic roots of c. anguria [ ] . the contents of naringenin and biochanin a were lower in concentrations in hairy roots than nontransgenic roots of c. anguria [ ] . myricetin, quercetin, catechin, kaempferol, rutin, biochanin a, and naringenin levels were higher in hairy roots compared to nontransgenic roots of m. charantia [ ] . naringin was presented in nontransgenic roots, but it was absent in hairy roots of m. charantia [ ] . quercetin, kaempferol, catechin, and rutin levels were higher in hairy roots compared to nontransgenic roots of m. dioica [ ] . myricetin, naringenin, and biochanin a contents were lower in concentrations in hairy roots than nontransgenic roots of m. dioica [ ] . kaempferol, myricetin, naringin, quercetin, and rutin have antimicrobial activity against human pathogenic microorganisms with some mechanisms of action such as inhibition of nucleic acid synthesis, cytoplasmic membrane function, and energy metabolisms [ ] . caffeic acid and chlorogenic acid were major hydroxycinnamic acid derivatives in hairy roots and nontransformed roots compared to p-coumaric acid, ferulic acid, ocoumaric acid, and t-cinnamic acid in c. anguria [ ] . caffeic acid, ferulic acid, ocoumaric acid, and t-cinnamic acid levels decreased in hairy roots compared to nontransformed roots of c. anguria [ ] . chlorogenic acid and p-coumaric acid contents were higher in hairy roots than nontransformed roots of c. anguria [ ] . chlorogenic acid containing plant materials have been shown to have antiviral, antifungal, and strong antibacterial activities [ ] . chlorogenic acid, p-coumaric acid, and ferulic acid levels were higher in hairy roots compared to nontransformed roots of m. dioica [ ] . caffeic acid, o-coumaric acid, and t-cinnamic acid contents were lower in hairy roots than nontransformed roots of m. dioica [ ] . caffeic acid, p-coumaric acid, o-coumaric acid, chlorogenic acid, and m-coumaric acid levels were higher and ferulic acid content was lower in hairy roots than nontransgenic roots of m. charantia [ ] . protocatechuic acid, β-resorcylic acid, syringic acid, gentisic acid, and salicylic acid levels were higher and gallic acid, p-hydroxybenzoic acid, and vanillic acid were lower in hairy roots than nontransgenic roots of c. anguria [ ] . gallic acid, p-hydroxybenzoic acid, gentisic acid, and salicylic acid levels were higher and protocatechuic acid, β -resorcylic acid, and vanillic acid were lower in hairy roots compared to nontransformed roots of m. dioica [ ] . gentisic acid has an effective role in the anticarcinogenetic activity [ ] . gallic acid, protocatechuic acid, β-resorcylic acid, vanillic acid, syringic acid, gentisic acid, and salicylic acid levels were higher and p-hydroxybenzoic acid was lower in hairy roots compared to nontransformed roots of m. charantia [ ] . veratric acid was higher and vanillin, hesperidin, and homogentisic acid were lower in hairy roots compared to nontransformed roots of c. anguria [ ] . vanillin was higher and veratric acid, hesperidin, and homogentisic acid levels were lower in m. charantia and m. dioica [ , ] . previous studies have revealed that polyphenolic compounds are commonly found in both edible and nonedible plants and that they have multiple biological effects, including antioxidant activity [ ] . flavonoids and other phenolic substances may play a preventive role in the development of cancer and heart disease [ ] . biological activities related to antibacterial and antioxidant activities may be correlated with total polyphenol and flavonoid contents [ ] . the total phenolic and flavonoid contents were higher in hairy roots compared to untransformed roots of c. anguria, m. charantia, and m. dioica [ ] [ ] [ ] . gypenosides (gyp) are the major components of gynostemma pentaphyllum makino, a chinese medicinal plant. phytochemical studies of g. pentaphyllum have identified approximately dammarane-type saponin glycosides, known as gypenosides, which are responsible for its pharmacological activities [ ] . saponins are a class of chemical compounds found in particular abundance in various plant species. more specifically, they are amphipathic glycosides grouped phenomenologically by the soap-like foaming they produce when shaken in aqueous solutions and structurally by having one or more hydrophilic glycoside moieties combined with a lipophilic triterpene derivative. triterpenoid saponins are triterpenes which belong to the group of saponin compounds. triterpenes are a type of terpene containing carbon atoms. triterpenes are assembled from a five-carbon isoprene unit through the cytosolic mevalonate pathway to make a thirty-carbon compound. cucurbitacins are triterpenoids that confer a bitter taste in cucurbits such as cucumber, melon, watermelon, squash, and pumpkin. these compounds discourage most pests on the plant and have also been shown to have antitumor properties [ ] . gypenoside content was higher compared to roots of control parent plant of gynostemma pentaphyllum hairy root cultures [ ] which was significantly higher than that previously reported for hairy root cultures [ ] . transformed roots can synthesize and store significant quantities of secondary metabolites. although the hairy roots under these conditions produced approximately % to % less gypenosides than commercial sources of g. pentaphyllum, the growing time was much shorter when compared to field-grown plants. with hairy root cultures, product quality and quantity are easy to control because natural variances in seasonal climates and geographical environments are excluded and culture conditions and process variables are easily optimized [ ] . the hairy root cultures have been considered as a potential alternative for production of gypenosides. several strategies for the enhancement of biomass and gypenosides have been adopted like the effects of medium compositions, culture conditions, and elicitations [ ] . ribosome-inactivating proteins (rips) are widely distributed plant enzymes that inhibit protein synthesis by virtue of their n-glycosidic activity, selectively cleaving an adenine residue from a highly conserved and surface-exposed stem loop structure in the s rrna [ ] . this cleavage prevents the binding of the ef- /gtp complex, with the subsequent arrest of protein synthesis leading to autonomous cell death [ ] . rips are either enzymatically active single polypeptides (type i) or heterodimers (type ii). a type ii rip consists of an a chain, functionally equivalent to a type i rip, which is attached to a sugar-binding b chain [ ] . besides rna n-glycosidase activity, some rips have ribonuclease, dnase, dna glycosylase, and apurinic/apyrimidic lyase activities [ , ] . in addition, rips from trichosanthes kirilowii cell cultures have been demonstrated to possess chitinase activity [ ] . certain type i rips display a variety of antimicrobial activities, including antifungal, antibacterial [ ] , and broad-spectrum antiviral effects against different plant and animal viruses [ ] , including human immunodeficiency virus [ ] . rips have been studied as potential tumor cytotoxic agents, both in their native form and after conjugation with monoclonal antibodies. rip activity of l. cylindrica plantlets, grown in vitro on ms medium, was evaluated in crude extracts from different parts and organs and compared to the inhibitory activity shown by extracts from seeds and from transformed roots [ ] . the inhibitory activity, as far as normal, nontransformed tissues are concerned, is in agreement with what was already known from previous reports of l. cylindrica [ , ] . rip-producing hairy roots promise to be much more stable than conventional in vitro grown calluses and cell suspensions [ ] . this study tested the sc-rip extracts from the seeds and hairy root tissue cultures of luffa cylindrica (established by transformation with agrobacterium rhizogenes strain ) for inhibitory effects on the growth of in vitro melanotic and amelanotic human melanoma cell lines [ ] . the results reported that rips can be produced and purified from hairy root cultures, in good agreement with what has been recently reported [ ] for hairy root lines of trichosanthes kirilowii. ribosomeinactivating proteins (rips) from plants catalytically damage eukaryotic ribosomes, making them unable to perform the elongation step of protein synthesis. type rips are single-chain proteins, whereas type rips consist of two polypeptide chains and possess a galactose-specific binding domain to cell surfaces. type rips are more common and have been identified and purified from more than plants. interest in type rips has been growing due to their widespread physiological activities as abortifacient agents and immunotoxins [ ] . the antiviral activity of rips has also focused attention on their potential use as anti-hiv agents [ ] . there have been few reports on the production of rips by plant tissue or cell cultures. a low level of trichosanthin was reported to accumulate in transformed hairy root cultures of trichosanthes kirilowii var. japonica [ ] . trichosanthin was also identified in cell extracts of the transformed callus tissues resulting from infection by agrobacterium rhizogenes but not in the untransformed callus of t. kirilowii [ ] . the major protein in the basic protein fraction was tentatively identified as a class iii chitinase based on the n-terminal amino acid sequence. this is consistent with the report [ ] , who identified two major extracellular basic proteins and one intracellular basic protein produced by t. kirilowii var. japonica hairy roots as class iii chitinases. however, the n-terminal sequence of hr-pb was very similar to but not identical with the sequence of any of these proteins. a molecule of charantin consists of aglycone or a steroidal portion, which is highly soluble in relatively nonpolar solvent such as chloroform and dichloromethane. however, the glucosides attached to its molecules make it slightly soluble in polar organic solvents such as ethanol or methanol. conventionally, isolation of this compound involves extraction with mixtures of these solvents using soxhlet apparatus. chloroform is highly toxic and carcinogenic, and its use has now been replaced with its much less toxic relative, dichloromethane, which still carry some health risks. chronic exposure to dichloromethane has been linked to cancer of lungs, liver, and pancreas in laboratory animals. it is a mutagen and may cause birth defect if women were exposed to it during pregnancy [ ] . this compound could be used to treat diabetes and can potentially replace treatment by injection of insulin which has not been successful in stimulating the pancreas of the diabetic patients to lower blood sugar to the desired level [ ] . in some cases, the injected patient shows signs of side effects. plant derived compounds that show antidiabetic property such as charantin and others are now being widely accepted as an alternative medicine for diabetes mellitus, and they are free from side effects [ ] . charantin, a naturally occurring steroidal glycoside, is widely distributed throughout the plant of momordica charantia. the presence of charantin was confirmed by performing thin layer chromatography (tlc) in hairy roots as well as in fruit and leaf [ ] . the charantin content was lower in hairy roots compared to leaf and fruit of m. charantia [ ] . the typical cucumber flavor results from the enzymatic action of lox on linolenic and linoleic acids, which introduces molecular oxygen at c or c , forming -hydroperoxylinolenic acid ( -hpot) or -hydroperoxylinolenic acid ( -hpot). hpl cleaves -hydroperoxide ( -hpo) and -hpo to produce the c and c aldehydes that are responsible for the cucumber flavor [ ] . these aldehydes can then be reduced to the corresponding c alcohols by alcohol dehydrogenase (adh). studies have reported that only the oxylipin metabolic pathway contributes to aldehyde and alcohol content and hence flavor [ ] . to date, volatile compounds have been identified in cucumber fruits, including aldehydes, alcohols, esters, alkanes, furfurans, and others [ ] , and (e,z)- , nonadienal and (e)- -nonenal are the main aroma compounds [ ] . the hairy roots could newly synthesize some essential oils such as (z)- -hexenol, (e)- hexenal, -nonanol, and (z)- -nonenol, which were reported to be important aroma volatiles in melon [ ] . the stable production of the fruity aroma volatiles by the kmh- was assessed by comparing the yields of the compounds from the hairy roots repeatedly subcultured for more than years. the data revealed that the essential oils for aroma scent were constantly synthesized with no relation to the increased number of times for subculture, and the constant production by this clone was successfully maintained in the hairy roots repeatedly subcultured of cucumis melo [ ] . the volatile compounds were extracted and identified by glc-mass spectrometry. some essential oils such as (z)- -hexenol, (e)- -hexenal, -nonanol, and (z)- -nonenol were stably synthesized by these hairy roots despite the increased number of subcultures. the productivity of these compounds by the best hairy root line was shown to be considerably higher than naturally ripened melon fruits [ ] . phenolic compounds have multiple additional roles in plants, including attracting insects for seed dispersion and pollination. they are also part of the natural defense system against insects, fungi, viruses, and bacteria, and they can act as plant hormone controllers. moreover, in recent years, phenolic compounds have been intensively investigated because of their potential health-promoting effects [ , ] . they have been reported to possess many useful properties for human health, including anti-inflammatory, enzyme inhibition, antimicrobial, antiallergic, vascular, and cytotoxic antitumor activity, but the most important action of phenolics is their antioxidant activity [ , , ] . it has been demonstrated recently that quercetin and kaempferol synergistically suppress cell proliferation in human gut cancer lines [ ] . the translational inhibitory activity found in extracts from our hairy root cultures is the highest that has been found in various tissues of l. cylindrica, including seeds [ ] . the antioxidant potential of hairy roots and nontransformed roots were determined using free radicals scavenging, reducing potential, phosphomolybdenum assays, and chelating effects on ferrous ions. the highest antioxidant activity was exhibited in hairy roots compared to nontransformed roots in c. anguria, m. charantia, and m. dioica [ ] [ ] [ ] . reducing capacity of extracts suggests that hairy roots were more potential when compared to untransformed roots in c. anguria, m. charantia, and m. dioica [ ] [ ] [ ] . the antioxidant capacity shown by phosphomolybdenum method was higher in the hairy root extract than nontransformed root extract of c. anguria, m. charantia, and m. dioica [ ] [ ] [ ] . the percentage of metal scavenging capacity of transgenic hairy roots was higher than nontransgenic roots of m. charantia, c. anguria, and m. dioica [ ] [ ] [ ] . hairy roots exhibited higher antioxidant activity in m. charantia [ ] . the hairy roots and nontransformed roots of c. anguria, m. charantia, and m. dioica revealed varying antibacterial activity, as exposed by the growth inhibition zones [ ] [ ] [ ] . the results from the disc diffusion method indicated that both hairy roots and nontransformed root extracts had comparable antibacterial effects against gram positive and gram-negative bacteria. hairy roots exhibited highest activity with both gram-positive and gram-negative bacteria compared to nontransformed roots of c. anguria, m. charantia, and m. dioica [ ] [ ] [ ] . gram-positive (s. aureus) bacteria exhibited greater inhibition compared to gram-negative (p. aeruginosa and e. coli) bacteria in m. charantia, c. anguria, and m. dioica [ ] [ ] [ ] . by using the disc diffusion method against the fungal strains, it can be seen that extracts of m. charantia, c. anguria, and m. dioica hairy roots and nontransformed roots exhibited good antifungal activity [ ] [ ] [ ] . hairy roots exhibited greater inhibition of fungus (f. oxysporum and a. niger) in hairy roots than nontransgenic roots of c. anguria, m. charantia, and m. dioica [ ] [ ] [ ] . hairy roots exhibited higher antibacterial and antifungal activity compared to nontransformed roots [ , ] . flavonoid derivatives have also been reported to possess antiviral activity against a wide range of viruses such as hsv, hiv, coxsackie b virus, corona virus, cytomegalovirus, poliomyelitis virus, rhinovirus, rotavirus, poliovirus, sindbis virus, and rabies virus [ ] . cytotoxicity activity and quantitative assay of virus yields using plaque assay were carried out for hairy roots and nontransgenic roots of m. dioica [ ] . hairy roots exhibited higher antiviral activity compared to nontransgenic root extracts of m. dioica [ ] . gypenosides (gyp) are compounds found in the crude extracts from g. pentaphyllum and they have been shown to exert various biological effects such as anti-inflammatory and antioxidative [ ] , antihyperlipidemic, anticardiovascular [ ] , and anticancer [ ] [ ] [ ] . our previous studies have shown that gypenosides induced apoptosis in human colon cancer colo cells [ ] and human tongue cancer scc- cells through endoplasmic reticulum stress and mitochondria-dependent pathways [ ] . although gypenosides have been shown to induce cell cycle arrest and apoptosis in several human cancer cell lines, there is no available information to address whether gypenosides induce dna damage or affects dna repair genes in sas human oral cancer cells. diabetes mellitus is an endocrine metabolic disorder in which the body does not produce sufficient insulin or lack of responsiveness to insulin, resulting in hyperglycemia (high blood glucose level). the classical symptoms include polyuria, polydipsia, weight loss, lethargy, polyphagia, visual blurring, frequent or recurring infections, cuts and bruises that are slow to heal, tingling and/or numbness in hands and/or feet, drowsiness, nausea, and decreased endurance during exercise [ ] . a number of potential medicinal components from bitter gourd, such as α and β momorcharin, momordin, and cucurbitacin b, have been isolated. a number of reported clinical studies have shown that bitter gourd extract from fruits, seeds, and leaves contain several bioactive compounds that have hypoglycemic activity in both diabetic animals and humans [ ] . fruits, seeds, and leaves extract of momordica charantia possess hypoglycemic activity in antihyperglycemic activity in alloxan [ ] or streptozotocin [ ] . the major compounds that have been isolated and identified as hypoglycemic agents include charantin, polypeptide-p, and vicine. charantin is a steroidal glycoside shown to possess powerful hypoglycemic properties when administered orally and intravenously in diabetic rabbits [ ] . hairy roots produced higher amount of charantin which used for antidiabetics [ ] . the vast potential of hairy root cultures as a stable source of biologically active chemicals has focused the attention of the scientific community for its exploitation. scaling up of hairy roots in novel bioreactors can provide the best conditions for optimum growth and secondary metabolite production, comparable to or higher than that in native roots. though the need for developing bioreactors suitable for the hairy root cultivation has long been recognized, root cultures present unique challenges [ ] . the complex fibrous structure of the roots makes the growth analysis and development of a large-scale culture system difficult. hairy root growth is not homogeneous, which affects the reactor performance. furthermore, the hairy root morphology is quite plastic as the roots respond to the changes in the local environment. changes in morphology, including changes in the density and length of the root hairs, directly affect the secondary metabolite production from hairy roots [ ] . thus, bioreactor design for root cultures is a balancing act between the biological needs of the tissues, without inducing an additional, undesirable biological response [ ] . reviews on hairy roots briefly discuss the importance of the use of bioreactors for hairy root cultures [ ] . mechanical agitation causes wounding of hairy roots and leads to callus formation. due to branching, the roots form an interlocked matrix that exhibits resistance to nutrient flow. hairy roots are hetrotrophic, respiratory organisms that rely on oxygen for energy generation and other metabolic functions. substantial progress has been made in understanding the mechanisms of oxygen limitation, one of the principle challenges for large-scale growth of hairy root cultures [ ] . because of the solid phase nature of the roots and the development of oxygen gradients within root tissues, relatively small reductions in the dissolved oxygen concentration in the medium can lead to a significant decrease in growth rate and may also affect the synthesis of certain secondary metabolites. in fact, hairy roots can be oxygen limited even in shake flask cultures [ ] . restriction of nutrient oxygen delivery to the central mass of tissue gives rise to a pocket of senescent tissues. mass transfer resistances near the liquid and solid boundary affect the oxygen delivery to the growing hairy roots. thus, exploitation of hairy root culture as a source of bioactive chemicals depends on the development of suitable bioreactor system where several physical and chemical parameters (nutrient availability, nutrient uptake, oxygen, and hydrogen depletion in the medium, mixing, and shear sensitivity) must be taken into account. the design of bioreactors for hairy root cultures should also take into consideration factors such as the requirement for a support matrix and the possibility of flow restriction by the root mass in certain parts of the bioreactor. several bioreactor designs have been reported for hairy root culture taking into consideration the above factors that permit the growth of interconnected tissue unevenly distributed throughout the culture vessel. reactors used to culture hairy roots can roughly be divided into three types: liquid-phase, gas-phase, or hybrid reactors that are a combination of both [ ] . previously, there are no reports on the large scale production of hairy roots using bioreactor system in cucurbits and the production of phenolic compounds. various biotic and abiotic elicitors applied to hairy root cultures and their stimulating effects on the accumulation of secondary metabolites. according to their origin, elicitors can be divided into different types: (a) biotic and (b) abiotic. abiotic elicitors can be considered as substances of nonbiological origin, being predominantly inorganic compounds such as salts or physical factors [ , ] . inorganic chemicals like salts or metal ions have been used to increase the production of bioactive compounds by their modification of plant secondary metabolism. among the many elicitors applied to hairy root cultures, the most common and effective elicitors are fungal cell extracts, polysaccharides from fungal and plant cells, and heavy metal salts. with the crude fungal cell extracts, it is essential to observe the preparation conditions carefully for achieving reproducible effects. in addition to the chemical agents, uv-radiation, hyperosmotic stress, and temperature shift have been shown effective for some plant species/metabolites. elicitor type, dose, and treatment schedule are major factors determining the effects on the secondary metabolite production. in addition to the accumulation of products in roots, elicitor treatments often stimulate the release of intracellular products. although elicitation is mainly effective to increase specific product yield on per unit mass of roots, the incorporation of nutrient feeding strategies can be applied to enhance the volumetric product yield. the integration of in situ product recovery from the roots/liquid medium is another synergistic strategy with the elicitor treatment to improve the process. so far, there are no reports on the elicitation of hairy roots and production of phenolic compounds from hairy root cultures of cucurbits. further, researchers can use the elicitation to improve the contents of secondary metabolites in cucurbits. hairy root technology has been significantly improved in various fields for past few years. overall, the major groups of secondary metabolites have already been produced from hairy roots of cucurbits. compared to plant cell suspension cultures, hairy root cultures appear to be potential systems for continuous production of valuable secondary metabolites because of their fast growth rates, ease of maintenance, genetic and biosynthetic stability, and ability to synthesize a vast array of compounds. environmental factors, such as light, oxygen, and temperature, as well as abiotic and biotic stress factors, such as phytohormones, heavy metals, and fungal elicitors, have all been applied to hairy roots for increased yield of phytochemicals. in addition to these external stimuli, secondary metabolic pathways have also been modified for enhanced metabolite production, such as overexpression of biosynthetic genes and transcription factors, and suppression of catabolic or competing pathway genes. a better understanding of the biosynthetic pathway and regulation architecture of valuable secondary metabolites is crucial for genetic engineering and fully realizing the biosynthetic potential of hairy roots. the discovery of new genes that participate in the metabolic pathways from hairy root studies increases the tremendous potential of such cultures. it is also predicted that this model of pharmaceutical production is relatively safe. driven by the demand for productive, robust, and stable hairy root cultures for the production of active agents for the food, cosmetics, and pharmaceutical industry, the development of a direct available measuring method for the biomass concentration of hairy root cultures in liquid medium still does not exist. transgenic hairy roots grew rapidly than nontransgenic roots in standardized liquid culture conditions and produced greater amount of biomass and phenolic compounds. the higher amount of secondary metabolites possibly contributes to greater biological activity of hairy roots in cucurbits. the genetic and biochemical stability of the hairy roots as well as its high productivity offers an effective platform for further studies on the biosynthetic pathways of phytochemicals. this prediction is strengthened by the observation that emerging private companies have converted this technology to allow production at a commercial scale. plant biologists can work closely with engineers to tackle the challenges with scaling up hairy root cultures, such as optimal biomass growth and adaptation of the extraction methods to industrial-scale metabolite production. looking forward, establishment of hairy roots guided by bioassays, augmented by elicitations and genetic manipulations, and coupled with efficient metabolite extractions will streamline the process and allow full exploitation of hairy roots as a production platform of valuable secondary metabolites. a new system of cucurbitaceae cucurbitaceae of bihar diversity and conservation the genus luffa in india: diversity and conservation a review on the medicinally important plants of the family cucurbitaceae ethnobotany in the nepal himalaya plant terpenoids: applications and future potentials recent advances in medicinal plant biotechnology elicitation a new window into plant chemodiversity and phytochemical drug discovery bioactive compounds in foods: their role in the prevention of cardiovascular disease and cancer biotechnology for agro-industrial residues utilization potentials for exploiting allelopathy to enhance crop production biochemical and physiological mechanisms mediated by allelochemicals elevated temperatures increase leaf senescence and root secondary metabolite concentrations in the understory herb panax quinquefolius (araliaceae) effect of secondary metabolites associated with anaerobic soil conditions on ion fluxes and electrophysiology in barley roots production of d'agropine par des racines transformes sous i'action d'agrobacterium rhizogenes souche a agrobacterium rhizogenes inserts t-dna into the genomes of the host-plant root cells individual and combined effect of rola, b and c genes on anthraquinone production in rubia cordifolia on transformed calli function of rol gene in plant secondary metabolism hairy root type plant in vitro systems as sources of bioactive substances diterpenoids and triterpenoids in hairy roots of salvia sclarea secondary metabolism of hairy root cultures in bioreactors agrobacterium rhizogenes mediated transformation of the medicinal plant decalepis arayalpathra and production of -hydroxy- -methoxy benzaldehyde approaches to understanding and manipulating the biosynthetic potential of plant roots production of plant secondary metabolites: a historical perspective transgenic hairy roots: recent trends and applications hairy root cultures of gynostemma pentaphyllum (thunb.) makino: a promising approach for the production of gypenosides as an alternative of ginseng saponins evaluation of phenolic compounds, antioxidant and antimicrobial activities from transgenic hairy root cultures of gherkin (cucumis anguria l.) establishment of momordica charantia hairy root cultures for the production of phenolic compounds and the determination of their biological activities induction of hairy roots by agrobacterium rhizogenes-mediated transformation of spine gourd (momordica dioica roxb. ex. willd) for the assessment of phenolic compounds and biological activities the influence of plant growth regulators on callus induction in pumpkin (cucurbita pepo l.) hairy roots a hairy root culture of melon produces aroma compounds expression of the human tissue-plasminogen activator in hairy roots of oriental melon (cucumis melo) expression analysis of the s camv promoter and its derivatives in transgenic hairy root cultures of cucumber (cucumis sativus) generated by agrobacterium rhizogenes infection roots induced on cucumber cotyledons by the agropine ri plasmid tr-dna exhibit the transformed phenotype production of ribosomeinactivating protein from hairy root cultures of luffa cylindrica (l.) roem hairy roots induction in oriental melon (cucumis melo) by a. rhizogenes and production of the root-knot nematode biosynthesis of defense-related proteins in transformed root cultures of trichosanthes kirilowii maxim. var japonicum (kitam basic proteins produced by hairy root cultures of trichosanthes kirilowii var. japonica bryonolic acid production in hairy roots of trichosanthes kirilowii max. var japonica kitam. transformed with agrobacterium rhizogenes and its cytotoxic activity agrobacterium rhizogenes-mediated hairy root induction of momordica charantia linn. and the detection of charantin, a potent hypoglycaemic agent in hairy roots transformation of cucumber (cucumis sativus l.) plants with agrobacterium rhizogenes molecular basis for novel root phenotypes induced by agrobacterium rhizogenes a on cucumber composite cucurbita pepo plants with transgenic roots as a tool to study root development host-tissue differences in transformation of pumpkin (cucurbita pepo l.) by agrobacterium rhizogenes biotransformation of protocatechuic aldehyde and caffeic acid to vanillin and capsaicin in freely suspended and immobilized cell cultures of capsicum frutescens phenolic compounds: from plants to foods flavone and flavonone pathway phenolic compounds in plants and agri industrial by-products: antioxidant activity, occurrence, and potential uses structure and function of enzymes involved in the biosynthesis of phenylpropanoids the nutritional and metabolic effects of boron in humans and animals the effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer antimicrobial properties of phenolic compounds from berries comparative chemical composition, free radical-scavenging and cytotoxic properties of essential oils of six stachys species from different regions of the mediterranean area antioxidant and antidiabetic activity of dangyuja (citrus grandis osbeck) extract treated with aspergillus saitoi effects of grape antioxidant dietary fiber in cardiovascular disease risk factors antioxidant, antimutagenic and antibacterial activities of curcumin-β-diglusoside antimicrobial activity of flavonoids in vitro antibacterial and antibiofilm activities of chlorogenic acid against clinical isolates of stenotrophomonas maltophilia including the trimethoprim/sulfamethoxazole resistant strain study on prevention of two-stage skin carcinogenesis by hibiscus rosa sinensis extract and the role of its chemical constituent, gentisic acid, in the inhibition of tumour promotion response and oxidative stress in mice antioxidant activity and phenolic compounds in selected herbs antioxidant activity of plant extracts containing phenolic compounds polyphenols as antimicrobial agents novel dammarane-type glycosides from gynostemma pentaphyllum plant science. biosynthesis, regulation, and domestication of bitterness in cucumber transformation of gynostemma pentaphyllum by agrobacterium rhizogenes and saponin production in hairy root cultures plant cell and tissue culture in liquid systems the rna n-glycosidase activity of ricin a-chain. the characteristics of the enzymatic activity of ricin a-chain with ribosomes and with rrna dual effects of the ricin a chain on protein synthesis in rabbit reticulocyte lysate. inhibition of initiation and translocation ribosome-inactivating proteins: a plant perspective trichosanthin, a potent hiv- inhibitor, can cleave supercoiled dna in vitro dna-nuclease activity of the single-chain ribosomeinactivating proteins dianthin , saporin and gelonin bifunctional plant defense enzymes with chitinase and ribosome inactivating activities from trichosanthes kirilowii cell cultures control of root formation by plant growth regulators. in: basra as (ed) plant growth regulators in agriculture and horticulture: their role and commercial uses effect of pokeweed antiviral protein (pap) on the infection of viruses inhibition of hiv replication by pokeweed antiviral protein targeted to cd + cells by monoclonal antibodies isolation and characterization of two luffins, protein-biosynthesis inhibitory proteins from the seeds of luffa cylindrica identification of lysine residue at or near active site of luffin-a, a ribosome inactivating protein from seeds of luffa cylindrica a ribosome-inactivating protein principle from hairy roots and seeds of luffa cylindrica (l) roem and its cytotoxicity on melanotic and amelanotic melanoma cell lines preliminary screening of some reputed abortifacient indigeneous plants extraction of insulin like compounds from bitter melon plants antihyperglycaemic and insulin release effects of aegle mamelos leaves in sterptozotocin-diabetic rats fatty acid -and -hydroperoxide lyases from cucumber workshop on taste and smell in the elderly: an overview aroma components and their contents in cucumbers from different genotypes the flavor of cucumbers additional aroma components of honeydew melon dietary phenolics: chemistry, bioavailability and effects on health flavanols and anthocyanins in cardiovascular health: a review of current evidence flavonoid content of several vegetables and their antioxidant activity synergistic antiproliferative action of the flavonols quercetin and kaempferol in cultured human cancer cell lines antibacterial activity of hairy-root cultures of maytenus senegalensis production of anthraquinones, phenolic compounds and biological activities from hairy root cultures of polygonum multiflorum thunb cytotoxicity, antiviral and antimicrobialactivities of alkaloids, flavonoids, and phenolic acids anti-viral protein of momordica charantia l. inhibits different subtypes of influenza a. evid based complement alter nat med development of a potent in vitro source of phyllanthus amarus roots with pronounced activity against surface antigen of the hepatitis b virus obtaining of hairy root: callus and suspension cell cultures of carrot (daucus carota l.) able to accumulate human interferon alpha- b gypenosides protect primary cultures of rat cortical cells against oxidative neurotoxicity cardiovascular effects of the aqueous extract of gynostemma pentaphyllum makino regulation of bcl- family molecules and activation of caspase cascade involved in gypenosides-induced apoptosis in human hepatoma cells the effect of gynostemma pentaphyllum mak (gp) on carcinogenesis of the golden hamster cheek pouch induced by dmba a preliminary observation of preventive and blocking effect of gynostemma pentaphyllum (thunb) makino on esophageal cancer in rats gypenosides induced apoptosis in human colon cancer cells through the mitochondria-dependent pathways and activation of caspase- gypenosides induced g /g arrest via chk and apoptosis through endoplasmic reticulum stress and mitochondria-dependent pathways in human tongue cancer scc- cells evaluation of the hypoglycemic activity of cucumis metuliferus (cucurbitaceae) fruit pulp extract in normoglycemic and alloxaninduced hyperglycemic rats slow acting protein extract from fruit pulp of momordica charantia with insulin secretagogue and insulinomimetic activities comparative evaluation of ypoglycaemic activity of some indian medicinal plants in alloxan diabetic rats amelioration of experimental diabetic neuropathy and gastropathy in rats following oral administration of plant (eugenia jambolana, mucuna pruriens and tinospora cordifolia) extracts anti-diabetic properties and phytochemistry momordica charantia l. (curcurbitaceae) fermentation studies of transformed root cultures characterization of fluid-flow resistance in root cultures with a convective flow tubular bioreactor the growth of single roots of artemisia annua in nutrient mist reactors advances and challenges in bioreactor design for the production of chemicals from plant tissue culture the extent to which external oxygen transfer limits growth in shake flask culture of hairy roots elicitation: an underutilized tool in the development of medicinal plants as a source of therapeutic secondary metabolites plant cell elicitation for production of secondary metabolites: a review acknowledgements this paper was supported by the ku research professor program of konkuk university, seoul, republic of korea. key: cord- -xvc wx authors: wink, michael title: chapter allelochemical properties or the raison d'être of alkaloids date: - - journal: nan doi: . /s - ( ) - sha: doc_id: cord_uid: xvc wx this chapter provides evidence that alkaloids are not waste products or functionless molecules as formerly assumed, but rather defense compounds employed by plants for survival against herbivores and against microorganisms and competing plants. these molecules were developed during evolution through natural selection in that they fit many important molecular targets, often receptors, of cells, which are seen in molecules that mimic endogenous neurotransmitters. the chapter discusses that microorganisms and herbivores rely on plants as a food source. since both have survived, there must be mechanisms of adaptations toward the defensive chemistry of plants. many herbivores have evolved strategies to avoid the extremely toxic plants and prefer the less toxic ones. many herbivores have potent mechanisms to detoxify xenobiotics, which allow the exploitation of at least the less toxic plants. in insects, many specialists evolved that are adapted to the defense chemicals of their host plant, in that they accumulate these compounds and exploit them for their own defense. alkaloids function as defense molecules against insect predators in the examples studied, and this is further support for the hypothesis that the same compound also serves for chemical defense in the host plant. it needs more experimental data to understand fully the intricate interconnections between plants, their alkaloids, and herbivores, microorganisms, and other plants. organisms. we must also consider that plants compete with other plants (of the same or different species) for light, water, and nutrients. how do plants defend themselves against microorganisms (including bacteria, fungi, and viruses), herbivores, and plants? because plants do rather well in nature, this question has often been overlooked. we are well aware of the defensive strategies of higher animals against microbes and predators ( , , , , , , ) . the complex immune system with its cellular and humoral components is a well-studied area in the context of vertebrate-microbe interactions. against predating animals, nature evolved weapons, armor, crypsis, thanatosis, deimatic behavior, aposematism, flight, or defense chemicals (usually called "poisons") ( ). it is evident that most of these possibilities are not available for plants with their sessile and "passive" life-style. what then is their evolutionary solution? we can distinguish the following defense mechanisms in plants ( , , , , ) ; the mechanisms are not independent and may act cooperatively and synergistically. we should be aware that many species have additionally evolved specialized traits in this context. . mechanical protection is provided by thorns, spikes, trichomes, glandular hairs, and stinging hairs (which are often supported by defense chemicals). . formation of a thick bark on roots and stems can be considered as a sort of armor, and the presence of hydrophobic cuticular layers as a penetration barrier directed against microbes. b. cell walls are biochemically rather inert with reduced digestibility to many organisms because of their complex cellulose, pectin, and lignin molecules. callose and lignin are often accumulated at the site of infection or wounding ( , ) and form a penetration barrier. c. synthesis of inhibitory proteins (e.g., lectins, protease inhibitors) or enzymes (e.g., chitinase, lysozyme, hydrolases, nucleases) that could degrade microbial cell walls or other microbial constituents would be protective, as well as synthesis of peroxidase and phenolase, which could help inactivate phytotoxins produced by many bacteria and fungi. these proteins are either stored in the vacuole . allelochemical properties of alkaloids or are secreted as exoenzymes into the cell wall or the extracellular space ( , ) . these compounds are thus positioned at an "advanced and strategically important defense position." in addition, storage proteins (of cereals and legumes) are often deficient in particular essential amino acids, such as lysine or methionine. d. as a widely distributed and important trait, secondary metabolites with deterrenthepellent or toxic properties against microorganisms, viruses, and/or herbivores may be produced ( - , - ) . these allelochemicals can be constitutively expressed, they may be activated by wounding (e .g., cyanogenic glycosides, glucosinolates, coumaryl glycosides, alliin, ranunculin), or their de ~o u o synthesis may be induced by elicitors (so-called phytoalexins), infection, or herbivory ( , , [ ] [ ] [ ] . these products are often synthesized and stored at strategically important sites [epidermal tissues or in cells adjacent to an infection ( , )] or in plant parts that are especially important for reproduction and survival [flowers, fruits, seeds, bark, roots ( , , ) ]. in animals, we can observe the analogous situation in that many insects and other invertebrates (especially those which are sessile and unprotected by armor), but also some vertebrates, store secondary metabolites for their defense which are often similar in structure to plant allelochemicals ( , , , , , - , [ ] [ ] [ ] ) . in many instances, the animals have obtained the toxins from their host plants ( , , , , - ). hardly any zoologist or ecologist doubts that the principal function of these secondary metabolites (which are often termed ''toxins" in this context) in animals is that of defense against predators or microorganisms ( , , , [ ] [ ] [ ] . these defense compounds are better known as natural products or secondary metabolites. the latter expression originally meant compounds which are not essential for life, and thus distinct from primary metabolites ( , , ) . unfortunately the term "secondary" has also a pejorative meaning, indicating perhaps that the compounds have no importance for the plant. as discussed in this chapter, just the opposite is true. more than , natural products have been reported from plants so far ( , , ) . owing to the sophistication in phytochemical methods, such as chromatography (hplc, glc) and spectroscopy (nmr, ms) , new products are reported at rapid intervals. because only - % of all higher plants, which consist of over , species, have been analyzed phytochemically in some detail, the overall real number of secondary products is certainly very large. it is a common theme that an individual plant does not produce a single natural product, but usually a moderate number of major metabolites and a larger number of minor derivatives. within a taxon secondary metabolites often share a common distribution pattern and are therefore of some importance for phytochemical systematics. classic taxonomy, however, has taken little account of alkaloid distribution: if the same alkaloid is present in two plants of the same taxon, this is interpreted as evidence for a relationship, but its occurrence in two plants of nonrelated taxa is taken as evidence of independent evolution. because secondary metabolites are also derived characters that were selected during evolution, their general value for taxonomy and systematics is certainly smaller than formerly anticipated ( ). for many years, secondary metabolites were considered as waste products or otherwise functionless molecules, merely illustrating the biochemical virtuosity of nature ( , ) . in and , errera and stahl ( , , ) published the idea that natural products are used by plants for chemical defense against herbivores. since the leading plant physiologists of that time were mostly anti-darwinian, they were not willing to accept the defense argument, which was too much in line with the darwinian concept. therefore, this early defense concept was negated and remained forgotten for nearly years. in , fraenkel( ) reopened the debate in a review article and presented new data supporting the view that secondary metabolites serve as chemical defense compounds against herbivores. during the next three decades this concept was improved experimentally, and we can summarize the present situation as follows although the biological function of many plant-derived secondary metabolites has not been studied experimentally, it is now generally assumed that these compounds are important for the survival and fitness of a plant and that they are not useless waste products, as was suggested earlier in the twentieth century ( , ) . in many instances, there remains a need to analyze whether a given compound is active against microorganisms (viruses, bacteria, fungi), against herbivores (molluscs, arthropods, vertebrates), or against competing plants (so-called allelopathy). in some instances, additional functions are the attraction of pollinating or seed-dispersing animals, for example, by colored compounds such as betalains (within the centrospermae), anthocyanins, carotenoids, and flavonoids or by fragrances such as terpenes, amines, and aldehydes ( , ) . physiological roles, such as uv protection [by flavonoids or coumarins ( , )], nitrogen transport or storage ( , , ) , or photosynthesis (carotenoids), may be an additional function. allelochemicals are often not directed against a single organism, but generally against a variety of potential enemies, or they may combine the roles of both deterrents and attractants (e.g., anthocyanins and many essential oils can be attractants in flowers but are also insecticidal and antimicrobial). thus, many natural products have multiple functions, a fact which is easily overlooked since most scientists usually specialize on a narrow range of organisms (i.e., a microbiologist will usually not check whether an antibiotic alkaloid also deters the feeding of caterpillars). to understand all the interactions we need to adopt a holistic, that is, interdisciplinary, approach. it might be argued that the defense hypothesis cannot be valid since most plants, even those with extremely poisonous metabolites (from the human point of view), are nevertheless attacked by pathogens and herbivores. however, we have to understand and accept that chemical defense is not an absolute process. rather, it constitutes a general barrier which will be effective in most circumstances, that is, most potential enemies are repelled or deterred. plants with allelochemicals at the same time represent an ecological niche for potential pathogens and herbivores. during evolution a few organisms have generally been successful in specializing toward that niche (i.e., in a particular toxic plant) in that they found a way to sequester the toxins or become immune to them ( , , ) . this is especially apparent in the largest class of animals, the insects (probably with several million species on earth), which are often highly host plant specific. the number of these "specialists" is exceedingly small for a given plant species as compared to the number of potential enemies that are present in the ecosystem. we can compare this situation with our immune system: it works against the majority of microorganisms but fails toward a few viruses, bacteria, fungi, and protozoa, which have overcome this defense barrier by clever strategies. nobody would call the immune system and antibodies useless because of these few adapted specialists! we should adopt the same argument when we consider plants' defenses by secondary metabolites ( ) . since secondary metabolites have evolved in nature as biologically active compounds with particular properties in other organisms, many of them are useful to mankind as pharmaceuticals, fragrances, flavors, colors, stimulants, or pesticides. in addition, many allelochemicals provide interesting lead structures that organic medicinal chemists can develop into new and more active compounds. about - % of higher plants accumulate alkaloids ( , ). the incidence of alkaloid production varies between taxa to some degree; for example, about - % of species of the solanaceae and apocynaceae are michael wink alkaloidal, whereas other families contain few alkaloid-producing species. some alkaloids have a wide distribution in nature: caffeine occurs in the largest number of families, lycorine in the largest number of genera and berberine in the largest number of species. alkaloids are not restricted to higher plants (although they are here most numerous); they are also present in club mosses (lycopodium), horsetails (equisetum), fungi, and animals such as marine worms (e.g., nereidae), bryozoans, insects (e.g., coccinellidae, solenopsidae), amphibians (toads, frogs, salamanders), and fishes. alkaloids thus represent one of the largest groups of natural products, with over , known compounds at present, and they display an enormous variety of structures, which is due to the fact that several different precursors find their way into alkaloid skeletons, such as ornithine, lysine, phenylalanine, tyrosine, and tryptophan ( ) ( ) ( ) . in addition, part of the alkaloid molecule can be derived from other pathways, such as the terpenoid pathway, or from carbohydrates ( [ ] [ ] [ ] . whereas the structure elucidation of alkaloids and the exploration of alkaloid biosynthetic pathways have always commanded much attention, there are relatively few experimental data on the ecological function of alkaloids. this is the more surprising since alkaloids are known for their toxic and pharmacological properties and many are potent pharmaceuticals. alkaloids were long considered to be waste products [even by eminent alkaloid researchers such as w. . james and kurt mothes ( , , )l. because nitrogen is a limiting nutrient for most plants, a nitrogenous waste product would be a priori unlikely. the waste product argument probably came from animal physiology: carnivorous animals take up relative large amounts of proteins and nucleic acids, containing more nitrogen than needed for metabolism, which is consequently eliminated as uric acid or urea. a similar situation or need, however, is not applicable for plants. in fact, many plants remobilize their nitrogenous natural products (including alkaloids) from senescing organs such as old leaves ( , , ). if alkaloids were waste products, we would expect the opposite, namely, accumulation in old organs which are shed. on the other hand, the alkaloids produced by animals were never considered to be waste products by zoologists, but rather regarded as defense chemicals ( , , ) . thus, the more plausible hypothesis is that alkaloids of plants, microorganisms, and animals, like other allelochemicals, serve as defense compounds. this idea is intuitively straightforward, because many alkaloids are known as strong poisons for animals and homo sapiens. as a prerequisite for an alkaloid to serve as a chemical defense compound we should demand the following criteria. ( ) the alkaloid should have significant effects against microbes and/or animals in bioassays. ( ) the compounds should be present in the plant at concentrations that are of the same order (or, better, even higher) as those determined in the bioassays. ( ) the compound should be present in the plant at the right time and the right place. ( ) evidence should be provided that a particular compound is indeed important for the fitness of a plant. although more than , alkaloids are known, only few (- - %) have been analyzed for biochemical properties, and even fewer for their ecophysiological roles. in most phytochemical studies only the structures of alkaloids have been elucidated, so that often no information is available on their concentrations in the different parts and through the ontogenetic development of a plant, or on their biological activities. furthermore, the corresponding studies were usually designed to find useful medicinal or sometimes agricultural applications of alkaloids, not to elucidate their evolutionary or ecological functions. these objections have to be kept in mind, because an alkaloid is sometimes termed "inactive" in the literature, which usually means less active than a standard compound already established as a medicinal compound (such as penicillins in antimicrobial screenings). in many medicinal experiments relatively low doses are applied because of the toxic properties of many alkaloids. if the same compound would have been tested at relevant (which normally means elevated) concentrations that are present in the plant, an ecologically relevant activity might have been detected. another restriction is that the activities of alkaloids have been tested with organisms that are sometimes irrelevant for plants but medicinally important. however, if a compound is active against escherichia coli, it is likely that is is also active against other gram-negative and plant-relevant bacteria. nevertheless, most of the data obtained in these studies (tables i-viii ) provide important information which at present permits extrapolation to the function of alkaloids in plants. in this chapter the focus is on the biological activity of alkaloids (the information available on the pharmacological properties of alkaloids is mostly excluded), and we try to discuss these data from an ecological perspective. in the following, the possible functions of alkaloids in plant-animal, plant-plant, and plant-microbe interactions are discussed in more detail. it is nearly impossible to cover the literature exhaustively. therefore, an overview of the allelochemical properties of alkaloids is presented. because of the large amount of data (literature up to is included), the selection of examples must remain subjective to some degree. nevertheless, the author would be grateful to receive information or publications about relevant omissions. because homo supiens and domestic animals are to some degree herbivores, a large body of empirical knowledge has accumulated on the toxic properties of alkaloids (tables i through v) and alkaloid-containing plants. previously, the toxic properties of alkaloids in vertebrates was part of the definition (as a common denominator) for this group of natural products ( , ) . in the following, the toxic or adverse effects of alkaloids are separately discussed for invertebrates (mainly insects) and vertebrates. among the invertebrates, insects have been extremely successful from the evolutionary point of view, and they form the largest class of organisms on our planet as far as the number of both individuals and species is concerned. entomologists estimate that the number of insects is at least million, but tropical rain forests may harbor up to - million species, many of which are still unknown and, owing to the fast extinction of this ecosystem, will probably also disappear without having been discovered and studied by scientists. most insects are herbivores, and adaptation to host plants and their chemistry is often very close and complex ( i , , , , , - , , ) . whereas insects rely on plants for food, many plants need insects for pollination and seed dispersal. in the latter context we often find that plants attract insects by chemical means (colors, fragrances, sugars, amino acids). at the same time, other secondary metabolites are employed to discourage the feeding on flowers and seeds. the close association between plants, especially the angiosperms, and insects evolved during the last million years. some scientists have called this phenomenon a "coevolutionary" process, but it has to be recalled that the associations seen today are not necessarily those in which the chemical interactions originally evolved ( , , ). applications of synthetic insecticides have shown that resistance to these new compounds can occur rapidly, sometimes encompassing only a dozen generations. times can also be much longer. if plant species are introduced to a new continent or island, it usually takes a long time before new pathogens or herbivores become adapted and specialized to this new species. for example, lupinus polyphyllus from north america has a number of specialized herbivores, but is rarely attacked by herbivores in europe. this lupine left its enemies behind when it was transferred to europe three centuries ago. about years ago, however, the north american lupine aphid (macrosiphum albifrons) was introduced to europe accidentally. this aphid is specialized to alkaloid-rich lupines with lupanine as a major alkaloid. at present, this aphid has spread over most of europe and is now colonizing its former host, l. polyphyllus ( , ) . insect herbivores can be divided into two large groups whose strategies with respect to the plant's defense chemistry differ substantially ( ). the polyphagous species can exploit a wide range of host plants, whereas the mono-/oligophagous insects are often specialized on one or a small number of (often systematically related) hosts. polyphagous insects, namely, species which feed on a wide variety of food plants, are usually endowed with fantastic and powerful olfactory receptors ( ) that allow the distinction between plants with high or low amounts of "toxins." the receptors also allow insects to ascertain the quality of the essential products present, such as lipids, proteins, or carbohydrates ( ). these "generalists," as we can also call this subgroup of herbivores, are usually deterred from feeding on plants which store especially noxious metabolites and select those with less active ones (such as our crop species, where man has bred away many of the secondary metabolites that were originally present; see table xi ). alternatively, they change host plants rapidly and thus avoid intoxication. in addition, most polyphagous species have evolved active detoxification mechanisms, such as microsomal oxidases and glutathione peroxidase, which lead to the rapid detoxification and elimination of dietary secondary products ( , , , ). in contrast, mono-and oligophagous species often select their host plants with respect to the composition of the nutrients and secondary metabolites present. for these "specialists" the originally noxious defense compounds are often attractive feeding and oviposition stimulants. these insects either tolerate the natural products or, more often, actively sequester and exploit them for their own defense against predators or for other purposes ( , , - , , , , , - ). these observations seem to contradict the first statement, that secondary metabolites are primarily defense compounds, and a number of renowned authors have fallen into this logical pit, such as mothes ( ) and robinson ( ). however, these specialized insects are exceptions to the general rule. for these specialists, the defense chemistry of the host plant is usually not toxic, but they are susceptible to the toxicity of natural toxins from non-host plants ( ) . as compared to the enormous number of potential herbivores, the number of adapted monophagous species is usually very small for a particular plant species. quite a number of alkaloids have been tested toward herbivorous insects (table i ). in general it is observed that many alkaloids can act as feeding deterrents at higher concentrations (>i%, w/w). given the choice, insects tend to select a diet with no or only a small dose of alkaloids. also, specialists avoid most "toxins" except those of their host plants. these data indicate that under natural conditions plants with a high content of alkaloids should be safe from most herbivorous insects, with the exception of particular monophagous species or a few very potent polyphagous ones. if insects have no choice or if they are very hungry, the deterrency threshold value is much reduced, and they often feed on a diet with alkaloids that they would normally avoid ( , ). in this case we have the chance to test the toxicity of an ingested alkaloid. if insects do not take up alkaloid-containing food, alkaloid toxicity can be assessed to some degree by topical application or by injection ( table i) . as can be seen from table i a substantial number of alkaloids display significant insect toxicity, including nicotine, piperine, lupine alkaloids, caffeine, gramine, strychnine, berberine, ephedrine, and steroidal alkaloids. only the specialists can tolerate the respective alkaloids. the tobacco hornworm (manduca sexta), for example, can grow on a diet with more than % nicotine without any adverse effects. most of the nicotine is either degraded or directly eliminated via the malpighian tubules and in feces ( ). because nicotine binds to the acetylcholine (ach) receptor, it is likely that in manduca this receptor has been modified in such a way that ach can still bind, but not nicotine (so-called target site modification). the toxic effects of alkaloids in insects (table i) can be caused by their interference with diverse cellular and intracellular targets. since most mechanisms have not yet been elucidated for insects, this issue is discussed below in the section on vertebrate toxicity (see table iv ). with some caution we can extrapolate to insect toxicity. because homo sapiens and domestic animals are largely herbivores, a voluminous body of information on the adverse effects of secondary metabolites has accumulated over the centuries. many allelochemicals and alkaloids are feeding deterrents for vertebrates, owing to their bitter or pungent taste or bad smell, and instinctively a foul-smelling, bitter, or pungent diet is normally avoided. examples of bitter alkaloids (at least for man) are quinine, strychnine, brucine, and sparteine, and for pungent alkaloids are capsaicin, and piperine. it should be recalled that these taste properties are not identical for all animals. for example, geese, which are obligate herbivores, hardly avoid food with alkaloids or smelly compounds (amines, mercaptoethanol) that man would hardly touch ( ). conversely, fragrances that are attractive to us are highly repellent to geese ( ). even within a given population taste can differ significantly. it has been observed that a substantial proportion of homo sapiens cannot detect the smell of hcn, whereas others are highly sensitive. furthermore, olfactory sensitivity can differ with age, sex, and hormonal cycles. bitterness varies with the chemical structure of an alkaloid. with the quinolizidine alkaloids (qas) the following scale was assessed for man: mean detection levels are . % for sparteine, . % for lupanine, and . % for hydroxylupanine ( ). whereas we know a few parameters of olfactory qualities in homo sapiens, often much less or hardly anything is known for most other vertebrates. alkaloids are famous for their toxic properties in vertebrates, and plants that produce alkaloids are often classified by man as poisonous or toxic plants. for a number of alkaloids the respective ld,, values have been determined with laboratory animals, especially mice, but also rats, guinea pigs, cats, rabbits, dogs, or pigeons. table i presents an overview for alkaloids, including the very poisonous alkaloids aconitine, coniine, atropine, brucine, curarine, ergocornine, physostigmine, strychnine, colchicine, germerine, veratridine, cytisine, delphinidine, and nicotine. toxicity is usually highest if the alkaloids are applied parenterally [intravenously (i.v.), intraperitoneally (i.p.), and subcutaneously (s.c.)] as compared to oral application [per s (p.o.)]. also, some of the alkaloids which are made or stored by animals are strong vertebrate poisons, including batrachotoxin, batrachotoxinin a, anabasine, glomerine, maitotoxin, nereistoxin, palytoxin, saxitoxin, and tetrodotoxin ( , , , ) . although the general toxicity of alkaloids differs from species to species, the data in table i generally show that many alkaloids are more or less toxic to vertebrates. the toxic effects observed with intact animals has its counterpart in the cytotoxic effect, which has been recorded for nearly alkaloids (table ). these data have been obtained by screening many natural products for anticancer activity. however, an alkaloid that can kill a cancer cell is usually also toxic for "normal" cells. therefore, the data shown in table i are another indication of the general toxicity of alkaloids toward animals. because this toxicity applies also for herbivores, the production of alkaloids by plants can certainly be interpreted as a potent antiherbivore mechanism. for a number of alkaloids the mechanisms underlying the toxic effects have already been elucidated in some detail. we can distinguish molecular targets and processes that are important for all cells, such as synthesis of dna, rna, and proteins, replication, transcription, translation, membrane assembly and stability, electron chains, or metabolically important enzymes or proteins including receptors, hormones, and signal compounds (table iv ). in the following we discuss some of these toxic effects. a. cellular targets nucleic acids. dna, the macromolecule which holds all the genetic information for the life and development of an organism, is a highly vulnerable target. it is not surprising that a number of secondary metabolites have been selected during evolution which interact with dna or dnaprocessing enzymes. some alkaloids bind to or intercalate with dna/rna (table iv) and thus affect replication or transcription, or cause mutations, leading to malformations or cancer (table v) : -methoxyellipticine, dictamnine, ellipticine, harmane alkaloids, melinone f, quinine and related alkaloids, skimmianine, avicine, berberine, chelerythrine, coptisine, coralyne, fagaronine, nitidine, sanguinarine, pyrrolizidine alkaloids (pas), cycasin, olivacine, etc. many of the intercalating molecules are planar, hydrophobic molecules that fit within the stacks of at and gc base pairs. other alkaloids act at the level of dna and rna polymerases, such as vincristine, vinblastine, avicine, chelilutine, coralyne, fagaronine, nitidine, amanitine, hippeastrine, and lycorine, thus impairing the processes of replication and transcription. whereas these toxins usually cause a rapid reaction, some alkaloids cause long-term effects in vertebrates in that they are mutagenic or carcinogenic (table v) . besides basic data obtained in salmonella or drosophila, there are a few reports which illustrate the potent mutagenic effect of alkaloids on vertebrates. anagyrine, anabasine, and coniine cause "crooked calf disease" if pregnant cows or sheep feed on these alkaloids during the first period of gestation ( , , , , , ) . the offspring born show strong malformation of the legs. some of the steroid alkaloids (e.g., cyclopamine, jervine, and veratrosine), which are produced by veratrum species, cause the formation of a central large cyclopean eye ( - , an observation that was probably made by the ancient greeks and thus led to the mythical figure of the cyclops. it is likely that any herbivore which regularly feeds on plants containing these alkaloids will suffer from reduced productivity and reduced fitness in the long term. in effect, the plants which contain these alkaloids are usually avoided by vertebrate herbivores. another long-term effect caused by alkaloids with carcinogenic properties has been discovered only recently (tables iv and v) . the alkaloid aristolochic acid, which is produced by plants of the genus aristolochia, is carcinogenic. the mechanism of action of this alkaloid is believed to be similar to the well-known carcinogen nitrosamine ( , ) , because of its no, group. pyrrolizidine alkaloids and their n-oxides, which are abundantly produced by members of the asteraceae and boraginaceae but also occur in the families apocynaceae, celestraceae, elaeocarpaceae, euphorbiaceae, fabaceae, orchidaceae, poaceae, ranunculaceae, rhizo- esterified ( , ) . after oral intake, the n-oxides are reduced by bacteria in the gut. the lipophilic alkaloid base is resorbed and transported to the liver, where it is "detoxified" by microsomal enzymes. as a result, a reactive alkylating agent is generated, which can be considered as a pyrrolopyrrolidine. the alkaloid can then cross-link dna and rna and thus cause mutagenic or carcinogenic effects (especially in the liver) ( ). thus, pyrrolizidine alkaloids represent highly evolved and sophisticated antiherbivore compounds, which utilize the widespread and active detoxification system of the vertebrate liver. the pa story is very intriguing, since it shows how ingenious nature was in the "arms race." the herbivores invented detoxifying enzymes, and nature the compound which is activated by this process. a herbivore feeding on pa-containing plants will eventually die, usually without reproducing properly. only those individuals which carefully avoid the respective bitter-tasting plants maintain their fitnes and thus survive. the protection due to pa can easily be seen on meadows, where senecio and other pa-containing plants are usually not taken by cows and sheep, at least as long other food is available. protein biosynthesis is essential for all cells and thus another important target. indeed, a number of alkaloids have already been detected (although few have been studied in this context) that inhibit protein biosynthesis in uitro (table iv) , such as vincristine, vinblastine, emetine, tubulosine, tyramine, sparteine, lupanine and other quinolizidine alkaloids, cryptopleurine, haningtonine, homohamngtonine, haemanthamine, isohamngtonine, lycorine, narciclasine, pretazettine, pseudolycorine, tylocrebrine, tylophorine, and tylocrepine. for lupine alkaloids, it was determined that the steps which are inhibited are the loading of acyl-trna with amino acids, as well as the elongation step. the inhibitory activity was strongly expressed in heterologous systems, that is, protein biosynthesis in the producing plants, such as lupines, was not affected ( ). electron chains. the respiratory chain and atp synthesis in mitochondria demand the controlled flux of electrons. this target seems to be attacked by ellipticine, pseudane, pseudene, alpinigenine, sanguinarine, tetrahydropalmatine, ch,-(ch ),,- , -methyl-piperidines, capsaicin, the hydroxamic acid dimboa, and solenopsine. as mentioned before, however, only a few alkaloids have been evaluated in this context (table v) . biomembranes and transport processes. a cell can operate only when it is enclosed by an intact biomembrane and by a complex compartmenta- tion that provides separated reaction chambers. because biomembranes are impermeable for ions and polar molecules, cells can prevent the uncontrolled efflux of essential metabolites. the controlled flux of these compounds across biomembranes is achieved by specific transport proteins, which can be ion channels, pores, or carrier systems. these complex systems are also targets of many natural products (table iv) . disturbance of membrane stability is achieved by -methoxyellipticine, ellipticine, berbamine, cepharanthine, tetrandrine, steroidal alkaloids, irehdiamine, and malouetine. steroidal alkaloids, such as solanine and tomatine, which are present in many members of the solanaceae, can complex with cholesterol and other lipids of biomembranes; cells are thus rendered leaky. cells carefully control the homeostasis of their ion concentrations by the action of ion channels (na+,k+, ca + channels) and through na+,k+-atpase and ca +-atpase. these channels and pumps are involved in signal transduction, active transport processes, and neuronal and neuromuscular signaling. inhibition of transport processes (ion channels, carriers) is achieved by (table iv) acronycine, ervatamine, harmaline, quinine, reserpine, colchicine, nitidine, salsolinol, sanguinarine, stepholidine, caffeine, sparteine, monocrotaline, steroidal alkaloids, aconitine, capsaicine, cassaine, maitoxin, ochratoxin, palytoxin, pumiliotoxin, saxitoxin, solenopsine, and tetrodotoxin. a special class of ion channels in the central nervous system and involved in neuromuscular signal transfer are coupled with receptors of neurotransmitters such as noradrenaline (na), serotonin, dopamine, glycine, and acetylcholine (ach). we can distinguish two types. type is a ligand-gated channel (i.e., a receptor), which is part of an ion-channel complex, such as the nicotinergic ach-receptor. in type the receptor is an integral protein. when a neurotransmitter binds, the receptor changes its conformation and induces a conformational change in an adjacent gprotein molecule, which consists of three subunits. the a subunit then activates the enzyme adenylate cyclase, which in turn produces camp from atp. the camp molecule is a second messenger which activates protein kinases or ion channels directly, which in turn open for milliseconds (e.g., the muscarinergic ach receptor). a number of alkaloids are known whose structures are more or less similar to those of endogenous neurotransmitters. targets can be the receptor itself, the enzymes which deactivate neurotransmitters, or transport processes, which are important for the storage of the neurotransmitters in synaptic vesicles. alkaloids relevant here include (table iv) brucine, ergot alkaloids, eseridine, serotonin, physostigmine, gelsemine, p-carboline alkaloids, strychnine, yohimbine, berberine, bicuculline, bulbocapnine, columbamine, coptisine, coralyne, corlumine, ephedrine, ga- lanthamine, laudanosine, nuciferine, palmatine, papaverine, thebaine, cytisine and other quinolizidine alkaloids, heliotrine, chaconine and other steroidal alkaloids, cocaine, atropine, scopolamine, anabaseine, arecoline, dendrobine, gephyrotoxin, histrionicotoxin, methyllycaconitine, muscarine, nicotine, pilocarpine, psilocin, psilocybin, morphine, mescaline, and reserpine. a number of these alkaloids are known hallucinogens, which certainly decrease the fitness of an herbivore feeding on them regularly. cytoskeleton. many cellular activities, such as motility, endocytosis, exocytosis, and cell division, rely on microfilaments and microtubules. a number of alkaloids have been detected which can interfere with the assembly or disassembly of microtubules (table iv) , namely, vincristine, vinblastine, colchicine, maytansine, maytansinine, and taxol. colchicine, the major alkaloid of colchicum autumnale (liliaceae), inhibits the assembly of microtubules and the mitotic spindle apparatus. as a consequence, chromosomes are no longer separated, leading to polyploidy . whereas animal cells die under these conditions, plant cells maintain their polyploidy, a trait often used in plant breeding because polyploidy leads to bigger plants. because of this antimitotic activity, colchicine has been tested as an anticancer drug; however, it was abandoned because of its general toxicity. the derivative colcemide is less toxic and can be employed in the treatment of certain cancers ( ). also, cellular motility is impaired by colchicine; this property is exploited in medicine in the treatment of acute gout, in order to prevent the migration of macrophages to the joints. for normal cells, and thus for herbivores, the negative effects can easily be anticipated, and colchicine is indeed a very toxic alkaloid which is easily resorbed because of its lipophilicity . another group of alkaloids with antimitotic properties are the bisindole alkaloids, such as vinblastine and vincristine, which have been isolated from catharanthus roseus (apocynaceae). these alkaloids also bind to tubulin ( ). both alkaloids are very toxic, but are nevertheless important drugs for the treatment of some leukemias. from taxus baccata (taxaceae) the alkaloid taxol has been isolated. taxol also affects the architecture of microtubules in inhibiting their disassembly ( ). nonalkaloidal compounds to be mentioned in this context include the lignan podophyllotoxin ( ). in conclusion, any alkaloid which impairs the function of microtubules is likely to be toxic, because of their importance for a cell, and, from the point of view of defense, a wellworking and well-shaped molecule. enzyme inhibition. the inhibition of metabolically important enzymes is a wide field that cannot be discussed in full here (see table iv ). briefly, inhibition of camp metabolism (which is important for signal transduction and amplifications in cells), namely, inhibition of adenylate cyclase by anonaine, isoboldine, tetrahydroberberine and inhibition of phosphodiesterase by -ethyl-p-carboline, p-carboline- -propionic acid, papaverine, caffeine, theophylline, and theobromine are some examples. inhibition of hydrolases, such as glucosidase, mannosidase, trehalase, and amylase, is specifically achieved by some alkaloids (table iv) b. action at organ level. whereas the activities mentioned before are more or less directed to molecular targets present in or on cells, there are also some activities that function at the level of organ systems or complete organisms, although, ultimately, they have molecular targets, too. central nervous system and neuromuscular junction. a remarkable number of alkaloids interfere with the metabolism and activity of neurotransmitters in the brain and nerve cells, a fact known to man for a thousand years (table iv) . the cellular interactions have been discussed above. disturbance of neurotransmitter metabolism impairs sensory faculties, smell, vision, or hearing, or they may produce euphoric or hallucinogenic effects. a herbivore that is no longer able to control its movements and senses properly has only a small chance of survival in nature, because it will have accidents (falling from trees, or rocks, or into water) and be killed by predators. thus euphoric and hallucinogenic compounds, which are present in a number of plants, and also in fungi and the skin of certain toads, can be regarded as defense compounds. some individuals of homo sapiens use these drugs just because of their hallucinogenic properties, but here also it is evident that long-term use reduces survival and fitness dramatically. the activity of muscles is controlled by ach and na. it is plausible that an inhibition or activation of neurotransmitter-regulated ion channels will severely influence muscular reactivity and thus the mobility or organ function (heart, blood vessels, lungs, gut) of an animal. in the case of inhibition, muscles will relax; in the case of overstimulation, muscles will be tense or in tetanus, leading to a general paralysis. alkaloids which activate neuromuscular action (so-called parasympathomimetics) include nicotine, arecoline, physostigmine, coniine, cytisine, and sparteine. inhibitory (or parasympatholytic) alkaloids include hyoscyamine and scopolamine, (see above) ( ) . skeletal muscles as well as muscle-containing organs, such as lungs, heart, circulatory system, and gut, and the nervous system are certainly very critical targets. the compounds are usually considered to be strong poisons, and it is obvious that they serve as chemical defense compounds against herbivores, since a paralyzed animal is easy prey for predators or, if higher doses are ingested, will die directly (compare ld,, values in table ). inhibition of digestive processes. food uptake can be reduced by a pungent or bitter taste in the first instance, as mentioned earlier. the next step may be the induction of vomiting, diarrhea, or the opposite, constipation, which negatively influences digestion in animals. the ingestion of a number of allelochemicals such as emetine, lobeline, morphine, and many other alkaloids causes these symptoms ( ). another mode of interference would be the inhibition of carriers for amino acids, sugars, or lipids, or of digestive enzymes. relevant alkaloids are the polyhydroxyalkaloids, such as swainsonine, deoxynojirimycin, and castanospermine, that inhibit hydrolytic enzymes, such as glucosidase, galactosidase, trehalase (trehalose is a sugar in insects which is hydrolyzed by trehalase), and mannosidase selectively (table iv) . nutrients and xenobiotics (such as secondary metabolites) are transported to the liver after resorption in the intestine. in the liver, the metabolism of carbohydrates, amino acids, and lipids takes place with the subsequent synthesis of proteins and glycogen. the liver is also the main site for detoxification of xenobiotics. lipophilic compounds, which are easily resorbed from the diet, are often hydroxylated and then conjugated with a polar, hydrophilic molecule, such as glucuronic acid, sulfate, or amino acids ( ). these conjugates, which are more water soluble, are exported via the blood to the kidney, where they are transported into the urine for elimination. both liver and kidney systems are affected by a variety of secondary metabolites, and the pyrrolizidine alkaloids have been discussed earlier (tables iv and v) . the alkaloids are activated during the detoxification process, and this can lead to liver cancer. also, many other enzyme or metabolic inhibitors (e.g., amanitine), discussed previously, are liver toxins. many alkaloids and other allelochemicals are known for their diuretic activity ( ). for an herbivore, an increased diuresis would also mean an augmented elimination of water and essential ions. since na' is already limited in plant food (an antiherbivore device?), long-term exposure to diuretic compounds would reduce the fitness of an herbivore substantially. disturbance of reproduction. quite a number of allelochemicals are known to influence the reproductive system of animals, which ultimately reduces their fitness and numbers. antihormonal effects could be achieved by mimicking the structure of sexual hormones. these effects are not known for alkaloids yet, but have been confirmed for other natural products. estrogenic properties have been reported for coumarins, which di-merize to dicoumarols, and isoflavones ( , ) . insect molting hormones, such as ecdysone, are mimicked by many plant sterols, which include ecdysone itself, such as in the fern polypodium uulgare, or azadirachtin from the neem tree ( , ) . juvenile hormone is mimicked by a number of terpenes, present in some coniferae. spermatogenesis is reduced by gossypol from cottonseed oil ( ) . the next target is the gestation process itself. as outlined above, a number of alkaloids are mutagenic and lead to malformation of the offspring or directly to the death of the embryo ( table v) . the last step would be the premature abortion of the embryo. this dramatic activity has been reported for a number of allelochemicals, such as mono-and sesquiterpenes and alkaloids. some alkaloids achieve this by the induction of uterine contraction, such as the ergot and lupine alkaloids ( ) . the antireproductive effects are certainly widely distributed, but they often remain unnoticed under natural conditions. nevertheless, they are defense strategies with long-term consequences. blood and circulatory system. all animals need to transport nutrients, hormones, ions, signal compounds, and gas between the different organs of the body, which is achieved by higher animals through blood in the circulatory system. inhibitors of the driving force for this process, the heart muscle, have already been discussed. however, the synthesis of red blood cells is also vulnerable and can be inhibited by antimitotic alkaloids such as vinblastine or colchicine ( ) . some allelochemicals have hemolytic properties, such as saponins. if resorbed, these compounds complex membrane sterols and make the cells leaky. steroidal alkaloids from solanum or veratrum species display this sort of activity as well as influencing ion channels (table iv) . allergenic effects. a number of secondary metabolites influence the immune system of animals, such as coumarins, furanocoumarins, hypericin, and helenalin. common to these compounds is a strong allergenic effect on those parts of the skin or mucosa that have come into contact with the compounds ( , , ) . activation or repression of the immune response is certainly a target that was selected during evolution as an antiherbivore strategy. the function of alkaloids in this context is hardly known. this selection of alkaloid activities, though far from complete, clearly shows that many alkaloids inhibit central processes at the cellular, organ, or organismal level, an important requisite for a chemical defense compound. however, most of the potential targets for the , alkaloids known at present remain to be established. if no activity has been reported, it often means that nobody looked into this question scientifically, and not that a particular alkaloid is without a certain biological property. summarizing this section, it is safe to assume that most alkaloids can affect animals and thus herbivores significantly. dead plants easily rot due to the action of bacteria and fungi, whereas metabolically active, intact plants are usually healthy and do not decay ( ) . how is this achieved? the aerial organs of terrestrial plants have epidermal cells that are covered by a more or less thick cuticle, which consists of waxes, alkanes, and other lipophilic natural products ( , ) . this cuticle layer is water repellent and chemically rather inert, and it thus constitutes an important penetration barrier for most bacteria and fungi. in perennial plants and in roots we find another variation of this principle in that plants often form resistant bark tissues. the only way for microbes to enter a healthy plant is via the stomata or at sites of injury, inflicted by herbivory, wind, or other accidents. at the site of wounding, plants often accumulate suberin, lignin, callose, gums, or other resinous substances which close off the respective areas ( , ) . in addition, antimicrobial agents are produced such as lysozyme and chitinase, lytic enzymes stored in the vacuole which can degrade bacterial and fungal cell walls, protease inhibitors which can inhibit microbial proteases, or secondary metabolites with antimicrobial activity. secondary metabolites have been routinely screened for antimicrobial activities by many researchers, since the corresponding assays are relatively easy to perform. these studies have usually been directed toward a pharmaceutical application, and they often employ the routine methods for screening microbial or fungal antibiotics. it may happen that these tests do not detect an antibacterial activity of a compound because the wrong test species or a nonrelevant concentration was assayed. in the pharmaceutical context we search for very active compounds which can be employed at low concentrations. therefore, the higher concentrations, which would be more meaningful ecologically, are often not tested. these precautions have to be kept in mind when screening the literature for data on the antimicrobial activity of alkaloids. secondary compounds known for their antimicrobial activity include many phenolics (e.g., flavonoids, isoflavones, and simple phenolics), glucosinolates, nonproteinogenic amino acids, cyanogenic glycosides, acids, aldehydes, saponins, triterpenes, mono-and disesquiterpenes, and last but not least, alkaloids ( , , , , ) . in table vi alkaloids are tabulated for which antibacterial activities have been detected. the alkaloids usually affect more gram-positive than gram-negative bacteria. especially well represented are alkaloids which '-hydroxytabernamine hydroxytetrahydrosecamine tetrandrine thalicarpine thalicerbine thalidasine thalidezine thaliglucinone thalistine thalistyline thalmelatine thalmirabine thalphenine thalrugosaminine thalrugosidine thalrugosine tubocurarine ( i +. active; -, no activity observed in the concentration range tested (many alkaloids were only assayed in low concentrations as microbial antibiotics); ad, agar diffusion, al, agar dilution; bg, biogram; ld, liquid culture; mic, minimal inhibitory concentration; pd, paper disk; sp, suspension; tlc, tlc disk test according to wolters and eilert ( ) . if more than one value is given, the data refer to different bacterial species tested. derive from tryptophan (indole alkaloids) and phenylalaninehyrosine, which may be due to the fact that these alkaloids have obtained considerable scientific attention since the discovery of many medicinally important compounds within these groups ( , , , , , , - ) . some of these alkaloids are highly antibiotic, with similar activities as fungal antibiotics, namely, cinchophylline ( ), dictamnine ( , fagarine ( ), stemmadine ( ), yuehchukene ( ), liriodenine ( , lysicamine ( ), oxonantenine ( ), sanguinarine ( ), solacasine ( , ) , rutacridone epoxide ( ), tryptanthrine (i@#), and tuberin ( , ) (table vi) . in many instances, when alkaloids are assessed for their antibacterial activity, they are often also tested for antifungal properties. usually yeasts and candidu are used as test organisms (table vii) . table vii lists ( i , ), thaliglucinone ( ), demissidine ( , ), solacasine ( ), soladulcidine ( , ), solasodine ( , )tidine ( , ), tomatine ( , ) , verazine ( ), cryptopleurine ( ) hydroxyrutacridone epoxide ( , tryptanthrine ( ), and tuberin ( ) . whereas the mode of action and targets of antibiotics of fungal and bacterial origin have been elucidated in many instances (see table iv ), relevant information for plant-derived compounds is scant. however, the molecular targets of some alkaloids have been determined at the general level, but not specifically for bacterial or fungal systems (table iv) that may be responsible for the antibiotic effects observed. the following interactions of alkaloids having antimicrobial properties with molecular targets of bacterial or fungal cells are likely (compare tables vi and vii with tables iv and v) . protein biosynthesis in ribosomes is affected by sparteine ( , , lupanine, angustifoline, -tigloyloxylupanine, and hydroxylupanine ( , , , , , ) . intercalation or binding to dna is influenced by fagaronine, dictamnine ( ), harman alkaloids ( , ) [binding to dna is light dependent ( )], berberine ( - , chelerythrine ( ), and sanguinarine ( , ) ; these compounds may thus inhibit important processes such as dna replication and rna transcription that are also vital for microorganisms. the stability of biomembranes may be disturbed by cepharanthine, tetrandrine, and steroidal alkaloids such as solamargine ( , solanine ( , , ), and solasonine ( ) , thus leading to an uncontrolled flux of metabolites and ions into microbial cells. inhibition of metabolically important enzymes is affected by berber- ine ( ), chelerythrine ( , ), chelidonine ( ), palmatine ( ), sanguinarine ( , ), solacongestidine ( ) , and papaverine. in contrast to antibiotics of microbial origin that could be classified as alkaloids from a chemical point of view in many instances, and which often interfere with the biosynthesis or maintenance of the cell wall (murein) (table iv) , such an interaction has not been described for plantderived compounds. since this topic has not been studied in detail it remains open whether this complex is another target for alkaloids. we can distinguish between secondary metabolites that are already present prior to an attack or wounding, so-called constitutive compounds, and others that are induced by these processes and made de now. inducing agents, which have been termed "elicitors" by phytopathologists, can be cell wall fragments of microbes, the plant itself, or many other chemical constituents ( , , - ) . the induced compounds are called "phytoalexins," which is merely a functional term, since these compounds often do not differ in structure from constitutive natural products. in another way this term is misleading, since it implies that the induced compound is only active in plant-microbe interactions, whereas in reality it often has multiple functions that include antimicrobial and antiherbivoral properties (see below). many of the antimicrobial alkaloids found are constitutively expressed and accumulated, that is, they are already present before an infection. using plant cell cultures, it was observed that some cultures start to produce new secondary metabolites when challenged with bacterial or fungal cell walls, culture fluids, or other chemical factors ( , , - ) . among the compounds found to be inducible are alkaloids such as sanguinarine and hydroxyrutacridone epoxide (see table xi ). quinolizidine alkaloids display some antimicrobial properties, besides their main role in antiherbivore defense ( ) (see table i ). on wounding, qa production is enhanced, thus increasing the already high alkaloid concentration in the plant; in other words, the antimicrobial and herbivoral effect is further amplified (table xi) ( , , ) . the reactions leading to the induction and accumulation of phytoalexins with phenolic structures have been studied in molecular detail ( , , - ) . these studies revealed that plants can detect and react rapidly to environmental problems, such as wounding or infection: within min of elicitation, mrnas coding for enzymes that catalyze the reactions leading to the respective defense compounds are increasingly generated, leading to the accumulation of the respective enzymes and consequently the production of the secondary metabolites ( , , - ) . similar processes are likely for alkaloids, but so far the mechanisms have not been elucidated. we assume that a substantial number of the , alkaloids have antimicrobial properties (which remain to be tested in most cases) that are directed against the ubiquitous and generalist microbes which have not table vi . if a range is given, the first value gives a % inhibition, the second value a % inhibition. specialized on a particular host plant. however, alkaloid production does not necessarily have to be involved with antimicrobial defense. for example, phytophthora or fusarium will attack alkaloid-rich plants of nicotiana, solanum esculentum, and s . tuberosum. cladosporium and fusarium can develop in nutrient-containing media enriched with alkaloids, and aspergillus niger can utilize alkaloids as a nitrogen source ( ). in addition, most plant species are known to be parasitized or infected by at least a few specialized bacteria or fungi which form close, often symbiotic, associations. in these circumstances an antimicrobial effect expected from the secondary metabolites present in the plant can often no longer be observed. we suggest that these specialists have adapted to the chemistry of their host plants. mechanisms may include inhibition of biosynthesis of the respective compounds, degradation of the products, or alteration of the target sites, which are then no longer sensitive toward a given compound (so-called target site modification). these mechanisms need to be established for most of the microbial specialists living on alkaloid-producing plants. some associations between plants and fungi are symbiotic in nature, such as rhizobia in root nodules of legumes or microrhizal fungi in many species. in lupines, nitrogen-fixing rhizobia are present both in alkaloid-rich and alkaloid-free plants. they must therefore be able to tolerate the alkaloids, which are also present in the root. alkaloid production in lupines is more or less unaffected whether or not the plants harbor rhizobia ( , ) . an ecologically important symbiosis between plants and fungi can be observed in fungal species that produce ergot alkaloids. graminaceous species that are infected by ergot suffer much less from herbivory because of the strong antiherbivoral alkaloids produced by the fungi ( ). a similar relationship may occur for other fungal species of plants, many of which produce secondary metabolites possessing animal toxicity. from the pharmaceutical point of view, few alkaloids are interesting as antibiotics, because many are highly toxic to vertebrates (tables i and ). since many alkaloids are antibacterial and antifungal (tables vi and vii) and are present in plants at relatively high concentrations (section iila), it seems likely that from an ecological perspective alkaloids, besides their prominant role in antiherbivore strategies, may play an important role also in the defense against microbial infections. it should be recalled that even alkaloid-producing plants synthesize antimicrobial proteins, such as chitinase and lysozyme, and other antimicrobial secondary products, such as simple phenolics, flavonoids, anthocyanins, saponins, and terpenes ( - , ) . a cooperative, or even synergistic, process could thus be operating. c. antiviral properties plants, like animals, are hosts for a substantial number of viruses, which are often transmitted by sucking insects such as aphids and bugs (heteroptera). resistance to viral infection can be achieved either by biochemical mechanisms that inhibit viral development and multiplication or by warding off vectors such as aphids in the first place. the assessment of antiviral activity is relatively difficult. as a result, only a few investigators have studied the influence of alkaloids on virus multiplication. nevertheless, at least alkaloids have been reported with antiviral properties (table viii) . only sparteine ( ) and cinchonidine ( ) have been tested for antiviral activities against a plant virus, the potato x virus. all other evidence for antiviral activities (table viii) table viii are difficult to interpret at present. polyhydroxy alkaloids, such as swainsonine, can block the action of endoplasmic reticulum-and golgi-localized glucosidases and mannosidases, which are important for the posttranslational trimming of viral envelope proteins. because alkaloids often deter the feeding of insects, such as aphids and bugs (table i ), viral infection rates may be reduced in alkaloid-rich plants. such a correlation exists for alkaloid-rich lupines (so-called bitter lupines) and low-alkaloid varieties (the so-called sweet lupines) (see table xii) . plants often compete with other plants, of either the same or different species, for space, light, water, and nutrients. this phenomenon can be intuitively understood when the flora of deserts or semideserts is analyzed, where resources are limited and thus competition intense ( , , - ) . a number of biological mechanisms have been described, such as temporal spacing of the vegetation period in which some species flower at an earlier season, when others are still dormant or ungerminated. it was observed by molisch in ( ) that plants can also influence each other by their constituent natural products, and he coined the term "allelopathy" for this process. secondary products are often excreted by the root or rhizosphere to the surrounding soil, or they are leached from the surface of intact leaves or from decaying dead leaves by rain ( , ) . both processes will increase the concentration of allelochemicals in the soil surrounding a plant, where the germination of a potential competitor may occur. allelopathy, namely, the inhibition of germination or of the growth of a seedling or plant by natural products, is well documented at the level of controlled in v i m experiments ( , , , - ) , but how it operates in ecosystems is still often a matter of controversy. it is argued, for example, that soil contains a wide variety of microorganisms which can degrade most organic compounds. thus allelochemicals might never reach concentrations high enough to be allelopathic. allelopathic natural products have been recorded in all classes of secondary metabolites. few research groups have studied the effect of alkaloids in this context, but at least alkaloids have been reported with allelopathic properties (table ix) . as can be seen from table ix , allelopathic activities can be found within nearly all structural types of alkaloids. at higher alkaloid concentrations, a marked reduction in the germination rate can be recorded regularly. more sensitive, however, is the growth of the radicle and hypocotyl. they respond to alkaloids at a much lower level, and usually a reduction in growth can be observed but sometimes also the opposite, either of which reduces the fitness of a seedling. in species which produce the compounds, the inhibitory effects can be absent, as was reported for quinolizidine alkaloids in lupines and colchicine in colchicum autumnale ( , ) . it is likely that autotoxicity is prevented either by a special modification of cellular target sites or by other mechanisms. alkaloids ( , , ), berberine ( - ), sanguinarine ( , ) and veratrum alkaloids]; inhibition of protein biosynthesis [e.g., emetine ( ) and quinolizidine alkaloids ( , , - , ) tables iv and ix) . the inhibitory action of quinolizidine alkaloids should be explained in this context ( , ) . they are very abundant in lupine seeds (up to - % dry weight). during germination, -hydroxylupanine is converted to ester alkaloids, such as -tigloyloxylupanine. the latter compound is predominantly excreted via the roots of young seedlings and in germination assays proved to be the most allelopathic qa. these alkaloids influence only heterologous systems, not the germination of lupine seeds themselves. when lupine and lepidium seeds were grown together in the same pot, growth of the lepidium seedlings was much reduced and inhibited, indicating that qas may also be relevant in the ecological context ( ) . although the number of alkaloids with known allelopathic properties is not large, owing to the limited number of studies conducted, it is clear from table ix that alkaloids can be toxic to plants, probably by interfering with basic metabolic or molecular processes. although comparably few alkaloids have been studied for their biological activities in detail, and considering that our data collection (tables i-ix) is far from complete, we can safely state that alkaloids have potent deterrent or poisonous properties in herbivorous animals, and also affect bacteria, fungi, viruses, and plants. the next question will be whether all the adverse activities of alkaloids, which are often assayed in in uitro systems only, are meaningful in nature. because most of the allelochemical activities are dose dependent (others may be synergistic, additive, etc.), the question is whether the amounts of alkaloids produced and stored in plants are high enough to be ecologically meaningful. it is difficult, and also dangerous, to make a general statement concerning alkaloid levels in plants. we must remember that alkaloid composition and levels are often tissue or organ specific ( , , ) . they may vary during the day [a diurnal cycle has been observed for qas and tropane alkaloids ( , , )l or during the vegetation period ( . , ) . furthermore, as in all biological systems, there are differences at the level of individual plants and between populations and subspecies. unfortunately, many phytochemical reports do not contain any quantitative information, or these data are given for the whole plant without realizing the above-mentioned variables. in addition, concentrations are usually given on a dry weight basis, which is appropriate in the chemical or pharmaceutical context. however, herbivores or pathogens do not feed on the dry plant in general, but on the "wet" fresh material. in the context of chemical ecology we urgently need data on a fresh weight basis. as an approximation, in this chapter we use a conversion factor of to convert dry weight to fresh weight data if only the dry weight data are available. summarizing the relevant phytochemical literature, we find that alkaloid levels are between . and % (dry weight), which is equivalent to o.oi-ls%fresh weight, or . - mg/gfresh weight. for plantscontaining quinolizidine alkaloids, actual alkaloid contents are given for a number organs or parts (table x ) , which fall in the range deduced before. we have evaluated the situation for quinolizidine alkaloids and found that the actual concentrations of alkaloids in the plant are usually much higher than the concentrations needed to inhibit, deter, or poison a microorganism or herbivore ( , , , ) . this means that plants obviously play safe and have stored more defense chemicals than actually needed. if we look at the ed,, and ld,, values given in tables through ix, it is likely that the situation is similar for other alkaloid-producing plants, but these correlations need to be experimentally established in most instances. it seems trivial that plants not only synthesize but also store their secondary products, which makes sense only in view of their ecological functions as defense compounds, since they can fulfil these functions only if the amounts stored are appropriate. achieving and maintaining the high levels of a defense compound are very demanding from the point of view of physiology and biochemistry. most allelochemicals would probably interfere with the metabolism of the producing plant if they would accumulate in the compartments where they are made ( ). whereas biosynthesis takes place in the cytoplasm, or in vesicles (berberine) or organelles such as chloroplasts (qas, coniine), the site of accumulation of water-soluble alkaloids is the central vacuole, and that of lipophilic compounds includes latex, resin ducts, or glandular hairs (e.g., nicotine) ( , ) . in this context it should be recalled that many alkaloids are charged molecules at cellular ph and do not diffuse across biomembranes easily. during recent years, evidence has been obtained that at least some alkaloids pass the tonoplast with the aid of a carrier system. the next problem is determining how the uphill transport, that is, the accumulation against a concentration gradient, is achieved. proton-alkaloid antiport mechanisms and ion trap and chemical trap mechanisms have been postulated and partially proved experimentally ( , , ) . thus, the sequestration of high amounts of alkaloids in the vacuole is a complex and energy-requiring task, which would certainly have been lost during evolution were it not important for fitness. as a rule of thumb, we can assume that all parts of an alkaloidal plant contain alkaloids, although the site of synthesis is often restricted to a particular organ, such as the roots or leaves. translocation via the phloem, xylem, or apoplastically must have therefore occurred. phloem transport has been demonstrated for quinolizidine, pyrrolizidine, and indolizidine alkaloids, and xylem transport for nicotine and tropane alkaloids ( , , ) . if the plant relies on alkaloids as a defense compound, these molecules have to be present at the right place and at the right time. alkaloids are often stored in specific cell layers, which can differ from the site of biosynthesis ( , , ) . in lupines, but also in other species ( , , alkaloids are preferentially accumulated in epidermal and subepidermal cell layers, reaching local concentrations between and mm (table x) , which seems advantageous from the point of view of chemical ecology, since a pathogen or small herbivore encounters a high alkaloid barrier when trying to invade a lupine. the accumulation of many alkaloids in the root or stem bark, such as berberine, cinchonine, and quinine, can be interpreted in a similar way. a number of plants produce laticifers filled with latex. for example, isoquinoline alkaloids in the family papaveraceae are abundant in the latex ( ), where they are sequestered in many small latex vesicles. in latex vesicles of chelidonium mujus the concentration of protoberberine and benzophenanthridine alkaloids can be in the range of . - . m, which is achieved by their complexation with equal amounts of chelidonic acid ( ). if a herbivore wounds such a plant, the latex spills out immediately. besides gluing the mandibles of an insect, the high concentration of deterrent and toxic alkaloids will usually do the rest, and, indeed, chelidonium plants are hardly attacked by herbivores. in addition, as these alkaloids are also highly antimicrobial (table iv) , the site of wounding is quickly sealed and impregnated with natural antibiotics. other well-known plants that have biologically active alkaloids in their latex belong to the families papaveraceae (genera papauer, macleya, and sanguinaria) and campanulaceae (genus lobelia) ( ) . it is intuitively plausible that a valuable plant organ must be more protected than others. alkaloid levels are usually highest during the time of flowering and fruit/seed formation. in annual species actively growing young tissue, leaves, flowers, and seeds are often alkaloid-rich, whereas in perennial ones, like shrubs and trees, we find alkaloid-rich stem and root barks in addition. all these plant parts and organs have in common that they are important for the actual fitness or for the reproduction and thus the long-term survival of the species. spiny species, which invest in mechanical defense, accumulate fewer alkaloids than soft-bodied ones ( ); examples are isoquinoline alkaloids in cacti or qas in legumes ( ) . if a plant produces few and large seeds, their alkaloid levels tend to be higher than in species with many and small seeds ( , ); thus. a plant with few and big seeds is generally a rich source of alkaloids, which makes sense in view of the defense hypothesis. these few examples show that accumulation and storage of alkaloids have been optimized in such a way that they are present at strategically important sites where they can ward off an intruder at the first instance of attack. thus, specialized locations must be regarded as adaptive. alkaloid concentrations can fluctuate during the vegetation period, or even during a day ( , ). but in biochemical terms their biosynthesis and accumulation are constitutive processes. this ensures that a certain level of defensive compounds is present at any time. furthermore, continuous turnover is a common theme for molecules of the cells whose integrity is important, such as proteins, nucleic acids, and signal molecules. the same seems to be true for a defense compound. an alkaloid which mimics a neurotransmitter, such as hyoscyamine, nicotine, or sparteine, could be oxidized or hydrolyzed in the cell by chance, and thus would be automatically inactivated. only by replacing these molecules continuously can the presence of the active compounds be guaranteed. for example, it was suggested that nicotine has a half-life of hr in nicotiana plants, and that more than % of the co, fixed passes through this alkaloid ( ). in other groups of natural products it was possible to show that plants can react to infection by microbes or to wounding by herbivores by inducing the production of new defense compounds. these compounds are termed "phytoalexins" in phytopathology ( ) ( ) ( ) . classic examples of phytoalexins include isoflavones, phenolics, terpenes. protease inhibitors, coumarins, and furanocoumarins. using plant cell cultures it could be shown that a similar process can be observed with some alkaloidal plants, which start to produce alkaloids with antimicrobial properties (e.g., sanguinarine, canthin- -one, rutacridone alkaloids) when challenged with elicitors from bacterial or fungal cell walls (table xi) . but what is the situation after herbivory? when plants are eaten by large herbivores, a de nouo synthesis would be almost useless for a plant (except maybe trees), since this would not be quick enough. the situation is different, however for small herbivores such as insects or worms, which may feed on a particular plant for days or weeks. here the de nouo production of an allelochemical would be worthwhile. there are indeed some preliminary experimental data that support this view. in liriodendron rirlipifera several aporphine alkaloids accumulate after wounding, which are otherwise not present ( ). in tobacco the produc- " cc, cell culture; pl, plant tion of nicotine, in lupines that of qas, and in atropci belleidonnu that of hyoscyamine are induced by wounding, thus increasing the already high levels of alkaloids by up to a factor of . whereas the response was seen after - hr in lupines, it took days in nicotiunu and in atropei (table xi) . we suggest that the wound-induced stimulation of alkaloid formation is not an isolated phenomenon, but rather an integral part of the chemical defense system. the induced antimicrobial and antiherbivoral responses show that plants can detect environmental stress and that secondary metabolism is flexible and incorporated in the overall defense reactions. many details on how a plant perceives and transmits information remain to be disclosed, but this will surely be a stimulating area of research in the future. although the physiology and metabolism of most alkaloids are extremely intricate ( ) and often not known, the available data suggest that they are organized and regulated in such a way that alkaloids can fulfill their ecological defense function. in other words, the alkaloids are present at the right time, the right place, and the right concentration. the aforementioned arguments strongly support the hypothesis that alkaloids serve as defense compounds for plants. besides circumstantial evidence, we would welcome critical experiments which clearly prove that alkaloids are indeed important for the fitness and survival of the plants producing them. we suggest that if a plant species which normally produces alkaloids is rendered alkaloid-free, it should have a reduced fitness because it is much more molested by microorganims and herbivores than its alkaloid-producing counterpart. for one group of alkaloids, the quinolizidine alkaloids, these experiments have already been performed ( , , , , ) . as mentioned before, qas constitute the main secondary products of many members of the leguminosae, especially in the genera lirpinus, genistu, cyfisiis, bccptisiu, thrrmopsis, sophoru, ormosici, and others ( ). lupines have relatively large seeds which contain up to - % protein, up to % lipids, and - % alkaloids. to use lupine seed for animal or human nutrition, homo scipiens, for several thousand years, used to cook the seeds and leach out the alkaloids in running water. this habit has been reported for the egyptians and greeks in the old world, and for the indians and incas of the new world. the resulting seeds taste sweet, in contrast to the alkaloid-rich ones which are very bitter. in mediterranean countries people still process lupines in the old way, and sometimes the seeds are salted afterward and served as an appetizer, comparable to peanuts. at the turn of the twentieth century, german plant breeders set out to grow alkaloid-free lupines, the so-called sweet lupines. although sweet lupines are extremely rare in nature ( in > . ), the efforts were largely successful, and at present, sweet varieties with an alkaloid content lower than . % exist for lupinus albus, l. mutabilis, l . luteus, l. angustifolius, and l . polyphyllus. as far as we know, the sweet varieties differ from the original bitter wild forms only in the degree of alkaloid accumulation. this offers the chance to test experimentally whether bitter lupines have a higher fitness than sweet ones with regard to microorganisms and herbivores. the results of these experiments were clearcut ( , , , ) (table xu). in the greenhouse, where plants are protected from herbivores or pathogens, no clear advantage was seen. when lupines were planted in the field, without being fenced in and without man-made chemical protection, however, a dramatic effect was regularly encountered, especially with regard to herbivores ( , , , ) . rabbits (cuniculus europaeus) and hares (lepus europaeus) clearly prefer the sweet plants and leave the bitter plants almost untouched, at least as long as there was an alternative food source. before dying rabbits will certainly try to eat bitter lupines. a similar picture was seen for a number of insect species, such as aphids, beetles, thrips, and leaf-mining flies (table xii) , namely, the sweet forms were attacked, whereas the alkaloid-rich ones were largely protected. the alkaloid-poor variety of l . luteus also became a host of acyrthosiphon pisii ( ). in poland, where the sweet yellow lupine is one of the more important fodder plants, the invasion of the aphids became a serious problem not only because the aphid enfeebles the plants by sucking its phloem sap, but also because it transfers a viral disease. the disease, known as lupine narrow leafness, decreases seed production in infected plants, and the infection takes place early, that is, prior to the plants' blossoming. thus, a mixed population of sweet and bitter lupines can, after a few generations, lose all sweet forms. infestation by the aphid and the following viral infection accelerate the elimination of alkaloid-poor plants, which, even without infection, are already inferior in seed production ( ). this observation again stresses the importance of alkaloids for the fitness of lupines. plant breeders have also observed that bacterial, fungal, and viral diseases are more abundant in the sweet forms, but this effect has not been documented in necessary detail. these experiments and observations clearly prove the importance of qas for lupines, but it should not be forgotten that other secondary metabolites, such as phenolics, isoflavones, terpenes, saponins, stachyose, erucic acid, and phytic acid, are also present in lupines and may exert additional or even synergistic effects. the lupine example also tells us about the standard philosophy and problems of plant breeding. with our present knowledge on the ecological importance of qas for the fitness of lupines, it seems doubtful whether the selection of sweet lupines was a wise decision. in order to grow them we have had to build fences and, worse, to employ man-made chemical pesticides, which have a number of well-documented disadvantages. it can be assumed that similar strategies, namely, breeding away unwanted chemical traits, have been followed with our other agricultural crops, with the consequence that the overall fitness was much reduced ( ). we can easily observe the reduced fitness by trying to leave crop species to themselves in the wild: they will quickly disappear and not colonize new habitats. there are, however, alternatives. taking lupines as an example, we could devise large-scale technological procedures to remove alkaloids from the seeds after harvest (similar to sugar raffination from sugar beets). at present a few companies are actively exploring these possibilities. one idea is to produce pure protein, lipids, dietary fibers from bitter seeds. a spin-off product would be alkaloids, which could be used either in medicine (sparteine is exploited as a drug to treat heart arrhythmia) or in agriculture as a natural plant protective, that is, as an insecticide ( , ) . it is evident, however, that each plant has developed its own strategy for survival. if all plants would follow the same strategy, it would be an easy life for herbivores and pathogens, since being adapted to one species would mean adapted to all species. this specialization becomes evident if we analyze the qualitative patterns of secondary metabolite profiles present in the plant. we regularly see one to five main alkaloids in a plant, but also several (up to ) minor alkaloids. this qualitative pattern is not constant, but differs among organs, developmental stages, individuals, populations, and species. normally, we classify the compounds as belonging to one or two chemical groups. this does not mean, however, that their biological activities are identical. on the contrary, the addition of a lipophilic side chain to a molecule seems to be a small and insignificant variation from the chemical point of view, but this may render the compound more lipophilic, and thus more resorbable. in consequence, its toxicity may be higher (see qas in table i ). thus, a herbivore or pathogen has to adapt not only to one group of chemicals but to the individual compounds present. as the composition of these chemicals changes, it is even more difficult for them to cope. therefore, we suggest that structural diversity and continuous variation are means by which nature counteracts the adaptation of specialists. in medicine, we do a similar thing if we want to control microbial diseases. to overcome or to prevent resistance of bacteria toward a particular antibiotic, very often mixtures of structurally different antibiotics are applied, whose molecular targets often differ. if only one antibiotic were given to all patients, the development of resistance would be much favored. it has been argued that alkaloids cannot have a significant role in plants because not all plant species produce alkaloids (only % of all plants do). these authors, such as robinson ( , have overlooked the fact that if all plants would produce one single alkaloid, even a very toxic alkaloid such as colchicine, it could be certain that nearly all herbivores would have developed a resistance toward this alkaloid. only the variation of secondary metabolites, and thus of the targets which they affect, provides a means to develop efficient defense compounds. the arguments of robinson would be correct if there were higher plants without any secondary metabolites, which, nevertheless, would thrive in nature; however, these plants are not known. from an evolutionary perspective it is not important whether the defense chemical is an alkaloid or a terpene; it is only essential that it affect certain and important targets in herbivores or pathogens. although the biological activities of many alkaloids have not yet been studied and their ecological functions remain to be elucidated or proved, we can nevertheless safely say that alkaloids are neither waste nor functionless molecules, but rather they are important fitness factors, probably mostly antiherbivore compounds. since nature obviously favored multitasking, additional activities, such as allelopathic or antimicrobial activities, are plausible. for quinolizidine and pyrrolizidine alkaloids, these multiple functions are already well documented (tables i-x) . plants that defend themselves effectively constitute an ecological niche almost devoid of herbivores and pathogens. it is not surprising that during evolution a number of organisms evolved which have specialized on a particular host plant species and found ways to tolerate, or even to exploit, the defense chemistry of their hosts ( , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . as compared to the huge number of potential enemies, the number of adapted specialists is usually small, and in general a "status quo" or equilibrium can be observed between the specialists (or parasites) and their hosts. a specialist is not well advised to kill its host, since this would destroy its own resources; a mutualism is more productive for survival. host plant-specific specialists occur within bacteria, fungi, and herbivores. the interaction of the former two groups is a central topic for plant pathologists. they often find that susceptible and nonsusceptible microbe strains exist. in most cases, it is not known how these microbial specialists achieved a relationship with the host plant chemistry, for example, whether they degrade secondary metabolites or whether they simply toler-ate them. many phytopathogenic bacteria and fungi produce their own secondary metabolites, which are often toxic to plants. it is assumed that these phytotoxins serve to weaken the host plants' defense, but may be this is not the whole story. many grasses are infected with fungi that produce ergot alkaloids. it has been assumed that these fungi (e.g., clauiceps) are proper parasites. in recent years, however, experimental evidence suggests that the relationship between grasses and ergot may be of a symbiotic nature ( ). ergot alkaloids are strong vertebrate toxins (tables i-iv) ; they mimic the activity of several neurotransmitters, such as dopamine, serotonin, and noradrenaline (table iv) . in fact, the impact of herbivores on populations which were highly infected by fungi was more reduced than those without. this means that the fungi exploit the nutrients of their host plants and supply them with strong poisons, which are not produced by the plants themselves. since the fungi do not kill their hosts, this close interrelationship seems to be of mutual interest. we expect that similar relationships are likely to be detected in the future. as mentioned earlier, a large number of mono-and oligophagous insects exist which have adapted to their host plants and the respective defense chemistry in complex fashions. in general, we can see the following main schemes ( , , , , , ) . in type adaptations, a species "learns" (or, as we should say, during evolution variants have been selected by natural selection which can tolerate a noxious defense compound) (a) by finding a way to avoid its resorption in the gut; (b) if resorption cannot prevented, by eliminating the toxin quickly via the malpighian tubules or degrading it by detoxifying microsomal and other enzymes; and (c) by developing a target site that is resistant to the toxin, such as a receptor which no longer bind the exogenous ligand. alternatively, in type strategies a species not only tolerates a plants' defense compound, but exploits it for its own defense or for other purposes, such as pheromones i , , ) . examples of type include manduca sexra, whose larvae live on nicoriana and other solanaceous plants. the alkaloids present in these plants, such as nicotine or hyoscyamine, are not stored but are degraded or directly eliminated with the feces ( ). in addition, it has been postulated that nicotine may either not diffuse into nerve cells or that the acetylcholine recpetor no longer binds nicotine as in "normal" animals ( ). the potato beetle (leptinotarsa decernlineata) lives on solanurn species containing steroid alkaloids, which are tolerated, but not stored, by this species, the bruchid beetle callosohruchus fasciarus predates seeds of qa-rich plants, such as laburnum anagyroides; this beetle eliminates most of the dietary cytisine with the feces ( ). examples of type are to some degree more interesting. in a number of plants alkaloids are translocated via the phloem ( ). when aphids live on these plants they are in direct contact with the alkaloids present. a number of examples are known at present which show that adapted aphids can store the dietary alkaloids. examples are the quinolizidines in aphis cytisorum, a. genistae, and macrosiphum albifrons, the pyrrolizidines in aphis jacobaea, a . cacaliaster, and aconitine in aphis aconiti ( , ) . for alkaloid-storing m . albifrons it was shown experimentally that the qas stored provide protection against carnivorous beetles, such as carabus problematicus or coccinella septempunctata ( , ) . acyrthosiphon spartii prefers sparteine-rich cytisus scoparius plants ( ); although it is likely that this species also stores qas, it has not been demonstrated to do so. larvae of the pyralid moth uresiphita reversalis live on qa-producing plants, such as teline monspessulana. the larvae store some of the dietary alkaloids, especially in the integument and also the silk glands. the uptake is both specific and selective and is achieved by a carrier mechanism. whereas alkaloids of the -oxosparteine type dominate in the plant, it is the more toxic cytisine that is accumulated by the larvae, with the oxosparteines being eliminated with the feces ( , ) . the larvae gain some protection from storing qas, as was shown in experiments with predatory ants and wasps. when the larvae pupate, most of the alkaloids stored are used to impregnate the silk of the cocoon, thereby providing defense for this critical developmental stage ( , ). the emerging moth lives cryptically, has no aposematic coloring, and does not contain alkaloids. in contrast the alkaloid-rich larvae are aposematically colored and live openly on the plants ( , ) . the larvae of the blue butterfly (plebejus icaroides) feed only on lupines, rich in alkaloids. as far as we know, the larvae do not sequester or store the dietary alkaloids ( ). helopeltis feeds on cinchona bark, which is rich in cinchonine-like alkaloids; it stores and uses them for its own defense ( ). larvae of the butterflies pachlioptera aristolochiae, zerynthia polyxena, ornithoptera priamus, and battus philenor live on arisrolochia plants and were shown to take up and sequester aristolochic acid, a carcinogenic alkaloid discussed earlier, as an effective defense compound ( , , ) . the best-studied group of acquired alkaloids are the pyrrolizidines, which are produced by plants, especially in the families asteraceae and boraginaceae ( ). some arctiid larvae of tyria jacobaea, cycnia mendica, amphicallia bellafrix, arginia cribaria, and arctia caja were shown to store the dietary pas and exploit them for their own defense ( , , , , - , ) . in tyria jacobaea, arctia caja, diacrisia sannio, phragmatobia fuligonosa, and callimorpha dominula pas are taken up and stored in the integument ( ). monarch butterflies (e.g., danaus plexipus) combine two sets of natural compounds. larvae feed on plants rich in cardiac glycosides and use them as chemical defense compounds. adult butterflies visit plants with pas, where they collect pas that are converted to pheromones or transferred to their eggs ( ,f , , , f, ) . a similar pa utilization scheme was observed with larvae of the moth utetheisa ornatrix ( , ) , where the compounds were shown to be deterrent for spiders and birds ( , ) . the chrysomelid beetle oreina feeds on pa-containing plants, such as adenostyles, and stores the dietary pas in the defense fluid ( , ). in the arctiid creatonotos transiens was observed an advanced exploitation of pas ( , , , - ) . the alkaloids are phagostimulants for larvae, which are endowed with specific alkaloid receptors. dietary pyrrolizidine n-oxides are resorbed by carrier-mediated transport. after resorption, free pas are converted to the respective n-oxides and ( s)-heliotrine to ( r)-heliotrine. the latter form is later converted to a male pheromone, ( r)-hydroxydanaidal. pas are stored in the integument, where they serve as defense compounds and are not lost during metamorphosis. in the adult moth, however, the pas are mobilized. in the female adult, pas are translocated into the ovary and subsequently into the eggs. in the male, pas are necessary for the induction of abdominal scent organs and concomitantly for the biosynthesis of pa-derived pheromones, which are dissipated from these coremata. in addition, pas are transferred into the spermatophore and thus donated to the female. a significant amount of pas is further transferred to the eggs, which thus obtain chemical protection from the pas previously acquired by both male and female larvae. marine dinoflagellates produce a number of toxins, such as saxitoxin, surugatoxin, tetrodotoxin, and gonyautoxin, that affect ion channels (table iv). these algae are eaten by some copepods, fish, and molluscs that also store these neurotoxins ( , , , , , ) . as a consequence, these animals have acquired chemical defense compounds, which they can use against predators. this discussion is not meant to be complete, but should illustrate that a number of insect herbivores exploit the chemistry of their food plants. these insects are adapted and have evolved a number of molecular and biochemical traits that can be considered as prerequisites. however, many of the respective plant-insect interactions have not yet been studied, and it is therefore likely that the acquisition of dietary defense compounds is even more widely distributed in nature than anticipated. whereas insect herbivores are often highly host plant specific, vertebrate herbivores tend to be more of the polyphagous type, although some specialization may occur. for example, grouse (lagopus lagopus) or capercaillies (tetra urogallus) prefer plants of the families of ericaceae or coniferae, and crossbills seeds of picea and abies species, which are rich in terpenes. the australian koala is oligophagous and prefers terpene-rich species of the genus eucalyptus. for approximately million years, the only true herbivorous vertebrates have been the mammals. the mesozoic reptiles disappeared following the mesophytic flora. birds, though a few species feed on seeds and berries, seldom eat leaves (except geese and grouse), and they frequently use insects, in addition to plant parts, as a food source ( ). although a single plant can be a host for hundreds of insect larvae, hundreds of plants comprise a daily menu for a larger mammal. the strategies of the polyphagous species include the following. avoidance of plants with very toxic vertebrate poisons (these species are usually labeled toxic or poisonous by man) by olfaction or taste discrimination. often such compounds may be described as bitter, pungent, bad smelling, or in some other way repellent. . sampling of food from a wide variety of sources and thus minimizing the ingestion of high amounts of a single toxin. . detoxification of dietary alleochemicals, which can be achieved by symbiotic bacteria or protozoa living in the rumen or intestines, or by liver enzymes which are specialized for the chemical modification of xenobiotics. this evolutionary trait is very helpful for homo sapiens, since it endowed us with a means to cope with our man-made chemicals which pollute the environment. carnivorous animals, such as cats, are known to be much more sensitive toward plant poisons ( ). it was suggested that these animals, which d o not face the problem of toxic food normally, are thus not adapted to the handling of allelochemicals. some animals, such as monkeys, parrots, or geese, ingest soil. for geese ( ) it was shown that the ingested soil binds dietary allelochemicals, especially alkaloids ( ). this procedure would reduce the allelochemical content available for resorption. . animals are intelligent and can learn. the role of learning in food and toxin avoidance should not be underestimated, but it has not been studied in most species. for most vertebrate herbivores, the ways they manage to avoid, tolerate, or detoxify their dietary allelochemicals have not been explored. sometimes, only domesticated animals were used in experiments, but they tend to make more mistakes in food choice than the wild animals. more evidence on this subject is available for homo sapiens, who has evolved a number of "tricks," some of them obviously not anticipated by evolution. first, man tends to avoid food with bitter, pungent, or strongly scented ingredients. as a prerequisite he needs corresponding receptors in the nose or on the tongue which evolved during the long run of evolution as a means to avoid intoxication. second, our liver still contains a set of detoxifying enzymes which can handle most xenobiotics. furthermore, some of these enzymes, such as cytochrome p. oxidase, is inducible by dietary xenobiotics. third, besides these biological adaptations, man has also used his brain to avoid plant allelochemicals. (a) many fruits or vegetables are peeled. as many alkaloids and other compounds are stored in the epidermis, for example, steroid alkaloids in potato tubers or cucurbitacins in cucurbits, peeling eliminates some of these compounds from consumption. (b) most food is boiled in water. this leads to the thermal destruction of a number of toxic allelochemicals, such as phytohaemagglutinins, protease inhibitors, and some esters and glycosides. many watersoluble compounds are leached out into the cooking water and are discarded after cooking (e.g., lupines or potatoes). (c) south american indians ingest clay when alkaloid-rich potato tubers are on the menu. since clay binds steroidal alkaloids, geophagy is thus an ingenious way to detoxify potential toxins in the diet ( ) . (d) man has modified the composition of allelochemicals in his crop plants, in that unpleasant taste components have been reduced by plant breeding. from the point of view of avoidance, this strategy is plausible, but, as was discussed earlier, it is deleterious from the point of view of chemical ecology. these plants often lose their resistance against herbivores and pathogens, which then has to be replaced by man-made pesticides. in general, only a few plants are exploited by man as food, as compared to the , species present on our planet. this means that even homo sapiens with all his ingenuity has achieved only a rather small success, indicating the importance and power of chemical plant defenses. in this context, it is worth recalling that a number of animals are able to synthesize their own defense compounds, among them several alkaloids ( , , , - ) . these animals have the common feature that they are usually slow-moving, soft-bodied organisms. marine animals, such as mol-luscs, sponges, zooanthids, and fishes, have been shown to contain a variety of alkaloids, such as acrylcholine, neosaxitoxin, murexin, pahutoxin, palytoxin, petrosine, and tetramine, that are toxic to other animals ( . , , , , , , , , , ) . a number of nemertine worms, such as amphiporus or nereis, produce alkaloids such as , -bipyridyl, anabaseine, nemertelline, or nereistoxin, which are toxic to predators such as crayfish ( , , , ,) . arthropod-made alkaloids include glomerine and homoglomerine in glomerus ( ) , adaline in adalia ( ), coccinelline, euphococcinine, and derivatives in coccinella, epilachna, and other coccinellid beetles ( , , , ) , and stenusine in stenus ( ) , which are considered to be antipredatory compounds ( , , , - ) . solenopsis ants produce piperidine alkaloids which resemble the plant alkaloid coniine. these alkaloids are strong deterrents and inhibit several cellular processes, such as electron transport chains (table iv) ( , ) . many insects indicate the content of toxic natural products by warning colors (aposematism) or by the production of malodorous pyrazines ( , , , ) . not only are lower animals able to synthesize alkaloids, but also vertebrates, especially in the class amphibia. tree frogs of the genus dendrobates accumulate steroidal alkaloids, such as batrachotoxin, pumiliotoxins a-c, gephyrotoxin, and histrionicotoxin, in their skin, which are strong neurotoxins (table iv) ( , , ) . natives have used the alkaloids as arrow poisons. similar alkaloids (i.e., homobatrachotoxin) have recently been detected in passerine birds of the genus pitohui ( ) . salamanders, salamandra maculosa, which are aposematically colored, produce the toxic salamandrine and derivatives, alkaloids of the steroidal group ( , , ). salamandrine is both an animal toxic (paralytic) and an antibiotic. toads (bufonidae) produce in their skin cardiac glycosides of the bufadienolide type, but also a set of alkaloids, such adrenaline, noradrenaline, adenine, bufotenine, or bufotoxin ( , , ). except for bufotoxin, the other chemicals are, or mimic, neurotransmitters. these examples show that alkaloids found in animals can either be derived from dietary sources (see section ,d, ) or be made endogenously. common to both origins is their use as chemical defense compounds, analogous to the situation found in plants. in animals we can observe the trend that sessile species, such as sponges and bryozoans, or slow-moving species without armor, such as worms, nudibranchs, frogs, toads, and salamanders, produce active allelochemicals ( , , , ) , but not so those with weapons, armor, or the possibility for an immediate flight. plants merely developed a similar strategy as these "unprotected" animal species. in this context it seems amazing that hardly anybody has doubted the defensive role of alkaloids in animals, whereas people did, and still do, where alkaloids in plants are concerned. evidence is presented in this overview that alkaloids are not waste products or functionless molecules as formerly assumed ( , ), but rather defense compounds employed by plants for survival against herbivores and against microorganisms and competing plants. these molecules were obviously developed during evolution through natural selection in that they fit many important molecular targets, often receptors, of cells (i.e. they are specific inhibitors or modulators), which can clearly be seen in molecules that mimic endogenous neurotransmitters (table iv; section ii,a, ,a). on the other hand, microorganisms and herbivores rely on plants as a food source. since both have survived, there must be mechanisms of adaptations toward the defensive chemistry of plants. many herbivores have evolved strategies to avoid the extremely toxic plants and prefer the less toxic ones. in addition, many herbivores have potent mechanisms to detoxify xenobiotics, which allows the exploitation of at least the less toxic plants. in insects, many specialists evolved that are adapted to the defense chemicals of their host plant, in that they accumulate these compounds and exploit them for their own defense. alkaloids obviously function as defense molecules against insect predators in the examples studied, and this is further support for the hypothesis that the same compound also serves for chemical defense in the host plant. the overall picture of alkaloids and their function in plants and animals seems to be clear, but we need substantially more experimental data to understand fully the intricate interconnections between plants, their alkaloids, and herbivores, microorganisms, and other plants. defense in animals introduction to ecological biochemistry defense mechanisms in plants herbivores: their interaction with secondary okologische biochemie allelochemicals: role in agriculture and forestry cell culture and somatic cell genetics of plants" (f. constabel and . m. wink gifttiere und ihre waften perspectives in chemoreception behavior biochemie und physiologie der sekundaren pflanzenstoffe plant metabolites biosynthese der alkaloide biochemistry of alkaloids the alkaloids: the fundamental chemistry antiseptika baerheim svendsen lloydia . n. m. rojas hernandez vallejos and . a. roveri the merck index proc. rd inf. lupin conf insect biology in the future micromolecular evolution, systematics and ecology phytochernical ecology: allelochernicals, mycotoxins and insect pheromones phytochem biogene gifte rozniki nauk rolnikzych die gift-und arzneipflanzen in mitteleuropa antibiotics: mechanism of action of antimicrobial and antitumour agents antimicrob. agents chemo pharmazeutische biologie , biogene arzneistoffe pharmazeutische biologie drug use in pregnancy the alkaloids handbook of enzyme inhibitions cold spring harbor conf primary and secondary metabolism of plant cell cultures chemical defenses of arthropods der einfluss einer pflanze auf die andere-allelopathie the science of allelopathy allelopathy lectures on insect olfaction focus on insect-plant interactions alkaloid biology and metabolism in plants molecular aspects of insect plant associations methods of plant biochemistry secondary products in plant tissue culture proc. natl the alkaloids the work of the author was supported by the deutsche forschungsgemeinschaft. i thank dr. th. twardowski for reading an earlier draft of the manuscript. key: cord- -bn x authors: cox-georgian, destinney; ramadoss, niveditha; dona, chathu; basu, chhandak title: therapeutic and medicinal uses of terpenes date: - - journal: medicinal plants doi: . / - - - - _ sha: doc_id: cord_uid: bn x terpenes, also known as terpenoids are the largest and most diverse group of naturally occurring compounds. based on the number of isoprene units they have, they are classified as mono, di, tri, tetra, and sesquiterpenes. they are mostly found in plants and form the major constituent of essential oils from plants. among the natural products that provide medical benefits for an organism, terpenes play a major and variety of roles. the common plant sources of terpenes are tea, thyme, cannabis, spanish sage, and citrus fruits (e.g., lemon, orange, mandarin). terpenes have a wide range of medicinal uses among which antiplasmodial activity is notable as its mechanism of action is similar to the popular antimalarial drug in use—chloroquine. monoterpenes specifically are widely studied for their antiviral property. with growing incidents of cancer and diabetes in modern world, terpenes also have the potential to serve as anticancer and antidiabetic reagents. along with these properties, terpenes also allow for flexibility in route of administration and suppression of side effects. certain terpenes were widely used in natural folk medicine. one such terpene is curcumin which holds anti-inflammatory, antioxidant, anticancer, antiseptic, antiplasmodial, astringent, digestive, diuretic, and many other properties. curcumin has also become a recent trend in healthy foods and open doors for several medical researches. this chapter summarizes the various terpenes, their sources, medicinal properties, mechanism of action, and the recent studies that are underway for designing terpenes as a lead molecule in the modern medicine. terpenes, also known as isoprenoids are the largest and most diverse group of naturally occurring compounds that are mostly found in plants but larger classes of terpenes such as sterols and squalene can be found in animals. they are responsible for the fragrance, taste, and pigment of plants. terpenes are classified on the basis of organization and number of isoprene units it contains (see footnote ). an isoprene unit is a building block of terpenes that is a gaseous hydrocarbon that contains the molecular formula c h (see footnote ). terpenes and terpenoids are terms that are often used interchangeably but the two terms have slight differences; terpenes are an arrangement of isoprene units that are naturally occurring, volatile, unsaturated -carbon cyclic compounds that give off a scent or a taste to defend itself from organisms that feed off of certain types of plants (see footnote ). terpenes have many functions in plants such as a thermoprotectant, signaling functions, and not limited to, pigments, flavoring, and solvents but also have various medicinal uses (yang et al. ) . table . shows the different types of terpenes discussed in this chapter along with an example of that terpene. terpene is a natural compound with various medical properties and found in both plants and animals (gershenzon ) . among natural products that mediate antagonistic and beneficial interactions within the organism, terpene play a variety of roles (gershenzon ) . terpene protects many living organisms like microorganisms, animals and plants from abiotic and biotic stresses (gershenzon ) . terpene can ward off pathogens, predators, and competitors. living organisms use terpene for multiple reasons like medicinal purposes and communications about food, mates, or enemies (gershenzon ) . it is impressive how different organisms use terpene for common purposes even though terpene contain many forms and varieties (gershenzon ) . so far only a small percentage of terpene is investigated (franklin et al. ). cannabis is one of the most common sources for the medicinal terpene (franklin et al. ). this plant contains many medicinal properties like anticancer, antimicrobial, antifungal, antiviral, antihyperglycemic, analgesic, anti-inflammatory, and antiparasitic (franklin et al. ) . terpene is also used to enhance skin penetration, prevent inflammatory diseases (franklin et al. ) . nowadays modern medication use large scales of terpene for various treatment drugs (franklin et al. ) . there are commonly used plants like tea (melaleuca alternifolia), thyme, cannabis, salvia lavandulifolia (spanish sage), citrus fruits (lemon, orange, mandarin) etc. that provide wide range of medicinal values (perry et al. ) . tea tree oil has increased in popularity in recent years when it comes to alternative medicine (perry et al. ) . tea tree oil is a volatile essential oil and is famous for its antimicrobial properties, and acts as the active ingredient that is used to treat cutaneous infections (carson et al. ) apart from the flavor that gives to food, essential oil contain antimicrobial properties (bound et al. ) . thyme is one of plants that synthesize terpene alcohols and phenols which contain powerful antibacterial and antifungal properties (bound et al. ) . terpene synthesized from cannabis also long served as medicines (perry et al. ) . they also contain psychoactive properties and used against many infectious diseases (perry et al. ) . salvia lavandulifolia is famous for anti-dementia (current memory-enhancing) drugs by enhancing james and dubery ( ) cholinergic activity via inhibition of cholinesterase (perry et al. ) . in vitro examination method was used to study the effects of constituent terpenes on human erythrocyte acetylcholinesterase (perry et al. ) . some of the medicinal properties of terpenes are listed in table . . important properties associated with terpene are difficult to overstress (franklin et al. ). there are many important uses with terpene and these include antiinsect properties, antimicrobial properties and anti-herbivore properties (franklin et al. ) . terpene can be extracted through plants and thorough some insects (franklin et al. ) . without using harsh chemicals that could potentially contain side effects, terpene is a healthy alternative to ward off insects (franklin et al. ). there have been many pesticides made for killing domestic pests like lice, or mites (franklin et al. ) . in these cases, it is very important to make sure that these pesticides do not affect humans in harmful ways (franklin et al. ). there are many options like shampoo, sprays, lotions that were manufactured against pests that include one or more terpenes that are employed in the instant invention (franklin et al. ). these naturally occurring terpenes are generally not modified they were used in their raw form and the environment protection agency in the usa classified as "gras" which mean generally regards as safe (franklin et al. ). certain terpene is highly effective against both lice and lice eggs and there is a less than significant chance of resistance developing against this terpene based pesticides; reason for this is their observed modes of action (franklin et al. ). silva et al. ( ) unlike other types of pediculosis medication this terpene based instant inventions are not neurotoxins (franklin et al. ) terpenes are also used combined with terpene aldehyde called citral. citral derives from an essential oil that is extracted from lemongrass (cymbopogon citratus) (franklin et al. ) . citral possesses antibacterial and antifungal properties, while lemongrass possesses anti-insect properties (franklin et al. ) . a series of anti-insect formulation contain many terpenes (franklin et al. ) most of these pesticides are a mix of terpene and citral (franklin et al. ). table . consists of what these terpenes include. antimicrobial properties or the ability to kill or stop growth of a microorganism in terpenes are commonly used in traditional and modern medicine (himejima et al. ). there are many terpenes with antimicrobial activities (himejima et al. ). the following plants produce terpenes which have antimicrobial properties: pinus ponderosa (pinaceae), spices (sage, rosemary, caraway, cumin, clove, and thyme), cretan propolis, helichrysum italicum, rosmarinus officinalis, and so on (himejima et al. ) . these antimicrobial terpenes can also be used against food borne pathogen like escherichia coli, staphylococcus aureus, and bacillus cereus (himejima et al. ) . pinus ponderosa cell extract contain wide-ranging antimicrobial activities (himejima et al. ) . after steaming and distillation from pinus ponderosa cell extract, a distillate and a residue are obtained (himejima et al. ) . the distillate consists of monoterpenes and some sesquiterpenes while the residue consists of four diterpene acids (himejima et al. ) . it was also reported that when a physical damage is caused to the pine tree or any other terpene containing tree from insect attacks, resin which contains terpene secret to protect the tree from further damage (himejima et al. ) . five different kinds of terpene can be isolated from cretan propolis, they are, the diterpenes, , -dinor- -oxo- ( )-labden- -oic acid and a mixture of labda- ( ), e-dien- -carboxy- -yl oleate, palmitate and triterpene (popova et al. ). spectroscopic analysis and chemical evidence has been used to establish the structures of the different compounds (popova et al. ). these compounds that were isolated from terpene was tested for its antimicrobial activity against bacteria like gram positive and gram negative (popova et al. ). it was all tested for human pathogenic fungi which has broad-spectrum antimicrobial activity (popova et al. ). helichrysum italicum essential oil was analyzed using gas chromatography and mass spectrometry to fraction into terpene and terpenoid. fifty two compounds, including hydrocarbons of the oil; α-pinene ( . %), α-cedrene ( . %) aromadendrene ( . %), β-caryophyllene ( . %), and limonene ( . %), neryl acetate ( . %), -methylcyclohexyl pentanoate ( . %), -methylcyclohexyl octanoate ( . %), and geranyl acetate ( . %) were identified (mastelic et al. ). the smallest of terpenes are monoterpenes. they contain the compound c h , come from different flowers, fruits and leaves and are known as the main component of essential oils, fragrances and many structural isomers (see footnote ). monoterpenes are also the most fragrant of all the classes of terpenes (see footnote ). examples for the types of monoterpenes found in natural scents are α-pinene, which imparts scent to pine trees, and limonene from citrus plants (see footnote ). what is thought to be one of the main purposes of monoterpenes is to attract pollinators or to serve the purpose of repelling other organisms from feeding off of plants. they also may be related to the flowering process of the plants (loreto et al. ) . they are isolated from their plant sources by distillation with steam and have a boiling points in the range of °c to °c (see footnote ). monoterpenes are purified using fractional distillation at pressures that are reduced or use another process in order to form a crystalline derivative (see footnote ). many studies test the hypothesis of high emissions of monoterpenes under high temperatures using the leaves of quercus ilex, also known as evergreen oak (table . ). the evergreen tree is native to the mediterranean area where it has to survive under hot and dry conditions and synthesis of these monoterpenes may have been an adaptive mechanism for the plants to survive under heat stress. this tree does not emit isoprenes but it emits monoterpenes and is able to handle different environmental stresses such as drought, salt, and heat (see footnote ). a particular study done by loreto et al. ( ) were conducted to visualize monoterpene production in response to high temperatures and to see if thermotolerance is increased with monoterpenes (loreto et al. ) . in this study, the leaves were exposed in °c intervals ranging from the temperatures °c to °c and leaves were kept under conditions in which inhibited or allowed monoterpenes to synthesize (loreto et al. ) . the results that were found in this experience was a discovery of seven most abundant monoterpenes which was emitted at the maximum temperature of °c and decreased its abundance over time as the temperatures increased and α-pinene had the greatest abundance of emittance at °c as well as other terpenes but greatly reduced over higher temperatures (loreto et al. ) . at °c the monoterpenes, myrcene and limonene had higher emission rates compared to temperatures around °c (loreto et al. ) . photosynthesis was also decreased when the leaves were exposed to any temperature that was higher than °c and at °c showed a loss of co and recovery occurred around °c (loreto et al. ) . overall, the monoterpenes showed that their optimal temperature for emission was around - °c (loreto et al. ) . researchers prove that the emission of monoterpenes is under enzymatic control due to their optimal temperatures (loreto et al. ) . sesquiterpenes, containing the chemical formula c h , are much larger compounds than monoterpenes and are much more stable in comparison. they are isolated by distillation with steam or by extraction and purified by methods such as vacuum fractional distillation or gas chromatography (see footnote ). oxidation or rearrangement of isoprene units that are made to sesquiterpenes produce the corresponding sesquiterpenoids (see footnote ). sesquiterpenes are naturally occurring and found in plants, fungi, and insects and act as a defensive mechanism or attract mates with pheromones in insects (see footnote ). acyclic compounds of sesquiterpenes such as farnesans can be used as a natural pesticide for insects and also as pheromones for some insects and mammals such as elephants, to attract mates or to mark their territory (see footnote ). sesquiterpenes have a vital role in plant growth hormones and signaling properties in response to its environment (giraudat ) . abscisic acid has a role in plants such as development, germination, cell division, and synthesis of protein storage and signalling (giraudat ) . it also plays a role in plants in response to various environmental stresses. it regulates the closure of the stoma by regulating ion channels and exchange of water across the plasma membrane (giraudat ) . cyclic adp-ribose signals abscisic acid in response to drought-stressing conditions from the environment (giraudat ) . abscisic acid is not unique to plants, it has shown to be present in the central nervous system of other organisms such as pigs and may play a role in humans as a pro-inflammatory cytokine and stimulator of insulin release in the human pancreas (chadwick et al. ) . gossypol is a sesquiterpene that is found in cotton plants. it has anticancer properties and can potentially inhibit fertility in male humans which is why it must be removed from essential oils and various other products before human use or consumption. avarol, a sesquiterpenoid that has shown to have antimicrobial and antifungal uses, is effective against the aids virus in humans (see footnote ). the medicinal properties of sesquiterpenes typically come from flowering plants that are included in the asteraceae family, which include, but not limited to sunflowers, marigolds, and daisies. this family of flowers is a significant resource for potent sesquiterpene lactones, which are usually found in the leaves and the flower portion of plants and are constantly being produced at high levels (chadwick et al. ) . the role of sesquiterpenes in these flowering plants are not solely made for human use but for the purpose of protecting the plant from predators and are produced de novo in response to microbial attack and ultraviolet ray protection (chadwick et al. ) . their bitter taste is a defense mechanism against herbivores from feeding on them but some have sweet tastes or tastes that are pleasant to certain organism for the purpose of spreading their seeds and being fertilized in different areas (chadwick et al. ) . sesquiterpenes have many uses in traditional, western medicine because they contain so many anticancer, antiplasmodial, and anti-inflammatory activities (chadwick et al. ) . sesquiterpenes lactones are able to reduce stomach ulcers in some people and are also present in powerful antimalarial drugs (chadwick et al. ). artemisinin, a metabolite produced from artemisia annua, which contains sesquiterpene lactone produced in the roots and shoots of the plants, is used in drugs to treat malaria (chadwick et al. ) . other uses of this family of flowers is for treatment of bacterial infections, migraines, and to improve skin (chadwick et al. ) . lettuce opium has been used for many years as a painkiller (chadwick et al. ). diterpenes are naturally occurring chemical compounds that contain the molecular formula, c h . diterpenes have physiologically active groups such as vitamin a activity well as plant growth hormones that regulate germination, flowering and switch reproductive cycles (from asexual to sexual reproduction) of plants (lee et al. ) . they can also be classified as a phytol, which is an oxygenated acyclic diterpene. over diterpenoids have been isolated from euphorbia plants, which is a very diverse genus of flowering plants (popova et al. ). diterpenes have many therapeutic benefits such as antitumor, cytotoxic, and anti-inflammatory (vasas and hohmann ) . they are present in anticancer drugs such as taxol, and the tumor promoter, phorbol (vasas and hohmann ) . tanshinones are a class of diterpenes that are isolated from dried roots or rhizomes of an herb in traditional chinese medicine called salvia miltiorrhiza also known as danshen or tanshen (zhang et al. ) . tanshinones were first isolated in the s, and since then, more than chemicals have been identified and split up into two groups: lipophilic and hydrophilic compounds (zhang et al. ) . tanshinones have recently been extensively researched for their anticancer properties in vitro and in vivo (zhang et al. ). their potential use as an anticancer drug comes from their broad range of activities such as anti-proliferation and inhibiting adhesion, migration, and invasion (zhang et al. ) . analogues of tanshinone have been synthesized in many clinical trials because they have many anticancer attributes (lee et al. ) . this herb has been used in many asian countries for preventative and therapeutic solutions to many diseases such as heart disease, vascular diseases, and arthritis (zhang et al. ) . tanshinones may also reduce inflammation and increase immune responses (zhang et al. ) . cafestol and kahweol are diterpene alcohols that are found in the oil derived from coffee beans. these chemical structures are very similar but only differ by an extra double bond that is present in kahweol's chemical structure. researchers have reported that coffee lowers the risk of depression in women, prostate cancer in men, stroke, diabetes, and some cancers (see footnote ). it is thought that the antiinflammatory and antioxidant properties of these particular diterpenes are responsible for such events (see footnote ). coffee benefits the liver as well by lowering liver enzymes that are in response to inflammation and damage and may offer some protection against liver cancer as well (see footnote ). the adverse result of these diterpenes is that they raise cholesterol level, but it seems to be limited to coffee that has been unfiltered and has oily droplets of cafestol and kahweol (see footnote ). filtered coffee may not have much impact on cholesterol levels (see footnote ). triterpenes are composed of three or six isoprene units and have the chemical formula c h which includes steroids and sterols with squalene being the biological precursor of all triterpenes (see footnote ). triterpenes are produced by animals, plants, and fungi. they play a role as precursors to steroids in animal and plant organisms, and are derived from mevalonic acid (see footnote ). saponins come from the skins of many plants and have emulsion like properties that make them excellent detergents in the human digestive system (see footnote ). chemical structures of steroid saponins are similar to hormones that are produced in the human body (see footnote ). the medicinal uses of triterpenes are not quite as recognized as other different types of terpenes but their uses are being continuously investigated by researchers. their properties have been studied for anticancer, antioxidant, antiviral, and anti-atherosclerotic activities (nazaruk and borzym-kluczyk ) . some studies have shown that there is promising potential for the use of triterpenes for people with diabetes by aiming to reduce glucose levels and also by reducing sweetness inhibitors in sweet and high calorie foods (nazaruk and borzym-kluczyk ) . saponins have detoxification properties and act as a diuretic for the kidneys and wound healing properties (nazaruk and borzym-kluczyk ) . tetraterpenes are also known as carotenoids that have the molecular formula c h and can be in the category of terpenes because they are made from isoprene units. most carotenoids are highly unsaturated and for this reason, they are extremely difficult to isolate and purify (see footnote ). they are found in all different types of fungi, bacteria, and plants and mainly responsible for red, yellow, or orange fatsoluble plant and animal pigments (see footnote ). one of the most crucial and common tetraterpene is beta-carotene that contributes to the yellow pigment in carrots. it is important to mammals especially because it is a precursor in producing vitamin a and other important terpenoids for vision (see footnote ). higher order terpenes have been shown to increase thermotolerance (singsaas ) . the permeability of the thylakoid membranes increase at higher temperatures and this happens by an increase in cyclic photophosphorylation around photosystem ii (singsaas ) . when the temperature of the atmosphere continues to rise, the photophosphorylation system is not able to keep up with protons leaking, which causes the transmembrane gradient to drop and a reduction in atp synthesis occurs (singsaas ) . all these events can potentially cause lowering in the rubisco activation state due to an inhibition of rubp regeneration (singsaas ) . the mep pathway, also known as the non-mevalonate pathway or methylerythritol phosphate pathway, is a metabolic pathway for isoprenoid biosynthesis that creates the products isopentenyl pyrophosphate (ipp) and dimethylallyl pyrophosphate (dmapp). this pathway occurs in the chloroplasts and produce monoterpenes, specific sesquiterpenes, diterpenes, and carotenoids (zhang et al. ) . the vital application of this pathway is to develop antimicrobial agents to target diseases such as malaria and sexually transmitted diseases (hunter ) . since this pathway does not occur in humans, it is a valuable resource to develop antibacterial and antiparasitic drugs (seemann et al. ). the first steps of this pathway involve pyruvate and d-glyceraldehyde -phosphate to produce doxp which is catalyzed by -deoxy-d-xylulose- -phosphate (dxs) (hunter ) . -deoxy-d-xylulose- -phosphate reductoisomerase, otherwise known as ispc, coverts doxp to mep. from mep, it reacts with ctp to create -diphosphocytidyl- c-methyl-d-erythritol (hunter ) . a phosphate is released in this reaction and then reacts with atp-dependent ispe to make -dipho sphocytidyl- c-methyl-d-erythritol -phosphate and adp and then reacts with the enzyme ispf to create c-methyl-d-erythritol , -cyclodophosphate (hunter ) . the enzyme requires metal cations. then finally, in the least understood step of the reaction, the two enzymes, ispg and isph make the two products, ipp and dmapp by using a two-electron reduction (hunter ) . the pathway is regulated by control of repression or activation of gene expression via feedback loops within the pathway or by effector molecules which target an enzyme or downstream activities (hunter ) . the mva pathway or mevalonic acid pathway occurs in the cytosol. it is responsible for the synthesis of sterols, specific sesquiterpenes, and also may play a role in the synthesis of transhinones (zhang et al. ) . in gram-positive bacteria, the genes in the metabolic pathways such as mva are organized into operons and are thought to be regulated by transcription (hunter ) . the use of cannabis is increasing for medicinal uses that commonly treat pain, the side effects of chemotherapy in cancer patients such as nausea, anxiety and depression, and its uses and benefits are continuously being researched by scientists (cathcart et al. ) . there are at least compounds that come from the cannabis plant that are regarded as cannabinoids that cause psychotropic effects in the human brain due to cb receptors (klein et al. ) . the main active ingredient, delta- tetrahydrocannabinol, otherwise known as thc, is a psychoactive agent and is a focus for controversy in society because it binds to the human endocannabinoid receptors in areas of the brain such as the hippocampus and the frontal cortex, which are responsible for memory, cognition and attention. how thc works is by taking the place of endocannabinoids, naturally occurring chemicals in the human body (see footnote ). one of the most common and well known molecules that thc replaces in the human body is called anadamide (see footnote ). to this day, scientists are researching to discover the exact role of this molecule in the human body. cannabidiol, or cbd is also a common ingredient in cannabis but compared to thc, it is a non-psychoactive and it can potentially reduce the effects of thc (klein et al. ). cbd does not bind to the same receptors as thc does in the human body and it works by inhibiting faah or the enzyme fatty acid amide hydroxyls (see footnote ). this enzyme is responsible for degrading anadamide in the body and by inhibiting faah, cbd increases natural endocannabinoids already in the human system (klein et al. ) . cbd is thus an agent that works for depression, anxiety and neuroprotective effects (klein et al. ) . what are major components in cannabis are the monoterpenes that are responsible for many different medicinal properties. one of the main uses for thc is the potential for cancer treatment and can play a role in reducing size of tumors (see footnote ). thc can also reduce inflammation caused by certain diseases in patients. other conditions that thc can help but are not limited to are adhd, arthritis, migraines, and glaucoma (see footnote ). it can also improve the symptoms in individuals that suffer from hiv by helping their appetite and thus causing weight again, improving their depression symptoms and their quality of life (lutge et al. ). terpenes have been shown to have a favorable antiplasmodial activity. with the rising malarial infections and drug resistance, terpenes have gained more attention towards it through antiplasmodial activity (nogueira and lopes ) . the interesting mechanism behind the terpene activity is that it binds to the hemin part of infected erythrocytes and kills the parasite just like the famous antimalarial drug chloroquine (orjih et al. ; kayembe et al. ) . hemin is made of iron which is necessary for the plasmodium development in the erythrocytes. though hemin breaking enzymes are not yet found in plasmodium, it could be one reason why hemin binding accounts for parasite lysis (ginsburg and demel ) . another study suggests that drug-hemin complex binds to phospholipid layers thereby disrupting the respective membrane structure and causing cell lysis (ginsburg and demel ) . moreover, it is also known that hemin can affect the carbohydrate metabolism of the parasites, which could lead to lysis of parasites (rodriguez and jungery ) . thus, terpenes can be designed to be promising drugs for malaria. different kinds of terpenes show different effects on the parasites. for instance, beta-myrcene the most common terpenes, is proven to have in vitro antiplasmodial activity (kpoviessi et al. ). beta-myrcene from cannabis sativa, the plant which is high in terpenes, does not show an anti plasmodial effect but extracts from stem, leaves, and seeds of clove basil showed a good antiplasmodial activity (small ; kpoviessi et al. ). additionally, it was also reported to have antitrypanosomal activity when tested against trypanosoma brucei brucei (habila et al. ) . this data leads to the fact that terpenes are effective against pathogenic protista. limonene regarded as the second most commonly found terpene, also possesses antiplasmodial activity against plasmodium falciparum. limonene achieves its goal by targeting the intermediates of the active isoprenoid pathway of the parasite. isoprenoid pathway plays a major role in parasite survival by mediating cell signaling, protein translation and several other biological processes (jordão et al. ) . specifically, the isoprenic products that are inhibited from being synthesized are dolichol and ubiquinone (goulart et al. ). the isoprenoid pathway of parasites is distinct from that found in mammals, which makes limonene a reliable constituent of antimalarial drug (goulart et al. ). thus, the host cell pathway will not be affected by the administration of the drug. pinene, commonly found monoterpene in pine trees is composed of two classesalpha-pinene and beta-pinene. both the classes of pinene were reported to be effective against the w strain of plasmodium falciparum, which is resistant to chloroquine (boyom et al. ) . of particular interest is the increase in antiplasmodial activity of pinene in cumin seed oil with increase in the distillation time. the study concluded that the optimal distillation time for increased antimalarial activity is - and - . min (zheljazkov et al. ) . further investigation is needed to ascertain if distillation time is just increasing the yield of pinenes in the oil or improving the bioactivity of pinenes. the next most abundant terpene, caryophyllene has the ability to both prevent and cure malaria. caryophyllene is an active component of insect repellents especially for mosquitoes and other blood-feeding diptera (maia and sarah ) . recent studies ensured that silver nanoparticles synthesized from caryophyllene are highly effective against plasmodium falciparum (kamaraj et al. ). thus, terpenes could be a safer and a cost effective alternative for malarial treatment. the emerging viral diseases have necessitated the research for new effective antiviral agents such as terpenes. as a result, scientists evaluated various terpenes for their properties, among which monoterpenes showed a good result. monoterpenes are terpene classes that possess two isoprene units. they form a major constituent of essential oils in plants which indicates monoterpenes play a major role in defense for plants (grabmann ) . a study evaluated the in vitro antiviral activity of several essential oils extracted from south american plants (duschatzky et al. ) . the oil extracts were tested against three major human viruses-herpes simplex virus- (hsv ), dengue virus type , and junin virus. the oils that were proved to be virucidal were mainly composed of monoterpenes, namely, carvone, carveol limonene, alphaand beta-pinene, caryophylene, camphor, beta-ocimene, and one sesquiterpene which is germacrene (duschatzky et al. ) . a similar study in analyzed the essential oils of seven plants from lebanon for in vitro antiviral activity (loizzo et al. ). the viruses under investigation were hsv and severe acute respiratory syndrome corona virus (sars cov). the results were positive for antiviral effects, and the major constituents were alpha-and beta-pinene, beta-ocimene, and , -cineole (loizzo et al. ) . following this, a study on salvia cedronella also had similar results which suggested , -cineole, α-pinene, caryophyllene oxide, and sabinene to be the major components of virucidal oils (alim et al. ). functional data from these studies reveal that a few monoterpenes are shared by various plants for antiviral properties (alim et al. ). these shared monoterpenes could be of importance as they are present universally. of particular interest is the single main monoterpene that is contributing to the virucidal activity. this was studied by astani et al. ( ) using eucalyptus, tea tree, and thyme essential oil extracts (astani et al. ). they suggested that monoterpene hydrocarbons have a slightly higher virulent activity compared to the monoterpene alcohols against hsv- . the monoterpenes with the highest virucidal activity were identified to be alpha-pinene and alpha-terpineol (astani et al. ). the mechanism behind the virucidal activity was suggested to be direct inactivation of free viral particles. however, the study concluded that more than isolated single monoterpenes, a mixture of monoterpenes are more effective and possessed lesser toxicity to host cells (astani et al. ). this was further bolstered by another study which evidenced the virucidal property of a combination of monoterpenes obtained from melaleuca alternifolia (zamora et al. ). the activity was tested against a human flavivirus west nile virus. the results were positive both in vivo and in vitro. the underlying mechanism was predicted to be induced cell cycle arrest at g or g phase. this indicates that a mixture of monoterpenes could act as a better antiviral agent rather than a single monoterpene (zamora et al. ) . recent studies have shown that triketone-terpene adducts also exert antiviral, antimicrobial and antitumor activity (chen et al. ). these adducts are obtained from myrtaceae as secondary metabolites in the form of sesquiterpenes called myrtucomvalones a, b, and c. the terpene adducts successfully inhibited the respiratory syncytial virus (rsv) (chen et al. ) . the bioactive terpenes present in various plants have shown various results for antiviral property. it would therefore be important to look for various plant source rather than various monoterpenes for therapeutic purposes. researchers are also focusing on synthesizing terpene hybrid from fungal sources as they are presumed to have antiviral and uv protective properties (yuan et al. ) . terpene synthesis from fungi can lead to cost effective and limited labor methods (yuan et al. ). the medicinal benefits of terpenes are not limited to pathogenic diseases. terpenes are widely acclaimed for their anticancer activity too. an early study concluded that a combination of monoterpenes, diterpenes and sesquiterpenes can be effectively used to treat cancers that occur in colon, brain, prostate gland, and bones. it also claimed that administration of terpenes in humans inhibited the growth of prostate cancer cells and sensitized the tumor in such a way it becomes susceptible to radiotherapy (see footnote ). the major advantage of this treatment was that, the drug can be administered through several routes among which oral and topical were most preferred (see footnote ). among the different kinds of terpenes, limonene is well recognized as an anticancer agent. limonene is a bioactive food component found in citrus peels, orange peels, and several other citrus fruits (jirtle et al. ) . studies have reported limonene to exhibit strong cancer inhibition activity both in vitro and in vivo. the mechanism behind limonene activity is still under investigation. a study by jirtle et al. ( ) reported that limonene acts through induction of transforming growth factor b- and mannose- -phosphate/insulin-like growth factor ii receptors (jirtle et al. ). in contrast a study by bishayee and rabi ) structural studies on limonene reported that they are lipophilic and have the tendency to be deposited in fatty tissues when administered orally. this indicates that limonene can act as an excellent chemopreventive drug for cancer as it can be deposited in the body (miller et al. ) . another study in concluded that limonene acts by suppressing the expression of breast tumor cyclin d (miller et al. ) . this lead to cell cycle arrest and mitigated proliferation of cancer cells in women with early stages of breast cancer (miller et al. ) . recent study showed that limonene from pinecones can kill lung cancer cells in vitro by apoptotic mechanism that is activated through caspase- pathway (lee et al. ) . these findings indicate a novel application of limonene towards fighting and preventing cancer. not just limonene, but also its metabolite perillyl alcohol is also said to exhibit antitumor activity in pancreatic cell lines through apoptotic mechanisms (sobral et al. ; dalessio et al. ) . apart from limonene, the terpene thymoquinone has all been widely studied for its chemoprotective and chemotherapeutic activity. thymoquinone is found to be an active constituent of the volatile oils of an annual herbaceous plant called nigella sativa (black cumin) (majdalawieh et al. ). the pathways affected by thymoquinone to exert its antitumor properties are p , pparγ, mapk, nf-κb, pi k/akt, and stat signaling pathways (majdalawieh et al. ) . thymoquinone has been proved to be anticancerous against several cancers such as breast cancer, skin cancer, non-small cell lung cancer, bile duct cancer, and brain cancer. the basic mechanisms underlying the cancer inhibition is apoptosis and cell cycle arrest (sobral et al. ; khader and eckl ) . most of the cancer related studies were performed using thermoquinone obtained from the n. sativa extracts. a study showed that thermoquinone can be obtained in larger amounts from the mint family, namely, monarda didyma and monarda media (taborsky et al. ) . thus, thermoquinone from alternative sources has to be tested for its precious potential in cancer therapy. other terpenes that have reported cytotoxic effects on cancer cells include alloocimene, camphor, beta-myrcene, pinene, alpha-and gamma-thujaplicin, terpinene, thymohydroquinone, carvone, camphene, and cymene (sobral et al. ) . terpenes being natural compounds are unlikely to affect the healthy cells or create a side effect, which attracts many researchers to exploit its capability in cancer treatment. diabetes is one of the widely prevalent diseases in the world. it is affecting both children and adults in both developing and developed nations (you and henneberg ; narayan et al. ) . the social and economic burden of diabetes continues to grow and it is expected to rise rapidly in developing countries (sarwar et al. ) . in usa, diabetes is one of the leading causes for visual impairment, limb amputation, renal diseases, heart diseases and death (saddinne et al. ) . diabetes can be of two types-type (where the immune system of the body acts against the insulin-producing organs) and type (where the insulin produced cannot be used by the body or insulin is produced in low amounts). although there are several medications available, their use is limited due to their adverse effects. some of the commonly found side-effects include low blood sugar, vomiting, nausea, diarrhea, bloating, and weight gain. this led to the research for natural products to be used as effective antidiabetic medication. phytochemicals from the medicinal plants have been recommended for treating type diabetes, of which terpene forms a major constituent (jung et al. ) . medicinal plants of oriental morocco were studied for their antidiabetic property in rats. the report showed that terpenes, terpene diols, and terpene diol glucosides form major components of the extracts of plants under study (bnouham et al. ) . a similar study on medicinal plant and their natural products that were reported from - was conducted by jung et al. . this study was focused on non-insulin-dependent diabetes mellitus (type ), and it proved that terpenes along with few other secondary metabolites such as alkaloids and flavonoids exhibit antidiabetic potential (jung et al. ) . the most promising terpene compound for treating diabetes is called andrographolide which is a diterpenoid lactone (brahmachari ) . this compound forms the major component of the leaves of the small herbaceous plant andrographis paniculata. a. paniculata is an asian plant that has already been reported to be used in traditional medicines for its therapeutic nature (brahmachari ) . the terpenoid acts by reducing the plasma glucose and increasing the utilization of glucose by the body in diabetes mellitus rats (gupta et al. ). the actual mechanism by how it does this is it activates the alpha-adrenoreceptors to increase the release of an opioid peptide beta-endomorphin (brahmachari ) which is reported to be secreted in low amounts in diabetic rats (forman et al. ) . this increased secretion in turn activates the opioid μ-receptors. these receptors can effectively curb the hepatic gluconeogenesis (glucose synthesis from non-carbohydrate precursors) and elevate the utilization of glucose by muscles. finally, this results in a reduced plasma glucose concentration (brahmachari ) . andrographolide is also observed to prevent the secondary complications of diabetes such as diabetic retinopathy, a condition that will lead to blindness (brahmachari ) . it significantly weakens the retinal angiogenesis and inflammation during the development of the disease (brahmachari ) . moreover, it can also fix the impaired or extended estrous cycle in diabetic rats (reyes et al. ) . andrographolide was orally administered in all the above studies. this indicates its efficiency for being used as a lead molecule in the future drugs designed for treating diabetes mellitus. another widely known terpene is curcumin obtained from curcuma longa which commonly called turmeric (nabavi et al. ) . it exhibits high antidiabetic property and acts by quashing the oxidative stress and inflammation. by regulating the polyol pathway, it can also reduce the plasma glucose and levels of glycosylated hemoglobin (nabavi et al. ) . moreover, curcumin is also reported to activate the enzymes present in the liver that are essential for glycolysis, gluconeogenesis, and lipid metabolism (zhang et al. ) . alike andrographolide, curcumin is also reported to reduce the complications of diabetes (nabavi et al. ) , for example, liver disorder which is a common manifestation of diabetes type (zhang et al. ) . curcumin treats these disorders by reducing the liver weight and lipid peroxidation products. further, it is also reported to normalize the levels of fetuin-a in serum that contributes to insulin resistance and fatty liver in diabetic rats (zhang et al. ) . other complications that can be attenuated by curcumin include diabetes associated-retinopathy, microangiopathy, neuropathy, and nephropathy (zhang et al. ) . these findings confirm that curcumin is likely to be used in the future for diabetes treatment. depression has become a serious health concern by contributing to the emerging mental and emotional disorders throughout the world. it is hitting both the developed and developing countries. depression can pave way to various health issues from alcoholism to heart diseases (holden ) . it is also said to increase the rate of mortality significantly in breast cancer patients (hjerl et al. ) . moreover, depression immobilizes its victims thereby leading to economic loss (holden ) . by analyzing the social and economic burdens caused by depression, researchers have stepped out towards finding novel stress-relieving drugs. synthetic drugs have serious side-effects and unintended interactions with the body that negatively affects the treatment outcome (jawaid et al. ) . hence this necessitated the need for natural drugs. terpenes serves as one of the most relevant bioactive compound for treating depression and therefore can open doors for designing natural or synthetic antidepressant drugs (bahramsoltani et al. ) . twenty-five percentage of antidepressant drugs prescribed by doctors are obtained from herbs through various extracts (saki et al. ) . to estimate the important compounds contributing to the antidepressant effect, saki et al. ( ) performed an electronic database based study. the results revealed that terpenes formed a major part of the extracts of medicinal plants that exerted antidepressant effects (saki et al. ) . thus, scientists focused on identifying the active principles of plant extracts contributing to the antistress effects. different plant had different acting compounds. among the several terpenes, linalool and beta-pinene are commonly found to be active principles (both guzmán- gutiérrez et al. ; guzmán-gutiérrez et al. ) . they were discovered from the extracts of medicinal plants litsea glaucescens and tagetes lucida and flowers of lavender (appleton ; guzmán-gutiérrez et al. ; guadarrama-cruz et al. ). these monoterpenes act by interacting with the ht a receptors of the serotonergic pathway. serotonins are important in the fact that their release and re-uptake levels can be altered to overcome stress (chaouloff ; guzmán-gutiérrez et al. ) . they also interact with adrenergic receptors of the body that play a major role in stress-induced behavioral changes (pandey et al. ; guzmán-gutiérrez et al. ) . another interesting finding is the interaction of beta-pinene with dopaminergic receptors namely d receptors. this is the mechanism followed by most of the antidepressant drugs available in the market (guzmán- gutiérrez et al. ) . a more interesting study would be to examine the beta-pinene and linalool efficiency through inhalation tests. this is because these monoterpenes are aromatic compounds that generally have an enhanced activity when inhaled as they can directly hit the central nervous system (guzmán gutiérrez et al. ) . apart from monoterpenes, sesquiterpenes also exhibit antidepressant effects. one striking example is beta-caryophyllene which was proved to ameliorate the depressive symptoms in mice (bahi et al. ) . the underlying mechanism of this compound is binding to a receptor called cb and activating it. cb is found in the brain and immune cells and plays a major role in regulating depressive-related disorders (bahi et al. ) . thus beta-caryophyllene curbs depression by acting as a cb receptor agonist (bahi et al. ) . other terpenes that have effective antidepressant properties include hyperforin which is present in the extracts of hypericum perforatum (subhan et al. ) . it has been shown that the extracts of h. perforatum differ in their antidepressant potential with the difference in concentration of hyperforin present in the extract (laakmann et al. ). similar to many other antidepressants hyperforin acts by inhibiting the neuronal uptake of mood regulators such as serotonin, dopamine and norepinephrine. in addition, it also has its own unique mechanism of controlling depression by inhibiting the neurotransmitters gaba and l-glutamate uptake (müller et al. ) . another fascinating antidepressant plant is valeriana wallichii, which is a short perennial herb. this plant not only reduces the stress and anxiety levels but also improves the symptoms of depression in humans (bhattacharyya et al. ). the major components of valeriana extracts are terpenoids called maaliol, patchouli alcohol, and -acetoxypatchouli alcohol (subhan et al. ) . the terpenoid-less extract of valeriana was found to be devoid of antidepressant activity which indicates that terpenes are the active components involved in reducing the depression (subhan et al. ). folk medicine has always been an eye-opener for designing novel drugs for diseases. to be more specific, almost three-fourths of the plant-based drugs were created based on the knowledge of folk medicine (table . ) (efferth et al. ) . realizing this fact, western worlds are now turning back into old medicines and bioactive plant components to treat modern diseases (efferth et al. (efferth et al. , . this has boosted the export rates of chinese medicinal products (based on traditional chinese medicine) from china to other developed nations. plants used in traditional chinese medicine (tcm) are being extensively studied for their secondary metabolites and their therapeutic properties (efferth et al. ). one of the active principles of tcm products is terpenes (liu and jiang ) . due to their large availability and diversity, terpenes contribute the most to industrial and medicinal applications among all the secondary metabolites of plants (zwenger and basu ) . paclitaxel is one of the most successful terpenes available in the market today (efferth et al. ) . it is made out of yew trees which is a medicinal tree used in tcm. raw material from yew contains taxol (brand name of paclitaxel) which is used in the treatment of cancers in breast, lung, ovary, pancreas, cervix, and blood (see footnote ). , two variations of this drug are used now in chemotherapyconventional paclitaxel and albumin-bound paclitaxel (see footnote ). the advantage of the latter is that concentration increases in tumor cells at a rate higher than that of the former (see footnote ). the mechanism of anticancer activity is described as disruption of microtubules in the mitotic spindle, which will lead to incomplete chromosome separation thereby causing cell death (see footnote ). in tcm and ayurveda (herbal medicinal science mainly developed in india), healers used the twigs and barks of the tree to make a special kind of tea that can be given to patients suffering from cancer. however due to the slow growing nature of yew tree, paclitaxel nowadays is produced by coalescing the products of endophytic fungus that grows under the tree and the bark of the tree , (heinig et al. one more common terpene present in the drugs used in tcm is pinene (wu et al. ) . pinene exhibits therapeutic properties such as anti-inflammatory, antiseptic, anticancer, and antibiotic properties. , the source for pinene is eucalyptus and other related coniferous trees (see footnote , sartorelli et al. ) in olden days, the juice from the bark of eucalyptus was collected and mixed in water, milk or wine to be used as a drug (see footnote ). currently, they are extracted in the form of oil and sold in the form of syrups and lozenges. as eucalyptus oil contains several monoterpenes, a study analyzed the different constituents of eucalyptus oil for its effectiveness against bacteria. here it was concluded that alpha-pinene is the best monoterpene with the highest inhibitory activity (sartorelli et al. ) . recently scientists are studying another primary terpene in eucalyptus called cineole. cineole is reported to improve the memory power, cognitive performance and attenuate the symptoms of alzheimer's disease in humans (see footnote ; moss and oliver ) . in addition, studies also showed that cineole is capable of improving the health of bronchitis patients by reducing their cough (fischer and dethlefsen ) . this is in agreement with the fact that eucalyptus oil was used as an expectorant in ayurvedic medicine. it is also known that local brazilians used the eucalyptus leaves to treat several human diseases such as cancer (mathias et al. ) . further reports also suggest that eucalyptus oil has been involved in ancient indian ayurvedic and greco-european medicine systems (see footnote ). ayurveda is a popular medicine system which originated about years ago in india. the ayurvedic medicines are based on medicinal herbs, minerals, and metals (see footnote ) along with diet regimes such as vegetarianism (caldecott ) . this system of medicine has proven to cure chronic disorders that could not be treated by western medicine (sharma et al. ) . interestingly a lot of medicinal plants used by ayurvedic practitioners owe their therapeutic property to their terpene contents. one good example is turmeric, a family of ginger which is regarded as "golden goddess" by medical practitioners (see footnote ). it has numerous therapeutic properties that includes anti-inflammatory, antioxidant, anticancer, antiseptic, antiplasmodial, astringent, digestive, diuretic, and many more (see footnote ). recently, scientists discovered that most of the turmeric's properties are laid out by the yellow-colored terpene-curcumin (kocaadam and Şanlier ) . studies are now trying to create curcumin analogues to improve the effects and activity of natural curcumin (kocaadam and Şanlier ) . another popular example is clove which was used by both ayurveda and tcm as a painkiller in dental cases. it was applied topically on cavities to relieve toothache and abdomen to treat digestive problems (alqareer et al. ) . the essential oil of clove is mostly composed of eugenol, a bioactive terpene that is responsible for clove's aroma (alqareer et al. ) . eugenol by itself is said to enhance the blood circulation in the body and improve metabolism (see footnote ). thus, based on the above data we can conclude that various terpenes have been in use even before their discoveries by modern science, due to their amazing medicinal properties. a schematic summary of different terpenes and their medicinal uses, that we discussed, is provided below in fig. . . (zetter ; keklikoglou and palma ) in vitro antimicrobial and antiviral activities of the essential oil and various extracts of salvia cedronella boiss the effect of clove and benzocaine versus placebo as topical anesthetics lavender oil for anxiety and depression comparative study on the antiviral activity of selected monoterpenes derived from essential oils β-caryophyllene, a cb receptor agonist produces multiple behavioral changes relevant to anxiety and depression in mice phytochemical constituents as future antidepressants: a comprehensive review initial exploratory observational pharmacology of valeriana wallichii on stress management: a clinical report d-limonene sensitizes docetaxel-induced cytotoxicity in human prostate cancer cells: generation of reactive oxygen species and induction of apoptosis antidiabetic effect of some medicinal plants of oriental morocco in neonatal non-insulin-dependent diabetes mellitus rats synthesis and antibacterial properties of , -dideoxyglucosides of terpene alcohols and phenols antiplasmodial volatile extracts from cleistopholis patens engler & diels and uvariastrum pierreanum engl. (engl. & diels) (annonaceae) growing in cameroon discovery and development of antidiabetic agents from natural products natural product drug discovery obtaining of carotenoid extract from lycium chinense and characterization using spectometrical analysis ayurveda: the divine science of life. mosby, maryland heights carson cf et al ( ) melaleuca alternifolia (tea tree) oil: a review of antimicrobial and other medicinal properties cannabis and cancer: reality or pipe dream? sesquiterpenoids lactones: benefits to plants and people serotonin, stress and corticoids geraniol-a review of a commercially important fragrance material myrtucomvalones a-c, three unusual triketone-sesquiterpene adducts from the leaves of myrtus communis 'variegata' skin repair properties of d-limonene and perillyl alcohol in murine models evaluation of chemical and antiviral properties of essential oils from south american plants molecular target-guided tumor therapy with natural products derived from traditional chinese medicine phytochemistry and pharmacogenomics of natural products derived from traditional chinese medicine and chinese materia medica with activity against tumor cells antibacterial and antifungal activity of juniper berry oil and its selected components efficacy of cineole in patients suffering from acute bronchitis: a placebo-controlled double-blind trial diabetes induced by streptozocin results in a decrease in immunoreactive beta-endorphin levels in the pituitary and hypothalamus of female rats terpene based pesticide treatments for killing terrestrial arthropods including, amongst others, lice, lice eggs, mites and ants addictive drugs and their relationship with infectious diseases the function of terpene natural products in the natural world interactions of hemin, antimalarial drugs and hemin-antimalarial complexes with phospholipid monolayers abscisic acid signaling terpenes arrest parasite development and inhibit biosynthesis of isoprenoids in plasmodium falciparum terpenoids as plant antioxidants. plant hormon, vitamin hormon antidepressant-like effects of tagetes lucida cav. in the forced swimming test an overview of indian novel traditional medicinal plants with anti-diabetic potentials medicinal plants for the treatment of "nervios", anxiety, and depression in mexican traditional medicine antidepressant activity of litsea glaucescens essential oil: identification of β-pinene and linalool as active principles linalool and β-pinene exert their antidepressant-like activity through the monoaminergic pathway evaluation of in vitro activity of essential oils against trypanosoma brucei brucei and trypanosoma evansi getting to the bottom of taxol biosynthesis by fungi antimicrobial terpenes from oleoresin of ponderosa pine tree pinus ponderosa: a defense mechanism against microbial invasion depression as a prognostic factor for breast cancer mortality mental health: global survey examines impact of depression the non-mevalonate pathway of isoprenoid precursor biosynthesis pentacyclic triterpenoids from the medicinal herb, centella asiatica (l.) urban a review on herbal plants showing antidepressant activity increased mannose -phosphate/insulin-like growth factor ii receptor and transforming growth factor beta levels during monoterpene induced regression of mammary tumors isoprenoid biosynthesis in the erythrocytic stages of plasmodium falciparum antidiabetic agents from medicinal plants ag nanoparticles synthesized using β-caryophyllene isolated from murraya koenigii: antimalarial (plasmodium falciparum d ) and anticancer activity (a and hela cell lines) in vitro antimalarial activity of terpenes isolated from ocimum gratissimum and cassia alata leaves. screening of their binding affinity with haemin metastasis risk after anti-macrophage therapy thymoquinone: an emerging natural drug with a wide range of medical applications cannabidiol potentiates Δ -tetrahydrocannabinol (thc) behavioural effects and alters thc pharmacokinetics during acute and chronic treatment in adolescent rats curcumin, an active component of turmeric (curcuma longa), and its effects on health a review on therapeutic uses of ocimum sanctum linn (tulsi) with its pharmacological actions in vitro antitrypanosomal and antiplasmodial activities of crude extracts and essential oils of ocimum gratissimum linn from benin and influence of vegetative stage johns wort in mild to moderate depression: the relevance of hyperforin for the clinical efficacy identification of plant compounds that disrupt the insect juvenile hormone receptor complex pinecone of pinus koraiensis inducing apoptosis in human lung cancer cells by activating caspase- and its chemical constituents in: dietary chinese herbs: chemistry, pharmacology and clinical evidence phytochemical analysis and in vitro antiviral activities of the essential oils of seven lebanon species salvia (sage): a review of its potential cognitive-enhancing and protective effects on the monoterpene emission under heat stress and on the increased thermotolerance of leaves of quercus ilex l. fumigated with selected monoterpenes the medical use of cannabis for reducing morbidity and mortality in patients with hiv/aids plant-based insect repellents: a review of their efficacy, development and testing anti-cancer properties and mechanisms of action of thymoquinone, the major active ingredient of nigella sativa composition and antimicrobial activity of helichrysum italicum essential oil and its terpene and terpenoid fractions in vitro cytotoxic potential of essential oils of eucalyptus benthamii and its related terpenes on tumor cell lines compositions comprising citrus flavonoids and quaternary ammonium salts for treating head lice antifungal and antimicrobial activity of beta-ionone and vitamin a derivative limonene: a bioactive food component from citrus and evidence for a potential role in breast cancer prevention and treatment human breast tissue disposition and bioactivity of limonene in women with early-stage breast cancer plasma , -cineole correlates with cognitive performance following exposure to rosemary essential oil aroma hyperforin-antidepressant activity by a novel mechanism of action curcumin: a natural product for diabetes and its complications diabetes-a common, growing, serious, costly, and potentially preventable public health problem the role of triterpenes in the management of diabetes mellitus and its complications hemin lyses malaria parasites ) β-adrenergic receptor subtypes in stress-induced behavioral depression in-vitro inhibition of human erythrocyte acetylcholinesterase by salvia lavandulaefolia essential oil and constituent terpenes terpenes with antimicrobial activity from cretan propolis anti-diabetic potentials of momordica charantia and andrographis paniculata and their effects on estrous cyclicity of alloxan-induced diabetic rats a protein on plasmodium falciparum-infected erythrocytes functions as a transferrin receptor prevalence of self-rated visual impairment among adults with diabetes-united states the effect of most important medicinal plants on two importnt psychiatric disorders (anxiety and depression)-a review chemical composition and antimicrobial activity of the essential oils from two species of eucalyptus diabetes mellitus, fasting blood glucose concentration, and risk of vascular disease: a collaborative meta-analysis of prospective studies. emerging risk factors collaboration isoprenoid biosynthesis via the mep pathway: isoprenoid biosynthesis via the mep pathway in-vivo moessbauer spectroscopy identifies a [ fe- s] + center with unusual coordination sphere in the lytb protein utilization of ayurveda in health care: an approach for prevention, health promotion, and treatment of disease. part -ayurveda in primary health care antifungal activity of the lemongrass oil and citral against candida spp antitumor activity of monoterpenes found in essential oils terpenoid content of valeriana wallichii extracts and antidepressant-like response profiles identification of potential sources of thymoquinone and related compounds in asteraceae, cupressaceae, lamiaceae, and ranunculaceae families euphorbia diterpenes: isolation, structure, biological activity, and synthesis volatility-dependent d ir correlation analysis of traditional chinese medicine 'red flower oil' preparation from different manufacturers enhancing production of bio-isoprene using hybrid mva pathway and isoprene synthase in e. coli type diabetes prevalence increasing globally and regionally: the role of natural selection and life expectancy at birth polyketide-terpene hybrid metabolites from an endolichenic fungus pestalotiopsis sp the in vitro and in vivo antiviral properties of combined monoterpene alcohols against west nile virus infection the scientific contributions of m. judah folkman to cancer research novel norsesquiterpenoids from the roots of phyllanthus emblica tanshinones: sources, pharmacokinetics and anti-cancer activities curcumin and diabetes: a systematic review distillation time as tool for improved antimalarial activity and differential oil composition of cumin seed oil plant terpenoids: applications and potentials holy basil/ tulsi eugenol, β-elemene, β-caryophyllene and germacrenerestores functions of nervous system increases fertiltity used to treat asthma and cold key: cord- - yyv vuy authors: rybicki, ed title: history and promise of plant-made vaccines for animals date: - - journal: prospects of plant-based vaccines in veterinary medicine doi: . / - - - - _ sha: doc_id: cord_uid: yyv vuy plant-made vaccines are now a well-established and well-tested concept in veterinary medicine—yet the only product so far licenced was never produced commercially. this is puzzling, given the breadth of exploration of plant-made animal vaccines, and their immunogenicity and efficacy, over more than twenty years of research. the range of candidate vaccines that have been tested in laboratory animal models includes vaccines for e. coli, salmonella, yersinia pestis, foot and mouth disease virus, rabbit haemorrhagic disease virus, rabbit and canine and bovine papillomaviruses, mink enteritis and porcine circovirus, and lately also bluetongue virus, among many others. there are many proofs of efficacy of such vaccines, and regulatory pathways appear to have been explored for their licencing. this review will briefly explore the history of plant-made vaccines for use in animals, and will discuss the unique advantages of plant-made vaccines for use in a veterinary medicine setting in detail, with a proposal of their relevance within the “one health” paradigm. plant-produced vaccines for veterinary medicine are an exciting prospect, largely because of the possibilities of producing protein-based vaccines' including edible vaccines' at low cost, at almost any scale, and potentially locally and on demand. they have also been controversial because of the very real possibilities of contamination of the human food supply with vaccine-producing transgenic plants, and because of concerns around the possibility of immunological tolerance developing to oral or edible vaccines. however, one set of problems that many foresawregulatory and production problems-has not eventuated, and in fact the environment now seems primed very favourably for their introduction. the main justifications for plant-made vaccines are that vaccine antigen production in plants is safe; that it is both cheap and highly scalable; that plants produce and process eukaryote-derived proteins much better than can bacteria or even yeasts; that use of plants would allow for production of vaccines in the developing world where they are needed most; and of vaccines or therapeutics that will never be produced economically by other technologies. however, despite more than twenty years of development, there are still no plant-produced vaccines or biologics available for animals-although there are in fact products licenced for and in use in humans. this review will explore the early history of plant-produced vaccines with an emphasis on proofs of principle and of efficacy, what the recent development of robust, stable transient plant production systems for vaccine antigens could mean for veterinary medicine, and the potential of plant-produced vaccines to advance both animal and potentially human health' under the banner of the one health movement. while viral proteins have probably been the most common vaccine candidates made in plants (reviewed in rybicki ) , it was expression of a bacterial protein-escherichia coli heat labile enterotoxin (lt-b)-that first proved that veterinary-relevant antigens could be produced in plants, and provided the first proof of principle for edible vaccines. lt-b produced in transgenic tobacco or potatoes (haq et al. ) was functionally equivalent to e coli-produced protein in specific assays, and immunisation of mice by oral gavage with plant material elicited systemic and mucosal toxin neutralising antibodies. moreover, fresh potato containing lt-b was immunogenic in mice when eaten. an early virus vaccine candidate was one against mink enteritis virus (mev) disease: this was novel in that it comprised chimaeric cowpea mosaic virus (cpmv) virions incorporating a short linear epitope from mev vp capsid protein and displaying it on the surface of virions, produced by inoculation of bean plants with an infectious cdna clone of rcpmv (dalsgaard et al. ) . this conferred protection against clinical disease and virtually abolished virus shedding-and given that the epitope sequence used is found in mev, canine parvovirus, and feline panleukopenia virus, the same vaccine could potentially also protect against these viruses. another early virus vaccine candidate was against rabbit haemorrhagic disease virus (rhdv): this was made by expressing the whole rhdv vp capsid protein in transgenic potatoes; parenteral immunisation with plant extracts was protective in rabbits (castanon et al. ) . subsequently, another study demonstrated that an edible vaccine consisting of leaves of transgenic plants containing presumably partially-assembled vp subunits, was an effective priming vaccine for later baculovirus-derived parenterally-delivered vaccine (gil et al. ). the first report of a foot and mouth disease virus (fmdv) plant-made antigen was of expression in plant protoplasts of a vp -derived peptide of fmdv as an insertion into the minor coat protein of a replicating cpmv as a demonstration of antigen presentation (usha et al. ) . however, the first proof of efficacy was done using transgenic arabidopsis thaliana expressing whole vp : parenteral immunisation of mice with leaf extracts elicited antibodies that bound to vp and to intact fmdv particles, and all immunised mice were protected against virulent fmdv challenge (carrillo et al. ) . the wigdorovitz group went on to demonstrate that mice could be protected against fmdv challenge after oral or parenteral vaccination with extracts of transgenic alfalfa expressing vp (wigdorovitz et al. ) , or immunisation with leaf extracts of tobacco plants expressing vp via a recombinant tobacco mosaic virus vector (wigdorovitz et al. ) . a refinement of these achievements included transgenic expression in alfalfa of amino acid residues - of vp (vp - ) fused to glucuronidase (gus), which both allowed selection of strongest expressers by assay of enzyme activity, and was protective in mice (dus santos et al. ) . another novel application of carrier technology was the insertion of vp amino acids - (g-h loop) in an interior region of the hepatitis b virus core antigen gene (hbcag), and expression of the chimaera in transgenic nicotiana tabacum. the chimaeric protein formed virus-like particles (vlps) in the tobacco leaves, and mice immunised intraperitoneally with a soluble extract were protected against viral challenge (huang et al. ). an early attempt at showing the feasibility of making an anthrax vaccine was the expression in transgenic n. tabacum of the protective antigen (pa) protein of bacillus anthracis, possibly the best target for a subunit vaccine because it alone is protective (aziz et al. ) , although it went no further than showing cytolytic activity of the protein. soon after, the same group went on to express pa in transplastomic n. tabacum, with significant yield increases but still no efficacy trial (aziz et al. ) . another investigation of transplastomic tobacco by henry daniell's group was more thorough: yields were high ( . g/kg in fresh leaf tissue), the protein was protected in chloroplasts from protease cleavage and was stable when stored in leaves or as crude extracts, and was biologically active (watson et al. ). while they did not show immunogenicity or protection, the authors speculated that "with an average yield of mg of pa per plant using an experimental transgenic cultivar grown in a greenhouse, million doses of vaccine (free of contaminants) could be produced per acre". the daniell group subsequently showed that chloroplast-derived pa was equal in potency to the natural product from b anthracis, and that mice immunised subcutaneously with partially purified chloroplast-derived pa with adjuvant produced high igg titres and survived challenge with lethal doses of toxin (koya et al. ) . a different sort of approach to anthrax, and one of the first attempts at making a therapeutic antibody in plants, was taken by vidadi yusibov's group, who used the technique of transient agrobacterium infiltration-mediated expression in n. benthamiana to produce a human-derived pa-specific monoclonal antibody (hull et al. ) . the antibody neutralised toxin activity both in vitro and in vivo at a comparable level to hybridoma-produced antibodies. the yusibov group at what became fraunhofer usa center for molecular biotechnology later used the same transient expression technology to separately express artificial antigens comprising domain of pa or domain of b anthracis lethal factor (lf), fused in-frame with lichenase (lickm), a thermostable enzyme from clostridium thermocellum (chichester et al. ) . mice immunised with a combination of the two antigens produced high titres of mainly igg , and sera could neutralise the effects of anthrax lethal toxin (letx) in vitro. rabies vaccines made in plants included an early yet highly sophisticated candidate that was composed of the alfalfa mosaic virus (amv) cp fused to an artificial polypeptide containing rabies virus g protein amino acids - , and n protein amino acids - , and expressed either in n. tabacum plants transgenic for amv replicase, or via rtmv in either n. benthamiana or spinach (yusibov et al. ) . the plants made particles containing amv-derived rna, encapsidated with chimaeric cp: raw spinach leaves were orally immunogenic in mice and in human volunteers. a simpler candidate was the g protein alone, with plant signal peptide and er retention signal, made in transgenic n. tabacum (ashraf et al. ) . while yields were relatively low ( . % of total soluble leaf protein), purified protein injected peritoneally in mice elicited protective immunity against lethal intracerebral challenge with live rabies virus-an excellent proof of both principle and efficacy. plant-made animal rotavirus vaccines were an early target, with a stand-out study by yu and langridge ( ) providing evidence that transgenic potato could produce fusion proteins consisting of cholera toxin (ct) b and a subunits fused with murine rotavirus enterotoxin and enterotoxigenic e coli fimbrial antigen, respectively. fusion antigens assembled in potato tubers into cholera holotoxin-like structures that bound enterocytes, and elicited serum and intestinal antibodies after oral immunisation in mice. moreover, passively immunised mouse neonates were partially protected against diarrhoea after rotavirus challenge, demonstrating that combination vaccines for viral and bacterial pathogens may be made in plants. a simpler approach to rotavirus prevention was expression of a his-tagged vp * fragment of bovine rotavirus (brv) vp in n. benthamiana via recombinant tmv, purification of the antigen by ni + chromatography, and intraperitoneal immunisation of adult female mice ). eighty-five percent of suckling mice born from these mothers were protected from brv challenge, compared to % immunised with an irrelevant control antigen. the same group also showed that a fusion protein made in transgenic alfalfa consisting of a short peptide derived from brv vp fused to gus was immunogenic both when given intraperitoneally and orally to adult female mice, and their sucklings were protected against challenge ). another group used transgenic alfalfa to produce human rotavirus vp , and showed that female mice gavaged with alfalfa extract containing oligocpg as an adjuvant developed high titres of antibodies both systemically and mucosally, and their pups were partially protected against simian rotavirus challenge. the same animal model first used to show the efficacy of insect cell-made papillomavirus virus-like particle (vlp)-based vaccines (breitburd et al. ) was also used to demonstrate the efficacy of two very different plant-made papillomavirus vaccines, a few years after the demonstration that human papillomavirus l major capsid protein virus-like particles could be produced in transgenic tobacco or potato (biemelt et al. ; varsani et al. ; warzecha et al. ) . cottontail rabbit papillomavirus (crpv), the cause of the famous "jackalope" sightings in the usa, provides an excellent model system in domestic rabbits for investigation of prophylactic and therapeutic papillomavirus vaccines (breitburd et al. ) . accordingly, in the first study crpv l major capsid protein-containing extracts were prepared either from transgenic n. tabacum or n. benthamiana infected with recombinant tmv, and used with freund's incomplete adjuvant to immunise rabbits that were subsequently challenged with live virus (kohl et al. ) . although the vaccines appeared to contain small aggregates of crpv l rather than intact vlps, and immune rabbit sera failed to neutralise crpv infectivity in an in vitro assay, the rabbits were protected from wart development (kohl et al. ). in the second study, infectious recombinant tmv was used to surface display, via fusion to the capsid protein, a peptide consisting of amino acids - of the l minor capsid protein from either crpv or the rabbit oral papillomavirus (ropv) (palmer et al. ) . groups of rabbits received either or both vaccines, and were challenged with live crpv or ropv. immune rabbit sera reacted with whole l protein, and crpv-specific sera neutralised crpv pseudovirion infectivity. rabbits receiving the crpv or crpv + ropv vaccines were completely protected against crpv infection, and those receiving ropv alone were weakly protected against crpv. these studies demonstrated for the first time that plant-made papillomavirus vaccines based on l protein or l -derived peptide had real potential as prophylactic vaccines, for use in animals as well as in humans. strangely, given that bovine papillomaviruses (bpv) had been used for many years as model systems for anti-wart vaccination, it was not until , with transient agroinfiltration-mediated expression of bpv- vlps in n. benthamiana, that a candidate plant-made bpv l vlp-based vaccine was successfully made, although no efficacy trials were done (love et al. ) . expression of animal vaccine components in seeds of transgenic plants was attempted quite early on, with lamphear et al. ( ) in reviewing their own earlier work on maize seed expression of the b subunit of e coli heat-labile enterotoxin and the tgev s protein, with data on the potency, efficacy, and stability of these vaccines. another report followed in on the expression in maize seed of the s envelope protein of transmissible gastroenteritis coronavirus (tgev) of swine, and its protective efficacy in piglets fed with the seed (jilka ) . this followed an earlier demonstration of oral immunogenicity of the s protein n-terminal domain in transgenic potato tubers (gomez et al. ) . rabies too was a target for maize seed expression, with a report of g protein expression in transgenic maize seed to % of total soluble protein, and complete protection in a heterologous rabies strain challenge of mice orally immunized with one dose of * lg of g protein in seed extract (loza-rubio et al. ) . the same group later showed that sheep orally given a single dose of the transgenic maize seed containing * mg of the g protein were protected to the same extent as those immunized with a commercial inactivated vaccine ). the authors claimed that "this is the first study in which an orally administered edible vaccine showed efficacy in a polygastric model", which was an important development. maize was a popular target for both production and storage of recombinant proteins in early molecular farming times (see streatfield et al. ) ; however, other hosts were used too. for instance, the haemagglutinin (h) protein of rinderpest virus was expressed in transgenic pigeon pea to . % of total soluble protein (satyavathi et al. ) , and also in peanuts for a product that was both orally and parenterally immunogenic in mice (khandelwal et al. ); so too was glycoprotein b (gb) of human cytomegalovirus in seeds of transgenic tobacco (tackaberry et al. ) , the fusion (f) glycoprotein of newcastle disease virus in transgenic rice seed (yang et al. ) , and the serotype-specific vp protein of bluetongue virus in transgenic peanuts (athmaram et al. ) . most of these efforts were negated, however, by the one big scandal to have hit molecular farming as far as the use of food plants for vaccine production is concerned. in , aphis inspectors found volunteer tgev cp-expressing maize growing in soybean fields in two locations that were used to grow prodigene inc's tgev transgenic maize in the previous season (aphis )-and in one, the soybeans were harvested with the maize plants still standing and sent to a storage facility, where they were mixed with a large volume of other seeds. the company was fined and paid substantial cleanup costs, had to develop a new compliance implementation programme, and the us dept of agriculture issued new guidelines for trials of such products. this had an unfortunate knock-on effect for molecular farming, in that it resulted in an effectively voluntary moratorium on the use of food crops for recombinant protein production worldwide. the one major success story of early work on veterinary vaccines was the approval by the us department of agriculture's center for veterinary biologics of dow agrosciences' injectable newcastle disease virus (ndv) haemagglutin-based vaccine for poultry, that had been made in a suspension cultured n. tabacum cell line. sadly, the product was never sold: the company only wanted '… to demonstrate that our concert™ plant-cell-produced system is capable of producing a vaccine that is safe and effective and to demonstrate that it meets the requirements for approval under the rigorous usda regulatory system. ndv is well known and understood by the regulatory agency, so it served as an excellent model to prove this new technology' (rybicki ). the early historical account of molecular farming for veterinary vaccines given above gives an idea of the array of technologies available and used up to the mid- s: transgenic and transplastomic expression of subunit proteins; recombinant plant viruses either used to express whole vaccine candidate genes, or to display chosen peptides fused to their capsid proteins; fusion of vaccine protein genes to carrier proteins to improve immunogenicity, including by inherent adjuvant properties; candidate parenteral and oral vaccines to both viruses and bacteria; therapeutics for animals made in plants; use of plant cell cultures to make antigens. many proofs of principle were obtained, for candidate vaccines against a wide range of viral and bacterial disease agents; and proofs of efficacy for vaccines delivered orally or parenterally, in whole plant material or as extracts. while all of these aspects are still currently used in molecular farming, developments that have revolutionised the field were first, the widespread adoption of agrobacterium-mediated transient expression (agroinfiltration) of recombinant proteins; and second, the use of "deconstructed" plant virus-derived vectors delivered via agrobacterium to amplify expression (reviewed in rybicki ). these innovations enabled the advent of high-throughput testing of expression constructs, coupled with very rapid and generally higher yield production of vaccine antigens once optimal construct design had been determined. for example, our group investigated, via agroinfiltration techniques, three different codon usage schemes and three different intracellular localization strategies for optimization of human papillomavirus type l protein expression in n. benthamiana, in one large experiment over only days (maclean et al. ) . use of deconstructed tmv-based vectors delivered by agrobacterium routinely has allowed significant increases of antigen yield, up to grams per kilogram fresh tissue weight (gleba et al. ; klimyuk et al. ). the so-called tmv-based "launch vectors" of fraunhofer usa have also allowed significant yield increases and rapid production of antigens (chichester et al. ; shamloul et al. ) . improved non-replicating hyper-translational (ht) expression vectors derived from cowpea mosaic virus rna have also allowed significantly higher yields via agroinfiltration (sainsbury et al. (sainsbury et al. , and the possibility of multiple genes from the same vector (saxena et al. ) ; so too has the use of a ssdna geminivirus-derived set of vectors by different groups (huang et al. ; regnard et al. ) , and other ssdna plant (or other host) virus-derived vectors (rybicki and martin ) . the number of peptide display vectors/chimaeric protein fusion partners has multiplied: while self-replicating rtmv was once state of the art, now one may choose between tmv-and potato virus x (pvx)-based vectors (lico et al. ) , cucumber mosaic virus (cmv) cp (nemchinov and natilla ; zhao and hammond ) , bamboo mosaic virus (yang et al. ), pvx-vectored alternanthera mosaic virus (altmv) cp gene (tyulkina et al. ) , lichenase (lickm), cholera toxin b subunit (ctb), amv cp, and gus, as mentioned earlier. plant virus virions in particular are now seen as easily-made nanoparticles suitable for a number of vaccine-relevant purposes (steele et al. ) , including as selfadjuvanting peptide-based vaccine display vehicles (lebel et al. ; leclerc ) , and excellent inducers of cross-presentation by mhc receptors (hanafi et al. ) . the use of tags or small peptide fusion partners is now also considerably more sophisticated, with a variety of specialized tags to choose from. these include the now-ubiquitous xhis tag, used for ni + or other immobilised metal affinity chromatography (imac) protein purification technique; a new "cysta-tag" for the same purpose ; the n-terminal proline-rich domain of maize seed gamma zein (zera) that induces the formation of er-located protein bodies (torrent et al. ); elastin-like polypeptides (elps) with repeating pentapeptide 'vpgxg' sequences, or hydrophobins-small fungal proteins which alter the hydrophobicity of the fusion partner-both of which also form protein bodies (conley et al. ) . as examples, our group has recently successfully used elp fusion to the cp of beak and feather disease virus (bfdv) of parrots to aid in both accumulation and purification of the protein as a candidate vaccine (duvenage et al. ) . we have also used the zera tag as a protein body display vehicle for an ectopic m e moiety common to all influenzavirus a types, which could serve as a universal vaccine for these viruses (mbewana et al. ) . another potentially veterinary use of zera was in the enhancement of yersinia pestis f -v antigen fusion protein accumulation: this was * Â higher than f -v alone in three different host plant systems-namely, n. benthamiana, alfalfa and n. tabacum nt suspension-cultured cells (alvarez et al. ) . the expression vehicles themselves have also been subject to engineering: it is now possible to precisely control glycosylation of plant-made proteins. this can be done by knock-out modification via rna interference (rnai) technology of the plant glycosyltransferases beta , -xylosyltransferase (xylt) and core alpha , -fucosyltransferase (fuct). these enzymes are responsible for the transfer of beta , -linked xylose and core alpha , -linked fucose residues to glycoprotein n-glycans, which are plant-specific modifications not found in mammalian glycoproteins (strasser et al. ) . it is also possible to use transient co-expression technologies to modify glycosylation (castilho and steinkellner ) , as well as to achieve almost completely native sialylated recombinant proteins by expression of whole mammalian glycosylation pathways in plants (castilho et al. ; steinkellner and castilho ) . it is possible to abolish n-glycosylation entirely, by co-expression of bacterial pngase f (mamedov and yusibov ) . one can also control endogenous plant proteases that may limit recombinant protein accumulation: for example, transient co-expression of secreted a /s protease inhibitor tomato cathepsin d inhibitor (slcdi) significantly lowered a and s protease activities in the n. benthamiana apoplast, while increasing recombinant protein content by * % (goulet et al. ) . it was found that co-expression of tomato cystatin slcys , which inhibits c a proteases, increased the transient expression yield of a monoclonal antibody in n. benthamiana by nearly % (robert et al. ) . it is also possible to reduce protease activity in cell suspension cultures by expression of specific antisense rnas, resulting in significantly increased accumulation of recombinant antibodies (mandal et al. ) . while suspension-cultured plant cells have been used for many years for molecular farming-and in fact were used for the only usda-licenced plant-produced animal vaccine, against ndv-new developments have made them an even more attractive prospect for low-cost vaccine production. use of flow cytometry with cell sorting, formerly the province of mammalian cell culture work only, has allowed high-expressing mab-producing tobacco by- cell lines from a heterogeneous population of cells by selecting the co-expressed fluorescent marker protein dsred (kirchhoff et al. ). however, one of the most exciting recent developments with this technology is the advent of the "cell pack": this is a technique for getting highly efficient (up to %) agrobacterium-mediated transient transformation of suspension-cultured cells that have been captured by suction onto a filter (rademacher ) . cell packs can be tiny (eppendorf tube tips) or large (e.g.: centimetres deep in a cm buchner funnel); protein expression occurs in immobilised cells in the presence of minimal liquid media, and can continue for days (https://tinyurl.com/k da q). the technology is ideal for rapid and high-throughput screening of expression-and the possibility exists for taking cells back into culture and selecting for permanent transfection. these are important developments, because of the acceptability of the products of plant cell cultures for production of biologics to regulatory bodies (see below). another production host highly suited to industrial-scale production is microalgae: they are easier to establish and use than plant cell cultures, and share all the same advantages of scalability, contained growth, and consistent transgene expression levels (specht and mayfield ) . a very important development for molecular farming has been the development of protocols for increasing yields and implementing industrial-scale production and downstream processing of vaccines and biologics, without which no large-scale trials could take place, or routine manufacturing occur. a useful development was use of a transgenic n. tabacum/n. glauca hybrid that does not synthesize alkaloids, is highly vigorous, can easily be propagated by vegetative cuttings and does not produce viable pollen, which greatly aids biocontainment (ling et al. ). the application of techniques more familiar to chemical engineers is also advantageous: for example, it proved possible, by sequential use of fractional factorial designs and response surface methodology, to optimize culture media for mab production in transgenic tobacco by- cells, and to increase mab yields up to -fold after days of culture compared to use of standard media (vasilev et al. ). the fraunhofer ime group have described generic chromatography-based strategies focusing on the binding behaviours of host cell proteins to chromatography resins under varying conditions of ph and conductivity (buyel and fischer ) . another useful technique from that group is a comprehensive description of the use of heat treatment of either intact leaves or of plant extracts to facilitate the industrial-scale removal of host cell proteins, optimised by a design-of-experiments approach that will also be familiar to engineers (buyel et al. ). many of these and other strategies used to optimise yields in molecular farming are reviewed here (twyman et al. ) . the establishment by various companies and institutes of facilities suitable for manufacture of animal and clinical trial material is also a very welcome development. as examples, the long-established kentucky bioprocessing inc (kbp) is a contract manufacturer capable of production from transgenic plants or transiently transfected plants, using the u.s. food and drug administration's current good manufacturing practices (cgmp) for pharmaceuticals, at scales up to thousands of kilograms of plants per week (https://www.kentuckybioprocessing.com/). they have recently produced and stockpiled mabs against ebolaviruses. another contract manufacturing firm with large production capacity is ibio inc: like kbp, they have a wide range of patents on their proprietary gene expression technology (holtz et al. ) . they are also partnering with a range of agencies and companies, including with the brazilian oswaldo cruz foundation for plant-made yellow fever vaccine, and the us dept of defense and the bill & melinda gates foundation for influenza vaccines (http://www.ibioinc.com/). the fraunhofer usa center for molecular biotechnology (http://www.fhcmb. org/) is a not-for-profit research and development organisation, that offers "… plant-based protein production, purification, scale-up and gmp manufacturing to support the development of vaccines, therapeutics and diagnostics", also with proprietary expression platforms, and can take products right through to fill and finish. the fraunhofer ime in aachen also has a state-of-the-art mechanised plant production facility still under construction as of . the regulatory environment has changed for the better, even though it was not in truth as inimical as first supposed: this was borne out by the fact that as early as , the cuban regulatory agencies and the usda had approved plant-made mabs for the purification of an already-licenced yeast-made hepatitis b vaccine, and the tobacco cell-made ndv vaccines, respectively (rybicki ). as another early example, the fraunhofer ime molecular farming group published in that use of whole plants for biologics production lacks intrinsic benefits of cell culture techniques, such as precise control over growth conditions, batch-to-batch product consistency, sterile containment, and it being much harder to be in compliance with good manufacturing practice (gmp) (hellwig et al. ). they pointed out that plant cell suspension cultures have all the merits of microbial and animal cell cultures, have an established track record for secondary metabolite production, and are far cheaper to use. these justifications notwithstanding, the same group later noted, in a review on gmp issues for plant-made proteins in whole plants, that: "when [plant-derived] recombinant proteins are intended for medical use… they fall under the same regulatory guidelines for manufacturing that cover drugs from all other sources, and when such proteins enter clinical development this includes the requirement for production according to [gmp] . in principle, the well-characterized gmp regulations that apply to pharmaceutical proteins produced in bacteria and mammalian cells are directly transferrable to plants" . they subsequently were able to get gmp manufacturing authorisation from german authorities for making mabs from transgenic n. tabacum for a phase i clinical trial (ma et al. ) . other entities have also scaled and regularised production to allow production of materials for animal and clinical trial-and one of the most successful has been medicago inc., who presently has routine large-scale production of influenzavirus a haemagglutinin (ha)-based vlps for use in advanced human clinical trial (d'aoust et al. ) . in , medicago inc. succeeded in manufacturing million doses of an h n vlp-based influenza vaccine candidate in one month, by phase -appropriate cgmp, as part of the us defense advanced research projects agency (darpa)-funded challenge (darpa ) . a group in japan has also recently developed a gmp-compliant production process for a transgenic rice seed-based cholera vaccine-mucorice-ctb-which is simply polished, powdered seed, now in clinical trial (kashima et al. ) . as evidence of the increasing maturity of veterinary molecular farming, one of the editors of this book has co-authored a recent article on regulatory and commercial hurdles hampering the advance to market of plant-produced veterinary vaccines, covering developing business plans, assessing market opportunities, manufacturing scale-up, financing, protecting and using intellectual property, and regulatory approval (macdonald et al. ) . at first sight, molecular farming appears the ideal way to make recombinant protein-based veterinary vaccines: production of active ingredients is markedly cheaper per unit mass than by use of any animal tissue-culture system, and generally cheaper than yeast or bacterial culture (rybicki ); partially-purified or unprocessed extracts are highly unlikely to contain any animal pathogens; edible and oral vaccines appear highly feasible; the financial barrier to entry for manufacture appears far lower than for conventionally-made vaccines. it is possible to efficiently make bacterial proteins using bacterial-derived translational machinery in chloroplasts in transplastomic plants, as well as to make other proteins at very high yield; conventional transgenics have been used to make many vaccine candidates, with many proofs of efficacy; transient expression technologies have revolutionised the field in terms of providing high yields and very rapid development times from concept to product. and yet, only one product-dow's ndv vaccine-is registered for use, and that is not sold. it is possible that heavy investment by big industry players in conventional manufacturing technologies has stalled their uptake of molecular farming technology for veterinary vaccines and biologics: this has certainly been true for human biologics. however, perhaps developments from the human field could be used as a spur for uptake of veterinary vaccines and biologics: an example here is the licencing of protalix biotherapeutics' elelyso ® or glucocerebrosidase, a therapeutic for a genetic mitochondrial enzyme deficit called gaucher disease, made using transgenic carrot cell lines in litre plastic bag fermenters (http://protalix.com/ about/elelyso/). a contamination of genzyme's mammalian cell production facilities in with a mammalian calicivirus led to the fda allowing protalix to supply the drug to patients who needed it, and to accelerated licensure (bethencourt ). the company has also successfully tested oral administration of drugs in plant cells, which would be a highly welcome development: they claim that "oral delivery of protein therapies [is] possible due to the unique cellulose wall of plant cells that makes them resistant to degradation when passing through the digestive tract" (protalix ). another apposite example was the fortuitous availability of a plant-made anti-zaire ebolavirus mab cocktail known as zmapp™, at the height of the recent west african ebola disease outbreak (reviewed in rybicki ). this was made by transient expression in n. benthamiana, and only a few clinical trial doses were available: these were used under the humanitarian principle, and later the mabs were cleared for use by the fda in an efficacy trial just before the end of the epidemic (leafbio ). both these examples are of niche products that were not being made at large scale or for a large market by conventional techniques, and for which there was a sudden, pressing need that could not be supplied by other means. this could provide motivation for small companies to either develop inexpensive vaccines for emerging diseases, or to target niche vaccines or niche therapeutics, in the knowledge that large established entities are unwilling to take the risk. one example for the former possibility comes from the recent emergence of bluetongue virus (btv) disease in sheep and small ruminants in europe, due to northward spread of the insect vector with climate change (purse et al. ) : while attenuated live vaccines are available-south africa presently uses a cocktail of such viruses-concerns in europe about reassortment of virus dsrna genome components between vaccinated and naturally diseased animals, as well as of the safety of the vaccines in terms of possible under-attenuation which may result in disease development in certain sheep breeds (niedbalski ) , mean these are not being used. the irregular occurrence of outbreaks, and the limited number of strains involved, mean that stockpiling vaccines is desirable. however, killed vaccines still require growing potentially dangerous viruses, and while it is possible to make vlps in cell cultures and these are effective (pearson and roy ; roy et al. ) , the technology is too expensive for farm animal use. it is fortunate, therefore, that it is also possible to make btv- vlps via transient expression in n. benthamiana, and these are as effective in a single injected dose as the commercial vaccine (thuenemann et al. ) . there are currently no plans to manufacture this or other plant-produced btv vaccines for the european or other markets; however, this may soon change. an example for a niche vaccine product comes from ours and others' work on beak and feather disease virus (bfdv) vaccines: psittacines are highly valued companion animals; however, there are very few vaccines for their diseases, and none yet available for bfdv. while some recent work in this area has shown that recombinant cp can be made in e coli and in insect cells (heath et al. ; patterson et al. ; stewart et al. ) , that it appears to be protective (bonne et al. ) and that this can apparently form vlps (sarker et al. ) , it still appears that the protein is too expensive to produce for use as a vaccine. while initial work with plant production of bfdv cp was disappointing due to low yields, recent work from our group (duvenage et al. ) showed a significant increase in bfdv yield due to fusion with elastin-like polypeptide (elp), and good immunogenicity in mice. this, coupled with a very simple purification protocol enabled by elpylation (conley et al. ), could allow scalable, cheap production of bfdv vaccines. while therapeutics such as mabs or other biologics for veterinary use are generally limited to high-value companion animals, plant production could open up a hitherto neglected market niche. one excellent example is the manufacture in japan of canine interferon-a (tabayashi and matsumura ) : this is done via transgenic strawberries in a completely enclosed gmp-compliant facility, and the product is powdered strawberry extract given orally, to combat canine periodontal disease. another very recent example in dogs, albeit with them being used as a model for human disease, was the proof that lyophilised transplastomic lettuce leaves expressing ctb fusions of coagulation factor ix (fix) could be used orally in feed for > days in haemophilia b dogs with no ill effects-and that this treatment resulted in robust suppression of igg/inhibitor and ige formation against intravenously-provided fix, and a marked shortening of blood coagulation times (herzog et al. ). an example for agricultural use is the oral dosing of pigs with transgenic arabidopsis thaliana seeds containing designer igas against enterotoxigenic e coli (etec) (virdi et al. ) : this product consisted of dimeric llama-derived heavy chain variable region fused to the fc portion of a porcine iga and the porcine iga j chain and secretory component, which allowed production of dimeric secretory iga-like antibodies (vhh-iga). in a piglet feed-challenge experiment with etec, dosing piglets with mg/d per pig vhh-iga produced a progressive decline in bacterial shedding and a significantly higher weight gain than seen in control or other experimental pigs. a highly novel plant-made therapeutic product was the receptor binding domain of the tailspike protein gp from the p bacteriophage: this is known to reduce salmonella colonisation in the chicken gut (miletic et al. ) . purified elp-fused gp bound to salmonella enterica serovar typhimurium in vitro, and feeding lyophilized leaves containing gp -elp to newly hatched chickens showed that it has the potential to control salmonella contamination in commercially-raised fowl. these and other experiments are reviewed here (juarez et al. ; topp et al. ) , in articles that make an excellent case for plant-made immunotherapeutics for veterinary use. the one health concept has as one of its central themes the integration of opportunities for vaccine-based approaches for the prevention of zoonotic and emerging diseases across veterinary and human medicine (monath ) a set of disease agents which exemplify the potential strength of the one health approach are influenza viruses, and they have in fact been the focus of a number of international meetings and planning sessions (chien ; dwyer and kirkland ; kahn et al. ; ludwig et al. ; powdrill et al. ; short et al. ) . the unique mix of hosts that occurs in intensive agricultural environments that could give rise to pandemics-swine, birds and humans-is a major cause of international concern; so too is the development of suitable vaccines for the prevention of infection in domesticated birds, farmed swine, and humans. plants have been shown to be highly useful for the production of influenza vaccines, and indeed possibly the fastest ever production at scale of an influenzavirus a strain vaccine- month for million doses-was done by medicago inc. for h n pdm ha vlps in (rybicki ) . medicago also managed in , as an exercise to demonstrate preparedness, to produce grams of cgmp-grade plant-made h ha-only vlps only days after accessing the h ha gene cdna sequence, in response to an outbreak in china in the same year. the fact that plant-made influenza vaccines have worked very well in animal models means that they should be trialled extensively in domestic fowl and swine, to see if the maintenance of the viruses in these hosts can be curbed. as for companion animals, there is even a canine influenza vaccine candidate: following a h n outbreak in the us, a group in canada used the plant-derived filamentous malva mosaic virus (mamv) nanoparticles as a vaccine platform to display the highly conserved ectopic m e peptide and to increase its immunogenicity. together with the adjuvant ompc derived from salmonella typhi, the vaccine was protective against both the homologous virus and a heterosubtypic strain of influenza in mice, as well as eliciting antibodies reactive with m e peptides derived from h n , h n and h n strains and being immunogenic in dogs (leclerc et al. ) . given that brucellosis is listed as a one health priority, it is worth noting that a transgenic plant-produced b abortus outer membrane protein (u-omp ) was an effective oral vaccine in mice against a systemic challenge, eliciting an adaptive il- immune response (pasquevich et al. )-and that the protein has significant adjuvant activity, and oral vaccination of mice with u-omp plus salmonella antigens was protective against virulent challenge with s typhimurium (risso et al. ) . it is important to realise that, while vaccines are the target of this review, one health products can also be reagents to be used in more effective or cheaper diagnostic kits, and in particular for point-of-care devices, or for research laboratory use-and especially proteins that could be both a reagent and used as a candidate vaccine in animals and possibly humans. a few of the best potential one health targets for plant-made dual-function proteins would be proteins from middle eastern respiratory syndrome (mers) coronavirus (wirblich et al. ) , nipah and hendra viruses (landford and nunn ; mackenzie et al. ) , diagnostic/ vaccine candidate proteins from rift valley fever and crimean-congo haemorrhagic fever viruses (kortekaas ; monath ) . inexpensive and abundant proteins made from these agents could first serve as reagents in the development of cheap point-of-care diagnostics, and then as vaccine candidates in animals, if appropriate, and then possibly in humans. a useful example here is of the expression both by agroinfiltration in n. benthamiana as a reagent, and in transgenic n. tabacum roots and leaves as a vaccine, of a fused gcgn envelope glycoprotein-encoding gene from crimean-congo haemorrhagic fever virus (ghiasi et al. ). the protein yield was - mg/kg fresh plant weight. transgenic material was orally immunogenic, and elicited humoral and mucosal antibody responses, and antibodies bound inactivated virus used as a vaccine booster in some experiments. agroinfiltration-produced gngc was used as a reagent in elisa to detect immune responses. another study from our group was of the production of cchfv n protein in n. benthamiana by agroinfiltration specifically as a reagent for use in diagnostic tests (atkinson et al. ): a plant codon-optimised and xhis tagged n protein gene was found to accumulate best as a soluble protein in the cytoplasm, from which it could be easily purified by ammonium sulphate fractionation and immobilised ni + column chromatography. purified np was used in a validated indirect elisa to detect anti-cchfv igg in sera from convalescent human patients: this was successful for / samples, with no readings for samples from patients with no history of cchfv infection. the results were % concordant with those from a commercially available immunofluorescent assay. given that soluble n protein is hard to produce and difficult to purify from insect cell cultures, the plant-made product would seem to be a desirable replacement. while the same has been said in many venues over more than twenty years now, the field of molecular farming really does seem to be near to meeting its initial promise for veterinary use. all of the technology that is required for efficient, high-yield production of biologics is in place; downstream processing modalities have been well worked out by a number of near-and cgmp-compliant facilities; many candidate vaccines for a wide variety of pathogens have been tested; therapeutic biologics too for veterinary use are now feasible; regulatory agencies seem agreeable to considering plant-made products. the generally shorter regulatory path, the possibility of using less stringently purified products, and the very real possibility of using oral vaccines and therapeutics, should also be highly attractive for product developers. i sincerely hope, then, that realisation of the promise comes very soon. higher accumulation of f -v fusion recombinant protein in plants after induction of protein body formation noncompliance history high level expression of surface glycoprotein of rabies virus in tobacco leaves and its immunoprotective activity in mice integration and expression of bluetongue vp gene in somatic embryos of peanut through particle bombardment method plant-produced crimean-congo haemorrhagic fever virus nucleoprotein for use in indirect elisa expression of protective antigen in transgenic plants: a step towards edible vaccine against anthrax transformation of an edible crop with the paga gene of bacillus anthracis virus stalls genzyme plant production of human papillomavirus type virus-like particles in transgenic plants assessment of recombinant beak and feather disease virus capsid protein as a vaccine for psittacine beak and feather disease immunization with viruslike particles from cottontail rabbit papillomavirus (crpv) can protect against experimental crpv infection the rabbit viral skin papillomas and carcinomas: a model for the immunogenetics of hpv-associated carcinogenesis generic chromatography-based purification strategies accelerate the development of downstream processes for biopharmaceutical proteins produced in plants comparison of tobacco host cell protein removal methods by blanching intact plants or by heat treatment of extracts immunization with potato plants expressing vp protein protects against rabbit hemorrhagic disease virus in planta protein sialylation through overexpression of the respective mammalian pathway transient expression of mammalian genes in n. benthamiana to modulate n-glycosylation immunogenicity of a subunit vaccine against bacillus anthracis a plant-produced protective antigen vaccine confers protection in rabbits against a lethal aerosolized challenge with bacillus anthracis ames spores how did international agencies perceive the avian influenza problem? the adoption and manufacture of the 'one world, one health' framework optimization of elastin-like polypeptide fusions for expression and purification of recombinant proteins in plants protein body-inducing fusions for high-level production and purification of recombinant proteins in plants the production of hemagglutinin-based virus-like particles in plants: a rapid, efficient and safe response to pandemic influenza plant-derived vaccine protects target animals against a viral disease a novel methodology to develop a foot and mouth disease virus (fmdv) peptide-based vaccine in transgenic plants expression in tobacco and purification of beak and feather disease virus capsid protein fused to elastin-like polypeptides influenza: one health in action gmp issues for recombinant plant-derived pharmaceutical proteins mice orally immunized with a transgenic plant expressing the glycoprotein of crimean-congo hemorrhagic fever virus successful oral prime-immunization with vp from rabbit haemorrhagic disease virus produced in transgenic plants using different fusion strategies plant viral vectors for delivery by agrobacterium oral immunogenicity of the plant derived spike protein from swine-transmissible gastroenteritis coronavirus a protease activity-depleted environment for heterologous proteins migrating towards the leaf cell apoplast two distinct chimeric potexviruses share antigenic cross-presentation properties of mhc class i epitopes oral immunization with a recombinant bacterial antigen produced in transgenic plants the capsid protein of beak and feather disease virus binds to the viral dna and is responsible for transporting the replication-associated protein into the nucleus plant cell cultures for the production of recombinant proteins oral tolerance induction in hemophilia b dogs fed with transplastomic lettuce commercial-scale biotherapeutics manufacturing facility for plant-made pharmaceuticals immunogenicity of the epitope of the foot-and-mouth disease virus fused with a hepatitis b core protein as expressed in transgenic tobacco a dna replicon system for rapid high-level production of virus-like particles in plants human-derived, plant-produced monoclonal antibody for the treatment of anthrax an oral vaccine in maize protects against transmissible gastroenteritis virus in swine biomanufacturing of protective antibodies and other therapeutics in edible plant tissues for oral applications swine and influenza: a challenge to one health research good manufacturing practices production of a purification-free oral cholera vaccine expressed in transgenic rice plants systemic and oral immunogenicity of hemagglutinin protein of rinderpest virus expressed by transgenic peanut plants in a mouse model monoclonal tobacco cell lines with enhanced recombinant protein yields can be generated from heterogeneous cell suspension cultures by flow sorting production of recombinant antigens and antibodies in nicotiana benthamiana using 'magnifection' technology: gmp-compliant facilities for small-and large-scale manufacturing plant-produced cottontail rabbit papillomavirus l protein protects against tumor challenge: a proof-of-concept study one health approach to rift valley fever vaccine development plant-based vaccine: mice immunized with chloroplast-derived anthrax protective antigen survive anthrax lethal toxin challenge delivery of subunit vaccines in maize seed good governance in 'one health' approaches leafbio announces conclusion of zmapp™ clinical trial plant viruses as nanoparticle-based vaccines and adjuvants a novel m e based flu vaccine formulation for dogs plant viral epitope display systems for vaccine development the two-faced potato virus x: from plant pathogen to smart nanoparticle an interspecific nicotiana hybrid as a useful and cost-effective platform for production of animal vaccines in planta production of a candidate vaccine against bovine papillomavirus type induction of a protective immune response to rabies virus in sheep after oral immunization with transgenic maize, expressing the rabies virus glycoprotein influenza, a one health paradigm-novel therapeutic strategies to fight a zoonotic pathogen with pandemic potential regulatory approval and a first-in-human phase i clinical trial of a monoclonal antibody produced in transgenic tobacco plants bringing plant-based veterinary vaccines to market: managing regulatory and commercial hurdles managing emerging diseases borne by fruit bats (flying foxes), with particular reference to henipaviruses and australian bat lyssavirus optimization of human papillomavirus type (hpv- ) l expression in plants: comparison of the suitability of different hpv- l gene variants and different cell-compartment localization in vivo deglycosylation of recombinant proteins in plants by co-expression with bacterial inhibition of protease activity by antisense rna improves recombinant protein production in nicotiana tabacum cv. bright yellow (by- ) suspension cells production of h n influenza virus matrix protein ectodomain protein bodies in tobacco plants and in insect cells as a candidate universal influenza vaccine a plant-produced bacteriophage tailspike protein for the control of salmonella vaccines against diseases transmitted from animals to humans: a one health paradigm transient expression of the ectodomain of matrix protein (m e) of avian influenza a virus in plants bluetongue vaccines in europe protection of rabbits against cutaneous papillomavirus infection using recombinant tobacco mosaic virus containing l capsid epitopes an oral vaccine based on u-omp induces protection against b. abortus mucosal challenge by inducing an adaptive il- immune response in mice differential expression of two isolates of beak and feather disease virus capsid protein in escherichia coli genetically engineered multi-component virus-like particles as veterinary vaccines passive protection to bovine rotavirus (brv) infection induced by a brv vp * produced in plants using a tmv-based vector one health approach to influenza: assessment of critical issues and options invasion of bluetongue and other orbivirus infections into europe: the role of biological and climatic processes high level protein expression in plants through the use of a novel autonomously replicating geminivirus shuttle vector u-omp from brucella abortus is a useful adjuvant for vaccine formulations against salmonella infection in mice protection of recombinant mammalian antibodies from development-dependent proteolysis in leaves of nicotiana benthamiana long-lasting protection of sheep against bluetongue challenge after vaccination with virus-like particles: evidence for homologous and partial heterologous protection plant-produced vaccines: promise and reality plant-made vaccines for humans and animals plant-based vaccines against viruses virus-derived ssdna vectors for the expression of foreign proteins in plants expression of multiple proteins using full-length and deleted versions of cowpea mosaic virus rna- peaq: versatile expression vectors for easy and quick transient expression of heterologous proteins in plants a chimeric affinity tag for efficient expression and chromatographic purification of heterologous proteins from plants an efficient approach for recombinant expression and purification of the viral capsid protein from beak and feather disease virus (bfdv) in escherichia coli expression of hemagglutinin protein of rinderpest virus in transgenic pigeon pea [cajanus cajan (l.) millsp.] plants virus-derived vectors for the expression of multiple proteins in plants optimization and utilization of agrobacteriummediated transient protein production in nicotiana algae-based oral recombinant vaccines synthetic plant virology for nanobiotechnology and nanomedicine n-glyco-engineering in plants: update on strategies and major achievements baculovirus expression of beak and feather disease virus (bfdv) capsid protein capable of self-assembly and haemagglutination generation of glyco-engineered nicotiana benthamiana for the production of monoclonal antibodies with a homogeneous human-like n-glycan structure corn as a production system for human and animal vaccines forefront study of plant biotechnology for practical use: development of oral drug for animal derived from transgenic strawberry increased yield of heterologous viral glycoprotein in the seeds of homozygous transgenic tobacco plants cultivated underground a method for rapid production of heteromultimeric protein complexes in plants: assembly of protective bluetongue virus-like particles the case for plant-made veterinary immunotherapeutics protein body induction: a new tool to produce and recover recombinant proteins in plants optimizing the yield of recombinant pharmaceutical proteins in plants new viral vector for superproduction of epitopes of vaccine proteins in plants expression of an animal virus antigenic site on the surface of a plant virus particle expression of human papillomavirus type major capsid protein in transgenic nicotiana tabacum cv optimization of by- cell suspension culture medium for the production of a human antibody using a combination of fractional factorial designs and the response surface method orally fed seeds producing designer igas protect weaned piglets against enterotoxigenic escherichia coli infection oral immunogenicity of human papillomavirus-like particles expressed in potato expression of bacillus anthracis protective antigen in transgenic chloroplasts of tobacco, a non-food/feed crop induction of a protective antibody response to foot and mouth disease virus in mice following oral or parenteral immunization with alfalfa transgenic plants expressing the viral structural protein vp protection of mice against challenge with foot and mouth disease virus (fmdv) by immunization with foliar extracts from plants infected with recombinant tobacco mosaic virus expressing the fmdv structural protein vp protective lactogenic immunity conferred by an edible peptide vaccine to bovine rotavirus produced in transgenic plants one-health: a safe, efficient, dual-use vaccine for humans and animals against middle east respiratory syndrome coronavirus and rabies virus induction of protective immunity in swine by recombinant bamboo mosaic virus expressing foot-and-mouth disease virus epitopes expression of the fusion glycoprotein of newcastle disease virus in transgenic rice and its immunogenicity in mice a plant-based multicomponent vaccine protects mice from enteric diseases expression in plants and immunogenicity of plant virus-based experimental rabies vaccine development of a candidate vaccine for newcastle disease virus by epitope display in the cucumber mosaic virus capsid protein key: cord- - obr b authors: prasad, r.; sharma, m.; chatterjee, s.; chauhan, g.; tripathi, s.; das, a.; kamal, s.; rawat, a. k. s.; bhutani, k. k.; rai, m. k.; pushpangdan, p.; varma, a. title: interactions of piriformospora indica with medicinal plants date: journal: mycorrhiza doi: . / - - - - _ sha: doc_id: cord_uid: obr b the microbial world exerts a negative as well a positive impact on living plants and animals, and forms an association either pathogenic or symbiotic with the other partners of the living world. mycorrhiza refers to an association or symbiosis between plants and fungi that colonize the roots during periods of active plant growth. the intimate symbiotic relationships developed between mycorrhizal fungi and plants, since the colonization of land by the latter, have led to interdependence between these organisms for many basic processes. the fungi require plants to accomplish their life cycle. plants depend heavily on mycorrhizal fungi for many different functions, such as mineral nutrition and abiotic and biotic stress resistance. substantial evidence has accumulated in the recent past about how the use of the microsymbiont could significantly contribute in decreasing use of fertilizers and pesticides in agriculture, forestry and flori-hortriculture, especially if combined with other beneficial soil microorganisms. the most common and prevalent arbuscular mycorrhizal fungi play an indispensable role in upgrading plant growth, vigor and survival by a positive impact on the nutritional and hydratic status of the plant and on soil health, by increasing the reproductive potential, improving root performance, and providing a natural defence against invaders, including pests and pathogens. the described species of arbuscular mycorrhizal fungi mainly belong to zygomycetes placed in the order glomerales. however, the growing of arbuscular mycorrhizae in pure culture in the absence of living host roots is a matter of global concern. unfortunately, their biotechnological applications cannot be exploited to the level they deserve due to their axenically unculturable nature. the microbial world exerts a negative as well a positive impact on living plants and animals, and forms an association either pathogenic or symbiotic with the other partners of the living world. mycorrhiza refers to an association or symbiosis between plants and fungi that colonize the roots during periods of active plant growth. the intimate symbiotic relationships developed between mycorrhizal fungi and plants, since the colonization of land by the latter, have led to interdependence between these organisms for many basic processes. the fungi require plants to accomplish their life cycle. plants depend heavily on mycorrhizal fungi for many different functions, such as mineral nutrition and abiotic and biotic stress resistance. substantial evidence has accumulated in the recent past about how the use of the microsymbiont could significantly contribute in decreasing use of fertilizers and pesticides in agriculture, forestry and flori-hortriculture, especially if combined with other beneficial soil microorganisms. the most common and prevalent arbuscular mycorrhizal fungi play an indispensable role in upgrading plant growth, vigor and survival by a positive impact on the nutritional and hydratic status of the plant and on soil health, by increasing the reproductive potential, improving root performance, and providing a natural defence against invaders, including pests and pathogens. the described species of arbuscular mycorrhizal fungi mainly belong to zygomycetes placed in the order glomerales. however, the growing of arbuscular mycorrhizae in pure culture in the absence of living host roots is a matter of global concern. unfortunately, their biotechnological applications cannot be exploited to the level they deserve due to their axenically unculturable nature. in the present scenario, herbal medicines are once again gaining popularity as they are easily available, and have rare or no side effects. there is a resurgence in the demands for medicinal herbs. as a consequence, the herbs are now under pressure, also because of shrinking habitats, and are in a phase of extinction. it has, therefore, become necessary to cultivate medicinal plants on a large scale (rai ) . at least - medicinal herbs have been declared chronically endangered by the government of india. the main reason is increasing biotic pressure on forests and unscientific exploitation of medicinal plants. conservation of threatened species and promotion of high yielding varieties can be achieved by various modern techniques of biotechnology, such as tissue culture, micropropagation and protoplast culture. applying these techniques, endangered medicinal plants have been successfully multiplied and developed. our ancestors were well equipped with a vast knowledge regarding drugs of natural origin, but they had little knowledge of how to isolate and obtain pure chemical compound as active ingredients. "charak samhita" is the oldest text available having a wide resource of hundreds of herbs in the complete treatment of disorders including cholera, tuberculosis, leprosy, etc. "indian materia medica" deals with the detail identification, collection and therapeutic uses of thousands of medicinal plants (mazumder and mazumder ) . the earliest evidence of humans making use of plants for healing dates back to the neanderthal period. in the sixteenth century, botanical gardens were created to grow medicinal plants for medical schools. herbal medicine practice flourished until the seventeenth century when more "scientific" pharmacological remedies were favored. there are multiple reasons patients turn to herbal therapies. often cited is a "sense of control", a mental comfort from taking action, which helps explain why many people taking herbs have diseases that are chronic or incurable, such as diabetes, cancer, arthritis, or aids. in such situations, they often believe that conventional medicine has failed them. when patients use home remedies for acute, often self-limited, conditions, such as a cold, sore throat, or bee sting, it is often because professional care is not immediately available, too inconvenient, costly, or time consuming. in rural areas, there are additional cultural factors that encourage the use of botanicals. natural plant products are perceived to be healthier than manufactured medicines. additionally, reports of adverse effects of conventional medications are found in the lay press at a much higher rate than reports of herbal toxicities, in part because mechanisms to track adverse effects exist for conventional medicines whereas such data for self-treatment are harder to ascertain. even physicians often dismiss herbs as harmless placebos, and many consumers and physicians alike mistakenly believe that the us food and drug administration (fda) have not approved anything in a pill form (winslow and david ). different surveys conducted in various parts of india revealed that the majority of the people are suffering from different microbial diseases, ranging in severity from mild-like cough or fever to dreadful-like tuberculosis, leprosy, etc. researchers have found that tendency of self-medication; drug resistance, ignorance, poor health and hygiene are some of the factors responsible for the widespread occurrence of such diseases. considerable progress has been made during the past two centuries when chemists and biologists accepted the challenge of combating these dreadful diseases by synthesizing a wide plethora of organic compounds having the capacity to combat various pathogens. however, indiscriminate use of synthetic drugs has resulted in mutation of strains making them insensitive to the chemical agent leading to global hazard of drug resistance. the scientists of the twenty-first century are generally reviving our traditional knowledge and are screening various parts of plants scientifically used in folklore medicine in search of newer lead compounds to create alternative medicines (mazumder and mazumder ). since ancient times, plants have been an exemplary source of medicine. ayurveda and other indian literature mention the use of plants in treatment of various human ailments. india has about , plant species and, among them, several thousands have been claimed to possess medicinal properties (grover et al. ) . the therapeutic value of some of the medicinally important plants is given in table . researches conducted in the last few decades on plants mentioned in ancient literature or used traditionally for diabetes have shown anti-diabetic properties as observed by several experimental or clinical data showing anti-diabetic activity. (grover et al. ) . because of increased safety concerns about synthetic antioxidants, exploitation of cheaper and safer sources of antioxidants based on natural origins is the focus of research nowadays (iqbal et al. ) . plants as antioxidants: there are many plants as antioxidants. screening of plants is done by measuring the antioxidant activity through various in vitro models mice like , -diphenyl- -picryl-hydrazyl (dpph) free radical scavenging, scavenging of superoxide anion radical-generated non-enzymatic system, ferric thiocyanate method, reducing power, hydrogen peroxide scavenging and metal-chelating activities. for example, ocimum basilicum l. (lamiaceae) assayed by different methodologies (gülçin et al. ) , and black pepper (piper nigrum) (gülçin ). accidental (environmental or occupational) and self-inflicted (suicide) exposure to organophosphate (op) pesticides is encountered frequently in the emergency room, especially in the developing world. these perennial public health issues are compounded by a growing concern over the potential use of op nerve agents such as sarin as a means of terror and nonconventional warfare. ops disrupt neurotransmission by inhibiting synaptic acetylcholinesterase (ache-s), leading to an accumulation of acetylcholine in the synapse and neural overstimulation. the severity of the ensuing nicotinic and muscarinic symptoms is dose-dependent and can result in death due to cardiovascular and respiratory collapse. those surviving often suffer long-term sequelae, including op-induced delayed neuropathy, muscle weakness, permanent brain dismorphology, and social/behavioral deficits. for the production of protein pharmaceuticals, plant systems offer low production costs (with comparable purification and regulatory costs), production scalability and flexibility (with low capital investment), and improved safety (no concern of human pathogens and prions). nevertheless, mammalian-based production systems seem less promising for large-scale production of ache-r because of the low levels and relative instability of the protein and its cognate mrna in such systems. to solve these difficulties, plant-based production is being tried, though still in its experimental infancy, for example: nicotiana benthamiana and endoplasmic reticulum (er) retention of recombinant human ache-r. we report here the efficient production and purification of this novel therapeutic protein, a single administration of which provides prophylactic protection from otherwise lethal op challenges and attenuates the long-term serum ache-r excess and nmj damages caused by op poisoning (evron et al. ). various components in green and black tea, the beverages made by infusing appropriately processed dried leaves of camellia sinensis, notably simple catechins, have properties in vitro that suggest an anti-carcinogenic activity. these include: a direct bactericidal effect against streptococcus mutans and s. sobrinus; prevention of bacterial adherence to teeth; inhibition of glucosyl transferase, thus limiting the biosynthesis of sticky glucan; and inhibition of human and bacterial amylases. studies in animal models show that these in-vitro effects can translate into caries prevention. a limited number of clinical trials in man suggest that regular tea drinking may reduce the incidence and severity of caries. if substantiated, this could offer a very economical public health intervention (hamilton-miller ) . apart from this, withania somnifera dunal (solanaceae) is also under study for anticancer activity (mathur et al. ). meticillin-resistant staphylococcus aureus (mrsa) is recognized as a major nosocomial pathogen that has caused problems in hospitals worldwide, with the uk having one of the highest rates of mrsa in europe. by far the most important reservoir for mrsa, and hence the most important source for spread and subsequent infection, is patients who may be colonized without evidence of infection. the usual sites of mrsa colonization are areas of broken skin, the groin and the axillae, with mrsa infections occurring most frequently in areas of broken skin and in the bloodstream. it is common practice to attempt to clear mrsa colonization and infection in hospital patients with topical antimicrobials and antiseptics; mupirocin and chlorhexidine, for example, are currently employed as part of standard hospital mrsa decolonization protocols. however, resistance to these agents is increasing, with a marked increase in antibiotic resistance recently reported for bacterial strains isolated from superficial skin wounds and leg ulcers. alternative agents for mrsa decolonization are therefore required. tea-tree oil (tto), the essential oil of melaleuca alternifolia, has been suggested as a potential agent for mrsa decolonization, as it has been shown to be an effective broad-spectrum anti-microbial with good activity in vitro against a variety of bacteria including mrsa. furthermore, it has been shown that bacteria such as s. aureus that transiently colonize the skin were more susceptible to tto than bacteria such as coagulase-negative staphylococci (cons), which are regarded as part of the normal commensal skin flora. it has been suggested, therefore, that tto could be useful for removing transient skin flora while suppressing but still maintaining the resident flora, which acts as a natural defence against colonization by other pathogenic bacteria. studies comparing the activity of tto against planktonically grown clinical skin isolates of mrsa, meticillin-sensitive s. aureus (mssa) and cons using both a modified broth microdilution method and a quantitative in vitro time-kill test method have been carried out (pinto et al. ). fungal infections have been increasing in recent years due to a growing number of high-risk patients, particularly immunocompromised hosts. candida is the third-or fourth-most common isolate in nosocomial bloodstream infections in the usa. in addition, candidosis is the most common invasive fungal infection in critically ill nonneutropenic patients.the mortality rate due to invasive aspergillosis increased by % between and in the usa. dermatomycoses are common infections caused by members of the genus candida and by filamentous fungi, particularly the dermatophytes. superficial candidosis and dermatophytosis can be severe in immunocompromised patients. in spite of the introduction of new antifungal drugs, they are limited in number. the increase of fungal resistance to classical drugs, the treatment costs, and the fact that most available anti-fungal drugs have only fungistatic activity, justify the search for new strategies. aromatic plants have been widely used in folk medicine. it is known that most of their properties are due to their volatile oils. essential oils from many plants are known to possess antifungal activity, but only limited information exists about activity towards human fungal pathogens. they have been empirically used as antimicrobial agents, but the mechanisms of action are still unknown. according to our preliminary results some essential oils show an important antifungal activity against yeasts, dermatophyte fungi and aspergillus strains, which could predict therapeutic benefits, mainly for diseases with mucosal, cutaneous and respiratory tract involvement. several studies have shown that thyme oils, particularly those of thymus vulgaris and t. zygis possess antimicrobial activity, those of the phenol type being the most active. the limited occurrence of these phenols in nature is one of the reasons why thymus oils containing thymol and carvacrol have been of great interest for some time. thymus pulegioides is widely distributed on the european continent south of the mediterranean islands. in portugal, it grows in the northeast, and it is locally used as an antiseptic. previous results have demonstrated that this species is polymorphic, and that the thymol/carvacrol chemotype is one of the most abundant in portugal (pinto et al. ). the essential oils of origanum vulgare ssp. hirtum, mentha spicata, lavandula angustifolia, and salvia fruticosa exhibited antifungal properties against the human pathogens malassezia furfur, trichophyton rubrum, and trichosporon beigelii. of the four oils, o. vulgare ssp. hirtum oil showed the highest fungicidal activity and at a dilution of / , caused a % reduction in the number of metabolically active cells within h of exposure. among the main components of the four oils, carvacrol and thymol exhibited the highest levels of antifungal activity. the therapeutic efficacy of the o. vulgare ssp. hirtum essential oil was tested in rats experimentally infected with t. rubrum and yielded promising results. furthermore, the above essential oils were tested with the ames test and did not exhibit any mutagenic activity (adam et al. ). since early times, plants were used to control fertility, but now this knowledge is restored only to the tribal population. the prosecution of witches in early modern europe led to the decline of "wise women", who had for centuries transmitted the lore of contraception. by the seventeenth and eighteenth centuries, that knowledge was everywhere disappearing from europe, and it remained for researchers in the twentieth century to rediscover it. there is an absence of evidence of the widespread use of effective contraceptives before the modern era. it is assumed that, because of social constraints, knowledge of contraception remained a secret lore, which was transmitted orally or alluded to in written sources in coded form (riddle ) . but this knowledge is under study again and the following are a few of the plants which are being studied for their contraceptive property: ancistrophyllum secundiflorum (odesanmi et al. ) , tripterygium wilfordii, a chinese herbal plant (kutney et al. ) , neem oil from azadirachta indica (juneja et al. ) , emblia ribes (williamson ) , montanoa tomentosa (browner and bernard ) , carica papaya (lohiya et al. ) , trigonella foenum graecum (fenugreek) (kassem et al. ) , vicoa indica (banjauri) (dhall and dogra ) , and gloriosa superba (dixit et al. ). in , the fda began scrutinizing the herbal and supplement industry, which triggered a massive letter-writing campaign organized by health food stores. under pressure, the fda created the supplement category, which includes vitamins, minerals, and herbs, and created the dietary supplement and health education act (dshea), signed october . the dshea requires no proof of efficacy, no proof of safety, and sets no standards for quality control for products labeled as supplements. although the dshea requires that supplements do not promise a specific cure on the label, they may claim an effect. now, if questions arise, the burden lies with the fda to prove a product unsafe, rather than a company proving its product safe. manufacturers must put a message on the label stating that the fda has not reviewed claims, but this statement can be subtle. in contrast, regulating agencies in germany, france, the united kingdom, and canada enforce standards of herb quality and safety assessment on manufacturers. because of the lack of requirements for quality control, safety, and efficacy, consumers cannot determine if a herb's active ingredients are actually in the product, if the ingredient is bioavailable, if the dosage is appropriate, if the next bottle they buy will have the same components, or what else is in the pill besides the claimed ingredients (winslow and david ). the age-old system of medicine has been neglected mainly because of the rapid expansion of allopathic medical treatment. presently, the indian system of medicine uses over , medicinal plants and most of them are collected regularly from the wild, of which over five dozen species are said to be in great demand (mazumder and mazumder ). an upsurge in the use of products based on plants is booming. medicinal plants and their derivatives will continue to play a major role in medical therapy in spite of advances in chemical technology and the appearance of cheap, synthesized, complex molecules from simple ones through highly specific reaction mechanisms. the reactions involved are either difficult or expensive to duplicate by classical chemical methods. since production of drugs from medical plants is less expensive than chemical synthesis (mazumder and mazumder ) and other associated benefits, the use of herbal products is going to increase not just nationally but internationally, requiring a need for the conservation of natural flora. along with the flourishing herbal industry there is an urgent need to develop ethics for the use of herbs. the world is already facing issues like global warming and deterioration of the natural environment, so if the herbal industry is to be promoted to the desired level there will be a requirement to set up stringent rules and regulations for the conservation of flora and fauna. we are facing problems in the conservation of endangered species and, with the advent of massive herbal production, the current scenario is getting worse. there is a need to develop techniques to enhance the active ingredient from its usual quantity present in the plant, and to enhance the biomass and better growth of medicinally important herbs. varma and his collaborators, from the school of life sciences, jawaharlal nehru university, new delhi, have screened a novel endophytic root-colonizing fungus which mimics the capabilities of a typical am fungus. however, the unique feature is that this fungus is axenically culturable, and this is a golden lining for am fungi for the scientist dealing with the mycorrhizal research. the fungus has been named piriformospora indica based on its characteristic pear-shaped chlamydospores (fig. ) , and is related to the hymenomycetes of the basidiomycota (verma et al. ) . piriformospora indica tremendously improves the growth and overall biomass production of diverse hosts, including legumes , medicinal and economically important plants (rai et al. ; peškan-berghöfer et al. ; rai and varma ; shahollari et al. ; prasad et al. ) . a pronounced growth-promoting effect was seen with terrestrial orchids (blechert et al. ; bhatnagar and varma ) . a study suggested that p. indica is able to colonize the rhizoids of liverworts and that the thalli failed to grow under in situ conditions in the absence of this fungus . the fungus also provides protection when inoculated into the tissue culture-raised plants by overcoming the 'transient transplant shock' on transfer to the field, and provides almost % survival on transplant varma , ) . based on anatomical and genomic studies, p. indica has been attributed to the highly evolved hymenomycetes (basidiomycetes) (fig. ) . however, neither clamp connections nor sexual structures could be observed. the morphological features and s gene sequences certainly placed the fungus in the group. the septal pores consisted of dolipores with continuous parenthosomes. the dolipores were very prominent, with a multilayered cross wall. the parenthosomes were in contact with the er membranes, which were mostly found near the dolipore (verma et al. ) . the fungus colonizes the roots and improves the health, vigor and survival of a wide range of mono-and dicotyledonous plants. this fungus grows on a large varieties of inorganic, organic and polyphosphates, and thus serves as a good model organism to study phosphorus metabolism (malla et al. ). the molecular mass of denatured acid phosphatase (acpase) of p. indica was found to be kda on sds page. this fungus mediates uptake of phosphorus from the substratum and its translocation to the host by an energy-dependent active process, serves as a strong agent for biological hardening of tissue culture-raised plants, protecting them from "transplantation shock", and renders almost % survival rate on the hosts tested. this fungus is also a potential "bio-control agent" against potent root pathogens. thus, it displays immense potential to be utilized as a biological tool for plant promotion, protection from pests, and for relieving stress conditions such as those due to acidity, desiccation and heavy metal toxicity. thus, it may be concluded that this novel fungus has immense potential for biotechnological applications. recently, molecular techniques like polymerase chain reaction (pcr), molecular cloning, and sequencing showed that members of sebacinaceae have been involved in various mycorrhizal associations. proteomics and genomics data about this fungus has recently been described (peškan-berghöfer et al. ; kaldorf et al. ; shahollari et al. ) . however, sebacinoids were demonstrated recently to be ectomycorrhizal (selosse et al. ) . observations on ectomycorrhizae and basidiomes suggest that species of sebacinaceae are fairly common mycobionts in various ectomycorrhizal plant communities (urban et al. ) . the phylogenetic position of the sebacinaceae within the basidiomycota gives an overview of phylogenetic relationships inside this subgroup of hymenomycetes for which the new order sebacinales is proposed. the ultrastructural data also indicate that p. indica is a member of the hymenomycetes (basidiomycota), and studies on the molecular phylogeny will help to reveal the closest relatives of this species (fig. ) . immunological characterization showed its strong cross-reactivity with the members of zygomycota (glomerales) instead of basidiomycota singh et al. b) , which needs further critical appraisal. a neighbor-joining analysis on comparisons of partial s rdna sequences ( nucleotide position) placed p. indica close to the rhizoctonia solani group (ceratobasidiales) within the basidiomycota. a maximum-likelihood analysis on complete s rdna sequences ( , nucleotide positions) confirmed this finding. a comprehensive phylogenetic analysis of rhizoctonia using sequences from mitochondrial and nuclear rdna on more representatives may provide an insight into the evolution of this important group and its evolutionary relationship with p. indica within hymenomycetes. analysis of s rdna exhibited no change with respect to the taxonomic status of p. indica ). thus, based on the s and s rdna analysis and the ultrastructure of the septal pore, it is placed within the hymenomycetes (basidiomycota). the fungus p. indica associates with the roots of various plant species in a manner similar to mycorrhiza and promotes their growth singh et al. singh et al. , a pešken-berghöfer et al. ; pham et al. a; oelmüller et al. oelmüller et al. , shahollari et al. ; deshmukh et al. ) . the fungus possesses unique properties to act as biofertilizer, bioprotector and immunoregulator. fig. maximum-likelihood tree estimated by quartet puzzling method (strimmer and haeseler, ) on s rdna sequences showing phylogenetic relationships between p. indica and other representatives of basidiomycetes by verma et al. ( ) it also plays a key role in protecting roots from insects by increasing the tolerance of the host roots waller et al. ; serfling et al. ) . it also promotes the antifungal potential of the medicinal plant spilanthus calva due to an increase in spilanthol content after interaction (rai et al. ) . among the compounds released in root exudates infected with p. indica, flavonoids are found to be present. flavonoids have been suggested to be involved in stimulation of precontact hyphal growth and branching (gianinazzi-pearson et al. ; siqueira et al. ) , which is consistent with their role as signaling molecules in other plant-microbe interactions (giovannetti and sbrana, ) . cell wall degrading enzymes like cellulase, polygalactouronase and xylanase were found in significant quantities both in the culture filtrate and in the root exudates colonized by p. indica. p. indica showed profound effects on disease control when challenged with a virulent root and seed pathogen, gaeumannomyces graminis, by completely inhibiting the growth of this pathogen. it indicates that p. indica acted as a potential agent for biological control of root diseases, although the chemical nature of the inhibitory factor is still unknown ). p. indica colonizes the root cortex and forms inter-and intracellular hyphae. within the cortical cells, the fungus often forms dense hyphal coils or branched structures intracellularly. the fungus also forms spore-or vesicle-like structures within or between the cortical cells. like am, hyphae multiply within the host cortical tissues and never traverse through the endodermis. likewise, they also do not invade the aerial portion of the plant (stem and leaves). however, under certain modified cultural conditions, fungus may also invade the stem and leaves without damaging the plant. the characteristic features of p. indica are axenically culturable: no clamp connections, anastomosis present, hypha-hypha aggregation, no hyphal knots, simple septum with dolipores and continuous, straight parenthosomes, chlamydospores - μm in length and - μm in width and - nuclei per spore the host spectrum of p. indica is very much like am fungi: it has been calculated that am fungi interact with almost % of the terrestrial plants (bagyaraj and varma, ; giovannetti and sbrana ; smith and read, ; varma et al. ). however, only limited members of the plant community have failed to interact and these belong to the family of amaranthaceae, chenopodiaceae, cyperaceae, junaceae, proteaceae, or lupines and cruciferae, etc. (denison et al. ) . a careful perusal of the literature indicates that this statement may not be true (leake ; tester et al. ) . denison et al. ( ) have emphasized that model systems are also important as a new research tool to understand the co-operation between microbes and the plants. cruciferae includes the model plant, arabidopsis thaliana, that lacks symbiotic interactions such as mycorrhizae and rhizobia. however, most species of plants are normally infected by mycorrhizae, but some plant taxa do not usually form recognizable mycorrhization. p. indica colonizes the roots of host plants of diverse groups of economically important crops: medicinal (rai et al. (rai et al. , , horticultural, forest and ornamental plants . the similar host range of p. indica and am fungi suggests that this phenomenon may be correlated with some identical functional aspects as indicated by the serological data (elisa, western blotting, immunofluorescens and immunogold labeling) showing close similarities between amf and p. indica (singh et al. b; varma et al. ) . one of the striking differences is that, unlike am, the host range of p. indica also includes terrestrial orchids dactylorhiza purpurella (t. & t.a. stephenson), soo, d. incarnata (l.) soo, d. majalis (rchb.) p.f. hunt & summerh. and d. fuchsii (druce) soo (blechert et al. ; singh and varma ; singh et al. ; varma et al. ) (table ) . however, exceptions are those belonging to members of the cruciferae and some members of chenopodiaceae and amaranthaceae (read ; varma et al. ) . literature reports that the members of these group normally do not accept tectona grandis linn. f. (teak) am fungi. in vitro studies, on p. indica and s. vermifera sensu recorded that these two symbiotic fungi profusely interacted with the root system of the crucifer plants viz., mustard (brassica junacea), spinach (spinaceae oleracea), cabbage (brassica oleracea var capitata) (kumari et al. ) and arabidopsis thaliana (pham et al. a; peškan-berghöfer et al. ; shahollari et al. ) . it would be useful to assess the non hosts of am fungi with respect to their interaction with p. indica for its further functional characterization. in order to enlighten the molecular events that promote the root growth, the difference in protein expression was analyzed and modification arises due to the interaction with the fungus. membraneassociated proteins from roots were separated by two-dimensional gel-electrophoresis ( d-page) and identified by electrospray ionization mass spectrometry (esi-ms) and tandem mass spectrometry (ms-ms). p. indica consists of secondary metabolites like hydroxamic acids (diboa, dimboa) which act as natural pesticides ). p. indica tremendously improves the growth and overall biomass production of diverse hosts, including legumes , medicinal and other economically important plants (pham et al. b; rai et al. ; peškan-berghöfer et al. ; shahollari et al. ) . p. indica colonizes the roots of host plants of a diverse group of plants belonging to monocots, dicots including orchids (blechert et al. : pham et al. b , prasad et al. , herbs, shrubs and woody trees. the effect of p. indica interaction with various plants such as bacopa monniera, azadirachta indica, tridex procumbans, abrus precatorius, withania somnifera, chlorophytum borivilianum and spilanthes calva (rai et al. (rai et al. , have been tested in laboratory conditions as well as in the extensive field trial. spilanthes calva dc (family asteraceae), commonly known as toothache plant or virus blocker, is well known for enhancing immunity. because of its high medicinal value, it is costly and there is much demand of this plant in the market. it is cultivated in tribal pockets for herbal treatment in various diseases. this plant has antiageing properties and cures various diseases of tooth and gums including pyorrhoea. it is antimicrobial in nature and economically very useful as tooth powder, which is prepared from this plant (dey ) its leaves stimulate salivation, which is due to the presence of an active chemical spilanthol. manifold enhancement of the antifungal activity and quantity of spilanthol was recorded on cocultivation with p. indica (fig. ) . the chemical analysis of the roots of the plant revealed a slight increase in spilanthol content. adhatoda vasica nees (common name, malabar nut; family, acanthaceae) is an evergreen shrub. it is well known for preparation of medicine for bronchitis, asthma and other pulmonary infections. glycodin ® , a famous product used for the cure of bronchitis, is extracted from the leaves of this plant. it is also known for its antiarthritis, antiseptic, antimicrobial, expectorant, sedative and antituberculosis properties (dey ; singh and jain ) . in ayurveda, several medicines are manufactured from this plant. due to increasing demand for a. vasica by pharmacies, there is a need for its rapid multiplication. in the observations, cuttings of a. vasica were inoculated with p. indica to assess the growth-promoting property of p. indica on this important medicinal plant. profuse proliferation of roots of a. vasica after inoculation of p. indica was repeatedly recorded (fig. ) . root-colonization of a. vasica by p. indica increased with time from % after months to % after months (rai and varma ) . withania somnifera is also known as indian ginseng and belongs to the family solanaceae. more than pharmaceutical products are produced from the roots of this plant. multiple shoot cultures of withania somnifera were established from single shoot tip explants and their potential for the production of two principal withanolides, withaferin a and withanolide d, was investigated (ray and jay ; ganzera et al. ) . the plantlets were then transferred to pots and maintained in a greenhouse for months. % of these in vitro-propagated plantlets survived and showed normal growth. leaves from these plants were used for isolation of the withanolides. methanolic extract of leaves from plantlets growing in tissue culture and those transferred to the greenhouse were evaluated for immunomodulatory activity. while the extract from greenhouse samples showed potent immunosuppressive activity, those from tissue culture samples did not show any activity (furmanowa et al. ) . withaferin a acts as radiosensitizer from withania somnifera. ser (sensitizer enhancement ratio) increased with drug dose, but at higher doses the increased lethality appears to be due to two effects, drug toxicity and radiosensitization the applicability of this drug as a radiosensitizer in cancer therapy needs to be explored (devi et al. ; devi ) . the basal stem and leaf areas of treated plants were also enhanced. the lengths of the inflorescence and the number of flowers on inoculated s. calva plants were also increased relative to controls. similarly, the number of flowers on the flowers on the inoculated plants of w. somnifera was higher than on controls. seed counts were higher for treated than for control plants. the overall root biomass of the inoculated plants was higher than that of the corresponding controls. the fresh and dry weights of both underground and aboveground parts of w. somnifera inoculated plants were higher than controls (fig. ) . the net primary productivity of inoculated s. calva and w. somnifera plants was . and . g/plant/day, respectively. these values were higher than those of control plants ( . and . g/plant/day, respectively). safed musli, scientifically known as chlorophytum borivilianum, belongs to the liliaceae family and is endowed with rasayana (antiageing and immunoboosting), balya (performance-boosting) and vrishya (aphrodisiac) properties to keep one young and healthy with a well-tuned body for better handling of stress. it lives up to the description as recent clinical trials show that musli if taken on regular basis, it maintains health and youthfulness, keeps the person energetic and active, increases working capacity, increases tolerance to stress and strain, increases working capacity, gives sound sleep, develops a firm and muscular body, and improves conjugal capability. phytochemicals like saponins, carbohydrate and proteins are present in the root. this plant has got immense market potential, both domestic and international. peeled and dried roots are used for therapeutic purposes. both tubers and seeds are used, but the tubers are a more viable option because they offer better germination rate and tuber growth than seeds. they are normally used to maintain the equilibrium of all the systems of the body and keeps the "body-mind-soul complex" in a state of harmony. on interaction with p. indica, significant growth promotional effect on the plant has been observed besides early flowering in the crop and % survival on transplantation (fig. ) . bacopa monniera commonly known in india as brahmi is an important ancient ayurvedic medicinal plant in the scrophulariaceae family. in the traditional system of medicine, brahmi is a reputed nervine tonic. it is also used to treat asthma, insanity, epilepsy, hoarseness, enlargement of the spleen, snake bite, rheumatism, leprosy, eczema and ringworm, and as a diuretic, aperitive and cardiotonic (basu and walia ; basu et al. ; bhakuni et al. ; elangovan et al. ) . the main active ingredient of b. monniera is believed to be the bacosides. the fungus p. indica is documented to promote plant growth and protects the host against root pathogens and insects. fungus root colonization by the fungus p. indica promoted the plant growth and enhanced antioxidant activity as well as the active ingredient bacoside by several folds (fig. ) . tissue culture technology could play an important role in the clonal propagation, germplasm conservation and improvement of b. monniera. shoot regeneration has been reported from the distal ends of -to -mm-long internode segments of b. monniera cultured on growth regulator free medium, longer internodes being more conducive to regeneration (thakur et al. ; tiwari et al. ) . it was found that there was an inherent problem with the micropropagated plants during the time of transplantation from the laboratory to field. it was noticed that the rate of survival was very low, up to % in the field conditions. the salient reason could be the 'transient transplant shock' that resulted in the stunted growth. biological hardening has proved to be fruitful. it provided better results for the overall performance of the plants. for employing the technique, inoculation of micropropagated plantlets with active cultures of amf or mycorrhiza-like fungi appears to be critical for their survival and growth. these preacclimatized plantlets, when transferred to the field, overcame the transient transplant shock, and were able to cope with the changed environment of transplantation which also helped in their successful establishment. pre-establishment of mycorrhiza in the host roots also helps in the development of the synergistic effect with other rhizosphere microflora that compete in the ecosystem for successful survival ). medicinal plants are in great demand in modern civilization to extract various herbal drugs for human welfare. these products come from a labor-and capital-intensive activity, where chemical inputs play an essential role but bring with them a set of problems linked to the degradation of the natural environment and resource base. thus, the potential use of biological tools such as micropropagation and biological hardening with am, which ensure adequate level of production with satisfactorily reduction of chemical fertilizer and pesticides, like technologies needed for sustainable agriculture. among the soil inhabiting microorganisms, am and other mycorrhizae-like fungi acquire added importance due to their role in establishment, productivity and longevity of natural and man-altered ecosystems. am fungi share a distinct ecological niche in soil along with a variety of microorganisms including some which are pathogenic, some commensalistic and some which are symbiotic. a newly-described species piriformospora indica covers most of the characteristics of am. it improves the growth and overall biomass production of a diverse host including medicinal and other plants of economic importance. this fungus has another important feature that it has potency to grow axenically as an effective alternative to am. this fungus mediates uptake of phosphorus from the substratum and it is translocated to the host by an energy-dependent active process. the fungus serves as a strong agent for biological hardening of tissue cultureraised plants, protecting them from "transplantation shock", and rendering almost % survivals on the hosts tested. this fungus is also a potential "biological agent" against potent root pathogens. the growth promotion observed may have been caused by a greater absorption of water and mineral nutrients due to extensive root colonization and the proliferation of the mycelium into the soil. thus, it can be concluded that this root-colonizing fungus promotes growth of many plant species of medicinal and economical importance, including cereals, legumes, ornamental plants, oilseed and vegetables, is a potential candidate for the hardening of tissue culture-raised plants and exerts fungicidal and herbicidal resistance. in plant biotechnology, the emphasis is on the manpower rather than on expensive equipment in both developing and developed countries because of its vast impact on agriculture. today, micropropagation is the most widely and successfully used technology for the mass production of horticultural, ornamentals, fruits, vegetable, cereals, plantation crops, spices and medicinal plants. antifungal activities of origanum vulgare sub sp. hirtum, mentha spicata, lavandula angustifolia, and salvia fruticosa essential oils against human pathogenic fungi interaction between arbuscular mycorrhizal fungi and plants, and their importance in sustainable agriculture in arid and semi-arid tropics chemical examination of bacopa monniera wettst. part iii: bacoside b the chemical investigation of the leaves of herpestis monniera screening of indian plants for biological activity. part ii the healthy marriage between terrestrial orchids and fungi first remarks on the symbiotic interactions between piriformospora indica and terrestrial orchids herbal emmenagogues used by women in colombia and mexico cooperation in the rhizosphere and the "free rider the root endophytic fungus piriformospora indica requires host cell death for proliferation during mutualistic symbiosis with barley withania somnifera dunal (ashwagandha): potential plant source of a promising drug for cancer chemotherapy and radiosensitization withaferin a, a new radiosensitizer from the indian medicinal plant withania somnifera indian medicinal plants used in ayurvedic preparations clinical trials with vicoa indica (banjauri), an herbal medicine, as an antifertility agent possible antispermatogenic effect of g. superba in male gerberils in vitro studies on the anticancer activity of bacopa monnieri plant-derived human acetylcholinesterase-r provides protection from lethal organophosphate poisoning and its chronic aftermath in vitro propagation of withania somnifera and isolation of withanolides with immunosuppressive activity quantitative hplc analysis of withanolides in withania somnifera in vitro enhancement of spore germination and early hyphal growth of a vesicular-arbuscular mycorrhizal fungus by host root exudates and plant flavonoids meeting a non-host: the behaviour of am fungi medicinal plants of india with anti-diabetic potential the antioxidant and radical scavenging activities of black pepper (piper nigrum) seeds determination of antioxidant and radical scavenging activity of basil (ocimum basilicum l. family lamiaceae) assayed by different methodologies anti-cariogenic properties of tea (camellia sinensis) antioxidant properties of methanolic extracts from leaves of rhazya stricta neem oil inhibits two-cell embryo development and trophectoderm attachment and proliferation in vitro patterns of interaction between populus esch and piriformospora indica: a transition from mutualism to antagonism evaluation of the potential antifertility effect of fenugreek seeds in male and female rabbits colonization of cruciferous plants by piriformospora indica studies with tissue cultures of the chinese herbal plant, tripterygium wilfordii. isolation of metabolites of interest in rheumatoid arthritis, immunosuppression, and male contraceptive activity the biology of myco-hetrotrophic (saprophytic) plants anti-fertility effects of aqueous extracts of carica papaya seeds in male rats phosphorus solubilizing symbiotic fungus: piriformospra indica evaluation of the effect of withania somnifera root extracts on cell cycle and angiogenesis traditional knowledge, herbal remedies to anti bacterial therapy: its current status in the st century comparison of metabolic effects of ethanolic extracts of ancistrophyllum secundiflorum and menstrogen (orthodox contraceptive) on metabolic parameters in pregnant rabbits math-domain proteins represent a novel protein family in arabidopsis thaliana and at least one member is modified in roots in the course of a plant/microbe interaction association of piriformospora indica with arabidopsis thaliana roots represent a novel system to study beneficial plant-microbe interactions and involve in early plant protein modifications in the endocytoplasmic reticulum and in the plasma membrane interaction of piriformospora indica with diverse microorganisms and plants axenic cultures of piriformospora indica antifungal activity of the essential oil of thymus pulegioides on candida, aspergillus and dermatophyte species interaction of medicinal plants with plant growth promoting rhizobacteria and symbiotic fungi sebacinaceae: culturable mycorrhiza-like endosymbiotic fungi and their interaction with non-transformed and transformed roots. in: declerck s (ed) root organ culture of mycorrhizal fungi. soil biology series beneficial microorganisms: herbal and medicinal plants herbal medicines in india: retrospect's and prospects arbuscular mycorrhiza-like biotechnological potential of piriformospora indica, which promotes the growth of adhatoda vasica nees positive growth responses of withania somnifera and spilanthes calva were cultivated with piriformospora indica in field antifungal potential of spilanthes calva after inoculation of piriformospora indica production of withaferin a in shoot cultures of withania somnifera mycorrhiza: the state of art eve's herb: a history of contraception and abortion in the west piriformospora indica: a new biological hardening tool for micropropagated plants biological approach towards increasing the survival rates of micropropagated plants a new fungal phylum, the glomeromycota: phylogeny and evolution basal hymenomycetes belonging to sebacinaceae are ectomycorrhizal on temperate deciduous trees performance of the biocontrol fungus piriformospora indica on wheat under greenhouse and field conditions expression of a receptor kinase in arabidopsis roots is stimulated by the basidiomycete piriformospora indica and the protein accumulates in triton x- insoluble plasma membrane microdomains orchidaceous mycorrhizal fungi root endosymbiont: piriformospora indica -a boon for biotechnology importance of piriformospora indica-a novel symbiotic mycorrhiza-like fungus: an overview amf-likefungus: piriformospora indica-a boon for plant industry piriformospora indica-in vitro raised leguminous plants: a new dimension in establishment and phyto-promotion stimulation of vesicular-arbuscular mycorrhiza formation and growth of white clover by flavonoid compounds the phenomenon of "nonmycorrhizal" plants morphogenesis of organ differentiation in bacopa monnieri stem cultures shoot regeneration and somatic embryogenesis from different explants of brahmi (bacopa monniera) ectomycorrhizae involving sebacinoid mycobionts microbial-biotechnology: new paradigms and role in sustainable agriculture. in: rajak rc(ed)microbial biotechnology for sustainable development and productivity piriformospora indica: an axenically culturable mycorrhiza like endosymbiotic fungus piriformospora indica, a cultivable plant growth promoting root endophyte piriformospora indica, gen. et sp. nov., a new root colonizing fungus the endophytic fungus piriformospora indica reprograms barley to salt stress tolerance, disease resistance and higher yield ) major herbs of ayurveda key: cord- -yrw wrxj authors: mugford, sam t.; osbourn, anne title: saponin synthesis and function date: - - journal: isoprenoid synthesis in plants and microorganisms doi: . / - - - - _ sha: doc_id: cord_uid: yrw wrxj saponins are one of the most numerous and diverse groups of plant natural products. they serve a range of ecological roles including plant defence against disease and herbivores and possibly as allelopathic agents in competitive interactions between plants. some saponins are also important pharmaceuticals, and the underexplored biodiversity of plant saponins is likely to prove to be a vital resource for future drug discovery. the biological activity of saponins is normally attributed to the amphipathic properties of these molecules, which consist of a hydrophobic triterpene or sterol backbone and a hydrophilic carbohydrate chain, although some saponins are known to have potent biological activities that are dependent on other aspects of their structure. this chapter will focus on the biological activity and the synthesis of some of the best-studied examples of plant saponins and on recent developments in the identification of the genes and enzymes responsible for saponin synthesis. structures of plant saponins. triterpenoid saponins ( top panel ): avenacin a- from oat roots ( avena spp.), chromosaponin i from pea seed ( pisum sativum ), ginsenoside rg from ginseng roots ( panax spp.), soyasaponin i (also known as soyasaponin bb) from soya ( glycine max ), avicin d from acacia victoriae seed pods, glycyrrhizin from liquorice roots ( glycyrrhiza spp.) and qs from quillaja saponaria bark. examples of both monodesmosidic (one sugar chain) and bisdesmosidic (two sugar chains) saponins are shown. bottom panels : steroidal glycoalkaloid a -tomatine from tomato leaves ( solanum lycopersicum ); the steroidal saponin avenacoside a from oat leaves ( avena spp.) properties of these compounds. the highly polar sugar moieties together with the non-polar triterpene or sterol backbones result in a highly amphipathic compound. hence, these compounds produce stable foams, a feature often associated with aqueous extracts from saponin-accumulating plants (hostettmann and marston ) . indeed, the names of some plants originate from this property, such as soapwort ( saponaria of fi cinalis ), which was historically used as a source of detergent. saponins represent a sizable proportion of the number of known plant natural products, which is in excess of , (dixon ; hartmann ; osbourn et al. ) . while plant natural products used to be regarded as waste products of a "luxurious metabolism", they are now accepted as the products of natural selection with diverse biological activities and important ecological roles (dixon ; hartmann ) . the structural diversity of saponins is re fl ected in the array of different biological activities associated with these compounds, and these diverse compounds provide a signi fi cant resource for drug and agrochemical discovery. indeed, many plant-derived saponins are currently used as important pharmaceuticals in the treatment of a range of diseases in conventional and traditional medicine (arase et al. ; cinatl et al. ; jayatilake et al. ; germonprez et al. ; harada ) . research into the functions and synthesis of saponins has provided a wealth of information on the properties of this important group of compounds, both for human use and in plants. the basis of the diversity of saponins lies in several aspects of their structure (fig. . ) (hostettmann and marston ) . firstly, the aglycone triterpenes and sterols themselves encompass a wide range of structures, with variation in the degree and nature of cyclization and oxidation of the backbone. secondly, the nature of glycosylation is widely variable with respect to the number and type of sugar molecules, the types of inter-sugar linkages and the presence of one or more sugar chains. monodesmosidic saponins have a single sugar chain attached at the c- position, while bidesmosidic saponins have an additional sugar chain at the c- (for triterpenoid saponins) or c- (steroid saponins) position. further modi fi cations of the saponin backbone give rise to even greater structural diversity, such as the addition of acyl-or etherlinked groups derived from organic acids (e.g. avenacin a- , chromosaponin i, avicin d and qs ; fig. . ) (begley et al. ; kudou et al. ; yoshikawa et al. yoshikawa et al. , yoshikawa et al. , yoshikawa et al. , germonprez et al. ; zou et al. ) . squalene synthase (sqs) converts fpp to squalene, and squalene epoxidase (sqe) then oxidises squalene to produce , -oxidosqualene. , -oxidosqualene serves as the substrate for a range of oxidosqualene cyclase (osc) enzymes, including cycloartenol synthase for primary sterol synthesis and b -amyrin synthase. these enzymes are responsible for the synthesis of the major sterol and triterpene precursors of saponin biosynthesis, respectively. triterpenes and sterols derived from , oxidosqualene are further elaborated by oxidative and other modi fi cations, and by glycosylation, leading to the synthesis of saponins the function and synthesis of saponins in plants will be discussed in this chapter, with particular focus on triterpenoid saponins and on the oat root triterpenoid saponins known as avenacins. as might be expected from their chemical diversity, saponins collectively have a wide range of biological activities. many of these compounds have antimicrobial and/or anti-herbivore activity and so may have roles in plant defence ( osbourn ; morrissey and osbourn ; francis et al. ; friedman friedman , sparg et al. ) . saponins also have a range of important pharmaceutical properties, for example, antiin fl ammatory, antifungal, antibacterial, anti-parasitic, anti-cancer and antiviral activities (reviewed by sparg et al. ; podolak et al. ) . saponins have further applications in a range of industries extending beyond pharmaceuticals. their surfactant properties are important in the beverage and cosmetics industries, and saponins are used as foaming agents for a variety of purposes including in fi re extinguishers (hostettmann and marston ) . in addition, some saponins are used as fl avourings due to their intense sweetness or bitterness (price et al. ; grenby ; kitagawa ; heng et al. ) . for example, the sweetness of liquorice root is attributable to the presence of the triterpenoid saponin glycyrrhizin (kitagawa ) . saponins generally act by permeabilising plasma membranes. their amphipathic properties enable them to penetrate membranes, where they complex with sterols and cause pore formation (roddick ; roddick and drysdale ; steel and drysdale ; fenwick et al. ; armah et al. ) . while membrane permeabilisation is a common feature of saponins, these compounds are also likely to have further effects on cells, for example, by interfering with cellular processes, such as enzyme activities, transport, organelle integrity, redox-related functions and other signal transduction processes and through triggering apoptosis (e.g. mcmanus et al. ; ohana et al. ; sparg et al. ; haridas et al. a ; lemeshko et al. ) . for some saponins, it has been shown that biological activity does not depend on amphipathicity, making it unlikely that their mode of action is through membrane permeabilisation (oda et al. ; simons et al. ) . the biological properties of saponins in the context of their ecological functions and commercial applications are discussed below. avenacins are triterpenoid saponins that are found in the tips of oat roots (crombie et al. ; crombie and crombie ; hostettmann and marston ) . oats appear to be unique amongst the cereals in being able to synthesise saponins (ohmoto and ikuse ; osbourn et al. ) . there are four forms of avenacin. the major form (avenacin a- ) is shown in fig. . . avenacins are oleane-type triterpenoids derived from b -amyrin (begley et al. ; haralampidis et al. ) . b -amyrin is elaborated by addition of various functional groups including hydroxyls and an epoxide and by addition of a branched trisaccharide chain consisting of one l -arabinose and two d -glucose molecules. in addition, avenacins are acylated at the c- carbon of the triterpene with either n-methyl anthranilate (avenacins a- and b- ) or benzoate (avenacins a- and b- ). the n -methylanthraniloyl acyl group confers bright blue fl uorescence under uv illumination, and this fl uorescence can be readily seen in the root tip. avenacins are potent antifungal compounds and are effective against the fungal pathogen gaeumannomyces graminis var. tritici , which is the casual agent of take-all disease (papadopoulou et al. ) . take-all causes major yield losses in wheat crops throughout the world, and there is currently no effective means of control. in contrast, oats are highly resistant to infection by g. graminis . around half a century ago, it was suggested that the resistance of oats to this disease might be associated with the blue fl uorescent material in the root tips of oat plants, which was shown to be antifungal (goodwin and pollock ; turner ) . isolation of the antifungal components of oat roots then led to the puri fi cation and structural identi fi cation of the avenacins maizel et al. ) . osbourn et al. ( ) provided further evidence to highlight the importance of avenacins in resistance to take-all. avenacins are only found in the avena genus, and most oat species synthesise the compounds, suggesting that avenacins confer a selective advantage. one oat species, avena longiglumis , was found to lack avenacins in its roots and was also shown to be susceptible to g. graminis var. tritici , while all other oat accessions investigated produced avenacins and were resistant to this pathogen. further compelling evidence for a role for avenacins in plant defence came from the mutagenesis of a diploid avenacinproducing oat species ( avena strigosa ), and the demonstration that avenacin-de fi cient mutants (isolated by screening for reduced root fl uorescence) have enhanced susceptibility to a range of soil-borne fungal pathogens including g. graminis var. tritici (papadopoulou et al. ) . glycosylation of saponins is generally critical for antifungal activity (sandrock and van etten ; morrissey and osbourn ) . the loss of a single sugar from the oligosaccharide chain does not greatly reduce the amphipathicity of saponins but can impair the ability to complex with sterols (arneson and durbin ) . many fungi can hydrolyse sugars from saponins, thereby reducing antifungal activity (sandrock and van etten ; morrissey and osbourn ) . for example, the ability of an oat-attacking variant of the take-all fungus ( g. graminis var. avenae ) to infect oats is dependent on its ability to produce a saponin glycosyl hydrolase known as avenacinase (bowyer et al. ) . the various deglucosylated forms of avenacin have signi fi cantly reduced antifungal activity and reduced ability to complex membrane sterols. examples of saponin glycosyl hydrolases have been reported from various other plant pathogenic fungi, including pathogens of oat leaves (which encounter the steroidal avenacosides) and of tomato (which encounter the steroidal glycoalkaloid a -tomatine) (sandrock and van etten ; morrissey and osbourn ) (fig. . ). like many plant secondary metabolites, avenacins are localised in the plant vacuole. an interesting insight into the role of saponin glycosylation in self-protection in plants was made recently during investigation of oat mutants that fail to fully glycosylate avenacins (mylona et al. ) . these mutants have stunted roots, a root hair-de fi ciency phenotype and membranetraf fi cking defects. these defects were shown to be due to accumulation of the incompletely glucosylated avenacin intermediate. thus, although this intermediate is less toxic to fungi (turner ; bowyer et al. ) and has a reduced capacity to cause permeabilisation of fungal membranes, it is toxic to plants cells. glucosylation may be important for transport of avenacins to the vacuole. consistent with this, the incompletely glucosylated avenacin intermediate has an atypical subcellular distribution and is not appropriately targeted to the vacuole. this suggests that vacuolar sequestration is an important self-protection mechanism (mylona et al. ) . avenacins are synthesised and accumulate in the epidermal cells of the root tip (haralampidis et al. ) . they are also released into the soil (carter et al. ) , although it is not clear whether this is an active process or a consequence of sloughing of the root epidermis. carter et al. ( ) analysed fungi isolated from the roots of fi eld-grown oat plants and found that many of these fungi were resistant to the toxic effects of avenacins and most were able to degrade these saponins. thus, avenacins are likely to in fl uence the growth of microorganisms in and around oat roots. the release of avenacins into the soil also has implications for competitive interactions between plants. saponins from other plant species have been shown to have phytotoxic properties and as a consequence have been implicated in allelopathy (oleszek and jurzysta ; waller et al. ; hiradate et al. ; li et al. ) . avenacins are phytotoxic and may therefore also have functions in suppression of the growth of neighbouring plants (field et al. ) . oats are an important weed of other cereals, and understanding the basis of this competitive ability could lead to bene fi ts for agriculture. ecological roles for saponins have been identi fi ed in a range of other plant species. soyasaponins, like avenacins, have a pentacyclic oleane triterpene skeleton. members of the soyasaponin group of saponins are found in a variety of agriculturally important legumes (hostettmann and marston ; yoshiki et al. ; suzuki et al. suzuki et al. , agrell et al. ) , and several of these compounds have important pharmacological properties (konoshima et al. ; milgate and roberts ; dixon and sumner ; gur fi nkel and rao ) . soyasaponins are a diverse group of compounds that exist as monoand bisdesmosidic forms (hostettmann and marston ) . an example of a monodesmosidic soyasaponin is soyasaponin i (fig. . ). in some bisdesmosidic legume saponins, the terminal monosaccharide of the c- sugar chain is modi fi ed by the addition of a g -pyranoyl group (an ether-linked , -dihydro- , -dihydroxy- methyl- h -pyran- -one (ddmp) group: yoshiki et al. ; tsurumi et al. ) . one such g -pyronyl saponin, chromosaponin i ( fig. . ), sometimes also referred to as soyasaponin vi (hostettmann and marston ) , has been shown to have a growth-promoting effect on other plants (tsurumi and wada ; tsurumi and ishizawa ; tsurumi et al. ) and is believed to exert its effects through regulation of auxin in fl ux (rahman et al. ) . while chromosaponin i promotes plant growth, other legume saponins have been shown to suppress the growth of other plant species, an observation that is of particular relevance to organic farming methods in which these species are used as green fertiliser crops and that may explain why this practice can have a negative impact on subsequent crop yields (oleszek and jurzysta ; waller et al. ; hiradate et al. ; li et al. ) . accumulation of a variety of triterpenes including soyasaponin i ( fig. . ) (also known as soyasaponin b b ) and the related bidesmosidic saponin, medicagenic acid, occurs in response to fungal elicitors (suzuki et al. (suzuki et al. , , wounding and herbivory in medicago sativa (agrell et al. ) and has been linked with plant defence. similarly, soyasaponins have been shown to be major insecticidal and antifeedant components of pea seeds (taylor et al. ) and are likely to protect these plants from herbivory by insects. resistance to insect herbivores in the brassicaceae is known to be mediated by glucosinolates (bones and rossiter ; halkier and gershenzon ) . some specialist herbivores are not affected by glucosinolate toxicity and use the compounds as a signal to stimulate oviposition on the plant (huang et al. ) . however, the ability of one brassicaceous species, barbarea vulgaris , to resist attack by the specialist diamondback moth ( plutella xylostella ) was found to be associated with the accumulation of a triterpenoid saponin in the plant (shinoda et al. ) . the saponin is associated with antifeedant effects towards the larva of p. xylostella and causes toxicity at high concentrations. antifeedant activity and/or toxicity to insects have also been suggested for a number of other saponins (hlywka et al. ; agrell et al. ; taylor et al. ) and may be an important component of plant defence. saponins also impact on palatability for other animals including humans. for example, some saponins are used as sweeteners (hayashi et al. (hayashi et al. , a . conversely, other saponins have anti-sweet properties and are able to suppress the sweet taste of glucose (yoshikawa et al. (yoshikawa et al. , (yoshikawa et al. , . the fl avour properties of soyasaponins have been investigated through the stimulation of the glossopharyngeal nerve of frogs by direct application of pure saponins (yoshiki et al. ) . antiherbivore activity and bitterness can also have detrimental consequences for agriculture (francis et al. ) , and saponins from legumes can reduce the ability of ruminant mammals to digest plant material (milgate and roberts ; dixon and sumner ) . many solanaceous species produce antifungal steroidal glycoalkaloid saponins. tomato plants produce a -tomatine, a monodesmosidic steroidal glycoalkaloid with a tetrasaccharide side chain that accumulates in the leaves and immature fruit (friedman ) (fig. . ) . a -tomatine is fungitoxic and has been implicated in protection against fungal infection (sandrock and van etten ; morrissey and osbourn ; friedman ) . as with other saponins, a -tomatine is toxic to a wide range of fungal species. toxicity is generally ascribed to the ability of the saponin to complex with sterols and permeabilise fungal plasma membranes (arneson and durbin a ; roddick ; keukens et al. keukens et al. , . a -tomatine has recently also been shown to induce reactive oxygen-mediated programmed cell death in fungi (ito et al. ) . specialist pathogens of tomato generally have a higher level of resistance to a -tomatine when compared with fungi that do not infect tomato (arneson and durbin b ; steel and drysdale ; suleman et al. ; sandrock and van etten ; morrissey and osbourn ) . the tomato leaf spot fungus, septoria lycopersici , provides an interesting example of this plant-pathogen relationship. s. lycopersici is resistant to a -tomatine and is able to infect tomato plants. this fungus produces an a -tomatine-hydrolysing enzyme, tomatinase, which deglucosylates a -tomatine to the less toxic product b -tomatine (arneson and durbin ) . tomatinase-de fi cient mutants of s. lycopersici (generated by insertional inactivation of the tomatinase gene) are unable to degrade a -tomatine and have enhanced sensitivity to this saponin. such mutants are not compromised in their ability to cause disease on tomato leaves. however, they do trigger enhanced cell death and elevated expression of defence genes during early infection (martin-hernandez et al. ) . heterologous expression of s. lycopersici tomatinase in the phytopathogenic fungi cladosporium fulvum and nectria haematococca , both of which are normally unable to degrade a -tomatine, resulted in enhanced sporulation on tomato plants (melton et al. ; sandrock and van etten ) , providing further evidence for a role for tomatinase in virulence. another fungal pathogen of tomato, fusarium oxysporum f. sp. lycopersici, produces a tomatinase enzyme that has a different mode of action to that of s. lycopersici and that hydrolyses a -tomatine to give the tetrasaccharide lycotetraose and the aglycone tomatidine. gene silencing and targeted gene disruption experiments indicate that f. oxysporum f. sp. lycopersici tomatinase is required for full virulence on tomato (ito et al. ; pareja-jaime et al. ) . importantly, the a -tomatine hydrolysis products b -tomatine, tomatidine and lycotetraose have all been shown to suppress induced defence responses in tomato (bourab et al. ; ito et al. ito et al. , , suggesting that hydrolysis of a -tomatine may serve a dual function during infection of tomato plants by fungi, namely, detoxi fi cation of a preformed toxin and subversion of the hydrolysis products for suppression of induced defences. signi fi cantly, the a -tomatine aglycone, tomatidine, has been shown to inhibit sterol biosynthesis in yeast (simons et al. ) , although the mechanism by which tomatidine and other a -tomatine hydrolysis products interfere with induced plant defence responses is as yet unknown. saponins are exploited as important pharmaceuticals and for a variety of other industrial uses. the triterpenoid ginsenoside saponins (e.g. ginsenoside rb ; fig. . ) are the major bioactive components of ginseng, the roots of which are widely used in traditional chinese medicine. ginsenosides have multiple pharmacological properties, including anti-tumour, immunomodulatory and neurological activity (attele et al. ) . the triterpenoid saponin from liquorice, glycyrrhizin ( fig. . ) , also has wide-ranging medical uses. this compound has antiviral activity and is used in the treatment of hepatitis (arase et al. ) . glycyrrhizin is also active against the hiv and sars viruses (cinatl et al. ; harada ) and in addition has antiin fl ammatory (matsui et al. ) , immunomodulatory (takahara et al. ) and anti-ulcer activity (he et al. ) . the main use of glycyrrhizin globally is, however, as a sweetener in the food industry (kitagawa ) . avicins (e.g. avicin d; fig. . ) are triterpenoid saponins from the australian desert tree acacia victoriae that have anti-tumour activity (jayatilake et al. ) and are used in the treatment of cancer. avicins have a range of physiological effects in mammalian cells including induction of apoptosis (haridas et al. a ) , suppression of in fl ammatory responses (haridas et al. b ) , inhibition of cell proliferation (mujoo et al. ) and prevention of mutagenesis caused by environmental toxins (hanausek et al. ) , all of which may contribute to the anti-cancer properties of this compound. two modes of action have been identi fi ed. avicins permeabilise the mitochondrial outer membrane (lemeshko et al. ; haridas et al. ) ; they also covalently modify a transcription factor leading to modulation of responses to oxidative stress (haridas et al. ) . the main forms of avicin, such as avicin d, are acylated at the c- carbon with a group derived from two monoterpenes joined via a xylose ( fig. . ) (jayatilake et al. ) , and this group is essential for both modes of action (haridas et al. (haridas et al. , . an important adjuvant used to improve the effectiveness of vaccines is a saponin derived from the bark of the south american tree quillaja saponaria , known as qs (kensil et al. ) (fig. . ) . a number of saponins have been shown to act as adjuvants (barr et al. ; oda et al. ) . interestingly, the most effective adjuvant saponins are bidesmosidic (oda et al. ). this contrasts with other biological activities of saponins, which often depend on the amphipathic properties associated with monodesmosidic saponins. a comprehensive review of the pharmacological effects of saponins can be found in sparg et al. ( ) . saponins also have important dietary properties, and their presence in food crops has implications for human health. saponins ingested as part of the human diet have been linked with a variety of effects on health, including reducing blood cholesterol levels (milgate and roberts ; friedman ) . the major steroidal glycoalkaloids found in potato are a -chaconine and a -solanine, which are monodesmosides of the steroidal alkaloid aglycone solanidine that differ only in the nature of the carbohydrate chain (friedman ) . glycoalkaloids accumulate in potato tubers in response to insect damage (hlywka et al. ) and also during post-harvest deterioration after exposure to light or as a result of physical damage (mondy et al. ; dao and friedman ) . a -solanine and a -chaconine are inhibitors of acetylcholine esterase (abbott et al. ; roddick ) , which is also the mode of action of many insecticides and can result in neurological symptoms in animals. consumption of potatoes containing elevated levels of these glycoalkaloids can result in vomiting, diarrhoea, disorientation and death (hansen ; mcmillan and thompson ; korpan et al. ) , and these symptoms are associated with reduced serum cholinesterase activity (mcmillan and thompson ) . the tomato steroidal glycoalkaloid a -tomatine accumulates to high levels in immature tomato fruits (up to mg/kg of fresh fruit weight) (friedman ) . however, the consumption of immature tomatoes does not appear to cause symptoms, and likewise, tomato varieties that accumulate high levels of a -tomatine in the mature fruit do not appear to cause ill effects amongst the peruvians who eat them (rick et al. ) , indicating that a -tomatine is not so toxic to humans as the potato glycoalkaloids. in fact, the steroidal glycoalkaloids from these solanaceous species have been found to have healthpromoting effects. both tomato and potato steroidal glycoalkaloids have antiproliferative effects against human cancer cell lines in vitro (lee et al. ) and have also been shown to act as chemosensitisers, increasing the effectiveness of chemotherapeutic drugs by blocking their export through multi-drug resistance-type transport proteins (lavie et al. ) . in addition, a -tomatine has recently been shown to protect fi sh against tumours induced by an environmental toxin (friedman et al. ) . triterpenes and sterols are derived from a common precursor , -oxidosqualene, which is synthesised from acetyl-coa via mevalonic acid (mva) and isopentyl diphosphate (ipp) (haralampidis et al. ) . figure . shows an outline of saponin biosynthesis. the enzymes that convert , -oxidosqualene into the precursors of more elaborate sterols and triterpenes belong to the oxidosqualene cyclase (osc) family. the products of osc enzymes are diverse, varying principally in the degree of cyclization (haralampidis et al. ; phillips et al. ; lodeiro et al. ; vincken et al. ; abe ) . collectively, oscs are capable of cyclising , -oxidosqualene into a diverse range of different products, highlighting the importance of this single enzymatic step. triterpenoid skeletons alone account for more than different structures that have been described (segura et al. ; connolly and hill ) . the genes encoding the cycloartenol synthase enzyme (cas) are widely conserved across plant lineages, consistent with the role of this enzyme in the synthesis of essential membrane sterols (phillips et al. ) . however, the osc gene family has expanded and diversi fi ed in many plants, providing a molecular basis for triterpene diversity (suzuki et al. ; ebizuka et al. ; phillips et al. ; field and osbourn ) . some osc enzymes produce single cyclization products, while others are multifunctional and generate a variety of different products (kushiro et al. a ; segura et al. ; basyuni et al. ; phillips et al. ; lodeiro et al. .; shibuya et al. ; abe ) . indeed, a single osc enzyme from arabidopsis thaliana was found to be responsible for the synthesis of at least nine distinct triterpenes when heterologously expressed in yeast (kushiro et al. a ) . amongst the best-characterised members of the plant osc family are the sterol synthase, cycloartenol synthase and the triterpene synthase, b -amyrin synthase (haralampidis et al. ; abe ) . synthesis of b -amyrin is the fi rst committed step in the triterpene pathways leading to avenacins in oat, glycyrrhizin in liquorice and soyasaponins in soy (fig. . ) , and b -amyrin synthases have been cloned and characterised from these plant species (chung et al. ; hayashi et al. a ; haralampidis et al. ; shibuya et al. ) . numerous other plant osc enzymes have also been characterised by heterologous expression in yeast (e.g. kushiro et al. a ; hayashi et al. a, b ; kawano et al. ; ebizuka et al. ; zhang et al. ; suzuki et al. ; tansakul et al. ; xiang et al. ; shinozaki et al. a, b ; abe ) . although there is as yet no crystal structure for plant oscs, the structures of the related bacterial enzyme, squalene cyclase (lenhart et al. ) and the human osc lanosterol synthase (thoma et al. ) have been determined empirically. extensive work has also been carried out on investigating the mode of action of oscs through site-directed mutagenesis and directed evolution (dang and prestwich ; kushiro et al. b ; sato and hoshino ; meyer et al. ; wu and grif fi n ; segura et al. ; wu and chang ; wu et al. wu et al. , . in some cases, this knowledge has enabled the rational engineering of osc enzymes to give altered product pro fi les (e.g. kushiro et al. b ; meyer et al. ; lodeiro et al. ; wu and chang ) . the diversity of triterpene and sterol saponin skeletons is not solely due to the variety of cyclization reactions but also to the further elaboration of the structure by oxidative modi fi cations. all triterpenes and sterols have in common a c- hydroxyl group originating from the epoxide of , oxidosqualene, although some osc enzymes can utilise the unusual dioxidosqualene substrate resulting in the synthesis of a product bearing two hydroxyl groups (shan et al. ) (e.g. arabidiol, which is produced by the arabidopsis thaliana arabidiol synthase enzyme ). also, some osc enzymes can accept a range of arti fi cial substrates leading to the synthesis of unusual compounds bearing multiple functional groups (noma et al. ; abe, this volume) . many saponin skeletons include multiple oxidative modi fi cations that are introduced after cyclization, including further hydroxylation, desaturation and epoxidation (begley et al. ; yoshiki et al. ; jayatilake ; qi et al. ; seki et al. ) . oxidation of sterols and triterpenes can in fl uence their biological activity (ji et al. ) , although the functional signi fi cance of these types of modi fi cation for the action of saponins is apparent in only a few examples. the relevance of saponin skeleton modi fi cations has been studied through the chemical modi fi cation of these compounds. chemical modi fi cation of glycyrrhizin to alter the number of hydroxyl groups and positions of desaturations had signi fi cant impact on the inhibition of interleukins in human cell culture (matsui et al. ) . also, in some cases, the comparison of the biological activities of naturally occurring compounds that differ speci fi cally in these types of modi fi cation can reveal the importance of the modi fi cations for activity. for example, the legume saponins avicins d and g differ only by one hydroxyl group, but this difference is suf fi cient to affect their respective abilities to inactivate caspase (haridas et al. b ) , although the hydroxyl group in question is associated with the acyl group and not with the triterpene skeleton. in some instances, this type of approach may also reveal that certain modi fi cations have little impact on biological activity (yoshikawa et al. ) . the avenacin pathway downstream of b -amyrin synthase involves a number of steps, including extensive oxidative modi fi cation of the triterpene ring structure. while plant ocs enzymes are relatively well characterised, the enzymes that catalyse the subsequent functionalisation of the triterpene backbone are only poorly understood (haralampidis et al. ) . a cytochrome p from oat (sad ) has been shown to be required for avenacin synthesis and is likely to mediate oxygenation of b -amyrin at an as yet undetermined position (qi et al. ) . sad belongs to the ancient and highly conserved cyp family of cytochrome p s. prior to the characterisation of sad , cyp enzymes were only known to function in the sterol pathway as sterol demethylases. sad is the fi rst cyp enzyme to be identi fi ed that has a different function -in the synthesis of defence-related triterpene glycosides (avenacins). this unusual cyp is the founder member of a monocotspeci fi c divergent subfamily of cyp enzymes (de fi ned as the cyp h subfamily; nelson et al. ) . the functions of cyp h enzymes in other cereals and grasses await investigation. it is possible that these enzymes may also have roles in plant defence. two other cyp s that mediate modi fi cation of the triterpene backbones of saponins in other plant species have been identi fi ed through bioinformatics-based approaches. the soya cyp e enzyme was identi fi ed using a combination of large-scale expressed sequence tag (est) analysis and gene expression analysis to identify candidate genes involved in soyasaponin biosynthesis. this approach was facilitated by the fact that synthesis of soyasaponins can be induced by elicitor treatment . candidate cyp enzymes identi fi ed in these experiments were functionally characterised by heterologous expression in yeast. the cyp e enzyme was shown to catalyse the -hydroxylation of b -amyrin and also the formation of the -hydroxylated derivative, sophoradiol . a similar approach was used to characterise the triterpene-modifying cyp cyp d from liquorice. cyp d catalyses the oxidation of b -amyrin to -oxo-b -amyrin when expressed in yeast (seki et al. ) . glycosylation of saponins is generally important for the biological activity of these compounds, as discussed above. to date, few of the enzymes responsible for saponin glycosylation have been identi fi ed (bowles et al. ; townsend et al. ) . activity-based protein puri fi cation studies have been successfully applied to some saponin glycosyltransferases. for example, a udpgalactose:tomatidine galactosyltransferase has been puri fi ed from tomato leaves (zimowski ) , and a solanidine glycosyltransferase (sgt) from potato has also been puri fi ed (stapleton et al. ) . these enzymes both catalyse the transfer of sugar moieties onto steroidal glycoalkaloid aglycones. unlike triterpene aglycones, the steroidal alkaloid aglycones solanidine and tomatidine have potent antifungal activity (moehs et al. ; simons et al. ) . this property was exploited to clone a solanidine glycosyltransferase (sgt) gene from potato by expressing a potato cdna library in yeast, plating the transformants onto medium containing solanidine and looking for colonies that had increased solanidine tolerance (moehs et al. ) . this led to the cloning of the potato sgt enzyme, which catalysed the transfer of glucose to the solanidine aglycone (the fi rst committed step in the synthesis of the steroidal glycoalkaloid, a -chaconine) in vitro . the role of sgt in steroidal glycoalkaloid biosynthesis in planta was investigated through antisense rna-mediated gene silencing (mccue et al. ) . silencing of sgt did not result in the expected reduction in a -chaconine levels. however, the levels of a different steroidal glycoalkaloid, a -solanine, were substantially reduced in sgt -silenced plants, suggesting that sgt functions as a galactosyltransferase in planta and not as a glucosyltransferase. indeed, subsequent biochemical characterisation showed that the sgt enzyme has a preference for udpgalactose over udp-glucose in vitro (mccue et al. ) . a second potato glycosyltransferase sgt was subsequently identi fi ed as the primary udp-glucose:solanidine glucosyltransferase (mccue et al. ) . more recently, a rhamnosyltransferase implicated in the extension of the sugar chains of solanidine-derived steroidal glycoalkaloids has also been reported (mccue et al. ) . solanidine glycosyltransferases have been characterised from aubergine (eggplant; solanum melongena ) (paczkowski et al. ; zimowski ) . also, a steroidal saponin glucosyltransferase has been puri fi ed from the medicinal herb withania somnifera , and the corresponding cdna cloned (madina et al. ) . bioinformatics-based approaches to the identi fi cation of saponin glycosyltransferases have also been adopted. medicago truncatula accumulates a range of triterpene saponins in response to elicitor treatment (suzuki et al. (suzuki et al. , . transcriptome analysis has been used to identify transcripts for predicted glycosyltransferases on the basis of co-expression with a cloned b -amyrin synthase gene. subsequent biochemical characterisation revealed that two glycosyltransferases identi fi ed in this way were able to catalyse the glucosylation of triterpene aglycones (suzuki et al. (suzuki et al. , achnine et al. ) and a crystal structure has been obtained for one of these enzymes (shao et al. ) . however, these glycosyltransferases had very broad substrate speci fi city in vitro and were able to glycosylate phenolic compounds more effectively than saponins (suzuki et al. ; shao et al. ) . this highlights a common concern associated with analysis of the properties of enzymes in vitro -namely, that data gained from in vitro studies may not re fl ect the true properties of these enzymes in planta (suzuki et al. ; bowles et al. ) . comparison of the kinetic properties of enzymes in vitro with the availability of substrates in planta offers one means of providing further evidence of likely function in plants. however, the presence of other compounds in planta may modulate the substrate speci fi city of glycosyltransferases. for example, phospholipids of varying types, while not serving as substrates, have been shown to markedly alter the substrate preference of the aubergine solanidine glycosyltransferase (paczkowski et al. ) . it is interesting to note that the glycosyltransferases from m. truncatula that have been implicated in saponin glycosylation are phylogenetically distinct, and each shows similarity to different characterised glycosyltransferases from other species that act on quite different groups of compounds (gachon et al. ) . avenacins are acylated at the c- carbon with n-methyl anthranilate or benzoate (e.g. avenacin a- ; fig. . ). acylation at the c- position in particular has been suggested to be an important factor in the biological activity of saponins (podolak et al. ) . acylation of avenacins is catalysed by a serine carboxypeptidase-like (scpl) enzyme (asscpl ) which is encoded by the sad gene (mugford et al. ; mugford and osbourn ) . scpl acyltransferases have previously been identi fi ed in dicotyledonous species with roles in the acylation of a range of plant natural products (milkowski and strack ) . acyl groups have been identi fi ed in a number of saponins from a range of plant species (fig. . ) (warashina et al. ; kudou et al. ; yoshikawa et al. yoshikawa et al. , yoshikawa et al. , yoshikawa m et al. ; germonprez et al. ; zou et al. ) . examples of triterpenoid saponins acylated with aromatic side chains are found in purple salsify ( tragopogon porrifolius ) (warashina et al. ) , stephanotis lutchuensis (yoshikawa et al. (yoshikawa et al. , and the vietnamese medicinal species maesa balansae (germonprez et al. ) . warashina et al. ( ) isolated tragopogonsaponins -all glycosides of echinocystic acid acylated with the phenylpropanoids p -coumarate, ferulate, -hydroxyphenyl proponoate or -hydroxy, -methoxyphenyl proponoate. yoshikawa et al. ( yoshikawa et al. ( , have identi fi ed a number of anti-sweet acylated triterpenoid saponins -sitakisosides -including some that, like avenacin a- , are acylated with n-methyl anthranilate. germonprez et al. ( ) identi fi ed fi ve forms of triterpenoid saponins from maesa balansae , collectively known as maesabalides, which contain cinnamate and benzoate acyl groups. the maesabalides were found to exhibit anti-leishmanial activity. some saponin acyl groups have been ascribed a biological function through the comparison of acylated saponins and their unacylated counterparts. avenacin-de fi cient oat mutants that are defective in avenacin acylation have been identi fi ed (papadopoulou et al. ; qi et al. ) . these mutants have enhanced susceptibility to fungal pathogens, indicating that acylation is important for disease resistance, although the signi fi cance of this modi fi cation for the stability and antifungal activity of avenacins is not yet known. biological activity of theasaponins from tea ( camellia sinensis ) has been shown to be dependent upon acylation. theasaponins are acylated at both the c- and c- positions by angelate or tiglate (( z )-or ( e )- -methylbut- enoate, respectively) groups (yoshikawa et al. ) . yoshikawa et al. ( ) showed that the gastro-protective effect offered by these compounds against ethanol toxicity was dependent on the presence of these acyl groups. the a -pyranosyl triterpenoid saponin chromosaponin i from pea is conjugated with a , -dihydro- , -dihydroxy- -methyl- h-pyran- -one (ddmp) group by an ether linkage (fig. . ). in addition to its effects on plant growth and development ishizawa , ) , chromosaponin i also has strong antioxidative capacity (tsujino et al. ) . soya also produces triterpenoid saponins conjugated with ddmp, and the antioxidative capacity of these compounds has been shown to be largely due to the presence of the ddmp group (yoshiki et al. ) , suggesting an important contribution of ddmp modi fi cation to saponin activity. the different steps in the synthesis of saponins are likely to occur in different subcellular locations. the early steps in saponin synthesis (mediated by osc and cyp enzymes) are most probably associated with the endoplasmic reticulum (ruf et al. ; qi et al. ; seki et al. ) , while glycosyltransferases are typically found in the cytoplasm (bowles et al. ) . however, avenacins are sequestered in the vacuole (mylona et al. ) , suggesting that at least one transport step is required for their synthesis. future work should lead to the identi fi cation of transporters that are required for saponin synthesis and accumulation. while biological activities have been ascribed for many saponins, the demonstration of the importance of these compounds in planta is a dif fi cult matter to resolve, requiring isogenic (or near isogenic) lines that differ solely in ability/inability to produce saponins. so far, the application of reverse genetics-based approaches for investigation of saponin biosynthesis and function has been limited. transgenic potato plants that have reduced a -solanine content by antisense-mediated silencing of the solanidine glycosyltransferase gene sgt have been generated (mccue et al. ) . this work identi fi ed a different function for the enzyme than had been predicted from biochemical analysis in vitro , highlighting the importance of genetic tests of function in planta . these fi ndings are of commercial relevance since they open up opportunities for reducing steroidal glycoalkaloid levels in plants with ensuing bene fi ts for human health. the impact of this modi fi cation for broader environmental interactions between potato plants and other organisms was not investigated. the strong blue fl uorescence of avenacin a- in oat root tips under uv illumination enables the direct visualisation of the presence of the compound in planta and has provided a facile screen for isolation of avenacin-de fi cient oat mutants (papadopoulou et al. ) . a total of saponinde fi cient ( sad ) mutants with reduced root fl uorescence have been identi fi ed to date (papadopoulou et al. ; qi et al. ; qin et al. ) . these mutants, which represent at least six independent saponin biosynthesis ( sad ) loci, have enhanced susceptibility to disease, consistent with a role for avenacins in plant protection (papadopoulou et al. ) . sad encodes b -amyrin synthase, the osc that catalyses the fi rst committed step in avenacin synthesis (haralampidis et al. ) . remarkably, four of the other loci that have been de fi ned by genetic analysis as being required for avenacin synthesis co-segregate with sad , indicating that the avenacin biosynthetic genes are clustered (qi et al. ) . physical clustering of avenacin pathway genes was con fi rmed by the recent cloning of sad , (qi et al. ) and of sad (mugford et al. ) . the three genes are adjacent and lie within kb of each other. the fi nding that the genes for the avenacin pathway form an operon-like gene cluster was surprising, given our current understanding of eukaryotic genome organisation (osbourn ) . metabolic gene clusters are common amongst the fungi, where there are many examples of gene clusters for natural product pathways (keller and hohn ; bok et al. ; keller et al. ) . however, there are an increasing number of examples of gene clusters for metabolic pathways in plants. other examples include the cyclic hydroxamic acid ( , -dihydroxy- , -benzoxazin- -one, diboa) pathway in maize (frey et al. (frey et al. , gierl and frey ) and the diterpenoid momilactone cluster in rice (shimura et al. ) . both diboa and momilactones are implicated in plant defence. gene clusters for triterpenoid synthesis have also been recently discovered in the model plant arabidopsis thaliana (field and osbourn ; field et al. ) . signi fi cantly, the triterpenoid gene clusters in oat and arabidopsis have evolved recently and independently, suggesting that there is selection for clustering of genes for triterpenoid pathways (qi et al. ; field and osbourn ; osbourn ) . gene clustering will favour inheritance of the genes for the pathway in its entirety by minimising the chances of recombination occurring within the cluster during meiosis, which will provide a selective advantage if the pathway end products confer broad-spectrum disease resistance. there is also evidence to indicate that interference with the integrity of natural product gene clusters can lead to the formation of toxic intermediates (mylona et al. ) , so providing further selection pressure to maintain the cluster as a whole. similarly, the biosynthetic intermediates of some other plant saponins are known to exhibit toxicity to fungi at least and may also have phytotoxic effects (moehs et al. ; simons et al. ) . it has been observed that the early steps in several of the pathways encoded by known gene clusters are closely related to various hormone biosynthetic pathways and that this might be a factor in the evolution of the clusters (chu et al. ) . additionally, the clustering of genes may facilitate tight co-ordinate regulation of gene expression at the level of chromatin (qi et al. ; shimura et al. ; field and osbourn ) . the genes belonging to these metabolic clusters do show highly co-ordinated expression. for example, expression of the rice momilactone genes is co-ordinately induced upon treatment with elicitors or uv light (shimura et al. ) . the oat avenacin gene cluster and the a. thaliana thalianol gene cluster are both coordinately regulated in speci fi c cell types within the roots and are expressed during normal growth and development (qi et al. (qi et al. , field and osbourn ) . the genes within the , -dihydroxy- -methoxy- , -benzoxazin- -one (dimboa) gene cluster in maize are expressed in the shoots of young seedlings (frey et al. ) , although some members of this gene cluster are also expressed in other tissues (von rad et al. ) . however, one would expect the genes required for metabolic pathways to be co-ordinately regulated regardless of physical clustering, and there are many instances of co-ordinate regulation of genes that are not clustered but that belong to a single metabolic pathway (e.g. hemm et al. ) . thus, gene clustering is not essential for co-ordinate expression. research into the chemical diversity of saponins is well documented (hostettmann and marston ) , and the molecular genetics underlying the biosynthesis of these compounds is gaining ground. forward genetics, bioinformatics-based approaches and genome browsing have led to the characterisation of new saponin biosynthetic genes and enzymes (papadopoulou et al. ; suzuki et al. suzuki et al. , qi et al. qi et al. , achnine et al. ; seki et al. ) . the current acceleration in gene discovery will yield biotechnological toolkits (genes, enzymes, regulators, transporters) that will be invaluable in designing strategies for quantitative and qualitative manipulation of saponin content in plants and for production of high-value compounds for commercial use. future work will also shed further light on the ecological signi fi cance of saponins, on the relationship between structure and biological activity and on the mechanisms through which these compounds exert their effects on living cells. observation on the correlation of anticholinesterase effect with solanine content of potatoes enzymatic synthesis of cyclic triterpenes genomics-based selection and functional characterization of triterpene glycosyltransferases from the model legume medicago truncatula herbivore-induced responses in alfalfa ( medicago sativa ) the long term ef fi cacy of glycyrrhizin in chronic hepatitis c patients the membrane-permeabilizing effect of avenacin a- involves the reorganization of bilayer cholesterol hydrolysis of tomatine by septoria lycopersici : a detoxi fi cation mechanism studies on the mode of action of tomatine as a fungitoxic agent the sensitivity of fungi to a -tomatine ginseng pharmacology iscoms and other saponin based adjuvants molecular cloning and functional expression of a multifunctional triterpene synthase cdna from a mangrove species kandelia candel (l.) druce the isolation of avenacin a- , a- , b- and b- , chemical defences against cereal "take-all" disease. structure of their "aglycones", the avenestergenins, and their anhydro dimers secondary metabolic gene cluster silencing in aspergillus nidulans the myrosinase-glucosinolate system, its organisation and biochemistry a saponin-detoxifying enzyme mediates suppression of plant defences glycosyltransferases of lipophilic small molecules host range of a plant pathogenic fungus determined by a saponin detoxifying enzyme avenacin, an antimicrobial substance isolated from avena sativa isolation, characterization, and avenacin sensitivity of a diverse collection of cereal-root-colonizing fungi from hormones to secondary metabolism: the emergence of metabolic gene clusters in plants molecular characterization of the gmams gene encoding b -amyrin synthase in soybean plants glycyrrhizin, an active component of licorice roots, and replication of sars-associated coronavirus distribution of the avenacins a- , a- , b- and b- in oat roots: their fungicidal activity towards take-all fungus structure of the four avenacins, oat root resistance factors to takeall disease site-directed mutagenesis of squalene-hopene cyclase: altered substrate speci fi city and product distribution chlorophyll, chlorogenic acid, glycoalkaloid and protease inhibitor content of fresh and green potatoes natural products and disease resistance legume natural products: understanding and manipulating complex pathways for human and animal health functional genomics approach to the study of triterpene biosynthesis toxic substances in crop plants metabolic diversi fi cationindependent assembly of operon-like gene clusters in different plants first encountersdeployment of defence-related natural products by plants formation of plant metabolic gene clusters within dynamic chromosomal regions the biological action of saponins in animal systems: a review expression of a cytochrome p gene family in maize analysis of a chemical plant defense mechanism in grasses tomato glycoalkaloids: roles in the plant and in the diet potato glycoalkaloids and metabolites: roles in the plant and in the diet protective effect of dietary tomatine against dibenzo[a, l]pyrene (dbp)-induced liver and stomach tumors in rainbow trout plant secondary metabolism glycosyltransferases: the emerging functional analysis new pentacyclic triterpene saponins with strong anti-leishmanial activity from the leaves of maesa balansae evolution of benzoxazinone biosynthesis and indole production in maize studies on roots. i. properties and distribution of fl uorescent constituents in avena roots intense sweeteners for the food industry: an overview soyasaponins: the relationship between chemical structure and colon anticarcinogenic activity biology and biochemistry of glucosinolates avicins, a family of triterpenoid saponins from acacia victoriae (bentham), suppress h-ras mutations and aneuploidy in a murine skin carcinogenesis model two fatal cases of potato poisoning the broad anti-viral agent glycyrrhizin directly modulates the fl uidity of plasma membrane and hiv- envelope a new class of oxidosqualene cyclases directs synthesis of antimicrobial phytoprotectants in monocots biosynthesis of triterpenoid saponins in plants avicins: triterpenoid saponins from acacia victoriae (benthem) induce apoptosis by mitochondrial perturbation avicins, a family of triterpenoid saponins from acacia victoriae (bentham), inhibit activation of nuclear factor-k b by inhibiting both its nuclear localization and ability to bind dna avicinylation (thioesteri fi cation): a protein modi fi cation that can regulate the response to oxidative and nitrosative stress avicins, a novel plant-derived metabolite lowers energy metabolism in tumor cells by targeting the outer mitochondrial membrane from waste products to ecochemicals: fi fty years of research of plant secondary metabolism molecular cloning and characterization of two cdnas for glycyrrhiza glabra squalene synthase cloning and characterization of a cdna encoding b -amyrin synthase involved in glycyrrhizin and soyasaponin biosynthesis in licorice molecular cloning and characterization of isomulti fl orenol synthase, a new triterpene synthase from luffa cylindrica , involved in biosynthesis of bryonolic acid the in fl uence of commonly prescribed synthetic drugs for peptic ulcer on the pharmacokinetic fate of glycyrrhizin from shaoyao-gancao-tang light induces phenylpropanoid metabolism in arabidopsis roots bitterness of saponins and their content in dry peas three plant growth inhibiting saponins from duranta repens effects of insect damage on glycoalkaloid content in potatoes ( solanum tuberosum ) saponins: chemistry and pharmacology of natural products oviposition stimulants in barbarea vulgaris for pieris rapae and p. napi oleracea : isolation, identi fi cation and differential activity post-transcriptional silencing of the tomatinase gene in fusarium oxysporum f. sp lycopersici tomatidine and lycotetraose, hydrolysis products of a -tomatine by fusarium oxysporum tomatinase, suppress induced defense responses in tomato cells ) a -tomatine, the major saponin in tomato, induces programmed cell death mediated by reactive oxygen species in the fungal pathogen fusarium oxysporum isolation and structures of avicins d and g: in vitro tumor-inhibitory saponins derived from acacia victoriae polyoxygenated sterols and triterpenes: chemical structures and biological activities molecular cloning and functional expression of cdnas encoding oxidosqualene cyclases from costus speciosus metabolic pathway gene clusters in fi lamentous fungi fungal secondary metabolism -from biochemistry to genomics separation and characterisation of saponins with adjuvant activity from quillaja saponaria molina cortex dual speci fi city of sterolmediated glycoalkaloid induced membrane disruption molecular basis of glycoalkaloid induced membrane disruption licorice root. a natural sweetener and an important ingredient in chinese medicine anti-tumor-promoting activities of afromosin and soyasaponin i isolated from wisteria brachybotrys potato glycoalkaloids: true safety or false sense of security? isolation and structural elucidation of ddmp-conjugated soyasaponins as genuine saponins from soybean seeds a novel multifunctional triterpene synthase from arabidopsis thaliana mutational studies on triterpene synthases: engineering lupeol synthase into b -amyrin synthase inhibitory effect of steroidal alkaloids on drug transport and multidrug resistance in human cancer cells glycoalkaloids and metabolites inhibit the growth of human colon (ht ) and liver (hepg ) cancer cells avicins, natural anticancer saponins, permeabilize mitochondrial membranes crystal structure of a squalene cyclase in complex with the potential anticholesteremic drug ro - allelopathic activity of root saponins of alfalfa on wheat, corn and barnyardgrass oxidosqualene cyclase second-sphere residues profoundly in fl uence the product pro fi le an oxidosqualene cyclase makes numerous products by diverse mechanisms: a challenge to prevailing concepts of triterpene biosynthesis puri fi cation and characterization of a novel glucosyltransferase speci fi c to b -hydroxy steroidal lactones from withania somnifera and its role in plant stress responses avenacin, an antimicrobial substance isolated from avena sativa . i. isolation and antimicrobial activity effects of targeted replacement of the tomatinase gene on the interaction of septoria lycopersici with tomato plants glycyrrhizin and related compounds down-regulate production of in fl ammatory chemokines il- and eotaxin in a human lung fi broblast cell line metabolic compensation of steroidal glycoalkaloid biosynthesis in transgenic potato tubers: using reverse genetics to con fi rm the in vivo enzyme function of a steroidal alkaloid galactosyltransferase the primary in vivo steroidal alkaloid glucosyltransferase from potato potato glycosterol rhamnosyltransferase, the terminal step in triose side-chain biosynthesis an activator of calcium-dependent potassium channels isolated from a medicinal herb an outbreak of suspected solanine poisoning in schoolboys: examinations of criteria of solanine poisoning heterologous expression of septoria lycopersici tomatinase in cladosporium fulvum : effects on compatible and incompatible interactions with tomato seedlings directed evolution to generate cycloartenol synthase mutants that produce lanosterol the nutritional and biological signi fi cance of saponins serine carboxypeptidaselike acyltransferases cloning and expression of solanidine udp-glucose glucosyltransferase from potato changes in total phenolic, total glycoalkaloid, and ascorbic acid as a result of bruising fungal resistance to plant antibiotics as a mechanism of pathogenesis evolution of serine carboxypeptidase-like acyltransferases in the monocots a serine carboxypeptidase-like acyltransferase is required for synthesis of antimicrobial compounds and disease resistance in oats triterpenoid saponins from acacia victoriae (bentham) decrease tumor cell proliferation and induce apoptosis sad and sad are required for saponin biosynthesis and root development in oat comparative genomics of rice and arabidopsis. analysis of cytochrome p genes and pseudogenes from a monocot and a dicot enzymatic formation of an unnatural novel tetracyclic sesterterpene by b -amyrin synthase relationship between adjuvant activity and amphipathic structure of soyasaponins identi fi cation of a novel triterpenoid saponin from pisum sativum as a speci fi c inhibitor of the diguanylate cyclase of acetobacter xylinum triterpenoids of the gramineae the allelopathic potential of alfalfa root medicagenic acid glycosides and their fate in soil environments saponins and plant defence-a soap story secondary metabolic gene clusters: evolutionary toolkits for chemical innovation an oat species lacking avenacin is susceptible to infection by gaeumannomyces graminis var. tritici dissecting plant secondary metabolism-constitutive chemical defences in cereals the saponinspolar isoprenoids with important and diverse biological activities udp-glucose:solasodine glucosyltransferase from eggplant ( solanum melongena l. ) leaves: partial puri fi cation and characterization phospholipids modulate the substrate speci fi city of soluble udp-glucose:steroid glucosyltransferase from eggplant leaves compromised disease resistance in saponin-de fi cient plants tomatinase from fusarium oxysporum f. sp lycopersici is required for full virulence on tomato plants biosynthetic diversity in plant triterpene cyclization saponins as cytotoxic agents: a review the chemistry and biological signi fi cance of saponins in foods and feedstuffs a gene cluster for secondary metabolism in oat -implications for the evolution of metabolic diversity in plants a different function for a member of an ancient and highly conserved cytochrome p family: from essential sterols to plant defence high throughput screening of mutants of oat that are defective in triterpene synthesis chromosaponin i speci fi cally interacts with aux protein in regulating the gravitropic response of arabidopsis roots high a -tomatine content in ripe fruit of andean lycopersicon esculentum var . cerasiforme : developmental and genetic aspects complex formation between solanaceous steroidal glycoalkaloids and free sterols in vitro the acetylcholinesterase-inhibitory activity of steroidal glycoalkaloids and their aglycons destabilization of liposome membranes by the steroidal glycoalkaloid a -tomatine the monotopic membrane protein human oxidosqualene cyclase is active as monomer fungal sensitivity to and enzymatic degradation of the phytoanticipin a-tomatine the relevance of tomatinase activity in pathogens of tomato: disruption of the b -tomatinase gene in colletotrichum coccodes and septoria lycopersici and heterologous expression of the septoria lycopersici b -tomatinase in nectria haematococca , a pathogen of tomato fruit catalytic function of the residues of phenylalanine and tyrosine conserved in squalene-hopene cyclases arabidopsis thaliana lup converts oxidosqualene to multiple triterpene alcohols and a triterpene diol directed evolution experiments reveal mutations at cycloartenol synthase residue his that dramatically alter catalysis mutagenesis approaches to deduce structure-function relationships in terpene synthases licorice b -amyrin -oxidase, a cytochrome p with a key role in the biosynthesis of the triterpene sweetener glycyrrhizin enzymatic cyclization of dioxidosqualene to heterocyclic triterpenes crystal structures of a multifunctional triterpene/ fl avonoid glycosyltransferase from medicago truncatula identi fi cation of b -amyrin and sophoradiol -hydroxylase by expressed sequence tag mining and functional expression assay origin of structural diversity in natural triterpenes: direct synthesis of seco -triterpene skeletons by oxidosqualene cyclase identi fi cation of a biosynthetic gene cluster in rice for momilactones identi fi cation of a triterpenoid saponin from a crucifer, barbarea vulgaris , as a feeding deterrent to the diamondback moth , plutella xylostella dammaradiene synthase, a squalene cyclase, from dryopteris crassirhizoma nakai squalene cyclase and oxidosqualene cyclase from a fern dual effects of plant steroidal alkaloids on saccharomyces cerevisiae biological activities and distribution of plant saponins puri fi cation and characterization of solanidine glucosyltransferase from the potato electrolytic leakage from plant and fungal tissues and disruption of liposome membranes by a -tomatine variation in sensitivity to tomatine and rishitin among isolates of fusarium oxysporum f. sp. lycopersici , and strains not pathogenic on tomato a genomics approach to the early stages of triterpene saponin biosynthesis in medicago truncatula methyl jasmonate and yeast elicitor induce differential transcriptional and metabolic re-programming in cell suspension cultures of the model legume medicago truncatula lanosterol synthase in dicotyledonous plants effects of glycyrrhizin on hepatitis b surface antigen: a biochemical and morphological study dammarenediol-ii synthase, the fi rst dedicated enzyme for ginsenoside biosynthesis, in panax ginseng insecticidal components from fi eld pea extracts: soyasaponins and lysolecithins insight into steroid scaffold formation from the structure of human oxidosqualene cyclase saponin glycosylation in cereals antioxidative effects of dihydro-g -pyronyl-triterpenoid saponin (chromosaponin-i) involvement of ethylene in chromosaponin-induced stimulation of growth in lettuce roots chromosaponin-i stimulates the elongation of cortical-cells in lettuce roots a g -pyronyl triterpenoid saponin from pisum sativum effects of chromosaponin i and brassinolide on the growth of roots in etiolated arabidopsis seedlings the nature of the resistance of oats to the take-all fungus an enzymic basis for pathogenic speci fi city in ophiobolus graminis saponins, classi fi cation and occurrence in the plant kingdom two glucosyltransferases are involved in detoxi fi cation of benzoxazinoids in maize allelopathic activity of root saponins from alfalfa ( medicago sativa l.) on weeds and wheat novel acylated saponins from tragopogon porrifolius l. isolation and the structures of tragopogonsaponins a-r enzymatic formation of multiple triterpenes by mutation of tyrosine of the oxidosqualene-lanosterol cyclase from saccharomyces cerevisiae conversion of a plant oxidosqualene-cycloartenol synthase to an oxidosqualene-lanosterol cyclase by random mutagenesis histidine residue at position of oxidosqualene-lanosterol cyclase from saccharomyces cerevisiae simultaneously in fl uences cyclization, rearrangement, and deprotonation reactions tryptophan within oxidosqualene-lanosterol cyclase from saccharomyces cerevisiae in fl uences rearrangement and deprotonation but not cyclization reactions a new triterpene synthase from arabidopsis thaliana produces a tricyclic triterpene with two hydroxyl groups antisweet natural products. x: structures of sitakisosides i-v from stephanotis lutchuensis kodiz. var. japonica antisweet natural products. xii: structures of sitakisosides xi-xx from stephanotis lutchuensis kodiz. var. japonica antisweet natural products. xv: structures of jegosaponins a-d from styrax japonica sieb. et zucc bioactive saponins and glycosides. xxiii. triterpene saponins with gastroprotective effect from the seeds of camellia sinensis relationship between chemical structures and biological activities of triterpenoid saponins from soybean oxidosqualene cyclases from cell suspension cultures of betula platyphylla var. japonica : molecular evolution of oxidosqualene cyclases in higher plants characterization of udpgalactose:tomatidine galactosyltransferase from tomato ( lycopersicon esculentum ) leaves synthesis of g -chaconine and g -solanine are catalyzed in potato by two separate glycosyltransferases: udp-glucose:solanidine glucosyltransferase and udp-galactose:solanidine galactosyltransferase diastereoisomeric saponins from albizia julibrissin key: cord- - xn jtmh authors: sargin, seyid ahmet title: potential anti-influenza effective plants used in turkish folk medicine: a review date: - - journal: j ethnopharmacol doi: . /j.jep. . sha: doc_id: cord_uid: xn jtmh ethnopharmacological relevance: due to the outbreaks such as sars, bird flu and swine flu, which we frequently encounter in our century, we need fast solutions with no side effects today more than ever. due to having vast ethnomedical experience and the richest flora ( % endemic) of europe and the middle east, turkey has a high potential for research on this topic. plants that locals have been using for centuries for the prevention and treatment of influenza can offer effective alternatives to combat this problem. in this context, herbal taxa belonging to families were identified among the selected studies conducted in the seven regions of turkey. however, only ( . %) of them were found to be subjected to worldwide in vitro and in vivo research conducted on anti-influenza activity. quercetin and chlorogenic acid, the effectiveness of which has been proven many times in this context, have been recorded as the most common ( . %) active ingredients among the other active substances identified. aim of the study: this study has been carried out to reveal the inventory of plant species that have been used in flu treatment for centuries in turkish folk medicine, which could be used in the treatment of flu or flu-like pandemics, such as covid , that humanity has been suffering with, and also compare them with experimental studies in the literature. materials and methods: the investigation was conducted in two stages on the subject above by using electronic databases, such as web of science, scopus, sciencedirect, proquest, medline, cochrane library, ebsco, highwire press, pubmed and google scholar. the results of both scans are presented in separate tables, together with their regional comparative analysis. results: data obtained on taxa are presented in a table, including anti-influenza mechanism of actions and the active substances. rosa canina ( . %) and mentha x piperita ( . %) were identified as the most common plants used in turkey. also, sambucus nigra ( . %), olea europaea ( . %), eucalyptus spp., melissa officinalis, and origanum vulgare ( . %) emerged as the most investigated taxa. conclusion: this is the first nationwide ethnomedical screening work conducted on flu treatment with plants in turkey. thirty-nine plants have been confirmed in the recent experimental anti-influenza research, which strongly shows that these plants are a rich pharmacological source. also, with ( . %) taxa, detections that have not been investigated yet, they are an essential resource for both national and international pharmacological researchers in terms of new natural medicine searches. considering that the production of antimalarial drugs and their successful use against covid- has begun, this correlation was actually a positive and remarkable piece of data, since there are plants, including centaurea drabifolia subsp. phlocosa (an endemic taxon), that were found to be used in the treatment of both flu and malaria. plants have always been the primary choice for preventing and treating various diseases faced by human beings, and contain specific or broad-spectrum active compounds for almost any type of disease (alaoui-jamali, ) . people living in turkey have also benefited from plants in the prevention and treatment of various diseases for centuries. people living in rural areas still have an especially rich medicinal plant repertoire (ertuğ, ) . although herbal cures such as rosehip tea, peppermint-lemon tea and garlic-lemon tea, which are used to prevent and treat flu outbreaks, are well known by the local people, the vast majority of them and their antiinfluenza effects have not yet been adequately investigated in vitro by the related industries (bekut et al., ) . in virus classification, influenza viruses are rna viruses that comprise of the genera of the family orthomyxoviridae (kawaoka, ) , while human rhinoviruses (hrvs) are within the genus enterovirus and only english and turkish words were used in the search engines. if they exist, their english translations were reviewed for the studies conducted in different languages, such as chinese, korean and french. in this context, approximately articles conducted between january and february throughout turkey were excluded since they did not meet the inclusion criteria and a consensus has been provided among the works on the determination of medicinal plants used by local people for centuries. the list of selected plants from these studies is presented in table . the studies determined to be within the scope of plant screening were reviewed, compared and carefully selected according to the following criteria. accordingly, a study should: • be carried out in an area within the borders of turkey. • performed on ethnobotanical or ethnopharmacological concept layout. • include scientific names and local names of the plants used. in addition, the criterion for choosing the book sources was either the writer having an academic title or the work having been cited. if neither of these were in case, the work was not taken into consideration. the screening of the resulting plants in the world literature was carried out considering the following criteria. accordingly, a study should be: • an experimental (in vitro or in vivo) study, not a review. • included the scientific name of the plant in its title. in case of writing only the english name of the plant, it is obligatory to include the scientific name in the text. • carried out under the headings of "anti-flu, anti-influenza or antiviral activities against influenza". if it contains the active compound(s), it becomes preferable and the mechanism of action is recorded. table contains the scientific names of plants, their families, local names, english common names, parts used, forms used, and references. the validation of the scientific names of the specified plant taxa was provided by the book turkey plant list (vascular plants) (güner et al., ) , the international plant names index (ipni: http://www.ipni.org) and the plant list (http://www.theplantlist.org). english common names of the taxa are placed in the table using the following databases or search engines: eppo global database j o u r n a l p r e -p r o o f , lebanon flora (http://www.lebanon-flora.org), springer link (https://link.springer.com/article), flora of israel online (http://flora.org.il), altervista flora italiana (http://luirig.altervista.org/flora), and plants of the world online (http://www.plantsoftheworldonline.org). taxa for which common english names could not be found have been noted as endemic to turkey, or containing irano-turanian elements. finally, the plants were arranged in alphabetical order according to family names. in order to prove the scientific validity of the ethnobotanical data obtained, the research data of the experimental studies regarding the taxa in the list, as found in the world literature, are shown in a separate table (table ). in this table, the mechanism of action, active compounds and used parts are also included, in addition to the researched taxa and their references. great care has been taken to ensure that the findings obtained in these screening studies belong to experimental studies (in vitro or in vivo), not a review. after obtaining the total list of plants with anti-influenza potential in turkish folk medicine, a comparison was made to determine the similarity percentages in similar studies conducted in neighboring and nearby countries (table ) . to avoid distraction from the subject integrity, not all studies in those countries were included in our comparison. therefore, only the study with the richest content and the highest percentage of similarity from each country was included in the comparison list. studies with a similarity percentage > % were eliminated in the primary elections. ozturk et al. ( a) . southeastern anatolia sargin and büyükcengiz ( ) . mediterranean tuzlacı and doğan ( ) . eastern anatolia tuzlacı and erol ( ) . mediterranean ertuğ ( ) . aegean güneş and Özhatay ( ) . eastern anatolia kılıç ( ) . eastern anatolia kilic and bagci ( ) . eastern anatolia guzel and guzelsemme ( ) . mediterranean ozturk et al. ( b) . mediterranean saraç ( ) . all regions tetik et al. ( ) . eastern anatolia yeşilyurt et al. ( b) . marmara akgül et al. ( ) nacakcı and dutkuner ( ) . mediterranean Özçelik ( ) . mediterranean akan and bakır sade ( ) . southeastern anatolia akbulut et al. ( ) . aegean kurt and karaoğul ( ) . black sea paksoy et al. ( ) . central anatolia sargin et al. ( a) . aegean yılmaz ( ) . aegean demirci and Özhatay ( ) . southeastern anatolia kaval et al. ( ) . eastern anatolia kocabaş and gedik ( ) . mediterranean maranki and maranki ( ) . all regions tuzlacı and eryaşar-aymaz ( ) . marmara ugulu et al. ( ) . aegean tanker et al. ( ) the demand for new antimicrobial agents, especially antivirals, is constantly increasing. this demand arises from the lack of antiviral agents in the market and the emergence of resistant mutants to existing drugs (vijanyan et al., ) . throughout our existence, human beings have always been in search of healing from plants in the fight against winter diseases, but clinical studies have to this point been limited. although the following work is relatively new in turkey, they are promising for future study: duman et al. ( ) elicited in vitro antiviral activity of ribes uva-crispa l and ribes multiflorum kit ex schult, which are naturally grown in turkey, use the methanol and aqueous extracts of the leaves and fruits; dogan et al. ( ) revealed anti-rsv effects of ribes uva-crispa juicy fruit and leaf methanol extracts against the respiratory syncytial virus (rsv) (the cause of a worldwide viral infection), and emphasized their advantages to synthetic drugs; finally, adem et al. ( ) found that natural polyphenols, such as hesperidin, routine, diosmin and apiin were more effective than nelfinavir in treating covid- . the plants (table ) , which have been used by locals in turkey for centuries for the prevention and treatment of influenza and its adverse effects -from colds to sudden deaths from respiratory failure -need to be investigated in this way. today, much more research is needed, as outbreaks such as sars, avian influenza, swine influenza and covid- threaten the existence of human beings every year. distribution of studies by region was performed as follows: in the mediterranean ( . %), in eastern anatolia ( . %), in the marmara and aegean region ( . %), in the black sea ( %), in central and southeastern anatolia ( . %), and general studies across all regions ( . %). the regional distribution of total citations received was as follows: mediterranean: ( . %), eastern anatolia: ( . %), aegean: ( . %), marmara: ( . %), central and southeastern anatolia: ( . %), black sea: ( . %), and general studies covering all regions: ( . %). the reason why the studies conducted in the mediterranean and eastern anatolia regions were highly cited may be due to the fact that there are more plant options, which is the result of having a higher rate of biodiversity and endemism in these regions (güner et al., ) compared to others, that the locals can use in the treatment of influenza. in addition, the topographic structure of the region, and the fact that the region is isolated from city centers in winter conditions (doğanay and orhan, ) may have been a factor for the people living in these rural areas to choose mostly natural treatment methods. it has been determined that plants, selected from studies composing of articles, books, seven theses, three chapters and one congress report in total, belonging to families. these plant taxa most commonly belong to the lamiaceae ( taxa, . %), compositae ( taxa, . %), rosaceae ( taxa, . %), malvaceae ( taxa, . %), and other families ( taxa, . %). the most preferred outcome of the lamiaceae family may be due to the turkish people's preference for flu treatment, as it is the family that contains the highest dosage of essential oils (askun et al., ) . the second family, compositae, is known as turkey's most common family (guner et al., ) . infusions prepared from taxa with capitula flower structures such as its representative chamomile are widely used by local people. therefore, this was an expected result. according to studies conducted in different regions of turkey ( fig. ) , the most common genera are sideritis ( taxa, . %), salvia ( taxa, . %), thymus ( taxa, . %), and origanum ( taxa, . %). this finding may indicate that these genus members are more effective in anti-influenza treatment than other genera. in addition, they are the most favored medicinal tea for the locals of turkey, and even without natural nationwide distribution, it is possible to find these products in almost every public market, herbal and spice shop (ertug ; dogan ) . some species, such as thyme (thymus spp.), melissa (melissa officinalis), lavender (lavandula angustifolia), cassidony (lavandula stoechas) and sage (salvia officinalis), are today being grown in home gardens, balconies or on small farms by rural people for folk medicine use, or for trade and household income (güneş, ; ekşi et al., ) . like thyme, melissa, lavender, and sage, among the identified plants, were wild ( . %), were wild and cultivated ( . %), were endemic ( . %) and (allium cepa, allium sativa and malus domestica) were cultivated ( . %). these parameters are shown in a column in table ; wild taxa as "w", cultivated "c", cultivated & wild "cw" and endemic "e". most of the plant pieces used are aerial parts ( . %), flowers/flowering branches/petals ( . %), leaves ( . %), fruits ( . %), seeds/cones ( . %), roots/bulbs/tubers ( . %), and other parts (stems, buds, barks, whole parts, resins, tars, cupula, bracts, fruit stalks, essential oils and fixed oils) ( . %). those parts were mostly used as infusions ( . %), decoction/boiling ( . %), raw eating/swallowing/salad ( . %), molasses/jam/syrup/juice ( . %), lotion/drop/cataplasm/vapor compression ( . %) and other consumption types (roasting, mouthwash, tincture, mixture and pastes) ( . %) and powdered for spice use ( . %). the taxa having with the most usage types are citrus spp ( types, . %), rosa canina and rubus sanctus ( types, . %) and vitis vinifera ( types, . %), while the taxa with the maximum number of consumption parts belong to rosa canina and tilia tomentosa ( parts, . %), and juniperus oxycedrus ( parts, . %). additionally, rosa canina (with different types of use and different parts) have appeared as the most efficient plants in terms of the total of both part and usage type (table ) . taxa, such as rosa canina (with references and . %) and mentha x piperita (with references and . %) (fig. , red color), have been identified as the most frequently cited plants. the reason why these herbs are highly cited may be a reflection of their stronger protective and therapeutic effects against flu; this may be the result of the experience gained in turkish folk medicine for centuries. we would obviously see this when comparing similar studies between geographically close countries (fig. , blue color). the emergence of the data presented in table in a similar manner as in figure confirms the superior efficacy of these plants, with . % similarity. as a matter of fact, similar results were obtained from studies conducted in neighboring countries, comparing with the taxon list presented in the study, including especially rosa canina ( countries with . %), sambucus nigra ( countires with . %) and mentha x piperita ( countries with . %). while the similarity was seen mostly in iraq ( . %), bosnia and herzegovina ( . %), and cyprus ( . %), the least similarity was seen in montenegro ( . %) and israel ( . %). this may due to the fact that, besides the resemblance of landforms, climate and vegetation, we lived together with the cultures of those countries during the ottoman period for about years. the reason for the low similarity in israel and montenegro may be due to the geographical distance as well as the difference of social-cultural habits, religious rituals, topography and flora (table ) . it was not very surprising that matricaria chamomilla emerged as the plant used most in influenza treatment in countries ( . %) since the spreading area of this plant is very wide and it is very easy for the public to access and use (fig. ) . ( ) lamiaceae boiss. ( ) tuzlacı ( ) experimental research studies carried out in the world in terms of anti-influenza activities have been determined only for out of taxa ( . %). still, among these studies, the active substances were detected for only taxa ( . %); for the remaining taxa ( . %), it was observed that they had not been specified (table ). in table , only "the parts used in research" were given as an idea for these taxa for which active gradients had been "not specified". it is noteworthy that no investigation has been conducted for ( . %) taxa yet (they are highlighted in bold in table ). among these taxa, the most common active chemicals are quercetin and chlorogenic acid ( . %), mentofin ( . %) and , -cineole ( . %). the most preferred mechanisms in research are inhibition of viral replication by inhibiting viral nucleoprotein synthesis or polymerase and neuraminidase activity ( . % out of the mechanisms in total), blocking the receptor site of the viruses by inhibition of neuraminidase, reducing the hemagglutination, or blocking hemadsorption ( . %), inhibition of the virus-induced cytopathic effect by blocking hemadsorption ( . %), and stimulating and boosting of the immunity ( . %). the reason that the six taxa at the end of the list are shown as a line separated from the alphabetical sequence is that there was no significant result for virus inactivation in the experimental studies conducted for them (table ) . according to screening results found in the global literature, the most preferred plants in experimental antiinfluenza studies are sambucus nigra ( . %, out of taxa), olea europaea ( . %), followed by eucalyptus camaldulensis, e. globules, melissa officinalis and origanum vulgare ( . %). the reason for this may be that these plants are easily accessible in nature or from the virtual market environment, and can be obtained for less money. additionally, eucalyptus trees in turkey are also known as "malaria trees", as the infusion prepared from its leaves is used against malaria in traditional medicine (baytop, ; ertuğ, ) . although its effectiveness against covid- has not been fully proven by clinical trials, the widespread use and mass production of chloroquine and similar malaria drugs are permitted in many countries, and positive results continue to be achieved (millán-oñate et al., ; touret and de lamballerie, ) . this correlation of data has been positive and unexpected because there are fourteen more plants, including centaurea drabifolia subsp floccosa (an endemic taxon), which have been detected in this study to be used in the treatment of malaria. these fifteen plants are presented in table by adding the "*" sign to the end of their scientific names. the percentage of compatibility of the plant parts belonging to these ( . %) taxa found between the investigation results in the world literature and ethnobotanical results of the study was found to be . %. this result may prove the fact that for centuries, the locals have been equally justified in their preferences of plant usage. taxa containing quercetin, which has a typical polyphenol structure with anti-influenza activity, are hypericum perforatum, morus alba and papaver rhoeas (kim et al., ; liu et al., ; kim and chung, ) (table ) . it is not accidental that we detected quercetin and chlorogenic acid as the most common active gradients in our screening records, because these compounds are found to be the most effective compounds used in the treatment of influenza. supporting these findings, kumar et al. ( ) stated in a study of mice that quercetin ( fig. a) may be useful as a drug to reduce oxidative stress caused by influenza virus infection in the lungs, and to protect them from the toxic effects of free radicals. in another study, wu et al. ( ) stated that quercetin, which shows inhibitory activity in the early stage of influenza infection, offers a future therapeutic option for developing effective, safe and affordable natural products for the treatment and prophylaxis of influenza virus infections. moreover, nile et al. ( ) , in an investigation of the antiviral and cytotoxic effects of quercetin -glucoside (q g) from dianthus superbus, q g (fig. b) found that this substance showed strong antiviral activity against influenza a and b viruses. therefore, they emphasized that it could be developed and used as a natural anti-influenza drug. on the other hand, chlorogenic acid (cha) is a caffeoylquinic acid constituent (fig. c) found in many vegetables and fruits traditionally used in turkish folk medicine, such as cydonia oblonga, crataegus monogyna, morus alba, hypericum perforatum, eucalyptus globules (baytop, ; ding et al., ; kim and chung, ) . indeed, many researchers including ding et al. ( ) and ren et al. ( ) have pointed out that cha acts as a neuraminidase blocker to inhibit influenza a virus at both in vitro and in vivo levels, thus they stated that cha is potentially beneficial in the treatment of influenza. among the researches, the taxa containing the most active compounds in terms of anti-influenza activity were glycyrrhiza glabra ( chemicals with . % out of the ), papaver rhoeas ( ; . %), morus alba ( ; . %) and punica granatum ( ; . %) ( table ). glycyrrhiza glabra (licorice) is among the oldest and most popular traditional herbal medicines worldwide (grienke et al., ) . also, its roots are one of the most frequently used parts for treating respiratory tract infections in turkish folk medicine (baytop, ; ertuğ, ) . hence, the roots may have appeared to have the greatest number of active ingredients in the screening. this result overlaps with the findings of grienke et al. ( ) because they had emphasized that the accumulation of the plant components exhibits d similarities to known flu neuraminidase inhibitors (which are key enzymes in viral replication and the first-line drug target to fight influenza) according to their basis of a shape-focused virtual screening. therefore, this finding may be pointing out that this plant is more effective and specific than other taxa in terms of anti-influenza activity. active compounds identified (and used parts) alcea olea europaea l. carvacrol (essential oil) shows significant antiviral activity. olive oil was included in formulations to ameliorate its potential cytotoxic effects. vimalanathan and hudson ( ) j o u r n a l p r e -p r o o f olea europaea l. not specified (fruits-essential oil) both in influenza a/h n and hrv , replication cycle and progeny virus production were significantly decreased after the treatment with capeo (an essential oil combination based on three aromatic plants (thymbra capitata, origanum dictamnus and salvia fruticosa in extra-virgin olive oil) origanum vulgare l β-carotene and linoleic acid (aerial parts) decrease influenza virus activation by inhibiting the hemagglutination mancini et al. ( ) origanum vulgare l. carvacrol (essential oil) shows significant antiviral activity. olive oil was included in formulations to ameliorate its potential cytotoxic effects. vimalanathan and hudson ( ) origanum vulgare l. not specified (essential oil) linalool (essential oil) linalool (essential oil) reduce visible cytopathic effects of influenza a/ws/ virus activity by > . %. choi ( ) papaver rhoeas l. kaempferol- -sophoroside, kaempferol- neohesperidoside, kaempferol- -sambubioside, kaempferol- -glucoside, quercetin- -sophoroside, luteolin, chelianthifoline sambucus nigra l. not specified (fruits) exhibit a specific neuraminidase-inhibiting effect krawitz et al. ( ) silybum marianum (l.) gaertn. silymarin (seeds) reduces cytopathic effect (cpe) and inhibits viral mrna synthesis with no cytotoxicity song and choi ( ) thymbra capitata (l.) cav. carvacrol (essential oil) shows significant antiviral activity. olive oil was included in formulations to ameliorate its potential cytotoxic effects. thymbra capitata (l.) cav. apigenin, thymol (aerial parts-essential oil) both in influenza a/h n and hrv , replication cycle and progeny virus production were significantly decreased after the treatment with capeo (an essential oil combination based on three aromatic plants ( the results indicated that the prepared emulsions could elicit a little degree of immunity, but they could not inhibit the anamnestic response and infection. najjari et al. ( ) olea europaea l. * not specified (fruits) the results indicated that the prepared emulsions could elicit a little degree of immunity, but they could not inhibit the anamnestic response and infection. najjari et al. ( ) origanum acutidens (hand.-mazz.) ietsw. * none of the extracts inhibited the reproduction of influenza a/aichi virus in mdck cells sökmen et al. ( ) rosmarinus officinalis l. * carnosic acid (aerial parts) inhibit both a-and b-type hrsv, while it does not affect the replication of influenza a virus shin et al. ( ) teucrium polium l.* not specified (aerial parts) no significant effects on influenza virus infectivity derakhshan ( ) * the taxa that have no significant result for virus inactivation. j o u r n a l p r e -p r o o f in addition, medicinal exotic herbs were detected to have been traditionally used in the treatment of influenza and sold in herbal and public markets. zingiber officinale (ginger), curcuma longa (turmeric), syzygium aromaticum (cloves), piper nigrum (black pepper) and cinnamomum verum (cinnamon) are examples of these plants. information on which parts, methods, and how often these plants are used in flu treatment is given in table . the citrus species presented in table are actually exotic species. for several centuries, they have mainly exhibited a distribution in the aegean and mediterranean coasts in turkey's flora. citrus limon (lemon), c. sinensis (orange), c. reticulata (tangerine), c. paradisi (grapefruit) and c. x aurantium (citrus) are among these types. eucalyptus camaldulensis and e. globulus (eucalyptus trees), another plant that has settled in the flora, are of australian origin and have been used in forestry, roadside landscaping, drying of the marshes and folk medicine practices, such as combating malaria, since the ottoman era (Özgün, ) . the point we should especially emphasize here is that, while herbal products to be released for the treatment of influenza are determined by world health organisation (who) and the european phytotherapy scientific cooperative (escop), and controlled by the turkish government, these standard practices are not yet available for fresh or dried plant taxa that are traditionally consumed and sold in public markets and herbalist shops in turkey. besides, it can never be ignored that medicinal plants are very successful in preventing and treating influenza if used according to the prescriptions specified in their pharmacopoeia. thus, it is necessary to record traditional-empirical practices with proven trial-and-error methods urgently, to demonstrate their activities and active ingredients in vitro or in vivo studies, and to enlighten the public by adding optimal tariffs to their pharmacopoeia by the relevant official standard institutions. in our study, it was also determined that endemic plants were used effectively in influenza treatment and collected from nature. the unconscious collection of endemic and endangered species in the red list of the international association for nature conservation (iucn) should be more carefully monitored using laws, media and educational tools and methods, and the necessary precautions should be urgently taken. j o u r n a l p r e -p r o o f although the first choice for influenza control and reducing the effects of epidemics is a vaccine, it is also known that it is not the fastest and most effective option since modifications in viral proteins require annual adaptation of the influenza vaccine formulation, as noted by nachbagauer and palese ( ) . considering the side effects and complications of antiviral medicines, the search for more effective remedies for fast-spreading pandemic influenza strains continues intensively all over the world today. due to their easy production, low cost, water-solubility, low toxicity and selective effects, medicinal plants, especially herbal essential oils and antiviral compounds found in their aqueous extracts are the most studied natural ingredients in recent times (grienke et al., ) . therefore, natural products such as traditional herbs show great promise in the development of potentially effective new antiviral drugs. particularly, recent studies on phytochemicals, such as quercetin, chlorogenic acid, mentofin, and linalool abundantly found in many plants and vegetables, eliminate the efforts and huge costs of finding lots of antiviral vaccines that need to be renewed every year and allow us to be more optimistic about the successful management of the next influenza outbreaks. turkey has remarkable potential for serious research on this topic due to having vast ethnomedicinal experience and the richest flora of europe and the middle east. this study, conducted in this regard, is the first nationwide ethnomedical screening study conducted on flu treatment with plants in turkey. in particular, we would like to emphasize that the most common detected genus members, such as sideritis ( taxa; . %), salvia ( ; . %), thymus ( ; . %), and origanum ( ; . %) may be more efficient in terms of the anti-influenza targeting than other genera for the interest of the sectors that are researching new natural drug sources. through this study, we strongly recommend these ( . %) plants, which have proved their high antiinfluenza activities and inhibition potentials in the experimental studies, to be subject to clinical research and for widespread use in the near future. also, with ( . %) taxa detections that have not been investigated yet, it is an important resource for both national and international pharmacological researchers. clinical research and evaluation studies required for standard compliance for human use, starting especially with the fifteen plant taxa whose use records against both malaria and influenza were presented in this study, can be begun. with a possible mass production of one or more malaria-like drugs, a significant contribution can be provided to the indigenous people living in that region and to the national economy. therefore, more experimental studies are urgently needed to understand the true value of these plants. based on the data to be obtained, we believe that the future extension of anti-influenza studies, including plant taxa that are frequently used in turkish folk medicine, would be a more effective option. adem, s., eyupoglu, v., sarfraz, i., rasul, a., ali, m., . j o u r n a l p r e -p r o o f ( ) ( ) potential of selected lamiaceae plants in anti (retro) viral therapy ethnomedicinal uses of genus lavandula (lamiaceae) in turkish traditional medicine an ethnobotanical study of medicinal plants in acıpayam (denizli-turkey) an ethnobotanical study of medicinal plants in bayramiç an ethnobotanical study of medicinal plants in turgutlu (manisa-turkey) the preliminary ethnobotanical study of medicinal plants in uşak (turkey. marmara pharm medicinal and wild food plants of marmara island (balikesir-turkey) the medicinal and wild food plants of batman city and kozluk district medicinal and food plants of svaneti and lechkhumi, sakartvelo (republic of georgia) contributions of the ethnobotanical investigation carried out in amasya district of turkey ethnopharmacological survey of medicinal plants in maden (elazig-turkey) assessment of anti-influenza activity and hemagglutination inhibition of plumbago indica and allium sativum extracts sambucus nigra extracts inhibit infectious bronchitis virus at an early point during replication chemical constituents of essential oils possessing anti-influenza a/ws/ virus activity Çermik ilçesi ve köylerinin (diyarbakır) etnobotanik özellikleri traditional medicinal plants of ağrı province agathisflavone, a biflavonoid from anacardium occidentale l., inhibits influenza virus neuraminidase an ethnobotanical study in kahramanmaraş (turkey); wild plants used for medicinal purpose in andirin a research on the present uses of the medicinal plants in de materia medica written by dioscorides in eastern mediterranean region the effects of teucrium polium l. on human influenza virus antiviral activity of chlorogenic acid against influenza a (h n /h n ) virus and its inhibition of neuraminidase antiviral activity of ribes uva-crispa l. extracts in vitro traditionally used wild edible greens in the aegean region of turkey türkiye beşeri coğrafyası. pegem akademi yayıncılık cytotoxic and antiviral activity of ribes uva crispa linn. and ribes multiflorum kit. ex romer and schultes extracts garlic and onions: an eastern tale the aromatic-medicinal plant taxa of pure scots pine stands in sürmene-camburnu (trabzon) wild edible plants of the bodrum area buldan (denizli) etnobotanik alan araştırması tıbbi ve aromatik bitkilerin kullanım alanları ve etiği doğanın mucizesi şifalı bitkiler antiviral potential and molecular insight into neuraminidase inhibiting diarylheptanoids from alpinia katsumadae influenza neuraminidase: a druggable target for natural products computer-guided approach to access the anti-influenza activity of licorice constituents the current status of ethnopharmacobotanical knowledge in Çamlıdere (ankara, turkey) türkiye bitkileri listesi (damarlı bitkiler) (s. - . nezahat gökyiğit botanical garden and flora research association publication wild medicinal plants sold in balıkesir/turkey herbal markets and their using properties medicinal plants used in the uzunköprü district of edirne survey of wild food plants for human consumption in karaisalı (adana-turkey) folk medicine in düzce province (turkey) wild plants used as medicinal purpose in yalova (northwest turkey). turk descriptive study of contemporary status of the traditional knowledge on medicinal plants in bulgaria ethnobotanical features of güneysu (rize) district inhibitory activity of a standardized elderberry liquid extract against clinically-relevant human respiratory bacterial pathogens and influenza a and b viruses antiviral substances in plants of the mint family (labiatae. i. tannin of melissa officinalis effect of quercetin on lipid peroxidation and changes in lung morphology in experimental influenza virus infection bartın'da aktarlarda satılan tıbbi aromatik bitkiler ve Ülkemizdeki pazar payları influenza virology: current topics characterization of neuraminidase inhibitors in korean papaver rhoeas bee pollen contributing to anti-influenza activities in vitro ethnopharmacological survey of traditional drugs sold in israel at the end of the th century intranasal co-administration of , -cineole with influenza vaccine provide cross-protection against influenza virus infection antiviral activity of portulaca oleracea l. against influenza a viruses computational screen and experimental validation of anti-influenza effects of quercetin and chlorogenic acid from traditional chinese medicine evaluation of compounds from oregano (origanum vulgare) that inactivate the influenza virus in host animals kozmik bilim işığında Şifalı bitkiler in vivo anti-viral effect of melaleuca alternifolia (tea tree oil) and olea europaea (olive leaf extract) on vero cell adapted avian influenza virus ethnobotanical study on traditional uses of wild medicinal plants in prokletije mountains (montenegro) successful recovery of covid- pneumonia in a patient from colombia after receiving chloroquine and clarithromycin in vitro anti influenza virus activity, antioxidant potential and total phenolic content of twelve iranian medicinal plants pomegranate peel extract inhibits internalization and replication of the influenza virus: an in vitro study medical ethnobotany of the albanian alps in kosovo a cross-cultural comparison of folk plant uses among albanians, bosniaks, gorani and turks living in south kosovo kumluca (antalya)'da etnobotanik bir çalışma is a universal influenza virus vaccine possible? the effect of the hexanic extracts of fig (ficus carica) and olive (olea europaea) fruit and nanoparticles of selenium on the immunogenicity of the inactivated avian influenza virus subtype h n ethnobotanical study of medicinal plants of sirjan in kerman province probing the effect of quercetin -glucoside from dianthus superbus l against influenza virus infection-in vitro and in silico biochemical and toxicological screening ethnobotanical characteristics of arıcak (elazığ) antiviral activity and cytotoxicity of the lipophilic extracts of various edible plants and their fatty acids chapter : plant diversity of the drylands in southeast anatolia-turkey: role in human health and food security centre for science technology of the non-aligned and other developing countries determination of antiviral activity and cytotoxicity of selected sage (salvia l.) species burdur ili bitki envanteri (ekonomik, nadir ve endemik bitkileri). sistem ofset sağlıklı bir yaşamdır yabancı otlar: türk mutfak kültürü üzerine araştırmalar osmanlı ağaç kültüründe yeni ve egzotik bir tür: okaliptüs. Çağdaş türkiye tarihi araştırmaları dergisi ethnomedicinal uses of the wild vascular plants from european turkey (turkish thrace) ethnopharmacological survey of medicinal plants in ulukışla (niğde-turkey) local knowledge of medicinal plants and wild food plants among tatars and romanians in dobruja traditional uses of medicinal plants in solhan (bingöl-turkey) ethnobotanical study on medicinal plants in bingöl (city center) (turkey) antiviral activity of the oseltamivir and melissa officinalis l. essential oil against avian influenza a virus (h n anti-influenza a virus effect of hypericum perforatum l. extract identification of traditional medicinal plant extracts with novel anti-influenza activity docking and molecular dynamics: simulation of the inhibition of h n influenza virus traditional medicinal plant knowledge among albanians, macedonians and gorani in the sharr mountains (republic of macedonia) study on the effect of anti-influenza virus of the volatile oil of schizonepetae, menthone and pulegone oil-in-water emulsion formulated with eucalyptus leaves extract inhibit influenza virus binding and replication in vitro an ethnobotanical survey from yahyalı (kayseri) and tarsus (mersin) doğanın şifalı eli tıbbi bitkiler ve bitkisel sağlık rehberi. gün ofset an ethnobotanical study of medicinal plants used by the local people of alaşehir (manisa) in turkey ethnobotanical survey of medicinal plants in bozyazı district of mersin ethnomedicinal plants of sarigöl district (manisa), turkey ethnomedicinal plants of aydıncık district of mersin plants used in ethnomedicinal practices in gulnar district of mersin medical ethnobotany on the javor mountain (bosnia and herzegovina) interfering with lipid raft association: a mechanism to control influenza virus infection by sambucus nigra antiviral activity of carnosic acid against respiratory syncytial virus synthesis and in vitro study of novel borneol derivatives as potent inhibitors of the influenza a virus silymarin efficacy against influenza a virus replication in vitro antioxidant, antimicrobial, and antiviral activities of the essential oil and various extracts from herbal parts and callus cultures of origanum acutidens ethnobotanical researches in the southern districts of nevşehir farmasötik botanik traditional uses of some medicinal plants in malatya (turkey) of chloroquine and covid- antiviral effect of an essential oil combination derived from three aromatic plants (coridothymus capitatus (l.) rchb. f., origanum dictamnus l. and salvia fruticosa mill.) against viruses causing infections of the upper respiratory tract turkish folk medicinal plants. part ii: eğirdir (isparta) turkish folk medicinal plants, ix: ovacık (tunceli) the investigation and quantitative ethnobotanical evaluation of medicinal plants used around izmir province, turkey protective and antiviral activities of nigella sativa against avian influenza (h n ) in turkeys ethnobotanical research of medicinal plants in mihalgazi (eskişehir) the carbohydrate-binding plant lectins and the non-peptidic antibiotic pradimicin a target the glycans of the coronavirus envelope glycoproteins the first contribution to the ethnobotany of inland dalmatia: medicinal and wild food plants of the knin area the covid- epidemic antiviral activity of medicinal plants of nilgiris anti-influenza virus activities of commercial oregano oils and their carriers anti-influenza virus activity of essential oils and vapors anti-influenza agents from plants and traditional chinese medicine influenza virus-host interactomes as a basis for antiviral drug development quercetin as an antiviral agent inhibits influenza a virus (iav) entry İyileştiren bitkiler an ethnobotanical survey in selected districts of the black sea region (turkey) plants used as folk medicine in some settlements of the marmara region ethnobotanical features of datça peninsula (muğla) inhibition of several strains of influenza virus in vitro and reduction of symptoms by an elderberry extract (sambucus nigra l.) during an outbreak of influenza b panama eaten raw, infusion, decoction, jam, marmalate tuzlacı and erol ( ) , saraç ( ) , koçyiğit and Özhatay ( ) , Özhatay et al. ( Özhatay et al. ( ), ugulu et al. ( j o u r n a l p r e -p r o o f ( ) . black sea ozturk et al. ( a) . southeastern anatolia sargin and büyükcengiz ( ) . mediterranean tuzlacı and doğan ( ) . eastern anatolia tuzlacı and erol ( ) . mediterranean ertuğ ( ) . aegean güneş and Özhatay ( ) . eastern anatolia kılıç ( ) . eastern anatolia kilic and bagci ( ) . eastern anatolia guzel and guzelsemme ( ) . mediterranean ozturk et al. ( b) . mediterranean saraç ( ) . all regions tetik et al. ( ) . eastern anatolia yeşilyurt et al. ( b) . marmara akgül et al. ( ) . central anatolia bulut et al. ( a) . aegean cansaran and kaya ( ) . black sea güner and selvi ( ) . marmara nacakcı and dutkuner ( ) . mediterranean Özçelik ( ( ), güneş and Özhatay ( ) , İşler ( ) lamiaceae ( ) alcea rosea l. not specified ( kim and chung ( ) nigella sativa l. not specified (seeds) enhance immune responsiveness and suppress pathogenicity of influenza viruses in turkeys umar et al. ( ) olea europaea l. not specified (leaves) blokes the receptor site of the viruses mehmood et al. ( ) olea europaea l. carvacrol (essential oil) shows significant antiviral activity. olive oil was included in formulations to ameliorate its potential cytotoxic effects. vimalanathan and hudson ( ) j o u r n a l p r e -p r o o f sambucus nigra l. not specified (fruits) exhibit a specific neuraminidase-inhibiting effect krawitz et al. ( ) silybum marianum (l.) gaertn.silymarin (seeds) reduces cytopathic effect (cpe) and inhibits viral mrna synthesis with no cytotoxicity song and choi ( ) thymbra capitata (l.) cav. carvacrol (essential oil) shows significant antiviral activity. olive oil was included in formulations to ameliorate its potential cytotoxic effects. vimalanathan and hudson ( ) thymbra capitata (l.) cav. apigenin, thymol (aerial parts-essential oil)both in influenza a/h n and hrv , replication cycle and progeny virus production were significantly decreased after the treatment with capeo (an essential oil combination based on three aromatic plants ( cota tinctoria (l.) j.gay* not specified (aerial parts) no correlation was found between antiviral activity and fatty acid contents of the extracts. orhan et al. ( ) ficus carica l.* not specified (fruits) the results indicated that the prepared emulsions could elicit a little degree of immunity, but they could not inhibit the anamnestic response and infection. najjari et al. ( ) olea europaea l. * not specified (fruits) the results indicated that the prepared emulsions could elicit a little degree of immunity, but they could not inhibit the anamnestic response and infection. najjari et al. ( ) origanum acutidens (hand.-mazz.) ietsw. * none of the extracts inhibited the reproduction of influenza a/aichi virus in mdck cells sökmen et al. ( ) rosmarinus officinalis l. * carnosic acid (aerial parts) inhibit both a-and b-type hrsv, while it does not affect the replication of influenza a virus shin et al. ( ) teucrium polium l.* not specified (aerial parts) no significant effects on influenza virus infectivity derakhshan ( ) * the taxa that have no significant result for virus inactivation.j o u r n a l p r e -p r o o f key: cord- -pyb pt authors: newell-mcgloughlin, martina; re, edward title: the flowering of the age of biotechnology – date: journal: the evolution of biotechnology doi: . / - - - _ sha: doc_id: cord_uid: pyb pt nan the significance of developing genetic and physical maps of the genome, and the importance of comparing the human genome with those of other species. it also suggested a preliminary focus on improving current technology. at the request of the u.s. congress, the office of technology assessment (ota) also studied the issue, and issued a document in -within days of the nrc report -that was similarly supportive. the ota report discussed, in addition to scientific issues, social and ethical implications of a genome program together with problems of managing funding, negotiating policy and coordinating research efforts. prompted by advisers at a meeting in reston, virginia, james wyngaarden, then director of the national institutes of health (nih) , decided that the agency should be a major player in the hgp, effectively seizing the lead from doe. the start of the joint effort was in may (with an "official" start in october) when a -year plan detailing the goals of the u.s. human genome project was presented to members of congressional appropriations committees in mid-february. this document co-authored by doe and nih and titled "understanding our genetic inheritance, the u.s. human genome project: the first five years" examined the then current state of genome science. the plan also set forth complementary approaches of the two agencies for attaining scientific goals and presented plans for administering research agenda; it described collaboration between u.s. and international agencies and presented budget projections for the project. according to the document, "a centrally coordinated project, focused on specific objectives, is believed to be the most efficient and least expensive way" to obtain the -billion base pair map of the human genome. in the course of the project, especially in the early years, the plan stated that "much new technology will be developed that will facilitate biomedical and a broad range of biological research, bring down the cost of many experiments (mapping and sequencing), and finding applications in numerous other fields." the plan built upon the reports of the office of technology assessment and the national research council on mapping and sequencing the human genome. "in the intervening two years," the document said, "improvements in technology for almost every aspect of genomics research have taken place. as a result, more specific goals can now be set for the project." the document describes objectives in the following areas mapping and sequencing the human genome and the genomes of model organisms; data collection and distribution; ethical, legal, and social considerations; research training; technology development; and technology transfer. these goals were to be reviewed each year and updated as further advances occured in the underlying technologies. they identified the overall budget needs to be the same as those identified by ota and nrc, namely about $ million per year for approximately years. this came to $ billion over the entire period of the project. considering that in july , the dna databases contained only seven sequences greater than . mb this was a major leap of faith. this approach was a major departure from the single-investigator-based gene of interest focus that research took hitherto. this sparked much controversy both before and after its inception. critics questioned the usefulness of genomic sequencing, they objected to the high cost and suggested it might divert funds from other, more focused, basic research. the prime argument to support the latter position is that there appeared to be are far less genes than accounted for by the mass of dna which would suggest that the major part of the sequencing effort would be of long stretches of base pairs with no known function, the so-called "junk dna." and that was in the days when the number of genes was presumed to be - , . if, at that stage, the estimated number was guessed to be closer to the actual estimate of - , (later reduced to - , ) this would have made the task seem even more foolhardy and less worthwhile to some. however, the ever-powerful incentive of new diagnostics and treatments for human disease beyond what could be gleaned from the gene-by-gene approach and the rapidly evolving technologies, especially that of automated sequencing, made it both an attractive and plausible aim. charles cantor ( ) , a principal scientist for the department of energy's genome project contended that doe and nih were cooperating effectively to develop organizational structures and scientific priorities that would keep the project on schedule and within its budget. he noted that there would be small short-term costs to traditional biology, but that the long-term benefits would be immeasurable. genome projects were also discussed and developed in other countries and sequencing efforts began in japan, france, italy, the united kingdom, and canada. even as the soviet union collapsed, a genome project survived as part of the russian science program. the scale of the venture and the manageable prospect for pooling data via computer made sequencing the human genome a truly international initiative. in an effort to include developing countries in the project unesco assembled an advisory committee in to examine unesco's role in facilitating international dialogue and cooperation. a privately-funded human genome organization (hugo) had been founded in to coordinate international efforts and serve as a clearinghouse for data. in that same year the european commission (ec) introduced a proposal entitled the "predictive medicine programme." a few ec countries, notably germany and denmark, claimed the proposal lacked ethical sensitivity; objections to the possible eugenic implications of the program were especially strong in germany (dickson ) . the initial proposal was dropped but later modified and adopted in as the "human genome analysis programme" (dickman and aldhous ) . this program committed substantial resources to the study of ethical issues. the need for an organization to coordinate these multiple international efforts quickly became apparent. thus the human genome organization (hugo), which has been called the "u.n. for the human genome," was born in the spring of . composed of a founding council of scientists from seventeen countries, hugo's goal was to encourage international collaboration through coordination of research, exchange of data and research techniques, training, and debates on the implications of the projects (bodmer ) . in august nih began large-scale sequencing trials on four model organisms: the parasitic, cell-wall lacking pathogenic microbe mycoplasma capricolum, the prokaryotic microbial lab rat escherichia coli, the most simple animal caenorhabditis elegans, and the eukaryotic microbial lab rat saccharomyces cerevisiae. each research group agreed to sequence megabases (mb) at cents a base within years. a sub living organism was actually fully sequenced and the complete sequence of that genome, the human cytomegalovirus (hcmv) genome was . mb. that year also saw the casting of the first salvo in the protracted debate on "ownership" of genetic information beginning with the more tangible question of ownership of cells. and, as with the debates of the early eighties, which were to be revisited later in the nineties, the respondent was the university of california. moore v. regents of the university of california was the first case in the united states to address the issue of who owns the rights to an individual's cells. diagnosed with leukemia, john moore had blood and bone marrow withdrawn for medical tests. suspicious of repeated requests to give samples because he had already been cured, moore discovered that his doctors had patented a cell line derived from his cells and so he sued. the california supreme court found that moore's doctor did not obtain proper informed consent, but, however, they also found that moore cannot claim property rights over his body. the quest for the holy grail of the human genome was both inspired by the rapidly evolving technologies for mapping and sequencing and subsequently spurred on the development of ever more efficient tools and techniques. advances in analytical tools, automation, and chemistries as well as computational power and algorithms revolutionized the ability to generate and analyze immense amounts of dna sequence and genotype information. in addition to leading to the determination of the complete sequences of a variety of microorganisms and a rapidly increasing number of model organisms, these technologies have provided insights into the repertoire of genes that are required for life, and their allelic diversity as well as their organization in the genome. but back in many of these were still nascent technologies. the technologies required to achieve this end could be broadly divided into three categories: equipment, techniques, and computational analysis. these are not truly discrete divisions and there was much overlap in their influence on each other. as noted, lloyd smith, michael and tim hunkapiller, and leroy hood conceived the automated sequencer and applied biosystems inc. brought it to market in june . there is no much doubt that when applied biosystems inc. put it on the market that which had been a dream became decidedly closer to an achievable reality. in automating sangers chain termination sequencing system, hood modified both the chemistry and the data-gathering processes. in the sequencing reaction itself, he replaced radioactive labels, which were unstable, posed a health hazard, and required separate gels for each of the four bases. hood developed chemistry that used fluorescent dyes of different colors for each of the four dna bases. this system of "color-coding" eliminated the need to run several reactions in overlapping gels. the fluorescent labels addressed another issue which contributed to one of the major concerns of sequencing -data gathering. hood integrated laser and computer technology, eliminating the tedious process of information-gathering by hand. as the fragments of dna passed a laser beam on their way through the gel the fluorescent labels were stimulated to emit light. the emitted light was transmitted by a lens and the intensity and spectral characteristics of the fluorescence are measured by a photomultiplier tube and converted to a digital format that could be read directly into a computer. during the next thirteen years, the machine was constantly improved, and by a fully automated instrument could sequence up to , , base pairs per year. in three groups came up with a variation on this approach. they developed what is termed capillary electrophoresis, one team was led by lloyd smith (luckey, ) , the second by barry karger , and the third by norman dovichi. in molecular dynamics introduced the megabace, a capillary sequencing machine. and not to be outdone the following year in , the original of the species came up with the abi prism sequencing machine. the is also a capillary-based machine designed to run about eight sets of sequence reactions per day. on the biology side, one of the biggest challenges was the construction of a physical map to be compiled from many diverse sources and approaches in such a way as to insure continuity of physical mapping data over long stretches of dna. the development of dna sequence tagged sites (stss) to correlate diverse types of dna clones aided this standardization of the mapping component by providing mappers with a common language and a system of landmarks for all the libraries from such varied sources as cosmids, yeast artificial chromosomes (yacs) and other rdnas clones. this way each mapped element (individual clone, contig, or sequenced region) would be defined by a unique sts. a crude map of the entire genome, showing the order and spacing of stss, could then be constructed. the order and spacing of these unique identifier sequences composing an sts map was made possible by development of mullis' polymerase chain reaction (pcr), which allows rapid production of multiple copies of a specific dna fragment, for example, an sts fragment. sequence information generated in this way could be recalled easily and, once reported to a database, would be available to other investigators. with the sts sequence stored electronically, there would be no need to obtain a probe or any other reagents from the original investigator. no longer would it be necessary to exchange and store hundreds of thousands of clones for full-scale sequencing of the human genome-a significant saving of money, effort, and time. by providing a common language and landmarks for mapping, sts's allowed genetic and physical maps to be cross-referenced. with a refinement on this technique to go after actual genes, sydney brenner proposed sequencing human cdnas to provide rapid access to the genes stating that 'one obvious way of finding at least a large part of the important [fraction] of the human genome is to look at the sequences of the messenger rna's of expressed genes' (brenner, ) . the following year the man who was to play a pivotal role on the world stage that became the human genome project suggested a way to implement sydney's approach. that player, nih biologist j. craig venter announced a strategy to find expressed genes, using ests (expressed sequence tag) (adams, ) . these so called ests represent a unique stretch of dna within a coding region of a gene, which as sydney suggested would be useful for identifying full-length genes and as a landmark for mapping. so using this approach projects were begun to mark gene sites on chromosome maps as sites of mrna expression. to help with this a more efficient method of handling large chunks of sequences was needed and two approaches were developed. yeast artificial chromosomes, which were developed by david burke, maynard olson, and george carle, increased insert size -fold (david t. burke et al., ) . caltech's second major contribution to the genome project was developed by melvin simon, and hiroaki shizuya. their approach to handling large dna segments was to develop "bacterial artificial chromosomes" (bacs), which basically allow bacteria to replicate chunks greater than , base pairs in length. this efficient production of more stable, large-insert bacs made the latter an even more attractive option, as they had greater flexibility than yacs. in in a collaboration that presages the snp consortium, washington university, st louis mo, was funded by the pharmaceutical company merck and the national cancer institute to provide sequence from those ests. more than half a million ests were submitted during the project (murr l et al., ) . on the analysis side was the major challenge to manage and mine the vast amount of dna sequence data being generated. a rate-limiting step was the need to develop semi-intelligent algorithms to achieve this herculean task. this is where the discipline of bioinformatics came into play. it had been evolving as a discipline since margaret oakley dayhoff used her knowledge of chemistry, mathematics, biology and computer science to develop this entirely new field in the early sixties. she is in fact credited today as a founder of the field of bioinformatics in which biology, computer science, and information technology merge into a single discipline. the ultimate goal of the field is to enable the discovery of new biological insights as well as to create a global perspective from which unifying principles in biology can be discerned. there are three important sub-disciplines within bioinformatics: the development of new algorithms and statistics with which to assess relationships among members of large data sets; the analysis and interpretation of various types of data including nucleotide and amino acid sequences, protein domains, and protein structures; and the development and implementation of tools that enable efficient access and management of different types of information. paralleling the rapid and very public ascent of recombinant dna technology during the previous two decades, the analytic and management tools of the discipline that was to become bioinformatics evolved at a more subdued but equally impressive pace. some of the key developments included tools such as the needleman-wunsch algorithm for sequence comparison which appeared even before recombinant dna technology had been demonstrated as early as ; the smith-waterman algorithm for sequence alignment ( ); the fastp algorithm ( ) and the fasta algorithm for sequence comparison by pearson and lupman in and perl (practical extraction report language) released by larry wall in . on the data management side several databases with ever more effective storage and mining capabilities were developed over the same period. the first bioinformatic/biological databases were constructed a few years after the first protein sequences began to become available. the first protein sequence reported was that of bovine insulin in , consisting of residues. nearly a decade later, the first nucleic acid sequence was reported, that of yeast alanine trna with bases. just one year later, dayhoff gathered all the available sequence data to create the first bioinformatic database. one of the first dedicated databases was the brookhaven protein databank whose collection consisted of ten x-ray crystallographic protein structures (acta. cryst. b, ) . the year saw the creation of the genetics computer group (gcg) as a part of the university of wisconsin biotechnology center. the group's primary and much used product was the wisconsin suite of molecular biology tools. it was spun off as a private company in . the swiss-prot database made its debut in in europe at the department of medical biochemistry of the university of geneva and the european molecular biology laboratory (embl). the first dedicated "bioinformatics" company intelligenetics, inc. was founded in california in . their primary product was the intelligenetics suite of programs for dna and protein sequence analysis. the first unified federal effort, the national center for biotechnology information (ncbi) was created at nih/nlm in and it was to play a crucial part in coordinating public databases, developing software tools for analyzing genome data, and disseminating information. and on the other side of the atlantic, oxford molecular group, ltd. (omg) was founded in oxford, uk by anthony marchington, david ricketts, james hiddleston, anthony rees, and w. graham richards. their primary focus was on rational drug design and their products such as anaconda, asp, and chameleon obviously reflected this as they were applied in molecular modeling, and protein design engineering. within two years ncbi were making their mark when david lipman, eugene myers, and colleagues at the ncbi published the basic local alignment search tool blast algorithm for aligning sequences (altschul et al., ) . it is used to compare a novel sequence with those contained in nucleotide and protein databases by aligning the novel sequence with previously characterized genes. the emphasis of this tool is to find regions of sequence similarity, which will yield functional and evolutionary clues about the structure and function of this novel sequence. regions of similarity detected via this type of alignment tool can be either local, where the region of similarity is based in one location, or global, where regions of similarity can be detected across otherwise unrelated genetic code. the fundamental unit of blast algorithm output is the high-scoring segment pair (hsp). an hsp consists of two sequence fragments of arbitrary but equal length whose alignment is locally maximal and for which the alignment score meets or exceeds a threshold or cutoff score. this system has been refined and modified over the years the two principal variants presently in use being the ncbi blast and wu-blast (wu signifying washington university). the same year that blast was launched two other bioinformatics companies were launched. one was informax in bethesda, md whose products addressed sequence analysis, database and data management, searching, publication graphics, clone construction, mapping and primer design. the second, molecular applications group in california, was to play a bigger part on the proteomics end (michael levitt and chris lee). their primary products were look and segmod which are used for molecular modeling and protein design. the following year in the human chromosome mapping data repository, genome data base (gdb) was established. on a more global level, the development of computational capabilities in general and the internet in specific was also to play a considerable part in the sharing of data and access to databases that rendered the rapidity of the forward momentum of the hgp possible. also in edward uberbacher of oak ridge national laboratory in tennessee developed grail, the first of many gene-finding programs. in the first two "genomics" companies made their appearance. incyte pharmaceuticals, a genomics company headquartered in palo alto, california, was formed and myriad genetics, inc. was founded in utah. incyte's stated goal was to lead in the discovery of major common human disease genes and their related pathways. the company discovered and sequenced, with its academic collaborators (originally synteni from pat brown's lab at stanford), a number of important genes including brca and brca , with mary claire king, epidemiologist at uc-berkeley, the genes linked to breast cancer in families with a high degree of incidence before age . by a low-resolution genetic linkage map of the entire human genome was published and u.s. and french teams completed genetic maps of both mouse and man. the mouse with an average marker spacing of . cm as determined by eric lander and colleagues at whitehead and the human, with an average marker spacing of cm by jean weissenbach and colleagues at ceph (centre d'etude du polymorphisme humaine). the latter institute was the subject of a rather scathing book by paul rabinow ( ) based on what they did with this genome map. in , an american biotechnology company, millennium pharmaceuticals, and the ceph, developed plans for a collaborative effort to discover diabetes genes. the results of this collaboration could have been medically significant and financially lucrative. the two parties had agreed that ceph would supply millennium with germplasm collected from a large coterie of french families, and millennium would supply funding and expertise in new technologies to accelerate the identification of the genes, terms to which the french government had agreed. but in early , just as the collaboration was to begin, the french government cried halt! the government explained that the ceph could not be permitted to give the americans that most precious of substances for which there was no precedent in law -french dna. rabinow's book discusses the tangled relations and conceptions such as, can a country be said to have its own genetic material, the first but hardly the last franco-american disavowal of détente (paul rabinow, ) . the latest facilities such as the joint genome institute (jgi), walnut creek, ca are now able to sequence up to mb per day which makes it possible to sequence whole microbial genomes within a day. technologies currently under development will probably increase this capacity yet further through massively parallel sequencing and/or microfluidic processing making it possible to sequence multiple genotypes from several species. nineteen ninety-two saw one of the first shakeups in the progress of the hgp. that was the year that the first major outsider entered the race when britain's wellcome trust plunked down $ million to join the hgp. this caused a mere ripple while the principal shake-ups occurred stateside. much of the debate and subsequently the direction all the way through the hgp process was shaped by the personalities involved. as noted the application of one of the innovative techniques, namely ests, to do an end run on patenting introduced one of those major players to the fray, craig venter. venter, the high school drop out who reached the age of majority in the killing fields of vietnam was to play a pivotal role in a more "civilized" but no less combative field of human endeavor. he came onto the world stage through his initial work on ests while at the national institute of neurological disorders and stroke (ninds) from to . he noted in an interview with the scientist magazine in , that there was a degree of ambiguity at ninds about his venturing into the field of genomics, while they liked the prestige of hosting one of the leaders and innovators in his newly emerging field, they were concerned about him moving outside the nind purview of the human brain and nervous system. ultimately, while he proclaimed to like the security and service infrastructure this institute afforded him, that same system became too restrictive for his interests and talent. he wanted the whole canvas of human-gene expression to be his universe, not just what was confined to the central nervous system. he was becoming more interested in taking a whole genome approach to understanding the overall structure of genomes and genome evolution, which was much broader than the mission of ninds. he noted, with some irony, in later years that the then current nih director harold varmus had wished in hindsight that nih had pushed to do a similar database in the public domain, clearly in venter's opinion varmus was in need of a refresher course in history! bernadine healy, nih director in , was one of the few in a leadership role who saw the technical and fiscal promise of venter's work and, like all good administrators, it also presented an opportunity to resolve a thorny "personnel" issue. she appointed him head of the ad hoc committee to have an intramural genome program at nih to give the head of the hgp (that other larger than life personality jim watson) notice that he was not the sole arbitrator of the direction for the human genome project. however venter very soon established himself as an equally non-conformist character and with the tacit consent of his erstwhile benefactor. he initially assumed the mantle of a non-conformist through guilt by association rather than direct actions when it was revealed that nih was filing patent applications on thousands of these partial genes based on his ests catalyzing the first hgp fight at a congressional hearing. nih's move was widely criticized by the scientific community because, at the time, the function of genes associated with the partial sequences was unknown. critics charged that patent protection for the gene segments would forestall future research on them. the patent office eventually rejected the patents, but the applications sparked an international controversy over patenting genes whose functions were still unknown. interestingly enough despite nih's reliance on the est/cdna technique, venter, who was now clearly venturing outside the ninds mandated rubric, could not obtain government funding to expand his research, prompting him to leave nih in . he moved on to become president and director of the institute for genomic research (tigr), a nonprofit research center based in gaithersburg, md. at the same time william haseltine formed a sister company, human genome sciences (hgs), to commercialize tigr products. venter continued est work at tigr, but also began thinking about sequencing entire genomes. again, he came up with a quicker and faster method: whole genome shotgun sequencing. he applied for an nih grant to use the method on hemophilus influenzae, but started the project before the funding decision was returned. when the genome was nearly complete, nih rejected his proposal saying the method would not work. in a triumphal flurry in late may and with a metaphorical nose-thumbing at his recently rejected "unworkable" grant venter announced that tigr and collaborators had fully sequenced the first free-living organism -haemophilus influenzae. in november , controversy surrounding venter's research escalated. access restrictions associated with a cdna database developed by tigr and its rockville, md.-based biotech associate, human genome sciences (hgs) inc. -including hgs's right to preview papers on resulting discoveries and for first options to license products -prompted merck and co. inc. to fund a rival database project. in that year also britain "officially" entered the hgp race when the wellcome trust trumped down $ million (as mentioned earlier). the following year hgs was involved in yet another patenting debacle forced by the rapid march of technology into uncharted patent law territory. on june , hgs applied for a patent on a gene that produces a "receptor" protein that is later called ccr . at that time hgs has no idea that ccr is an hiv receptor. in december , u.s. researcher robert gallo, the co-discoverer of hiv, and colleagues found three chemicals that inhibit the aids virus but they did not know how the chemicals work. in february , edward berger at the nih discovered that gallo's inhibitors work in late-stage aids by blocking a receptor on the surface of t-cells. in june of that year in a period of just days, five groups of scientists published papers saying ccr is the receptor for virtually all strains of hiv. in january , schering-plough researchers told a san francisco aids conference that they have discovered new inhibitors. they knew that merck researchers had made similar discoveries. as a significant valentine in the u.s. patent and trademark office (uspto) grants hgs a patent on the gene that makes ccr and on techniques for producing ccr artificially. the decision sent hgs stock flying and dismayed researchers. it also caused the uspto to revise its definition of a "patentable" drug target. in the meantime haseltine's partner in rewriting patenting history, venter turned his focus to the human genome. he left tigr and started the for-profit company celera, a division of pe biosystems, the company that at times, thanks to hood and hunkapillar, led the world in the production of sequencing machines. using these machines, and the world's largest civilian supercomputer, venter finished assembling the human genome in just three years. following the debacle with the then nih director bernine healy over patenting the partial genes that resulted from est analysis, another major personality-driven event in that same year occurred. watson strongly opposed the idea of patenting gene fragments fearing that it would discourage research, and commented that "the automated sequencing machines 'could be run by monkeys.' " (nature june , ) with this dismissal watson resigned his nih nchgr post in to devote his full-time effort to directing cold spring harbor laboratory. his replacement was of a rather more pragmatic, less flamboyant nature. while venter maybe was described as an idiosyncratic shogun of the shotgun, francis collins was once described as the king arthur of the holy grail that is the human genome project. collins became the director of the national human genome research institute in . he was considered the right man for the job following his success (along with lap-chee tsui) in identifying the gene for the cystic fibrosis transmembrane (cftr) chloride channel receptor that, when mutated, can lead to the onset of cystic fibrosis. although now indelibly connected with the topic non-plus tout in biology, like many great innovators in this field before him, francis collins had little interest in biology as he grew up on a farm in the shenandoah valley of virginia. from his childhood he seemed destined to be at the center of drama, his father was professor of dramatic arts at mary baldwin college and the early stage management of career was performed on a stage he built on the farm. while the physical and mathematical sciences held appeal for him, being possessed of a highly logical mind, collins found the format in which biology was taught in the high school of his day mind-numbingly boring, filled with dissections and rote memorization. he found the contemplation of the infinite outcomes of dividing by zero (done deliberately rather than by accident as in einstein's case) far more appealing than contemplating the innards of a frog. that biology could be gloriously logical only became clear to collins when, in , he entered yale with a degree in chemistry from the university of virginia and was first exposed to the nascent field of molecular biology. anecdotally it was the tome, the book of life, penned by the theoretical physicist father of molecular biology, edwin schrodinger, while exiled in trinity college dublin in that was the catalyst for his conversion. like schrodinger he wanted to do something more obviously meaningful (for less than hardcore physicists at least!) than theoretical physics, so he went to medical school at unc-chapel hill after completing his chemistry doctorate in yale, and returned to the site of his road to damascus for post-doctoral study in the application of his newfound interest in human genetics. during this sojourn at yale, collins began working on developing novel tools to search the genome for genes that cause human disease. he continued this work, which he dubbed "positional cloning," after moving to the university of michigan as a professor in . he placed himself on the genetic map when he succeeded in using this method to put the gene that causes cystic fibrosis on the physical map. while a less colorful-in-your-face character than venter he has his own personality quirks, for example, he pastes a new sticker onto the back of his motorcycle helmet every time he finds a new disease gene. one imagines that particular piece of really estate is getting rather crowded. interestingly it was not these four hundred pound us gorillas who proposed the eventually prescient timeline for a working draft but two from the old power base. in meetings in the us in , john sulston and bob waterston proposed to produce a 'draft' sequence of the human genome by , a full five years ahead of schedule. while agreed by most to be feasible it meant a rethinking of strategy and involved focusing resources on larger centers and emphasizing sequence acquisition. just as important, it asserts the value of draft quality sequence to biomedical research. discussion started with the british based wellcome trust as possible sponsors (marshall e. ) . by a rough draft of the human genome map was produced showing the locations of more than , genes. the map was produced using yeast artificial chromosomes and some chromosomes -notably the littlest -were mapped in finer detail. these maps marked an important step toward clone-based sequencing. the importance was illustrated in the devotion of an entire edition of the journal nature to the subject. (nature : - ) the duel between the public and private face of the hgp progressed at a pace over the next five years. following release of the mapping data some level of international agreement was decided on sequence data release and databases. they agreed on the release of sequence data, specifically, that primary genomic sequence should be in the public domain to encourage research and development to maximize its benefit to society. also that it be rapidly released on a daily basis with assemblies of greater than kb and that the finished annotated sequence should be submitted immediately to the public databases. in an international consortium completed the sequence of the genome of the workhorse yeast saccharomyces cerevisiae. data had been released as the individual chromosomes were completed. the saccharomyces genome database (sgd) was created to curate this information. the project collects information and maintains a database of the molecular biology of s. cerevisiae. this database includes a variety of genomic and biological information and is maintained and updated by sgd curators. the sgd also maintains the s. cerevisiae gene name registry, a complete list of all gene names used in s. cerevisiae. in a new more powerful diagnostic tool termed snps (single nucleotide polymorphisms) was developed. snps are changes in single letters in our dna code that can act as markers in the dna landscape. some snps are associated closely with susceptibility to genetic disease, our response to drugs or our ability to remove toxins. the snp consortium although designated a limited company is a nonprofit foundation organized for the purpose of providing public genomic data. it is a collaborative effort between pharmaceutical companies and the wellcome trust with the idea of making available widely accepted, high-quality, extensive, and publicly accessible snp map. its mission was to develop up to , snps distributed evenly throughout the human genome and to make the information related to these snps available to the public without intellectual property restrictions. the project started in april and was anticipated to continue until the end of . in the end, many more snps, about . million total, were discovered than was originally planned. by the complete genome sequence of mycobacterium tuberculosis was published by teams from the uk, france, us and denmark in june . the abi prism sequencing machine, a capillary-based machine designed to run about eight sets of sequence reactions per day also reached the market that year. that same year the genome sequence of the first multicellular organism, c. elegans was completed. c. elegans has a genome of about mb and, as noted, is a primitive animal model organism used in a range of biological disciplines. by november the human genome draft sequence reached mb and the first complete human chromosome was sequenced -this first was reached on the east side of the atlantic by the hgp team led by the sanger centre, producing a finished sequence for chromosome , which is about million base-pairs and includes at least genes. according to anecdotal evidence when visiting his namesake centre, sanger asked: "what does this machine do then?" "dideoxy sequencing" came the reply, to which fred retorted: "haven't they come up with anything better yet?" as will be elaborated in the final chapter the real highlight of was production of a 'working draft' sequence of the human genome, which was announced simultaneously in the us and the uk. in a joint event, celera genomics announced completion of their 'first assembly' of the genome. in a remarkable special issue, nature included a -page article by the human genome project partners, studies of mapping and variation, as well as analysis of the sequence by experts in different areas of biology. science published the article by celera on their assembly of hgp and celera data as well as analyses of the use of the sequence. however to demonstrate the sensitivity of the market place to presidential utterances the joint appearances by bill clinton and tony blair touting this major milestone turned into a major cold shower when clinton's reassurance of access of the people to their genetic information caused a precipitous drop in celera's share value overnight. clinton's assurance that, "the effort to decipher the human genome will be the scientific breakthrough of the century -perhaps of all time. we have a profound responsibility to ensure that the life-saving benefits of any cutting-edge research are available to all human beings." (president bill clinton, wednesday, march , ) stands in sharp contrast to the statement from venter's colleague that " any company that wants to be in the business of using genes, proteins, or antibodies as drugs has a very high probability of running afoul of our patents. from a commercial point of view, they are severely constrained -and far more than they realize." (william a. haseltine, chairman and ceo, human genome sciences). the huge sell-off in stocks ended weeks of biotech buying in which those same stocks soared to unprecedented highs. by the next day, however, the genomic company spin doctors began to recover ground in a brilliant move which turned the clinton announcement into a public relations coup. all major genomics companies issued press releases applauding president clinton's announcement. the real news they argued, was that "for the first time a president strongly affirmed the importance of gene based patents." and the same bill haseltine of human genome sciences positively gushed as he happily pointed out that he "could begin his next annual report with the [president's] monumental statement, and quote today as a monumental day." as distinguished harvard biologist richard lewontin notes: "no prominent molecular biologist of my acquaintance is without a financial stake in the biotechnology business. as a result, serious conflicts of interest have emerged in universities and in government service (lewontin, ) . away from the spin doctors perhaps eric lander may have best summed up the herculean effort when he opined that for him "the human genome project has been the ultimate fulfilment: the chance to share common purpose with hundreds of wonderful colleagues towards a goal larger than ourselves. in the long run, the human genome project's greatest impact might not be the three billion nucleotides of the human chromosomes, but its model of scientific community." (ridley, ) . gene therapy the year also marked the passing of another milestone that was intimately connected to one of the fundamental drivers of the hgp. the california hereditary disorders act came into force and with it one of the potential solutions for human hereditary disorders. w. french anderson in the usa reported the first successful application of gene therapy in humans. the first successful gene therapy for a human disease was successfully achieved for severe combined immune deficiency (scid) by introducing the missing gene, adenosine deaminase deficiency (ada) into the peripheral lymphocytes of a -year-old girl and returning modified lymphocytes to her. although the results are difficult to interpret because of the concurrent use of polyethylene glycol-conjugated ada commonly referred to as pegylated ada (pgla) in all patients, strong evidence for in vivo efficacy was demonstrated. ada-modified t cells persisted in vivo for up to three years and were associated with increases in t-cell number and ada enzyme levels, t cells derived from transduced pgla were progressively replaced by marrow-derived t cells, confirming successful gene transfer into long-lived progenitor cells. ashanthi desilva, the girl who received the first credible gene therapy, continues to do well more than a decade later. cynthia cutshall, the second child to receive gene therapy for the same disorder as desilva, also continues to do well. within years (by january ), more than gene therapy protocols had been approved in the us and worldwide, researchers launched more than clinical trials to test gene therapy against a wide array of illnesses. surprisingly, a disease not typically heading the charts of heritable disorders, cancer has dominated the research. in cancer patients were treated with the tumor necrosis factor gene, a natural tumor fighting protein which worked to a limited extent. even more surprisingly, after the initial flurry of success little has worked. gene therapy, the promising miracle of failed to deliver on its early promise over the decade. apart from those examples, there are many diseases whose molecular pathology is, or soon will be, well understood, but for which no satisfactory treatments have yet been developed. at the beginning of the nineties it appeared that gene therapy did offer new opportunities to treat these disorders both by restoring gene functions that have been lost through mutation and by introducing genes that can inhibit the replication of infectious agents, render cells resistant to cytotoxic drugs, or cause the elimination of aberrant cells. from this "genomic" viewpoint genes could be said to be viewed as medicines, and their development as therapeutics should embrace the issues facing the development of small-molecule and protein therapeutics such as bioavailability, specificity, toxicity, potency, and the ability to be manufactured at large scale in a cost-effective manner. of course for such a radical approach certain basal level criteria needed to be established for selecting disease candidates for human gene therapy. these include, such factors as the disease is an incurable, life-threatening disease; organ, tissue, and cell types affected by the disease have been identified; the normal counterpart of the defective gene has been isolated and cloned; either the normal gene can be introduced into a substantial subfraction of the cells from the affected tissue, or the introduction of the gene into the available target tissue, such as bone marrow, will somehow alter the disease process in the tissue affected by the disease; the gene can be expressed adequately (it will direct the production of enough normal protein to make a difference); and techniques are available to verify the safety of the procedure. an ideal gene therapeutic should, therefore, be stably formulated at room temperature and amenable to administration either as an injectable or aerosol or by oral delivery in liquid or capsule form. the therapeutic should also be suitable for repeat therapy, and when delivered, it should neither generate an immune response nor be destroyed by tissue-scavenging mechanisms. when delivered to the target cell, the therapeutic gene should then be transported to the nucleus, where it should be maintained as a stable plasmid or chromosomal integrant, and be expressed in a predictable, controlled fashion at the desired potency in a cell-specific or tissue-specific manner. in addition to the ada gene transfer in children with severe combined immunodeficiency syndrome, a gene-marking study of epstein-barr virus-specific cytotoxic t cells, and trials of gene-modified t cells expressing suicide or viral resistance genes in patients infected with hiv were studied in the early nineties. additional strategies for t-cell gene therapy which were pursued later in the decade involve the engineering of novel t-cell receptors that impart antigen specificity for virally infected or malignant cells. issues which still are not resolved include nuclear transport, integration, regulated gene expression and immune surveillance. this knowledge, when finally understood and applied to the design of delivery vehicles of either viral or non-viral origin, will assist in the realization of gene therapeutics as safe and beneficial medicines that are suited to the routine management of human health. scientists are also working on using gene therapy to generate antibodies directly inside cells to block the production of harmful viruses such as hiv or even cancer-inducing proteins. there is a specific connection with francis collins, as his motivation for pursuing the hgp was his pursuit of defective genes beginning with the cystic fibrosis gene. this gene, called the cf transmembrane conductance regulator, codes for an ion channel protein that regulates salts in the lung tissue. the faulty gene prevents cells from excreting salt properly causing a thick sticky mucus to build up and destroy lung tissue. scientists have spliced copies of the normal genes into disabled adeno viruses that target lung tissues and have used bronchioscopes to deliver them to the lungs. the procedure worked well in animal studies however clinical trials in humans were not an unmitigated success. because the cells lining the lungs are continuously being replaced the effect is not permanent and must be repeated. studies are underway to develop gene therapy techniques to replace other faulty genes. for example, to replace the genes responsible for factor viii and factor ix production whose malfunctioning causes hemophilia a and b respectively; and to alleviate the effects of the faulty gene in dopamine production that results in parkinson's disease. apart from technical challenges such a radical therapy also engenders ethical debate. many persons who voice concerns about somatic-cell gene therapy use a "slippery slope" argument. it sounds good in theory but where does one draw the line. there are many issues yet to be resolved in this field of thorny ethics "good" and "bad" uses of the gene modification, difficulty of following patients in long-term clinical research and such. many gene therapy candidates are children who are too young to understand the ramifications of this treatment: conflict of interest -pits individuals' reproductive liberties and privacy interests against the interests of insurance companies or society. one issue that is unlikely to ever gain acceptance is germline therapy, the removal of deleterious genes from the population. issues of justice and resource allocation also have been raised: in a time of strain on our health care system, can we afford such expensive therapy? who should receive gene therapy? if it is made available only to those who can afford it, then a number of civil rights groups claim that the distribution of desirable biological traits among different socioeconomic and ethnic groups would become badly skewed adding a new and disturbing layer of discriminatory behavior. indeed a major setback occurred before the end of the decade in . jesse gelsinger was the first person to die from gene therapy, on september , , and his death created another unprecedented situation when his family sued not only the research team involved in the experiment (u penn), the company genovo inc., but also the ethicist who offered moral advice on the controversial project. this inclusion of the ethicist as a defendant alongside the scientists and school was a surprising legal move that puts this specialty on notice, as will no doubt be the case with other evolving technologies such as stem cells and therapeutic cloning, that its members could be vulnerable to litigation over the philosophical guidance they provide to researchers. the penn group principal investigator james wilson approached ethicist arthur caplan about their plans to test the safety of a genetically engineered virus on babies with a deadly form of the liver disorder, ornithine transcarbamylase deficiency. the disorder allows poisonous levels of ammonia to build up in the blood system. caplan steered the researchers away from sick infants, arguing that desperate parents could not provide true informed consent. he said it would be better to experiment on adults with a less lethal form of the disease who were relatively healthy. gelsinger fell into that category. although he had suffered serious bouts of ammonia buildup, he was doing well on a special drug and diet regimen. the decision to use relatively healthy adults was controversial because risky, unproven experimental protocols generally use very ill people who have exhausted more traditional treatments, so have little to lose. in this case, the virus used to deliver the genes was known to cause liver damage, so some scientists were concerned it might trigger an ammonia crisis in the adults. wilson underestimated the risk of the experiment, omitted the disclosure about possible liver damage in earlier volunteers in the experiment and failed to mention the deaths of monkeys given a similar treatment during pre-clinical studies. a food and drug administration investigation after gelsinger's death found numerous regulatory violations by wilson's team, including the failure to stop the experiment and inform the fda after four successive volunteers suffered serious liver damage prior to the teen's treatment. in addition, the fda said gelsinger did not qualify for the experiment, because his blood ammonia levels were too high just before he underwent the infusion of genetic material. the fda suspended all human gene experiments by wilson and the university of penn subsequently restricting him solely to animal studies. a follow-up fda investigation subsequently alleged he improperly tested the experimental treatment on animals. financial conflicts of interest also surrounded james wilson, who stood to personally profit from the experiment through genovo his biotechnology company. the lawsuit was settled out of court for undisclosed terms in november . the fda also suspended gene therapy trials at st. elizabeth's medical center in boston, a major teaching affiliate of tufts university school of medicine, which sought to use gene therapy to reverse heart disease, because scientists there failed to follow protocols and may have contributed to at least one patient death. in addition, the fda temporarily suspended two liver cancer studies sponsored by the schering-plough corporation because of technical similarities to the university of pennsylvania study. some research groups voluntarily suspended gene therapy studies, including two experiments sponsored by the cystic fibrosis foundation and studies at beth israel deaconess medical center in boston aimed at hemophilia. the scientists paused to make sure they learned from the mistakes. the nineties also saw the development of another "high-thoughput" breakthrough, a derivative of the other high tech revolution namely dna chips. in biochips were developed for commercial use under the guidance of affymetrix. dna chips or microarrays represent a "massively parallel" genomic technology. they facilitate high throughput analysis of thousands of genes simultaneously, and are thus potentially very powerful tools for gaining insight into the complexities of higher organisms including analysis of gene expression, detecting genetic variation, making new gene discoveries, fingerprinting strains and developing new diagnostic tools. these technologies permit scientists to conduct large scale surveys of gene expression in organisms, thus adding to our knowledge of how they develop over time or respond to various environmental stimuli. these techniques are especially useful in gaining an integrated view of how multiple genes are expressed in a coordinated manner. these dna chips have broad commercial applications and are now used in many areas of basic and clinical research including the detection of drug resistance mutations in infectious organisms, direct dna sequence comparison of large segments of the human genome, the monitoring of multiple human genes for disease associated mutations, the quantitative and parallel measurement of mrna expression for thousands of human genes, and the physical and genetic mapping of genomes. however the initial technologies, or more accurately the algorithms used to extract information, were far from robust and reproducible. the erstwhile serial entrepreneur, al zaffaroni (the rebel who in founded alza when syntex ignored his interest in developing new ways to deliver drugs) founded yet another company, affymetrix, under the stewardship of stephen fodor, which was subject to much abuse for providing final extracted data and not allowing access to raw data. as with other personalities of this high through put era, seattle-bred steve fodor was also somewhat of a polymath having contributed to two major technologies, microarrays and combinatorial chemistry, the former has delivered on it's, promise while the latter, like gene therapy, is still in a somewhat extended gestation. and despite the limitations of being an industrial scientist he has had a rather prolific portfolio of publications. his seminal manuscripts describing this work have been published in all the journals of note, science, nature and pnas and was recognized in by the aaas by receiving the newcomb-cleveland award for an outstanding paper published in science. fodor began his industrial career in yet another zaffaroni firm. in he was recruited to the affymax research institute in palo alto where he spearheaded the effort to develop high-density arrays of biological compounds. his initial interest was in the broad area of what came to be called combinatorial chemistry. of the techniques developed, one approach permitted high resolution chemical synthesis in a light-directed, spatially-defined format. in the days before positive selection vectors, a researcher might have screened thousands of clones by hand with an oligonucleotide probe just to find one elusive insert. fodor's (and his successors) dna array technology reverses that approach. instead of screening an array of unknowns with a defined probe -a cloned gene, pcr product, or synthetic oligonucleotide -each position or "probe cell" in the array is occupied by a defined dna fragment, and the array is probed with the unknown sample. fodor used his chemistry and biophysics background to develop very dense arrays of these biomolecules by combining photolithographic methods with traditional chemical techniques. the typical array may contain all possible combinations of all possible oligonucleotides ( -mers, for example) that occur as a "window" which is tracked along a dna sequence. it might contain longer oligonucleotides designed from all the open reading frames identified from a complete genome sequence. or it might contain cdnas -of known or unknown sequence -or pcr products. of course it is one thing to produce data it is quite another to extract it in a meaningful manner. fodor's group also developed techniques to read these arrays, employing fluorescent labeling methods and confocal laser scanning to measure each individual binding event on the surface of the chip with extraordinary sensitivity and precision. this general platform of microarray based analysis coupled to confocal laser scanning has become the standard in industry and academia for large-scale genomics studies. in , fodor co-founded affymetrix where the chip technology has been used to synthesize many varieties of high density oligonucleotide arrays containing hundreds of thousands of dna probes. in , steve fodor founded perlegen, inc., a new venture that applied the chip technology towards uncovering the basic patterns of human diversity. his company's stated goals are to analyze more than one million genetic variations in clinical trial participants to explain and predict the efficacy and adverse effect profiles of prescription drugs. in addition, perlegen also applies this expertise to discovering genetic variants associated with disease in order to pave the way for new therapeutics and diagnostics. fodor's former company diversified into plant applications by developing a chip of the archetypal model of plant systems arabidopsis and supplied pioneer hi bred with custom dna chips for monitoring maize gene expression. they (affymetrix) have established programs where academic scientists can use company facilities at a reduced price and set up 'user centers' at selected universities. a related but less complex technology called 'spotted' dna chips involves precisely spotting very small droplets of genomic or cdna clones or pcr samples on a microscope slide. the process uses a robotic device with a print head bearing fine "repeatograph" tips that work like fountain pens to draw up dna samples from a -well plate and spot tiny amounts on a slide. up to , individual clones can be spotted in a dense array within one square centimeter on a glass slide. after hybridization with a fluorescent target mrna, signals are detected by a custom scanner. this is the basis of the systems used by molecular dynamics and incyte (who acquired this technology when it took over synteni). in , incyte was looking to gather more data for its library and perform experiments for corporate subscribers. the company considered buying affymetrix genechips but opted instead to purchase the smaller synteni, which had sprung out of pat brown's stanford array effort. synteni's contact printing technology resulted in dense -and cheaper -arrays. though incyte used the chips only internally, affymetrix sued, claiming synteni/incyte was infringing on its chip density patents. the suit argued that dense biochips -regardless of whether they use photolithography -cannot be made without a license from affymetrix! and in a litigious congo line endemic of this hi-tech era incyte countersued and for good measure also filed against genetic database competitor gene logic for infringing incyte's patents on database building. meanwhile, hyseq sued affymetrix, claiming infringement of nucleotide hybridization patents obtained by its cso. affymetrix, in turn, filed a countersuit, claiming hyseq infringed the spotted array patents. hyseq then reached back and found an additional hybridization patent it claimed that affymetrix had infringed. and so on into the next millennium! in part to avoid all of this another california company nanogen, inc. took a different approach to single nucleotide polymorphism discrimination technology. in an article in the april edition of nature biotechnology, entitled "single nucleotide polymorphic discrimination by an electronic dot blot assay on semiconductor microchips," nanogen describes the use of microchips to identify variants of the mannose binding protein gene that differ from one another by only a single dna base. the mannose binding protein (mbp) is a key component of the innate immune system in children who have not yet developed immunity to a variety of pathogens. to date, four distinct variants (alleles) of this gene have been identified, all differing by only a single nucleotide of dna. mbp was selected for this study because of its potential clinical relevance and its genetic complexity. the samples were assembled at the nci laboratory in conjunction with the national institutes of health and transferred to nanogen for analysis. however, from a high throughput perspective there is a question mark over microarrays. mark benjamin, senior director of business development at rosetta inpharmatics (kirkland, wa), is skeptical about the long-term prospects for standard dna arrays in high-throughput screening as the first steps require exposing cells and then isolating rna, which is something that is very hard to do in a high-throughput format. another drawback is that most of the useful targets are likely to be unknown (particularly in the agricultural sciences where genome sequencing is still in its infancy), and dna arrays that are currently available test only for previously sequenced genes. indeed, some argue that current dna arrays may not be sufficiently sensitive to detect the low expression levels of genes encoding targets of particular interest. and the added complication of the companies' reluctance to provide "raw data" means that derived data sets may be created with less than optimum algorithims thereby irretrievably losing potentially valuable information from the starting material. reverse engineering is a possible approach but this is laborious and time consuming and being prohibited by many contracts may arouse the interest of the ever-vigilant corporate lawyers. over the course of the nineties, outgrowths of functional genomics have been termed proteomics and metabolomics, which are the global studies of gene expression at the protein and metabolite levels respectively. the study of the integration of information flow within an organism is emerging as the field of systems biology. in the area of proteomics, the methods for global analysis of protein profiles and cataloging protein-protein interactions on a genome-wide scale are technically more difficult but improving rapidly, especially for microbes. these approaches generate vast amounts of quantitative data. the amount of expression data becoming available in the public and private sectors is already increasing exponentially. gene and protein expression data rapidly dwarfed the dna sequence data and is considerably more difficult to manage and exploit. in microbes, the small sizes of the genomes and the ease of handling microbial cultures, will enable high throughput, targeted deletion of every gene in a genome, individually and in combinations. this is already available on a moderate throughput scale in model microbes such as e. coli and yeast. combining targeted gene deletions and modifications with genome-wide assay of mrna and protein levels will enable intricate inter-dependencies among genes to be unraveled. simultaneous measurement of many metabolites, particularly in microbes, is beginning to allow the comprehensive modeling and regulation of fluxes through interdependent pathways. metabolomics can be defined as the quantitative measurement of all low molecular weight metabolites in an organism's cells at a specified time under specific environmental conditions. combining information from metabolomics, proteomics and genomics will help us to obtain an integrated understanding of cell biology. the next hierarchical level of phenotype considers how the proteome within and among cells cooperates to produce the biochemistry and physiology of individual cells and organisms. several authors have tentatively offered "physiomics" as a descriptor for this approach. the final hierarchical levels of phenotype include anatomy and function for cells and whole organisms. the term "phenomics" has been applied to this level of study and unquestionably the more well known omics namely economics, has application across all those fields. and, coming slightly out of left field this time, the spectre of eugenics needless to say was raised in the omics era. in the year american and british scientists unveiled a technique which has come to be known as pre-implantation genetic diagnosis (pid) for testing embryos in vitro for genetic abnormalities such as cystic fibrosis, hemophilia, and down's syndrome (wald, ) . this might be seen by most as a step forward, but it led ethicist david s. king ( ) to decry pid as a technology that could exacerbate the eugenic features of prenatal testing and make possible an expanded form of free-market eugenics. he further argues that due to social pressures and eugenic attitudes held by clinical geneticists in most countries, it results in eugenic outcomes even though no state coercion is involved and that, as abortion is not involved, and multiple embryos are available, pid is radically more effective as a tool of genetic selection. the first regulatory approval of a recombinant dna technology in the u.s. food supply was not a plant but an industrial enzyme that has become the hallmark of food biotechnology success. enzymes were important agents in food production long before modern biotechnology was developed. they were used, for instance, in the clotting of milk to prepare cheese, the production of bread and the production of alcoholic beverages. nowadays, enzymes are indispensable to modern food processing technology and have a great variety of functions. they are used in almost all areas of food production including grain processing, milk products, beer, juices, wine, sugar and meat. chymosin, known also as rennin, is a proteolytic enzyme whose role in digestion is to curdle or coagulate milk in the stomach, efficiently converting liquid milk to a semisolid like cottage cheese, allowing it to be retained for longer periods in a neonate's stomach. the dairy industry takes advantage of this property to conduct the first step in cheese production. chy-max™, an artificially produced form of the chymosin enzyme for cheese-making, was approved in . in some instances they replace less acceptable "older" technology, for example the enzyme chymosin. unlike crops industrial enzymes have had relatively easy passage to acceptance for a number of reasons. as noted they are part of the processing system and theoretically do not appear in the final product. today about % of the hard cheese in the us and uk is made using chymosin from geneticallymodified microbes. it is easier to purify, more active ( % as compared to %) and less expensive to produce (microbes are more prolific, more productive and cheaper to keep than calves). like all enzymes it is required only in very small quantities and because it is a relatively unstable protein it breaks down as the cheese matures. indeed, if the enzyme remained active for too long it would adversely affect the development of the cheese, as it would degrade the milk proteins to too great a degree. such enzymes have gained the support of vegetarian organizations and of some religious authorities. for plants the nineties was the era of the first widespread commercialization of what came to be known in often deprecating and literally inaccurate terms as gmos (genetically modified organisms). when the nineties dawned dicotyledonous plants were relatively easily transformed with agrobacterium tumefaciens but many economically important plants, including the cereals, remained inaccessible for genetic manipulation because of lack of effective transformation techniques. in this changed with the technology that overcame this limitation. michael fromm, a molecular biologist at the plant gene expression center, reported the stable transformation of corn using a high-speed gene gun. the method known as biolistics uses a "particle gun" to shoot metal particles coated with dna into cells. initially a gunpowder charge subsequently replaced by helium gas was used to accelerate the particles in the gun. there is a minimal disruption of tissue and the success rate has been extremely high for applications in several plant species. the technology rights are now owned by dupont. in some of the first of the field trials of the crops that would dominate the second half of the nineties began, including bt corn (with the bacillus thuriengenesis cry protein discussed in chapter three). in the fda declared that genetically engineered foods are "not inherently dangerous" and do not require special regulation. since , researchers have pinpointed and cloned several of the genes that make selected plants resistant to certain bacterial and fungal infections; some of these genes have been successfully inserted into crop plants that lack them. many more infection-resistant crops are expected in the near future, as scientists find more plant genes in nature that make plants resistant to pests. plant genes, however, are just a portion of the arsenal; microorganisms other than bt also are being mined for genes that could help plants fend off invaders that cause crop damage. the major milestone of the decade in crop biotechnology was approval of the first bioengineered crop plant in . it represented a double first not just of the first approved food crop but also of the first commercial validation of a technology which was to be surpassed later in the decade. that technology, antisense technology works because nucleic acids have a natural affinity for each other. when a gene coding for the target in the genome is introduced in the opposite orientation, the reverse rna strand anneals and effectively blocks expression of the enzyme. this technology was patented by calgene for plant applications and was the technology behind the famous flavr savr tomatoes. the first success for antisense in medicine was in when the u.s. food and drug administration gave the go-ahead to the cytomegalovirus (cmv) inhibitor fomivirsen, a phosphorothionate antiviral for the aids-related condition cmv retinitis making it the first drug belonging to isis, and the first antisense drug ever, to be approved. another technology, although not apparent at the time was behind the second approval and also the first and only successful to date in a commercial tree fruit biotech application. the former was a virus resistant squash the second the papaya ringspot resistant papaya. both owed their existence as much to historic experience as modern technology. genetically engineered virus-resistant strains of squash and cantaloupe, for example, would never have made it to farmers' fields if plant breeders in the 's had not noticed that plants infected with a mild strain of a virus do not succumb to more destructive strains of the same virus. that finding led plant pathologist roger beachy, then at washington university in saint louis, to wonder exactly how such "cross-protection" worked -did part of the virus prompt it? in collaboration with researchers at monsanto, beachy used an a. tumefaciens vector to insert into tomato plants a gene that produces one of the proteins that makes up the protein coat of the tobacco mosaic virus. he then inoculated these plants with the virus and was pleased to discover, as reported in , that the vast majority of plants did not succumb to the virus. eight years later, in , virus-resistant squash seeds created with beachy's method reached the market, to be followed soon by bioengineered virus-resistant seeds for cantaloupes, potatoes, and papayas. (breeders had already created virusresistant tomato seeds by using traditional techniques.) and the method of protection still remained a mystery when the first approvals were given in and . gene silencing was perceived initially as an unpredictable and inconvenient side effect of introducing transgenes into plants. it now seems that it is the consequence of accidentally triggering the plant's adaptive defense mechanism against viruses and transposable elements. this recently discovered mechanism, although mechanistically different, has a number of parallels with the immune system of mammals. how this system worked was not elucidated until later in the decade by a researcher who was seeking a very different holy grail -the black rose! rick jorgensen, at that time at dna plant technologies in oakland, ca and subsequently of, of the university of california davis attempted to overexpress the chalcone synthase gene by introducing a modified copy under a strong promoter.surprisingly he obtained white flowers, and many strange variegated purple and white variations in between. this was the first demonstration of what has come to be known as post-transcriptional gene silencing (ptgs). while initially it was considered a strange phenomenon limited to petunias and a few other plant species, it is now one of the hottest topics in molecular biology. rna interference (rnai) in animals and basal eukaryotes, quelling in fungi, and ptgs in plants are examples of a broad family of phenomena collectively called rna silencing (hannon ; plasterk ) . in addition to its occurrence in these species it has roles in viral defense (as demonstrated by beachy) and transposon silencing mechanisms among other things. perhaps most exciting, however, is the emerging use of ptgs and, in particular, rnai -ptgs initiated by the introduction of double-stranded rna (dsrna) -as a tool to knock out expression of specific genes in a variety of organisms. nineteen ninety one also heralded yet another first. the february , issue of science reported the patenting of "molecular scissors": the nobel-prize winning discovery of enzymatic rna, or "ribozymes," by thomas czech of the university of colorado. it was noted that the u.s. patent and trademark office had awarded an "unusually broad" patent for ribozymes. the patent is u.s. patent no. , , , claim of which reads as follows: "an enzymatic rna molecule not naturally occurring in nature having an endonuclease activity independent of any protein, said endonuclease activity being specific for a nucleotide sequence defining a cleavage site comprising single-stranded rna in a separate rna molecule, and causing cleavage at said cleavage site by a transesterification reaction." although enzymes made of protein are the dominant form of biocatalyst in modern cells, there are at least eight natural rna enzymes, or ribozymes, that catalyze fundamental biological processes. one of which was yet another discovery by plant virologists, in this instance the hairpin ribozyme was discovered by george bruening at uc davis. the self-cleavage structure was originally called a paperclip, by the bruening laboratory which discovered the reactions. as mentioned in chapter , it is believed that these ribozymes might be the remnants of an ancient form of life that was guided entirely by rna. since a ribozyme is a catalytic rna molecule capable of cleaving itself and other target rnas it therefore can be useful as a control system for turning off genes or targeting viruses. the possibility of designing ribozymes to cleave any specific target rna has rendered them valuable tools in both basic research and therapeutic applications. in the therapeutics area, they have been exploited to target viral rnas in infectious diseases, dominant oncogenes in cancers and specific somatic mutations in genetic disorders. most notably, several ribozyme gene therapy protocols for hiv patients are already in phase trials. more recently, ribozymes have been used for transgenic animal research, gene target validation and pathway elucidation. however, targeting ribozymes to the cellular compartment containing their target rnas has proved a challenge. at the other bookend of the decade in , samarsky et al. reported that a family of small rnas in the nucleolus (snornas) can readily transport ribozymes into this subcellular organelle. in addition to the already extensive panoply of rna entities yet another has potential for mischief. viroids are small, single-stranded, circular rnas containing - nucleotides arranged in a rod-like secondary structure and are the smallest pathogenic agents yet described. the smallest viroid characterized to date is rice yellow mottle sobemovirus (rymv), at nucleotides. in comparison, the genome of the smallest known viruses capable of causing an infection by themselves, the single-stranded circular dna of circoviruses, is around kilobases in size. the first viroid to be identified was the potato spindle tuber viroid (pstvd). some species have been identified to date. unlike the many satellite or defective interfering rnas associated with plant viruses, viroids replicate autonomously on inoculation of a susceptible host. the absence of a protein capsid and of detectable messenger rna activity implies that the information necessary for replication and pathogenesis resides within the unusual structure of the viroid genome. the replication mechanism actually involves interaction with rna polymerase ii, an enzyme normally associated with synthesis of messenger rna, and "rolling circle" synthesis of new rna. some viroids have ribozyme activity which allow self-cleavage and ligation of unit-size genomes from larger replication intermediates. it has been proposed that viroids are "escaped introns". viroids are usually transmitted by seed or pollen. infected plants can show distorted growth. from its earliest years, biotechnology attracted interest outside scientific circles. initially the main focus of public interest was on the safety of recombinant dna technology, and of the possible risks of creating uncontrollable and harmful novel organisms (berg , ) . the debate on the deliberate release of genetically modified organisms, and on consumer products containing or comprising them, followed some years later (nas, ) . it is interesting to note that within the broad ethical tableau of potential issues within the science and products of biotechnology, the seemingly innocuous field of plant modification has been one of the major players of the 's. the success of agricultural biotechnology is heavily dependent on its acceptance by the public, and the regulatory framework in which the industry operates is also influenced by public opinion. as the focus for molecular biology research shifted from the basic pursuit of knowledge to the pursuit of lucrative applications, once again as in the previous two decades the specter of risk arose as the potential of new products and applications had to be evaluated outside the confines of a laboratory. however, the specter now became far more global as the implications of commercial applications brought not just worker safety into the loop but also, the environment, agricultural and industrial products and the safety and well being of all living things. beyond "deliberate" release, the rac guidelines were not designed to address these issues, so the matter moved into the realm of the federal agencies who had regulatory authority which could be interpreted to oversee biotechnology issues. this adaptation of oversight is very much a dynamic process as the various agencies wrestle with the task of applying existing regulations and developing new ones for oversight of this technology in transition. as the decade progressed focus shifted from basic biotic stress resistance to more complex modifications the next generation of plants will focus on value added traits in which valuable genes and metabolites will be identified and isolated, with some of the later compounds being produced in mass quantities for niche markets. two of the more promising markets are nutraceuticals or so-called "functional foods" and plants developed as bioreactors for the production of valuable proteins and compounds, a field known as plant molecular farming. developing plants with improved quality traits involves overcoming a variety of technical challenges inherent to metabolic engineering programs. both traditional plant breeding and biotechnology techniques are needed to produce plants carrying the desired quality traits. continuing improvements in molecular and genomic technologies are contributing to the acceleration of product development in this space. by the end of the decade in , applying nutritional genomics, della penna ( ) isolated a gene, which converts the lower activity precursors to the highest activity vitamin e compound, alpha-tocopherol. with this technology, the vitamin e content of arabidopsis seed oil has been increased nearly -fold and progress has been made to move the technology to crops such as soybean, maize, and canola. this has also been done for folates in rice. omega three fatty acids play a significant role in human health, eicosapentaenoic acid (epa) and docosahexaenoic acid (dha), which are present in the retina of the eye and cerebral cortex of the brain, respectively, are some of the most well documented from a clinical perspective. it is believed that epa and dha play an important role in the regulation of inflammatory immune reactions and blood pressure, treatment of conditions such as cardiovascular disease and cystic fibrosis, brain development in utero, and, in early postnatal life, the development of cognitive function. they are mainly found in fish oil and the supply is limited. by the end of the decade ursin ( ) had succeeded in engineering canola to produce these fatty acids. from a global perspective another value-added development had far greater impact both technologically and socio-economically. a team led by ingo potrykus ( ) engineered rice to produce pro-vitamin a, which is an essential micronutrient. widespread dietary deficiency of this vitamin in rice-eating asian countries, which predisposes children to diseases such as blindness and measles, has tragic consequences. improved vitamin a nutrition would alleviate serious health problems and, according to unicef, could also prevent up to two million infant deaths due to vitamin a deficiency. adoption of the next stage of gm crops may proceed more slowly, as the market confronts issues of how to determine price, share value, and adjust marketing and handling to accommodate specialized end-use characteristics. furthermore, competition from existing products will not evaporate. challenges that have accompanied gm crops with improved agronomic traits, such as the stalled regulatory processes in europe, will also affect adoption of nutritionally improved gm products. beyond all of this, credible scientific research is still needed to confirm the benefits of any particular food or component. for functional foods to deliver their potential public health benefits, consumers must have a clear understanding of, and a strong confidence level in, the scientific criteria that are used to document health effects and claims. because these decisions will require an understanding of plant biochemistry, mammalian physiology, and food chemistry, strong interdisciplinary collaborations will be needed among plant scientists, nutritionists, and food scientists to ensure a safe and healthful food supply. in addition to being a source of nutrition, plants have been a valuable wellspring of therapeutics for centuries. during the nineties, however, intensive research has focused on expanding this source through rdna biotechnology and essentially using plants and animals as living factories for the commercial production of vaccines, therapeutics and other valuable products such as industrial enzymes and biosynthetic feedstocks. possibilities in the medical field include a wide variety of compounds, ranging from edible vaccine antigens against hepatitis b and norwalk viruses (arntzen, ) and pseudomonas aeruginosa and staphylococcus aureus to vaccines against cancer and diabetes, enzymes, hormones, cytokines, interleukins, plasma proteins, and human alpha- -antitrypsin. thus, plant cells are capable of expressing a large variety of recombinant proteins and protein complexes. therapeutics produced in this way are termed plant made pharmaceuticals (pmps). and non-therapeutics are termed plant made industrial products (pmips) (newell-mcgloughlin, ) . the first reported results of successful human clinical trials with their transgenic plant-derived pharmaceuticals were published in . they were an edible vaccine against e. coli-induced diarrhea and a secretory monoclonal antibody directed against streptococcus mutans, for preventative immunotherapy to reduce incidence of dental caries. haq et al. ( ) reported the expression in potato plants of a vaccine against e. coli enterotoxin (etec) that provided an immune response against the toxin in mice. human clinical trials suggest that oral vaccination against either of the closely related enterotoxins of vibrio cholerae and e. coli induces production of antibodies that can neutralize the respective toxins by preventing them from binding to gut cells. similar results were found for norwalk virus oral vaccines in potatoes. for developing countries, the intention is to deliver them in bananas or tomatoes (newell-mcgloughlin, ) . plants are also faster, cheaper, more convenient and more efficient than the principal eukaryotic production system, namely chinese hamster ovary (cho) cells for the production of pharmaceuticals. hundreds of acres of protein-containing seeds could inexpensively double the production of a cho bioreactor factory. in addition, proteins can be expressed at the highest levels in the harvestable seed and plant-made proteins and enzymes formulated in seeds have been found to be extremely stable, reducing storage and shipping costs. pharming may also enable research on drugs that cannot currently be produced. for example, croptech in blacksburg, va., is investigating a protein that seems to be a very effective anticancer agent. the problem is that this protein is difficult to produce in mammalian cell culture systems as it inhibits cell growth. this should not be a problem in plants. furthermore, production size is flexible and easily adjustable to the needs of changing markets. making pharmaceuticals from plants is also a sustainable process, because the plants and crops used as raw materials are renewable. the system also has the potential to address problems associated with provision of vaccines to people in developing countries. products from these alternative sources do not require a so-called "cold chain" for refrigerated transport and storage. those being developed for oral delivery obviates the need for needles and aspectic conditions which often are a problem in those areas. apart from those specific applications where the plant system is optimum there are many other advantages to using plant production. many new pharmaceuticals based on recombinant proteins will receive regulatory approval from the united states food and drug administration (fda) in the next few years. as these therapeutics make their way through clinical trials and evaluation, the pharmaceutical industry faces a production capacity challenge. pharmaceutical discovery companies are exploring plant-based production to overcome capacity limitations, enable production of complex therapeutic proteins, and fully realize the commercial potential of their biopharmaceuticals (newell-mcgloughlin, ) . nineteen ninety also marked a major milestone in the animal biotech world when herman made his appearance on the world's stage. since the palmiter's mouse, transgenic technology has been applied to several species including agricultural species such as sheep, cattle, goats, pigs, rabbits, poultry, and fish. herman was the first transgenic bovine created by genpharm international, inc., in a laboratory in the netherlands at the early embryo stage. scientist's microinjected recently fertilized eggs with the gene coding for human lactoferrin. the scientists then cultured the cells in vitro to the embryo stage and transferred them to recipient cattle. lactoferrin, an iron-containing anti-bacterial protein is essential for infant growth. since cow's milk doesn't contain lactoferrin, infants must be fed from other sources that are rich in iron -formula or mother's milk (newell-mcgloughlin, ) . as herman was a boy he would be unable to provide the source, that would require the production of daughters which was not necessarily a straightforward process. the dutch parliments permission was required. in they finally approved a measure that permitted the world's first genetically engineered bull to reproduce. the leiden-based gene pharming proceeded to artificially inseminate cows with herman's sperm. with a promise that the protein, lactoferrin, would be the first in a new generation of inexpensive, high-tech drugs derived from cows' milk to treat complex diseases like aids and cancer. herman, became the father of at least eight female calves in , and each one inherited the gene for lactoferrin production. while their birth was initially greeted as a scientific advancement that could have far-reaching effects for children in developing nations, the levels of expression were too low to be commercially viable. by , herman, who likes to listen to rap music to relax, had sired calves and outlived them all. his offspring were all killed and destroyed after the end of the experiment, in line with dutch health legislation. herman was also slated for the abattoir, but the dutch public -proud of making history with herman -rose up in protest, especially after a television program screened footage showing the amiable bull licking a kitten. herman won a bill of clemency from parliament. however, instead of retirement on a comfortable bed of straw, listening to rap music, herman was pressed into service again. he now stars at a permanent biotech exhibit in naturalis, a natural history museum in the dutch city of leiden. after his death, he will be stuffed and remain in the museum in perpetuity (a fate similar to what awaited an even more famous mammalian first born later in the decade). the applications for transgenic animal research fall broadly into two distinct areas, namely medical and agricultural applications. the recent focus on developing animals as bioreactors to produce valuable proteins in their milk can be catalogued under both areas. underlying each of these, of course, is a more fundamental application, that is the use of those techniques as tools to ascertain the molecular and physiological bases of gene expression and animal development. this understanding can then lead to the creation of techniques to modify development pathways. in a european decision with rather more far-reaching implications than hermans sex life was made. the first european patent on a transgenic animal was issued for a transgenic mouse sensitive to carcinogens -harvard's "oncomouse". the oncomouse patent application was refused in europe in due primarily to an established ban on animal patenting. the application was revised to make narrower claims, and the patent was granted in . this has since been repeatedly challenged, primarily by groups objecting to the judgement that benefits to humans outweigh the suffering of the animal. currently, the patent applicant is awaiting protestors' responses to a series of possible modifications to the application. predictions are that agreement will not likely be forthcoming and that the legal wrangling will continue into the future. bringing animals into the field of controversy starting to swirl around gmos and preceding the latter's commercialization, was the approval by the fda of bovine somatotropin (bst) for increased milk production in dairy cows. the fda's center for veterinary medicine (cvm) regulates the manufacture and distribution of food additives and drugs that will be given to animals. biotechnology products are a growing proportion of the animal health products and feed components regulated by the cvm. the center requires that food products from treated animals must be shown to be safe for human consumption. applicants must show that the drug is effective and safe for the animal and that its manufacture will not affect the environment. they must also conduct geographically dispersed clinical trials under an investigational new animal drug application with the fda through which the agency controls the use of the unapproved compound in food animals. unlike within the eu, possible economic and social issues cannot be taken into consideration by the fda in the premarket drug approval process. under these considerations the safety and efficacy of rbst was determined. it was also determined that special labeling for milk derived from cows that had been treated with rbst is not required under fda food labeling laws because the use of rbst does not effect the quality or the composition of the milk. work with fish proceeded a pace throughout the decade. gene transfer techniques have been applied to a large number of aquatic organisms, both vertebrates and invertebrates. gene transfer experiments have targeted a wide variety of applications, including the study of gene structure and function, aquaculture production, and use in fisheries management programs. because fish have high fecundity, large eggs, and do not require reimplantation of embryos, transgenic fish prove attractive model systems in which to study gene expression. transgenic zebrafish have found utility in studies of embryogenesis, with expression of transgenes marking cell lineages or providing the basis for study of promoter or structural gene function. although not as widely used as zebrafish, transgenic medaka and goldfish have been used for studies of promoter function. this body of research indicates that transgenic fish provide useful models of gene expression, reliably modeling that in "higher" vertebrates. perhaps the largest number of gene transfer experiments address the goal of genetic improvement for aquaculture production purposes. the principal area of research has focused on growth performance, and initial transgenic growth hormone (gh) fish models have demonstrated accelerated and beneficial phenotypes. dna microinjection methods have propelled the many studies reported and have been most effective due to the relative ease of working with fish embryos. bob devlins' group in vancouver has demonstrated extraordinary growth rate in coho salmon which were transformed with a growth hormone from sockeye salmon. the transgenics achieve up to eleven times the size of their littermates within six months, reaching maturity in about half the time. interestingly this dramatic effect is only observed in feeding pins where the transgenics' ferocious appetites demands constant feeding. if the fish are left to their own devices and must forage for themselves, they appear to be out-competed by their smarter siblings. however most studies, such as those involving transgenic atlantic salmon and channel catfish, report growth rate enhancement on the order of - %. in addition to the species mentioned, gh genes also have been transferred into striped bass, tilapia, rainbow trout, gilthead sea bream, common carp, bluntnose bream, loach, and other fishes. shellfish also are subject to gene transfer toward the goal of intensifying aquaculture production. growth of abalone expressing an introduced gh gene is being evaluated; accelerated growth would prove a boon for culture of the slowgrowing mollusk. a marker gene was introduced successfully into giant prawn, demonstrating feasibility of gene transfer in crustaceans, and opening the possibility of work involving genes affecting economically important traits. in the ornamental fish sector of aquaculture, ongoing work addresses the development of fish with unique coloring or patterning. a number of companies have been founded to pursue commercialization of transgenics for aquaculture. as most aquaculture species mature at - years of age, most transgenic lines are still in development and have yet to be tested for performance under culture conditions. extending earlier research that identified methylfarnesoate (mf) as a juvenile hormone in crustaceans and determined its role in reproduction, researchers at the university of connecticut have developed technology to synchronize shrimp egg production and to increase the number and quality of eggs produced. females injected with mf are stimulated to produce eggs ready for fertilization. the procedure produces percent more eggs than the traditional crude method of removing the eyestalk gland. this will increase aquaculture efficiency. a number of experiments utilize gene transfer to develop genetic lines of potential utility in fisheries management. transfer of gh genes into northern pike, walleye, and largemouth bass are aimed at improving the growth rate of sport fishes. gene transfer has been posed as an option for reducing losses of rainbow trout to whirling disease, although suitable candidate genes have yet to be identified. richard winn of the university of georgia is developing transgenic killifish and medaka as biomonitors for environmental mutagens, which carry the bacteriophage phi x as a target for mutation detection. development of transgenic lines for fisheries management applications generally is at an early stage, often at the founder or f generation. broad application of transgenic aquatic organisms in aquaculture and fisheries management will depend on showing that particular gmos can be used in the environment both effectively and safely. although our base of knowledge for assessing ecological and genetic safety of aquatic gmos currently is limited, some early studies supported by the usda biotechnology risk assessment program have yielded results. data from outdoor pond-based studies on transgenic catfish reported by rex dunham of auburn university show that transgenic and non-transgenic individuals interbreed freely, that survival and growth of transgenics in unfed ponds was equal to or less than that of non-transgenics, and that predator avoidance is not affected by expression of the transgene. however, unquestionably the seminal event for animal biotech in the nineties was ian wilmut's landmark work using nuclear transfer technology to generate the lambs morag and megan reported in (from an embryonic cell nuclei) and the truly ground-breaking work of creating dolly from an adult somatic cell nucleus, reported in february, (wilmut, ) . wilmut and his colleagues at the roslin institute demonstrated for the first time with the birth of dolly the sheep that the nucleus of an adult somatic cell can be transferred to an enucleated egg to create cloned offspring. it had been assumed for some time that only embryonic cells could be used as the cellular source for nuclear transfer. this assumption was shattered with the birth of dolly. this example of cloning an animal using the nucleus of an adult cell was significant because it demonstrated the ability of egg cell cytoplasm to "reprogram" an adult nucleus. when cells differentiate, that is, evolve from primitive embryonic cells to functionally defined adult cells, they lose the ability to express most genes and can only express those genes necessary for the cell's differentiated function. for example, skin cells only express genes necessary for skin function, and brain cells only express genes necessary for brain function. the procedure that produced dolly demonstrated that egg cytoplasm is capable of reprogramming an adult differentiated cell (which is only expressing genes related to the function of that cell type). this reprogramming enables the differentiated cell nucleus to once again express all the genes required for the full embryonic development of the adult animal. since dolly was cloned, similar techniques have been used to clone a veritable zoo of vertebrates including mice, cattle, rabbitts, mules, horses, fish, cats and dogs from donor cells obtained from adult animals. these spectacular examples of cloning normal animals from fully differentiated adult cells demonstrate the universality of nuclear reprogramming although the next decade called some of these assumptions into question. this technology supports the production of genetically identical and genetically modified animals. thus, the successful "cloning" of dolly has captured the imagination of researchers around the world. this technological breakthrough should play a significant role in the development of new procedures for genetic engineering in a number of mammalian species. it should be noted that nuclear cloning, with nuclei obtained from either mammalian stem cells or differentiated "adult" cells, is an especially important development for transgenic animal research. as the decade reached its end the clones began arriving rapidly with specific advances made by a japanese group who used cumulus cells rather than fibroblasts to clone calves. they found that the percentage of cultured, reconstructed eggs that developed into blastocysts was % for cumulus cells and % for oviductal cells. these rates are higher than the % previously reported for transfer of nuclei from bovine fetal fibroblasts. following on the heels of dolly, polly and molly became the first genetically engineered transgenic sheep produced through nuclear transfer technology. polly and molly were engineered to produce human factor ix (for hemophiliacs) by transfer of nuclei from transfected fetal fibroblasts. until then germline competent transgenics had only been produced in mammalian species, other than mice, using dna microinjection. researchers at the university of massachusetts and advanced cell technology (worcester, ma) teamed up to produce genetically identical calves utilizing a strategy similar to that used to produce transgenic sheep. in contrast to the sheep cloning experiment, the bovine experiment involved the transfer of nuclei from an actively dividing population of cells. previous results from the sheep experiments suggested that induction of quiescence by serum starvation was required to reprogram the donor nuclei for successful nuclear transfer. the current bovine experiments indicate that this step may not be necessary. typically about embryos needed to be microinjected to obtain one transgenic cow, whereas nuclear transfer produced three transgenic calves from reconstructed embryos. this efficiency is comparable to the previous sheep research where six transgenic lambs were produced from reconstructed embryos. the ability to select for genetically modified cells in culture prior to nuclear transfer opens up the possibility of applying the powerful gene targeting techniques that have been developed for mice. one of the limitations of using primary cells, however, is their limited lifespan in culture. primary cell cultures such as the fetal fibroblasts can only undergo about population doublings before they senesce. this limited lifespan would preclude the ability to perform multiple rounds of selection. to overcome this problem of cell senescence, these researchers showed that fibroblast lifespan could be prolonged by nuclear transfer. a fetus, which was developed by nuclear transfer from genetically modified cells, could in turn be used to establish a second generation of fetal fibroblasts. these fetal cells would then be capable of undergoing another population doublings, which would provide sufficient time for selection of a second genetic modification. as noted, there is still some uncertainty over whether quiescent cells are required for successful nuclear transfer. induction into quiescence was originally thought to be necessary for successful nuclear reprogramming of the donor nucleus. however, cloned calves have been previously produced using non-quiescent fetal cells. furthermore, transfer of nuclei from sertoli and neuronal cells, which do not normally divide in adults, did not produce a liveborn mouse; whereas nuclei transferred from actively dividing cumulus cells did produce cloned mice. the fetuses used for establishing fetal cell lines in a tufts goat study were generated by mating nontransgenic females to a transgenic male containing a human antithrombin (at) iii transgene. this at transgene directs high level expression of human at into milk of lactating transgenic females. as expected, all three offspring derived from female fetal cells were females. one of these cloned goats was hormonally induced to lactate. this goat secreted . - . grams per liter of at in her milk. this level of at expression was comparable to that detected in the milk of transgenic goats from the same line obtained by natural breeding. the successful secretion of at in milk was a key result because it showed that a cloned animal could still synthesize and secrete a foreign protein at the expected level. it will be interesting to see if all three cloned goats secrete human at at the identical level. if so, then the goal of creating a herd identical transgenic animals, which secrete identical levels of an important pharmaceutical, would become a reality. no longer would variable production levels exist in subsequent generations due to genetically similar but not identical animals. this homogeneity would greatly aid in the production and processing of a uniform product. as nuclear transfer technology continues to be refined and applied to other species, it may eventually replace microinjection as the method of choice for generating transgenic livestock. nuclear transfer has a number of advantages: ) nuclear transfer is more efficient than microinjection at producing a transgenic animal, ) the fate of the integrated foreign dna can be examined prior to production of the transgenic animal, ) the sex of the transgenic animal can be predetermined, and ) the problem of mosaicism in first generation transgenic animals can be eliminated. dna microinjection has not been a very efficient mechanism to produce transgenic mammals. however, in november, , a team of wisconsin researchers reported a nearly % efficient method for generating transgenic cattle. the established method of cattle transgenes involves injecting dna into the pronuclei of a fertilized egg or zygote. in contrast, the wisconsin team injected a replication-defective retroviral vector into the perivitelline space of an unfertilized oocyte. the perivitelline space is the region between the oocyte membrane and the protective coating surrounding the oocyte known as the zona pellucida. in addition to es (embryonic stem) cells other sources of donor nuclei for nuclear transfer might be used such as embryonic cell lines, primordial germ cells, or spermatogonia to produce offspring. the utility of es cells or related methodologies to provide efficient and targeted in vivo genetic manipulations offer the prospects of profoundly useful animal models for biomedical, biological and agricultural applications. the road to such success has been most challenging, but recent developments in this field are extremely encouraging. with the may announcement of geron buying out ian wilmuts company roslin biomed, they declared it the dawn of an new era in biomedical research. geron's technologies for deriving transplantable cells from human pluripotent stem cells (hpscs) and extending their replicative capacity with telomerase was combined with the roslin institute nuclear transfer technology, the technology that produced dolly the cloned sheep. the goal was to produce transplantable, tissue-matched cells that provide extended therapeutic benefits without triggering immune rejection. such cells could be used to treat numerous major chronic degenerative diseases and conditions such as heart disease, stroke, parkinson's disease, alzheimer's disease, spinal cord injury, diabetes, osteoarthritis, bone marrow failure and burns. the stem cell is a unique and essential cell type found in animals. many kinds of stem cells are found in the body, with some more differentiated, or committed, to a particular function than others. in other words, when stem cells divide, some of the progeny mature into cells of a specific type (heart, muscle, blood, or brain cells), while others remain stem cells, ready to repair some of the everyday wear and tear undergone by our bodies. these stem cells are capable of continually reproducing themselves and serve to renew tissue throughout an individual's life. for example, they continually regenerate the lining of the gut, revitalize skin, and produce a whole range of blood cells. although the term "stem cell" commonly is used to refer to the cells within the adult organism that renew tissue (e.g., hematopoietic stem cells, a type of cell found in the blood), the most fundamental and extraordinary of the stem cells are found in the early-stage embryo. these embryonic stem (es) cells, unlike the more differentiated adult stem cells or other cell types, retain the special ability to develop into nearly any cell type. embryonic germ (eg) cells, which originate from the primordial reproductive cells of the developing fetus, have properties similar to es cells. it is the potentially unique versatility of the es and eg cells derived, respectively, from the early-stage embryo and cadaveric fetal tissue that presents such unusual scientific and therapeutic promise. indeed, scientists have long recognized the possibility of using such cells to generate more specialized cells or tissue, which could allow the generation of new cells to be used to treat injuries or diseases, such as alzheimer's disease, parkinson's disease, heart disease, and kidney failure. likewise, scientists regard these cells as an important -perhaps essential -means for understanding the earliest stages of human development and as an important tool in the development of life-saving drugs and cell-replacement therapies to treat disorders caused by early cell death or impairment. geron corporation and its collaborators at the university of wisconsin -madison (dr. james a. thomson) and johns hopkins university (dr. john d. gearhart) announced in november the first successful derivation of hpscs from two sources: (i) human embryonic stem (hes) cells derived from in vitro fertilized blastocysts (thomson ) and (ii) human embryonic germ (heg) cells derived from fetal material obtained from medically terminated pregnancies (shamblott et al. ) . although derived from different sources by different laboratory processes, these two cell types share certain characteristics but are referred to collectively as human pluripotent stem cells (hpscs). because hes cells have been more thoroughly studied, the characteristics of hpscs most closely describe the known properties of hes cells. stem cells represent a tremendous scientific advancement in two ways: first, as a tool to study developmental and cell biology; and second, as the starting point for therapies to develop medications to treat some of the most deadly diseases. the derivation of stem cells is fundamental to scientific research in understanding basic cellular and embryonic development. observing the development of stem cells as they differentiate into a number of cell types will enable scientists to better understand cellular processes and ways to repair cells when they malfunction. it also holds great potential to yield revolutionary treatments by transplanting new tissue to treat heart disease, atherosclerosis, blood disorders, diabetes, parkinson's, alzheimer's, stroke, spinal cord injuries, rheumatoid arthritis, and many other diseases. by using stem cells, scientists may be able to grow human skin cells to treat wounds and burns. and, it will aid the understanding of fertility disorders. many patient and scientific organizations recognize the vast potential of stem cell research. another possible therapeutic technique is the generation of "customized" stem cells. a researcher or doctor might need to develop a special cell line that contains the dna of a person living with a disease. by using a technique called "somatic cell nuclear transfer" the researcher can transfer a nucleus from the patient into an enucleated human egg cell. this reformed cell can then be activated to form a blastocyst from which customized stem cell lines can be derived to treat the individual from whom the nucleus was extracted. by using the individual's own dna, the stem cell line would be fully compatible and not be rejected by the person when the stem cells are transferred back to that person for the treatment. preliminary research is occurring on other approaches to produce pluripotent human es cells without the need to use human oocytes. human oocytes may not be available in quantities that would meet the needs of millions of potential patients. however, no peer-reviewed papers have yet appeared from which to judge whether animal oocytes could be used to manufacture "customized" human es cells and whether they can be developed on a realistic timescale. additional approaches under consideration include early experimental studies on the use of cytoplasmic-like media that might allow a viable approach in laboratory cultures. on a much longer timeline, it may be possible to use sophisticated genetic modification techniques to eliminate the major histocompatibility complexes and other cell-surface antigens from foreign cells to prepare master stem cell lines with less likelihood of rejection. this could lead to the development of a bank of universal donor cells or multiple types of compatible donor cells of invaluable benefit to treat all patients. however, the human immune system is sensitive to many minor histocompatibility complexes and immunosuppressive therapy carries life-threatening complications. stem cells also show great potential to aid research and development of new drugs and biologics. now, stem cells can serve as a source for normal human differentiated cells to be used for drug screening and testing, drug toxicology studies and to identify new drug targets. the ability to evaluate drug toxicity in human cell lines grown from stem cells could significantly reduce the need to test a drug's safety in animal models. there are other sources of stem cells, including stem cells that are found in blood. recent reports note the possible isolation of stem cells for the brain from the lining of the spinal cord. other reports indicate that some stem cells that were thought to have differentiated into one type of cell can also become other types of cells, in particular brain stem cells with the potential to become blood cells. however, since these reports reflect very early cellular research about which little is known, we should continue to pursue basic research on all types of stem cells. some religious leaders will advocate that researchers should only use certain types of stem cells. however, because human embryonic stem cells hold the potential to differentiate into any type of cell in the human body, no avenue of research should be foreclosed. rather, we must find ways to facilitate the pursuit of all research using stem cells while addressing the ethical concerns that may be raised. another seminal and intimately related event at the end of the nineties occurred in madison wisconsin. up until november of , isolating es cells in mammals other than mice proved elusive, but in a milestone paper in the november , issue of science, james a. thomson, ( ) a developmental biologist at uw-madison reported the first successful isolation, derivation and maintenance of a culture of human embryonic stem cells (hes cells). it is interesting to note that this leap was made from mouse to man. as thomson himself put it, these cells are different from all other human stem cells isolated to date and as the source of all cell types, they hold great promise for use in transplantation medicine, drug discovery and development, and the study of human developmental biology. the new century is rapidly exploiting this vision. when steve fodor was asked in "how do you really take the human genome sequence and transform it into knowledge?" he answered from affymetrix's perspective, it is a technology development task. he sees the colloquially named affychips being the equivalent of a cd-rom of the genome. they take information from the genome and write it down. the company has come a long way from the early days of venter's ests and less than robust algorithms as described earlier. one surprising fact unearthed by the newer more sophisticated generation of chips is that to percent of the non-repetitive dna is being expressed as accepted knowledge was that only . to percent of the genome would be expressed. since much of that sequence has no protein-coding capacity it is most likely coding for regulatory functions. in a parallel to astrophysics this is often referred to in common parlance as the "dark matter of the genome" and like dark matter for many it is the most exciting and challenging aspect of uncovering the occult genome. it could be, and most probably is, involved in regulatory functions, networks, or development. and like physical dark matter it may change our whole concept of what exactly a gene is or is not! since beadle and tatum's circumspect view of the protein world no longer holds true it adds a layer of complexity to organizing chip design. depending on which sequences are present in a particular transcript, you can, theoretically, design a set of probes to uniquely distinguish that variant. at the dna level itself there is much potential for looking at variants either expressed or not at a very basic level as a diagnostic system, but ultimately the real paydirt is the information that can be gained from looking at the consequence of non-coding sequence variation on the transcriptome itself. and fine tuning when this matters and when it is irrelevant as a predicative model is the auspices of the affymetrix spin-off perlegen. perlegen came into being in late to accelerate the development of high-resolution, whole genome scanning. and they have stuck to that purity of purpose. to paraphrase dragnet's sergeant joe friday, they focus on the facts of dna just the dna. perlegen owes its true genesis to the desire of one of its cofounders to use dna chips to help understand the dynamics underlying genetic diseases. brad margus' two sons have the rare disease "ataxia telangiectasia" (a-t). a-t is a progressive, neurodegenerative childhood disease that affects the brain and other body systems. the first signs of the disease, which include delayed development of motor skills, poor balance, and slurred speech, usually occur during the first decade of life. telangiectasias (tiny, red "spider" veins), which appear in the corners of the eyes or on the surface of the ears and cheeks, are characteristic of the disease, but are not always present. many individuals with a-t have a weakened immune system, making them susceptible to recurrent respiratory infections. about % of those with a-t develop cancer, most frequently acute lymphocytic leukemia or lymphoma suggesting that the sentinel competence of the immune system is compromised. having a focus so close to home is a powerful driver for any scientist. his co-founder david cox is a polymath pediatrician whose training in the latter informs his application of the former in the development of patient-centered tools. from that perspective, perlegen's stated mission is to collaborate with partners to rescue or improve drugs and to uncover the genetic bases of diseases. they have created a whole genome association approach that enables them to genotype millions of unique snps in thousands of cases and controls in a timeframe of months rather than years. as mentioned previously, snp (single nucleotide polymorphism) markers are preferred over microsatellite markers for association studies because of their abundance along the human genome, the low mutation rate, and accessibilities to high-throughput genotyping. since most diseases, and indeed responses to drug interventions, are the products of multiple genetic and environmental factors it is a challenge to develop discriminating diagnostics and, even more so, targetedtherapeutics. because mutations involved in complex diseases act probabilisticallythat is, the clinical outcome depends on many factors in addition to variation in the sequence of a single gene -the effect of any specific mutation is smaller. thus, such effects can only be revealed by searching for variants that differ in frequency among large numbers of patients and controls drawn from the general population. analysis of these snp patterns provides a powerful tool to help achieve this goal. although most bi-alleic snps are rare, it has been estimated that just over million common snps, each with a frequency of between and %, account for the bulk of the dna sequence difference between humans. such snps are present in the human genome once every base pairs or so. as is to be expected from linkage disequilibrium studies, alleles making up blocks of such snps in close physical proximity are often correlated, resulting in reduced genetic variability and defining a limited number of "snp haplotypes," each of which reflects descent from a single, ancient ancestral chromosome. in cox's group, using high level scanning with some old-fashioned somatic cell genetics, constructed the snp map of chromosome .the surprising findings were blocks of limited haplotype diversity in which more than % of a global human sample can typically be characterized by only three common haplotypes (interestingly enough the prevalence of each hapolytype in the examined population was in the ratio : : . ).from this the conclusion could be drawn that by comparing the frequency of genetic variants in unrelated cases and controls, genetic association studies could potentially identify specific haplotypes in the human genome that play important roles in disease, without need of knowledge of the history or source of the underlying sequence, which hypothesis they subsequently went on to prove. following cox et al. pioneering work on "blocking" chromosome into characteristic haplotypes, tien chen came to visit him from university of southern california and following the visit his group developed discriminating algorithms which took advantage of the fact that the haplotype block structure can be decomposed into large blocks with high linkage disequilibrium and relatively limited haplotype diversity, separated by short regions of low disequilibrium. one of the practical implications of this observation is as suggested by cox that only a small fraction of all the snps they refer to as "tag" snps can be chosen for mapping genes responsible for complex human diseases, which can significantly reduce genotyping effort, without much loss of power. they developed algorithms to partition haplotypes into blocks with the minimum number of tag snps for an entire chromosome. in they reported that they had developed an optimized suite of programs to analyze these block linkage disequilibrium patterns and to select the corresponding tag snps that will pick the minimum number of tags for the given criteria. in addition the updated suite allows haplotype data and genotype data from unrelated individuals and from general pedigrees to be analyzed. using an approach similar to richard michelmore's bulk segregant analysis in plants of more than a decade previously, perlegen subsequently made use of these snp haplotype and statistical probability tools to estimate total genetic variability of a particular complex trait coded for by many genes, with any single gene accounting for no more than a few percent of the overall variability of the trait. cox's group have determined that fewer than total individuals provide adequate power to identify genes accounting for only a few percent of the overall genetic variability of a complex trait, even using the very stringent significance levels required when testing large numbers of dna variants. from this it is possible to identify the set of major genetic risk factors contributing to the variability of a complex disease and/or treatment response. so, while a single genetic risk factor is not a good predictor of treatment outcome, the sum of a large fraction of risk factors contributing to a treatment response or common disease can be used to optimize personalized treatments without requiring knowledge of the underlying mechanisms of the disease.they feel that a saturating level of coverage is required to produce repeatable prediction of response to medication or predisposition to disease and that taking shortcuts will for the most part lead to incomplete, clinically-irrelevant results. in hinds et al. in science describe even more dramatic progresss. they describe a publicly available, genome-wide data set of . million common singlenucleotide polymorphisms (snps) that have been accurately genotyped in each of people from three population samples. a second public data set of more than million snps typed in each of people has been generated by the international haplotype map (hapmap) project. these two public data sets, combined with multiple new technologies for rapid and inexpensive snp genotyping, are paving the way for comprehensive association studies involving common human genetic variations. perlegen basically is taking to the next level fodor's stated reason for the creation of affymetrix, the belief that understanding the correlation between genetic variability and its role in health and disease would be the next step in the genomics revolution. the other interesting aspect of this level of coverage is, of course, the notion of discrete identifiable groups based on ethnicity, centers of origin and such breaks down and a spectrum of variation arises across all populations which makes the perlegen chip, at one level, a true unifier of humanity but at another adds a whole layer of complexity for hmos! at the turn of the century, this personalized chip approach to medicine received some validation at a simpler level in a closely related disease area to the one to which one fifth of a-t patients ultimately succumb when researchers at the whitehead institute used dna chips to distinguish different forms of leukemia based on patterns of gene expression in different populations of cells. moving cancer diagnosis away from visually based systems to such molecular based systems is a major goal of the national cancer institute. in the study, scientists used a dna chip to examine gene activity in bone marrow samples from patients with two different types of acute leukemia -acute myeloid leukemia (aml) and acute lymphoblastic leukemia (all). then, using an algorithm, developed at the whitehead, they identified signature patterns that could distinguish the two types. when they cross-checked the diagnoses made by the chip against known differences in the two types of leukemia, they found that the chip method could automatically make the distinction between aml and all without previous knowledge of these classes. taking it to a level beyond where perlegen are initially aiming, eric lander, leader of the study said, mapping not only what is in the genome, but also what the things in the genome do, is the real secret to comprehending and ultimately curing cancer and other diseases. chips gained recognition on the world stage in when they played a key role in the search for the cause of severe acute respiratory syndrome (sars) and probably won a mcarthur genius award for their creator. ucsf assistant professor joseph derisi, already famous in the scientific community as the wunderkind originator of the online diy chip maker in pat brown's lab at stanford, built a gene microarray containing all known completely sequenced viruses ( , of them) and, using a robot arm that he also customized, in a three day period used it to classify a pathogen isolated from sars patients as a novel coronavirus. when a whole galaxy of dots lit up across the spectrum of known vertebrate cornoviruses derisis knew this was a new variant. interestingly the sequence had the hottest signal with avian infectious bronchitis virus. his work subsequently led epidemiologists to target the masked palm civet, a tree-dwelling animal with a weasel-like face and a catlike body as the probable primary host. the role that derisi's team at ucsf played in identifying a coronavirus as a suspected cause of sars came to the attention of the national media when cdc director dr. julie gerberding recognized joe in march , press conference and in when joe was honored with one of the coveted mcarthur genius awards. this and other tools arising from information gathered from the human genome sequence and complementary discoveries in cell and molecular biology, new tools such as gene-expression profiling, and proteomics analysis are converging to finally show that rapid robust diagnostics and "rational" drug design has a future in disease research. another virus that puts sars deaths in perspective benefitted from rational drug design at the turn of the century. influenza, or flu, is an acute respiratory infection caused by a variety of influenza viruses. each year, up to million americans develop the flu, with an average of about , being hospitalized and , to , people dying from influenza and its complications. the use of current influenza treatments has been limited due to a lack of activity against all influenza strains, adverse side effects, and rapid development of viral resistance. influenza costs the united states an annual $ . billion in physician visits, lost productivity and lost wages. and least we still dismiss it as a nuisance we are well to remember that the "spanish" influenza pandemic killed over million people in and , making it the worst infectious pandemic in history beating out even the more notorious black death of the middle ages. this fear has been rekindled as the dreaded h n (h for haemaglutenin and n for neuraminidase as described below) strain of bird flu has the potential to mutate and recognise homo sapiens as a desirable host. since rna viruses are notoriously faulty in their replication this accelerated evolutionary process gives then a distinct advantage when adapting to new environments and therefore finding more amenable hosts. although inactivated influenza vaccines are available, their efficacy is suboptimal partly because of their limited ability to elicit local iga and cytotoxic t cell responses. the choices of treatments and preventions for influenza hold much more promise in this millennium. clinical trials of cold-adapted live influenza vaccines now under way suggest that such vaccines are optimally attenuated, so that they will not cause influenza symptoms but will still induce protective immunity. aviron (mountain view, ca), biochem pharma (laval, quebec, canada), merck (whitehouse station, nj), chiron (emeryville, ca), and cortecs (london), all had influenza vaccines in the clinic at the turn of the century, with some of them given intra-nasally or orally. meanwhile, the team of gilead sciences (foster city, ca) and hoffmann-la roche (basel, switzerland) and also glaxowellcome (london) in put on the market neuraminidase inhibitors that block the replication of the influenza virus. gilead was one of the first biotechnology companies to come out with an anti-flu therapeutic. tamiflu™ (oseltamivir phosphate) was the first flu pill from this new class of drugs called neuraminidase inhibitors (ni) that are designed to be active against all common strains of the influenza virus. neuraminidase inhibitors block viral replication by targeting a site on one of the two main surface structures of the influenza virus, preventing the virus from infecting new cells. neuraminidase is found protruding from the surface of the two main types of influenza virus, type a and type b. it enables newly formed viral particles to travel from one cell to another in the body. tamiflu is designed to prevent all common strains of the influenza virus from replicating. the replication process is what contributes to the worsening of symptoms in a person infected with the influenza virus. by inactivating neuraminidase, viral replication is stopped, halting the influenza virus in its tracks. in marked contrast to the usual protracted process of clinical trials for new therapeutics, the road from conception to application for tamiflu was remarkably expeditious. in , gilead and hoffmann-la roche entered into a collaborative agreement to develop and market therapies that treat and prevent viral influenza. in , as gilead's worldwide development and marketing partner, roche led the final development of tamiflu, months after the first patient was dosed in clinical trials in april , roche and gilead announced the submission of a new drug application to the u.s. food and drug administration (fda) for the treatment of influenza. additionally, roche filed a marketing authorisation application (maa) in the european union under the centralized procedure in early may . six months later in october , gilead and roche announced that the fda approved tamiflu for the treatment of influenza a and b in adults. these accelerated efforts allowed tamiflu to reach the u.s. market in time for the - flu season. one of gilead's studies showed an increase in efficacy from % when the vaccine was used alone to % when the vaccine was used in conjunction with a neuraminidase inhibitor. outside of the u.s., tamiflu also has been approved for the treatment of influenza a and b in argentina, brazil, canada, mexico, peru and switzerland. regulatory review of the tamiflu maa by european authorities is ongoing. with the h n birdflu strain's relentless march (or rather flight) across asia, in through eastern europe to a french farmyard, an unwelcome stowaway on a winged migration, and no vaccine in sight, tamiflu, although untested for this species, seen as the last line of defense is now being horded and its patented production right's fought over like an alchemist's formula. tamiflu's main competitor, zanamivir marketed as relenza™ was one of a group of molecules developed by glaxowellcome and academic collaborators using structure-based drug design methods targeted, like tamiflu, at a region of the neuraminidase surface glycoprotein of influenza viruses that is highly conserved from strain to strain. glaxo filed for marketing approval for relenza in europe and canada. the food and drug administration's accelerated drug-approval timetable began to show results by , its evaluation of novartis's gleevec took just three months compared with the standard - months. another factor in improving biotherapeutic fortunes in the new century was the staggering profits of early successes. in , $ . billion of the $ . billion in revenue collected by genentech in south san francisco came from oncology products, mostly the monoclonal antibody-based drugs rituxan, used to treat non-hodgkin's lymphoma, and herceptin for breast cancer. in fact two of the first cancer drugs to use the new tools for 'rational' design herceptin and gleevec, a small-molecule chemotherapeutic for some forms of leukemia are proving successful, and others such as avastin (an anti-vascular endothelial growth factor) for colon cancer and erbitux are already following in their footsteps. gleevec led the way in exploiting signal-transduction pathways to treat cancer as it blocks a mutant form of tyrosine kinase (termed the philadelphia translocation recognized in 's) that can help to trigger out-of-control cell division. about % of biotech companies raising venture capital during the third quarter of listed cancer as their primary focus, according to online newsletter venturereporter. by according to the pharmaceutical research and manufacturers of america, medicines were in development for cancer up from in . another new avenue in cancer research is to combine drugs. wyeth's mylotarg, for instance, links an antibody to a chemotherapeutic, and homes in on cd receptors on acute myeloid leukemia cells. expertise in biochemistry, cell biology and immunology is required to develop such a drug. this trend has created some bright spots in cancer research and development, even though drug discovery in general has been adversely affected by mergers, a few high-profile failures and a shaky us economy in the early 's. as the millennium approached observers as diverse as microsoft's bill gates and president bill clinton predicted the st century wiould be the "biology century". by the many programs and initiatives underway at major research institutions and leading companies were already giving shape to this assertion. these initiatives have ushered in a new era of biological research anticipated to generate technological changes of the magnitude associated with the industrial revolution and the computerbased information revolution. complementary dna sequencing: expressed sequence tags and human genome project basic local alignment search tool high-tech herbal medicine: plant-based vaccines asilomar conference on recombinant dna molecules potential biohazards of recombinant dna molecules hugo: the human genome organization chimeric plant virus particles administered nasally or orally induce systemic and mucosal immune responses in mice the human genome: the nature of the enterprise orchestrating the human genome project separation and analysis of dna sequence reaction products by capillary gel electrophoresis nutritional genomics: manipulating plant micronutrients to improve human health helping europe compete in human genome research genome project gets rough ride in europe construction of a linkage map of the human genome, and its application to mapping genetic diseases separation of dna restriction fragments by high performance capillary electrophoresis with low and zero crosslinked polyacrylamide using continuous and pulsed electric fields preimplantation and the 'new' genetics a history human genome project it aint necessarily so: the dream of the human genome and other illusions high speed dna sequencing by capillary electrophoresis a strategy for sequencing the genome years early expression of norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice rapid production of specific vaccines for lymphoma by expression of the tumor-derived single-chain fv epitopes in tobacco plants generation and analysis of , human expressed sequence tags national academy of sciences. introduction of recombinant dna-engineered organisms into the environment: key issues functional foods and biopharmaceuticals: the next generation of the gm revolution in let them eat precaution biotechnology: a review of technological developments, publishers forfas vitamin-a and iron-enriched rices may hold key to combating blindness and malnutrition: a biotechnology advance french dna: trouble in purgatory genome: the autobiography of a species in chapters harper collins derivation of pluripotent stem cells from cultured human primordial germ cells production of correctly processed human serum albumin in transgenic plants high-yield production of a human therapeutic protein in tobacco chloroplasts the common thread: a story of science, politics, ethics and the human genome capillary gel electrophoresis for dna sequencing. laser-induced fluorescence detection with the sheath flow cuvette production of functional human alpha -antitrypsin by plant cell culture genetic modification of oils for improved health benefits, presentation at conference, dietary fatty acids and cardiovascular health: dietary recommendations for fatty acids: is there ample evidence? stable accumulation of aspergillus niger phytase in transgenic tobacco leaves antenatal maternal serum screening for down's syndrome: results of a demonstration project viable offspring derived from fetal and adult mammalian cells key: cord- -vmqvropy authors: rukavtsova, e. b.; abramikhina, t. v.; shulga, n. ya.; bykov, v. a.; bur’yanov, ya. i. title: tissue specific expression of hepatitis b virus surface antigen in transgenic plant cells and tissue culture date: journal: russ j plant physiol doi: . /s sha: doc_id: cord_uid: vmqvropy the tobacco plants (nicotiana tabacum l.) carrying the hbsag gene controlled by (aocs)( )amaspmas, the hybrid promoter that includes regulatory elements of the agrobacterial octopine and mannopine synthase genes, as well as plants controlled by the same promoter and adh , maize alcohol dehydrogenase gene intron were obtained. the presence of the adh gene intron did not significantly change the level of expression of the hbsag gene in plants. the analysis of expression of hepatitis b virus surface antigen (hbs-antigen) in transformed plants expressing the hbsag under the control of different promoters was made. the level of hbs-antigen in plants carrying the hbsag gene controlled by (aocs)( )amaspmas, the hybrid agrobacterium-derived promoter, was the highest in roots and made up to . % of total amount of soluble protein. the level of hbs-antigen in plants carrying the hbsag gene controlled by the dual s rna cauliflower mosaic virus promoter was the same in all organs of the plant and made up to . % of the total amount of soluble protein. hairy root and callus cultures of plants carrying the hbsag gene and expressing the hbs-antigen were obtained. the transgenic plant-based technologies for production of subunit vaccines of a new generation, which may become cheaper and safer alternatives to traditionally obtained vaccine preparations, are now being developed. plant cells contain enzymatic systems of post-translational modification necessary for the assembling of monomer vaccine proteins they synthesize into the immunogenic multimer forms. plants are fully able to synthesize target antigens that can cause an active immune response [ ] of the host organism. the viral and bacterial antigens were shown to stimulate the production of immunoglobulines against the corresponding pathogens [ ] [ ] [ ] [ ] [ ] [ ] . various transgenic plants are now being produced and tested in research centers worldwide as potential producers of vaccines against infection agents causing various human diseases, including the hepatitis b virus [ , , ] . however, the transgenic plant-derived vaccine preparations are not yet commercially available. earlier we have obtained the tobacco plants expressing the synthetic gene of the hepatitis b surface antigen ( hbsag ) controlled by single and dual s rna cauliflower mosaic virus promoters (camv s and camv ss, respectively) [ , ] . the presence of the dual s promoter increased expression of the antigen to the level of . % of total amount of soluble protein. the transgenic potato plants expressing the hbsag gene controlled by the same promoter and also by the patatin promoter of potato tubers were also produced. the amount of the hbs-antigen in potato tubers exceeded µ g/g of tuber mass and was the highest in plants expressing the hbsag gene controlled by the dual camv ss promoter. to obtain plants with tissue-specific expression of the hepatitis b vaccine gene in tissue cultures and whole plants (as potential producers of the vaccine), tissue-specific promoters, especially of the hybrid agrobacterium-derived promoters (aocs) amaspmas , prove to be effective. these promoters consist of the regulatory elements of the octopine synthase ( ocs ) and mannopine syntase ( mas ) genes of agrobacterium tumefaciens. as was shown earlier, these promoters strongly induced the expression of the β -glucoronidase (gus) gene in plants up to the level greatly exceeding that in plants controlled by the dual s rna cauliflower mosaic virus promoter [ ] . the objective of this study was to obtain transgenic tobacco plants synthesizing the hepatitis b surface anti- gen controlled by ( aocs ) amaspmas promoters and regulated by the elements of agrobacterial octopine synthase and mannopine synthase genes and also to analyze the expression profile of the hbsag gene in different cells of the whole plant as well as that in callus and hairy root tissue cultures. materials and methods construction of plasmids for plant transformation. pss-hbsag, the recombinant plasmid, carrying a synthetic gene encoding the hbsag/mayw polypeptide [ ] , was used as a gene source. the two vectors both containing the ( aocs ) amaspmas promoter were used for cloning and plant transformation: pe and pe (courtesy of dr gelvin, purdue university, united states) [ ] . the extension of the hbsag gene with specific sequences necessary for molecular cloning was done by means of pcr with two primers containing the kpn i and sal i restriction sites: '-cggg-taccatggaaaacattactt and '-cggtcgac-ctatcattaaatgtaaac, respectively. the reaction mixture contained . µ g of pdes plasmid dna as a template, mm kcl, mm tris-hcl, ph . , at ° c, . mm mgcl , . % triton x- , mm -mercaptoethanol, . mm dntps mixture (usb, united states), . µ m of each primer and . units of taq dna polymerase (sibenzim, russia). the reaction volume was µ l. the reaction started with min at ° c and was followed by cycles: min at ° c, min at ° c, min at ° c, and ended with min at ° c. the gene amp pcr system (perkin-elmer, united states) was used for experiments. the amplified gene was cloned into a binary pe vector for transformation of plants between the restriction sites kpn i and sal i. for cloning purposes, the hbsag gene was cut out of the pss-hbsag plasmid between the xho i and cfr i restriction sites and incorporated into the pe vector between the same sites. the plasmid constructions obtained were used for transformation of escherichia coli strain hb . the constructions containing the hbsag gene downstream the promoter were transferred into strain lba (pal ) of a. tumefaciens [ ] by means of direct transformation [ ] . the analysis of dna of the obtained clones was done by southern hybridization with the p-labeled amplification product of the hbsag gene [ ] . transgenic plants. the nicotiana tabacum l., cv. samsun plants were cultivated in vitro in . -to -l cultivation containers on the agar-solidified phytohormone-free ms medium [ ] at to °ë , klx, and % relative humidity. the resulting agrobacterial strain culture was used for the infection of leaf disks according to a standard protocol [ ] . the leaf disks were co-cultivated with the overnight agrobacterial cultures for two days and then transferred onto the selection ms medium containing hormones ( mg/l of ba and . mg/l of naa), mg/l of kanamycin sulfate (km), and mg/l of cefotaxime. the regenerated shoots were passed onto the selection ms medium. calli were obtained from leaf explants of transformed plants on the ms medium containing % sucrose, . mg/l ba, mg/l naa, and mg/l km. the hairy root culture was grown on the hormone-free ms medium from disk leaves of transformed plants infected with strain a of a. rhizogenes [ ] . extraction of dna from tobacco leaves. the dna from tobacco leaves was extracted as described in [ ] . leaves were crushed in . ml eppendorf tubes, . ml of the extraction buffer was added (containing . m tris-hcl, ph . , . m nacl, mm edta, and . % sds); the mixture was incubated for h at room temperature and then centrifuged at rpm for clarification. an equal volume of isopropanol was added, and the dna precipitate was dissolved in µ l of the te buffer ( mm tris-hcl and mm edta, ph . ). the plant dna obtained was used as a template for pcr. pcr analysis of the hbsag gene. for the pcr analysis of hbsag gene, . to µ g of plant genomic dna was used. the reaction mixture and cycle conditions were the same as above. immunoassay of the surface antigen level. the immunoassay of the hepatitis b virus surface antigen level in transformed plants was done as earlier described by us [ ] with minor modifications. leaves, roots, and calli of tested plants were ground in liquid nitrogen, and the extraction buffer ( . m sodium phosphate buffer, ph . , . m nacl, mm edta, . % tween , . mm phenylmethanesulfonyl fluoride, and . % sodium ascorbate). the extract obtained was centrifuged for min at rpm, the supernatant was transferred to . -ml eppendorf tubes and recentrifuged for min at rpm. the "vektohep b-hbs-antigen" test systems (jsc vektor-best, russia) were used for measuring the hbs-antigen level in the supernatant. the recombinant yeast cell-derived hbsantigen [ ] was used as a positive control. the assay was carried out according to the manufacturer's instructions. the total amount of soluble protein was measured according to bradford [ ] . mechanical leaf wounding for induction of hbsag gene expression. leaves were immersed into the liquid ms medium, cut with a scalpel into stripes to mm wide, and incubated at to °ë for h. the injured leaves were used for immunoassay. the recombinant plasmids used for transformation of plants are shown in fig. . these plasmids are based on the pe and pe vectors containing the ( aocs ) amaspmas promoter [ ] . both plasmids are carrying hbsag , a synthetic gene [ ] . the difference between the two vectors is that the pe is carrying the adh the maize alcohol dehydrogenase gene intron, rukavtsova et al. but is lacking the tl enhancer. the obtained plasmid constructs carrying the hbsag gene under the ( aocs ) amaspmas promoter were used for the direct transformation of strain lba (pal ) of a. tumefaciens . separate agrobacterial colonies were collected for extraction of plasmid dna and its visualization in agarose gel electrophoresis. the transformed agrobacterial strains were used for the infection of tobacco leaf disks. to lines of transformed plants of each type were selected for subsequent molecular, genetic, and biochemical analysis. the hbsag gene presence in transformed plants was confirmed by means of pcr. the target dna, which corresponds to the hepatitis b virus surface antigen, was found in all transgenic plants tested (fig. ) . the immunoassay was carried out to study the expression profile of the surface antigen hbsag in obtained plants. the hbs-antigen was found in different amounts in plants of all transgenic tobacco lines tested (see the table). the expression level of this antigen in the in vitro grown transgenic tobacco plants was up to . % of total amount of soluble protein. maximum expression of the surface hbsag antigen was observed in roots. the genetic construct pe -ag used for some of our experiments contained the alcohol dehydrogenase gene intron. the inclusion of introns into plant cells may enhance the expression of foreign genes [ ] . however, in our case the presence of the adh gene intron did not increase the expression of the target gene. the enhancement effect seems to be dependent on the location of the intron in the genetic construct carrying the target gene. the table shows the expression levels of the hbsantigen in plants controlled by different promoters. thus, the expression level of the hbs-antigen in plants controlled by ( aocs ) amaspmas, the hybrid agrobacterial promoter, reached the maximum of . % of total amount of soluble protein in roots. the expression level of the hbs-antigen in plants controlled by the dual s rna cauliflower mosaic virus promoter was the same in all organs of the plant, accounting for up to . % of total amount of soluble protein. therefore, (aocs) amaspmas, the hybrid agrobacterial promoter, can be regarded as a tissue-specific element of the hbsag gene expression, maximum expression being induced in roots. the activity of the agrobacterial mannopine synthase gene can be very different in different tissues and organs of the plant, maximum expression being observed in roots and calli [ , ] . the expression level of the hbs-antigen in leaves of transgenic plants controlled by the (aocs) amaspmas promoter substantially increased after wounding and reached to . % of total amount of soluble protein. these results correspond with the available data on the induction of foreign gene expression in leaves of transgenic plants con-trolled by the mannopine synthase promoter under similar wounding conditions [ , ] . eight lines of transgenic plants with the highest level of expression of the hbs-antigen were selected to obtain so-called hairy root tissue cultures by means of infection of leaf disks with strain a of a. rhizogenes culture. hairy roots, being effectively plant tumors transformed by a. rhizogenes [ ] , are a convenient system for production of secondary metabolites and recombinant proteins due to their genetic stability and fast growth in the hormone-free medium. as a result of retransformation of plants carrying the hbsag gene with strain a of a. rhizogenes, hairy root cultures were obtained also carrying the hbs-antigen (fig. ) . the level of expression of the hbs-antigen in different lines of hairy root cultures remained the same as that in parent transgenic plants making up to . % of total soluble protein. leaf explants of the transformed plants expressing the hbs-antigen controlled by the (aocs) amaspmas promoter were used to obtain callus cultures. the hbsantigen expression in these cultures was also observed (fig. ) . this could be explained by the induction of activity of the mannopine synthase and octopine synthase promoters by a higher level of auxins in callus culture cultivated in the auxin-containing medium [ ] . it should be noted that the results of our study do not confirm the existing data on more efficient expression of foreign genes controlled by the (aocs) amaspmas hybrid promoters that those controlled by the dual camv ss promoter [ ] . in those study, the activity of the (aocs) amaspmas promoter was judged on the basis of gus staining assay. another data, however, are available [ ] when the researchers did not notice a considerable difference between the bt-cry ia , an amaspmas promoters. the attempts have also been made to use the (aocs) amaspmas hybrid promoter for transformation of plants that could potentially produce the vaccine against sars (severe acute respiratory syndrome) [ ] . the expression of sars-cov, the target s protein, was the highest in roots of transformed tobacco and immature fruits of the transformed tomato plants. (aocs) amaspmas, the hybrid promoter, can be considered as a potential tool for tissue specific expression of various pharmaceutically important proteins in transformed plants and their cell cultures. ( - ) tobacco plants transformed with pe -ag (lines , , , , ) ; ( - ) tobacco plants transformed with pe -ag (lines , , , ) . average values are presented for three independent experiments; standard error in each of them did not exceed . %. transgenic plants as vaccine production systems expression of hepatitis b surface antigen in transgenic plants edible vaccines expression of immunogenic glycoprotein s polypeptides from transmissible gastroenteritis coronavirus in transgenic plants plant expression systems for the production of vaccines transgenic plants as edible vaccines immunogenicity of porcine transmissible gastroenteritis virus spike protein expressed in plants expression of human hepatitis b virus surface antigen gene in transgenic tobacco polypeptides of hepatitis b surface antigen produced in transgenic potato production of transgenic tobacco plants expressing the gene encoding of hepatitis b virus surface antigen analysis of transgenic tobacco plants carring the gene for hepatitis b virus surface antigen strength and tissue specificity of chimeric promoters derived from the octopine and mannopine synthase genes expression of hepatitis b virus surface antigen in transgenic potato plants and its characteristics transfer of octopine t-dna segment to plant cells mediated by different types of agrobacterium tumouror root-inducing plasmids: generality of virulence systems binary vectors molecular cloning: a laboratory manual, cold spring harbor: cold spring harbor lab revised medium for rapid growth and bioassays with tobacco tissue cultures transformation of dicotyledonous plant cells using the ni plasmid of agrobacterium tumefaciens and ri plasmid of a. rhizogenes, plant genetic transformation and gene expression. a laboratory manual, draper transformation of several species of higher plants by agrobacterium rhizogenes. sexual transmission of the transformed genotype and phenotype a simple and rapid method for the preparation of plant genomic dna for pcr analysis recombinant plasmid dna pdes codes hepatitis b virus surface antigen (hbsag/mayw), yeast strain s. cerevisiae dan- /pdts -producer of hepatitis b virus antigen and vaccine on its base: rf patent no. a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding the effect of intron location on intron-mediated enhancement of gene expression in arabidopsis dual promoter of agrobacterium tumefaciens mannopine synthase genes is regulated by plant growth hormones combined usage of regulated promoters and grafting technique in gene fusions to lacz reveal new expression patterns of chimeric genes in transgenic plants evaluation of bt-cry ia (cryv) transgenic potatoes on two species of potato tuber moth, phthorimaea operculella and symmetrischema tangolias (lepidoptera: gelechiidae) in peru severe acute respiratory syndrome (sars) s protein production in plants: development of recombinant vaccine the authors thank s.v. chernyshov for assistance in the development of the pe -ag genetic construct.this study was supported by the presidium of russian academy of sciences, the program "dynamics of plant, animal, and human genofonds" and also by russian foundation for basic research (projects nos. - - and - - ). key: cord- -bveks t authors: butnariu, monica; butu, alina title: plant nanobionics: application of nanobiosensors in plant biology date: - - journal: plant nanobionics doi: . / - - - - _ sha: doc_id: cord_uid: bveks t nanobiosensors (nbss) are a class of chemical sensors which are sensitive to a physical or chemical stimulus (heat, acidity, metabolism transformations) that conveys information about vital processes. nbss detect physiological signals and convert them into standardized signals, often electrical, to be quantified from analog to digital. nbss are classified according to the transducer element (electrochemical, piezoelectric, optical, and thermal) in accordance with biorecognition principle (enzyme recognition, affinity immunoassay, whole sensors, dna). nbss have varied forms, depending on the degree of interpretation of natural processes in plants. plant nanobionics uses mathematical models based on qualitative and less quantitative records. nbss can give information about endogenous concentrations or endogenous fluxes of signaling molecules (phytohormones). the properties of nbss are temporal and spatial resolution, the ability of being used without significantly interfering with the system. nbss with the best properties are the optically genetically coded nbss, but each nbs needs specific development efforts. nbs technologies using antibodies as a recognition domain are generic and tend to be more invasive, and there are examples of their use in plant nanobionics. through opportunities that develop along with technologies, we hope that more and more nbss will become available for plant nanobionics. the main advantages of nbss are short analysis time, low-cost tests and portability, real-time measurements, and remote control. plants are a fascinating research topic if we relate to environmental stress. because they are physically stuck in specific spots, the plants have to handle in that site, regardless of the environmental conditions. moving to another place is not an option. but what plants can do is to modify the internal "environment," and plants are "true masters" of manipulating their metabolism to deal with environmental disturbances. this feature is one of the reasons why plants are useful in various research; we can rely on them as "sensitive indicators" of environmental changes, even in completely new environments. in the absence of normal conditions, plants cannot use the classical pathways of metabolism, so they need to identify other solutions. this is what happens when plants adjust their metabolism for regulating gene activation, thus producing more or less proteins that are useful or not in the new environmental conditions. the different parts of the plant come with their own genetic regulation strategies. a number of genes that are involved in the creation and remodeling of cell walls are activated differently in growing plants. other genes with a role in identifying light, which are normally active on the leaves, are active at the root level. in leaves, many genes associated with transmission of hormonal information are suppressed, and genes associated with insect protection are more active. these trends are also seen in the (higher) number of proteins involved in message transmission, cell wall metabolism, and plant protection. these patterns of gene and protein functioning indicate that in unfavorable conditions, the plants respond by weakening the cell walls and creating new ways to understand the environment. it is possible to monitor changes in the genes in real time by labelling certain proteins with fluorescent elements. plants modified with fluorescent proteins can give useful information about how they respond to the environment. these modified plants act as biological sensors (nbss). specialized cameras and microscopes allow us to monitor how the plant uses these fluorescent proteins (hamers et al. ) . chemical or biological nbs functions on the principle of signal emission (voltage or electrical, photonic) in response to a chemical reaction involve a chemical or biological receptor, r (macrocyclic ligand, antibody enzyme), that binds to a specific target molecule of a sample to be studied, the analyte, a. signal transmission is achieved by coupling with a transducer t that interfaces nbs processes with the processing-transform unit in a measurable signal. analysis of signals in plant nanobionics aims at processing signals recorded by measurements in order to extract the maximum of useful information for diagnostics and these devices are mostly used in genetic engineering in agriculture, where it is necessary to know the mechanisms of reaction and the affinity of enzymes and microorganisms for different substrates of interest and signaling molecules. a biochip is a device that contains a structure of individual sensory elements (nbss) interconnected by functions and recognition specifications, integrated on a chip. the number of nbss on a chip may be of the order of units. in this degree of integration, a set of distinct tests can be performed extremely quickly and efficiently. in contrast to microchips, biochips are not electronic structures (they contain different electronic structures coupled to nanobiosensory elements). each nbs can be thought of as a "microreactor" that performs a specific chemical reaction with an analyte. nbss from biochips can be designed to detect a wide variety of analytes, including dna, antibodies, proteins, and biomolecules. the advantages are multiple: sensors can be produced in batches or sequences that can be assembled in parallel or serial, providing a high manufacturing yield; sensors can be assembled on very small areas with reduced distances between them; d structures can be generated providing high signals besides d structures; any type of biochemical reaction may be incorporated; nbss can be produced separately and subsequently assembled according to the specificity and nature of the application. the important features regardless of industry or technology are selectivity, sensitivity, and stability in the design of sensory systems integrated with structures and arrangements of sensory elements (wan salim et al. ) . one of the integrated systems including rotational aseptic sampling is a robotic fluid and reusable electrodes formed by ink-jet printing injection system. the system contains an enzyme electrode with immobilized gox in gel, and the detection of hydrogen peroxide is carried out on a rhodinised carbon electrode (rh coating). although the enzyme electrode has stability and efficiency characteristics, the problem of automated sample monitoring and sampling in an integrated system requires multiparameter optimization due to reciprocal interferences. there is a requirement on specific domains (environment and genetic engineering) of highly performing integrated systems that work in in vivo conditions, such as dialysis, the use of biointerfaces, evanescent techniques, and atomic force microscopy to grasp in the depth of the biological phenomena (identifying and understanding the interaction of proteins). the in vivo exploitation of detection systems for both glucose and lactate was confirmed by the efficacy of using phospholipid copolymers and improving hemocompatibility. immunosensors provide an example for the development of integrated systems where microseparations, chromatographic methods, and electrochemical couplings with optical detectors are incorporated, which ultimately lead to a miniaturized system. there are examples where the level of integration and miniaturization becomes more pronounced (dna-nanotube or biochips-biointerfaces). nbss are expected to be widely used in plant nanobionics where physiological/biochemical parameters should be identified. advanced ink-jet technology has developed methods for analyzing nanoliter fractions on a three-dimensional nbs surface at a speed of m/s. it is expected to produce one million nbss/cm areas using photolithography, contact fingerprinting or self-assembling techniques, and adsorption/desorption under the laser beam that allows "writing" proteins on the surface to be analyzed with great precision. laser techniques, maple (matrix assisted pulsed laser evaporation) or dw (direct writing) approached for immobilizing biological materials on substrates, are in the laboratory stage but have prospects for use as molecular imprinting methods (potocký et al. ). whether nbss are individuals, in integrated systems, or areas of nbss, all are characterized by unique parameters such as sensitivity and detection limit for a range of analytes. trace detection of various analytes (indicators, additives, contaminants) with sufficient sensitivity and safety is the basic criteria of an nbs to be used. the detection limit in the laboratory is pushed to an atom when the atomic force microscopy is used. thus, the enzymatic electrodes, studied and continuously perfected, use palliative such as concentrating the analyte of interest, which leads to major design and miniaturization difficulties. nbss for phenol vapors were identified, where the phenoloxidase was immobilized on a glycerol gel with a range of interdigitated electrodes. phenol vapors are directly partitioned into the gel and oxidized to quinone. signal amplification was improved by redox amplification of the quinone/catechol couple to obtain a reasonable sensitivity resulting in a detection limit of ppb phenol. this principle can be extended to other carbon compounds up to the ppt (parts per trillion) limits. dna structures have been studied as potential receptors. sandwich structures of liquid crystal dispersions and dna-polycation complexes have been studied with relatively good success in identifying different analytes. the polycation with the role to maintain structural integrity of dna and dna-protamine complexes allows detection of hydrolysis of the trypsin enzyme to the detection limit of − m. elimination of the polycation leads to an increase in the distance between the two dna strands resulting in the appearance of an intensive band in the circular dichroism spectrum due to the texture modification (espinoza et al. ). from a practical standpoint, the disadvantage is their inherent instability. different strategies have been approached to improve longevity and preserve the structure of biological receptors. immobilization in matrices by sol-gel technique for glucose nbss is one of the strategies; fluorescence indicators are used: hexahydrate chloride ( , ′-bipyridyl) ruthenium (ii) and -hydroxypyrene- , , trisulfonic acid. in addition to the optical property improvement of the gel, the stability of the gox enzyme has also improved. other examples are the case of monooxygenase used in hydrocarbon detection; detection of organic halides with metalloporphyrins; and detection of carbon tetrachloride, haloalkane (perchloroethylene), and insecticides (ddt) (kazakova et al. ). improving the selectivity of an nbs can be approached on two levels: through direct transducer-biological receiver interfacing to reduce interference and new receptors with improved affinity or new affinity capabilities. selectivity is a key parameter that requires the performance of an nbs. pyrroloquinoline is used as a mediator in a glucose oxidase enzyme electrode for the measurement of glucose in the raw or elaborated cavity. alternatively, the electrocatalytic detection of the reaction products resulting from the enzymatic reactions can be improved by chemically modified electrodes such as rhodinised electrodes or hexacyanoferrate modified carbon electrodes. prussian blue is used to modify the electrode surface at amperometric detection of oxygenated water at both oxidation and reduction potentials for the enzyme electrode in lactate and glucose detection. one solution is to identify redox centers of the enzyme via a molecular wire to perform the electron transfer to the electrode (enzymes linked by molecular wires), but the concerns have focused on immobilized mediators on different polymer chains. molecular wires are regarded as intermediates in long-range electron transfer, consisting of two pyridine groups linked by thiophenes with different lengths. such wires can be used in conjunction with self-assembly techniques to produce an isolated electrode that transfers electrons to predetermined molecular pathways (jones et al. ). an ideal nbs is a device that will detect an "analyte," the subject under analysis and which is present in a given sample. most samples also contain other analytes which may interfere with the nbs response. it must have a specific selectivity to identify the target analyte. it is necessary to design nbss with selectivity for an analyte with the ability to discriminate the interferences produced by the other components in the analyzed sample. specific identification and selectivity capacity are the key components of molecular recognition. molecular recognition is accomplished by the sensor component of a host molecule (host-chemoreceptor) that binds selectively to the "guest" target molecule/guest molecule that needs to be identified. for each "host-guest" system, there is a specific chemical reaction from the multitude of possible reaction channels. when the host-guest response was identified, the host molecule is immobilized/incorporated into nbss, typically on a transducer-contacting membrane/ contact electrode. finally, a way to signal that the bindings/recognition event has occurred (transducer to transducer) has to be found (rodríguez-sevilla et al. ). one of the requirements for molecular recognition is the existence of groups or centers with specific reactivity in the host molecule that can "close" or bind ions, atoms, molecules, and biomolecules. all living organisms use enzymes, which are proteins that contain "pockets," active centers, designed to recognize a specific analyte. this means that only a specific analyte is able to enter the enzyme pocket. enzymes can be used in nbss as host receptors with molecular recognition capability but are unstable. to design host molecules that can be used in an nbs, the following criteria are considered: the host molecule must be stable under the conditions in which it is to be used, must be able to selectively bind the analyte in the sample, must be able to be immobilized in a film/membrane that is in contact with the sample, must signal that a host-host binding response has occurred, and must ideally release the analyte after detection so the host is free to be reusable. biological receptors include antibodies, membrane receptors, signaling molecules, enzymes, ribosomes, lectins, phytohormones, etc. they bind analytes using "lock-and-key" molecular recognition mechanisms (key-lock, identification-immobilization). biological receptors are not practical solutions for many applications because the specificity, sensitivity, and stability cannot be optimized. artificial receivers are immobilization environments that can be optimized by molecular design for any type of application. synthetic receptor design and synthesis are based on tools developed by proteomics and genetic engineering, producing recognition components that can respond to the occurrence and identification of metabolic deficiencies in plant nanobionics. there are platforms and areas of artificial receptors based on combinatorial mathematical techniques, interface biology, and surface chemistry. they have induced the development of various artificial receptor environments with rapid and diversified selection for any target analyte. the current technique for producing synthetic receptors is called cara (combinatorial array of receptor analysis) (fang et al. ) . supramolecular chemistry has developed a wide range of synthetic macrocycles. the most common feature for macrocycle classes is that they contain cavities that act as host pockets for guest molecules. the selectivity of the hosts can be done in "read mode" by varying the size of the preformed cavities. -crown- has a small cavity ideal for the binding of small ions such as li + , while -crown- has a large cavity that fits better with larger ions such as k + . the size of the cavities is important for the selectivity of the host, but the question remains what attracts an ion or molecule into a preformed cavity and which factors stabilize the host-guest complex. in enzymes, weak noncovalent interactions (hydrogen bonding, electrostatics, dipole-dipole, van der waals, etc.) are used to link the guest into the enzyme pocket (interactions stabilize host-guest interaction). macrocycles contain polar functionalities, capable of interacting with guests via hydrogen bonding, electrostatic interactions, and dipole-dipole interactions. it is desirable that bonding in the cavity is not strong, because it is important that the analyte, the guest, is released from the host after it has been detected and measured. crown ethers and calixarene are ideal for bonding metal cations, based on the size of their cavity, but also on the high density of electrons present on the oxygen atoms in the cavity. the base compound selectively binds li + to other metallic cations; the modified version of the base macrocycle has a good selectivity for na + . by synthetic modification it is possible to increase the capacity of the host cavity, and new functionalities can be introduced that will favor the binding of specific molecules and ions (da silva et al. ) . other modified calixarenes, which demonstrate this principle, are the group of tetraphosphine oxide of the calix[ ]arene. by changing binding groups on the same template, calix[ ]arene from esters in phosphorus hydrogen oxides selectivity changes from na + to ca + . by increasing the number of repeatable units in esters and phosphorus hydrogen oxides at six, the cavity increases, and the selectivity changes in favor of the higher cs + and pb + cations, respectively. some host compounds have been developed for the selective detection of low molecular weight compounds. an example involves the use of the tetra (s-propanol) calix[ ]arena containing four lateral chiral halves for selective differentiation of the phenylalanyl enantiomers. other techniques of supramolecular chemistry may be involved in the synthesis of synthetic receptors that simulate the properties of enzymes. the basic structures that can be modified are porphyrins, semiconductor polymers of the tetrathiafulvalene (ttf) class, and ppvs. other patterns can be considered modified polysaccharides. a linear archetype is the polyanilines containing the two types of redox states (meyer et al. ). among the multiple nbs classifications, bioaffinity has a range of applications, and antigen-antibody interactions (ag-ab) play a role and are considered to be an instrument in the development of molecular recognition principles. in vivo ag-ab interactions are reversible. factors that condition the ag-ab interaction are the structural complementarity between the antigenic determinant and the antibody combining site; this is the exclusive factor of the specificity of the reaction; the structural complementarity involves the conformational adaptation of the two reacting groups and was conceived in structural terms on the key-lock principle; the chemical complementarity of the reaction groups is the consequence of structural complementarity and signifies the entry into action of intermolecular forces that stabilize and consolidate the interaction of the two groups. the formation of intermolecular bonds requires the existence of atomic groups sufficiently close to the two molecules. the distance between them is inversely proportional to the degree of complementarity. although structural complementarity is not strictly obligatory, higher spatial matching is more conducive to interaction. it is expressed by the congruent of contact surfaces that provide intermolecular attraction forces that stabilize the complex. the ag-ab interaction involves the following types of noncovalent bonds: h bonds, electrostatic forces, van der waals linkages, and hydrophobic bonds. all are nonspecific forces of low value and their nature makes the reaction reversible. h bonds form when two atoms share an atomic h nucleus (one proton). the common proton is found between two atoms of n or o or between n and o. the h nucleus is covalently bonded to one of the two atoms (n or o). the h bond has a binding energy of - kcal/mol. intermolecular forces are involved in ag-ab complex formation. the action of these forces requires close contact between the two reaction groups. the h bonds result from the formation of an h bridge between two nearby atoms. the electrostatic forces are due to the attraction of the ionic groups with opposite charges located at the periphery of the two protein chains. the van der waals forces result from the interaction between different electron clouds, represented in the form of oscillating dipoles. the van der waals relationships, the weakest interaction forces, are active at very small distances between the reaction groups. the binding energy is - kcal/mol. van der waals' links are not based on a permanent separation of electrical charges but on their fluctuations induced by the proximity of molecules. at intermolecular distance, instantaneous electric fields are formed, with a polarizing effect on neighboring molecules. between the nearby atoms, there is a mutual attraction force induced by fluctuating dipole load, which a dipole induces in the neighboring dipole (dispersion forces). their intensity depends on the distance between the groups involved and is inversely proportional to the seventh power of the distance. their value is optimal at - Å. hydrophobic bonds, which can contribute with half of the ag-ab binding force, are produced by the association of nonpolar and hydrophobic groups, whereby water molecules are excluded. the optimum distance between the reactive groups varies with the type of bond. the electrostatic forces (coulomb or ionic) are the result of the attraction between atoms or groups of atoms with the opposite electric charge located on the two reacting groups: between a cation (na + ) and an anion (cl − ) or between coo − and nh + (agrawal et al. ) . the binding energy of these forces is significant at very small (less than Å) distances from the reaction groups. exact juxtaposition of ions favors the action of these forces. the binding energy is kcal/mol and varies inversely with the square of the distance between the two reaction groups ( /d ). hydrophobic (or a polar) linkages occur between nonpolar (nonionized) groups in aqueous solutions and are the consequence of the tendency to exclude the ordered molecule of water molecules between the antigen and antibody molecules. these linkages are favored by amino acids with a polar group that tends to associate, reducing the number of water molecules in their vicinity. by removing the water molecules between the reaction groups, the distance between the active sites decreases much, and the value of the stabilizing forces increases. taken each one on their own, space complementarity or intermolecular forces are not sufficient to form stable relationships. for the stability of the ag-ab interaction, both conditions are required. the higher the binding energy of the reactants, the stable the ag-ab complexes. the interaction of the antigen-and antibody-reactive groups is defined by two parameters: the affinity and avidity of the antibodies. measurement of antibody affinity can be achieved by dialysis at equilibrium. the ag-ab interaction is reversible. within the dialysis bag, the hapten is partially free and partially bound to the antibodies, depending on the affinity of the antibodies. through the membrane of the dialysis bag, only the free hapten can be diffused, and its external concentration will equal the concentration of the free hapten inside the bag (kersten and feilner ) . measurement of the concentration of the hapten in the dialysis bag allows for the calculation of the amount of antibody-bound hapten. the constant renewal of the buffer results in total dissociation and loss of hapten in the dialysis bag, which indicates the reversible nature of ag-ab binding. affinity of antibodies measures the binding force between an antigenic determinant and the complementary binding site of a specific antibody. affinity is the result of attraction and rejection forces that mediate the interaction of the two reactants. the strength of these interactions is measured in the reaction between a monovalent antigen (hapten) and specific antibodies. a high affinity interaction requires perfect complementary structures, while the imperfect complementarity of the reaction groups causes a low affinity, since the attraction forces are active only at very small distances and are diminished by the rejection forces. complexes formed by antibodies with high affinity are rapidly eliminated from the circulation without adverse effects on renal function. the ag-ab interaction is permanently characterized by the formation and cancellation of various types of intermolecular bonds. in vivo, probably all ag-ab reactions are reversible, but secondary in vitro reactions (agglutination, precipitation) , under the conditions of reagent balance, are irreversible (sakamoto et al. ) . it is essential that the host-guest binding event (the receptor-analyte interaction, r-a) is detected. it is thus necessary to have a way of identifying and transducing the signal from the receptor-analyte interaction to the outside to be processed. it is generally defined as a transducer. the transducer must be in contact with the receiver or the diaphragm that immobilized the handset. electrode and interfacial interactions are determinant in capturing the signal from an r-a interaction and transforming it into an electrical or photonic signal. there are ways of identifying the r-a event, collecting the signal and transducing it as an external signal. the way of identifying the signals and their transduction defines the type of nbss. this means immobilizing the receiver on an electrochemical transducer that measures a current (amperometric method) or a voltage (potentiometric) between two electrodes. if r is immobilized on an optical component, then we will define optical nbss (optical fiber, fluorescence, absorption, surface plasmon resonance (spr)). for detection, at most electrochemical nbss, it is necessary for the membranes containing the host molecule to be placed on a surface of an electrode that leads to an electrochemical response to the binding of the guest. the approach works well when target analysts are loaded species such as metal cations. neutral molecules cannot be detected from the point of view of electrochemical transduction, so the optical detection methods have been successfully used. a chiral host in calixarene contains naphthyl, fluorescent units. upon binding to the guest, fluorescence is attenuated as a result of interaction between the naphthyl-phenyl groups in the host or analyte. the fluorescence attenuation is proportional to the concentration of the analyte. optical methods are used because they offer greater sensitivity than electrochemical techniques. in the absence of the guest analyte, this compound does not exhibit fluorescence because the pyrenyl substituent cannot come in contact with the adjacent nitrophenyl substituent (and the fluorescence attenuation occurs due to their interaction). however, in the presence of na + ions, fluorescence is observed, because the na + ion enters the cavity and binds to the oxygen in the phenoxy and carbonyl groups in the host. this binding induces a more rigid conformation by removing the nitrophenyl groups of pyrene to prevent fluorescence attenuation (gaggeri et al. ). nbs is a bioelectronic analysis system that combines a transducer with a biological component that is in a specific interdependence. nbss use biological systems with different levels of recognition of the substances to be determined. the first step in this interaction is the formation of the specific complex of the biologically active, immobilized substance r (the receptor, the substrate with the sensitive biological component) with the analyte a (often defined as the chemical signal). table . summarizes specific patterns of nbss in relation to the nature of the receptor and the chemical/biochemical signal. there are two general classes of nbss that are based on the bioaffinity response between r and a that alters the distribution of electrical charges that can be measured with specific transducers or consuming the substrate by a specific reaction. the biological constituent of the molecular recognition element (r) is represented by various active species that can be enzymes or enzymatic systems, antibodies (ab) or antigens (ag), receptors, populations of bacteria or eukaryotic cells, tissue fragments, and sometimes even signaling molecules. analytes or substances that can be analyzed (a) are glucose or other sugars, amino acids, alcohols, lipids, and nucleotides. they can be identified by their specific interaction, or their concentration can be measured by various methods. both r and a represent distinct molecular species with high macromolecular specialization (antibodies, antigens, enzymes, receptors, etc.) or are complex systems (cells, tissues, etc.) (kersten and feilner ) . after the active biological component, they can be grouped as follows: • enzymatic nbss: enzymes are energy proteins characterized by their catalytic function. modified substrate molecules lead to oxidation, reduction, and hydrolysis reactions that can be measured by enzymatic nbss. enzymatic nbss produce a linear response depending on the substrate concentration. • immunosensors: antibodies are glycoproteins produced by the immune system when an external substance, antigen, is involved. it is theoretically possible to produce antibodies without identifying an antigen. an immunosensor is a high sensitivity nbs. the principle of operation is based on the ag-ab interaction of molecular recognition. • nbss with receptors: the regularity of biological processes is ensured by high sensitivity molecular processes based on the specialization of structural proteins (receptors) capable of recognizing a number of physiological signals. this is the case for neurotransmitters, whose action is mediated through the presence of receptors in the plasma membrane, in sites or cell targets. activation of the bio- logically active site is via the ion channels. the acetylcholine receptor is the first known receptor in neurotransmitting phenomena. • nbss based on cells or tissues: measurement of molecular species is not limited to interaction with the compounds to be analyzed; the transformations that occur can be measured as resulting products. it is desirable to operate with cell populations whose major metabolic pathways are known. relevant is nbss' l-arginine, which associates populations of bacterial cells of streptococcus faecium in combination with an ammonia electrode. arginine is metabolized by microorganisms. it is difficult to obtain complex reactions outside the cellular structures. similar to the use of cell populations as sensitive elements, fragments or parts of plant tissues may be used. the advantage is greater because there is no extra effort to keep the cells viable in a natural arrangement. for adenosine nbss, a tissue biosensitive element has been proposed. for dopamine nbss, specialists have focused on the pulp of banana fruit, considering that it has remarkable biocatalytic properties. • nbss with redox proteins: the redox proteins are involved in biochemical processes such as cellular respiration and photosynthesis reaction (kersten and feilner ) . nbs catalysts use enzymes, microorganisms, or cells to catalyze a reaction with a target substance. nbss own an affinity on using antibodies, receptors, and nucleic acids that bind to a target substance. reactions are quantified by electrochemical, optical, evanescence transducers, etc. the main types of known redox proteins are cytochromes, containing iron in the prosthetic group, and cytochrome "c" is involved in the transfer of electrons into mitochondria; ferredoxins contain iron and sulfur ions in dimeric combinations of chloroplasts ( fe- s) ferredoxin and tetrameric combinations of bacterial ferredoxin ( fe- s) involved in photosynthesis and transfer of fixed nitrogen ions, respectively; blue proteins contain copper linked to the smallest cysteine residue involved in a tetrahedral structure such as plastocyanin and azurin that mediates electron transfer in photosynthesis and possibly in nitrite reduction; flavoproteins, containing a prosthetic group and an organic conjugate, are involved in the transfer of proteins such as flavotoxins (agrawal et al. ) . these proteins play a role in nature, due to the location on their surface of the redox centers. the subtle architecture of molecules offers selectivity and specificity to these molecules in their interaction with other proteins or enzymes, such as the cytochrome c structure. porphyrin iron (heme) is located at the center of the molecule and is well covered or hidden, being exposed to solvents in a small proportion of . % of the total molecular surface. from those presented above and from table . , we can see that nbss can be classified into two groups according to the biological component. the protein has a positive potential of + mv due to excess lysinic base debris. there is a debye dipole moment, which produces an imbalance in the spatial distribution of the acidic chain balance. a number of lysine residues are distributed around the solvent to which the center of the heme that interacts with the redox proteins is exposed (nelsen et al. ). nbss are classified in three generations. at the first-generation sensors, the biocatalyst is attached to the surface of the membrane, and then this arrangement is fixed to the surface of the transducer. the adsorption or covalent attachment of the biologically active component to the surface of the transducer allows the elimination of the semipermeable membrane, which is the second generation. the direct linking of the biocatalyst to the electronic device that translates and amplifies the signal, such as the compact transistor, is the basis of the third-generation nbs miniaturization. depending on the nature of the immobilization and the interaction between the three components, the a-r membrane contact with the electrodes to the transducer, and the processes in the nbss have evolved over a generation. first, the specificity and selectivity are dominated by the biological component and are directly related to its nature: enzymes, antibodies, and microorganisms. the specificity derives from the binding of the analyte to the biological component used as the receptor. at the base of this dominant sequence is the a-r biochemical reaction and the collision process between a and r. second, the transport of the analyte to and through the surface considered to r is also an important factor. this process is related to the transport of a physical size through typical diffusion, migration, and convection mechanisms. third, the nbs signal is dependent on the a-r reaction assumed to be at a constant speed. transient states and biochemical pseudo-equilibrium conditions are dominated by the reaction kinetics, the nature of the transport, which in turn is coupled with the immobilized a-r interface substrate reactions. even in the case of a real equilibrium, the reaction speed near the steady state will be important in determining the response time. the kinetics of these processes require additional conditions (agrawal et al. ) . the new types of nanoscale materials with different levels of biocompatibility, the new generation of biocompound cells based on a better understanding of metabolism, the manipulation of information stored at the molecular level all have led to a generation of nbss with a high level of integration. molecular information initially stored in the base molecular components can be expressed directly to a higher level called "supramolecular" where interactions between molecules are performed by preestablished algorithms, leading to adaptive, functional, and intelligent materials. materials are built on conceptional, supramolecular, and combinatorial principles. separation, storage, and detection techniques are developed using "biomimetic" membranes that function according to biological models or precise physicochemical principles ). electrochemical nbs with dna is the result of medical diagnosis requirements to quickly and accurately determine the segments of a dna sequence. results from genetics, molecular biology, and nanotechnology have led to one of the most accurate detection methods: electrochemical nbss with dna (combining the principle of nbs isfet with molecular wires from nanotubes). the operating principle consists of collecting the signal between two electrodes-one working electrode and another reference electrode. the auxiliary electrodes have a specified role in their turn. the sensing mechanism consists in modifying the i-v characteristic (current-voltage) in the presence of a target molecule. carbon nanotubes are exceptional for the work electrode with high electron transfer velocity and excellent spatial resolution. target in nbs dna is an unknown sequence of dna (or oligonucleotides) attached by functionalization to the carboxyl or amino cnt groups. researchers reported the developing plants' ability to capture % more energy by implanting carbon nanotubes into chloroplasts and plant organisms where photosynthesis takes place. they managed to modify plants so as to detect nitric oxide by implanting another type of carbon nanotubes. "the plants are suitable for the role of a technological platform. they heal themselves, they are durable, they resist the harsh environments and they have their own sources of energy and water." the transformation of plants to photon devices, with own energy, such as explosive detectors or chemical weapons, is expected (panchal and upadhyay ) . external or surface electrodes are metal electrodes that contact the nbss' bioactive component either directly (dry or solid electrodes) or through an electrolyte solution (liquid electrodes). solid or dry electrodes are made in silver, platinum, gold, and nickel. internal electrodes are made of thin wires made of durable metal: stainless steel, platinum, and tungsten. the active part of the electrode can be covered with a metallic conductor layer (gold, silver) and the inactive one with an insulating layer (polymer/thin film). nbss' active contact surfaces are large in size compared to cell sizes and are used for extracellular recordings. microelectrodes are internal electrodes, but they are built to measure potentials in direct contact with the receiver in nbss. the contact surface with r is micronized. microelectrodes can be solid, compounds which can be achieved by depositing a conductive layer (platinum, gold) on a glass support having a particularly thin peak, and another constructive variant consists of inserting a metallic or carbon fiber conductor into an epoxy resin support mixed with a conductive paste; they are used in cellular samples and consist of a glass pipette having a micronized tip filled with an electrolytic solution containing potassium chloride. in the electrolyte solution, a conducting wire is immersed to pick up the electrical potential (wu et al. ). it was thought that it is not possible to transfer electrons directly between the electrode and the proteins due to their distortion. several practical considerations have led to the conclusion that the active center of the heme is irreversibly adsorbed when resulting in protein denaturation in contact with the electrode. changing the surface of the gold electrode by surface adsorption of , ′-bipyridyl resulted in the modification of the electrode surface configuration for interaction with cytochrome "c." , ′ bipyridyl is not an electroactive substance in the potential region and therefore does not play a role as a mediator. this electrochemical addition was possible due to the quasireversible binding of cytochrome "c" to the modified gold electrode with . ′ bipyridyl, thereby resulting in the hydrogen linkages in the lysine residues to bound to the nitrogen of the pyridyl which modified the surface of the electrode. transmutation through complex protein electrode rapidly directs the transfer of electrons, which is accomplished by the following scheme: cytochrome diffusion "c" on the electrode, protein binding on the surface, electron transfer, and protein desorption. following this process, more than surface changes were possible for electrochemistry of proteins and the gold electrode. using a bifunctional reagent (x-y), wherein the x group is the n, p or s bonded electrode and the y group, which must be bonded "represent also examples of patterns developed" (agrawal et al. ). electrochemistry of proteins has been extended to the carbon electrode. pyrolytic carbon-graphite forms, vitreous carbon and mesocarbon, are structures in which graphene plans are arranged in ababa hexagonal mesh or disordered in different turbulent forms. the base graphene plan is hydrophobic, but existing or induced defects lead to free c-c bonds, and there is an increase in c-o linkages by oxidation. the direct electrochemistry of positively charged proteins can therefore be performed on the edges of the graphite plans of the carbon electrode. the direct transfer of electrons to negatively charged proteins, such as plastocyanin with graphite electrode (edges or plane edges), can be aided with mn, ca, cr complex cations complexed with amino compounds, cr (am ) + , used as promoters of reactions. in this context, the promoters are inactive redox species in solutions but allow the transfer of electrons to the redox proteins. microminiaturized electrodes have specific advantages among which we mention the improvement of polarization and contact with biological material at the active sites. specificity and selectivity depend on the nbs receptor biological component and its affinity for the analyte. affinity is a specific feature of enzymes, antibiotics, and receptors being used in functions in living organisms. affinity is based on the chemical coupling between a component and its complementary partner. in the case of high-affinity components, the diffusion process is rapid, leading to the formation of the ag-ab type of complex. the association reaction specific to molecular recognition will be characterized by the first-order response rate constant. in nbs measurements it is essential to consider the concentration of a constant component and other variables. the results of electrochemistry of proteins have been extended to amino acids and peptides (barroso et al. ) . the addition of enzymes to solutions containing substrate molecules is the essential condition in enzyme catalysis reactions. extracting the necessary information from the enzyme science to be applied to the development of nbss such as the enzyme electrode is an extremely difficult task. references will be used to outline some of the enzyme properties necessary to describe enzymatic nbss. consider a simple reaction, with a single substrate s, that combines with enzyme e to form the enzyme-substrate intermediate complex, es. this unstable complex undergoes a new reaction resulting in product p. the formation and consumption rates of the complex are equal. as soon as e and s enter the reaction, the system becomes unbalanced, the concentration of the complex will be zero, and the formation speed of the complex is much higher than the rate of its consumption. as the reaction unfolds, es increases and implicitly increases the rate of disappearance of the complex relative to its rate of formation. initially, the excess of the substrate determines the consumption of the enzyme, and during the course of the reaction, the enzyme's constant regeneration begins to reach its steady state. the analysis of these reactions results in two important conclusions: • at a low concentration of the substrate, the rate of the reaction is proportional to the substrate concentration and inversely proportional to the rate of the formation and extinction rate of the complex or the dissociation reaction rate in the initial reactants plus the decomposition reaction rate in products. • at a high concentration of the substrate, maximum speed is limited by enzyme concentration. thus, the two sequences correspond to the two processes that can control the overall reaction rate (stein et al. ). when the reaction takes place in homogeneous solutions at a uniform rate, the same in the entire environment, it is necessary to consider the change in the concentration of the components over time. three mechanisms of mass transport occur in solution: diffusion, convection, and migration. an electrochemical nbs is an autonomous, self-contained device capable of providing specific quantitative or semiquantitative analytical information using as a molecular recognition element, a biochemical receptor (biological identification element) that is in direct spatial contact with an electrochemical transducer. electrochemical nbss are distinguished only by the nature of the transducer regardless of the nature of the biological component according to the classification in table . . due to their ability to be calibrated in a repetitive manner, an electrochemical nbs is distinguished by a bioanalytical system requiring additional processing steps, such as the addition of reagent. nbss for a single type of measurement, or unable to continuously monitor concentration analysis or not to be rapidly and reproducibly regenerated, are defined as "disposable." nbss are classified according to their biological specificity-with reference to the mechanism or to the interpretation of the physical-chemical signal (the transducer) (barroso et al. ) . the biological recognition element is based on a catalyzed chemical reaction or an equilibrium reaction with macromolecules that have been previously isolated or synthesized in their original biological environment. in the case of reversible reactions, the steady state can be reached if there is no net consumption by the agent of in addition to quantitative determination of analytes, nbss are also used to detect and quantify microorganisms: the receptors are bacteria, yeasts, or oligonucleotides coupled with electrochemical, piezoelectric, optical, or calorimetric transducers the immobilized biocomplex and incorporated into the nbss. electrochemical nbss can be classified according to the analyses and reactions they monitor: direct monitoring of the concentration of the analytes or their production or consumption reactions and, alternatively, an indirect monitoring of the inhibitor or activator of the biological recognition element (the biochemical receptor). criteria include calibration characteristics (sensitivity, linearity, operational range of concentration, quantitative determination limits, and specific detection), selectivity, equilibrium state and response time, reproducibility, lifetime, and stability (fang et al. ) . the notion of recognition is used in nbss or in nanobiosensory systems by association with the sensory systems of the plants. sensations such as smell or taste are made up of systems that contain an identification receiver cell coupled with neurotransmitter signal-processing pathways. such phenomena also occur in biochemosensors but at a much-simplified level compared to the complexity of molecular recognition in living systems (barroso et al. ) . examples of single or multiple transfer signals, limited to the main biochemosensors, are shown in table . . for the receptor types shown in table . , different electrodes and measurement methods can be selected from table . to form an electrochemical nbs. nbss are classified by the recognition element (table . ) or by the transduction mode (table . ). nbss irrespective of the type of classification should be treated unitarily as a microsystem, the biological recognition element being in direct contact with the transducer element. an electrochemical nbs is an nbs with electrochemical transducer (table . ). it is considered to be a chemically modified electrode (cme), the electric conductor nbss may use several other types of non-electrochemical transducer: (a) piezoelectric nbss; (b) nbs-saw, measures surface acoustic waves in a resonance circuit (shear and surface acoustic wave); (c) thermometric nbss (the active element is coupled with a thermistor); (d) optical nbss, uses optical phenomena: planar wave guide, optical fiber, surface plasmon resonance) spr nbss use the immobilized analyte-receptor interaction on a metal film deposited on an optical prism measuring the variance of the refractive index due to changes induced in the metal's electrical charge that transmits the electrons from the interaction process to the outside in the electronic measuring system. the electrode may be a metal, a semiconductor, or an ionic conductive material coated with a biochemical or bioactive film. electrochemical nbs is an integrated transducer microsystem capable of providing selective, quantitative, or semiquantitative analytical information using a biological identification element. it can be used to monitor biological and nonbiological elements. chemical nbss that incorporate nonbiological components as receptors, although used to monitor biological processes (ph or nbss of oxygen), are not nbss. the clark electrode is of importance in the nbss' measuring range. similar physical nbss used in biological environments such as those measuring pressure, etc. are not considered nbss (jacoby et al. ) . electrochemical nbss according to the terminology set out in tables . and . can be classified according to their biological specificity, by mechanism or mode of signal transmission, or alternatively, the combination of two. they can be amperometric, potentiometric, field effect (fet), or nmss' conductometric (electrical conduction measurement) respectively impedance metrics. alternatively, they may be called enzymatic amperometric nbss to specify the nature of the receptor and the transducer. the first nbss that were studied are the enzymes and immunosensors (fang et al. ) . nbss are based on a catalyzed reaction of biomacromolecules present in the original biological medium that is preisolated or synthetically produced. the reaction is monitored by an integrated detector (transducer) that measures the stationary or transition states or the final reaction product via the immobilized biocidal product in nbss. types of commonly used biocatalysts are enzymes (simple or enzymatic complexes)-most commonly used as recognition systems, cells, microorganisms (bacteria, fungi, eukaryotic cells, or yeasts), cellular organs, or component (cell walls, mitochondria) sections of plant or animal tissues. nbss with biocatalyst recognition elements are the best known and studied since the beginning of their approach by clark and lyons. one or more analytes, commonly called s and s′ substrates, react in the presence of one or more enzymes, cells, etc., to produce the p and p′ products (fang et al. ) . there are four strategies whereby the associated transducer can monitor the consumption of s-analyte through the biocatalytic reaction: • detection of s′ cosubstrate consumption, oxygen depletion through the oxidaseinduced reaction chain, bacteria, or yeast. the measured signal is the decrease in cosubstrate consumption compared to the initial value. • recycling of the reaction product p such as peroxyhydrogen, h + , co , and nh , in oxidoreductase reduction schemes, hydrolysis, lysis, etc. the signal from the transducer will be amplified. • detection of active centers in the biocatalyst: redox, cofactors, prosthetic groups evolving in the presence of substrate s by using an immobilized mediator. it reacts quickly with the biocatalyst and is easily detected in the transduction chain. various ferrocene derivatives, such as tetrathiafulvalene, tetracyanochinodimetane (ttf + tcnq), organic salts, quinones, quinone dyes, ru, or os complexes in polymeric matrices, can be used as mediators. • direct electron transfer is made between the enzymatic redox reactive site and the electrochemical transducer. the third strategy eliminates, partially or totally, the dependence of the nbss' response on the cosubstrate concentration, s′, which decreases the influence of interference between species. the use of mediators leads to the decrease of the substrate concentration together with the reaction chains by using a suitable membrane, whose permeability favors the transport of the cosubstrate. when enzymes are immobilized within the same reaction chains, it can improve the performance and abilities of nbss. three possibilities are commonly used: • some enzymes facilitate biological identification by sequentially converting the products of the enzyme reaction series into an electroactive final form: this way allows for a wide range of nbs analysis. • the enzymatic complex, applied in series, can regenerate the cosubstrate of the first enzyme and amplify the nbs output signal by regenerating another cosubstrate of the first enzyme. • the parallel enzyme complex improves selectivity of nbss by lowering the local concentration of electrochemical interfering substrate: this sequence is an alternative to the use of a permselective membrane or a sequential method (e.g., interpretation of an output signal generated by an nbs and a reference nbs without biorecognition element). the operation of nbss is based on the interaction between the analyte and the macromolecules or organized molecular assemblies. upon reaching the balance, there is no consumption of analyte by the biocomplex agent immobilized in the substrate. the response to the biocomplex analyte-reagent reaction is monitored by an integrator detector. in some cases, the biocomplex reaction is self-monitored by a complementary biocatalytic reaction. the integrator detector monitors stationary or transient states. antibody-antigen interactions, the most relevant examples of nbss using biocomplex receptors, are based on immunochemical reactions, e.g., binding an antigen (ag) to the characteristic antibody (ab). complexes formed by ab-ag can be detected provided that other nonspecific reactions are minimized, for each determination of ag corresponds to a certain ab that must be isolated, purified, etc. some recent studies have analyzed direct monitoring of ag-ab formation using ion-selective field effect transistors (isfets). increasing the sensitivity of immunological nbss is achieved by adding specific enzymes to ag-ab couples, but this requires additional synthesis steps. as the binding strength or affinity constant varies widely, these systems operate irreversibly (disposable nbss) or are coupled to flow injection analysis (fia) systems; then ab can be regenerated from dissociation of the complex with agents such as glycine-hcl to ph . (kurien et al. ). recently, they have been used as molecular recognition systems in conductometric analysis, isfet, or optical nbss with receptors with ion channels, membranes, or protein structures. a transporting protein, lactose-permease (lp), can be incorporated into a liposomal bilayer that permits protonic carbohydrate transport with a stoichiometric ratio of : . this mechanism was identified through the ph-dependent fluorescence of a fluorophore immobilized in liposomes. liposomes with lp were incorporated into a lipid bilayer deposited on a ph-sensitive isfet. preliminary results show that this modified isfet is capable of irreversibly detecting lactose from an fia system. protein receptor nbss have been recently discovered. binding of analytes, here called agonists, to immobilized receptor proteins is monitored by changing the flow of ions through these channels. glutamate, as an agonist, can be determined in the presence of other agonists that can interfere with the determination of na + or ca + streams using conductometric method or ion-selective electrodes. due to the dependence of the ionic channel on the nature of the linkages, it produces an independence toward the enzyme nature in order to achieve the desired sensitivity. two methods have been approached. the first refers to oligonucleotide duplex interleaving during the formation of the double helix structure of the dna of a molecule that is electrically active. the second method is the direct detection of guanine which is electroactive. some of these nbss cannot operate through analytical-sensing membrane separation membranes. the sensitive layer often has to be in contact with the biological environment where the analytes are located (fang et al. ) . nbss have been developed for indirect monitoring of organic pesticides or inorganic compounds (heavy metals, fluorides, cyanides) that inhibit the biocatalytic properties of enzymes used in the construction of nbss (devices are irreversible). in immunosensors, the initial biological activity can only be regenerated by chemical treatment and therefore is not part of the reconditioned or reusable nbs class. their application potential is to warn and not to accurately monitor a specific analyte (considered as disposable). nbss with cyanide (i.g. inhibition of cytochrome c oxidase) that are used as inhibitor to cytochromoxidase are regenerated by washing with a phosphate buffer at ph . (armstrong and beckett ). with the development of enzymatic glucose nbss, an experiment in which glucose oxidase is immobilized between two membranes, literature has emerged about techniques for immobilizing biological receptors. enzymes, antibodies, cells, or tissues with high biological activity can be immobilized in a thin film on the surface of a transducer through a variety of methods. the following immobilization procedures of biological receptors are used: • immobilization on the membrane on the surface unexposed to the analyte: an enzymatic solution, a cell or tissue suspension, rests between the permeable membrane to the analyte and the measuring electrode (electrochemical detector). • retaining of biological receptors in a polymeric matrix, polyacrylonitrile, agar gel, polyurethane (pu) or polyvinyl alcohol (apv), redox hydrogels with redox centers such as [os (bpy) cl] +/ + . • retaining of biological receptors between self-assembled layers (sam) or in membranes from the double lipid layer (blm). • the covalent binding of membrane surface receptors through bifunctional groups: glutaraldehydes, carbodiimides, sams, avidin-biotin silanized. • modification of the entire electrode structure (modified carbon paste with enzymes or graphite in epoxy resins). receptors are modified either alone or mixed with other proteins, such as bovine serum albumin (bsa), either directly on the surface of the transducer or in the polymer membrane. reactivated membranes can be used directly to immobilize enzymes or antibodies without chemical modification. the covalent binding and cross-linking are more difficult than immobilization or the retaining of receptors on the membrane. in the case of microsensor structures where the membrane is directly deposited on the transducer, the covalent bonding is safer and more stable (muñoz et al. ). besides reactive layers or membranes with immobilized receptors, many nbss, those for clinical or biological applications, incorporate one or more internal or external separation auxiliary membranes with three important functions: the barrier, the outer diffusion barrier for the substrate, and the biocompatible surfaces. for any nbs built on the principle of molecular recognition, it is important to characterize it by its response, which is related to operating parameters and limiting reaction speeds. accuracy, precision, sensitivity, and reproducibility are basic criteria for estimating nbs performance. these parameters are in direct relation to the reaction mechanisms, the transport phenomena, and the kinetics of the processes in the volume at the interface. most criteria have been developed for enzymatic nbss, being the most studied in the literature. in the case of immunosensors, the key element is the ability to capture the surface, i.e., the number of surface molecules that is active. one method of checking this parameter is to measure the specific activity, meaning the ratio of the number of active molecules to the number of immobilized molecules. this estimation is dependent on the immobilization mode (molecular orientation, number of attachment points or active sites), and the ratio ranges between . and . , rarely reaching the unit. capture capability becomes important when the surface decreases as in microfluidic applications. a problem encountered in immunosensors is that of regenerating the surface without significant loss of activity. there was a lack of rigor in the performance criteria (affi et al. ) . the response signal is corrected for background noise, the reference concentration is usually estimated in mol/l although this high value is never used when measuring ranges refer to − mol/l, and currently sensitivities of the order nmol/l and pmol/l have been reached. transient response is important for dynamic assay analysis and sampling techniques but is less significant in continuous monitoring. the transient response is estimated by the slope (dr/dt) max after the addition of the analyte in the measuring cell. one evaluation method is to introduce nbss into an fia system for sequential sample analysis in a specified hydrodynamic regime. the sensitivity and linear range of measurement of stationary concentration are determined through graphical representation. this method is more concise than the current calibration curves used to plot the response corrected to the baseline based on its concentration or logarithm. parameters are estimated in the linear response range of nbss. any electrochemical nbs has a superior linearity of the response. this limit is directly related to the biocatalytic or biocomplex properties of the biochemical or biological receptor. more in the case of nbss with enzymes, this limit is significantly influenced by membranes and immobilized substrates where the diffusion barriers and secondary kinetics play a role. the local concentration of the analyte in the reaction layer may be two orders of magnitude smaller than the volume of the solution (michelini and roda ) . enzymatic kinetics are described by michaelis-menten mechanisms and expressed by km and vm parameters. for the kinetics of the enzyme in the solution phase, km is usually determined from the lineweaver-burk graphical representation. for any electrochemical nbss, the number of standards used and how the standard sample matrix can be simulated or duplicated should be set, being required to specify the procedures for each type of nbss related to its application. these are important for the disposable nbss' case using immunoaffinity or inhibition reactions. sensitivity is the slope of the curve and should not be confused with the quantified detection limit (lod) relative to the baseline or noise signals. the range of work concentrations is determined by lower or higher detection limits (fang et al. ) . selectivity and safety are determined as any kind of amperometric or potentiometric nbss. they depend on the choice of receiver and transducer. most enzymes are specific, but there are also nonselective enzyme classes, such as alcohol oxidases, the group of oxidases sugars, peroxidases, lactases, tyrosinases, ceruloplasmin, alcohol dehydrogenases, glucose dehydrogenases, nadh dehydrogenases, etc. they have been used to develop nbss to determine environmental phenols or to monitor food quality. on the other hand, oxygen electrodes, ph electrodes, and isfets have a pronounced selectivity, the same as metal electrodes that are sensitive to many substances. their selectivity may be changed when these transducers are associated with receptors. enfet is ph sensitive to the buffer and protonation but its selectivity is not altered. when the transducer interferes with other substances, known as ascorbate or urease, to glucose nbss based on hydrogen peroxide detection, these side effects may be restricted by the use of outer or inner permselective membranes. alternatively, nbss with and without biological receptors that work by differential nbss are designed. safety in operation of nbss depends on the selectivity and reproducibility and accuracy of the measurements (heyl et al. ). the clark construction principle studied electrochemically oxygen as a reducing gas and platinum as a metal electrode. platinum used for detecting electrochemical oxygen is known as the clark electrode. the electrode has an organic membrane covering the electrolyte layer and two metal electrodes. oxygen diffuses through the membrane and is electrochemically reduced to the cathode. between the cathode and the anode, a fixed voltage is applied, for which the oxygen reduction reaction takes place. temperature greatly influences reaction speed and solubility. this is a polarographic electrode used to measure the concentration of oxygen in body fluids or gases. the sample is in contact with a membrane (polypropylene or teflon) through which the oxygen diffuses into a measuring chamber containing % saturated potassium chloride solution. inside the room are two electrodes, one is reference, ag/agcl, and the other is platinum, coated in the glass. the electrical current at the polarization potential of - mv is proportional to the oxygen concentration in the solution. for reverse polarization at + mv, hydrogen measurements can be made. reactions are very sensitive to temperature and should be maintained at ± . °c. the electrode is calibrated using a mixture of the two gas-oxygen and hydrogen-known concentrations. oxygen electrode or clark electrode has proven to be an analyzer of raw gas or developed gas when performing chemistry analyses in the clinical laboratory and in the field of medical care, ambulatory, or intensive care (on the surface of the platinum electrode an enzyme reacts with oxygen). the enzymes are placed in a closed membrane to the surface, which can be recognized as the simplest model of nbss. the oxygen concentration curve was proportional to the glucose concentration. it was the first nbs built, which helped the progress of laboratory analyses a lot. oxygen diffuses through the membrane and is electrolytically reduced to the cathode. the higher the partial oxygen pressure, the more oxygen diffuses at a time. the temperature nbss attached to the sample allow the membrane to compensate for the diffusion and solubility rate. the measuring instruments record cathode current, sample temperature, membrane temperature, barometric pressure, and salinity. with this information one can calculate the oxygen contained in the sample, either in parts per million (ppm) or in percent of oxygen saturation. the geometric configuration of the clark electrode is of great importance. in particular, the thickness of the electrolytic layer between the cathode and the membrane must have a certain limit, to ensure linearity and decrease of the drift current. calibrating a polarographic system is a must. proportionality between current and oxygen concentration must be ensured, with errors below % (biological samples role and air parameters are essential). air, as a gas mixture that has a constant oxygen content of about . %, when in contact with water, the dissolved amount depends on several factors: the optimal time for oxygen dissolution, homogeneity of the water solution, water temperature, air pressure, salts contained in water, and other water-soluble substances that are oxygen-consuming. oxygen contained in water is determinant for biological and chemical processes, so measurement of dissolved oxygen in water is important to find the partial pressure of dissolved oxygen; it must be saturated in pure water at a certain temperature (wolfbeis ) . the enzymatic electrode (in some references known as the enzyme electrode) is a combination of an electrochemical probe of any type (amperometric, potentiometric, or conductometric) with a thin layer ( - microns) of immobilized enzyme. in these devices the function of the enzyme is to provide selectivity in virtue of its biological affinity for a substrate of molecules. an enzyme can catalyze a reaction of a given substrate for a specific isomer from a plurality of substrates with different isomers. typically, the degree of advancement of an enzymatic reaction (directly related to the concentration of the analyte) is monitored by the rate of product formation or the disappearance of a reagent. if the product or reagent is electrically active, then the response can be directly monitored by amperometry, i.e., the variation of the current for a given applied potential. the main considerations are: does the enzyme contain active redox groups? are the biochemical reaction products electroactive? is one of the substrates or cofactors electrically active? what is the speed and response time? what is the final application of nbss? if the enzyme does not contain redox groups, then nbss are limited to measuring the product or substrate consumption by their reaction to the transduction electrode. the electrical current is directly related to the analyte concentration. nbss are based on electrochemical response due to h o . most common enzymes used in the design of enzyme electrodes are those that contain redox groups that change their redox state during the biochemical reaction. redox enzymes are oxidases and dehydrogenases, pyrroloquinoline quinone (pqq). they act by oxidizing the substrate, accepting electrons during the process, and further transforming in a reduced state. these enzymes return to the oxidized active state by transferring electrons to the oxygen molecule resulting in hydrogen peroxide (h o ). both oxygen and peroxide being electrochemically active, they continue by reducing to cosubstrate (oxygen) or oxidation of peroxide (reaction product). the method based on the reduction of oxygen to the o electrode is one of the simplest methods but suffers from several disadvantages, namely, slow response, miniaturization difficulties, low accuracy, and reproducibility. measurements on peroxide oxidation overcome the above difficulties and are currently the most popular method. mediator systems-a major limitation of the above-described hydrogen permeation system is the high operating potential (about . v against the ag/ agcl reference electrode) required for oxidation of peroxide which leads to increased interference. the use of mediators (molecules that can carry electrons between the enzyme redox center and the electrode) can minimize this inconvenience. depending on the nature of the mediators, the potential applied may be reduced below the limit of interferences of species such as ascorbate, urate, and paracetamol. a large number of compounds are able to act as mediators in the enzyme electrode. of these, the most popular are the metallic complexes. representatives for organometallic complexes are ferrocenes and their derivatives because they have redox potentials and are independent of ph. bienzymatic systems' recent works have focused on the direct communication of electron transfer between enzymes and electrodes. successes in the field are the peroxidase enzyme hrp (horseradish peroxidase) that catalyzes the reduction of hydrogen peroxide for a number of organic compounds. when the enzyme is immobilized on the electrode, the need for the organic reducer is prevented by the electrode itself providing the reducing equivalences. the coupling of peroxidase with an enzyme oxidase allows for the construction of bienzymatic systems through which the peroxide produced by the oxidase is detected by the electrode-peroxidase system which operates at lower potentials relative to a simple platinum electrode. this is where the minimization of active species interferences results from (frederickson matika and loake ). optical fiber as a nanobiosensor can be placed in the surface or inside the plant to directly measure parameters. nbss with optical fiber are proposed to be used in many and rapid medical determinations, and its applications are continuously expanding. it can be attached inside a hollow-like tubular instrument, serving to dilate a hole or channel, and inserted into the tissue, performing a minimal monitoring where it is needed. nbss with optical fiber are nontoxic, chemically inert, and can be successfully used inside the plant. it can be associated with plant monitoring equipment. it's easy to handle with negligible weight. the evolution of fiber-optic nbss is based on multiple performance and biocompatibility. biocompatibility is the first step in the plant's comfort; nbss should not affect the physiological parameters of the plant, but its functionality must not be compromised by plant disassimilation products. fiber-optic nbss can be classified as extrinsic, fiber acts as a way for signal and intrinsic, interactions occur in the fiber itself. there are two types of fobs: minimally invasive nbss that are introduced into the cavities of the plants and invasive nbss that are introduced into the organs or in wood conductive tissue (liu et al. ) . in the last decade, optical fiber is a product that is widely used in all the cutting-edge fields of advanced science and technology. given the ease with which it can be manipulated, unlimited sterilization possibilities, and reduced costs, it can be estimated that this product will increasingly gain market. the following applications are known to have used fiber-optic nbss: in epidermis and vascular tissues, for analysis of raw sebaceous elements, saturation in oxygen, raw sewage gas analysis, sap ph; in plant breeding monitoring; easy ph determination with a microabsorbent indicator and ph-modulator, acid-alkaline; in vegetal tissues, when it is intended to monitor the temperature, or to diagnose small and very small injuries that are difficult to reach; in epidermis for can test the quality and integrity of the layers, so, small lesions can be detected, can be used to stimulate tissues, a fobs based on oxygen demand (bod) can be used; in the stem can identify very small injuries that are inappropriate. another possible application is to appreciate the color or integrity of the vascular tissues. optic-fiber nbss can now monitor electrolytes from raw or elaborated sap as well potassium, sodium, and calcium. it takes the form of a tubular instrument, able to expand an optic-fiber channel ( . mm diameter) that can be inserted into vascular tissues. it can measure the gas concentration and the ph of the raw or elaborated sap and oxygen saturation also. the materials that make up the chemical transducers are ionophores that can be reversibly attached to the electrolyte by a molecular separator (spacer) and fluorophore, respectively. the degree of fluorescence, through excitation with electroluminescent diodes in purple, is modulated by ionophores proportional to the analyte concentration. nbss are used either extracorporeally in the external raw or elaborated sap gas analysis circuit or intracorporeally for continuous blood gas monitoring in critical situations. the chemical parameter capable of monitoring cell state is ph because lactic acid, formed when cell tissue dies, produces a decrease in ph. any drop in the ph of the raw or elaborated sap from . indicates cell death (mclamore et al. ) . achievements in the domain are invasive ph nbss that determine the state of the cell. nbss are composed of a fluorescent dye encapsulated in a gel matrix (polyacrylamide) attached at the end of the optical fiber. the dye is characterized by the emission of the acidic form centered on nm and the alkaline form centered on nm. the two forms are ph sensitive. excitation occurs at nm for both forms. separation of emission is done directly through optical filters, and sensitivity is . ph units far below acceptable clinical standards ( . ph). a bacterial disorder is a multifactorial condition that is characterized by demineralization of the inorganic portion and an organic destruction of the substance. each bacterial perturbation has as an etiological agent as pathogenic species. the content of the raw or elaborated sap in terms of bacterial load is about /ml raw or elaborated sap. therefore, raw or elaborated sap can be considered as a selective medium for bacterial growth. a significant correlation has been demonstrated between the number of pathogens in the raw or elaborated sap and their prevalence in the problems. a simple method has been adapted for detecting and counting the pathogen; it is a device consisting of a special support made with an agar culture medium containing % sucrose. it is inoculated with raw or elaborated sap, and the density of growth of the pathogen is assessed after incubation for hours at °c. next comes the morphological identification of distinct colonies on selective and nonselective agar, on distinct cell form, visible in the light of the microscope. the technique also has some disadvantages that it takes time for bacterial growth and also requires additional laboratories. to monitor pathogen activity in the sap, a fobs was developed that monitors the pathogen-mediated sucrose reactions through a photosensitive indicator immobilized in a porous glass coating. the surface of the optical fiber core is treated or coated in a porous glass film using the sol-gel technique (kozan et al. ) . spectroscopic analysis showed that there are two phases in light absorption at nm over a duration of min: between - min and - min. the investigation shows the potential of nbss in monitoring plant activity. the sol-gel technique is used to immobilize the photosensitive indicator, and it is simpler than compared with the principle of selective medium which takes time and is laborious. criteria at the base of the experiment: pathogens are partially anaerobic with opti-mal growth at °c; glucosyltransferases and fructosyltransferases from pathogens catalyze the synthesis of water-insoluble glucan and fructanic polymers in sucrose to form lactic acid found in acidic sap; pathogenic agents in the sap synthesize both extracellular and intracellular polysaccharides from sucrose; extracellular polysaccharides help the adhesion of bacteria to the surface, while intracellular polysaccharides are stored for bacterial energy; polysaccharides, intracellularly, help the bacterium to continue fermentation even when there is no exogenous form of food. acid tolerance of pathogens causes their activity to continue even at a low ph; a ph indicator, photosensitive, produces a characteristic color, according to a color gradient, depending on the ph of the raw or elaborate sap and is used in the fobs construction (miranda et al. ) . on the basis of these considerations, an experimental assembly was designed and performed with a double beam uv spectrometer in which optical fiber was introduced instead of a cuvette. in the reference well, the blue bromophenol buffer solution was used for different ph values, from to . the initial experiment helped determine the wavelength and peak characteristic to the buffer indicator and for different ph values in the sap, induced by bacterial activity. uv spectroscopic analysis at a ph of and of the blue bromophenol solution showed slightly prominent at nm wavelength; peak intensity decreases from ph to . comparison with literature data showed a good concordance, observing that in the medium with sap, sucrose and bromophenol blue, the absorption was stable at nm wavelength for a time interval of min, min, h, and h. for each set of corroded and processed fibers set, it requires independent calibration due to the in homogeneities resulting from the optic-fiber preparation steps. fobs proves to be a rapid quantity measurement test of pathogen activity in raw or elaborated sap. this test can also be adapted to other plant nanobionics where bacterial activity is involved in cellular or tissue destruction. the method of forming a biosensor from an optical fiber for the observation and detection of the pathogen, the experiments of this study followed the phase of biochemical recognition of the signal and the phase of the spectroscopic analysis. the idea of nanobionic plants has evolved to create solar cells that heal themselves from plant cells. the next step was the desire to try to amplify photosynthesis in isolated chloroplasts in plants to be used in solar cells. chloroplasts host everything they need for two-step photosynthesis. in the first step, pigments such as chlorophyll absorb light, which generates the stimulation of electrons that circulate through the chloroplast tilacloids. the plant captures this electrical energy and uses it to fuel the second stage of photosynthesis, creating glucose. chloroplasts also have these reactions after they have been removed from the plants, but after a few hours, they break down because light and oxygen destroy their photosynthetic proteins. normally, unlike extracted chloroplasts, plants can repair this damaging process. to prolong chloroplast productivity, researchers have attached ceric oxide nanoparticles. these particles are, in fact, powerful antioxidants that remove oxygen radicals and other high reactivity molecules produced by light and oxygen, protecting the decomposition chloroplasts. the researchers introduced nanoparticles into chloroplasts using a new technique called leep (lipid exchange envelope penetration). by wrapping the particles into polyacrylic acid, a heavily charged molecule allowed the particles to penetrate the lipid hydrophobic membranes surrounding the chloroplasts. in these chloroplasts, the level of decomposition of molecules has decreased tremendously. using the same technique, the researchers introduced semiconductor carbon nanotubes embedded in negatively charged dna into chloroplasts. plants use only one tenth of the available sunlight, but carbon nanotubes have functioned as artificial antennas that have allowed chloroplasts to capture unusual light waves such as green, ultraviolet, and near infrared. when carbon nanotubes functioned as prosthetic photoabsorbents, photosynthesis, measured by the activity of electrons in tilacloids, was % more intense than in chloroplasts isolated without attached nanotubes. when cerium oxide was joined with carbon nanotubes, the chloroplasts remained active for the next few hours (nikolelis et al. ) . researchers went to live plants and used a technique called vascular infusion to attach nanoparticles to arabidopsis thaliana, a small flower plant. using the above method, the researchers applied a nanoparticle solution on the lower side of the leaf, penetrating the stomata that usually allow the carbon dioxide to get in and the oxygen out. in these plants, the nanotubes have penetrated into chloroplasts and have increased the photonic electron circuit by about %. it is also to be discovered how these percentages influence the sugar production of plants. scientists have been able to transform the arabidopsis thaliana plant into a chemical nbs by implanting nanotubes that detect nitric oxide, a pollutant produced by combustion (hines et al. ) . nbss have been created on the basis of carbon nanotubes for several chemicals, including hydrogen peroxide, trinitrotoluene, and sarin gas. when molecules attach to polymers encased in nanotubes, the fluorescence of these nanotubes is altered. carbon nanotubes can be used to create nbss that detect real-time particle free radicals or signal the presence of molecules with a very low level of concentration and too difficult to detect. this is a tremendous demonstration of how nanotechnology can be combined with synthetic biology to modify and enhance the functions of living organisms of plants. the way that nanoparticles arrange themselves can be used to enhance plant photosynthetic capacity, being used as nbss and stress reducers. by adapting nbss to targets, researchers hope to develop plants that could be used to monitor environmental pollution, pesticides, fungal infections, or exposure to bacterial toxins. attempts to incorporate electron nanomaterials into plants, such as graphene, are currently being made. researchers have long tried to find the best way to transmit information in a timely manner, focusing on electronics and mechanics of nbss for their tasks. nbss used for agricultural purposes are not new. recombinant dna technology now offers the possibility of obtaining new biological insecticides that preserve the benefits of "classic" biological control agents, plus some new features. these technologies are not accessible to all possible users, especially if they are poor, and furthermore, they have also generated a series of public debates about their usefulness and effects on organisms other than the target or the environment. obtaining pest-resistant plants is perhaps the most spectacular field of genetic engineering applied to plants, since it allowed the regeneration of plants containing genes of bacterial origin that provide protection against harmful insects. this ensures, on the one hand, the obtaining of richer harvests and, on the other hand, the reduction of farmers' costs for pesticides (lukács et al. ; prasad et al. prasad et al. , . a series of new genes for resistance to insect attack, transferable to plants (genes encoding δ-endotoxin production from b. thuringiensis) has been discovered; genes for the synthesis of enzymes or enzyme inhibitors; plant genes encoding the synthesis of specific lectins; genes that cause induction of synthesis of plant compounds such as phytoalexins. the development of cloning methodology was based on the observation that there is a group of gram-positive bacteria belonging to b. thuringiensis species that produce a toxin called δ-endotoxin or crystalline protein capable of killing a wide range of insects (coleoptera, lepidoptera, diptera), depending on the bacterial strain. of great interest is strain b. thuringiensis var. tenebrionis that synthesizes an effective δ-endotoxin against the colorado beetle. the genes involved in the synthesis of this protein are localized, on most bacterial strains, to large plasmids ( kb), the production of the toxin occurring during sporulation. it has been shown that crystalline protein (δ-endotoxin) is normally expressed as a large inactive protoxin, which undergoes proteolytic processing in the intestine of the sensitive insect, becoming an active toxin. it recognizes the specific receptors in the intestinal cells and blocks the functions of these cells, leading to the death of the insects. studies on genes that encode inhibitory proteins produced by b. thuringiensis led to their grouping into four types based on target species specificity and nucleotide sequence: type i cry genes, encode specific kda proteins for lepidopteran larvae; type ii cry genes, encode active kda proteins on dipterous and lepidopteran larvae; type iii cry genes, encode kda specific activity on coleopteran larvae; and type iv cry genes, encode inhibitory proteins for diptera larvae. the number of genes identified that are coding for δ-endotoxin-like proteins is high: genes specific for lepidopteran, coleopteran, and diphtheria (genfa et al. ) . it has been achieved to obtain crystalline protein genes from several strains of b. thuringiensis by genetic amplification (pcr). because the whole gene for the crystal protein was found to be very poor in the transformed plant cells, a modified gene was created, containing only the n-terminal portion of the protein (amino acids - ). to increase gene expression in plants, the natural sequence for amino acids - rich in at was replaced by a synthetic sequence, rich in gc, containing the preferred codons for plant cells. these recombinant genes were introduced into ti plasmid-derived vectors (binary vectors containing the duplicate camv promoter, which increases the transcriptional and fivefold transcription and selection marker genes for antibiotic resistance or phosphinothricin herbicide) transferred to cells by a. tumefaciens containing ti disarmed plasmids. the size of recombinant plasmids obtained ranges between and , pb, depending on the size of the bacterial gene and the promoter sequence integrated into the vector. recombinant bacterial strains were then used to infect test plants (potato, tobacco, cotton). selection was first performed according to vector-borne selection markers (antibiotic resistance, gus test, herbicide resistance, etc.), and finally regenerated plants were subjected to insect attack (takakusagi et al. ) . regenerated plants have shown resistance to attack by insect pests, the specific character being maintained and expressed in experiments in the field. the first transgenic plant that manages the insect attack resistance belongs to the nicotiana tabacum species, expressing the whole or truncated cry a gene, cloned under the control of a constitutive promoter, so that the inhibitory protein represents . % of the total plant protein (leaf). there were obtained cotton plants into which the modified cry a (c) gene, cloned under the control of the camv s promoter or under the control of a promoter and a sequence for a chloroplast transit peptide isolated from arabidopsis so that the expression level of the gene of interest led to a high level of toxin: . % of total protein, %, respectively. another variant of cloning the bacterial toxin gene was that of using genetic elements that ensure expression of the gene of interest exclusively in the green portions of the plant (the promoter derived from the gene for pepc) or pollen (by using a gene derived promoter for a calcium dependent protein kinase (cdpk). a similar methodology has been used to transform rice plants, and by cloning a synthetic cry iii gene, they have obtained tobacco and potato plants resistant to the attack of colorado beetle (vigneux et al. ) . a modified a (b) modified gene was used for cloning under the control of the camv promoter and obtained sugarcane plants with resistance to diatraea saccharalis larvae. given the practical significance of plant resistance to harmful insects, research has been extended to other plant species, producing eggplant plants resistant to the attack of coleoptera, broccoli with resistance to certain lepidopteran species, maize with resistance to b. fusca, etc., as well as a number of advances in leguminous plants. toxicity studies conducted on plants expressing the gene for δ-endotoxin have shown that the existence of the transgene does not alter the normal features of the plants, except resistance to insect attack. in addition to δ-endotoxin produced by b. thuringiensis strains, other bacterial species have also been identified that produce insecticidal proteins (liu et al. ) . this is the case for b. cereus strains producing two vip and vip proteins with effect on insects, their mode of action being similar to δ-endotoxin. expression by plants of enzymes such as chitinase, cholesterol oxidase, lipoxygenase, phenol oxidase, peroxidase, or isopentenyl transferase (ipt) could be an alternative to using the δ-endotoxin gene. of the enzymes that can provide plant protection against insect attack, a particular place is occupied by chitinases, enzymes that act on chitin, a basic component of insect coatings. tobacco plants expressing genes for chitinase isolated from insects or beans exhibit increased resistance to lepidopterans. it has been observed that by cloning the isolated chitinase gene from the s. marcescens bacterium, a synergistic effect of endocytinase produced by plants containing the endotoxin gene in addition to s. littoralis larvae has been revealed. another enzyme of bacterial origin that exhibits insecticidal action is cholesterol oxidase. the introduction and expression of cho a gene for cholesterol oxidase from streptomyces sp. in tobacco plants have led to increased plant resistance to a. grandis larvae. the use for cloning the gene for the enzyme bacterial isopentenyl transferase (ipt) (involved in cytokine biosynthesis) by fusion with the protease inhibitor ii (pi-iik) gene promoter determined the production of n. plumbaginifolia lepidopteran-resistant plants (m. sexta or m. persicae). another embodiment was that of introducing into the plant cells the cpt gene that encodes a trypsin inhibitory protein isolated from vicia faba. this protein has antimetabolic effect, protecting plants from the attack of pests. similarly, other genes encoding protease inhibitors (kunitz trypsin inhibitor, bowman-birk proteinase inhibitor) or lectins have been cloned into different hosts, and encoded proteins may be true "defense guns" for the plants that contain them. it is known that insects, such as lepidopterans, depend on serine proteases (trypsin, chymotrypsin, or elastase), these being the first enzymes to digest (alvarez-fernandez ). a series of genes encoding different inhibitors for serine proteases have been isolated from various sources (plants, microorganisms) and cloned into various plant species, such as m. sativa, tobacco, corn, etc.; the plants obtained showed increased resistance to various insect pests compared to normal non-gm plants. in some cases, it has been noted that the insertion of additional genes to plants for protease inhibitors to join endogenous genes causes an increased level of pathogen resistance of transformed plants. however, the use of protease inhibitors for controlling insect pests requires thorough studies of plant and insect interactions because it has been observed that for some inhibitors such as serine proteases, the insecticide effect also manifests on useful insects (e.g., on bees). insect carbohydrate metabolism is another target for inhibitory agents tested in numerous studies. genes for different α-amylase inhibitors (wheat and beans) were isolated and characterized; after cloning the isolated gene from wheat in tobacco plants, an increase in lepidopteran larval mortality of up to % was observed. in the case of cloning the α-amylase inhibitor gene from beans in pea plants under the control of the pha gene promoter, an increased expression of the foreign gene in the seeds was obtained, which resulted in a higher resistance to callosobruchus sp. (ramirez and spears ) . vegetable lectins are a special group of glycoproteins that have protective functions against a number of harmful organisms. studies on these glycoproteins have shown that they produce strong effects on the development of various types of insects. the first example of plants containing genes for nonspecific lectins that show toxicity to pests is tobacco plants in which the lectin genes from the pea have been cloned. however, many of the vegetable lectins also have a toxic effect on mammals, which limits their use as agents to increase pest resistance. special attention has been given by many specialists to lectin specific for mangosteen from guinea pigs and concanavalin a: the genes for these lectins have been cloned into different plant species (tobacco, tomato, potato, sugarcane, rice, wheat), and the heterologous proteins synthesized by them have determined a reduced sensitivity to the attack of harmful insects (lepidopteran, aphids, coleopteran larvae). the results suggest that the use of plants containing insecticide genes (such as for lecithin) together with integrated management represents promising possibilities for controlling pests from many plant species, including wheat or rice (richard et al. ). contrary to the remarkable results achieved so far, the genes used to transform crop plants are either too specific or only partially effective on the targets of insect pests. to use plants as true "weapons" for pest control, it would be necessary to have genes at their level to determine the synthesis of compounds with different actions on the same target. the researchers are relatively recent and aim mainly to combine genes for a b. thuringiensis δ-endotoxin with another inhibitory gene in the same host gene: for example, the gene for the v. faba trypsin inhibitor (cpti) or for serine proteases and the protein gene in the potato virus y coating. another interesting approach is that which introduced the cry a (c) gene into a p. fluorescens strain able to colonize the sugarcane by means of two plasmids, pder and pkt , in which the gene is found in and children, respectively. testing of recombinant bacterial strains on sugarcane-specific insect pests revealed greater resistance of plants treated with the respective bacteria than untreated. also, although pest-resistant plants have been obtained for several plant species, fewer results have been reported for cereals, vegetables, and oleaginous plants (ibrahim et al. ) . plant resistance to various diseases (microbial, viral, and nematode phytopathogens) has been a subject for long-term studies, identifying a relatively large number of resistance genes. although it was thought that endogenous resistance genes would provide a sustainable effect for the appropriate plants, this was true in very few cases. in the case of potato, the control program for certain diseases, such as the rot caused by phytophthora infestans, had to be abandoned because the resistance to this disease of potato plants obtained by transferring the resistance genes on the basis of crossbreeds with the solanum demissum species proved to be of short duration. identifying genes of resistance in the genome of different plant species and transferring them to other crop plants are extremely difficult and time-consuming if traditional methods (intra-and interspecific hybridizations) are used. the process is considerably accelerated by the use of molecular markers generated by restriction fragment length polymorphism (rflp) techniques, randomized amplified polymorphic dna (rapd), single sequence repeat (ssr), or single nucleotide polymorphism (snp). the application of molecular markers allowed the isolation of nearly resistance genes (r-genes) from genetically well-characterized plant species, proving that many of them are grouped into specific chromosomal regions (they form clusters). of these resistance genes, some have been transferred to other plant species than to their origin through molecular cloning techniques, with the help of specific vectors that ensure the transfer of large fragments, revealing that in this way, the r-genes act synergistically and provide lasting resistance to some diseases. as it has already been mentioned, few r-genes have been shown to provide a lasting control of plant diseases. this is the case for pepper bs genes and rice xa which provide resistance to different phytopathogenic strains of x. campestris or x. oryzae in the case of species in which the genes have been cloned (e.g., tomatoes). resistance to these genes is due to the recognition of proteins produced by bacteria or phytopathogenic fungi, resulting in the occurrence of a plant hypersensitivity phenomenon and necrosis of affected tissues. another example of the long-acting r-gene is the barley recessive mlo gene which provides the resistance of the plants containing it to all e. graminis strains, through the accumulation in plant cells of a phenolic antifungal compound named p-coumaroylhydroxyagmatine. it is expected that this gene will be used for suppressing the antisense technique of the dominant mlo gene from wheat or other plant species susceptible to erysiphe sp. in vitro processing of the r-genes and then introducing them into new hosts provide new possibilities for resistance. this is the case for the tomato avrpto gene which, after cloning under the control of a strong promoter such as s to camv, determines the resistance of transformed tomato plants to p. syringae pv. strains, tomato, and to unrelated pathogens such as x. campestris and c. fulvum. researchers' efforts to obtain resistance systems applicable to a larger number of plant species are not limited to r-genes but also include systemic acquired resistance mechanisms (sar). several genes involved in sar have been isolated and characterized, of which the npr gene encoding a transcriptional regulator is a key gene in this system. overexpression of this gene increases the resistance of arabidopsis thaliana or rice plants to a wide variety of pathogens (knecht et al. ). an interesting behavior has been observed in the myb gene which is induced by vmt infection of resistant tobacco plants and which causes the synthesis of a transcription factor that binds to the promoter of a gene involved in pathogenesis (the pr a gene). modifying the expression level of the myb gene in tobacco plants leads to increased resistance to viral infection (with vmt) as well as to r. solani pathogenic fungus (raymond et al. ) . along with this, another recently cloned gene, pad , isolated from arabidopsis thaliana, proves to be extremely interesting for the development of broad-spectrum resistance: overexpression of the gene in plants increases resistance to phytopathogens. numerous biotech companies and universities have begun to assess the performance of plants that express antifungal proteins through both "in vitro" and field experiments. both plants containing r-genes or genes involved in sars, as well as genes such as those which encode the glucosidase (ago) from aspergillus sp., defensins, h o -generating enzymes, glucanases or chitinases, have been examined. although at the laboratory level potato plants containing the fungal gene for glucose oxidase showed an increased resistance to phytopathogens, the results were inconclusive when they were put into the field. for other genes such as chitinase and intracellular α- , -glucanase, overexpression of these in tomato plants resulted in significant resistance to fusarium oxysporum (werner et al. ) . companies have created nbss that farmers can use to detect information such as air pollution, soil humidity, and so on. however, given that plants are really good nbss and that they can naturally react to external stimuli and changes, they can be used instead. this is the idea behind the latest nbss initiative called advanced plant technologies. the idea is that, through genetic manipulation, researchers will be able to create self-sustaining plants, which in turn enable them to act as a kind of nbs when it comes to detecting chemical substances, pathogens, etc. this is not the first time this idea with plants as nbss appears, but before that, resources that plants needed to survive were used, which in turn reduced their resistance. this new idea indicates that nbss can be sustained by themselves, which means they can work longer in isolated parts. in the future, plants could be used to detect when a biological attack will take place. in addition, because they are plants, it also means that they can be placed anywhere and nobody will think twice about their presence and that they might be some nbss. through such examples, nature teaches us to optimize by exploring diversity. in this context, integrated nanoscale systems (nbss), energy sources (biocombustible cells) that use plants metabolism, manipulators for nanoscale surgery, and drug reservoirs embedded in intelligent polymers are explored. all of these are virus-sized. the proliferation of these types of nbss leads to a large number of applications, and combinations of these in the future will lead to microminiaturization, versatility, and functionality. plant species present a great diversity of genetics, and wild ones constitute a large genetic reservoir, from which genes that are important from a practical point of view can be obtained. plant genetic engineering research has a great theoretical significance, facilitating the knowledge of how genes of these organisms act, the effects of phytohormones on plant development, genes inactivation mechanisms, etc. by applying molecular biology techniques, useful information can be obtained on the genome of plants used for amelioration, the localization of genes of interest, the degree of relationship between different species, etc. as far as practical applications are concerned, a number of significant results have been obtained so far, some of which have already been applied, such as virus-resistant plants, plants resistant to the attack of pests, herbicide-resistant plants, plants of horticultural interest (ornamental plants with new phenotypes, plants producing softening resistant fruits), plants capable of synthesizing secondary metabolites in increased amounts, and plants producing "edible" antibodies, and enumeration could continue. numerical modeling of the dynamic response of a bioluminescent bacterial biosensor rapid detection of cadmium-resistant plant growth promotory rhizobacteria: a perspective of elisa and qcm-based immunosensor patented applications of gene silencing in plants: manipulation of traits and phytopathogen resistance experimental and modelling data contradict the idea of respiratory down-regulation in plant tissues at an internal [o ] substantially above the critical oxygen pressure for cytochrome oxidase d-nanostructured au electrodes for the event-specific detection of mon transgenic maize development of biosensor for phenolic compounds containing ppo in β-cyclodextrin modified support and iridium nanoparticles detection of glycoalkaloids using disposable biosensors based on genetically modified enzymes proteomic dissection of plant responses to various pathogens redox regulation in plant immune function chiral flavanones from amygdalus lycioides spach: structural elucidation and identification of tnf alpha inhibitors by bioactivity-guided fractionation the screening and isolation of an effective anti-endotoxin monomer from radix paeoniae rubra using affinity biosensor technology development of fret biosensors for mammalian and plant systems application of genetically engineered microbial whole-cell biosensors for combined chemosensing properties, functions and evolution of cytokinin receptors tracking transience: a method for dynamic monitoring of biological events in arabidopsis thaliana biosensors the influence of different nutrient levels on insect-induced plant volatiles in bt and conventional oilseed rape plants assessment of respiration in isolated plant mitochondria using clark-type electrodes in vivo biochemistry: applications for small molecule biosensors in plant biology chemosensors and biosensors based on polyelectrolyte microcapsules containing fluorescent dyes and enzymes generation of plant protein microarrays and investigation of antigenantibody interactions expression of bvglp- encoding a germin-like protein from sugar beet in arabidopsis thaliana leads to resistance against phytopathogenic fungi biosensing hydrogen peroxide utilizing carbon paste electrodes containing peroxidases naturally immobilized on coconut (cocos nucifera l.) fibers heat-solubilized curry spice curcumin inhibits antibody-antigen interaction in in vitro studies: a possible therapy to alleviate autoimmune disorders plant growth enhancement and associated physiological responses are coregulated by ethylene and gibberellin in response to harpin protein hpa portable optical aptasensor for rapid detection of mycotoxin with a reversible ligand-grafted biosensing surface effect of bt broccoli and resistant genotype of plutella xylostella (lepidoptera: plutellidae) on development and host acceptance of the parasitoid diadegma insulare (hymenoptera: ichneumonidae) measurement of the optical parameters of purple membrane and plant light-harvesting complex films with optical waveguide lightmode spectroscopy self-referencing optrodes for measuring spatially resolved, real-time metabolic oxygen flux in plant systems redox-sensitive gfp in arabidopsis thaliana is a quantitative biosensor for the redox potential of the cellular glutathione redox buffer staying alive: new perspectives on cell immobilization for biosensing purposes colorimetric bacteria sensing using a supramolecular enzyme-nanoparticle biosensor versatile strategy for the synthesis of biotin-labelled glycans, their immobilization to establish a bioactive surface and interaction studies with a lectin on a biochip complex regulation of the immunoglobulin mu heavy-chain gene enhancer: microb, a new determinant of enhancer function development of an electrochemical biosensor for the rapid detection of naphthalene acetic acid in fruits by using air stable lipid films with incorporated auxin-binding protein receptor boron nitride nanotube-based biosensing of various bacterium/ viruses: continuum modelling-based simulation approach live-cell imaging of phosphatidic acid dynamics in pollen tubes visualized by spo p-derived biosensor nanotechnology in sustainable agriculture: recent developments, challenges, and perspectives. front microbiol : nanotechnology in sustainable agriculture: present concerns and future aspects stem nematode counteracts plant resistance of aphids in alfalfa, medicago sativa host plant and population determine the fitness costs of resistance to bacillus thuringiensis fine mapping of co-x, an anthracnose resistance gene to a highly virulent strain of colletotrichum lindemuthianum in common bean electrochemical quantification of the antioxidant capacity of medicinal plants using biosensors enzyme-linked immunosorbent assay for the quantitative/qualitative analysis of plant secondary metabolites kinetic models for detection of toxicity in a microbial fuel cell based biosensor mapping a disordered portion of the brz -binding site on a plant monooxygenase, dwarf , using a quartz-crystal microbalance biosensor-based t phage display the xaxab genes encoding a new apoptotic toxin from the insect pathogen xenorhabdus nematophila are present in plant and human pathogens multi-analyte biochip (mab) based on allsolid-state ion-selective electrodes (assise) for physiological research belowground communication: impacts of volatile organic compounds (vocs) from soil fungi on other soil-inhabiting organisms luminescent sensing and imaging of oxygen: fierce competition to the clark electrode comparative quantification of oxygen release by wetland plants: electrode technique and oxygen consumption model key: cord- - i kktl authors: santra, hiran kanti; banerjee, debdulal title: natural products as fungicide and their role in crop protection date: - - journal: natural bioactive products in sustainable agriculture doi: . / - - - - _ sha: doc_id: cord_uid: i kktl seeking solutions from nature for solving one and all problems is the age-old practice for mankind, and natural products are proved to be the most effective one for keeping up the balance of development as well as the “healthy, wealthy, and well” condition of mother nature. fungal pathogens are proved to be a common and popular contaminant of agroecosystem that approximately causes – % of total microbial crop loss. to meet the proper global increasing need of food products as a result of population explosion, managing agricultural system in an eco-friendly and profitable manner is the prime target; thus the word “sustainable agriculture” plays it part, and this package is highly effective when coupled with nature-derived fungicidal products that can minimize the event of fungal infections in agrarian ecosystem. present study enlists the most common and effective natural products that might be of plant or microbial origin, their mode of action, day-by-day development of phytopathogenic resistance against the prevailing fungicides, and also their role in maintenance of sustainability of agricultural practices with special emphasis on their acceptance over the synthetic or chemical one. a large number of bioactive compounds ranging from direct plant (both cryptogams algae and moss and phanerogams)-derived natural extracts, essential oil of aromatic plants, and low-molecular-weight antimicrobial compounds known as phytoalexins to secondary metabolites that are both volatile and nonvolatile organic compounds of microbes (fungal and actinobacterial members) residing inside the host tissue, called endophyte, are widely used as agricultural bioweapons. the rhizospheric partners of plant, mycorrhizae, are also a prime agent of this chemical warfare and protect their green partners from fungal invaders and emphasize the concept of “sustainable agriculture.” natural products are the best weapon for the survival of any type of problems regarding infection, pathogenesis, or protection from diseases. due to their degradability in nature, they are the first options to be used by agriculturalists and plant biologist for combating fungal pathogenesis. the eukaryotic organism fungi have a separate kingdom in whittaker's five kingdom classification and are prime member of this ecosystem as a potent decomposer. in spite of their heavy and multidimensional applications in agricultural, medicinal, and industrial field ranging from the production of life-saving medicines to food supplements, they are the cause of huge global crop loss each year and lead to economic exhaustion. macro-and microscopic fungi-producing fruit bodies on different portions of a plant body (stem, leaf, fruit, root) lead to the death and decline of the crop species. several methods have already been tried since the start of civilization for crop protection but because of plant and fungal coevolution, fungi have dominated on the green eukaryotes and caused significant reduction in the crop yield. particularly in a country like india where the central gdp largely depends on agricultural output, the fungal pathogenesis has been a matter of grave concern for the agriculture department and policy makers. huge amount of money and manpower is invested to fight against the fungal diseases for ensuring higher and qualitative yield, but still it has been a burning problem of today's conditions. the problem with chemical and synthetic tools for combating the parasitic infections includes their toxicity leading to quality deterioration and environmental pollution accompanied with side effects on human health. in a case study, it has been reported that the extreme use of antibiotics in agricultural field and their direct consumption by humans through their daily meal have led to resistance of those antibiotics in human fungal pathogens. so we are in search of bioactive agents that will be of biological origin, selective to their host, and produce no secondary symptoms with least negative impact. the problem with fungi is their secretion of various types of mycotoxins (aflatoxins, ochratoxins, patulin, fumonisin, zearalenone, deoxynivalenol, etc.) in the stored food products, causing postharvest loss of cereals, pulses, dry fruits, and spices. mycotoxins are not only food spoilers but also potent disease-causing agents in humans leading to cancer, liver damage, kidney failure, and paralysis (miller ) . the severe effects of fungal crop loss are visible largely in tropical or subtropical regions where the temperature is moderately higher than the other parts of the world. fungal devastation occurs in two phases: firstly, when the crops are growing on the field and, secondly, when they are stored for further transportation, postharvest loss. the third type of contamination occurs when the microscopic airborne pathogens like molds grow on cooked foods and lead to food spoilage. at each and every level, scientists have developed techniques to minimize fungal food loss. on a gross annual estimate, almost % of agricultural food items are of no use due to fungal contamination (pittet ) . the major issues with fungi-related crop loss are deterioration as a result of increase of fatty acid conditions, change of color and texture of food items, poor nutritional conditions, and poor germinability of stored seeds (dhingra et al. ) . reports from asia and africa include death of humans and animals due to consumption of mycotoxin-contaminated foods (reddy and raghavender ) . fungal pathogens are sometimes dependent on more than one host for their successful completion of life cycle and disease development (puccinia graminis var. tritici, causal agent of black stem rust of wheat that requires berberis aristata for successful infection other than there main target wheat plant). so physical controls like eradication of secondary or collateral host and burning of the old livestocks and remnants of the field are the primary measures adopted by the farmers for disease-free crop production. so maintaining the sustainability along with less pathogenic infection is the deep ecological movement for crop maintenance. there are reports of resistance developed against the common and widely used antibiotics of agricultural importance. blasticidin s, an antibiotic obtained from streptomyces sp. (a type of actinobacteria predominantly present in soil samples), interacts with the protein synthesis and causes the death of the rice blast pathogens. development of resistance of this antibiotic is reported to be present in some fungal pathogens that detoxify it by deamination (dayan et al. ). compounds of bacterial and fungal origin from both soil and endophytic sources are used as an alternative source over the chemical ones. plant extracts especially essential oils from plant taxa of lamiaceae family are of immense importance and are used as fungicidal or fungistatic. most of the active ingredients act upon the fungal cell wall by either blocking the cellular processes like respiration, cell wall and cell membrane synthesis, ergosterol biosynthesis, protein synthesis, or dna replication. not only the secondary metabolites of plant and microbial origin but also the direct application of microorganisms in terms of biocontrol agent could be used as potent antifungals. other than these, plants' own defense molecules, known as the phytoalexins, could provide a strong line of defense against mycorrhizae; the root symbionts of higher plants can physically, biologically, and biochemically protect the plant root from pathogenic invasion and provide an enhanced resistance conditions to their hosts. this study includes the role of these compounds as natural agents of antifungal property and their role in disease prevention. mycorrhizae as a biocontrol agent mycorrhiza being the perfect example of symbiosis is known to be the oldest association between higher plant (both angiosperm and gymnosperm, monocot and dicot plants) and fungi and is an astonishing phenomenon of nature. the mycorrhizal association is one of nature's privileges for maintaining the sustainability of agriculture. in present day's changing environment, haphazard use of pesticides (fungicides) and chemicals poses a great risk to the existence and survival of mycorrhizal species in its complete biologically active form. there is a need to increase awareness in order to save mycorrhizal fungi from extinction. plants form beneficial association with other variants of life forms (animals, bacteria, or fungi) to complete their life processes, to fight against pathogenic microorganisms, and most importantly to thrive in adverse environmental situations. the plant root and its associated living microbial flora are together called "rhizosphere," particularly the area of mycorrhizal occurrence. the term mycorrhiza is derived from two greek words: mycos which means fungus and rhiza which means roots. in nature, more than % of angiosperms and almost all of gymnosperms are known to have mycorrhizal associations. the common two types of mycorrhizal associations that exist in nature are endomycorrhizae, also called arbuscular mycorrhizae (am), for example, endogone sp. and rhizophagus sp., and ectomycorrhizae (em), for instance amanita muscaria and laccaria bicolor. mycorrhizal associations support its host plants to survive in untimely soil conditions and drought situations by increasing the surface area of root and efficiency of mineral uptake. environmental threats including problems of temperature increase, climate changing, drought, and infertility of soil are some of the major challenges in agriculture and have to be mitigated to ensure global food security. in this context, mycorrhiza-based crop production is one of the key components of sustainable agriculture practices. in most of the cases, am fungi-mediated suppression of root pathogenic fungi is achieved by either morphological, physiological, or biochemical alterations of the host. several experiments on fungistatic activity of the mycorrhizal species have been done, and fruitful results are found against pathogenic fungi such as aphanomyces spp., botrytis fabae, chalara (thielaviopsis) basicola, dothiorella gregaria, fusarium oxysporum, gaeumannomyces graminis var. tritici, ganoderma pseudoferreum, pythium ultimum, p. splendens, phytophthora parasitica, p. cactorum, p. vignae, rhizoctonia solani, r. bataticola, and sclerotium rolfsii (lioussanne et al. ; bagyaraj ; bagyaraj and chawla ) . the most common outcome of am fungal colonization is seen as an increase in number of branches, resulting in a relatively larger proportion of higher-order roots in the root system. thickening of the cell walls due to lignification and production of polysaccharides in mycorrhizal plants are the common mode of prevention of penetration and growth of pathogens like fusarium oxysporum and phoma terrestris. a huge percentage of am-root pathogen interaction studies have been conducted in crop plants of agricultural and horticultural importance. but the information available on forest tree species is scanty. mycorrhizal technology can thus play an important role in production of low-cost quality seedlings and provide plant protection. like other methods of biological control, am fungi are not able to offer complete immunity against the infection caused by plant pathogens. they could only impart a degree of resistance against soilborne plant pathogens. however, the possibility of biologically controlling soilborne plant pathogens looks promising. am fungi play a protective role for plants by activating the defense mechanisms for the better resistance of crop plants and thus may protect the host plant from further fungal pathogenic attack, thus working as a potent biocontrol agent. researchers have proved that am symbiosis triggers the activation of several defense-related genes and also expression of pathogenesis-related proteins. evidences are drawn from modern techniques like molecular biology methods and immunological and histochemical analysis that strongly supports this concept. am fungi first act as a biotrophic agent, and before entering the host plant's root cell, they cause a sharp change in endogenous salicylic acid that is reflected in quick accumulation of reactive oxygen species (ros), a wide range of hydrolytic enzymes, and also the activation of phenylpropanoid biosynthetic pathway (güimil et al. , paszkowski , roman et al. . research findings have proved that the amount of defense-related compounds (essential enzymes like pal, phenylalanine ammonialyase, a product of phenylpropanoid pathway, enzymes needed for flavonoid or isoflavonoid biosynthesis like chalcone isomerase) that act for the protection of plant from fungal and bacterial pathogen is higher in the case of mycorrhiza-inoculated plant than in the uninoculated ones (volpin et al. (volpin et al. , . host's physiological and biochemical processes are greatly influenced by the mycorrhizal association in terms of decreased root exudation, higher concentration of phenylalanine and serine contents, ortho-dihydroxy phenols, increased membrane phospholipid content, etc. (smith et al. ) . when the phospholipid contents are high, it reduces the chances of root pathogenic attack, and higher concentrations of ortho-dihydroxy phenols show inhibitory activity against root rot pathogen sclerotium rolfsii (causal agent of southern blight), whereas the non-mycorrhizal plants are affected by the southern blight disease. tomato plants when inoculated with g. fasciculatum show inhibitory activity against root knot nematodes. host-am association leads to the formation of defense-related compounds like phytoalexins, chitinases (chi), β- , -glucanase (glu) (enzyme related to hydrolysis of fungal cell wall), peroxidases (pox), hydroxyproline-rich glycoproteins, and phenolics (st arnaud and vujanovic ) . synergistic effect of pgprs along with am fungi is proved to be a system of better protection than the use of am fungi alone (linderman ; bagyaraj and chawla ) . fungal wilt of common medicinal plant indian coleus (coleus forskohlii) caused by fusarium chlamydosporium could be minimized by the joint action of am fungus and trichoderma viride and cause a sharp increase in root yield and root forskolin concentration and may also reduce the severe disease conditions (singh et al. ). am causes a drastic change in the rhizospheric microbiota and intentionally either removes directly the pathogenic microorganisms or stimulates the accumulation of potent microbial partners especially fungus that are heavily antagonistic to the plant pathogenic ones. plants with mycorrhizal association harbor higher population of rhizospheric microorganisms, thus making it impossible for the pathogen to compete and invade the root. in the case of phytophthora cinnamomi, the numbers of sporangia and zoospores are found to be reduced when rhizospheric soil extracts of am plants are applied; it means the am fungi are able to alter the microbial population and particular functional groups of rhizospheric microorganisms (meyer and linderman ; larsen and bodker ) . they cause qualitative and quantitative changes in the fungal community by several factors like changed exudation patterns; altered root size and architecture; different physiological and biochemical parameters like sugar, organic acids, and amino acids; and also putative direct am fungal effects (toljander et al. ; ahmed et al. ; vigo et al. ) . fungistatic siderophore (low-molecular-weight chelating agents having higher affinity for ferric ion)-producing microorganisms are found to be crowded in mycorrhiza-infected roots and rhizospheric regions. mycorrhizal plants are to be reported with more actinomycetes and bacterial (gram-positive paenibacillus sp. against phytophthora parasitica) flora antagonistic to soilborne root pathogens (azcon-aguilar and barea ; budi et al. ). apart from providing biochemical and physiological defense strategies, arbuscular mycorrhizal species also exhibit physical barrier of defense by changing the root anatomy, morphology, and even architecture in terms of increased nutrient uptake, meristematic and nuclear activities of root cell, higher rate of growths, and branching patterns (atkinson et al. ; gamalero et al. ; gutjahr and paszkowski ) . thus responses of root morphology as a result from afm colonization seem to depend on plant characters, tap root system, etc. more benefits are seen in tap root system than fibrous root system in terms of gained biomass and nutrient acquisition. though there is a gap of knowledge in how increased root branching caused by mycorrhizal infection help the plant to defend fungal pathogenesis, synergism is seen as something that can balance the suppressed root growth caused by several root pathogens and restore the root health. mycorrhiza-mediated strengthening of the vascular system allows the higher rate of flow of nutrients, increased mechanical strength, and also inactivation of vascular pathogens. in conditions of limited resource such as carbon requirement and space for inoculation, a competition between the symbiotic partner (mycorrhizae) and pathogenic fungi is very common and expected (vos et al. ). in the direct warfare, mycorrhizae win over the pathogenic one and thus obtain higher amount of nutrients (almost - % of total assimilated carbon by host plant) and occupy large areas of available root cortical cells (jung et al. ; vierheilig et al. ) . defeating the pathogenic fungi in terms of nutrient uptake and providing a little or no room for infection are probably the mightiest cause of biocontrol ability of am fungus (hammer et al. ) . output of am and phytophthora interaction indicates that the pathogen does not penetrate cortical arbuscular cells, suggesting that localized competition for infection site does occur between the pathogenic fungi and the am fungus. not only fungi but also plant-invading nematodes are in the queue for colonization and nutrient uptake (smith ) . the infection of southern root-knot nematode (meloidogyne incognita, m. exigua) is reduced when the roots are priorly inoculated with symbiotic partners like in the case of coffee plants also (alban et al. ; dos et al. ) . reports have suggested that the number of infected sites is reduced within mycorrhizal root system than in the uninoculated one and thus strongly supports the mycorrhizal role as a biofungicide (vigo et al. ) . am fungi can help the plant uptake of nutrients like phosphate, nitrogen, minerals, microelements (zinc), and water at a higher rate than the uninfected one (baum et al. ; parniske ) , and as a result, they are provided with photosynthetic carbon (smith and smith ) . the plants capable of absorbing higher amount of nutrients in terms of am fungal association have the potential to tolerate pathogenic infections (karagiannidis et al. ) . though the improved nutrition and increased tolerance are not involved in a cause-effect relationship, proofs are there that higher uptake of phosphate results in remarkable reduction in pathogenic infection in mycorrhizal plant but not in non-mycorrhizal plant (bodker et al. ) . tomato plants already infected with rhizophagus irregularis are not colonized by the pathogen a. solani, whereas non-mycorrhizal plant is affected by the pathogen (fritz et al. ) . mixed action of arbuscular mycorrhizal fungi (amf) glomus intraradices and trichoderma harzianum as a biocontrol agent significantly reduces the damping off disease caused by rhizoctonia solani in the case of tomato seedlings (amer and seud ) . in order to combat parasitic (fungal, bacterial, viral, nematoidal, and insectal) infection like mammalian cells, plant cells also develop defense systems that mediate the release of low-molecular-weight and short-lived (generally - h of existence) antimicrobial compounds or molecules known as the phytoalexins (braga ; echiverri et al. ; paxton ) . these secondary metabolites help the plant to withstand biotic and abiotic stress (grayer and kukubun ) . most of them being lipophilic compounds can cross the plasma membrane and act inside the fungal cell causing cytoplasmic granulation of the infecting fungal cells, disorganization of the cellular components, rupture of the plasma membrane, and inhibition of the fungal enzymes and mycelial growth (cavalcanti ) . mode of action of phytoalexins against fungal pathogenesis varies from species to species (table . ). metabolism of phytoalexin mediated by fungus involves the tendency for its increased polarity by addition of hydroxyl group (oxygenation), removal of methyl group (demethylation), etc. (jeandet et al. ) . muller and borger first enlightened the concept of phytoalexins almost year ago (muller and borger ). the first reported case analyzed with the concept of phytoalexin was potato tuber infection by the different strains of causal organism of "late blight of potato," phytophthora infestans. this pathogenic fungus initiated the hypersensitive reactions that lead to the formation of some "plant secondary metabolite" that inhibited further infection of the same plant when infected with another strain of the same genus of phytophthora. muller and his coworker named this "principle" as "phytoalexins" that have protected the plant from secondary infection (deverall ) . accumulation of phytoalexins in the green plant tissue clearly indicates the presence of remarkable amount of fungal and bacterial infections in the host tissue (stoessl ) . phytoalexins are naturally occurring products secreted and accumulated temporarily by plants in response to pathogenic attack or abiotic stress and agents like heavy metal toxicity, uv radiation, and wounds on tissue (naoumkina et al. ). the inducer agent may be of two types, elicitor and elicitin. the elicitors are commonly the oligosaccharides from fungal cell origin (like hepatosaccharide from soja cell wall) (sharp et al. pezet and pont ( ) , adrian et al. ( ) , adrian and jeandet ( ) camalexin induction of the fungal programmed cell death (pcd) by apoptotic mechanisms et al. ( ) ). the elicitin types of molecules are generally a type of glycoproteins secreted by the fungal cells (cordelier et al. ) . reports on detailed investigations about phytoalexins have covered a very few families (leguminosae and solanaceae) of the green world (ingham ; kuc ) . though investigations on some selected number of species and genera are made from plant families including both monocotyledonous (amaryllidaceae, orchidaceae, poaceae) and dicotyledonous plants (apiaceae, asteraceae, convolvulaceae, chenopodiaceae, euphorbiaceae, linaceae, moraceae, piperaceae, rosaceae, rutaceae) and even gymnospermic taxa (ginkgoaceae) (coxon ) , cash crops like members of poaceae (focusing on maize and rice), vitaceae, and malvaceae (cotton) have been studied for their phytoalexin production (schmelz et al. ; langcake and pryce ; jeandet et al. ; sunilkumar et al. ) . though till date a lot of researches have already been performed regarding phytoalexins, a natural weapon against mycopathogens, but still to increase the fungitoxic effectivity of these stress metabolites, further advancement in design and genetic control is needed ). phytoalexin synthesis not only is dependent on pathogenic attack but also could be influenced by various abiotic factors such as temperature, humidity, and water availability ( fig. . ). there are evidences that different parts of the plant like leaves, flowers, stems, seeds, and root tubers are site of phytoalexin biosynthesis (mikkelsen et al. ) . different biochemical pathways are used for producing various types of phytoalexins. the three most common pathways include (i) the phenylpropanoic-polymalonic acid pathway, (ii) the methylerythritol phosphate (mep) and geranylgeranyl diphosphate (ggdp) route, and (iii) the indole phytoalexin (ip) pathway (jeandet et al. ). it is not always obvious that phytoalexins could be categorized not only by their chemical structure or biosynthetic pathway but also by their function and tissue specificity. examples include the occurrence of momilactone a on different plant parts of rice plant (lee et al. ; cartwright ) . momilactone a is known to be residing in rice husks and rice stems constitutively, but they are also a phytoalexin of rice leaves. further studies by toyomasu and his coworkers conclude that momilactone a is constitutively synthesized and oozed out from root of rice plants. still there is no sufficient data available to consider phytoalexins as ubiquitous throughout the whole plant kingdom. a lot of studies have revealed their complex biochemical synthetic machinery that involves their de novo synthesis, regulation, and mode of action (jeandet et al. ahuja et al. . regulatory mechanisms involve defense-related marker genes, calcium sensors, hormone signaling, phosphorylation cascades, and also their antipathogenic activity. there are reports on genetic engineering-mediated manipulation of phytoalexin production and increased disease resistance in the case of plants (delaunois et al. ; jeandet et al. jeandet et al. , . phytoalexins are secondary or stress metabolites that are produced when the host plant is infected with pathogenic fungus. phytoalexin-mediated defense response includes the expression of lytic enzymes such as chitinases and glucanases and a number of pathogenesis-related (pr) proteins, oxidizing agents, and lignification of cell walls (dixon and lamb ) . mode of action of phytoalexin involves the coordinated synergism between several defense factors for the effective inhibition of the fungal pathogen (purkayastha ; mansfield ) . in the case of sorghum plant, significant infection caused by fusarium proliferatum and fusarium thapsinum stimulates the production of -deoxyanthocyanidin, apigeninidin, and luteolinidin and also the concentration of defense-related proteins like peroxidases, β- , glucanases, and chitinases that help to fight the pathogenic infection (huang and backhouse (koga et al. ; fukuta et al. ). there are several ways of blocking the fungal infection in host plant tissues by phytoalexin-mediated response. that includes inhibition of fungal spore on the leaf surface and inhibition during and after penetration to host cell (usman et al. ) . the occurrence of fungal germ tube on the leaf surface and diffusion of fungal metabolites through the leaf cells cause the accumulation of phytoalexins by the underlying cells and provide the first line of induced chemical defense (vanwees et al. ) . phytoalexins may be located on papillae or cell walls, thereby producing a localized, fungitoxic barrier to penetration (friend ) . examples include occurrence of fungitoxic (against erysiphe graminis) flavonoid (thought to be phytoalexin) on papillae of resistant barley leaves. phytoalexins are known to be solely produced as a result of induction or stimulus by external agents. fruitful evidences could be drawn regarding this fact. induction of disease resistance in plants is developed through the direct and indirect involvement of elicitors. extracts of fungal basidiocarp, essential oils of aromatic plants (walters et al. ) , and also synthetic chemicals like aminobutyric acid, salicylic acid, jasmonic acid, and acibenzolar-s-methyl ( (matiello and bonaldo ) , hymenolobium petraeum, qualea albiflora, and corymbia citriodora (matiello et al. ) that act as the elicitors of deoxyanthocyanidins and glyceolin production. homeopathic preparations of species of calcarea (c. citriodora and calcarea carbonica), essential oils of eucalyptus globulus (telaxka et al. ; oliveira et al. ) , and mild concentrations of salicylic acid (durango et al. ) are major elicitins of pistain production and accumulation in cotyledons of common bean (phaseolus vulgaris). silicon-mediated enhancement of disease resistance by peroxidase (pox), polyphenol oxidase (ppo), chitinases (chi), β- , -glucanases (glu), and phenylalanine ammonia-lyase (pal) is found in the case of leaf spot of cotton plant caused by ramularia areola (curvêlo et al. ). southern amazonian amphibian family bufonidae represents the true toads, and their cutaneous secretions are of diverse source of bioactive compounds which can be fruitful as new chemical weapons for agrochemical development. use of elicitors in the case of crop protection nowadays is becoming a very popular method of inducing response which are proved to be durable and broad-spectrum disease control mechanism where the plant's own resistance is used. a group of seven brazilian scientists (deice et al. ) evaluated the possibilities of methanolic extracts of cutaneous secretions of two species of bufonidae, rhaebo guttatus and rhinella marina, on synthesis of phytoalexins named glyceolin (soybean plant), deoxyanthocyanidins (sorghum plants), and phaseolin (mung plant) in soybean cotyledons, sorghum mesocotyls, and bean hypocotyls, respectively. there is a direct relationship between the phytoalexins production and defense ability of the host plant against the fungal pathogenesis. studies reveal that when the phytoalexin glyceolin is produced in higher amount in the soybean plant (cultivar tmg rr) as a result of methanolic extracts of amphibian's (r. guttatus) cutaneous secretion (at a concentration of . mg/ml), stimulates the enzyme β- , -glucanase that can cause the hydrolysis of the fungal cell wall along with other defense-related enzymes (chitinase) is also produced in higher amount, but when suppression of glyceolin occurs, that particular enzyme is also not produced. there are evidences in the case of glycine max that the effectivity of phytoalexins varies from cultivar to cultivar. application of r. marina (amphibian) methanolic extracts induced glyceolin production in tmg rr and monsoy cultivars ipro but did not induce tmg rr cultivars to synthesize these defense-related compounds. less toxicity of phytoalexins than chemical fungicides is the reason for their universal acceptance. for over years, phytoalexins have been a detailed area of study for its antimicrobial activity, especially antifungal properties. several investigations include the in vivo bioeffectivity of the phytoalexins against serious plant pathogenic fungi (table . ). phytoalexin synthesizing genes have also been genetically modified to cope up with the pathogenic evolution. still reports are there that include examples of cruciferous phytoalexins detoxification by fungal enzymes (pedras and abdoli ) . modification of pathogen to overcome phytoalexinmediated damage includes curved germ tubes as a result of asymmetric growth of the germ tube. phytoalexins are natural products of diverse chemical nature, for example, alkaloids, coumarins, isoflavonoid (coumestans, isoflavans, isoflavones, isoflavanones, pterocarpans, pterocarpenes), lignans, polyacetylenes, pterocarpons (pisatin, phaseolin, glyceollin, medicarpin, and maackiain), terpenes, and non-isoflavonoid compounds (furanoacetylenes and stilbenes) ( fig. . ) (grayer and kokubun ) . both in vitro and in vivo fungicidal activity are shown by sakuranetin (rice phytoalexins) against the blast fungus (hasegawa et al. ). reduction of green mold (caused by penicillium digitatum) infections is achieved by the action of coumarin type of phytoalexin (scopoletin) of orange (sanzani et al. ). the loss of apples production caused by penicillium expansum and accumulation of patulin is minimized by the action of phenolic phytoalexins like resveratrol, scopoletin, scoparone, and umbelliferone (sanzani et al. ). in the case of medicago sativa (alfalfa), the isoflavonoid -o-methyltransferase provides increased resistance against phoma medicaginis by synthesizing maiackiain (he and dixon ) . for soybean plants, transformation of resveratrol to pterostilbene , langcake and pryce ( ) , coxon ( ) , and harding and heale ( ) (continued) (zernova et al. ) . scientists have proven that not only fungal infection acts as the stimulus for phytoalexin synthesis but also the hormone levels; phosphorylation cascades play a major role in this purpose. cytokinin overexpression in nicotiana tabacum is directly associated with its resistance against p. syringe by higher concentration of capsidiol and scopoletin (grosskinsky et al. .) the fungitoxicity of the phytoalexin could be enhanced by methylation or presence of electron-attracting groups on aromatic rings that is directly involved in affinity with membrane proteins being an uncoupler of ets system. endophytes are a type of hidden beneficial microorganisms that reside within the host plant causing no visible disease symptoms and syndrome and promote the plant to maintain its existence in typical harsh conditions. sometimes they could be latent pathogens at a very distant path of the host's life cycle but are simply a unique area of research where plant science and their microbial association get new definition. endophytes have been a constant and reliable source of exploration of bioactive compounds, but extensive search has not been performed till date, and that has given the endophyte biologists a great opportunity to search endophytic fungal and actinobacterial flora for the establishment of novel bioactive compounds. selection of plant for endophytic isolation is the most vital part of this study. exploitation of the proper isolates accelerates this search and opens up new angle of research. the search for uncommon products of agrochemical importance is a common demand of todays' world. the safer the antifungal agents become, the more it is well accepted in the scientific community as well as agricultural market. in general, the screening of thousands of natural products ends up giving only one commercial product. so indeed it's a tough job to end the search of new antibiotics with a hopeful result. a total of out of of the popular prescribed medicines are of fungal origin, and it is a fact that % of the fungi have been described till date (hawksworth (hawksworth , . so fungi serve as a continuous dependable source of new natural products. the intelligent screening procedure includes the selection of fungal flora of endophytic sources to open up the untapped potential of secondary metabolites synthesized by fungi. microorganisms grown in the petri plates or culture broth constitute minimal growth medium needed for their survival. any kind of stress or transfer of microorganisms on selective media acts as a stimulation for production of their secondary bioactive compounds. these secondary metabolites are produced for their survival in odd environments and strictly act as the selection force for the expression of their antimicrobial-producing genes. these crude by-products of microbial cultures are filtered and purified for their industrial, medicinal, and agricultural exploitation. soil microorganisms have been exploited for a long time for production of antibiotics, but microorganisms inhabiting plants are a new source in that respect. plants are selected usually with potent medicinal applications. here the knowledge of ethnobotany and folk taxonomy contributes a lot in this selection procedure. a strong literature survey supports the plant selection. the plants are surface sterilized and plated in nutrient-less solid plates. the fungi emerge out from different explants using the decaying plant parts as their primary growth substance. the isolates are identified by microscopic structures focusing on their conidial morphology, spore sculpturing, and colony characters. confirmatory identification includes s rrna analysis. endophytic fungi are tested for their antifungal activity against phytopathogenic fungi by one-to-one inhibition assay or antagonistic test ( fig. . ). two agar portions containing fungal hypha of endophyte and pathogen are placed on opposite sides of the plate. if the growth of pathogenic one is arrested partially or completely, that endophytic isolate is marked as antifungal agent and selected for further studies. another way of screening includes separating the agar plate into two equal halves, and two fungi are placed on two separate sides of the discontinuous plate. this test aims to screen the endophytes that produce volatile antifungals. if the isolate is potent enough to produce volatile organic compounds with fungi static or cidal activity, this will cease the growth of the pathogenic strain. then that isolate would be qualitatively and quantitatively measured for their volatile emissions using gc-ms as the master equipment ( fig. . ). liquid extracts of endophytic fungi are also tested for antifungal potentials by agar well-diffusion method. the fungal extract having antibiotic property shows clear zone of inhibition of growth of the pathogenic fungus surrounding the area of application of that fungal liquid. the potent isolate will be mass cultured, and the bioactive molecules will be extracted using organic solvents like ethyl acetate, ethyl ether, and n-hexane. steps include purification of that fungal extract by column chromatography, detection of the compound by thin-layer chromatography, and analysis of compounds by hplc and mass chromatography. field experiment includes the synergistic effect of a pure compound with mixture of natural compounds, and the effectivity of a newly applied antifungal agent strictly depends on the host plant and pathogenic microorganism's interaction, environmental condition, and development of drug resistance by that organism. application of pellets soaked in fungal extracts also is a method of determination of antifungal activity. secondary metabolites are itself diverse in nature. a variety of bioactive secondary metabolites are produced at significant concentrations by the endophytic microbial flora. the major components include quinones, phenols, phenolic esters, steroids, terpenoids, cytochalasins, benzopyranone, alkaloids, isocoumarins, and chromones. till date, a large number of plants have been studied for their endophytic flora as antifungal agents (table . ). alkaloid was the first ever reported insecticidal bioactive product. cryptocin was isolated from endophyte of tripterygium wilfordii, a plant of celastraceae family. the inner barks of the stem were used as explant, and cryptosporiopsis cf. quercina was isolated as a potent endophyte active against pyricularia oryzae and some other phytopathogenic fungi (li et al. ) . colletotrichum sp. produces -isoprenylindole- -corboxylic acid having inhibitory action against phytophthora capsici, a pathogen of cucurbitaceae, fabaceae, and solanaceae, and also other phytopathogens rhizoctonia cerealis and gaeumannomyces graminis var. tritici, a common pathogen of poaceae family . epoxycytochalasin h and cytochalasins n and h were isolated as chloroform and methanolic extracts of (fu et al. ) . a lot of endophytes have been explored for their antifungal production, but only a few of them were positive for antifungal metabolites categorizing in alkaloids. the common alkaloids acting as the antifungal agents of endophytic fungal origin are gliotoxin, cryptocanadin, tyrocidine a, fumigaclavine c, fumitremorgin c, -n-methyl albonoursin, and phomapsichalasin. the terpenoids, usually called isoprenoids, are large and diverse group of naturally occurring organic compounds derived from terpenes that are multicyclic. sixty percent of all the known natural products are terpenoids in nature. some endophytic fungicidal products are of terpenes by their native chemical structure. endophytic isolates (hormonema sp.) of gymnospermous plant juniperus communis were reported to be antifungal producers of a triterpene glycoside enfumafungin (pelaez et al. ) . known antifungal sterols of endophytic origin are β-hydroxyergosta- -ene, -oxoergosta- , , , -tetraene, etc. the sterols are strong inhibitors of helminthosporium sativum (present name: bipolaris sorokiniana), the asexual stage of cochliobolus sativus, a common root rot pathogen of wheat and barley crops which also infects leaf and stems of poaceae plants . sesquiterpenes are reported to be the growth inhibitors of cladosporium phlei (causal organism of leaf spot disease of timothy grass, phleum pratense). this is a unique example where the host plant (phleum pratense) itself harbors the endophyte (epichloe typhina) that inhibits the growth of its leaf spot pathogen (cladosporium phlei). from the point of view of organic chemistry, isocoumarins are defined as the isomer of coumarin where the orientation of the lactone is reversely arranged. zhang and his coworkers in the year isolated an endophytic fungus named microdochium bolleyi from fagonia cretica (also known as virgin's mantle of zygophyllaceae family), a herb of semiarid regions of gomera. isocoumarins were identified as the active compounds having antifungal activity against microbotryum violaceum (previously known as ustilago violacea), an obligate parasite of basidiomycete group and a common infectant of members of caryophyllaceae causing smut of anther. the four isolated and identified isocoumarins are monocerin, -oxo epimers of monocerin, and open-ring derivative compounds of monocerin. the compounds are obtained as mixtures by column chromatography followed by sephadex lh- chromatography techniques. preparative tlc further differentiated the four compounds. monocerin and its analogues were previously reported as antifungal compounds from fungal sources of drechslera monoceras, exserohilum monoceras, helminthosporium monoceras, exserohilum turcum, and fusarium larvarum (aldridge and turner ; robeson and strobel ; grove and pople ; claydon et al. ) . these secondary metabolites act on pathogens by interfering stages of divisional phases of cell cycle. the second isocoumarin was colorless oil. the third and fourth one are represented by the empirical formula of c h o and c h o . the fourth one is structurally correlated to fusarentin , -dimethyl ether. both the compounds are of heptaketide in their origin, and it is revealed that fusarentins are the probable precursors of the active compounds monocerins (scott et al. ; axford et al. ). dihydroisocoumarins, mellein (an isocoumarin derivative), (r)- -hydroxymellein, and fonsecinone were reported from species of xylaria (endophyte of piper aduncum), pezicula, penicillium (alibertia macrophylla), and aspergillus (cynodon dactylon), respectively (oliveira et al. ; schulz et al. ; song et al. ). phenols (popularly known as phenolics) represent a class of chemical compounds characterized with a hydroxyl group attached to an aromatic hydrocarbon group. phenol (or carbolic acid) is a colorless crystalline solid, aromatic compound having benzene rings. they are predominantly found in the plant kingdom as a response to stress and are of utmost importance. endophytic culture extracts are also known to be rich sources of phenolics; usually they are directly proportional to the antioxidative property of any fungal isolate, but in some particular cases, they are characterized with their antifungal potentials against phytopathogenic fungus. usually the liquid culture extracts of the endophytic isolates are subjected to solvent extraction using ethyl acetate, n-hexane, ethyl ether, etc. those organic solvents are believed to extract the phenolics from the water-based culture broth. those extracted compounds are further screened for their antifungal efficiency. ethyl acetate extracts of endophytic phoma sp. are reported to contain tetralone metabolites (derivatives of α-tetralone, , , -trihydroxy-α-tetralone) inhibiting the growth of two common broad phytopathogenic fungus fusarium oxysporum and rhizoctonia solani. griseofulvin is known to be the first antifungal compound isolated from penicillium griseofulvum. later it is isolated from several species of fungi including endophytic penicillium canescens and xylaria sp. (member of xylariaceae family). griseofulvin from endophytic p. canescens of popular chinese medicinal plant polygonatum cyrtonema (polygonaceae) showed strong inhibitory effectivity against phytopathogenic botrytis cinerea, sclerotinia sclerotiorum, colletotrichum orbiculare, and didymella bryoniae . other than penicillium, endophytic xylaria sp. isolated as an endophyte of abies holophylla yields griseofulvin and dechlorogriseofulvin for in vitro and in vivo effectivity against pathogenic magnaporthe grisea, corticium sasakii, blumeria graminis (park et al. ) . the ascomycete fungus pestalotiopsis is known to be a common plant pathogen but also has been reported many times because of their endophytic existence in the host plants. the two common species pestalotiopsis microspora (host: tropical plant terminalia morobensis) and p. fici are reported to be producing antifungal metabolites isopestacin and pestalofones d-e (harper et al. ; liu et al. a, b) . chlorogenic acid and colletotric acids are antifungal phenolics of colletotrichum gloeosporioides and sordariomycetes sp., respectively zou et al. ) . they were isolated from medicinal plants of china (artemisia mongolica and eucommia ulmoides) and effective against fungi imperfecti helminthosporium sativum. orcinol is used for the production of a dye called orcein used randomly for the staining of cells and chromosomes. orcinol is popularly known for its antifungal activity too and has been isolated as a product of endophytic origin of penicillium sp. from alibertia macrophylla (a plant of rubiaceae) showing bioactivity against cladosporium cladosporioides and cladosporium sphaerospermum (oliveira et al. ) . endophytic phomopsis sp., dothiorella sp., and diaporthe sp. have also been tested for their antifungal production and antifungal compounds that were detected (brady et al. ; xu et al. ; huang et al. ). volatile organic compounds (vocs) are said to be a type of organic low-molecularweight carbon-containing small compounds (up to c ) that have a high vapor pressure with low molecular mass ( - daltons) at room temperature. the high vapor pressure results from a low boiling point of that chemical compound, which causes a huge quantity of molecules to evaporate from the liquid, solid, or semisolid form of the compound and gets released into the surrounding environment. the endophytes are unique in their volatile emissions. the term mycofumigation that is very much popular with the treatment of agricultural phytopathogens is actually the output of vocs that originated from endophytic isolates. the first ever reported volatile antibiotic producer was muscodor albus (xylariaceae family), an endophyte of guazuma ulmifolia (a plant of sterculiaceae family collected from tropical forest of sw ecuador), isolated by gary strobel and his co-workers ). the major compounds isolated by gcms are known to be involved in antifungal, antibacterial activity. compounds include butanoic acid, -methyl-; butanoic acid, -methyl-; -butenal, -methyl-; butanoic acid, -methylbutyl ester; -buten- -ol, -methyl; guaiol; -octene, -ethyl-; formamide, n-( -methylpropyl); azulene and naphthalene derivatives; caryophyllene; phenylethyl alcohol; acetic acid, -phenylethyl ester; bulnesene; and various propanoic acid, -methyl-derivatives. these compounds were tested against a number of phytopathogenic fungi (botrytis cinerea, mycosphaerella fijiensis, pythium ultimum, phytophthora cinnamomi) showing partial or complete death or growth inhibition of those pathogens after or days of incubation. muscodor albus was reported from a diverse type of host plants, i.e., myristica fragrans, terminalia prostrata, cinnamomum zeylanicum, and ginkgo biloba, by several workers (worapong et al. ; sopalun et al. ; ezra and strobel ; mercier et al. ; ezra et al. a, b; atmosukarto et al. ; lacey and naven ; lacey et al. ; strobel et al. ; banerjee et al. a, b; corcuff et al. ; alpha et al. ) . the mycofumigants are effective against pathogen fusarium culmorum, causal agent of seedling blight, foot rot, ear blight, stalk rot, and common rot of cereals. sexual stage (teleomorph) of glomerella cingulata, a fungus of glomerellaceae, is a potent pathogen causing anthracnose-like symptoms of water-soaked, sunken spots and necrotic lesions on fruits of forest trees. this phytopathogen is strictly inhibited by the volatile emissions of this novel endophyte. banerjee et al. ( a, b) first reported muscodor albus strain gba from the usa as an isolate of ginkgo biloba (first isolate of m. albus from g. biloba) and tested the biological efficacy of its volatile mixtures against agricultural pathogens and also evaluated its promises to be used as a commercial mycofumigant agent for controlling the fungal diseases in storage fruits and vegetables, that is, agricultural productions and during food transportation. the strain gba in comparison to other strains of muscodor e and cz completely inhibits and potentially kills the member of phycomycetes, pythium ultimum after days of exposure of the mixture of volatiles. the organic compounds include alcohols, acids, esters, ketones, and lipids as their active components. -butanol, -methyl-, acetate was found in significant quantities. vitrine, a terpenoid, was first isolated from muscodor albus strain gab. the volatile mixture is artificially produced by the mixture of the pure compounds, and that mixture is again checked for antifungal activity. a positive mycocidal or mycostatic effect similar to the effect of endophyte's volatile emission will confirm establishment of the endophyte and its mixture as the biocontrol or antifungal agent. myrothecium inundatum, an endophyte of herbaceous acalypha indica (euphorbiacea member collected from northeastern part of india), produces unique mixture of volatile components having -octanone, -octanol, -octen- -ol, sesquiterpenes, organic acids, methyl esters, naphthalene, -octanoic acid, heptanoic acid, etc. this endophyte produces foam in its liquid culture predominant with long-chain carbon compounds like octane, , -cyclohexadiene, -methyl-and cyclohexane, and -ethylpropyl. (meshram et al. (meshram et al. , suwannarach et al. ; saxena et al. ; suwannarach et al. suwannarach et al. , suwannarach et al. , kudalkar et al. ; mitchell et al. ; worapong et al. ; daisy et al. ; siri-udom et al. postharvest fungal disease is one of the prime causes of agricultural loss of crops. use of biological agent to minimize this loss is one of the vital targets of agriculturalists, horticulturalists, and plant biologists. several chemical agents have been already tested for practical applications, but endophytes are less explored organisms in this arena. volatiles from endophytic source open up new scope of utilization of unique mixtures of chemicals to be used as mycofumigant agents. a large number of endophytes have already been screened for their postharvest disease management ability (table . ). the volatiles of the endophyte could be considered as the natural fungicides. muscodor vitigenus, an endophyte of hevea brasiliensis, was analyzed in gc-ms for their volatile production. the isolates produce a unique mixture of myroxylon balsamum , -cyclohexadiene, -methyl-; , -pentadiene and cyclohexene, -methyl- -( methylethenyl)-; alkyl alcohols starting with -butanol- -methyl, -propanol- -methyl, cinnamomum bejolghota. this isolate was known to produce azulene, a new compound detected first from any muscodor species. this species was tested in vitro and in vivo for antifungal activity against a common worldwide devastating pathogen rhizoctonia solani (causal agent of damping off). the vocs produced by this fungi include (s)-(+)- -methyl- -heptanol; ethyl acetate; propanoic acid, -methyl-, methyl ester; cis- , -dimethylthiane; s,s-dioxide; cyclopentane; butanoic acid, -methyl-, methyl ester; -butanol, -methyl-, acetate; β-humulene; azulene, , , , , , , , a-octahydro- , -dimethyl- -( -methylethenyl)-; s-( .α., .α., a.β); and eudosma- ( ), -diene , , , , , , -heptamethyl- , -bis(trimethylsiloxy) tetrasiloxane. rhizoctonia solani-infected seedlings were treated with volatile mixtures to assess the mycofumigation property. in vivo experiment was conducted on four seedlings of bird pepper, bush bean, garden pea, and tomato. it was concluded that gm of muscodor cinnamomi prepared on rye grain solid media is the minimum dose required for inhibition of rhizoctonia infection and total control and elimination of damping off symptoms. muscodor cinnamomi-infected soil does not show any seed germination inhibition in comparison to rhizoctonia solani-infected soil. so it is a type of pioneer study of using endophytic species as potent agents of fumigation and biocontrol. candida intermedia strain c (saccharomycetaceae) was isolated as an endophyte of strawberry (fragaria ananassa), and the volatile emission was known to be a mixture of organic compounds including esters, alcohols, alkenes, alkanes, alkynes, organic acids, ketones, and aldehydes of which , , , -cyclooctatetraene and -methyl- -butanol were the most dominant (huang et al. ). volatiles of strawberry endophyte were itself useful as postharvest control agent for the host plant against botrytis fruit rot. other compounds include , , , -cyclooctatetraene; -methyl- -butanol; -nonanone; pentanoic acid, -methyl-, ethyl ester; -methyl- -butanol, acetate; acetic acid, pentyl ester; and hexanoic acid, ethyl ester that were found to be extremely inhibitory to conidial germination (reproductive growth) and also vegetative (mycelial) proliferation of b. cinerea. when the fruits are exposed to c. intermedia synthetic volatiles or itself to the fungus, the incidence of botrytis fruit rot reduces significantly. strawberry fruits inoculated directly with the endophyte also remain disease-free. so mixtures of candida intermedia c , the unique natural products, are useful as mycofumigation technique or for postharvest disease management by biological control policies. tangerine fruit (citrus tangerine), the commercial citrus crop of northern thailand, faces huge postharvest losses due to pathogenesis of green mold (penicillium digitatum). the pathogen is the prime cause of worldwide deterioration of tangerine fruits by mycopathogenesis. out of detected compounds, the most predominant were -methylpropanoic acid and -methylbutan- -ol. other compounds include carbitol, octanoyl chloride, azulene, -methylhexane, -methylpropan- -ol, , -butanediol, caryophyllene, -methylbutyric acid, ethyl -hydroxyproponate, etc. the pathogen was treated both in vitro and in vivo for their inhibition by endophytic volatile components. in both cases, pathogen growth was restricted. during transportation of the fruits, fungus causes huge crop loss by infecting the fruits; when fruits were inoculated with gm of rye grain culture of m. suthepensis ( month old), the disease development is ceased. so it is a classic example of mycofumigation by the biocontrol agent of tangerine fruit for the control of rot lesions caused by p. digitatum infection. the in vivo application requires the proper surface sterilization (using sodium hypochlorite) of the targeted parts where the inoculation is going to be done, for example, fruit, stem, root, and leaf. usually infection on fruits for assessing the biocontrol potential is the most common and popular method. the seeds will be washed in distilled water, and using sterile needle, uniformly the whole area would be done, and the whole area would be infected or inoculated with the endophytic liquid extracts containing spore suspensions. muscodor albus vocs are potent enough to cause a significant reduction of in vitro spore germination of the tilletia species t. horrida, t. indica, and t. tritici. endophytic nodulisporium spp., trichoderma spp., phomopsis spp., and oxyporus latemarginatus are reported to produce vocs that inhibit mycelial growth of phytopathogenic fungi (lee et al. ; park et al. ; ajith and lakhsmidevi ; amin et al. ) . black sigatoka disease (also known as leaf spot or black leaf streak disease) of banana (musa paradisiaca) is caused by mycosphaerella fijiensis (ascomycete fungus). this phytopathogen is inhibited by the volatile emissions of muscodor sutura, an endophytic isolate of prestonia trifidi. the volatiles are effective also against ceratocystis ulmi, the causal agent of dutch elm disease of american elm (ulmus americana). the volatile mixtures include thujopsene, chamigrene, isocaryophyllene, and butanoic acid, -methyl-that are potent inhibitors of the common anthracnose pathogen of cucumber, muskmelon, and watermelon (members of cucurbits), colletotrichum lagenarium. so this unique endophyte and its chemical mixtures are potent mycofumigants and ensure crop protections against destructive pathogens like c. ulmi and c. lagenarium, sclerotinia sclerotiorum (causing white mold, cottony rot, water soft rot, stem rot, drop, crown rot, and blossom blight diseases of the host), and also phytophthora palmivora (oomycete fungi), the causal agent of bud root of palms and areca nut predominantly occurring in regions of south india (kudalkar et al. ) . liarzi and his coworkers tested the biological control efficacy of the endophytic daldinia cf. concentrica, isolated from olive tree (olea europaea l.) of israel against phytopathogens, and the unique mixtures of volatile were effective against the phytopathogenic mycelial growths. the mixtures include a variety of organic compounds: -methyl- -butanol, -methyl- -butanol, -methyl- , -cyclohexadiene, -methyl- , -cyclohexadiene, -heptanone, isoamyl acetate, -heptyn- -ol, -octenal, octanal, β-elemene, α-guaiene, β-selinene, α-selinene, α-bulnesene, germacrene a, etc. the unique mixtures having broad-spectrum antifungal property could be used for fumigation for eliminating the pathogenic infections of aspergillus niger (mold-causing organism on fruits of economic importance). so the endophytic d. cf. concentrica opens up opportunities for fungal disease control in food and agricultural industries (liarzi et al. ) . nodulisporium sp. strain gs d ii (hypoxylon anthochroum) and hypoxylon anthochroum strain blaci are potent enough to be used as biopesticide against fusarium oxysporum, a common contaminant of solanum lycopersicum var. cerasiforme (cherry tomato) causing a great percentage of crop loss globally. six vocs of alcohols' mixture, phenylethyl alcohol, -methyl- -butanol, -methyl- -butanol, eucalyptol, ocimene, and terpinolene, were detected and applied together with synergistic effect and individually both in vitro and in vivo. inoculation of pathogen on the cherry tomato fruits yields significant reduction in fusarium contamination. both agar dilution techniques and gas test were done to assess the in vitro antifungal activity, and the endophytic volatile mixtures were effective in both the cases. volatiles kill the pathogens probably by interfering cell membrane permeability, hyphal morphology, and respiratory activity of the pathogenic fusarium oxysporum. so it is a great opportunity to use the unique mixture of volatile organic compounds of the endophytic isolate to reduce the crop loss caused by the pathogenic infection on the commercially valuable plant of cherry tomato worldwide. endophytic phoma sp. (didymellaceae) and phomopsis sp. (valsaceae) were isolated from larrea tridentata and odontoglossum sp. singh et al. ) . the volatiles detected are effective against phytopathogens verticillum sp., ceratocystis sp., cercospora sp., sclerotinia sp. sclerotinia sp., and botrytis sp. algae are diverse group of autotrophs and the leading producers of o in the ecosystem. they range from prokaryotic unicellular to eukaryotic complex multicellular forms involved in the marine and terrestrial food chain. antifungal activity of the seaweed (members of phaeophyceae and rhodophyceae) is a major weapon for natural fungicides along with their antibacterial, anti-protozoan, and antiviral activities. algal seaweeds are potent holders of large number of secondary metabolites including phenolics, terpenes, alkaloids, and lectins which are not directly involved in photosynthesis and reproduction and thus fall under the category of secondary metabolites. they are common antimicrobial of algal origin that act on the target organisms by altering the microbial cell permeability accompanied with the loss of internal macromolecules or sometimes interfere with the membrane function causing cellular disintegrity ultimately leading to cell death (abu-ghannam and rajauria ). several studies include antifungal activity of algal members against human pathogens; a very few studies include their efficacy against plant pathogens (cheung et al. ; singh et al. ; stirk et al. ; padmakumar and ayyakkannu ; ismail et al. ; genovese et al. ; lopes et al. ). padmakumar and ayyakkannu tested species of algae against a variety of bacterial and fungal pathogens. out of the all screened organisms, % exhibited antibacterial efficiency, and only . % inhibited fungal growth. polysaccharides found in the cell wall and deposited in terms of storage food from red and brown algal sources include ulvans (obtained from ulva sp.), alginates and fucans (from fucus sp.), laminarin (laminaria sp.), and carrageenans that can induce defense responses in plants against phytopathogens by pathogen-associated molecular patterns (maps) and are capable of inducing plant resistance (vera et al. ) . polysaccharides stimulate regular cellular changes associated with pathogen perception and defense activation by change in ca + concentration and burst due to oxidative stress activation of salicylate, ethylene, and jasmonate biosynthetic pathways and by activating pathogenesis-related proteins (prps) (jaulneau et al. ; zhao et al. ) . as a result of the depolymerization of the polysaccharides, the obtained oligosaccharides induce protection against a variety of fungal, viral, and bacterial diseases by accumulation of the antimicrobial compounds in the cell. algal polysaccharides as an alternative weapon over the synthetic agricultural drugs for controlling plant disease have been widely studied (stadnik and freitas ; hahn et al. ). brown algae laminaria digitata, a genus of phaeophyceae, is commonly called seaweeds and known to be the potent producers of kelp, an iodine-rich substance needed for the normal functioning of thyroid gland. laminaria produces laminarin, glucan polysaccharide-containing , -linked β-d-glucose moiety, a reserve food material found on the vacuoles of the vegetative cells of this genus. β-glucans are involved as a major part of daily diet and obtained from the brands of common cereals. they are involved in the defense responses of agricultural crops like tomato (lycopersicon esculentum), eggplant (solanum melongena), pepper (piper nigrum), watermelon (citrullus lanatus), grape (vitis vinifera), apple (malus sp.), and pear (pyrus communis). elicitation of defense response by laminarin against causal agents of gray mold (botrytis cinerea) and downy mildew (plasmopara viticola) in grapevine plants remarkably suppresses their infection up to % and %, respectively (copping et al. ). so, natural product from brown algae laminaria sp. known as laminarin or laminaran can act as the biofungicide or biocontrol compounds. use of laminarin significantly reduces the mycelial growth and aflatoxin production in aspergillus flavus and ensures its use as a fungicide (liangbin et al. ) . the advantage of using laminarin over other products is that as it breaks down finally to glucose molecules, it has no maximum residue limit (mrl) on the plant treated with this product. so, there is no need of preharvest interval constraint. this has been a prime cause why laminarin has substituted five popular fungicides involved in the treatment of apple scab (venturia inaequalis) in france (mery et al. ). this phyto-pharmaceutical is used widely in france and some countries of europe in the name of vacciplant (major active constituent is laminarin). laminarin has broad-spectrum applicability on fire blight of apples and pears in greece, france, belgium, switzerland, portugal, and also morocco. it is effective for apple scab disease in france and belgium and for curing storage diseases of apples caused by gloeosporium sp. in belgium. laminarin comes out as a fungicide of natural origin after being eligible in tests between and in several parts of europe, for example, france, belgium, italy, and poland, on natural contamination of orchards on several sensitive strains of scab fungus including golden delicious, golden smoothie, read cheaf, galaxy, gala, and pink lady. laminarin is applied widely against secondary scab (to minimize secondary scab during summer and up to harvest) as a result of its uniqueness in its mode of action. it does not involve cell death of the host plant or hypersensitivity induction in the host organism but rather stimulates plants' natural resistance (klarzynski et al. ) . aziz et al. ( ) reported its effectiveness in tobacco plants, wheat, strawberries, apples, and vines. the application of laminarin and alginate reduced the development of wilt symptoms caused by verticillium dahliae on olive twigs, stimulating its phenolic metabolism (salah et al. ) . moreover, alginates reduced pathogen growth in vitro. laminarin induces the release of h o in cells of tobacco plants and leads to the increase in pal activity (phenylalanine ammonia-lyase) and causes the accumulation of pr- , pr- (glucanase), pr- (chitinase), and pr- . concerning red algae polysaccharides, carrageenans induced protection against a broad range of pathogens such as tobacco mosaic virus (tmv), b. cinerea, and e. carotovora on tobacco (vera et al. ) . again on tobacco, mercier et al. ( ) showed that carrageenan infiltrated the leaves and increased the expression of genes coding for a sesquiterpene cyclase involved in the synthesis of the antimicrobial terpenoid capsidiol, pr- proteins (basic chitinases), and proteinase inhibitor with antipathogenic activity. an adequate percentage of growth and spore germination inhibition of botrytis cinerea was mediated by the hexane extracts of laminaria digitata and undaria pinnatifida. porphyra umbilicalis, laverbread, is an edible seaweed (corato et al. ) . other than b-glucan polysaccharides (laminarin) of laminaria, other secondary metabolites (phenols, terpenes) of phaeophycean algae (sargassum sp.) showed effectivity against common pathogens fusarium solani, rhizoctonia solani, aspergillus spp., fusarium oxysporum, penicillium spp., and botrytis cinerea (khallil et al. ; ibraheem et al. ; mabrouk et al. ; liu et al. ). cyanophycean blue-green algae are abundant all over the world and ranging from pond ecosystem to oceanic system. though they have been reported to produce a large number of toxins and involved in death and disease of cattle and human being, they are of serious interest from the point of view of natural fungicidal products. drawing the similarities with bacteria, they are characterized with a mucilaginous or gelatinous sheath composed of polysaccharides which are the weapon against fungal pathogenesis. cyanobacterial polysaccharides (pol) show higher disease resistance against b. cinerea when they are applied on the intact fruit (preharvest conditions when fruit is attached to the plant) rather than the fruit detached (postharvest conditions) from the plant (zheng et al. ; feliziani et al. ; yao and tian ) . polysaccharides are involved in elicitation as elicitors for development of local and systemic disease resistance and expression of defense enzyme synthesis, for example, chitinases and glucanases that are involved directly in antifungal responses (paulert et al. ; reymond and farmer ; sharma et al. ) . water extracts of common bga anabaena sp., ecklonia sp. (common edible marine algae of japan and korea), and corallina sp. (hard seaweed of corallinaceae family) exhibit antifungal activity against podosphaera xanthii (causal agent of powdery mildew of cucurbits) on zucchini plant, cucurbita pepo, of cucurbitaceae (roberti et al. (roberti et al. , . in the recent past, fungi inhibitory ability of algal members has been reported by several workers (righini et al. ; corato et al. ; khallil et al. ; ibraheem et al. ) . in vitro growth inhibition of aspergillus oryzae and penicillium notatum has been seen by cyanophycean anabaena laxa (frankmölle et al. ) . devastating plant pathogens pythium sp., fusarium sp., and rhizoctonia sp. were restricted by extracts of anabaena sp. (moon et al. ; manjunath et al. ) . the use of bga extract as the growth inhibitor of pathogenic chaetomium globosum, cunninghamella blakesleeana, aspergillus oryzae, rhizoctonia solani, fusarium sp., pythium sp., and sclerotinia sclerotiorum is reported. the extracts of phormidium fragile and nostoc muscorum (rizk bryophytes, the simplest member of the broad umbrella of embryophyta, are situated between algae and pteridophytes, are known to be plant amphibians growing in the marshy or shady habitat, and require water for their fertilization and for the perfect swimming motility of their sperms. they have been evaluated for their antimicrobial activity for a long time. it has been proved that these cryptograms are rich source of bioactive secondary metabolites and can easily be exploited as an alternative source of fungicidal compounds. as they grow in marshy habitats and can protect themselves from biotic (ultraviolet rays, heat stress, and predation) and abiotic stress (fungal or bacterial attack), they are store house of diverse bioactive chemicals (xie and lou ) . members of hepaticopsida and mosses (the evolved members of bryophytes) are known to possess antifungal activity and are rich source of flavonoids, terpenoids, bibenzyls, and fatty acids of therapeutic importance (krzaczkowski et al. ) . bryophytes are known to possess antibiotic property (banerjee and sen ; banerjee ; singh et al. ; shirzadian et al. ; savaroglu et al. ) . their antibiosis has been evaluated against a large number of plant and human pathogenic fungus (mekuria et al. ) . antimicrobial compounds from bryophyte can cure the problems of conventional antibiotic resistance (vanden bossche et al. ) . the antifungal efficacy is tested by disc diffusion assay and microdilution method ( fig. . ). different concentrations of the extracts are prepared and checked for their antifungal efficacy against phytopathogenic fungi. they may be fungicidal or fungistatic in nature, interfering at cellular, genetic level and creating blockage at metabolic pathways. extracts are made on several organic solvents or water extractions and also mixture of one or two organic solvents. the solvents popularly used are ethanol, methanol, chloroform, ether, dimethyl sulfoxide (dmso), acetone, chloroform, and hexane (table . ). sporophytes and gametophytes of different bryophytes at different stages of growth and at a different amount are first surface sterilized and then crushed on the organic solvents and used as antifungals in vitro against the fungal pathogens (wolters (sabovljevic et al. ; pejin et al. ; veljic et al. ; gahotri and chaturvedi ; alam et al. ; deora and jain ; dey and de ; deora and suhalka ) . actinobacteria are a group of gram-positive filamentous bacteria that are called as the branched bacteria or ray fungi (from greek actis, ray beam, and mykes, fungus) and are characterized with the high g + c content occurring in mostly aerobic conditions but occasionally being anaerobes (ludwig and klenk ; olanrewaju and babalola ). their morphology varies from forming branching filaments or mycelial growth to external spores. they are ubiquitous in nature ranging their distribution from soil and human microbiota to plant and even animal kingdom. they are predominant in aquatic as well as terrestrial ecosystem playing a major part in mineralization and recycling of organic matters leading to soil formation (sharma et al. ). they are not only free-living members of the ecosystem but also a plant symbiont or endophyte, contributing to the plants' survival in extreme conditions and pursuing several bioactivities in vivo and in vitro. actinomycetes produce a diverse range of secondary metabolites, for example, antibiotics, antitumor, insectrepellent, and immunosuppressive agents, and plant growth-promoting regulators (pgprs) that are of immense pharmaceutical and agricultural importance. they are the prime producers of diverse antibiotics after the landmark discovery of penicillin in the year . the single genus of streptomyces sp. itself produces % of the total known bioactive ( , are produced by actinobacteria out of , produced by microorganisms, almost %) compounds from actinobacterial and riccia gangetica curvularia lunata deora and suhalka ( ) , guhil ( , ) bacterial source (berdy ) and is known to be the prime organism in the pharmaceutical world. they are equally profitable when isolated from plant source and designated as endophytic actinomycetes. so exploitation of the actinobacterial novel bioactive compounds both from endophytic and non-endophytic source is the ultimate way to fight against human and plant diseases. here we focus only on actinobacterial compounds' antifungal activity and role in plant protection from deadly diseases caused by severe phytopathogens leading to irreparable crop loss and economic breakdown of agricultural sectors. actinomycetes from soil source are selected based on the enrichment culture technique and are plated on selective media for isolation. antifungal agents, for example, nystatin and cycloheximide, are supplemented for the inhibition of fungal contamination. for isolation of endophytic actinobacteria from plant source, plants are first selected and surface sterilized for the elimination of the epiphytic contaminants and finally plated on the selective growth media like starch casein nitrate agar (scna), chitin-vitamin b, tap water yeast extract agar (twya), soybean, humic acid-vitamin b (hv), yeast extract casamino acid (yeca), modified gausse, and glycine-glycerol (ivantiskaya et al. ; küster ; küster and williams ; williams and davies ; hayakawa and nonomura ; crawford et al. ) . international streptomyces project (isp) medium is also popular media used for isolation, and they are supplemented with amino acids (l-asparagine for isp , tryptone for isp ), inorganic trace salts, starch or carbohydrate sources (malt extract for isp ), and agar as solidifying agent. ph set at near to optimum or slightly basic is mandatory for proper isolation techniques using isp medium. the actinomycete isolates are grown in solid or liquid medium for their antifungal bioactivity detection. antagonistic activities of the potent isolates are tested by growing them on both sides of the fungal hyphae, and isolate having anti-phytopathogenic activity will inhibit the growth of the pathogens. actinobacterial aqueous-or solvent-based extracts will be evaluated for either fungistatic or fungicidal activity by agar welldiffusion techniques. soluble bioactive compounds of antifungal importance will be extracted using wide range of organic solvents followed by purification by column and thin-layer chromatographic techniques. hplc analysis will be the most useful method for the detection of the purity of the compound, and further nmr studies are needed for the proper identification of the bioactive compound. cell line studies are made with the coupling of bioinformatics tools for the proper knowledge about their mode of action. actinobacteria can be a part of plant as endophyte, rhizospheric soil as symbiont for plant growth-promoting substance producer, and organisms' normal microbial flora as gut microorganism. so they are ubiquitous in their distribution. out of several biologically potent compound produced from the actinobacterial source, antibiotics are the major contribution of these microorganisms toward human civilization. all the known antibiotics (blasticidin, mildiomycin, natamycin, validamycin, kasugamycin) are of actinobacterial (most of them are the streptomyces sp.) source showing protective activity for the plants against agricultural fungal pathogens (tables . and . ). as human are dependent completely on nature and more particularly natural components of agricultural origin and importance, dependence on agricultural crops is of a known fact. but the problem arises when the crops are affected most by the fungal pathogens leading to huge crop loss, and thus the search for novel antibiotics is on, and the search has shifted to actinobacterial source, and endophyte plays an important role in this respect. there are significant reports of antifungal compounds from bacterial origin, but now the focus has shifted to microbes of endophytic origin (table . ). till date, a huge number of antibiotics are already reported and have minimized the crop loss to a notable amount ( fig. . ). antibiotics and other antifungal compounds include munumbicins a, b, c, d, e- , and e- , vanillin, saadamycin, , -dimethoxy- -p-methoxyphenyl coumarin, coronamycin, and fistupyrone isolated from different strains of streptomyces (shan et al. ; costa et al. ; igarashi et al. ; tian et al. ; zin et al. ) and are protecting a large number of cereals and other important cash crops from being affected by these common contaminants. endophytic actinobacteria directly counteract with fungal plant pathogens not only by producing bioactive compounds but also by enhancing the plant's growth through the production of plant growth promoters and making the plant less susceptible to pathogenic invasion. they are efficient agent of reducing the symptoms that arise due to exposure to environmental stress (shimizu ) . enhanced production of indole acetic acid (iaa) was mediated by streptomyces sp. (isolated from centella asiatica) and nocardiopsis sp. (dochhil et al. ; shutsrirung et al. ; gangwar et al. ) . experimental trials on cucumber indicate positive result as the isolates actinoplanes campanulatus, micromonospora chalcea, and streptomyces spiralis enhanced plant growth and improved yield conditions (el-tarabily et al. ) . other than auxin, auxin-like similarly functioning molecules named as pteridic acids a and b are found to be inducers of adventitious root proliferation in kidney bean plants at very minute concentrations of mm (igarashi et al. ) . chitin is a major fungal cell wall polysaccharide (the second most abundant polysaccharide in nature after cellulose) component and is the first line of defense of fungal cells. actinobacteria antagonize the fungal cell by producing chitinases (an enzyme capable of hydrolyzing fungal cell wall) and break the glycosidic bonds in chitin and lead to the death of the pathogenic cell. endophytic kitasatosporia sp. (isolate of catharanthus roseus) and kibdelosporangium sp. (isolate of achillea fragrantissima) are reported to be chitinase producers (el-shatoury et al. ; mini priya ) . actinoplanes missouriensis isolated from lupinus sp., a member of fabaceae family, produces chitinase causing hyphal cell lysis and reducing the conidial germination rate and protects the plant from pathogenic attack of plectosporium tabacinum, the causal agent of lupin root rot in egypt (el-tarabily ; el-tarabily and sivasithamparam ) . siderophores are soluble, small, high-affinity iron carriers produced by bacterial or fungal members and are involved in the transportation of iron (fe + ) across the cell membrane. they have caught sudden attention due to their involvement in plant growth promotion as well as antagonistic ability against phytopathogens (cao et al. ; tan et al. ; rungin et al. ) . endophytic actinobacteria from aloe vera, mentha arvensis, and ocimum sanctum are known to be producers of hydroxymate type and catechol type of siderophores, and the isolate saccharopolyspora o is known to be the potent inhibitor howell and stipanovic ( ) , homma et al. ( ) , thomashow et al. ( ) and smith et al. ( ) harpin proteins (erwinia amylovora), trade name: harpin αβ (proact) induction of systemic acquired resistance (sar) and less susceptibility to fungal and bacterial disease wei et al. ( ) strobilurin and oudemansin (members of basidiomycete grows on dead wood) commercial synthetic analogues: azoxystrobin and kresoxim-methyl of alternaria brassicicola, botrytis cinerea, and fusarium oxysporum (gangwar et al. ; el-shatoury et al. ). endophytic isolates of cucumis sativus (cucumber), identified as actinoplanes campanulatus, micromonospora chalcae, and streptomyces spiralis, are reported to control the growth and development of damping off, crown rot, and root rot pathogen pythium aphanidermatum. they are known to promote plant growth and to protect seedlings and mature plants. a novel bioactive compound identified as -prenylindole was isolated from endophytic streptomyces sp. showing strong antifungal activity against a broad range of phytopathogens: alternaria brassicicola and fusarium oxysporum (igarashi ) . another new prenylated indole derivative from endophytic actinobacterial source inhibited the growth of colletotrichum orbiculare, phytophthora capsici, corynespora cassiicola, and fusarium oxysporum (zhang et al. ) . naphthomycins a and k isolated from streptomyces sp. cs have antifungal activity against penicillium avellaneum shen , ) . biocontrol ability of fistupyrone has made it a useful tool to minimize the crop loss of brassica due to black leaf spot disease caused by alternaria brassicicola (igarashi ) . interest on actinomycetes of endophytic origin as an alternative tool for antifungal agent is increasing day by day (table . ). since the beginning of human civilization, whenever human race has faced any turbulence in its path of existence, they have rushed to their green friends, trees, for the ultimate solution. search for bioactive products of medical importance has been a thirst area from time immemorial. whether it is a concern of human or plant health, trees have given answers in all aspects. in the recent past, phytopathogenic infection has pushed the agricultural productive parameters to a real challenge, and plant extracts in its crude and purified form are applied as biocontrol methods (table . ). the existing synthetic chemicals are facing problem of immediate or delayed drug resistance and also issues of nephrotoxicity (the gold standard; amphotericin b), biomagnification, or quality assurance of the food products and thus are inconsistent in their business (goa and barradell ; cuenca-estrella et al. ) . so green plant extracts are the novel, safest, and the best effective treatment tool in this arena. plants are mysterious in their chemical nature and in respect to their secondary metabolite production. the faith is consistent on green plants due to the fact that plants protect themselves from fungal or bacterial diseases specially for the taxa that occur in marshy shady or water-logged or stress conditions (gurgel et al. ) . so the search is primarily made on the wild native taxa or invasive species that have higher potential of antimicrobial production. the knowledge of ethnobotany comes in this context, and tribal people are imitated for the gathering of crude knowledge. the problem of fungal pathogenesis is mainly faced by plants of economic importance, that is, cash crops. a single event of pathogenic attack can affect seriously the demand and supply ratio; thus the sustainability is lost, and restoring the good health of crops is a basic need of agricultural sectors but in an efficient way not hampering the soil health, ecosystem characters, and human health and also should be budget friendly. the search is strictly focused on plants of ethnomedicinal importance as history indicates the ability of medicinal plant extracts in human and animal mycoses and antifungal ability (mathias-mundy and mccorkle secondary metabolites are plants' best weapon against phytopathogenic invasion. several plant extracts have been assessed for their antifungal activity against a variety of phytopathogens of serious agricultural threats (table . ). the metabolites are divided into terpenoids, saponins, phenolic compounds, flavones, flavonoids, flavonols, alkaloids, and coumarins (table . ). plant extracts are primarily tested for antifungal efficacy and further are purified by solvent extraction and chromatographic procedures leading to discovery of new antifungal agents. terpenoids, also called as isoprenoids (under the chemical subclass of prenyllipids), are known to be the oldest group of widespread molecular compounds produced by plants. scher et al. ( ) reported a variety of six sesquiterpenes of antifungal importance against the causal organisms of bunch rot (botrytis cinerea) on grapes, scab of cucurbits (cladosporium cucumerinum), potato blight (phytophthora infestans), rice blast (pyricularia oryzae), and blotch of wheat (septoria tritici). sesquiterpene isolated from polygonum punctatum (dotted knotweed of knotweed family polygonaceae) named after the chemical polygodial is an effective control agent of zygosaccharomyces bailii (a common food spoilage yeast). scab of cucurbits is a common and devastating fungal pathogenic disease in agricultural fields, and this disease is to some extent prevented by the use of clerodane diterpenes extracted from detarium microcarpum, a plant of leguminosae family (cavin et al. ) . skaltsa ( ) reported fungi inhibitory (cunninghamella echinulata) activity of costunolide and eudesmane derivatives isolated from centaurea plants. other than terpenes, saponins (triterpene ad steroidal saponins) are also effective antifungals reported from plant sources. tea is one of the most vital cash crops in terms of foreign money earning and the most popular beverage having antioxidative properties. pathogenic infection by pestalotia longiseta causes a huge loss of tea production. nagata et al. in the year isolated triterpenoid saponins camelids i and ii from the leaves of camellia japonica (japanese camellia) that inhibited the tea pathogen p. longiseta. cucurbitacins i, a, b, q, and e isolated from cucurbitacins (ecballium elaterium) have antifungal activity against botrytis cinerea (har-nun and meyer ) . phenolics are odorous compounds having antifungal compounds and are also responsible for the plant pigment production. phenolics cover a large number of chemical compounds, for example, alkylated phenols, anthraquinones, coumarins, phenolic acid, phenols, phenylpropanoids, quinines, xanthones, hydroxycinnamic acid, p-coumaric acid, ferulic acid, and chlorogenic acid. phenol derivatives like crassinervic acid (p. crassinervium), aduncumene (p. aduncum), hostmaniane (p. hostamannianum), and gaudichaudanic acid (p. gaudichaudianum) are effective against strawberry blossom blight pathogen cladosporium cladosporioides (lago et al. ). -acetyl- -acetoxyacetophenone showed antifungal activity against spendley et al. ( ) , potterat et al. ( ) , martson et al. ( ) , marston et al. ( ) , viturro et al. ( ) , cavin et al. ( a) , and dhatwalia et al. ( ) pinocembrin from leaves of populus deltoides (salicaceae) shain and miller ( ) , hoof et al. ( ) cladosporium fruit and leaf rot and bitter root (cladosporium gloeosporioides) long-chain alcohol from peels of young fruit of persea americana from lauraceae, methylripariochromene a from roots of eupatorium riparium (asteraceae) prusky et al. ( ) , ratnayake bandara et al. ( ) pine needle pathogen (dothistroma pini) stearic acid from needles of pinus radiata (pinaceae) franich et al. ( ) pathogen of corn, sorghum, apple (helminthosporium carbonum) luteone and wighteone from leaf surface of lupinus albus (leguminosae) ingham et al. ( ) black and brown spot of banana (colletotrichum musae) dopamine from unripe banana fruit (musa sp.) muirhead and deverall ( ) powdery mildew of grains (erysiphe graminis) gramine from leaves of hordeum vulgare (poaceae) wippich and wink ( ) leaf spot, rots, and blights (alternaria alternata), disease of cereal (penicillium verrucosum) alizarin and emodin from root of rubia tinctorum of rubiaceae, alkylated phenols of peel and pulp of mangifera indica (anacardiaceae) cojocaru et al. ( ) , manojlovic et al. ( ) maize rot (fusarium moniliforme), epidemic outbreak of glume and kernel discoloration (curvularia lunata) flavan- -ols of root bark of sorghum cultivars of poaceae jambunathan et al. ( ) (continued) kobayashi et al. ( ) , endo et al. ( ) , cho et al. ( ) blue mold of tobacco (peronospora tabacina) diterpenoids from nicotiana tabacum of solanaceae reuveni et al. ( ) leaf and fruit pathogen (cladosporium cladosporioides) canaliculatol from bark of stemonoporus canaliculatus and long-chain alcohol from persea americana, phenylethanone from euodia lunuankenda, sinharine and methylsinharine from glycosmis cyanocarpa, illukumbin from glycosmis mauritiana (rutaceae), phenylethanone from euodia lunuankenda (lauraceae), benzoquinone from croton lacciferus (euphorbiaceae) bokel et al. ( ) , ratnayake bandara and wimalasiri ( ) , kumar et al. ( ) , greger et al. ( ) , pacher et al. ( ) , springob and kutchan ( ) miles et al. ( ) , miles et al. ( ) , lee et al. ( ) , deng and nicholson ( ) and yoganandam et al. ( ) (continued) sclerotinia sp. phenolic structures when contain a carbonyl group are known to be flavones, and the addition of an extra -hydroxyl group indicates flavonol. flavonoids are also known to hydroxylated phenolics but occurring as a c -c unit linked to aromatic ring. not only plant samples directly but also plant derivatives like porpolis (galangin isolated from the bee glue or resinous mixture produced as a result of the mixture of tree buds, sap, botanical extracts, and bee exudates) are shown to be antifungal against green rot or mold of tangerine (pathogens penicillium digitatum, p. italicum) and also control postharvest disease of cereal grains, legumes, and tree nuts caused by a. flavus (afolayan and meyer ) . flavones ( , , ′-trihydroxy- , ′dimethoxyflavone, , ′-dihydroxy- , ′, ′-trimethoxyflavone) from artemisia giraldi are effective against a. flavus infections (cowan ) . leaf wax of arrabidaea brachypoda (brazilian medicinal plant from bignoniaceae) contains herger et al. ( ) and abdu-allah and elyousr ( ) cassia tora (dealcoholized extract of leaves) mukherjee et al. ( ) thymol, carvacrol, citronellol, geraniol, citral, perillyl, menthol, eugenol, , - cirsiliol, cirsimaritin, and hispidulin and is showed to be effective against cladosporium sphaerospermum (alcerito et al. ) . galeotti et al. ( ) against fusarium oxysporum f. sp. dianthi (galeotti et al. ) . fusarium culmorum, a serous pathogen of seedling blight, foot rot, ear blight, stalk rot, and common rot of cereals and grasses, is found to be inhibited by six commercial coumarins: bergapten, herniarin, umbelliferone, xanthotoxin, and scopoletin. tithonia diversifolia, the source of tithoniamarin, is effective against the anther smut fungus microbotryum violaceum, earlier known as ustilago violacea (yemele-bouberte et al. ) . berberine and jatrorrhizine (alkaloids) are isolated from mahonia aquifolium (a plant of berberidaceae family commonly called as oregon grape and native to western north america) and are effective against human pathogenic candida species. pathogens of mango (c. gloeosporioides), anthracnose of lupin species, postbloom fruit drop of citrus, valencia and navel oranges in florida (caused by c. acutatum), and strawberry (caused by colletotrichum fragariae) are inhibited by findersine, anhydroevoxine, and haplamine (cantrell et al. ) . roots of cyathobasis fruticulosa are source of beta-carboline, tryptamine, and phenylethylamine-derived alkaloids and are antifungal in nature (bahceevli et al. ). essential oils (eos) of aromatic and medicinal plant origin are reported to possess antifungal properties and are of wide spectrum in their application for the control of agricultural pathogen (table . ). eos are mainly categorized under the plants' secondary metabolites and may fall under the category of terpenes, ketones, esters, aromatic phenols, ethers, alcohols, oxides, etc. (fig. . ) . they act by inhibiting the fungal hyphal growth either by accumulating in the fungal cell membrane or by crossing the cell membrane and entering into the eukaryotic cell. being lipophilic in their chemical nature, they can easily cross the cell and interrupt in sterol biosynthesis leading to growth retardation and finally cell death. as sterols are the maintenance, compounds of cellular integrity treatment with eo cause fungal cell death. metabolic processes like respiration, replication, transcription, and translation are inhibited. membrane permeability is drastically changed as they cause swelling and disruption of protein-lipid-protein membrane. leakage of useful ions like ca + and k + causes cell death. thymol, carvacrol, eugenol, and related phenolic compounds cause h + and k + leakage and water imbalance and deplete intracellular high-energy molecule (atp). essential oils are extracted from almost every parts of a plant, for example, roots, fruits, barks, twigs, leaves, seeds, and flowers, by several extraction procedures that include hydro and steam distillation, cold pressing, and zataria multiflora lamiaceae fermentation. the antifungal efficacy is checked by direct contact of the essential oil components and fungal hypha and poison food method, following micro or broth dilution techniques, or in vivo fumigation assay is also performed in case of field trials. essential oils from leaves of chenopodium ambrosioides, a member of amaranthaceae family, are effective against storage fungi aspergillus flavus, a. glaucus, a. niger, a. oryzae, colletotrichum gloeosporioides, c. musae, fusarium oxysporum, and fusarium semitectum (jardim et al. ) . lemongrass oil from cymbopogon citratus and cymbopogon martini are potent inhibitors of botrytis cinerea, rhizoctonia solani, aspergillus tamari, a. fumigatus, and a. conicus (tzortzakis and economakis ; mishra et al. ) . the members of lamiaceae family are well known for their pungent odor and are tested for their antifungal activity by agar and broth dilution methods (roby et al. ; omidbeygi et al. ). essential oils extracted from laurus nobilis, syzygium aromaticum, and origanum vulgare are effective antifungal compounds against two pathogens of rice, fusarium culmorum and fusarium verticillioides (rosello et al. ) . essential oils from cymbopogon exhibited antifungal activities against rot molds (soundharrajan et al. ) . antifungal activities of peppermint and sweet basil were tested against plant pathogenic fungi s. sclerotiorum, rhizopus stolonifer, and mucor sp. (edris and farrag ) . antifungal activity of β-dolabrin, γ-thujaplicin, and -acetyltropolone was tested against pythium aphanidermatum ifo (morita et al. ) . boyraz and ozcan ( ) tested the antifungal activity of the essential oils isolated from wild turkish summer savory (satureja hortensis). essential oils (carvacrol, thymol, p-cymene) extracted from origanum acutidens are effective against phytopathogens. growth of a. humicola, colletotrichum gloeosporioides, rhizoctonia solani, and phytophthora cactorum was inhibited by the essential oil of asarum heterotropoides var. mandshuricum (dan et al. ) . though there are several reports of essential oils being potent anti-phytopathogenic (penicillium purpurogenum, rhizopus stolonifer, spondylocladium austral, penicillium digitatum, penicillium luteum, monilinia laxa, curvularia lunata, etc.) in nature, still there are some problems regarding their maximum use and optimum effectivity. that includes their volatile natures, requirement of close systems, and degradation of eos by oxidation due to presence of extreme amount of hydrogenated compounds (kim et al. ). we are nourished by mother nature. so it is our prime duty to keep up the normal equilibrium of natural parameters. but in a way to seek solutions, some steps taken toward success may have negative impact on our environment. to fight against the fungal pathogens for the ensuring of better crop productivity, use of chemical fungicide is just another example of that fact. but we must emphasize on products from direct natural origin over the chemically synthesized one. natural products are the best weapon to fight fungal pathogenic diseases on economically important crop species. they are less toxic, stable, and of no side effects when used in crop fields. the crying need of modern era is obtaining pathogen-free crop species in one hand and assurance of environmental sustainability on the other. fungal and bacterial products are already used in large scales followed by the plants' secondary metabolites. phytoalexins as internal molecules are the plants' own defense system. the detailed biochemical analysis of the phytoalexins and study of their regulatory mechanisms are opening up new horizons for universal use of phytoalexin inducing elicitors as plant defense enhancers. mycorrhizae provide the basic line of physical barrier against pathogenic invasion, and reports include their ability to enhance plant growth, thus making the plant nonsusceptible to fungal attack. endophyte on the other hand can enhance the plants' defense system by direct incorporation and open up popular angles of green immunization or plant vaccination. researches on these fields are still scanty, but in the near future, they could lead to the ultimate solution of fungal pathogenic crop loss. effect of certain plant extracts and fungicides against powdery mildew disease of grapevines in upper egypt antimicrobial activity of compounds isolated from algae flavonoid and other constituents of bauhinia manca effects of resveratrol on the ultrastructure of botrytis cinerea conidia and biological significance in plant/pathogen interactions biological activity of resveratrol, a stilbenic compound from grapevines, against botrytis cinerea, the causal agent for gray mold the antimicrobial activity of , , -trihydroxyflavone isolated from the shoots of helichrysum aureonitens extraction and identification of bioactive compounds (eicosane and dibutyl phthalate) produced by streptomyces strain kx for the biological control of rhizoctonia solani ag- strain kx to control target spot disease in tobacco leaf - phytoalexins in defense against pathogens effect of volatile and non-volatile compounds from trichoderma spp. against colletotrichum capsici incitant of anthracnose on bell peppers in vitro antifungal efficacies of aqueous extract of dumortiera hirsuta (swaegr.) nees against sporulation and growth of postharvest phytopathogenic fungi evaluation of streptomyces griseorubens e g for the biocontrol of fusarium oxysporum f. sp. lycopersici: ultrastructural and cytochemical investigations interactions between a root-knot nematode (meloidogyne exigua) and arbuscular mycorrhizae in coffee plant development (coffea arabica) foliar epicuticular wax of arrabidaea brachypoda: flavonoids and antifungal activity metabolites of helminthosporium monoceras: structures of monocerin and related benzopyrans trans-trans- , -tridecadiene , , -triyne- , -diol, an antifungal polyacetylene from diseased safflower (carthamus tinctorius) mycofumigation by the volatile organic compound-producing fungus muscodor albus induces bacterial cell death through dna damage mycorrhizal fungi and trichoderma harzianum as biocontrol agents for suppression of rhizoctonia solani damping off disease of tomato effect of volatile metabolites of trichoderma species against seven fungal plant pathogens in vitro production of gliotoxin on natural substrates by trichoderma virens studies on antagonistic effect against plant pathogenic fungi from endophytic fungi isolated from houttuynia cordata thunb. and screening for siderophore and indole- -acetic acid production effect of mushroom extracts in the induction of phytoalexins and in the control of soy oidium in a greenhouse impact of mycorrhizal colonisation on root architecture, root longevity and the formation of growth regulators isolation and characterization of muscodor albus i- . s, a volatile antibiotic producing fungus influence of seed priming on the development of pearl millet downy mildew (sclerospora graminicola) synthesis and incorporation of the first polyketide synthase free intermediate in monocerin biosynthesis laminarin elicits defense responses in grapevine and induces protection against botrytis cinerea and plasmopara viticola streptomyces sanglieri which colonised and enhanced the growth of elaeis guineensis jacq. seedlings was antagonistic to ganoderma boninense in in vitro studies current status of biological control of plant diseases using antagonistic organisms in india status and prospects for enhancing the uptake of antagonistic organisms for nematode management in india alkaloids and aromatics of cyathobasis fruticulosa (bunge) aellen antimicrobial activities of bryophytes a review antibiotic activity of bryophytes an endophytic myrothecium inundatum producing volatile organic compounds muscodor albus strain gba, an endophytic fungus of ginkgo biloba from united states of america, produces volatile antimicrobials increasing the productivity and product quality of vegetable crops using arbuscular mycorrhizal fungi: a review seaweed polysaccharides as bio-elicitors of natural defenses in olive trees against verticillium wilt of olive thoughts and facts about antibiotics: where we are now and where we are heading in vitro screening of bryophytes for antimicrobial activity effect of phosphate and the arbuscular mycorrhizal fungus glomus intraradices on disease severity of root rot of peas (pisum sativum) caused by aphanomyces euteiches canaliculatol, an antifungal resveratrol trimer from stemonoporous canaliculatus induction and identification of sativan and vestitol as two phytoalexins from lotus corniculatus the cytosporones, new octaketide antibiotics isolated from an endophytic fungus phytoalexins induction in rubiaceae the camalexins: new phytoalexins produced in the leaves of camelina sativa (cruciferae) anti-fungal effects of cocoa tannin on the witches' broom pathogen crinipellis pernicious applied and environmental microbiology the chemical composition, antifungal, antioxidant and antimutagenicity properties of bioactive compounds from fungal endophytes associated with thai orchids isolation and identification of antifungal and antialgal alkaloids from haplophyllum sieversii isolation and characterization of endophytic streptomyces antagonists of fusarium wilt pathogen from surface sterilized banana roots isolation and characterization of two phytoalexins from rice as momilactones a and b munumbicins, wide-spectrum antibiotics produced by streptomyces nrrl , endophytic on kennedia nigriscans munumbicins e- and e- : novel broad-spectrum antibiotics from streptomyces nrrl aspectos bioquímicos e moleculares da resistência induzida bioactive diterpenes from the fruits of detarium microcarpum in vitro evaluation of fungicides, plant extracts and biocontrol agents against brown leaf spot of paddy bioactive diterpenes from the fruits of detarium microcarpum crop diseases and their management. phi learning private limited antifungal activity and action mechanism of ginger oleoresin against pestalotiopsis microspora isolated from chinese olive fruits studies on a chlorogenic acid-producing endophytic fungi isolated from eucommia ulmoides oliver antifungal activity of cinnamaldehyde and eugenol congeners against wood-rot fungi antifungal and antiviral products of marine organisms cytotoxic and antifungal triterpene glycosides from the patagonian sea cucumber hemoiedema spectabilis antimicrobial activity of -hydroxybenzoic acid and trans -hydroxycinnamic acid isolated and identified from rice hull diversity and antifungal activity of fungal endophytes of asparagus racemosus willd insecticidal secondary metabolic products from the entomogenous fungus fusarium larvarum -heptadecenyl)-resorcinol, the major component of the antifungal activity in the peel of mango fruit antifungal activity of neo-clerodane diterpenoids from scutellaria the manual of biocontrol agents effect of water activity on the production of volatile organic compounds by muscodor albus and their effect on three pathogens in stored potato biological and molecular comparison between localized and systemic acquired resistance induced in tobacco by phytophthora megasperma glycoprotein elicitin biological control of phytopathogenic fungi by endophytic actinomycetes isolated from maize (zea mays l) plant products as antimicrobial agents identification of three hydroxyflavan phytoalexins from daffodil bulbs phytoalexins from other plant families isolation and characterization of actinomycete antagonists of a fungal root pathogen susceptibility of fluconazole-resistant clinical isolates of candida spp. to echinocandin ly , itraconazole and amphotericin b biochemical defense mechanisms in cotton plants against ramularia leaf spot mediated by silicon in vitro antifungal activity of -( , -dimethyl- , -dihydro- h-pyrrol- -yl)- -methylethyl pentanoate, a dihydro -pyrrole derivative phytoalexin accumulation in tissues of brassica napus inoculated with leptosphaeria maculans naphthalene, an insect repellent, is produced by muscodor vitigenus, a novel endophytic fungus activities of essential oils from asarum heterotropoides var. mandshuricum against five phytopathogens antagonist actinomycetes metabolites against plant pathogens fungi of agricultural importance induction of phytoalexins and proteins related to pathogenesis in plants treated with extracts of cutaneous secretions of southern amazonian bufonidae amphibians natural products in crop protection bioassay-guided isolation of allelochemicals from avena sativa l.: allelopathic potential of flavone c-glycosides antifungal activity of crude extracts from brown and red seaweeds by a supercritical carbon dioxide technique against fruit postharvest fungal diseases rhizospheric streptomycetes as potential biocontrol agents of fusarium and armillaria pine rot and as pgpr for pinus taeda geldanamycin, a new antibiotic induction of fusarium solani mutants insensitive to tomatine, their pathogenicity and aggressiveness to tomato fruits and pea plants molecular engineering of resveratrol in plants antifungal properties of surangin b, a coumarin from mammea longifolia phytochemical analysis and antifungal activity of moss bryum cellulare against some phytopathological fungi studies on antifungal potential of bryum cellulare against spore germination of fungus curvularia lunata in vitro antifungal activity of plagiochasma appendiculatum against alternaria solani evaluation of bryophyte for green fungicides as alternative treatment to control plant pathogen oxidative ring contraction of the phytoalexin cyclobrassinin: a way to brassilexin brassilexin, a novel sulphur-containing phytoalexin from brassica juncea l., (cruciferae) secondary metabolites production by actinomycetes and their antifungal activity antifungal bryophytes: a possible role against human pathogens and in plant protection isolation, characterization and antimicrobial activity at diverse dilution of wheat puroindoline protein free fatty acid accumulation and quality loss of stored soybean seeds invaded by aspergillus ruber molecular communication in interactions between plants and microbial pathogens seed germination enhancing activity of endophytic streptomyces isolated from indigenous ethno-medicinal plant centella asiatica interactions between an arbuscular mycorrhizal fungus (scutellospora heterogama) and the root-knot nematode (meloidogyne incognita) on sweet passion fruit (passiflora alata) application of plant extracts as inducers to challenge leaf rust of wheat inhibitory effect and mechanism of tagetes erecta l. fungicide on fusarium oxysporum f evaluating novel microbe amended composts as biocontrol agents in tomato biological activity summary for cocoa (theobroma cacao l.) effect of salicylic acid and structurally related compounds in the accumulation of phytoalexins in cotyledons of common bean phenylphenalenone phytoalexins, will they be a new type of fungicide? antifungal activity of peppermint and sweet basil essential oils and their major aroma constituents on some plant pathogenic fungi from the vapor phase production and genetic improvement of a novel antimycotic agent, saadamycin, against dermatophytes and other clinical fungi from endophytic streptomyces sp. hedaya antimicrobial activities of actinomycetes inhabiting achillea fragrantissima (family: compositae) an endophytic chitinase-producing isolate of actinoplanes missouriensis, with potential for biological control of root rot of lupine caused by plectosporium tabacinum performance of three endophytic actinomycetes in relation to plant growth promotion and biological control of pythium aphanidermatum, a pathogen of cucumber under commercial field production conditions in the united arab emirates nonstreptomycete actinomycetes as biocontrol agents of soil-borne fungal plant pathogens and as plant growth promoters structures of antifungal diarylheptenones, gingerenones a, b, c and isogingerenone b, isolated from the rhizomes of zingiber officinale coronamycins, peptide antibiotics produced by a verticillate streptomyces sp. (msu- ) endophytic on monstera sp antifungal, anti-oomycete and phytotoxic effects of volatile organic compounds from the endophytic fungus xylaria sp. strain pb f isolated from haematoxylum brasiletto new endophytic isolates of muscodor albus, a volatileantibiotic-producing fungus effect of substrate on the bioactivity of volatile antimicrobials produced by muscodor albus antifungal volatile organic compounds from the endophyte nodulisporium sp. strain gs d ii a: a qualitative change in the intraspecific and interspecific interactions with pythium aphanidermatum preharvest treatments with chitosan and other alternatives to conventional fungicides to control postharvest decay of strawberry fungistatic effects of pinus radiata needle epicuticular fatty and resin acids on dothistroma pini blue-green alga anabaena laxa. i isolation and biological properties plant phenolics, lignification arbuscular mycorrhiza reduces susceptibility of tomato to alternaria solani antifungal metabolites from phomopsis sp. by , an endophytic fungus in gossypium hirsutum comparative efficacies in vitro of antibacterial, fungicidal, antioxidant, and herbicidal activities of momilactones a and b antifungal and antibacterial potential of methanol and chloroform extracts of marchantia polymorpha l flavonoids from carnation (dianthus caryophyllus) and their antifungal activity interactions between a fluorescent pseudomonad, an arbuscular mycorrhizal fungus and a hypo virulent isolate of rhizoctonia solani affect plant growth and root architecture of tomato plants diversity and biopotential of endophytic actinomycetes from three medicinal plants in india the diversity, plant growth promoting and antimicrobial activities of endophytic actinomycetes isolated from emblica officinalis gaertn agriculture and bioactives: achieving both crop yield and phytochemicals isolation and characterization of endophytic actinomycetes from mangrove plant for antimicrobial activity two phytoalexins from sugar beet (beta vulgaris) leaves lass-florl c ( ) the mediterranean red alga asparagopsis taxiformis has antifungal activity against aspergillus species introduction of some new endophytic bacteria from bacillus and streptomyces genera as successful biocontrol agents against sclerotium rolfsii fluconazole: an update of its pharmacodynamic and pharmacokinetic properties and therapeutic use in major superficial and systemic mycoses in immunocompromised patients plant-fungal interactions: the search for phytoalexins and other antifungal compounds from higher plants sulfur containing cinnamides with antifungal activity from glycosmis cyanocarpa phytoalexin emit indolstruktur aus kohlrabi (brassica oleracea var. gongylodes) cytokinins mediate resistance against pseudomonas syringae in tobacco through increased antimicrobial phytoalexin synthesis independent of salicylic acid signaling metabolic products of fusarium larvarum fuckel. the fusarentins and the absolute configuration of monocerin potential of horsetail (equisetum sp.) preparations in the synthesis of defense metabolites in soy (glycine max l.) cotyledons and the effect on the growth of rhizoctonia solani kuhn, in vitro comparative transcriptomics of rice reveals an ancient pattern of response to microbial colonization chemical composition, antifungal and antitumor properties of ether extracts of scapania verrucosa heeg. and its endophytic fungus chaetomium fusiformis in vitro antifungal activity of dragon's blood from croton urucurana against dermatophytes multiple control levels of root system remodelling in arbuscular mycorrhizal symbiosis antifungal effect of five aqueous plant extracts on mycelial growth of penicillium expansum isolated from rotted yam tubers in storage alterations in root exudation of intercropped tomato mediated by the arbuscular mycorrhizal fungus glomus mosseae and the soil borne pathogen fusarium oxysporum f.sp. lycopersici host-pathogen interactions: xix. the endogenous elicitor, a fragment of a plant cell wall polysaccharide that elicits phytoalexin accumulation in soybeans tit for tat? a mycorrhizal fungus accumulates phosphorus under low plant carbon availability the accumulation of inhibitory compounds in the induced resistance response of carrot root slices to botrytis cinerea cucurbitacins protect cucumber tissue against infection by botrytis cinerea pestacin: a , -dihydro isobenzofuran from pestalotiopsis microspora possessing antioxidant and antimycotic activities the effect of post-infectional potato tuber metabolites and surfactants on zoospores of oomycetes the isolation of xanthoxylin from the bark of phytophthora and hendersonula-infected citrus lemon and its fungitoxic effect analysis on blast fungus-responsive characters of a flavonoid phytoalexin sakuranetin; accumulation in infected rice leaves, antifungal activity and detoxification by fungus the fungal dimension of biodiversity: magnitude, significance, and conservation the magnitude of fungal diversity: the ± million species estimate revisited efficacy of artificial humic acid is a selective nutrient in hv agar used for the isolation of actinomycetes genetic manipulation of isoflavone -o-methyltransferase enhances biosynthesis of ′-o-methylated isoflavonoid phytoalexins and disease resistance in alfalfa die wirkung von auszigen aus dem sachalin-staudenknoterich reynoutria sachalinensis (f. schmidt) nakai gegen plizkrankheiten, insbesondere echte mehltauplize production of antibiotics by pseudomonas cepacia as an agent for biological control of soilborne plant pathogens screening of poplar trees for antibacterial, antifungal and antiviral activity suppression of pythium ultimum induced damping -off of cotton seedlings by pseudomonas fluorescens and its antibiotic, pyoluterin effects of fusarium species on defence mechanisms in sorghum seedlings control of post harvest botrytis fruit rot of strawberry by volatile organic compounds of candida intermedia biodiversity of endophytic fungi associated with traditional chinese medicinal plants novel acidic sesquiterpenoids constitute a dominant class of pathogeninduced phytoalexins in maize antimicrobial activities of some brown macroalgae against some soil borne plant pathogens and in vivo management of solanum melongena root diseases antifungal and antiproliferative activities of endophytic fungi isolated from the leaves of markhamia tomentosa screening of novel bioactive compounds from plant-associated actinomycetes isolation of actinomycetes from live plants and evaluation of anti phytopathogenic activity of their metabolites isolation and identification of endophytic actinomycetes and their antifungal activity phytoalexins from the leguminosae fungitoxic isoflavones from lupinus albus and other lupinus species the polyoxins: pyrimidine nucleoside peptide antibiotics inhibiting fungal cell wall biosynthesis validoxylamines as trehalase inhibitors suppression of damping-off disease in host plants by the rhizoplane bacterium lysobacter sp. strain sb-k is linked to plant colonization and antibiosis against soilborne peronosporomycetes antibacterial and antifungal activities of brown alga zonaria tournefortii (jv lamouroux) direct isolation of micromonospora on selective media with gentamicin polyphenol concentrations in grain, leaf and callus tissues of mold-susceptible and mold-resistant sorghum cultivars composition and antifungal activity of the essential oil of the brazilian chenopodium ambrosioides l ulvan, a sulfated polysaccharide from green algae, activates plant immunity through the jasmonic acid signaling pathway modulation of phytoalexin biosynthesis in engineered plants for disease resistance metabolic engineering of yeast and plants for the production of the biologically active hydroxystilbene, resveratrol biosynthesis, metabolism, molecular engineering and biological functions of stilbene phytoalexins in plants deciphering the role of phytoalexins in plant-microorganism interactions and human health phytoalexins produced in the leaves of capsella bursapastoris (shepherd's purse) xanthotoxin: a phytoalexin of pastinaca sativa root mycorrhiza-induced resistance and priming of plant defenses isolation of endophytic actinomycetes from catharanthus roseus (l.) g. don leaves and their antimicrobial activity. iranian effect of verticillium wilt (verticillium dahliae kleb.) and mycorrhiza (glomus mosseae) on root colonization, growth and nutrient uptake in tomato and eggplant seedlings the possible association of phytoalexins with resistant gene expression in flax to melampsora lini antifungal potential in crude extracts of five selected brown seaweeds collected from the western libya coast in vitro antifungal, anti-elastase and anti-keratinase activity of essential oils of cinnamomum-, syzygium-and cymbopogon-species against aspergillus fumigatus and trichophyton rubrum insecticidal activities of aromatic plant extracts and essential oils against sitophilus oryzae and callosobruchus chinensis anthraquinones isolated from cassia tora (leguminosae) seed show an antifungal property against phytopathogenic fungi recent development in the use of blasticidin s, a microbial fungicide, as a useful reagent in molecular biology linear b- , glucans are elicitors of defense responses in tobacco-france induce systemic resistance and promotion of plant growth by bacillus spp antifungal activity of pisiferic acid derivatives against the rice blast fungus functional moiety for the antifungal activity of phytocassane e, a diterpene phytoalexin from rice phytoalexin induction in the sapwood of plants of the maloideae (rosaceae): biphenyls or dibenzofurans structural and functional characterization of gene clusters directing non-ribosomal synthesis of bioactive lipopeptides in bacillus amyloliquefaciens strain fzb evaluation of essential oils and their components for broad-spectrum antifungal activity and control of late leaf spot and crown rot diseases in peanut bryophytes, a potent source of drugs for tomorrow's medicine? a plant extract acts both as a resistance inducer and an oomycide against grapevine downy mildew outline of a comparative study of criteria used in characterization of the actinomycetes selection of media for isolation of streptomycetes phytoalexins from the solanaceae muscodor sutura, a novel endophytic fungus with volatile antibiotic activities endophytic fungi isolated from oil-seed crop jatropha curcas produces oil and exhibit antifungal activity identification of antifungal principle in the solvent extract of an endophytic fungus chaetomium globosum from withania somnifera isolation, characterization, and bioactivity of endophytic fungi of tylophora indica an endophytic nodulisporium sp. producing volatile organic compounds having bioactivity and fuel potential two fungicidal phenylethanones from euodia lunu-ankenda root bark antifungal and insect antifeedant -phenylethanol esters from the liverwort balantiopsis cancellata from chile efficacy of the biofumigant fungus muscodor albus (ascomycota: xylariales) for control of codling moth (lepidoptera: tortricidae) in simulated storage conditions the potential of the fungus, muscodor albus, as a microbial control agent of potato tuber moth (lepidoptera: gelechiidae) in stored potatoes benzoic acid derivatives from piper species and their fungitoxic activity against cladosporium cladosporioides and c. sphaerospermum the production of resveratrol by vitis vinifera and other members of the vitaceae as a response to infection or injury interactions between pea root-inhabiting fungi examined using signature fatty acids mycosubtilin overproduction by bacillus subtilis bbg enhances the organism's antagonistic and biocontrol activities momilactones a and b in rice straw harvested at different growth stages antibacterial activity of oriental medicinal plant extracts toward helicobacter pylori mycofumigation with oxyporus latemarginatus ef for control of postharvest apple decay and rhizoctonia root rot on moth orchid screening for endophytic fungi with antitumour and antifungal activities from chinese medicinal plants cryptocin, a potent tetramic acid antimycotic from the endophytic fungus cryptosporiopsis cf. quercina antifungal activity of camptothecin, trifolin, and hyperoside isolated from camptotheca acuminata effect of laminarin on aspergillus flavus growth and aflatoxin production use of the endophytic fungus daldinia cf. concentrica and its volatiles as bio-control agents mycorrhizae and plan health isoquinoline alkaloids from macleaya cordata active against plant microbial pathogens pestalofones a-e, bioactive cyclohexanone derivatives from the plant endophytic fungus pestalotiopsis fici bis( , -dibromo- , -dihydroxybenzyl) ether, a marine algae derived bromophenol, inhibits the growth of botrytis cinerea and interacts with dna molecules screening of a marine algal extract for antifungal activities a new macrolide antibiotic with antitumor activity produced by streptomyces sp. cs, a commensal microbe of maytenus hookeri a novel ansamycin, naphthomycin k from streptomyces sp new bioactive metabolites produced by colletotrichum sp., an endophytic fungus in artemisia annua overview: a phylogenetic backbone and taxonomic framework for prokaryotic systematics inhibitory activities of some marine algae on aflatoxin accumulation naphthoquinone spiroketal with allelochemical activity from the newly discovered endophytic fungus edenia gomezpompae plant disease control: understanding the roles of toxins and phytoalexins in host-pathogen interaction plant extracts, bau-biofungicide and fungicides in controlling some important diseases of rice cv. brri dhan biocontrol potential of cyanobacterial metabolites against damping off disease caused by pythium aphanidermatum in solanaceous vegetables bioprospecting for endophytes from australian flora with mycofumigation potential antifungal activity of rubia tinctorum, rhamnus frangula and caloplaca cerina antimicrobial compounds and resistance: the role of phytoalexins and antianticipins fungicidal and molluscicidal saponins from dolichos kilimandscharicus xanthones from polygala nyikensis ethnoveterinary medicine and development: a review of the literature elicitor activity of phytoalexins in soy and sorghum by extracts and tinctures of medicinal plant species synthesis of phytoalexins in soy and sorghum by extracts and tinctures from three forest species structures of ent-herbertane sesquiterpenoids displaying antifungal properties from the liverwort herberta adunca endophytic fungi associated with monarda citriodora, an aromatic and medicinal plant and their biocontrol potential bioactivity of bryophyte extracts against botrytis cinerea, alternaria solani and phytophthora infestans control of fungal decay of apples and peaches by the biofumigant fungus muscodor albus the algal polysaccharide carrageenans can act as an elicitor of plant defense laboratoires goëmar. parc technopolitain atalante muscodor ghoomensis and muscodor indica: new endophytic species based on morphological features, molecular and volatile organic analysis from northeast india muscodor camphora, a new record from cinnamomum camphora muscodor kashayum sp. nov. -a new volatile antimicrobial producing endophytic fungus muscodor strobelii, a new endophytic species from south india response of subterranean clover to dual inoculation with vesicular-arbuscular mycorrhizal fungi and a plant growth-promoting bacterium modulation oh cyp genes and glucosilate profiles in arabidopsis by defense pathways potential agrochemicals from leaves of wedelia biflora -trihydroxydihydrochalcone from psidium acutangulum fungi and mycotoxins in grain: implications for stored product research endophytic actinomycetes from indian medicinal plants as antagonists to some phytopathogenic fungi chemically characterized cymbopogon martinii essential oil for shelf life enhancer of herbal raw materials based on antifungal, antiaflatoxigenic, antioxidant activity and favorable safety profile volatile antimicrobials from muscodor crispans, a novel endophytic fungus volatile plant metabolites for postharvest crop protection -methoxybrassinin, a sulphur-containing phytoalexin from brassica oleracea brassicanal c and two dioxindoles from cabbage brassicanal a and b, novel sulfur-containing phytoalexins from the chinese cabbage brassica campestris l. ssp pekinensis dehydro- -methoxycyclobrassinin, a sulfur-containing phytoalexin isolated from turnip brassica campestris l. ssp. rapa calophycin, a fungicidal cyclic decapeptide from the terrestrial blue-green alga calothrix fusca biological activity of β-dolabrin, γ-thujaplicin, and -acetyltropolone, hinokitiol-related compounds bacillomycin d: an iturin with antifungal activity against aspergillus flavus chemical composition and fungitoxic properties to phytopathogenic fungi of essential oils of selected aromatic plants growing wild in turkey evaluation of , -dihydroxybenzaldehyde, dopamine and its oxidation products as inhibitors of colletotrichum musae (berk. and curt.) arx in green banana fruits antifungal activities of the leaf extract of cassia tora linn experimentelle untersuchungen über die phytophthora resistenz der kartoffel camellidins, antifungal saponins isolated from camellia japonica different mechanisms for phytoalexin induction by pathogen and wound signals in medicago truncatula glyceollin, a soybean phytoalexin with medicinal properties an endophytic actinomycete, streptomyces sp. aok- , isolated from mountain laurel and its antifungal activity antimicrobial activities of vernonia tenoreana streptomyces: implications and interactions in plant growth promotion dihydro isocoumarins produced by xylaria sp. and penicillium sp., endophytic fungi associated with piper aduncum and alibertia macrophylla activation of biochemical defense mechanisms in bean plants for homeopathic preparations inhibition of protein biosynthesis by mildiomycin, an antimildew substance antifungal activity of thyme, summer savory and clove essential oils against aspergillus flavus in liquid medium and tomato paste activity of fungal endophytes against four maize wilt pathogens efficacy of some agricultural wastes in controlling root rot of glycine max l. induced by rhizoctonia solani effect of seed inoculation with bacillus subtilis and streptomyces griseus on the growth of cereals and carrots stress induced carbazole phytoalexins in glycosmis species seasonal variation of antibacterial and antifungal activities of the extracts of marine algae from southern coasts of india griseofulvin from xylaria sp. strain f , and endophytic fungus of abies holophylla and its antifungal activity against plant pathogenic fungi arbuscular mycorrhiza: the mother of plant root endosymbiosis potential of the volatile producing fungus nodulisporium sp. cf for the control of postharvest diseases of apple isolation, abundance and phylogenetic affiliation of endophytic actinomycetes associated with medicinal plants and screening for their in vitro antimicrobial biosynthetic potential distribution and identification of endophytic streptomyces species from schima wallichii as potential biocontrol agents against fungal plant pathogens mutualism and parasitism: the yin and yang of plant symbioses effects of sulfated polysaccharide and alcoholic extracts from green seaweed ulva fasciata on anthracnose severity and growth of common bean (phaseolus vulgaris l.) biological control in greenhouse systems a new working definition of the term "phytoalexin biotransformation of the brassica phytoalexin brassicanal a by blackleg fungus phytoalexins from brassicas: overcoming plants' defenses phytoalexin accumulation and antifungal compounds from the crucifer wasabi pathogen inactivation of cruciferous phytoalexins: detoxification reactions, enzymes and inhibitors antimicrobial activity of rhodobryum ontariense. hemijska industrija the discovery of enfumafungin, a novel antifungal compound produced by an endophytic hormonema species biological activity and taxonomy of the producing organisms ultrastructural observations of pterostilbene fungitoxicity in dormant conidia of botrytis cinerea pers natural occurrence of mycotoxins in foods and feeds -an update review relation between the chemical structure and biological activity of hydroxystilbenes against botrytis cinerea two new antifungal naphthoxirene derivatives and their glucosides from sesamum angolense welw potential of plant extracts and fungicides for managing fusarium oxysporum f. sp lycopersici further evidence for the involvement of a pre-formed antifungal compound in the latency of colletotrichum gloeosporioides on unripe avocado fruits progress in phytoalexin research during the past years antifungal potential and defense gene induction in maize against rhizoctonia root rot by seed extract of ammi visnaga (l.) lam diterpene alcohols from croton lacciferus an antifungal chromene from eupatorium riparium outbreaks of aflatoxicoses in india removal of duvatrienediols from the surface of tobacco leaves increases their susceptibility to blue mold jasmonate and salicylate as global signals for defense gene expression use of algae in strawberry management antimicrobial activity of essential oils and ethanol natural products from plants and fungi as fungicides extract of phlomis fruticosa l. (lamiaceae) growth activities of the sugar beet pathogens sclerotium rolfsii sacc. rhizoctonia solani kühn. and fusarium verticillioides sacc. under cyanobacterial filtrates stress induction of defense responses in zucchini (cucurbita pepo) by anabaena sp. water extract activity of seaweed and cyanobacteria water extracts against podosphaera xanthii on zucchini antioxidant and antimicrobial activities of essential oil and extracts of fennel (foeniculum vulgare l.) and chamomile elicitation of foliar resistance mechanisms transiently impairs root association with arbuscular mycorrhizal fungi antifungal activity and potential use of essential oils against fusarium culmorum and fusarium verticillioides plant growth enhancing effects by a siderophore producing endophytic streptomycete isolated from a thai jasmine rice plant (oryza sativa l. cv. kdml ) bioactivities of extracts from some axenically farmed and naturally grown bryophytes antimicrobial activity of bryum argenteum screening of antimicrobial and antioxidant secondary metabolites from endophytic fungi isolated from wheat (triticum durum) structure biological activity relationships in triterpenic saponins: the relative activity of protobassic acid and its derivatives against plant pathogenic fungi pantoea agglomerans strain eh produces two antibiotics that inhibit erwinia amylovora in vitro effectiveness of phenolic compounds against citrus green mould control of penicillium expansum and patulin accumulation on apples by quercetin and umbelliferone determination of antimicrobial and antiproliferative activities of the aquatic moss fontinalis antipyretica hedw muscodor tigerii sp. nov.-volatile antibiotic producing endophytic fungus from the northeastern himalayas muscodor darjeelingensis, a new endophytic fungus of cinnamomum camphora collected from northeastern himalayas bioactivity guided isolation of antifungal compounds from the liverwort bazzania trilobata biosynthesis, elicitation and roles of monocot terpenoid phytoalexins biologically active secondary metabolites of endophytic pezicula sp pinocembrin: an antifungal compound secreted by leaf glands of eastern cottonwood endophytic actinomycetes from tea plants (camellia sinensis): isolation, abundance, antimicrobial, and plant-growth-promoting activities isolation of , -diacetylphloroglucinol from a fluorescent pseudomonad and investigation of physiological parameters influencing its production plant bio-stimulants: a review on the processing of macroalgae and use of extracts for crop management to reduce abiotic and biotic stresses purification and partial characterization of a b-glucan fragment that elicits phytoalexin accumulation in soybean diversity and antimicrobial activity of culturable endophytic fungi isolated from moso bamboo seeds in: maheshwari dk (ed) bacteria in agrobiology: plant growth responses studies on endophytic actinomycetes (i) streptomyces sp. isolated from rhododendron and its antifungal activity introduction study of antifungal activities of bryophyte extracts anti-apoptotic machinery protects the necrotrophic fungus botrytis cinerea from host-induced apoptotic-like cell death during plant infection diversity of endophytic actinomycetes in mandarin grown in northern thailand, their phytohormone production potential and plant growth promoting activity diversity and antifungal activity of the endophytic fungi associated with the native medicinal cactus opuntia humifusa (cactaceae) from the united states antifungal activity of securinine against some plant pathogenic fungi antimicrobial activity of some indian mosses an endophytic phomopsis sp. possessing bioactivity and fuel potential with its volatile organic compounds existence of muscodor vitigenus, m. equiseti and m. heveae sp. nov. in leaves of the rubber tree (hevea brasiliensis müll. arg.), and their biocontrol potential casbene: an antifungal diterpene produced in cell-free extracts of ricinus communis seedlings sesquiterpene lactones from centaurea thessala and centaurea attica: antifungal activity mechanisms of resistance to plant diseases what is the significance of the arbuscular mycorrhizal colonisation of many economically important crop plants? the role of phosphorous nutrition in interactions of vesicular arbuscular mycorrhizal fungi with soil borne nematodes and fungi suppression of cottony leak of cucumber with bacillus cereus strain uw management of mycorrhiza in agriculture, horticulture and forestry endophytic naphthopyrone metabolites are co-inhibitors of xanthine oxidase, sw cell and some microbial growths a record of muscodor albus, an endophyte from myristica fragrans in thailand antifungal activity of some essential oils two novel antifungal alka- , -dienals from triticum aestivum plant-derived natural products synthesis, function, and application streptomyces sp. p as effective biocontrol against chilli soilborne fungal phytopathogens algal polysaccharides as source of plant resistance inducers mycorrhizae in crop production an endophytic gliocladium sp. of eucryphia cordifolia producing selective volatile antimicrobial compounds seasonal variation in antifungal, antibacterial and acetyl cholinesterase activity in seven south african seaweeds phytoalexins -a biogenetic perspective the role of phytoalexins in the seedling resistance to leptosphaeria maculans in some crucifers synergism among volatile organic compounds resulting in increased antibiosis in oidium sp an endophytic/ pathogenic phoma sp. from creosote bush producing biologically active volatile compounds having fuel potential an endophytic nodulisporium sp. from central america producing volatile organic compounds with both biological and fuel potential the production of mycodiesel hydrocarbons and their derivatives by the endophytic fungus gliocladium roseum (nrrl ) muscodor albus e- , an endophyte of guazuma ulmifolia making volatile antibiotics: isolation, characterization and experimental establishment in the host plant antifungal activities of a steroid from pallavicinia lyellii, a liverwort engineering cottonseed for use in human nutrition by tissue-specific reduction of toxic gossypol muscodor cinnamomi, a new endophytic species from cinnamomum bejolghota evaluation of muscodor suthepensis strain cmu-cib as a postharvest biofumigant for tangerine fruit rot caused by penicillium digitatum molecular and morphological evidence support four new species in the genus muscodor from northern thailand biocontrol of rhizoctonia solani ag- , the causal agent of damping-off by muscodor cinnamomi cmu-cib antitumor activity of -arylcoumarins from endophytic streptomyces aureofaciens cmuac identification of streptomyces sp. tc , an endophyte in alpinia galanga, and the isolation of actinomycin d structures of moracins e, f, g and h, new phytoalexins from diseased mulberry isolation of three novel sulphur-containing phytoalexins from the chinese cabbage brassica campestris l. ssp. pekinensis (cruciferae) novel sulfur-containing phytoalexins from the chinese cabbage brassica campestris l. ssp. pekinensis (cruciferae) mechanism of kasugamycin action on polypeptide synthesis isolation of endophytic actinomycetes from different cultivars of tomato and their activities against ralstonia solanacearum in vitro isolation, purification and characterization of trichothecinol-a produced by endophytic fungus trichothecium sp. and its antifungal, anticancer and antimetastatic activities antibacterial activity and induction of phytoalexins in bean plants by homeopathic preparations of essential oil of eucalyptus globulus antibiotic production by soil and rhizosphere microbes in situ study on the communities of endophytic fungi and endophytic actinomycetes from rice and their antipathogenic activities in vitro identification of volatile metabolites from fungal endophytes with biocontrol potential towards fusarium oxysporum f. sp. cubense race influence of arbuscular mycorrhizal mycelial exudates on soil bacterial growth and community structure antifungal activity of lipopeptides from bacillus xt cect against botrytis cinerea hypoxylon sp., an endophyte of persea indica, producing , -cineole and other bioactive volatiles with fuel potential diterpene phytoalexins are biosynthesized in and exuded from the roots of rice seedlings antifungal activity of lemongrass (cymbopogon citratus l.) essential oil against key postharvest pathogens antifungal drug resistance in pathogenic fungi phytotoxic and antimicrobial activity of volatile and semi-volatile organic compounds from the endophyte hypoxylon anthochroum strain blaci isolated from bursera lancifolia (burseraceae) volatile organic compounds from endophytic fungi as innovative postharvest control of fusarium oxysporum in cherry tomato fruits studies on the mode of action of the phytoalexin phaseolin characterization of the early response of arabidopsis to alternaria brassicicola infection using expression profiling antimicrobial activity of methanol extracts of fontinalis antipyretica, hypnum cupressiforme and ctenidium molluscum sea weed polysaccharides and derived oligosaccharides stimulate defense responses and protection against pathogens in plants endophytic actinomycetes from azadirachta indica a. juss.: isolation, diversity, and anti-microbial activity the biocontrol effect of mycorrhization on soil borne fungal pathogens and the autoregulation of the am symbiosis: one mechanism, two effects? biocontrol of the pathogen phytophthora parasitica by arbuscular mycorrhizal fungi is a consequence of effects on infection loci -methylcoumaranones from mutisia friesiana and their bioactivity a vesicular arbuscular mycorrhizal fungus (glomus intraradix) induces a defense response in alfalfa roots suppression of an isoflavonoid phytoalexin defense response in mycorrhizal alfalfa roots fungal (−like) biocontrol organisms in tomato disease control controlling crop diseases using induced resistance: challenges for the future antifungal activities of essential oils and their constituents from indigenous cinnamon (cinnamomum osmophloeum) leaves against wood decay fungi antifungal activity screening of soil actinobacteria isolated from inner mongolia isolation and identification of an endophytic fungus of polygonatum cyrtonema and its antifungal metabolites loroglossol: an orchid phytoalexin evaluation of fungicides, bio-agents and plant extracts against pyricularia oryzae temporal synthesis and radiolabelling of the sorghum -deoxyanthocyanidin phytoalexins and the anthocyanin, cyanidin -dimalonyl glucoside peptide synthetase gene in trichoderma virens use of antibiotics for selective isolation and enumeration of actinomycetes in soil biological properties of alkaloids. influence of quinolizidine alkaloids and gramine on the germination and development of powderly mildew, erysiphe graminis f. sp. hordei die verbreitung antifungaler eigenschaften bei moosen the role of stilbenes in resistance of sitka spruce (picea sitchensis (bong) carr) to entry of fungal pathogens muscodor roseus anam. sp. nov., an endophyte from grevillea pteridifolia muscodor albus anam. sp. nov., an endophyte from cinnamomum zeylanicum chemical constituents from the chinese bryophytes and their reversal of fungal resistance metabolites from mangrove endophytic fungus dothiorella sp tetran or triterpenoids from chisocheton paniculatus effects of pre-and post-harvest application of salicylic acid or methyl jasmonate on inducing disease resistance of sweet cherry fruit in storage endophytic fungi harbored in the root of sophora tonkinensis gapnep: diversity and biocontrol potential against phytopathogens tithoniamarin and tithoniamide: a structurally unique isocoumarin dimer and a new ceramide from tithonia diversifolia evaluation of wedelia biflora (linn) d.c for anthelmintic and antimicrobial activity recent trends in studies on botanical fungicides in agriculture effect of polyacetylenic acids from prunella vulgaris on various plant pathogens potent in vivo antifungal activity against powdery mildews of pregnane glycosides from the roots of cynanchum wilfordii fungitoxic non-glycosidic iridoids from alibertia macrophylla diversity and antifungal activity of endophytic fungi associated with camellia oleifera potential of endophytic fungi isolated from cotton roots for biological control against verticillium wilt disease natural plant products as eco-friendly fungicides for plant diseases control-a review regulation of plant immunity through modulation of phytoalexin synthesis a new prenylated indole derivative from endophytic actinobacteria streptomyces sp. neau-d studies on chemical constituents in root tuber of cynanchum auriculatum muscodor fengyangensis sp. nov. from southeast china: morphology, physiology and production of volatile compounds bioactive isocoumarins isolated from the endophytic fungus microdochium bolleyi effects of yeast polysaccharide on growth and flavonoid accumulation in fagopyrum tataricum sprout cultures actinobacteria associated with chinaberry tree are diverse and show antimicrobial activity the diversity and anti-microbial activity of endophytic actinomycetes isolated from medicinal plants in panxi plateau china preharvest l -arginine treatment induced postharvest disease resistance to botrytis cinerea in tomato fruits neoverataline a and b, two antifungal alkaloids with a novel carbon skeleton from veratrum taliense bioactive endophytic streptomycetes from the malay peninsula camalexin accumulation in arabis lyrata metabolites of colletotrichum gloeosporioides, an endophytic fungus in artemisia mongolica key: cord- -ruf vvz authors: sohrab, sayed sartaj title: an edible vaccine development for coronavirus disease : the concept date: - - journal: clin exp vaccine res doi: . /cevr. . . . sha: doc_id: cord_uid: ruf vvz a novel coronavirus was emerged in december from wuhan city, china and has now become a global threat to human health. currently, the coronavirus disease (covid- ) has spread to more than countries with , deaths and , confirmed cases. currently, there is no vaccine available against covid- . the traditional vaccines development requires more time and high cost and due to this, the disease outbreaks becomes more challenging. now a days, plants have become more attractive platform for edible vaccine production than the other system. the development of an edible vaccine in a selected plant system has many significant advantages such as; easy and efficient oral delivery, low cost with higher scale production, avoidance of any trained medical personnel for delivery, lack of any pathogenic infection, multicomponent expression in a single plant, and so forth. in this manuscript, the concept, development, and importance of an edible vaccine have been discussed. by using this plant-based platform, an edible vaccines can be produced in many crops like banana, cucumber, carrot, lettuce, and tomato against various diseases. due to increasing cases globally with covid- , there is an urgent requirement to develop an ideal vaccine and antiviral therapy against this virus to control the disease worldwide. china with , deaths and , laboratory confirmed cases [ , ] . based on medical and healthcare workers, the medical supplies are getting low due to the high number of cases in wuhan city. to manage the cases, china has announced to build two new hospitals with , beds within days which is functional now. the outbreak caused by covid- in wuhan is an epidemic threat to global health [ , ] . currently, there are many systems available such as bacteria, yeast, and mammalian cell lines to express the recombinant subunit vaccines and therapeutic proteins but all the above systems have some disadvantages like high cost, safety, and target integrity. while, by using plant-based system is very useful and avoids all the issue as compared to other system and provides low cost and better safety with high scalability without harm with any pathogenic infections. this is the most recent and alternative technology for vaccine development. the expected protein folding and post-translational modifications of the proteins can be produced in the correct form in plant-based systems with the desired biological functions [ ] . plant-based edible vaccines have been recently introduced for vaccine production. the main goals of plant-based edible vaccines are the transformation and production of antigens into plants and the oral consumption of such vaccine to induce antigen specific immune responses. currently, the use of plant-based expression system platform have been extensively utilized for the expression and purification of vaccines, recombinant proteins, enzymes, and many bio-pharmaceuticals in a variety of plant species, including potato, corn, tomato, carrot, lettuce, and spinach and have reached at advanced stage of pre-clinical and clinical evaluation. the oral administration of edible vaccines is a preferable route of vaccination for being a simple and safe route of administration; the low production cost allows for local production and minimal plant material processing; natural bio-encapsulation and hence, stability in the gastrointestinal (gi) tract; and protective immunogenicity at the gi mucosa. the current status of plant-based vaccine and their clinical evaluation are summarized in table [ , ] . the plant-based vaccine development idea was started years ago and since then many vaccine proteins have been produced in plants for human and animal diseases [ ] [ ] [ ] [ ] [ ] . since more than years, plant science has grown tremendously and shown great importance towards molecular pharming and have significant achievements in large scale with low cost production of recombinant proteins, enzymes, and pharmaceuticals compounds. currently, various pharmaceutical compounds, antigen, antibodies, hormones, enzymes, and growth regulators have been developed into multiple plants system [ ] [ ] [ ] [ ] . the main objective of this review is to provide the latest information about the use of plant-based platform for the development of an edible vaccine. for plant-based vaccine development, the desired plant with efficient and quick regeneration and transformation properties should be selected. the desired gene can be delivered into plant cells to express desired proteins/pharmaceuticals compounds with low cost and high scale [ , , ] . this technology has attracted the researchers because of elimination of a specific animal requirement to conduct the experiment. the edible vaccines can be designed in such a way that, the expressed and produced proteins should not be pathogenic. the production of conventional vaccines is very expensive, and they require purification and refrigeration. apart from that, the plant-based vaccines are the most suitable for children as an oral delivery. currently, world health organization has suggested new technologies for the vaccine development. the specific proteins can be expressed into desired plants with very less cost and can be grown to the required locations so that, an edible vaccine can be available to the needy population globally, especially in the developing countries. due to high cost, storage, refrigeration, transportation, and requirements of trained medical personnel, an injectable vaccine cannot be easily taken in developing countries. additionally, various pathogenic organism, bacterial and viral diseases can be easily transmitted by re-use of needles. mono-clonal antibodies are being used in the treatment of arthritis and cancer. the monoclonal antibodies can be easily produced in desired transgenic plants with very less cost and time duration. it has been reported that the developed transgenic rice was stable at room temperature for months. because these vaccines are needle-free [ ] , they have the added advantage of eliminating the associated waste and potential for dissemination of blood borne-infections. antigens are released in the form of vaccine through bio-encapsulation which protects them from gastric enzymes. the released proteins were absorbed by m cells in the intestinal wall and passed on to the macrophages and antigen-presenting cells and local lymphocyte populations generating serum immunoglobulin (ig)g, ige, and local iga responses and memory cells which neutralize the attack by a real pathogen. the development and stability of edible vaccine as an antigen has been investigated in many recent reports. the current status of plant-based vaccines has been reviewed and presented [ ] . the concept of edible vaccine development has been presented in fig. . currently, the plant-based vaccine has many valuable advantages as compared to traditional vaccines which include. adjuvants require to enhance immune responses. ( ) orallyintroduced antigens elicit mucosal immunity. ( ) it is easy to bulk produce onsite, transport and stored with less cost and without refrigeration. ( ) no injection and need for trained medical person. ( ) easy to express, separate and purify the protein. ( ) they can be stored as seeds and oils and dried tissue without any refrigeration. ( ) they do not have any risk of contamination and disease spread. ( ) there is the possibility of enhanced compliance, especially in children [ ] . edible vaccines have received considerable attention from researchers in both academia and industry. the first plant-derived rabies vaccine was produced in tomatoes and offered the advantages of high biomass yields and the increased containment by growth in greenhouses. lettuce and bananas have also been utilized for the production of plant-based vaccines [ ] [ ] [ ] . the edible can be developed in many desired plants. recently, there are different crops like tomato, carrot, corn, cucumber, lettuce, and spinach plants that have been utilized as a green factory to express, and purify the desired protein, enzymes, antigens, and biopharmaceuticals compounds [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the most important advantage of plant expression systems is the low cost. tomato can be used as for vaccine production and the used as salad which facilitates the easy oral delivery of a particular vaccine. as an example, tomato has excellent biomass and easily transformed and generate the whole plant within short period and this provides an ideal choice for vaccine production. therefore, tomato is no doubt a green vaccine factory. currently, there is no approved vaccine or treatment with proven efficacy against newly emerged cov covid- . while previous work on sars-cov vaccines have shown that the spike glycoprotein (s) is the main inducer of neutralizing antibodies. the covs infection causes severe respiratory disease with clinical symptoms; specifically, patients develop acute respiratory symptoms. the spike protein can be used to develop a vaccine against the covid- . the spike (s) protein gene or subunit of spike like s subunit can be cloned into a plant expression vector and the desired plant like tomato, cu-cumber, or lettuce can be transformed. the developed transgenic plants can be used as salad and easily delivered orally and immunized the human against the newly emerged virus. several teams are working on a vaccine to protect against the new cov. recently, the therapeutic options for covid- has been discussed and suggested in a published paper [ ] . however, the development of a protective vaccine is of great importance to prevent and control the spread of the virus as well as any future outbreaks. the newly emerged cov has now become a global threat to human health. as on february , , this virus has been spread to more than countries including china with , deaths and , confirmed cases [ , ] . the fast spread of this virus has attracted the researchers towards the development of an urgent vaccine to control the virus infection and disease spread. the full-genome of covid- has provided the valuable information which can be used to design and develop an effective vaccine [ ] . the plant-based edible vaccine has now attracted many researchers and pharmaceutical companies to develop fast and cheaper vaccines against many pathogens. this technology has got more publicity in the last years and have proven the efficiency to express the desired protein either as antigens or pharmaceuticals therapeutic compounds in the desired plant cells. this novel technology provides the high and fast expression, purification, and better stability of desired proteins in to plant cells as well as their removal of refrigeration requirement and trained medical personnel for delivery. the edible vaccine also removes the risk factors and safety and regulatory issues related to living organisms. the edible vaccine can be grown at the site of requirement and orally delivered in the form of salad. therefore, this technology is emerging as a novel and alternative methods for large scale edible vaccine production, manufacturing, and processing as well as commercialization and easily availability to the needy persons globally. this technology will provide an effective vaccination against many diseases in human and animal globally. based on current information, it is concluded that there is an urgent need to develop a vaccine against this fast spreading novel cov by using various vaccine development technology. the edible vaccine technology will provide a fast and cheaper vaccine not only this new virus but also against many other pathogens. disease outbreak news (dons) [internet]. geneva: world health organization novel coronavirus ( -ncov) situation summary recent progress in the development of plant derived vaccines clinical development of plant-produced recombinant pharmaceuticals: vaccines, antibodies and beyond recent development and future prospects of plant-based vaccines plant-based vaccines against viruses plants as an alternative source of therapeutic proteins new strategies toward edible vaccines: an overview plant-made vaccines and reagents for the one health initiative production of tetravalent dengue virus envelope protein domain iii based antigens in lettuce chloroplasts and immunologic analysis for future oral vaccine development process development strategies in plant molecular farming farming of plant-based veterinary vaccines and their applications for disease prevention in animals hussein s. plant-based vaccines: production and challenges needle-free vaccine delivery therapeutic strategies in an outbreak scenario to treat the novel coronavirus originating in wuhan, china: version preliminary phylogenetic analysis of ncov- genomes key: cord- - h g s v authors: alam, fiaz; khan, gul nawaz; asad, muhammad hassham hassan bin title: psoralea corylifolia l: ethnobotanical, biological, and chemical aspects: a review date: - - journal: phytother res doi: . /ptr. sha: doc_id: cord_uid: h g s v psoralea corylifolia l. (leguminosae) is a well‐known traditional medicinal plant used from ancient times for treatment of various ailments. it is widely distributed and an important part of therapeutics in ayurveda and in chinese medicines. the aim of this review is to present comprehensive and most up to date report on its ethnobotanical, ethnopharmacological, clinical, phytochemical, and side effects. studies on the ethnobotanical, ethnopharmacological, clinical, phytochemical, and side effects of p. corylifolia were published until year and were searched using various scientific databases. the scientific literature searched revealed that these plant species has been extensively investigated in vivo and in vitro for various biological and phytochemical studies. it has cardiotonic, vasodilator, pigmentor, antitumor, antibacterial, cytotoxic, and anti‐helminthic properties and locally used for alopecia, inflammation, leukoderma, leprosy, psoriasis, and eczema. so far, about a hundred bioactive compounds have been isolated from seeds and fruits, and most important compounds identified belongs to coumarins, flavonoids, and meroterpenes groups. this review article summarized the most updated scientific literature on bioactive phytochemical and biological activities of p. corylifolia. this article will be a useful addition to providing information for future research, and more standard clinical trials are needed for the plant to be used as therapeutic agent. the family leguminosae contains about genera and is among the largest families of flowering plants. the plants of leguminosae are widely distributed, and in terms of number of specie, it is one of the largest terrestrial family of plants after orchidaceae and asteraceae (stevens, ) . the genera that belong to this family are important medicinally and contain a variety of biologically important molecules; p. corylifolia is an important part of therapeutics in ayurveda and in chinese medicines. the plant is cardio active and showed antimicrobial and cytotoxic properties. it is used as a pigmentor. the plant showed cytotoxicity against tumors and worms (baquar, ; rizvi, saeed, & zubairy, ; sharma, ) . in chinese medicines, the psoralea plant is considered warm by nature, and therefore shows many healing actions on kidney and spleen meridians (daiquan, (daiquan, - . the seeds of p. corylifolia are used in indigenous medicine systems for healing of various ailments. the seeds are diuretic, aphrodisiac, laxative, anti-helminthic, and are used in febrile conditions. in ayurveda, the seeds are used in the form of paste and as an ointment for external as well as internal use for treatment of different conditions such as alopecia, inflammation, leukoderma, leprosy, psoriasis, and eczema (huang, ; judd, campbell, kellogg, stevens, & donoghue, ; sjc, ) . in india, the powder of seeds is mixed with haratalabhasma (yellow arsenic) and converted to paste with the urine of cow. this paste is used to treat leukoderma lesions. in another formulation, the mixture of powdered seeds with buttermilk have been used externally for treating ringworm and scabies. the seed oil is taken orally with betel nut leaf for treatment of leprosy. another skin condition, dermatosis, has been treated with adjuvants therapy of bakuchi (p. corylifolia) with other local plants such as amalaki and khadira. similarly, the oils of bakuchi and karanja are mixed with vaseline to treat chronic skin diseases (khare, ) . the seeds of the plant are also considered useful in bilious disorder, snakebite, and in scorpion sting. the seeds are also used in the production of perfumes (deshaprabhu, ; maisch, ; sharma et al., ) . the root of p. corylifolia has shown its effectiveness in dental caries. the fruits are aphrodisiac and have laxative effect, and the leaves are antidiarrheal (baquar, ; sjc, ) . the plant is also valuable in treating alopecia areata (dua, kumar, pandey, & kumar, b) . in combination with haritka and gokshura, this plant is used for urinary frequency, and with ashwagandha and bala, it is used for treatment of reproductive diseases and cough. in chronic diarrhea and for treating cold symptoms, this plant is used in combination with nutmeg and haritaki (anderson & voorhees, ) . the seeds of bakuchi in powder form mixed with the decoction of bibhitaka (terminalia bellirica bark) and kaakodumbara (ficushispida) was effective to treat vitiligo. the infection of ringworm is treated with a combination of tila (sesame seeds) and bakuchi (gupta, dhar, & atal, ) . a combination of bakuchi with haratalabhasma provides relief against leukoderma when applied externally (khatune et al., ) . in japan, the plant extracted with alcohol has been used as an additive in processed foods and pickles (nadkarni, ) . when the fixed oils are removed, what is left after the seed cake are used as feed or manure due to the presence of nitrogen ( . %) and the minerals ( . %). volatile oils obtained from fruits have an irritant effect on the skin and mucous membranes and stimulate the voluntary muscles in high concentrations (chaudhuri, ; gidwani et al., ; huang, ) . p. corylifolia in the form of extracts have been used in various herbal formulations when combined with other herbs and used in handling psoriasis and many other skin disorders . this plant is also reported to be used in cardiac problems, asthma, and urinary discharge (kr, ) . it also has anti-leishmaniasis activity (ali, akhtar, sultana, baboota, & ahuja, ) . it is used to control watery stool, urinary frequency, and reproductive imbalances (pole, ) . in china, it is specially used against vitiligo disease (anderson & voorhees, ; khare, ) . p. corylifolia is available in the form of various ayurvedic marketed formulations in india and across the world, the most famous brands are safuf bars®, zimad kibrit®, svitrakaravati®, khadirarista®, algushadi yoga®, sarvangasundarigutika®, bhallatakawaleha®, maheshwa-raghrita®, ayorajodilepa®, brihatsomarajitaila®, somarajighrita®, bawchichurna®, etc. (ali et al., ; pole, ; qiao et al., ) . the investigation showed that p. corylifolia possessed a wide range of phytochemicals including flavones, coumarins, monoterpenes, chalcones, lipids, resins, stigmasteroids, and flavonoids. the volatile oils are also reported from this plant (zhang, zhao, wang, lu, & chen, ) . it is revealed that seasonal variation also effects the phytochemistry of p. corylifolia. mostly, the bioactive compounds are found to be concentrated in the seeds. the phytochemistry of this plant is discussed as follows. the whole plant of p. corylifolia was extracted with organic solvents such as petroleum ether and chloroform. the subsequent isolation methods lead to the purification of bioactive compounds, for example, psoralen, isopsoralen, corylifolin, corylin, and psoralidin (gupta, gupta, & gupta, ) . peng and colleague obtained a new compound identified as neo-psoralen from the whole plant of p. corylifolia in and elucidated its structure on the grounds of chemical indications and spectroscopic analysis (chaudhuri, ; gupta et al., ; table and figure ). it was already discussed that most of the active constituents isolated so far from p. corylifolia are part of the seed. sen and colleague extracted seed oil from p. corylifolia and found that the oil is unsaponifiable with boiling points - °c. during the experiment, another compound was also identified as methyl glucoside with melting points of - °c (chopra & chopra, ) . chopra and chaterjee, in , identified essential oil, a fixed oil and resin of dark brown color with some traces of alkaloids in p. corylifolia (chopra et al., ) . dymock studied the sugar contents, extractive matter, albumin concentraton, and ash value, and also found some traces of manganese in seeds of p. corylifolia. in continuation of research for compounds from the seeds of p. corylifolia, three more important components were fractionated and identified as bakuchiol a monoterpene phenol and the two novels dimeric monoterpenoids, bisbakuchiols a and b (panda, ) . a very important pharmacological compound known as bakuchiol has also been biosynthesized in , and it was concluded that it is a derivative of phenylpropane pathway (banerji & chintalwar, ) . similarly, bisbakuchiols a and b structures were evaluated, and it was noted that dimeric monoterpenoid skeleton contains two monoterpenes, which are connected through a dioxane bridge (wu et al., ) . the low polarity ether extract of p. corylifolia seeds was investigated, and it revealed the presence of various ketones and aldehydes containing compounds such as corylinal, c-formylated chalcone, and isoneobayachalcone. a new isoflavone compound known as psorlenal was also identified in the seeds (gupta et al., ) . the same compounds, psoralen and isopsoralen, were also isolated by applying other chromatographic techniques such as high-speed counter-current chromatography (liu, li, sun, & kong, ) . the seed's sample has been the main focus for the search of bioactive compounds, and such an isolation procedure involving spectroscopic methods and crystal x-ray diffraction resulted in isolation of five novel compounds. these compounds are named as psoracorylifols a-e and chalcone and bavachromanol (yin, fan, dong, & yue, ) . similarly, three new flavonoid compounds named as corylifols a, b, and c and bavachalcone were also fractionated from p. corylifolia seeds. another compound known as bakuchicin was also identified from the seeds (yin, fan, wang, dong, & yue, ) . the seeds are also reported to contain some other flavonoids such as bavachinin (bcn), bavachin, isobavachin, and isobavachalcone (ibc). the glycosides identified in seeds of p. corylifolia were psoralenoside and isopsoralenoside, which were of the benzofuran type. p. corylifolia also reported to contain some polar compounds, namely, neobavachalcone, -methyl bavachin, and bavachromene. these compounds were isolated and identified from the insoluble portion of the ethanolic extract. these compounds identified as (khatune et al., ) aryl coumarin coumarin seeds anticancer (limper et al., ) astragalin flavonoid seeds antioxidant bakuchiol meroterpene seeds/fruit anti-acne antibacterial (katsura et al., ; newton et al., ) antifungal (newton et al., ) , (hosamani et al., ; lau et al., ; lau et al., ; prasad et al., ; savoia, ; srinivasan & sarada, ; yang et al., ) retinal regeneration (seo et al., ) anti-aging (seo et al., ) estrogen receptor agonist, postmenopausal symptoms (lim et al., ) anti-diabetic (behloul & wu, ) lymphangiogenesis inhibition (jeong et al., ) anticancer (chen et al., ; li et al., ) bavachinin flavone seeds antibacterial (khatune et al., ) estrogen receptor agonist (lim et al., ) lymphangiogenesis inhibition (jeong et al., ) osteoporosis (liu et al., ) anti-alzheimer (chen et al., ) carboxylesterase inhibitors bakuisoflavone flavone fruit antibacterial (siva et al., ) bakuflavanone flavone fruit antibacterial (siva et al., ) bavachin flavnonoid seeds/fruit osteoblast bakuchicin coumarin seeds topoisomerase inhibitor (sun et al., ) bavachalcone chalcone seeds anticancer (shan et al., ) cvs protective effect (dang et al., ) bavachinone a flavonoid fruit antibacterial (won et al., ) bavachinone b flavonoid fruit antibacterial (won et al., ) bavacoumestan c flavonoid fruit antibacterial (won et al., ) corylifolinin chalcone seeds antibacterial (khatune et al., ) carboxylesterase inhibitors (sun et al., ) corylifols prenyl flavonoid seeds antibacterial (yin et al., ) corylifol a flavonoid seeds/fruit carboxylesterase inhibitors corylifol b flavonoid seeds carboxylesterase inhibitors corylifol c flavonoid seeds protein kinase inhibition (limper et al., ) anticancer (limper et al., ) corylifol d flavonoid seeds anticancer (stomach; yang et al., ; teschke et al., ) corylifol e flavonoid seeds anticancer (stomach; yang et al., ; teschke et al., ) coryfolin flavonoid whole plant antioxidant, anti-diabetic (behloul & wu, ) corylin flavonoid whole plant osteoblast wang et al., ) anticancer (shan et al., ) carboxylesterase inhibitors (sun et al., ) coryaurone a flavonoid fruit antibacterial (won et al., ) dadzin isoflavnoid fruit antioxidant (shinde et al., ) dadzein isoflavnoid fruit antioxidant (shinde et al., ) antidiabetic (behloul & wu, ) topoisomerase inhibitor (sun et al., ) dihydroxy coumestan essential oil component seeds insecticidal, genotoxic (khatune et al., ; dua et al., b) genistein isoflavone fruit ani-diabetic, anti-obesity (behloul & wu, ) , antioxidant (shinde et al., ) hydroxy bukuchiol meroterpene seeds lymphangiogenisis inhibition (jeong et al., ) hydroxypsoralenol a flavonoid fruit antibacterial (won et al., ) hydroxypsoralenol b flavonoid fruit antibacterial (won et al., ) types of esters were also studied in p. corylifolia, and psoralester and psorachromene were identified as two new metabolites. the lymphangiogenesis inhibition (jeong et al., ) anti-alzheimer (chen et al., ) carboxylesterase inhibitors isobavachin flavonoid seed/fruit osteoblast (li et al., ) isopsoralen furanocoumarin whole plant antiprotozoal neobavaisoflavone seeds antibacterial (khatune et al., ) psoralen furanocoumarin whole plant/root leucoderma, psoriasis anticancer (hao et al., ) , antioxidant , anti-alzheimer (somani et al., ) , collagengenesis psoralidin coumarin whole plant/seed estrogen receptor modulator (liu et al., ; lim et al., ) antioxidant (wang, yin, zhang, peng, & kang, b) , antibacterial (khatune et al., ) anti-diabetic (behloul & wu, ) , antiprotozoal anticancer (hao et al., ; limper et al., ; yang et al., ) , anti-depressent (farahani et al., ) psoracorylifol d flavonoid seed lymphangiogenesis inhibition (jeong et al., ) psoracoumestan coumestans seeds essential oil anti-cancer (limper et al., ) xanthoangelol chalcone seeds anticancer (limper et al., ) figure structures of important compounds isolated from psoralea corylifolia psoralester is a -membered lactone compound and the latter is an isomer of already known compound bayachromene (tewari & bhakuni, ) . khatune and colleague isolated new coumestan derivative -(- -methylbut- -enyl)- n- -dihydroxycoumestan while working on the chloroform soluble portion of the seeds of p. corylifolia (khatune et al., ) . coumestans- , ′-dihydroxy- ′, ′-dimethyldihydropyrano ( ′, ′: , ) coumestan, -hydroxy- ′-( hydroxy- -methylethyl) ′, ′-dihydrofuro ( ′, ′: , ) coumestan, and sophoracoumestan a have been fractionated from the seeds of p. corylifolia. lin and kuo isolated two new benzofuran derivatives; corylifonol and isocorylifonol along with astragalin, p-hydroxy benzoic acid from p. corylifolia seed extract (lin & kuo, ) . in , zaka and colleague investigated the lipid and fatty acid composition of p. corylifolia. the lipids recognized were triacylglycerols, diacylglycerols, free fatty acids, monoacylglycerols, hydrocarbons, wax esters, and polar lipids. the purified crude lipids of p. corylifolia seeds were analyzed by thin layer and gas chromatographic techniques. among the components, the major polar lipid was c : the sticky and oily pericarp constitute the fruit of p. corylifolia, and chemical investigation revealed some similar compounds as already isolated from seeds. a new isoflavone, corylinin, ( , ′-dihydroxy- ′- in another experiment, hplc protocol was applied to identify some isoflavonoids such as daidzein, genistein, and biochanin a (sehrawat, sangwan, & yadav, ) . further work on the fruit extracted with hexane showed the presence of a phenolic monoterpene known as bakuchiol (cui, taniguchi, kuroda, & hatano, ) . dried p. corylifolia fruit powder extracted with methanol and analyzed on hplc reverse column showed the presence of isoflavonoids known as genistein, daidzein, and biochanin a (hsu, wu, chen, yang, & wang, ) . raun and colleagues have isolated seven compounds from the fruit of p. corylifolia and established the structure of compounds after the spectroscopic analysis. the components identified were corylinin (new compound), psoralen, neobavaisoflavone, sophoracoumestan a, uracil, and daidzin (ruan et al., ) . in , two more new flavonoids, bakuisoflavone, and bakuflavanone were isolated from the fruit of p. corlylifolia with antimicrobial activities (lee, kim, baik, ryu, & lee, ) . in one study, six new flavonoid compounds and a meroterpenoid were isolated and identified by spectroscopic methods from p. corylifolia fruits and displayed medium activity against staph. mutans (won et al., ) . the dried fruits of the plant was investigated, and n-hexane- the roots of p. corylifolia has been investigated for bioactive compounds. it was found that furanocoumarins psoralen and isopsoralen isolated from a petroleum ether extract were responsible for the anti-feedant activity against instar spodoptera litura larvae (sah et al., ; figure ). the above discussions about phytochemical investigation of different parts of p. corylifolia clearly indicates that this plant is a very useful source of variety of bioactive constituents including flavonoids, terpenes, glycosides, alkaloids, coumarins, and others. p. corylifolia is a widely used herb and have many diverse ethnopharmacological and medicinal applications. the numerous chemical and pharmacological research that have been carried out have resulted in the isolation of the diverse bioactive compounds that are summarized below. p. corylifolia proved a promising agent in anti-acne formulations due to the presence of phenolic compounds bakuchiol. it proved to be safe and non-irritant and can be used for longer periods of the day because it showed no irritation and is non-sensitized . one of the bioactive isolated compound "soralen" found to have the ability to stimulate the development of melanin, and therefore it is employed for leucoderma treatment (kim, shim, ahn, & jung, ) . the plant is also used against the skin disease known as psoriasis (chen, yang, & zhang, ) . in one experiment, seeds of p. corylifolia was extracted with hexane and oil in water, cream was prepared with stearic acid as a base. in the next step, an open clinical trial was conducted on patients suffering from eczema for a period of days. after weeks of cream application, symptoms score reduced. final day observation revealed that the symptom scores for eczema reduced from . ± . to . ± . for length of the lesion, from . ± . to . ± . for exudation rate, and from . ± . to . ± . for the rate of itching. this formulation was compared with the placebo preparation, in which the formulated cream contained all the ingredients except for the hexane extract of p. corylifolia. this study concluded that this plant could be effectively used for the treatment of eczema (gidwani et al., ) . p. corylifolia has been tested for antibacterial activity. wang et al. nine compounds exhibited significant antibacterial activity against sa and s. epidermidis (yin et al., that showed . mm zone of inhibition (acharya et al., ) . p. corylifolia in the form of the mouth rinsing solution has prevented the growth of s. mutans bacteria in a very low concentration. along with mouth rinsing ability, the ethanol extract of p. corylifolia was also reported to inhibit the human gingival fibroblast. in the same series of experiments, it was concluded that the extract is safe to use and has no toxic effect in normal doses . in , two more new flavonoids, bakuisoflavone, and bakuflavanone were isolated from the fruit of p. corylifolia and were tested against the strains of mrsa (mrsa and mrsa ) and it was found that two compounds showed some good antibacterial effects with mic values of (> (> . ) and > (> . )) and (> (> . ) and > (> . μg/ml)), respectively . tissue culture studies showed that jasmonic acid treated plants of p. corylifolia enhanced psoralen content in leaves and roots of p. corylifolia. the methanol extract of root sample displayed effective antimicrobial activity against tested bacterial and fungal pathogens in range , , and μg/ml. most effective activity mm zone of inhibition was observed against p. aerugenosa at μg/ml (siva et al., ) . p. corylifolia was also tested against the microbes sa, pseudomonas aeruginosa, and candida albicans, which are known to contaminate the cosmetics products. in the -well microplate assay, it was found to have antimicrobial activity . the a phenolic compound bakuchiol extracted from p. corylifolia (seeds) exhibited antifungal activity against many strains of pathogenic fungi, (hosamani, lakshman, & sandeepkumar, ; lau et al., ; lau et al., ; newton et al., ; prasad, anandi, balasubramanian, & pugalendi, ; savoia, ; srinivasan & sarada, ; yang et al., ) . in another study, activity was found against other fungi such as alternari brassicae, aspergillus niger, fusarium oxysporum, and rhizoctonia cerealis, in which mycelial growth was inhibited (satish, raghavendra, & raveesha, ; vonshak et al., ; yang et al., ) . in one study, p. corylifolia significantly reduced the incidents of the anti-worm property of the seeds of p. corylifolia is clinically proven on roundworms and flatworms (gidwani et al., ) . the seeds and the leaves of p. corylifolia was extracted with water and alcohol, and were tested on the spontaneous movements of setaria cervi whole worm and again on the isolated nerve muscle preparations. the survival of the microfilariae was tested in vitro. the dose required to inhibit the movements of whole worm and nerve muscle preparations for alcohol extracts of leaves were , , and for seeds were , μg/ml (maurya, singh, & seth, ; mendam, kavitha, & naik, ; qamaruddin, parveen, khan, & singhal, ) . volatile oil extracted from the seeds of p. corylifolia displayed strong toxicity against both larvae and adult of the southern house mosquito, the bioactivity guided isolation from chloroform extract of seeds leads to the identification of pure compound, -(- -methylbut- enyl)- n- -dihydroxycoumestan and found active against larvae and adult cx. quinquefasciatus. the same compound has also showed activity against the red flour beetle, tribolium casteneum hebrst. in this detailed study, both the male and female adults were exposed to the compound for - hr period and a range of ld were calculated for dose starting from to ppm and doses were used in five replications. it was concluded that the isolated coumestan compound can be a useful botanical insecticide (dua, kumar, pandey, & kumar, a; khatune et al., ) . an external protozoan parasite ichthyophthirius multifiliis (also called "ich") has been reported to infest freshwater fish species. the p. corylifolia extracted with methanol showed excellent activity against i. multifiliis theronts in concentration of . mg/l or more when was exposed for a period of hr. the p. corylifolia extract at . mg/l concentration has caused % mortality of protomonts and . % of encysted tomonts. it was found that longer period ( hr) and higher concentration ( . mg/l) caused the significant reduction of the survival rate and reproduction of tomont of i. multifiliis, which were exited from the fish after in-bath handling in situ (ling et al., ) . p. corylifolia has been found to be an alternative to malachite green to control i. multifiliis, an external protozoan parasite. the screening showed that p. corylifolia extract have the maximum activity against i. multifiliis theronts. when the experiments were conducted in vivo, at . mg/l or more concentrations of methanol extract of p. corylifolia, it caused % mortality of theronts during the hr of exposure (ling et al., ) . this study showed the damaging effect of p. corylifolia against i. multifiliis trophont in situ. the same study leads to the evaluation of the activity of antiprotozoal compounds extracted form p. corylifolia against i. multifiliis. the two bioactivity guided isolated compounds, isopsoralen and psoralidin, were assessed. in comparison, study of psoralidin and isopsoralen, the inhibitory activity of psoralidin, was found more efficient. further, when experimented in vivo, the compound psoralidin at . mg/l, efficiently reduced the theronts numbers in infected fish over an exposure period of hr. this study showed that psoralidin could be a very good candidate for the formulation as a commercial drug against i. multifiliis . the compounds from p. corylifolia were found to have a protective effect when tested against retinal damage caused by oxidative stress. to evaluate the effects of bakuchiol on the cell viability of dif- (park et al., ) . a recent and very useful research on the same subject of hepatotoxicity shows that the isolated compound bakuchiol when administered at a dose of . and . mg/kg for weeks in rats, many abnormalities were observed in the bakuchiol-treated groups including suppression of weight gain and food intake, change of some parameters in serum biochemistry, and increased weight of liver. the mrna expression of cyp a , hmg-coa reductase, pparα, and srebp- decreased in bakuchiol-treated group, the expression of bsep increased in bakuchiol-treated low dosage, and the expression of bsep decreased in bakuchiol-treated high dosage. it was concluded that bakuchiol could induce cholestatic hepatotoxicity, suggesting potential hepatotoxicity. the mechanism may be related to effects on liver lipid metabolism (li et al., ) . in one study, six compounds isolated from p. corylifolia were studied for their binding affinities for estrogen receptors, erα and erβ, using the yeast transactivation test. among the compounds, bakuchiol was in another study, psoralidin activity as er (α and β) agonist was evaluated in endometrial and human breast cell lines. psoralidin at μm was able to get the highest reporter gene expression conforming to that of e -treated cells and such an activation of the ere-reporter gene by psoralidin was totally stopped by the treatment of a pure er antagonist, indicating that the biological actions of psoralidin are intermediated by er. psoralidin was also able to stimulate the endogenous estrogen-responsive gene (ps ) in mcf- , the human breast cancer cells. it was observed that activation of the classical er-signaling pathway by psoralidin is mediated via induction of er conformation by psoralidin and direct binding of the psoralidin-er complex of the eres present in the promoter region of estrogenresponsive genes. lastly, molecular docking of psoralidin to the ligand-binding pocket of the erα exposed that psoralidin is able to mimic the binding interactions of e , and therefore, in the cellular environment it could act as an er agonist (liu et al., ) . psoralen isolated from p. corylifolia fruits were investigated as an inhibitor of ache enzyme in an attempt to explore its potential for the management of alzheimer's disease. the concentration of psoralen used was - μg/ml. it inhibited the ache in a dose-dependent way in animal models. adult male wistar rats, weighing - g, were used in the study. while a molecular docking study was also carried out, which showed that psoralen binds well within the binding site of the enzyme showing interactions such as π-π stacking and hydrogen bonding (somani et al., ) . although the activity measured in this study was moderate when compared with the standard compound used, despite that, the compound could serve as lead for synthetic analog preparation to improve the inhibitory activity. p. corylifoia also found to possess antidepressant activity. marzieh sarbandi farahani and colleague mentioned the mechanism of action of the plants with antidepressant action and the chemical components isolated from them. they mentioned that psoralidin isolated from seeds of p. corylifolia modify the hypothalamic-pituitary-adrenal axis (farahani, bahramsoltani, farzaei, abdollahi, & rahimi, ) . a similar study was conducted on psoralidin by yi and colleague on the icr strain of male mice. the dose was administered orally in forced swimming test and they observed the increased levels of hydroxytryptamine and -hydroxyindoleacetic acid in the brain and an altered dopamine level. the mechanism for antidepressant activity was proposed to be through involvement of monoamine neurotransmitter and the hypothalamic pituitary adrenal axis systems (yi et al., ) . some previous studies are also available, for example, one study was conducted on mice models and it was concluded that furocoumarins were actually responsible for antidepressant activity. in this study, the well-established antidepressants were used as standards for comparison with the seed extract of p. corylifolia. the dose range used was . to mg/kg in comparison with amitriptyline ( and mg/kg) and fluoxetine ( mg/kg). this study was well designed and results indicate the potential of the seed extract as competitive antidepressant when compared to the conventional therapeutic agents (chopra et al., ) . p. corylifolia also has a wide range of antioxidant activity. different compounds isolated from p. corylifolia were tested for their antioxi- in the understanding of the reputation of this plant species in medicines now, attention has been given to produce callus culture. in one study, relationship between isoflavone and antioxidant activity of p. corylifolia cultures were experimented, and it was found that root-derived callus cultures produced more daidzein, whereas leafdervied callus produced more genistein, and this enhanced production was related to enhanced antioxidant activities (shinde, malpathak, & fulzele, ) . one of the isolated compound, psoralen showed the promising antioxidant activity (ic value = . ± . μg/ml) against the superoxide anion production by human neutrophils in response to formyl-lmethionyl-l-leucyl-l-phenylalanine/cytochalasin b (fmlp/cb; chen et al., ). p. corylifolia extract possesses anti-diabetic activity by its action as the protective effects on pancreatic β cells (behloul & wu, ) . a more detailed biochemical study was conducted on the aqueous extract of seed of p. corylifolia that caused a significant recovery in the activities of hexokinase, glucose- -phosphatase, and glucose- -phosphate dehydrogenase and antioxidant enzymes such as peroxidase, catalase, and superoxide dismutase, along with the lipid peroxidation level in liver tissue and serum transaminase, and corrected the fasting blood glucose level in streptozotocin-induced diabetic rats at a dose of mg/ . ml water/ gm body weight (ghosh, bera, chatterjee, ali, & debasis, ). p. corylifolia has been the part of many ayurvedic formulation that are used for the treatment of various central nervous system conditions such as for neurotropic activity and as central nervous system protective agent (goel & ojha, ) . based on such report, a study was con- in another study, it was revealed that ibc, a flavonoid from p. corylifolia, has the ability to ameliorate the neuronal injury in brain diseases related to inflammation, and this was accomplished through inhibition of lipopolysaccharide induced intercellular adhesion molecule- expression and leukocyte adhesion to brain endothelial cell by blocking toll-like receptor signaling . various studies on animals showed that genistein has the ability to decrease body weight by decreasing food intake. it also reduced the fat pad weight and enhanced the apoptosis of adipose tissues. for example, one such study was conducted on ovariectomised mice. this well-known trihydroxyflavone, genistein, has also been isolated from p. corylifolia, exhibited a potential anti-obesity and obesity related low grade inflammation activities through multiple mechanisms and cell signaling pathways. p. corylifolia extract possesses anti-obesity and antdiabetic activity by its action on adipocyte life cycle, obesity-related low-grade inflammation, and oxidative stress (behloul & wu, ) . the well-known chinese herb p. corylifolia l. (scurfpea fruit) has been employed for the treatment of bone fractures and also for joint diseases for thousands of years. p. corylifolia also improved the pathological bone condition, hyperosteoidosis, by increasing the serum inorganic phosphate level at a dose of mg/kg. it was observed previously that the extract markedly decreased osteoid volume and there has been improvement in bone calcification (miura, nishida, & linuma, ) . the flavonoids of corylin and bavachin has the osteoblastic proliferation stimulating activity in umr cell line cultured in vitro (cho et al., ; wang, li, & jiang, ) . p. corylifolia extract when administered orally to ovx rats, it was noted that there was a decrease in urinary calcium excretion and serum osteocalcin at a dose of - mg/kg body weight. the experiments in this study showed that the extract also increased the bone mineral density and bone formation at mg/kg bw (tsai et al., ) . such experiment, which were extended to months, it can be concluded that p. corylifolia extract can be used at postmenopause state to prevent the osteoporosis. a further in depth study is still needed to make a therapeutic candidate. this study leads to the isolation of the compound responsible for the mentioned activity by weng and colleagues. the isolated compounds from p. corylifolia, known as bakuchiol and bavachin, showed to prevent the estrogen deficiency by inducing the upregulation in primary human osteoblast differentiation (weng et al., ) . an advanced herbal formula containing psoraleae fructus has previously showed promising bone protecting effect when tested in rats, and later on showed excellent results in women with osteoporosis. this herbal formula could efficiently have promoted the osteogenesis and suppress the adipogenesis in mesenchymal stem cells (siu et al., ). an hplc analysis was carried out to study the important constituents in scurfpea fruit. in the method employed, the compounds were identified by comparing their retention indexes with standard substances and it resulted in the identification of compounds. the biological methods such as mtt and alp were employed to study the osteoblasts proliferation and differentiation activity. bavachin and isobavachin showed significant cell proliferation by stimulation, the compound bakuchiol displayed higher effect to boost osteoblasts differentiation. from the results, it was hypothesized that prenyl group as a side chain might be responsible for the activity mentioned, because this structural component was found as a common entity among the compounds tested for activity (li et al., ) . previous surveys revealed bakuchiol and bavachin, the two important components of p. corylifolia l., showed osteoblastic property. both the compounds displayed the prevention of bone loss caused by deficiency of estrogen in overiectomized animal models such as rats. one of in vitro study proposed that bavachin and bakuchiol caused the induction of primary human osteoblast differentiation by up regulating the wnt signaling pathway. this research proposes that boneprotective role makes these two compounds a promising and safe estrogen supplement for the estrogen replacement therapy (weng et al., ) . in one investigation, the compound psoralen with different concentrations ( , , and μm) was tested on chondrocytes isolated from rats at -and -day intervals. it was found that at the low dose concentration psoralen was safe toward chondrocytes; however, at higher dose suppression of chondrocytes, proliferation was observed. the compound psoralen also increased the synthesis of type ii collagen at μm, by . -fold on day and . -fold on day . this was a detailed study, including mts assay, alcian blue colorimetry, western blotting, and qrt-pcr. the compound psoralen was also tested for cytotoxicity and exhibited low cytotoxicity toward chondrocytes at a dose range of - μm. a much higher dose of μm suppression of chondrocytes proliferation was observed. the compound psoralen caused the inhibition of the type i collagen in gene expression and also in protein synthesis. it was concluded that psoralen could be an important biological compound for triggering the cartilaginous cellular functions of chondrocytes . the (jeong et al., ) . this was the first kind of study on p. corylifolia. the plant p. corylifolia extract neutralized the coagulation of caused by naja naja karachiensis snakebite when compared with the antidote used as a standard. the snake venom was experimented on human plasma (citrated) to evaluate its effect on activated partial thromboplastin time (aptt), prothrombin time (pt), and thrombin time (tt). snake venom ( μg/ml) was found to delay pt ( ± . to ± . sec), aptt ( ± . to ± . sec), and tt ( ± . to ± . sec). pt and tt were prolonged, and it suggested the occurrence of thrombin-like or plasminogen activating enzymes (asad et al., ; asad et al., ) . a further in depth study of this activity is still underway in the author's laboratory. the extract of seeds of p. corylifolia have been reported to have stimulant activity against natural killer cells when tested in mice. this study report that the extract also modulates the antibody dependent cellular toxicity. during tumor development, the seed extract also inhibited the antibody complement mediated cytotoxicity. the study was conducted on balb/c male mice. the dose of mg/kg was administered intraperitoneally. blood collected from punctured heart and serum was separated to study the antibody complement-mediated cytotoxicity. the natural killer cells were removed from spleen, and antibodydependent cellular cytotoxicity was assessed (latha, evans, panikkar, & jayavardhanan, ) . the isolated compounds from p. corylifolia including arylcoumarin and psoracoumestan showed strong anticancer potential by strongly hepatocarcinoma cells, it showed its inhibitory activity by inducing the mechanism of apoptosis (guo, liu, ye, & han, ; jiang & xiong, ; khan, iqbal, ahmed, & jamil, ; mohammadparast, rustaiee, rasouli, zardari, & agrawal, ; nehybova, smarda, & benes, ; rajan, tripathi, variyar, & pandey, ; tang et al., ; wong & rabie, ; yang et al., ) . similarly, two more compounds from the same specie identified as ibc and bcn attenuate aβ -induced cell toxicity. the investigation was carried out on yeast two-hybrid system (chen et al., ) . during this study, eight compounds were tested, and among them ibc ( μm) and bcn ( μm) were proved to be active at non-toxic concentrations. the results were confirmed by a standard tht fluorescence method. the compound ibc also exhibited strong inhibitory effect on tht fluorescence, and bcn showed more efficient activity, which was comparable with the reference. moreover, the determination of ic of ibc and bcn in the tht assay were about and μm, respectively. psoralidin in another study, caused the generation of reactive oxygen and it also thought to cause the inhibition of a cell proliferation. the method adopted was mtt assay. this study was designed to study the relationship of time and concentration used. the ic values obtained after -, -and -hr treatment were . , . , and . μm, respectively. (hao, zhang, zhao, & chen, ) . another evidence of anticancer activity came from the compound bakuchiol that suppressed the testosterone induced cell proliferation and gene expression in lncap cells. this study was designed to explore the bakuchiol action in the androgen-dependent pca cell line (lncap). mtt assay and real-time pcr method were employed. the ic of bakuchiol to androgen receptor was . × , which was similar to the standard flutamide ( . × ; miao et al., ) . these experiments showed that bakuchiol is a useful agent for drug development for androgen dependent pca. the same compound, bakuchiol, has also showed a strong anticancer action against human lung adenocarcinoma cell line a and showed much better results than its analogue resveratrol. ic of bakuchiol at hr was . ± . μmol/l, much lower than that of resveratrol ( . ± . μmol/l). bakuchiol triggered the process of apoptosis to a higher level, compared with resveratrol. it was also noted that oxygen species related apoptosis also contribute the cytotoxic properties of bakuchiol, and therefore, it also supports the use of bakuchiol against non-small-cell lung cancer. (chen et al., ) . bakuchiol also showed very selective clearance activity in hepatic stellate cells by a mechanism involving apoptosis. this study showed that p. corylifolia also has anticancer activity in liver cancer (chen et al., ; yang, paik, cho, cho, & kim, ) . in search of cytotoxic compounds from p. corylifolia, two isoflavnoids were isolated, named as corylifols d and e from the ethyl acetate extract. similarly, psoralidin was also found active against stomach carcinoma cell lines (teschke, wolff, frenzel, & schulze, ; yang et al., ) . bakuchiol is also an active ingredient of the dried ripe fruit of p. corylifolia that and corylin from p. corylifolia has been investigated against udp-glucuronosyltransferases and showed strong inhibition (shan et al., ) . it also inhibits lipopolysaccharide-induced endothelial-mesenchymal transition via down regulation of the nf-κb-snail signaling pathway (jung et al., ) . in one investigation against dna polymerase enzyme, p. corylifolia extract showed strong inhibitory activity. importance of isolated components from p. corylifolia is obvious from the fact that compounds such as psoralen is being produced from callus derived from various plant portions. in one experiment, it was found that in the in vitro conditions, cinnamic acid proved a very strong precursor of psoralen pathway that induced a maximum amount of psoralen (mohammadparast, rustaiee, rasouli, zardari, & agrawal, succeeding experiments with many kinase inhibitors propose that pf-mediated degradation of cyclin d and cdk is dependent on erk / and/or gsk β (park, sung, song, & jeong, ) . the crude extract of p. corylifolia with solvent ethanol was found a strong dna polymerase inhibitor of dna replication enzyme in an activity directed isolation assay that resulted in the purification of novel compound corylifolin, bakuchiol, neobavaisoflavone, and resveratrol. in a similar enzyme assay, some topoisomerase ii inhibitors were also isolated, namely, daidzein and bakuchicin (sun, woo, cassady, & snapka, ) . many studies have been designed so far to study the behavior of p. corylifolia extract and isolated compounds on important enzyme. additionally, the inhibition kinetics were calculated by dixon and lineweaver-burk plots for the inhibitory activities toward ces . the inhibition kinetic parameters (k i ) were calculated to be . , . , . , . , and . μm for neobavaisoflavone, corylifolinin, coryfolin, corylin and bcn, respectively. it was concluded that this inhibition by the constituents might be responsible for the possible adverse effects of fp through the disrupting ces -catalyzed metabolism of endogenous substances and xenobiotics (sun et al., ) . cytochrome p (cyp) is an assembly of heme-containing enzymes fixed essentially in the lipid bilayer of the endoplasmic reticulum, and helps metabolize several drugs and carcinogens. the plant p. corylifolia has been reported to be used in cardiac problems in traditional medicine (kr, ) . bavachalcone, a compound from p. corylifolia, has reported to have increased the luciferase activity of the manganese superoxide dismutase (mnsod) promoter and enhanced mnsod mrna and protein expressions. further, it was found that bavachalcone suppressed the mitochondrial superoxide production in endothelial cells. on the other hand, bavachalcone (in concentration range of , . , and μmol/l for hr) stimulated liver kinase b and ampkα phosphorylation in a converse manner. mrna interfering by using short hairpin rna (shrna) of amp-activated protein kinase (ampk) inhibited bavachalcone-induced mnsod expression. moreover, ampk reduced by shrna-ampk reversed the inhibition of bavachalcone on mitochondrial superoxide production in endothelial cells. these results showed that bavachalcone could shield the endothelial function by enhancing the ampk activity and mnsod expression and lowering the mitochondrial oxidative stress, which is considered to be the key etiological reason in cardiovascular diseases (dang et al., ) . many herbal remedies, including essential oils, contain psoralen, and methoxy psoralen are reported to cause photosensitivity in the form of erythema and blisters (koh & ong, ) . p. corylifolia has been the part of many herbal remedy used to treat vitiligo and patients using such treatments in the form of creams are also reported to experienced erythema when exposed to sunlight (maurice & cream, ) . p. corylifolia is frequently indicated for vitiligo in india, and it contains psoralen, isopsoralen, and psoralidin. its extracts when tested on guinea pig skin have been stated to possess potent sensitizing action (pathak, daniels, & fitzpatrick, ) . another study reported the gonadal toxicity. although the ethanol extract of p. corylifolia seeds are proposed to use in processed food preservation, but it showed toxicity when tested on male and female rats. the period of the experiment was days, and various concentrations were administered were %, . %, . %, . % or . %. histopathological investigation showed atrophy of seminiferous tubules, leydig cells, seminal vesicles, and prostate cells were observed in male rats administered with the . % and . %. with the same concentrations, the female rats showed a reduced number of corpora lutea in the ovaries and less frequent endometrial glands in the uterus. it was suggested that the extract caused the hypothalamus-pituitary-gonadal axis (takizawa et al., ) . in one investigation, p. corylifolia and its natural compounds (bavachin, corylifol a, neobavaisoflavone, ibc, and bcn) were evaluated for its potential toxicity, and results showed it had a potent inhibitory effect against human udp-glucuronosyltransferase a (ugt a ), and it is considered as main stimulant for p. corylifolia related toxicity, including hepatic injury and raised bilirubin levels . in another study p. corylifolia extract and fractionated compounds such as psoralen and isopsoralen were incubated with the recombinant cyp a enzyme or differentiated huh- and heparg cells. p. corylifolia extract, psoralen, and isopsoralen caused the inhibition of concentration cyp a activity in a dose dependent manner with different potency in vitro. it was also noted that none of the sample tested showed any toxicity (liu & flynn, ) . several compounds isolated from p. corylifolia have many industrial applications and are available commercially. some of the examples are the following: the compound angelicin (cas - - ) is available as antifungal compound (sardari, amin, sekhon, micetich, & daneshtalab, ) . the compound psoralen has been used as photochemical probe in studies of dna mutation and repair mechanisms (zarmouh, eyunni, & soliman, ) . methoxsalen ( -methoxypsoralen; cas - - ) has been known as a potent suicide inhibitor of cytochrome p- (toyooka & ibuki, ). bakuchiol (cas - - ) is available as a ptp b and dna polymerase inhibitor (choi et al., ) . bavachin (cas - - ) is available as a weak antioxidant that stimulates bone formation (jing et al., ) . corylifol c and, to a lesser extent, xanthoangelol are potent protein kinase inhibitors (inhibitory concentration % values for epidermal growth factor receptor; limper et al., ) . a well-known compound daidzin (cas - - ) isolated from many plant species is a potent inhibitor of human mitochondrial aldehye dehydrogenase that demonstrates chemopreventitive activities (keung & vallee, ) . the compound genistein (cas - - ) is available as a highly specific inhibitor of protein tyrosine kinase (zarmouh, messeha, elshami, & soliman, ) . these examples clearly show the importance of bioactive compounds isolated from p. corylifolia, an important traditional medicinal plant. the above-mentioned summary about p. corylifolia clearly showed that it is a very important plant from ethnobotanical, pharmacological, and chemical point of view. to date, more or less hundreds compounds have been separated from p. corylifolia, which was also mentioned by zhang et al., ( ) . we here presented the latest version of scientific literature on p. corylifolia, including the research work carried out in the recent years. p. corylifolia contains a wide variety of chemical constituents belonging to various groups, including flavonoids, coumarins, and meroterpenes, which are more dominant. p. corylifolia l. (fabacese) is a fortified source of biological active compounds, which gives the plant with great value for its use in pharmaceuticals, health, and body-care products. sehrawat and colleagues reported that p. corylifolia is a rare and endangered herbaceous medicinal plant. because most of the plant materials are collected from naturally occurring stands and are therefore being depleted rapidly and there is a possibility of extinction. therefore, it is suggested that there is a need for in vitro propagation of this species (sehrawat et al., ) . authors declare that there is no conflict of interest. anti microbial activity of different dosage forms of bakuchi (psoralea corylifolia linn.) taila, an ayurvedic formulation medicinal plants of bombay presidency efficacy of seed hydropriming with phytoextracts on plant growth promotion and antifungal activity in maize antipsoriatic microemulsion gel formulations for topical drug delivery of babchi oil (psoralea corylifolia) phytochemical studies and antimicrobial screening of non/less-polar fraction of psoralea corylifolia by using gc-ms psoralen photochemotherapy of cutaneous disorders compensatory effects of medicinal plants of pakistan upon prolongation of coagulation assays induced by naja naja karachiensis bite anti-venom potential of pakistani medicinal plants: inhibition of anticoagulation activity of naja naja karachiensis toxin biosynthesis of bakuchiol, a meroterpene from psoralea corylifolia medicinal and poisonous plants of pakistan genistein: a promising therapeutic agent for obesity and diabetes treatment systema naturae antibacterial activity of psoralea corylifolia l. seed and aerial parts with various extraction methods bakuchiol: a retinol-like functional compound, modulating multiple retinol and non-retinol targets new isoflavones and bioactive constituents from the fruits of psoralea corylifolia isobavachalcone and bavachinin from psoraleae fructus modulate aβ aggregation process through different mechanisms in vitro anti-tumor effects of bakuchiol, an analogue of resveratrol, on human lung adenocarcinoma a cell line bakuchiol: a hepatoprotective compound of psoralea corylifolia on tacrine-induced cytotoxicity in hep g cells prediction of drug-induced liver injury in hepg cells cultured with human liver microsomes psoralea corylifolia l. (buguchi)-folklore to modern evidence indigenous drugs of india ( nd edn.) un dhar and sons indigenous drugs of india natural products from plants constituents of psoralea corylifolia fruits and their effects on methicillin-resistant staphylococcus aureus extracts of canadian first nations medicinal plants, used as natural products, inhibit neisseria gonorrhoeae isolates with different antibiotic resistance profiles chinese medicine bavachalconeinduced manganese superoxide dismutase expression through the amp-activated protein kinase pathway in human endothelial cells the wealth of india: raw materials insecticidal and genotoxic activity of psoralea corylifolia linn.(fabaceae) against culex quinquefasciatus say insecticidal and genotoxic activity of psoralea corylifolia linn.(fabaceae) against culex quinquefasciatus say plant-derived natural medicines for the management of depression: an overview of mechanisms of action antidiabetic and antioxidative effects of aqueous extract of seed of psoralea corylifolia (somraji) and seed of trigonella foenum-graecum l.,(methi) in separate and composite manner in streptozotocin-induced diabetic male albino rat evaluation of a novel herbal formulation in the treatment of eczema with psoralea corylifolia ashtang ghrita: a noble ayurveda drug for central nervous system inhibitory effects of osthole, psoralen and aconitine on invasive activities of breast cancer mda-mb- bo cell line and the mechanisms. zhong xi yi jie he xue bao= quality standards of indian medicinal plants corylinal: a new isoflavone from seeds of psoralea corylifolia phytochemical analysis of methanol extracts of psoralea corylifolia psoralidin induces autophagy through ros generation which inhibits the proliferation of human lung cancer a cells antimicrobial activity of leaf extract of psoralea corylifolia l the presence of three isoflavonoid compounds in psoralea corylifolia the pharmacology of chinese herbs neuroprotective effects of psoralea corylifolia linn seed extracts on mitochondrial dysfunction induced by -nitropropionic acid cytotoxicity of corylifoliae fructus. ii. cytotoxicity of bakuchiol and the analogues studies on lymphangiogenesis inhibitors from korean and japanese crude drugs induction of apoptosis in human hepatocarcinoma smmc- cells in vitro by psoralen from psoralea corylifolia isobavachalcone attenuates mptp-induced parkinson's disease in mice by inhibition of microglial activation through nf-κb pathway medicinal plants. oxford and ibh publishing medicinal plants. tropical horticulture plant systematics: a phylogenetic approach aqueous extract of psoralea corylifolia l. inhibits lipopolysaccharideinduced endothelial-mesenchymal transition via downregulation of the nf-κb-snail signaling pathway handbook of ayurvedic medicinal plants: herbal reference library in vitro antimicrobial activities of bakuchiol against oral microorganisms. antimicrobial agents and chemotherapy daidzin: a potent, selective inhibitor of human mitochondrial aldehyde dehydrogenase antioxidant, hemolytic and mutagenic potential of psoralea corylifolia encyclopedia of indian medicinal plants indian medicinal plants: an illustrated dictionary pesticidal activity of a novel coumestan derivative isolated from psoralea corylifolia linn. against tribolium casteneum herbst. adults and larvae (coleptera: tenebrionidae) antibacterial compounds from the seeds of psoralea corylifolia phenolic phytochemical displaying sars-cov papain-like protease inhibition from the seeds of psoralea corylifolia protective effects of the compounds isolated from the seed of psoralea corylifolia on oxidative stress-induced retinal damage antimicrobial effect of commercially available mouth rinsing solutions and natural herbal extracts on streptococcus mutans selective inhibition of bakuchicin isolated from psoralea corylifolia on cyp a in human liver microsomes. evidence-based complementary and alternative medicine phytophotodermatitis due to the application of citrus hystrix as a folk remedy indian medicinal plants the wealth of india: raw materials: vol. viii. ph-re. the wealth of india: raw materials cytotoxicity of corylifoliae fructus. i. isolation of the effective compound and the cytotoxicity immunomodulatory and antitumour properties of psoralea corylifolia seeds two antifungal components isolated from fructus psoraleae and folium eucalypti globuli by bioassay-guided purification anti-dermatophytic activity of bakuchiol: in vitro mechanistic studies and in vivo tinea pedis-inhibiting activity in a guinea pig model isobavachalcone attenuates lipopolysaccharide-induced icam- expression in brain endothelial cells through blockade of toll-like receptor signaling pathways phytoestrogen bakuchiol exhibits in vitro and in vivo anti-breast cancer effects by inducing s phase arrest and apoptosis osteoblasts proliferation and differentiation stimulating activities of the main components of fructus psoraleae corylifoliae fructus psoraleae contains natural compounds with potent inhibitory effects towards human carboxylesterase bakuchiol contributes to the hepatotoxicity of psoralea corylifolia in rats estrogenic activities of psoralea corylifolia l. seed extracts and main constituents compounds isolated from psoralea corylifolia seeds inhibit protein kinase activity and induce apoptotic cell death in mammalian cells analysis of bakuchiol, psoralen and angelicin in crude drugs and commercial concentrated products of fructus psoraleae two new benzofuran derivatives, corylifonol and isocorylifonol from the seeds of psoralea corylifolia antiprotozoal screening of traditional medicinal plants: evaluation of crude extract of psoralea corylifolia against ichthyophthirius multifiliis in goldfish antimalarial activity of artemisia annua flavonoids from whole plants and cell cultures preparative isolation and purification of psoralen and isopsoralen from psoralea corylifolia by high-speed counter-current chromatography psoralidin, a coumestan analogue, as a novel potent estrogen receptor signaling molecule isolated from psoralea corylifolia cyp a inhibition by psoralea corylifolia and its major components in human recombinant enzyme, differentiated human hepatoma huh- and heparg cells useful plants of the genus psoralea granzyme a cleaves a mitochondrial complex i protein to initiate caspaseindependent cell death the dangers of herbalism potential medicinal plants for lymphatic filariasis: a review natural sources used for treatment and prevention of filariasis bakuchiol inhibits the androgen induced-proliferation of prostate cancer cell line lncap through suppression of ar transcription activity effect of crude fractions of psoralea corylifolia seed extract on bone calcification effect of crude fractions of psoralea corylifolia seed extract on bone calcification in vitro enhancement of psoralen as an important anticancer compound in psoralea corylifolia through precursor feeding indian materia medica with ayurvedic plant coumestans: recent advances and future perspectives in cancer therapy. anti-cancer agents in medicinal chemistry the evaluation of forty-three plant species for in vitro antimycobacterial activities; isolation of active constituents from psoralea corylifolia and sanguinaria canadensis phytochemical and pharmacognostic investigation of antidiabetic scoparia dulcis linn scrophulariaceae whole plant grown in nigeria herbs cultivation and medicinal uses protective effect of (s)-bakuchiol from psoralea corylifolia on rat liver injury in vitro and in vivo anti-cancer activity of psoralea fructus through the downregulation of cyclin d and cdk in human colorectal cancer cells the presently known distribution of furocoumarins (psoralens) in plants larvicidal property of essential oils against culex quinquefasciatus say (diptera: culicidae). industrial crops and products ayurvedic medicine: the principles of traditional practice antidermatophytic activity of extracts from psoralea corylifolia (fabaceae) correlated with the presence of a flavonoid compound potential antifilarial activity of the leaves and seeds extracts of psoralea corylifolia on cattle filarial parasite setaria cervi chemical fingerprint and quantitative analysis of fructus psoraleae by high-performance liquid chromatography mechanism of cytotoxicity by psoralea corylifolia extract in human breast carcinoma cells medicinal plants history, cultivation and uses studies on the chemical constituents of psoralea corylifolia l screening for inhibition activity of plant extracts on microorganism contaminating in cosmetics isolation and identification of furocoumarins from the seeds of psoralea corylifolia linn antifungal activity of diplotaenia damavandica antifungal potentiality of some plant extracts against fusarium sp plant-derived antimicrobial compounds: alternatives to antibiotics psoralea corylifolia l. an endangered medicinal plant with broad spectrum properties protective role of psoralea corylifolia l. seed extract against hepatic mitochondrial dysfunction induced by oxidative stress or aging comparison of the inhibitory potential of bavachalcone and corylin against udp-glucuronosyltransferases database on medicinal plants used in ayurveda agro-techniques of medicinal plants determination of isoflavone content and antioxidant activity in psoralea corylifolia l. callus cultures the effects of an antiosteoporosis herbal formula containing epimedii herba, ligustri lucidi fructus and psoraleae fructus on density and structure of rat long bones under tail-suspension, and its mechanisms of action optimization of elicitation condition with jasmonic acid, characterization and antimicrobial activity of psoralen from direct regenerated plants of psoralea corylifolia l systema naturae the netherlands in vitro acetylcholinesterase inhibition by psoralen using molecular docking and enzymatic studies in vitro and in vivo assessment of the effect of antiprotozoal compounds isolated from psoralea corylifolia against ichthyophthirius multifiliis in fish antifungal activity of phenyl derivative of pyranocoumarin from psoralea corylifolia l. seeds by inhibition of acetylation activity of trichothecene -o-acetyltransferase (tri ) pharmacognostic evaluation of the root of berberis asiatica fabaceae" angiosperm phylogeny website, ed. missouri botanical garden and university of inhibition behavior of fructus psoraleae's ingredients towards human carboxylesterase (hces ) dna polymerase and topoisomerase ii inhibitors from psoralea c orylifolia gonadal toxicity of an ethanol extract of psoralea corylifolia in a rat -day repeated dose study psoralen stimulates osteoblast differentiation through activation of bmp signaling review article: herbal hepatotoxicity-an update on traditional chinese medicine preparations new constituents from psoralea corylifolia histone deacetylase inhibitor sodium butyrate enhances the cell killing effect of psoralen plus uva by attenuating nucleotide excision repair psoralea corylifolia extract ameliorates experimental osteoporosis in ovariectomized rats screening south indian medicinal plants for antifungal activity against cutaneous pathogens osteoblastic proliferation stimulating activity of psoralea corylifolia extracts and two of its flavonoids chemical constituents from psoralea corylifolia and their antioxidant alphaglucosidase inhibitory and antimicrobial activities. zhongguo zhong yao za zhi= zhongguo zhongyao zazhi= chemical constituents from psoralea corylifolia and their antioxidant alphaglucosidase inhibitory and antimicrobial activities identification and characterization of naturally occurring inhibitors against udp-glucuronosyltransferase a in fructus psoraleae (bugu-zhi) indian medicinal plants. a compendium of species positive skeletal effect of two ingredients of psoralea corylifolia l. on estrogen deficiency-induced osteoporosis and the possible mechanisms of action bioactive metabolites from the fruits of psoralea corylifolia effect of psoralen on bone formation bisbakuchiols a and b, novel dimeric meroterpenoids from psoralea corylifolia effects of gaultheria yunnanensis on adjuvant arthritis in rats. zhongguo zhong yao za zhi= zhongguo zhongyao zazhi= psoralen activates cartilaginous cellular functions of rat chondrocytes in vitro psc-afp, an antifungal protein with trypsin inhibitor activity from psoralea corylifolia seeds resveratrol induces the suppression of tumor-derived cd + cd + regulatory t cells the cytotoxicity of psoralidin from psoralea corylifolia the osteoprotective effect of psoralen in ovariectomy-induced osteoporotic rats via stimulating the osteoblastic differentiation from bone mesenchymal stem cells antidepressant-like effects of psoralidin isolated from the seeds of psoralea corylifolia in the forced swimming test in mice psoracorylifols a-e, five novel compounds with activity against helicobacter pylori from seeds of psoralea corylifolia antibacterial prenylflavone derivatives from psoralea corylifolia, and their structure-activity relationship study lipid class and fatty acid composition of psoralia corylifolia seed the benzopyrone biochanin-a as a reversible, competitive, and selective monoamine oxidase b inhibitor evaluation of the isoflavone genistein as reversible human monoamine oxidase-a and-b inhibitor. evidence-based complementary and alternative medicine the chemical constituents and bioactivities of psoralea corylifolia linn.: a review psoralea corylifolia l: ethnobotanical, biological, and chemical aspects: a review key: cord- - k re y authors: daniell, henry; streatfield, stephen j; wycoff, keith title: medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants date: - - journal: trends in plant science doi: . /s - ( ) - sha: doc_id: cord_uid: k re y abstract the use of plants for medicinal purposes dates back thousands of years but genetic engineering of plants to produce desired biopharmaceuticals is much more recent. as the demand for biopharmaceuticals is expected to increase, it would be wise to ensure that they will be available in significantly larger amounts, on a cost-effective basis. currently, the cost of biopharmaceuticals limits their availability. plant-derived biopharmaceuticals are cheap to produce and store, easy to scale up for mass production, and safer than those derived from animals. here, we discuss recent developments in this field and possible environmental concerns. research in the past few decades has revolutionized the use of therapeutically valuable proteins in a variety of clinical treatments. because most genes can be expressed in many different systems, it is essential to determine which system offers the most advantages for the production of the recombinant protein. the ideal expression system would be the one that produces the most safe, biologically active material at the lowest cost. the use of modified mammalian cells with recombinant dna techniques has the advantage of resulting in products that are identical to those of natural origin; however, culturing these cells is expensive and can only be carried out on a limited scale. the use of microorganisms such as bacteria permits manufacture on a larger scale, but introduces the disadvantage of producing products that differ appreciably from the products of natural origin. for example, proteins that are usually glycosylated in humans are not glycosylated by bacteria. furthermore, human proteins that are expressed at high levels in e. coli frequently acquire an unnatural conformation accompanied by intracellular precipitation, owing to lack of proper folding and disulfide bridges. the production of recombinant proteins in plants has many potential advantages for generating biopharmaceuticals relevant to clinical medicine. first, plant systems are more economical than industrial facilities using fermentation or bioreactor systems. second, the technology is already available for harvesting and processing plants and plant products on a large scale. third, the purification requirement can be eliminated when the plant tissue containing the recombinant protein is used as a food (edible vaccines). fourth, plants can be directed to target proteins into intracellular compartments in which they are more stable, or even to express them directly in certain compartments (chloroplasts). fifth, the amount of recombinant product that can be produced approaches industrial-scale levels. last, health risks arising from contamination with potential human pathogens or toxins are minimized. in the decade since the expression and assembly of immunoglobulin (ig) heavy and light chains into functional antibodies was first shown in transgenic tobacco, plants have proven to be versatile production systems for many forms of antibodies. these include full-sized igg and iga, chimeric igg and iga, secretory igg and iga, single-chain fv fragments (scfv), fab fragments and heavy-chain variable domains. recently, this list has been extended to include bispecific antibodies, which are made by the genetic fusion of two different scfvs via a flexible peptide linker . plants have great potential as a virtually unlimited source of inexpensive monoclonal antibodies (dubbed 'plantibodies') for human and animal therapeutics (table ) . there is not yet a consensus as to the best plant species or tissue for commercial antibody production. most antibodies expressed to date have been in tobacco, although recently potatoes, soybean, alfalfa, rice and wheat have also been used successfully - . the major advantage of using green tissue (tobacco, alfalfa, soybean) is sheer productivity. both alfalfa and tobacco can support several crops (cuttings) per year, with potential annual biomass yields of tonne ha − and > tonne ha − , respectively. by contrast, the maximum yields of wheat, rice and corn seed are ~ tonne ha − , tonne ha − and tonne ha − , respectively. other advantages of tobacco include its relative ease of genetic manipulation, production of large numbers of seeds (up to a million per plant) and an impending need to explore alternate uses for this hazardous crop. however, seeds are likely to have fewer phenolic compounds and a less complex mixture of proteins and lipids than green leaves, which might be an advantage in purification. another advantage of seeds or tubers is their ability to be stored for long periods. levels of scfv in rice seeds did not show a significant decline after storage at room temperature for six months . potato tubers in cold storage for months lost only % of functional antibody . for short periods of time (five to seven days), dried tobacco and alfalfa leaves can also be stored with little loss of scfv (ref. ) or igg antibody . purification of antibody from stored plant material has the advantages that the processing facility need not be near the field and can be used continually all year, rather than for just a few large batches. to date, only four antibodies have been made in plants that are potentially useful as human therapeutics. only one of these has been tested in humans: a chimeric secretory igg-iga antibody against a surface antigen of streptococcus mutans, the primary causal agent of tooth decay. this tobaccoproduced antibody was applied topically to teeth and found to be as effective as an igg produced in a murine hybridoma at preventing recolonization by s. mutans . the second antibody, a humanized anti-herpes-simplex virus (hsv) antibody made in soybean, was effective in the prevention of vaginal hsv- transmission in a mouse model . its activity was indistinguishable both in vitro and in vivo from the monoclonal antibody produced in cell culture. a third antibody, against carcinoembryonic antigen (cea), has recently been expressed in rice and wheat . cea, a cell-surface glycoprotein, is one of the best-characterized tumor-associated antigens. antibodies against cea are used for in vivo tumor imaging, as well as in antibody-based cancer therapy. levels of scfv in seeds did not show a significant decline after storage at room temperature for six months. this same antibody has been expressed in a rice cell culture . the fourth antibody is an example of both a novel use of plant-produced antibodies and an alternative production system. a plant virus vector has been used to produce a tumor-specific vaccine transiently in tobacco for the treatment of lymphoma . the antibody genes for expression of an scfv were derived from a mouse b-cell lymphoma. the plantproduced scfv was used to immunize mice, which generated anti-idiotypic antibodies (antibodies against the binding portion of the antibody). these mice were protected against infection by the lymphoma that produced the original antibody. other groups have used modified plant viral vectors to produce therapeutically useful antibodies in plants, including an antibody against the colorectalcancer-associated antigen ga - (ref. ). although these vectors might find limited usefulness if the rapid production of an antibody is necessary (perhaps in greenhouse production), their acceptability to regulatory agencies (e.g. the us food and drug administration, dept of agriculture and environmental protection agency) has not been tested. there are no plantibodies yet in commercial production, therefore estimates of cost are difficult to find and involve many assumptions. the costs of producing an igg from alfalfa grown in a m greenhouse are estimated to be us$ - g − , compared with us$ g − for the hybridomaproduced antibody . planet biotechnology (mountain view, ca, usa) has compared the cost per gram of purified iga made by cell culture, transgenic goats, grain ( . tonne ha − ) and green biomass ( . tonne ha − ) (fig. ) . expression levels will have a significant impact on the costs but, at the best expression level reported [ µg g − leaf for a secretory iga (ref. )], the final cost should be well below us$ g − . this significantly undercuts the costs of cell culture (us$ g − ) or transgenic animal production systems (us$ g − ). the biggest component of cost with plantibodies will be purification. however, expression in seeds of rice and wheat opens up the possibility of oral administration of some therapeutic antibodies without the need for expensive purification. some of the properties of igs depend on their glycosylation (e.g. binding to monocyte fc receptors). there is one conserved n-glycosylation site in the ch domain of igg. the structures of n-linked glycans on plant-and murine-produced guy's (an igg ) have been determined and compared . the plantibody n-glycans were more structurally diverse, with % being of the high-mannose type. the other % of the plantibody oligosaccharides had β-( , )-xylose and α-( , )-fucose linked to the man glcnac core. these linkages are typical of plants but are not found in mammalian n-glycans. the plantibody also lacked sialic acid, which represented ~ % of the sugar content of the mouse monoclonal antibody. these differences in glycan structure appear to have no effect on antigen binding or affinity in vitro , , , and might not be significant in vivo either. an igg produced in alfalfa had a serum half-life in balb/c mice that was indistinguishable from that of the hybridoma-produced antibody . however, there is some concern about the potential immunogenicity and allergenicity of plantibodies used as human therapeutics. for mucosal applications, this is not likely to present problems for most people because plant glycoproteins are ubiquitous in the human diet. there has been no evidence of allergic reaction or of a human antimouse antibody (hama) response in patients receiving topical oral application of a secretory iga specific to s. mutans . proteins of microbial and viral pathogens were some of the earliest examples chosen to show the feasibility of transgenic plant expression systems - . the rationale was that key immunogenic proteins of major pathogens could be synthesized in plant tissues and then fed as edible . costs for plants compare green biomass ( . tonne ha − ) and seed production ( . tonne ha − ). cost differences are based primarily on production costs, and it was assumed that purification costs and losses during purification will be the same for all systems. subunit vaccines to humans or commercially important animals. the proof of this concept has since been shown using several bacterial and viral proteins ( table ). the practical aspects of choosing particular foodstuffs in which to deliver defined doses of a vaccine are being explored, and efforts are under way to establish clear regulatory paths for the development of edible vaccines. oral delivery of vaccines is an attractive alternative to injection, largely for reasons of low cost and easy administration. the chances of acquiring mucosal immunity against infectious agents that enter the body across a mucosal surface are also increased with oral vaccines. however, a major concern with oral vaccines is the degradation of protein components in the stomach and gut before they can elicit an immune response. to guard against degradation, several delivery vehicles have been developed to ferry intact proteins to the gut. these include recombinant strains of attenuated microorganisms, bioencapsulation vehicles such as liposomes and transgenic plant tissues. early work with plant-based subunit vaccines used the readily transformed species tobacco, potato and tomato - . however, the most attractive species for expressing subunit vaccine components should have high levels of soluble protein that is stable during storage; seed crops such as cereals are particularly suitable. the embryo fraction is rich in soluble protein and can easily be separated from other seed tissue to increase the concentration of antigen and thus decrease the dose size. the choice of crop defines the type of material to be fed. many plant tissues can be consumed raw but others must be processed. processing facilitates the creation of a homogeneous sample, enabling a defined dose size, but it is important that any heat or pressure treatments involved do not destroy the antigen. alternative processing steps have been applied to a candidate vaccine component against enterotoxigenic strains of e. coli that consists of the b subunit of the heat-labile toxin (lt-b) expressed in corn. a typical mg dose of lt-b could be delivered in an embryo fraction, to decrease the volume of the dose, or in a 'cooked' whole corn snack, to increase palatability and enhance stable storage (fig. ) . in this case, neither treatment degrades the antigen. for commercial animal vaccines, the relevant protein can be expressed in a plant tissue that constitutes a major proportion of the diet, and heat and pressure treatments are not necessary. some key examples that illustrate the range of candidate proteins under investigation and plant expression systems being used are given in table . plant-expressed antigens have been shown to able to induce mucosal and serum immune responses when administered parenterally or orally to experimental animals and, in some test cases, they have offered protection against a subsequent pathogen challenge or challenge model , , [ ] [ ] [ ] [ ] [ ] [ ] . a few of these vaccine candidates have been successfully tested in clinical trials or, where appropriate, in commercial or native animal trials [ ] [ ] [ ] [ ] [ ] [ ] . thus, edible vaccines delivered in plant tissues or processed plant products show great potential for efficacy in target organisms. the bioencapsulation of lt-b in transgenic corn material results in an increased mucosal immune response compared with that achieved with naked antigen when fed to mice . presumably, this is because the antigen is protected from degradation in the gut, and it augurs well for the development of plant-based edible vaccines. the quantity of plant tissue constituting a vaccine dose must be of a practical size for consumption. thus, achieving a high level of expression is crucial. the expression of vaccine components in plants has been increased by using a range of leader and polyadenylation signals and by optimizing codon usage for plants , , . expression could also be raised through crosses of transformed lines to various genetic backgrounds, an approach that has been successfully applied to boost protein production in corn. it is also important that any vaccine component should be present in its native form in the transgenic plant tissue. this has been assessed in several cases by examining the size of the synthesized protein, its ability to form higher-order complexes that mirror microbial or viral structures and, where relevant, by showing an enzymatic or receptor-binding activity , - , , , . the stability of heterologous proteins and the assembly of multisubunit structures depend on the cellular environment and therefore on the subcellular location. favored locations for the expression of selected subunit vaccine components are the cell surface and the endoplasmic reticulum and golgi body , , , , . as with antibodies, transient expression systems (in which candidate vaccine sequences are incorporated into plant viral surface proteins) have also been investigated extensively and high levels of expression have been achieved. a related strategy to that of edible vaccines uses transgenic plants expressing autoantigens, whereby a large oral dose of an autoantigen can inhibit the development of an autoimmune disease through the mechanism of oral tolerance. this approach has been successful in a mouse model for diabetes . generally, levels of pharmaceutical proteins produced in transgenic plants have been less than the % of total soluble protein that is needed for commercial feasibility if the protein must be purified . plantderived recombinant hepatitis-b surface antigen induced only a low level serum antibody response in a small human study, probably reflecting the low level of expression ( - ng g − fresh weight) in transgenic lettuce . in spite of recent improvements in expression levels in potato with a view to clinical trials , expression levels should be increased further for practical purposes. also, even though norwalk virus capsid protein expressed in potatoes caused oral immunization when consumed as food, expression levels are too low for large-scale oral administration ( . % of total soluble protein) , . expression of genes encoding other human proteins in transgenic plants has been disappointingly low: human serum albumin, . % total soluble protein; human protein c, . % total soluble protein; erythropoietin,~ . % total soluble protein; and human interferon-β, < . % fresh weight (table ) . a synthetic gene coding for the human epidermal growth factor was expressed only up to . % of total soluble protein in transgenic tobacco , . in spite of several successful reports of high-level expression of non-human proteins (e.g. phytase, glucanase) via the nuclear genome, there is a great need to increase expression levels of human blood proteins to enable the commercial production of pharmacologically important proteins in plants. one alternative approach is to express foreign proteins in chloroplasts of higher plants. foreign genes have been integrated into the tobacco chloroplast genome, giving up to copies per cell and resulting in the accumulation of recombinant proteins at up to % of the total soluble protein . chloroplast transformation uses two flanking sequences that, through homologous recombination, insert foreign dna into the spacer region between the functional genes of the chloroplast genome, thus targeting the foreign genes to a precise location. this eliminates the 'position effect' upon expression that is frequently observed in transgenic plants with genes inserted into the nuclear genome. in addition, gene silencing has not been observed with chloroplast transformation, whereas it is a common phenomenon with nuclear transformation. chloroplast genetic engineering is an environmentally friendly approach, minimizing several environmental concerns , . importantly, chloroplasts can process eukaryotic proteins, including enabling correct folding and the formation of disulfide bridges. chaperonin proteins are present in chloroplasts and might function in the folding and assembly of non-native proteins of both prokaryotic and eukaryotic origins. also, chloroplast proteins are activated by disulfide bond oxidation-reduction cycles using the plastid thioredoxin system or protein disulfide isomerase . accumulation of large quantities of a fully assembled form of human somatotropin with the correct disulfide bonds ( % total soluble protein) provides strong evidence for hyperexpression and assembly of pharmaceutical proteins using this approach. such folding and assembly of foreign proteins should eliminate the need for expensive in vitro processing of pharmaceutical proteins produced in recombinant organisms. for example, % of the total operating cost for the commercial production of human insulin in e. coli is associated with in vitro processing (formation of disufide bridges and cleavage of methionine) . purification is likely to represent most of the cost of biopharmaceutical production in plants. for the commercial production of insulin in e. coli, chromatography accounts for % of operating expenses and % of equipment costs . therefore, new approaches are necessary to minimize or eliminate chromatography in the production of pharmaceutical proteins. one successful recent approach is targeting pharmaceutical proteins to seed oil bodies. this was shown with hirudin, an anticoagulant first isolated from the leech hirudo medicinalis. an oleosin-hirudin fusion protein has been targeted to oil bodies of brassica napus seeds and purified by flotation centrifugation for commercial production in canada . another novel approach is the use of gvgvp as a fusion protein to facilitate single-step purification without the use of chromatography. gvgvp is a protein-based polymer encoded by synthetic genes. at low temperatures, it exists as an extended molecule but, upon raising the temperature above the transition range, the polymer hydrophobically folds into dynamic structures called β-spirals that further aggregate by hydrophobic association to form twisted filaments . using this approach, single-step purification of an insulin-polymer fusion has recently been shown. inverse temperature transition offers several advantages, including facilitating the scale-up of purification from grams to kilograms (o. carmona-sanchez and h. daniell, unpublished) . yet another recent approach is the use of a chaperonin protein to fold foreign proteins into cuboidal crystals, allowing their purification in a single step by centrifugation . one additional advantage of this method is the protection of foreign proteins from cellular proteases. plant-derived biopharmaceuticals should meet the same standards of safety and performance as other production systems. however, many herbal medicines are now exempt from such close scrutiny and are not required to meet the same standards because of their classification as nutritional supplements. because several environmental concerns have been raised by interest groups to confuse public perception, it is of paramount importance that regulating agencies distinguish between real and perceived public concerns (scientific versus non-scientific). if biopharmaceuticals that are potentially harmful are capable of persisting in the environment and might accumulate in non-target organisms, precautionary measures should be taken. induction of biopharmaceutical production after harvesting (as was done in the case of glucocerebrosidase ) might be one approach to minimize environmental exposure, provided that the use of viral vectors does not introduce additional environmental or regulatory concerns. expression of potentially harmful proteins in a form that must be treated for activation might minimize the risk of exposure. for example, hirudin is produced as a fusion protein and is inactive in this form; it is activated only after it is purified from seeds . another hotly debated environmental concern has been the outcrossing of transgenic pollen to weeds or related crops , . expression of harmful pharmaceutical proteins in non-target plants resulting from such outcrosses might create public concern and negative perception. several gene containment methods are currently being investigated, including apomixis, incompatible genomes, transgenic mitigation, control of seed dormancy or shattering, suicide genes, infertility barriers, male sterility and maternal inheritance. engineering foreign genes via the chloroplast genome has been shown to contain transgenes effectively, although there are a few exceptions in which the chloroplast genome shows biparental inheritance (e.g. pines) . as an example of an alternative strategy, rnase genes have been expressed under the control of a tissue-specific promoter to destroy the tapetum selectively during anther development, resulting in male sterile plants . there is also concern over the expression of harmful proteins in transgenic pollen. for example, the controversial observation of the toxic effect of bacillus thuringiensis (bt) corn pollen on milkweeds (asclepias spp.) fed to monarch butterfly larvae had a significant impact on public perception, even though the validity of this study has been repeatedly questioned. engineering biopharmaceuticals via the chloroplast genome might be a solution. although the cry protein of bt was expressed at high levels in leaves (up to % of total soluble protein), no toxicity was observed when milkweeds dusted with transgenic pollen were fed to monarch butterfly larvae . however, to date, chloroplast genetic engineering has been shown only in tobacco and potato. more recently, several academic and industrial laboratories have initiated projects to extend this technology to other useful crops. also, there are no reports of the production of glycoproteins in transgenic chloroplasts. another public concern is the presence of antibiotic resistance genes or their products (which are used as selective markers) in edible parts of genetically modified crops. however, several approaches are now available to generate plants with transgenes in their nuclear or chloroplast genomes without the use of antibiotic selection. practical considerations will dictate the choice of biopharmaceutical proteins and the crop in which they are to be produced. these include yield, storage conditions, containment properties, initial set up and running costs, purification strategies, size of the market, environmental concerns, public perception and competing technologies. access to several alternative approaches to optimize protein synthesis in plants in an environmentally sound manner augurs well for the safe production of biopharmaceuticals in transgenic plants and for greater availability of these proteins to populations requiring them. toxin b subunit oligomers in transgenic potato plants efficacy of a food plantbased oral cholera toxin b subunit vaccine protective immune response to foot-and-mouth disease virus with vp expressed in transgenic plants expression of immunogenic glycoprotein s polypeptides from transmissible gastroenteritis coronavirus in transgenic plants edible vaccine protects mice against escherichia coli heat-labile enterotoxin (lt): potatoes expressing a synthetic lt-b gene immunogenicity of transgenic plant-derived hepatitis b surface antigen induction of a protective antibody response to foot and mouth disease in mice following oral or parenteral immunization with alfalfa transgenic plants expressing the viral structural protein vp immunogenicity in humans of a recombinant bacterial antigen delivered in a transgenic potato human immune responses to a novel norwalk virus vaccine delivered in transgenic potatoes a plant-derived edible vaccine against hepatitis b virus immunization with potato plants expressing vp protein protects against rabbit hemorrhagic disease virus immunogenicity of porcine transmissible gastroenteritis virus spike protein expressed in plants plant-based vaccines: unique advantages production of hepatitis b surface antigen in transgenic plants for oral immunization development of biopharmaceuticals in plant expression systems: cloning, expression and immunological reactivity of human cytomegalovirus glycoprotein b (ul ) in seeds of transgenic tobacco transgenic plants expressing autoantigens fed to mice to induce oral immune tolerance production of recombinant proteins in transgenic plants: practical considerations application of transgenic plants as production systems for pharmaceuticals transgenic plants for therapeutic proteins: linking upstream and downstream technologies hyper-expression of the bt cry aa operon in chloroplasts leads to formation of insecticidal crystals gm crops: public perception and scientific solutions environmentally friendly approaches to genetic engineering regulation of chloroplast enzyme activities by thioredoxins: activation or relief from inhibition protein disulfide isomerase as a regulator of chloroplast translational activation high-yield production of a human therapeutic protein in tobacco chloroplasts computer-aided process analysis and economic evaluation for biosynthetic human insulin production: a case study molecular farming in plants: oil seeds as vehicles for production of pharmaceutical proteins hyperexpression of an environmentally friendly synthetic polymer gene containment of herbicide resistance through genetic engineering of the chloroplast genome induction of male sterility in plants by a chimaeric ribonuclease gene removing selectable marker genes: taking the shortcut engineering chloroplast genome without the use of antibiotic resistance genes transgenic plants as factories for biopharmaceuticals triple helix assembly and processing of human collagen produced in transgenic tobacco plants expression of full length bioactive antimicrobial human lactoferrin in potato plants key: cord- -qwqf rlf authors: labudda, mateusz; tokarz, krzysztof; tokarz, barbara; muszyńska, ewa; gietler, marta; górecka, mirosława; różańska, elżbieta; rybarczyk-płońska, anna; fidler, justyna; prabucka, beata; dababat, abdelfattah a.; lewandowski, mariusz title: reactive oxygen species metabolism and photosynthetic performance in leaves of hordeum vulgare plants co-infested with heterodera filipjevi and aceria tosichella date: - - journal: plant cell rep doi: . /s - - - sha: doc_id: cord_uid: qwqf rlf key message: defence responses of cyst nematode and/or wheat curl mite infested barley engage the altered reactive oxygen species production, antioxidant machinery, carbon dioxide assimilation and photosynthesis efficiency. abstract: the primary aim of this study was to determine how barley responds to two pests infesting separately or at once; thus barley was inoculated with heterodera filipjevi (madzhidov) stelter (cereal cyst nematode; ccn) and aceria tosichella keifer (wheat curl mite; wcm). to verify hypothesis about the involvement of redox metabolism and photosynthesis in barley defence responses, biochemical, photosynthesis efficiency and chlorophyll a fluorescence measurements as well as transmission electron microscopy were implemented. inoculation with wcm (apart from or with ccn) brought about a significant suppression in the efficiency of electron transport outside photosystem ii reaction centres. this limitation was an effect of diminished pool of rapidly reducing plastoquinone and decreased total electron carriers. infestation with wcm (apart from or with ccn) also significantly restricted the electron transport on the photosystem i acceptor side, therefore produced reactive oxygen species oxidized lipids in cells of wcm and double infested plants and proteins in cells of wcm-infested plants. the level of hydrogen peroxide was significantly decreased in double infested plants because of glutathione–ascorbate cycle involvement. the inhibition of nitrosoglutathione reductase promoted the accumulation of s-nitrosoglutathione increasing antioxidant capacity in cells of double infested plants. moreover, enhanced arginase activity in wcm-infested plants could stimulate synthesis of polyamines participating in plant antioxidant response. infestation with wcm (apart from or with ccn) significantly reduced the efficiency of carbon dioxide assimilation by barley leaves, whereas infection only with ccn expanded photosynthesis efficiency. these were accompanied with the ultrastructural changes in chloroplasts during ccn and wcm infestation. the changing climatic conditions, for example long, warm spring, hot summer, lack of the frost during winter and changed rainfall patterns in the temperate climate zone, are conducive to pests and pathogens gradation on crop plants. all these circumstances can contribute to a decrease in crop productivity (gregory et al. ). cyst nematodes and eriophyoid mites are significant pests affecting cereals. the cereal cyst nematode (ccn) heterodera filipjevi (madzhidov) stelter (nematoda: heteroderidae), is one of the global scale biotrophic phytopathogen of cereals (toumi et al. ) . upon the root infestation, infective larvae of ccn induce syncytia in the vascular cylinder. these multicellular structures are becoming the solely sources of nutrients drawing from the host plant. what is important, the parasitism of h. filipjevi specimens on roots of the colonised cereal plants may cause even to % grain yield losses (pariyar et al. ; dababat and fourie ) . the wheat curl mite (wcm), aceria tosichella keifer (acariformes: eriophyoidea), is around the world scattered eriophyoid pest of cereal plants (kuczyński et al. ) . wcm is a serious problem on cereals, including barley, because it damages leaves, what can impair overall physiology of plant, but until now there have been very little published reports regarding biochemical/physiological interactions with cereal hosts (skoracka et al. b) . furthermore, wcm can effectively spread plant pathogenic viruses such as the wheat streak mosaic virus (wsmv) from the family potyviridae. this is the most significant economic impact of wcm on cereal yielding. as a result of wcm feeding or wsmv transmission on cereal leaves, the grain yield losses may reach up to % (skoracka et al. (skoracka et al. , a aguirre-rojas et al. ) . it is well known that in plant organisms suffering from the pathogen or pest infestation, the altered metabolism of reactive oxygen species (ros) occurs woźniak et al. ) . ros are continuously produced in various metabolic pathways, including photosynthesis. besides, the ros metabolism and the regulation of photosynthesis are tightly linked (foyer ) . the chloroplastic ros have origins in photosystem i (psi) and photosystem ii (psii) and they are produced when the absorption of light quanta outbalances the efficiency of photosynthesis and the photoprotection responses are interrupted (khorobrykh et al. ) . ros have a dual role in plant physiology. on the one hand, they participate in various developmental processes under normal plant growth and act as signalling molecules in acclimatization to environmental stresses. on the other hand, the exaggerated ros level in plant cells can lead to the oxidation of proteins, lipids, nucleic acids, saccharides and pigments . to render the pauperization of plant cell functions impossible, the ros content in cells is mastered by enzymatic and non-enzymatic antioxidant mechanisms. the enzymatic machinery consists of superoxide dismutase (sod), catalase (cat), multifarious peroxidases and enzymes of the foyer-halliwell-asada cycle (labudda and azam ; kapoor et al. ). the non-enzymatic mechanisms are mainly based on the reduced glutathione (gsh) and ascorbate (asa) and an abundant group of the phenol metabolites (saxena et al. ; durak et al. ; muszyńska et al. ). among the phenols, salicylic acid (sa) is a phytohormonal molecule, which modulates many plant reactions to the environmental stresses (morkunas et al. (morkunas et al. , maruri-lópez et al. ; formela-luboińska et al. ) . while the knowledge about the physiological basis of plant responses to the infection by one pathogen or pest species is constantly increasing, the number of research reports attempting to explain the complex mechanisms of double biotic stress is extremely limited. in our research team, we have investigated the physiological response of plants to the cyst nematode infection for several years (labudda et al. a (labudda et al. , b, (labudda et al. , a labudda ). in addition, even though wcm is a very intensively studied mite on a global scale, little is known about the responses of host plants to wcm infection (skoracka et al. b ). our goal was to find out if a pest infection induces in barley leaves various but interconnected defence responses at different levels of plant organization (from biochemical, through physiological, to ultrastructural). we hypothesized that these responses against biotic stress factors could fit the concept of 'fanshaped' defence response engaging a holistic and complex but integrated plant stress response. this 'fan-shaped' type of plant defence response was originally proposed by sanità di toppi and gabbrielli ( ) for abiotic stress (cadmium exposure). however, so far such a hypothesis has not been considered in the literature in the context of cereal plant response to the stress caused by the infestation by two pests. therefore, we undertook to examine parameters of two primeval stress-sensitive metabolic areas, namely the redox balance and photosynthetic efficiency (Ślesak et al. ) . the research approach used by us seems to be particularly interesting because ccn attacks roots, while wcm infests leaves, what should cause consequences in the holistic plant response to biotic stressors. to learn about these, we implemented biochemical and enzymological methods, photosynthesis efficiency and chlorophyll a fluorescence measurements accompanied by observations under a transmission electron microscope. the spring barley hordeum vulgare l. cv. 'airway' seeds were washed in tap water for two hours. then, they were surface decontaminated in % sodium hypochlorite with . % polysorbate as a surfactant for min with stirring. next, they were rinsed under tap water for hour and an incubation, for hour in . % plant preservative mixture (ppm) (plant cell technologies, inc., washington dc, usa) to eliminate potential microbial contamination, was performed. the decontaminated seeds (embryos upwards) were put into petri dishes ( cm diameter) on a . % ppmsoaked filter paper and covered. after hours incubation in the fridge at °c in the dark, the seeds were kept in the dark at °c for days (labudda et al. b) . twelve germinated seeds were subsequently planted into a black plastic pot ( × / cm) with saucer. pot was filled with a commercial horticultural substrate consisted of mixed lowmoor and high-moor peats ( - mm fraction). substrate had no addition of mineral fertilizers, its ph in water was in the range of . - . and before planting it was autoclaved at °c, . mpa for min. aliquot of ml of sterile . × knop medium (ph . ) was added to the pots. plants were cultivated in a growth chamber mlr- (sanyo, tokyo, japan) at °c during the day and at °c at night with a h/ h day/night cycle under a photosynthetic photon flux density of ± μmol m − s − and at % humidity. every days, plants were watered with ml of water. the stock colony of aceria tosichella keifer was maintained for generations on h. vulgare plants growing in pots in laboratory of department of plant protection (section of applied entomology) at warsaw university of life sciences-sggw. a. tosichella biotype mt- (genbank: jf ) was used (skoracka et al. ) . wcm-infested plants were cultivated in a growth chamber mlr- at °c during the day and at °c at night with a h/ h day/night cycle under a photosynthetic photon flux density of ± μmol m − s − and at % humidity. each pot was put in rearing cages consisting of metal frames tightly covered with nylon mesh bags. the heterodera filipjevi (madzhidov) stelter cysts were collected from naturally cyst nematode-infested experimental triticum aestivum fields of the international maize and wheat improvement center (cimmyt) in yozgat ( ° ′ n, ° ′ e; altitude m a.s.l.) in the central anatolian plateau of turkey. cysts were extracted from rhizospheres and roots of triticum aestivum plants harvested at the end of the growing season. the modified extraction protocol was implemented (ashrafi et al. ) . hatching of h. filipjevi pre-parasitic juveniles was provoked by the incubation of cysts in sterile . m zncl at °c. freshly hatched pre-parasitic juveniles were washed six times and then suspended in sterile water. pots with -day-old plants were divided into four groups: nematode-uninoculated and wcm-uninoculated controls (c), ccn-inoculated (n), wcm-inoculated (wcm) and both ccn-inoculated and wcm-inoculated (n + wcm) plants. roots of plants from the n and n + wcm groups were inoculated with approximately freshly hatched pre-parasitic h. filipjevi juveniles (per pot) and plants were watered with ml of water. leaves of the plants from the wcm and n + wcm groups were inoculated with three females (per plant) of wcm adapted to feed on barley and plants were watered with ml of water. control plants were watered as well. each pot was put in rearing cages consisting of metal frames tightly covered with nylon mesh bags. control and infested plants were sampled after days of experiment. this sampling time point was chosen based on our previous published observations (labudda et al. b ) reflected the dynamics of growth and development of h. filipjevi larvae in spring barley (the same cultivar as tested in this article) roots under conditions of pot experiment. after days post-inoculation (dpi) but before dpi, most sedentary j larvae moulted to j larvae, which indicated that the -dpi syncytia met their nutritional demands. about dpi, the j larvae fed intensively to reach the j stage and sexual maturity after dpi and start reproducing. the sampling time at dpi was also rational and justified by the fact that at this time the barley plants showed clear signs of being infected by the wcm (leaf curl and yellowing) but the wcm population had not yet developed to the point that could cause the death of infested plants. for biochemical measurements, the leaf bulked samples were collected, and experiments were conducted in three biological replicates. assay of superoxide anions level was conducted according to doke's method (mai et al. ) . leaves ( mg) were immersed in millilitre of the . m k/na phosphate buffer (ph . ) containing . % nitro blue tetrazolium (nbt) and . m nan . samples were kept in the dark for an hour at room temperature (rt). next, reaction mixtures (without plant material) were incubated at °c for min and forthwith cooled on ice. the superoxide anions level was expressed as absorbance at nm per gram of fresh leaf weight (fw). the h o amount was measured according to the method by junglee et al. ( ) . leaves ( mg) were disintegrated in medium containing µl of . m k/na-phosphate buffer (ph . ), µl of . % trichloroacetic acid and µl of m ki. supernatants, collected after homogenate centrifugation ( °c, min, , ×g), were incubated in the dark for min at rt. next, the samples were centrifuged ( min, , ×g), and the absorbance was read at nm in nunc u-bottom -well plate (thermo scientific, waltham, ma, usa) on a varioskan lux multimode microplate reader (thermo scientific, waltham, ma, usa). the hydrogen peroxide amount was calculated from a standard curve and expressed per gram of fw. leaves ( mg) were crushed in a mortar with quartz sand and ml of ice-cold buffered mixture (ph . ) containing . m tris(hydroxymethyl)aminomethane hydrochloride (tris)-hcl, . m -mercaptoethanol, . m ethylenediaminetetraacetic acid (edta), % propane- , , -triol, . m phenylmethylsulfonyl fluoride, . m magnesium chloride, % polyvidone. enzymatic extracts were obtained by the centrifugation of homogenates ( °c, min, , ×g). the activity of superoxide dismutase (sod) was estimated by kostyuk and potapovich ( ) method. an assay mixture was made by mixing equal aliquots of . m edta and . m k/na phosphate buffer (ph . ). the ph value of assay mixture was precisely adjusted to with n,n,n′,n′-tetramethylethane- , -diamine. next, µl of assay mixture and one-hundred µl distilled water were pipetted to µl of enzymatic extract. the enzymatic reaction was initiated by the addition of µl of . × - m -( , -dihydroxyphenyl)- , , -trihydroxy- h-chromen- one suspended in (ch ) so. assays were made in nunc u-bottom -well plate on a varioskan lux multimode microplate reader. the absorbance at nm was recorded for min with reads every minute. the arbitrary unit of sod activity was ascertained as . decrease of absorbance after minute per gram of fw. the activity of catalase (cat) was estimated by aebi ( ) method. the enzymatic extract ( µl) was mixed with µl of . m tris-hcl buffer (ph . ) and µl of . % h o in the same buffer. assays were made at °c in uv-star -well plate (greiner, monroe, nc, usa) on a varioskan lux multimode microplate reader. the absorbance at nm was monitored for min with reads every minute. the cat activity was expressed as a breakdown of micromoles of h o per minute and gram of fw. the peroxidase activity (pod) was estimated by lück ( ) method. the enzymatic extract ( µl) was mixed with a reagent consisting of . % p-phenylenediamine and . % h o in . m tris-hcl buffer, ph . or . . assays were conducted at °c in nunc u-bottom -well plate on a varioskan lux multimode microplate reader. the absorbance at nm was read for min with reads every minute. the pod activity was expressed in arbitrary unit, separately for ph . (pod . ) and . (pod . ). the unit of pod activity was defined as . increase of absorbance after minute per gram of fw. the guaiacol peroxidase activity (gopx) was estimated by chance and maehly ( ) method. the enzymatic extract ( µl) was mixed with a reagent consisting of . m guaiacol and . m h o in . m acetic buffer, ph . . assays were performed at °c in nunc u-bottom -well plate on a varioskan lux multimode microplate reader. the absorbance at nm was measured for min with reads every minute. the gopx activity was expressed in micromoles of produced tetraguaiacol (ɛ = . mm − cm − ) per minute and gram of fw. the ascorbate peroxidase activity (apx) was estimated by nakano and asada ( ) method. the enzymatic extract ( µl) was mixed with a reagent consisting of . m tris-hcl buffer, ph . , . m asa, . m edta and . m h o . the apx activity was measured at °c in uv-star -well plate on a varioskan lux multimode microplate reader by following the rate of asa oxidation for min with absorbance reads every minute at nm. the apx activity was expressed in micromoles of asa decomposition (ɛ = . mm − cm − ) per minute and gram of fw. the dehydroascorbate reductase (dhar) activity was estimated using trümper et al. ( ) method. the enzymatic extract ( µl) was mixed with a reagent consisting of . m tris-hcl buffer, ph . , . m gsh and . m dehydroascorbic acid (dha). the dhar activity was measured at °c in uv-star -well plate on a varioskan lux multimode microplate reader by following the rate of dha reduction for min with absorbance reads every minute at nm. the dhar activity was expressed in micromoles of asa production (ɛ = mm − cm − ) per minute and gram of fw. the glutathione reductase (gr) activity was estimated by foyer and halliwell ( ) method. the enzymatic extract ( µl) was mixed with a reagent consisting of . m tris-hcl buffer, ph . , . m nicotinamide adenine dinucleotide phosphate (nadph), . m edta and . m oxidized glutathione (gssg). assays were performed at °c in nunc u-bottom -well plate on a varioskan lux multimode microplate reader and the change in absorbance at nm was noted for min with reads every minute. the gr activity was expressed as micromoles of oxidized nadph per minute and gram of fw. the nitrosoglutathione reductase (gsnor) activity was estimated by sakamoto et al. ( ) method. the enzymatic extract ( µl) was mixed with a reagent consisting of . m tris-hcl buffer, ph . , . m nicotinamide adenine dinucleotide (nadh), . m edta and . m s-nitrosoglutathione (gsno). assays were carried out at °c in nunc u-bottom -well plate on a varioskan lux multimode microplate reader and change in absorbance at nm was noted for min with reads every minute. the gsnor activity was expressed as micromoles of oxidized nadh per minute and gram of fw. the arginase (arg) activity in enzymatic extract was measured by labudda et al. ( a) method. after the activation of arg ( . m manganese dichloride, °c, min), the enzymatic extracts were kept at °c with . m l-arginine (ph . ). the reactions were terminated after min by adding the reagent consisting of sulphuric acid:orthophosphoric acid:distilled water ( : : , v/v/v). next, % α-isonitrosopropiophenone dissolved in % ethanol was added, and samples were incubated at °c for min. next, samples were placed for min in the dark at rt. the absorbance was recorded at nm in nunc u-bottom -well plate on a varioskan lux multimode microplate reader. the content of produced urea was estimated based on the standard curve and the arg activity was expressed as micromoles of urea per hour and gram of fw. leaves ( mg) were crushed in a mortar with quartz sand on ice-bath. the phenolic metabolites were extracted with ml of ice-cold % methyl alcohol and tissue homogenates were centrifuged for min at °c ( , ×g). the amount of total phenols, hydroxycinnamoyl tartaric acid esters, flavonols and anthocyanins was estimated by fukumoto and mazza ( ) method. the methyl alcohol extracts were mixed with . % hcl solution prepared in % ethanol and % hcl solution prepared in distilled water. then min after incubation in the dark at rt, the absorbance was noted in uv-star -well plate (greiner) on a varioskan lux multimode microplate reader. the absorbance at , , and nm reflected total phenol, hydroxycinnamoyl tartaric acid ester, flavonol and anthocyanin contents, respectively. the chlorogenic acid (total phenols), caffeic acid (hydroxycinnamoyl tartaric acid esters), quercetin (flavonols) and cyanidin (anthocyanins) were used as equivalents for assessment of phenolic metabolites. to measure the polyphenol content, the folin-ciocalteu method was used (labudda et al. b) . briefly, the methyl alcohol extract was mixed with distilled water and folin-ciocalteu reagent (poch, gliwice, poland). samples were kept at rt for min, and m saturated na co solution was pipetted and incubation at °c for min was carried out. the absorbance was noted at nm in nunc u-bottom -well plate on a varioskan lux multimode microplate reader and the polyphenol content was calculated as gallic acid equivalent. the results of the phenolic metabolite levels were expressed in milligrams of the respective equivalents per hundred grams of fw. the total salicylic acid (free and conjugated forms of sa) contents were measured by the reversed-phase highperformance liquid chromatography (rp-hplc) with detection of fluorescence according to szkop et al. ( ) method with minor modifications. leaves ( mg) were macerated in a mortar with quartz sand and . m dipotassium phosphate. samples were vigorously agitated for min at °c and centrifuged at , ×g for min. next, supernatants were mixed with m hcl and the hydrolysis at °c for min was conducted. subsequently, ethyl ethanoate was added to the hydrolysates and samples were vigorously agitated and centrifuged at , ×g for min. the organic phases were mixed with phosphate buffer (ph . ) and samples were vigorously agitated and centrifuged at , ×g for min. the aqueous phases were collected, filtered and pipetted into the vials. the hplc analysis was performed using system consisted of a binary pump (model , waters corporation, milford, ma, usa), a fluorometric detector (model , waters corporation) and an autosampler (model plus, waters corporation). separations were carried out at rt on a c column (symmetry . × mm, μm, waters corporation) guarded by a c precolumn (symmetry . × mm, μm, waters corporation) with a linear gradient elution. the content of sa was calculated based on the external standard curve prepared with the use of hplc-grade sa (sigma-aldrich, saint louis, mo, usa). the measurement of the -thiobarbituric acid reactive substances (tbars) content was estimated by hodges et al. ( ) method. two hundred microlitres of methyl alcohol extract (obtained as described in phenolic metabolites paragraph) was pipetted to µl . % -thiobarbituric acid prepared in % trichloroacetic acid. samples were incubated at °c for min and reactions were terminated on ice. samples were centrifuged ( , ×g) for min and the absorbance was read at , and nm in nunc u-bottom -well plate on a varioskan lux multimode microplate reader. tbars content was counted and expressed in micromoles per gramme of fw. to estimate protein carbonylation (carbonyl groups, c = o) content, leaves ( mg) were crushed with liquid nitrogen in mortar. two millilitres of extraction medium ( . m phosphate buffer ph . with . m edta, and . % triton x- ) was added into the macerated tissue. samples were centrifuged ( , ×g) for min at °c. the protein content was measured in supernatants with bradford reagent and bovine serum albumin (sigma-aldrich) as the protein standard. aliquots containing µg of soluble proteins were prepared, and next proteins were precipitated with cold propan- -one. the protein carbonylation was measured by derivatization of protein carbonyls with , -dinitrophenylhydrazine (dnph) using levine et al. ( ) method. briefly, . m dnph in . m hydrochloric acid was added to proteins and incubated at rt for hour in darkness with mixing every min. next, the protein pellets were washed three times with cold ethyl alcohol/ ethyl ethanoate ( : ) mixture. dinitrophenyl group (dnp)labelled proteins were solubilised in µl of rehydration medium containing m carbonic diamide, m thiocarbamide, % -[( -cholamidopropyl)dimethylammonio]- -propanesulfonate and . m cleland's reagent. samples containing µg of derivatized proteins were mixed ( : , v/v) with a sample buffer containing . m tris-hcl (ph . ), % propane- , , -triol, % sodium dodecyl sulphate (sds), % -mercaptoethanol and . % bromophenol blue. denatured protein samples were electrophoresed on % sds acrylamide gel in a . m tris, . m -aminoethanoic acid and . % sds running buffer (ph . ) at v for min followed by hour at a constant current of . a per gel until the blue dye front reached the bottom of the gel (mini-protean electrophoresis system; bio-rad, hercules, ca, usa). sds-polyacrylamide gel electrophoresis separated proteins were transferred to a nitrocellulose membrane (mini-protean electrophoresis system; bio-rad). after hour of blocking with % skimmed milk at rt, the membrane was incubated with anti-dnp polyclonal rabbit antibodies (sigma-aldrich; : ) in phosphate-buffered saline (pbs) (ph . ) with . % tween . alkaline phosphatase-conjugated goat antibodies against rabbit igg (sigma-aldrich; : , ) were used as the secondary antibodies. the blots were developed by nbt/ bromo- -chloro- ′-indolyphosphate (bcip) reagent containing . % of bcip and . % of nbt in . m tris-hcl buffer, ph . supplemented with . m sodium chloride and . m magnesium dichloride. the molecular weight (mw) of proteins was determined by spectratm multicolor broad range protein marker (thermo scientific). blots were digitalized with g:box ef (syngene, cambridge, uk) and the intensity of bands was quantified as % volume with free biovision software (vilber, collégien, france). the average intensity of all bands was ascertained. results were compared with c plants, to which a value of % has been ascribed. the photosynthetic pigments were estimated by lichtenthaler ( ) method. leaves ( mg) were homogenised in ice-cold % propan- -one with addition of calcium carbonate and centrifuged ( , ×g) for min at °c. the absorbance of propan- -one extracts was read at , and nm using double-beam spectrophotometer u- (hitachi high-technologies corporation, tokyo, japan). the chlorophyll a (chl a), chlorophyll b (chl b) and carotenoid (car) contents were calculated according to wellburn ( ) . total chlorophylls (chl a + b), the ratio of chlorophyll a to b (chl a:b) and the ratio of chl a + b to car (chl a + b:car) were also calculated. actual photosynthesis efficiency was evaluated by measuring gas exchange (h o and co ) and maximal efficiency of photosynthesis light reactions by photosynthetic light response curve measurement using a portable open gasexchange system (lcpro-sd; adc bioscientific ltd., hoddesdon, uk) equipped with a . cm cuvette and a mixed red/blue led light source head. measurements were carried out on fully developed leaves of five plants from each treatment. in gas exchange measurement, to allow photosynthesis to reach the steady state, each leaf was adapted for min in the cuvette. the measurements were performed in co saturated conditions ( μmol mol − ): μmol s − of airflow, - % relative humidity within the cuvette, °c leaf temperature and under the μmol m − s − red/blue light intensity. net photosynthesis [pn (μmol co m − s − )], stomatal conductance [gs (mmol h o m − s − )] and rate of transpiration [e (mmol h o m − s − )] were evaluated. photosynthetic light response curves were recorded on other leaf each of the same five plants. light source head was used for a stepwise reduction of photosynthetically active radiation (par) ranging from to μmol (quanta) m − s − (in , , , , , , , , , , and μmol (quanta) m − s − steps). the leaves were adapted to each of the light intensities for , , , , , , , , , , and min, respectively, before data point recording. air flow, relative humidity and co concentration inside the cuvette were the same as described for gas exchange measurement. chlorophyll a fluorescence measurement was carried out with handy-pea (hansatech, king's lynn, uk) fluorometer using standard procedures. ten leaves (two on each of five plants) from each treatment were dark-adapted for min. the fluorescence was induced by red light: max = nm, mol m − s − . selected functional and structural photosynthetic parameters were calculated (jiang et al. ; kalaji et al. ) (table ) . table was compiled according to piwowarczyk et al. ( ) . recorded curves were analysed using the fluorometer producer's softsware (pea-plus). evaluated parameters allow for assessment of photosystem ii (psii) efficiency. the leaf blade fragments (about × mm in size) were sampled from the central barley leaf part from each treatment. they were fixed in % glutaric dialdehyde and % polyoxymethylene dissolved in . m cacodylate buffer (ph . ) for h. after four times rinsing in . m cacodylate buffer, samples were post-fixed in % osmium tetroxide for h, dehydrated in increasing ethyl alcohol concentrations, propylene oxide and finally infiltrated with epon epoxy resin (fluka, buchs, switzerland). ultra-thin ( nm) sections were taken with a leica uct ultramicrotome (leica microsystems, wetzlar, germany) and stained with uranyl acetate and lead citrate. sections were examined in an fei d 'morgagni' (fei corp., hillsboro, or, usa) transmission electron microscope operating at kv and a sis 'morada' digital camera (olympus-sis, münster, germany) was used for acquisition of images. representative data were presented as the means ± sd. results were subjected to one-way analysis of variance (anova). the significant differences between experimental groups were determined using tukey's honest significant difference test at p < . . statistical analysis was performed using statistica program, version . (tibco software inc., palo alto, ca, usa). the level of superoxide anions was about . -fold lower in n, wcm and n + wcm plants than in c plants (fig. a) . the applied analytical method allowed to state that c plants had no h o in leaves (fig. b) . in contrast, plants under three stressful treatments presented the enhanced content of h o . n and wcm plants had its similar level about nmol g − . significant decrease in h o content to about nmol g − was noted in n + wcm plants in relation to n and wcm ones (fig. b) . the activity of sod was found to be . -fold lower in wcm than in c plants and . -fold higher in n + wcm plants in comparison to wcm plants and . -fold higher in n + wcm plants in comparison to n plants (fig. a) . the cat activity was up-regulated by . -fold in n plants in relation to control plants and down-regulated by about . -fold in wcm and n + wcm plants in relation to c plants. furthermore, the activity of this enzyme was . -fold lower in n + wcm plants in comparison with c ones (fig. b) . the activities of class iii peroxidases, including pod at ph . and . as well as gopx, had similar patterns. their activity decreased about . -fold in wcm and n + wcm plants in relation to c plants, while n + wcm plants presented about . -fold lower activities than n plants. the activity of pod . and pod . was % lower in wcm plants than in n plants and in the case of gopx by about % (fig. c-e) . the activity of apx, a member of class i peroxidases, was found to be decreased in n, wcm and n + wcm plants by . -, . -and . -folds, respectively, in comparison to c plants and its activity was significantly diminished by . -fold in n + wcm plants as against n and wcm plants (fig. f) . as fig. g presents, the dhar activity was slightly stimulated ( . -fold) in n plants in relation to c ones. the wcm plants had the lowest activity of this enzyme, which reached the level around µmol min − g − . however, the dhar activity was increased in n + wcm plants (about %) in comparison with wcm plants (fig. g) . in turn, the activity of gr was similar in c, wcm and n + wcm plants and it was kept at the level of µmol min − g − (fig. h) . as a result of wcm infestation, the gr activity was significantly down-regulated by . -fold and . -fold in relation to n + wcm and c barley plants, respectively (fig. h) . in leaves of barley plants separately infested with ccn or wcm the activity of the gsnor was increased by . -fold upon ccn and . -fold upon wcm infection in relation to the c plants, whereas in n + wcm plants the . -fold reduction of the gsnor activity was noted in comparison with both n and wcm plants (fig. i) . the highest activity of arg was observed in wcm plants (about µmol h − g − ) and the lowest (about µmol h − g − ) in n + wcm plants. in the comparison with c plants, the arg activity of n and n + wcm plants was diminished about . -and . -folds, respectively (fig. j ). the parameters including total phenols, hydroxycinnamoyl tartaric acid esters, flavonols, anthocyanins, polyphenols and salicylic acid differed significantly in the analysed four experimental conditions (fig. a-f ). the content of total phenols was decreased by about % in n plants in relation to the c plants and it increased by about % in n + wcm plants in comparison to n ones (fig. a) . it was found that the hydroxycinnamoyl tartaric acid ester content was about % significantly higher in wcm and about % higher in n + wcm plants than in n plants (fig. b) . the flavonol content was decreased by about % in n plants in relation to the c plants and it was increased by about % both in wcm and n + wcm plants in comparison with n plants (fig. c) . the anthocyanin content was decreased by about % in n plants in relation to the c plants and it was increased by about % both in wcm and n + wcm plants in comparison with n plants (fig. d) . the content of polyphenols was about % higher in n plants and about % higher both in wcm and n + wcm plants than in the c plants and it was about % higher both in wcm and n + wcm plants than in n plants (fig. e) . the level of salicylic acid was significantly enhanced by about % in wcm and by about % in n + wcm plants in comparison with c plants. furthermore, n + wcm plants had about % more and about % less salicylic acid in leaves as against n and wcm ones, respectively (fig. f ). the highest content of tbars was observed in n + wcm plants (about µmol g − ) and the lowest (about µmol g − ) in n plants. tbar level was . -fold diminished in n, and it was enhanced by . -and . folds in wcm and n + wcm plants in comparison with c plants. moreover, the amount of tbars was . -fold higher in n + wcm than in n plants and . -fold higher in n + wcm than in wcm plants (fig. ) . in addition to the tbars content, the intensity of the protein carbonylation processes, the protein marker of the oxidative damage in plants, was also ascertained (fig. ) . in response to the separate n and wcm inoculation, the level of protein carbonylation increased in comparison with the c plants. the n inoculation caused the increase in the total protein oxidation level by approximately %, and the wcm inoculation by % in comparison with c plants. however, the combination of n and wcm led to decrease in the intensity of carbonylation level by % against c plants. moreover, also patterns of oxidized (carbonylated) proteins changed in response to stresses. in the wcm and n + wcm plants carbonylated proteins with molecular weight ~ and kda were clearly visible, but they were not noticeable in c and n plants. the intensity of bands with mw ~ and kda was almost constant in all experimental conditions. the level of kda band was similar in c and wcm plants, but its intensity was lower in n plants, and the lowest in double-stressed barley specimens (n + wcm). also changes in bands with and kda differed between experimental variants. those two bands were the most intense in wcm plants, lower intensity was noticed in n and wcm ones and almost no carbonylation of these proteins was observed in control plants (fig. ). the photosynthetic pigment composition in leaves of various experimental plant groups was not significantly differed (fig. ). although differences in photosynthetic pigment contents were not at a statistically significant level, measurements of chl a fluorescence provided substantial and interesting results. actual photosynthesis efficiency did not change statistically between evaluated treatments as evidenced by unchanged net photosynthesis, stomatal conductance and the rate of transpiration (fig. a-c) . however, analysis of photosynthesis efficiency at higher radiation intensities revealed statistically significant differences in light reactions efficiency in plants under different treatments (fig. d) . between radiation of and µmol (quanta) m − s − , light reactions efficiency was significantly higher in plants inoculated with nematodes than in plants inoculated with mites and plants inoculated with both nematodes and mites. moreover, at the highest radiation ( µmol m − s − ), efficiency of light reactions was significantly lower in wcm plants than in c plants (fig. d) . furthermore, the measurement of chl a fluorescence also revealed some changes in psii efficiency between barley plants uninoculated and inoculated with different pests. values of psii parameters, presented on the radar chart, were normalized in relation to a control value (fig. e) . the rate of reduction of psii acceptor side (v i ) increased significantly in plants inoculated with wcm in comparison with control plants and nematodeinoculated plants. in turn, parameters related to the electron flows (ρ ro , δ ro ) and quantum yield for the reduction of end acceptors of psi per photon absorbed (φ ro ) decreased in wcm plants in comparison with c and n plants (fig. e) . also, total electron carriers per reaction centre (sm) were reduced in these plants in relation to n-inoculated plants (fig. e) . some changes were also noted in electron flow parameters (ρ ro , δ ro ) of n + wcm plants, which were reduced as compared with n plants (fig. e ). as some photosynthesis parameters differed between each experimental group, we used transmission electron microscopy to find out whether these changes go hand in hand with the changed ultrastructure of chloroplasts. chloroplasts of plants from treatments differed in the shape, the thylakoid structure, the stroma density and the presence of starch grains; however, their distribution and number in leaf cells were like each other (fig. ) . in the mesophyll of control plants, chloroplasts were ellipsoidal in shape and they had a regular structure of thylakoids, electrondense stroma and numerous plastoglobules. additionally, some small starch grains were occasionally observed (fig. a-c) . in leaves of n- (fig. d-f ) or wcm-infected plants (fig. g-i) , irregularly shaped chloroplasts with swollen ( fig. d-h) or electrontranslucent (fig. i ) stroma were found. apart from these malformations, chloroplasts of infested plants had rather regular arrangement of thylakoid membranes with welldeveloped grana. in turn, mesophyll cells of leaves from plants infected simultaneously with nematodes and mites contained chloroplasts with different degrees of degradations fig. j-l) . in these chloroplasts, the stroma was electrontranslucent, whereas the thylakoid system was well visible. interestingly, besides properly developed cells with chloroplasts similar to control ones, cells with far-reaching changes were found, probably in these leaf part where a. tosichella fed (fig. j, k) . in such degraded chloroplasts the dilated thylakoids forming many vesicles occurred apart from typically arranged ones (fig. l) . what is more, no starch grains were noted in all chloroplasts of infested plants, but numerous plastoglobuli were still observed. as sessile organisms, plants are equipped with sophisticated defence apparatus involving non-enzymatic and enzymatic antioxidants to cope with oxidative stress and to promote photosynthesis efficiency. regulation of ros and photosynthetic metabolism underlies plant successful responses against numerous environmental stressors (suzuki et al. ; choudhury et al. ; woźniak et al. ; morales et al. ) . the photosynthetic apparatus is one of the most sensitive sensors of redox changes in the plant, which allows it for effective acclimatization to altering environmental conditions (goltsev et al. ) . biotic and abiotic stress factors cause redox imbalance in the plant, leading to a reduction of photosynthesis efficiency, an activation of alternative fig. the contents of phenolic metabolites (a-f) in the leaves of the spring barley hordeum vulgare plants cultivated for days on commercial horticultural substrate after the cereal cyst nematode heterodera filipjevi and the wheat curl mite (wcm), aceria tosichella inoculations. results are shown as the means ± sd. asterisks indicate means which are significantly different at *p < . and **p < . according to one-way analysis of variance and a post-hoc tukey's test. control means the nematode-uninoculated and the wcm-uninoculated control plants metabolic pathways and a synthesis of secondary metabolites (piwowarczyk et al. ; tokarz et al. tokarz et al. , b makowski et al. ; rozpądek et al. ) . attack and colonization of plant by pests lead to an increased demand for primary metabolites, mainly carbohydrates, and imply the activation of a defence response requiring an increased production of secondary metabolites (blasi et al. ) . feeding mites secrete into the leaves specific mix consisting of elicitors and fatty and amino acids, which on the one hand enable the plant to recognize the pest, but on the other hand, lead to host tissue and cell damage (gilardoni et al. ) . as a result, an intensive production of ros occurs leading to membrane lipid peroxidation, dna degradation, redox and photosynthesis efficiency disorders (blasi et al. ) . the reduction of photosynthesis may be induced by either direct mesophyll damage and a significant reduction in stomatal conductance (fadini et al. ) or disorders in the transcription and translation of proteins associated with the photosynthetic apparatus (schmitt et al. ) or disruptions in the synthesis pathway of chlorophylls and carotenoids (bronner et al. ) . it was shown that reduced photosynthesis efficiency in rice plants can be caused not only by a decrease in chlorophyll content (buffon et al. (buffon et al. , but also by down-regulation of seven proteins synthesis related to nadph production and thus to adenosine triphosphate and glucose synthesis (blasi et al. ). in the presented experiment, however, no significant differences were observed in the efficiency of photosynthesis (pn), transpiration (e), as well as in stomatal conductance (gs) between barley plants inoculated and uninoculated with ccn and wcm. in addition, no differences were found in the content of photosynthetic pigments between these plants. however, the photosynthetic light response curve revealed that the presence of mites (apart from or with nematodes) significantly reduced the efficiency of co assimilation what some malformations in the chloroplast ultrastructure accompanied simultaneously. at the same time, inoculation with nematodes increased photosynthesis efficiency. therefore, to determine the real condition and efficiency of photosynthetic apparatus, the ojip fast-fluorescence test based on non-invasive chl a fluorescence measurement was applied. stress factors leading to redox imbalance disturb both photochemical and biochemical phases of photosynthesis (tokarz et al. b) . stressors reduce the number of electrons reaching the psii reaction center (rc) because of disturbances in the oxygen-evolving complex (oec) (nikkanen et al. ). this psii donor side limitation threatens with the appearance of the strongest biological oxidant the excited primary electron donor of psii (p *). on the other hand, redox homeostasis disorders on the psii acceptor side lead to a reduction in the rate and efficiency of electron transport between psii and psi and thus to psi oxidation (tokarz et al. a) . in contrast, the limitation on the psi acceptor side involves psi over-reduction (tokarz et al. a) . all these limitations lead to generation of ros, rns and organic radicals causing irreversible destruction of the photosynthetic apparatus tokarz et al. a) . kinetic of chl a fluorescence revealed that rice plants sensitive to mite feeding had a significantly lower number of open psii rc (f ) among all psii rc (vj) as well as significantly lower efficiency of trapped energy flux (tro/ cso) compared to resistant plants (buffon et al. ) . at the same time, sensitive cultivars dissipated more intensely part of the trapped energy (dio/rc) (buffon et al. ) . barley plants examined in this work, regardless of kind of pest inoculation, were characterized by the same efficiency of trapping and transporting radiation to psii rc as control plants. at the same time, no limitations on the psii donor side were observed indicating no damage of oec. on the other hand, inoculation with wcm and n + wcm caused a significant limitation in the efficiency of electron transport outside psii rc. limitation resulted from decreased pool of rapidly reducing plastoquinone pq (v i ) as well as the significantly diminished total electron carriers (sm). inoculation with wcm, apart from and together with nematode, significantly limited the electron transport on the psi acceptor side (ρ ro , δ ro , φ ro ); thus ros appeared in barley cells because of these limitations. we have previously presented enhanced superoxide anion and h o production in aerial parts of arabidopsis thaliana plants infested with heterodera schachtii (the beet cyst nematode) . what is more, khanna et al. ( ) meloidogyne incognita (root-knot nematode) and javadi khederi et al. ( ) documented enhanced level of h o in vitis vinifera leaves infested with colomerus vitis (the grape erineum mite). in our investigations discussed here, we noted a considerable decrease in superoxide anion accumulation in barley leaves because of separate ccn or wcm infection and of a double infection with ccn and wcm. diminished superoxide anion content partly arose from superoxide anions' dismutation into o and h o by the activity of sod. however, since the sod activity was not very strongly stimulated in stressful conditions, it can be argued that reduced amounts of superoxides were results of another metabolic event. nadph oxidase (nox) is an enzymatic source of superoxide anions in plants. nisimoto et al. ( ) presented that one of the nox, nox is a hydrogen peroxide sensor and its dehydrogenase domain reacts quickly to h o in cytosol to modulate the activity of nox . thus, reduced content of superoxide anions in barley plants under three stressful conditions may also result from nox down-regulation through exaggerated accumulation of h o . as already mentioned, increased content of h o fig. photosynthetic pigment contents and their ratios (a-f) in the leaves of the spring barley hordeum vulgare plants cultivated for eighteen days on commercial horticultural substrate after the cereal cyst nematode heterodera filipjevi and the wheat curl mite (wcm), aceria tosichella inoculations. results are shown as the means ± sd. control means the nematode-uninoculated and the wcm-uninoculated control plants fig. photosynthesis (a-d) and photosystem ii (psii) efficiency (e) in the leaves of the spring barley hordeum vulgare plants cultivated for eighteen days on commercial horticultural substrate after the cereal cyst nematode heterodera filipjevi and the wheat curl mite (wcm), aceria tosichella inoculations. results are shown as the means ± sd. asterisks indicate means which are significantly different at *p < . and **p < . according to one-way analysis of variance and a post-hoc tukey's test. control means the nematodeuninoculated and the wcm-uninoculated control plants; pn net photosynthesis, gs stomatal conductance, e rate of transpiration is different than the plant defence reaction to one stress taking place independently. consequently, the combination of two biotic stressors (cyst nematode incepting nutrients from roots and eriophyoid mite from leaves) imposed on spring barley plants a special physiological acclimatization to two biotic stress factors. plants protect themselves against oxidative stress consequences induced by pests and pathogens among others through antioxidative enzymes. class iii peroxidases (pod and gopx) and cat had the same activity patterns in plants khanna et al. ). in the present results, elevated activities of gopx and cat were not reflected in the reduced content of h o . this can suggest that the h o production was greater than the ability to scavenge h o by gopx and cat. the increased activities of gopx and cat corresponded to a reduced content of phenols, including flavonols. the peroxidase-flavonoid mechanism of the ros deactivation often occurs in plant cells, and during this gopx degrades phenolic molecules with simultaneous neutralisation of h o (takahama and oniki ) . furthermore, cat can act as a bifunctional enzyme; on the one hand, cat catalyses decomposition of h o , but on the other hand it can oxidize hydrogen donors such as phenolic molecules with consumption of peroxides (chen et al. ) . therefore, it can be assumed that both above described systems (peroxidase-flavonoids and catalase-phenols) participated in controlling h o level in leaves of nematodeinfected barley. interestingly, the postulated catalase-phenol activity corresponded well to the lower lipid peroxidation in nematode-infected plants that might partly be due to consumption of lipid peroxides by cat. as described above, plants have to counteract tear ros homeostasis down during stress, but phytophagous mites seek to colonise effectively of plant hosts among others owing to secreting salivary protein effectors into leaves to modulate/curtail plant defence mechanisms (jonckheere et al. ) . attention is drawn to the inhibited activity of pods and gpox in wcm and n + wcm plants in this context. hemetsberger et al. ( ) showed that ustilago maydis secreted effector pep (protein essential during penetration- ) into the maize leaves to inhibit of host peroxidase activity and to establish of a biotrophic interaction. although this type of effector has not yet been identified in mites, our results may suggest that wcm deliberately modulated the peroxidase activity to prevent the activation of class iii peroxidase-dependent defence responses such as lignin and suberin production, the cell wall components cross-linking or formation of phytoalexins (almagro et al. ; minibayeva et al. ) . apx, belonging to class peroxidases, takes part in h o neutralisation by foyer-halliwell-asada pathway consisting of gsh and asa as a key non-enzymatic antioxidant compounds and enzymes such as dhar, gr and monodehydroascorbate reductase (labudda ) . our results indicate that the decomposition of h o in the leaves of c plants depended on apx activity, because its stimulated activity was noted. however, regarding our main research question (how does barley react to the double infestation?), our finding that the apx activity was diminished in n + wcm plants in relation to n and wcm plants indicates that h o scavenging in n + wcm plants was re-routed to non-enzymatic h o decomposition during direct reaction with asa. decreased apx activity ensured asa molecules that reacted with h o and scavenged them (grinstead ) . the product of the h o reaction with asa was its oxidized form, dha, which could be a substrate for dhar. this antioxidant mechanism from foyer-halliwell-asada pathway was in operation efficiently because the activity of dhar in n + wcm plants was on the same level than in c plants; hence the recovery of asa by dhar still occurred. one more discovery supports our interpretation. dhar tapped two molecules of gsh to produce one molecule of asa but gssg was equally formed. the increase of gr activity in n + wcm plants was noted. since gr transforms gssg to gsh, regenerated gsh pool was constantly achievable and/or gsh could anew begin reaction of dhar. gsh could be also oxidized to gssg by the direct reaction with h o , which in this way was beneficially removed from cells (abedinzadeh et al. ; labudda ; ding et al. ) . summarizing, observed by us, decreased number of h o in n + wcm plants was presumably due in no small part to the foyer-halliwell-asada pathway. in addition, this mechanism could be supported by polyphenols, which through reaction with ros/rns restricted oxidative damage in stressed barley plants (hussain et al. ) . reactive nitrogen species (rns), including nitric oxide (no) that is in the leading position amid rns, are important regulatory molecules during undisturbed plant ontogenesis. they also participate in the cellular signalling under stressful conditions (corpas ) . moreover, no can reversibly bind the sulfhydryl groups of cysteines, so s-nitrosothiols (snos) are produced, including gsno, a biochemical stock of no in cells (jahnová et al. ) . metabolism of gsno in cells is controlled by gsnor, catalysing reduction (reliant on nadh) of gsno to gssg and ammonia (nh ) (corpas and barroso ) . kovacs et al. ( ) and begara- morales et al. ( ) presented claims that ros can inhibit the activity of gsnor resulting in the accumulation of gsno and significantly increased h o neutralisation through the foyer-halliwell-asada pathway. other research showed gsno-dependent irreversible inhibition of the enzyme activity of apx (clark et al. ) . this apx inhibition by gsno strengthens our explanation regarding down-regulated activity of apx in n + wcm plants. our results are in accordance with kovacs et al. ( ) and begara- morales et al. ( ) and indicate that antioxidant capacity of n + wcm barley plants to some extent came from the inhibition of gsnor and the accumulation of gsno. moreover, jahnová et al. ( ) proposed that diminished gsnor activity with simultaneous increase in the gsno level can lead to improved plant resistance to pest/pathogen infections. regardless of this, the up-regulated gsnor activity in n and wcm plants was documented, thus scavenging the intolerable amount of no through the activity of gsnor took place. therefore, barley plants under separately ccn and wcm infestation were protected from effects of the high concentrations of rns that are toxic for plants (corpas and barroso ) . our earlier published article indicated that alterations in nitrogen metabolism occurred in plants infected with the beet cyst nematode (labudda et al. a) . arginase (arg) is not an enzyme that has direct influence on ros deactivation, but it provides ornithine for the synthesis, e.g. polyamines, important metabolites during plant diseases (walters ) . ornithine decarboxylase initiates the arg-dependent polyamine synthesis pathway, so ornithine is decarboxylated to putrescine. from putrescine spermidine synthase produces spermidine, and spermine synthase synthesizes spermine from spermidine. putrescine, spermidine and spermine are positively charged metabolites; therefore, they can bind to negatively charged compounds and neutralize ros (hasanuzzaman et al. ). it cannot be excluded that enhanced activity of arg in wcm plants can promote synthesis of polyamines and indirectly improve non-enzymatic antioxidant apparatus in these plants. however, another mechanism is also possible. the enhanced arg activity, and thus possible stimulated polyamine synthesis, in combination with the observed accumulation of salicylic acid, a master mediator of plant defence (palmer et al. ) , may point to defence strategy induced by barley plants to prevent excessive development of wcm populations on infested plants. in reaction to devouring pests, attacked plants synthesize and emit herbivore-induced plant volatiles 'as a call for help' to attract carnivorous natural enemies of herbivores. due to these volatiles, plants can indirectly decrease the number of herbivores by more than % (kessler and baldwin ) . the exogenous treatment with putrescine, spermidine and spermine led to emission of volatiles in phaseolus lunatus leaves, among others methyl salicylate, and its content was particularly high after treatment with putrescine (ozawa et al. ). these authors also presented that p. lunatus leaves treated with spermine + jasmonic acid at the same time attracted more predatory mites phytoseiulus persimilis than those treated with jasmonic acid singly. it has been suggested that spermine has an important role in the regulation of synthesis of volatiles induced by tetranychus urticae (ozawa et al. ). furthermore, shimoda et al. ( ) proved that two predators of t. urticae, thysanopteran scolothrips takahashii and coleopteran oligota kashmirica benefica exhibited preference for methyl salicylate or methyl salicylate + jasmonic acid-treated p. lunatus leaves when compared with t. urticae-uninfested leaves. taken together, our results can suggest that stimulated polyamine synthesis through arg reaction and co-existing enhanced level of free and conjugated forms of salicylic acid in wcm-infected barley plants can indicate that barley plants try to cope with herbivores through increased synthesis of volatiles to lure carnivorous insects. such defence response of barley plants has been shown in article by ninkovic et al. ( ) . these authors proved that volatiles synthetized by the bird cherry-oat aphid (rhopalosiphum padi)-infested barley plants attracted adults of the seven-spotted ladybird (coccinella septempunctata). ninkovic et al. ( ) concluded that aphid-induced barley volatiles had an important role in food-searching behaviour of ladybirds and this behaviour can be inborn or effect of associative learning of c. septempunctata imagines. in our research, we implemented two biochemical markers for the assessment of oxidative damage in barley cells. we began with scrutiny of tbars level for the detection of lipid oxidation products, such as -hydroxy- -nonenal and malondialdehyde and other toxic aldehydes, alkenals and hydroxyalkenals (alché ) . the observed lowest level of tbars in n plants (even lower than in c plants) indicates that the antioxidative mechanisms were efficient enough to limit the oxidative damage of cell membranes, as we have previously suggested to some extent this may be due to catalase activity against peroxides through the pathway catalase-phenols (chen et al. ) . a high level of tbars was observed in barley plants on whose leaves wcm individuals were feeding. we suppose it could have a double effect on physiological processes in these plants. on the one hand, negative because the integrity of some cell membranes of wcm and n + wcm plants was destroyed what corresponded with different degrees of degradations of chloroplasts in these plants. however, the positive side of this state is also possible, with a direct impact on increased barley defence mechanisms against wcm. first, the increased accumulation of lipid oxidation products in wcminfested leaves makes them a less favourable nutrient source for wcm. a similar observation was made by shukle and murdock ( ) . it was observed that the plant diets containing the soybean prooxidant enzyme lipoxygenase (lox) (initiating various oxygenated compounds production) led to retarded manduca sexta larval growth, the insect pest of plants from the family solanaceae. second, wounds induced by wcm feeding on barley leaves could stimulate releasing of fatty acids from membrane lipids such as linolenic acid and next lox could convert linolenic acid to jasmonic acid and further volatile aldehydes can be produced (mosblech et al. ; rahimi et al. ). this would be consistent with the above postulated role of arginase in the production of polyamines and stimulated by them the emission of volatiles. moreover, hildebrand et al. ( ) presented that because of t. urticae infestation on soybean leaves, a large increase in tbar content and a significant enhancement in lox activity co-occurred. the second marker of oxidative stress was the carbonylation of proteins expressed in amount of the c = o groups incorporated into proteins because of predominantly lysine arginine, proline and threonine residues oxidation (kalemba and pukacka ) . the above presented antioxidant mechanisms operating together indicated efficient antioxidant capacity of n + wcm plants because the combination of n and wcm stresses led to the lowest intensity of carbonylation level against other plant groups. therefore, it can be assumed that wastage of protein functions was significantly diminished. however, upon separate nematode or mite inoculation, the number of c = o groups in proteins was enhanced in comparison with the control plants. our results are consistent with the research presented by dworak et al. ( ) , who showed that the protein carbonylation level was increased in leaves of zea mays plants infested with t. urticae or under water deficit in comparison with control plants. however, the combination of t. urticae and water deficit resulted in significantly decreased amount of c = o groups in proteins in comparison to singly t. urticae or water deficit-stressed plants as well as control ones. our results and dworak et al. ( ) can indicate that the double stress, regardless of whether these are two biotic stresses or a combination of abiotic and biotic stresses, plants react in a similar way. in the future, it would be interesting to examine whether such plant response takes place also in other experimental systems and whether it goes beyond species from the poaceae family. this discovery can set new research paths because issues related to posttranslational modification of proteins during stress combination are virtually unexplained. to conclude, the obtained results increase the knowledge of redox metabolism and photosynthesis of cereal plants infested simultaneously with two pests. we unravelled for the first time how barley reacts to stress arisen by cereal cyst nematode and wheat curl mite infestation. it was found that the ros production and oxidative damage were increased in nematode and mite-infested leaves and prominent enzymatic and non-enzymatic antioxidants were activated. furthermore, infestation with mites (apart from or with nematodes) significantly decreased the efficiency of co assimilation by leaves of barley plants, but infection only with nematodes increased photosynthesis efficiency. our investigations point out tight relatedness between the ros metabolism and the regulation of photosynthesis in leaves of barley plants colonised by nematodes and mites. summarizing, to manage the stress induced by pest infestation, barley plants can induce a multi-component model of stress response in the form of biochemical-physiological 'fan-shaped' defence response (fig. ). kinetic study of the oxidation mechanism of glutathione by hydrogen peroxide in neutral aqueous medium catalase in vitro barley varieties stoneham and sydney exhibit mild antibiosis and antixenosis resistance to the wheat curl mite, aceria tosichella (keifer) a concise appraisal of lipid oxidation and lipoxidation in higher plants class iii peroxidases in plant defence reactions ijuhya vitellina sp. nov., a novel source for chaetoglobosin a, is a destructive parasite of the cereal cyst nematode heterodera filipjevi the function of s-nitrosothiols during abiotic stress in plants high infestation levels of schizotetranychus oryzae severely affects rice metabolism alterations in rice, corn and wheat plants infested by phytophagous mite enhanced peroxidase activity associated with the hypersensitive response of solanum dulcamara to the gall mite aceria cladophthirus (acari: eriophyoidea) unraveling rice tolerance mechanisms against schizotetranychus oryzae mite infestation physiological and molecular alterations promoted by schizotetranychus oryzae mite infestation in rice leaves assay of catalases and peroxidases subcellular localization of a plant catalase-phenol oxidase, accatpo, from amaranthus and identification of a non-canonical peroxisome targeting signal reactive oxygen species, abiotic stress and stress combination nitric oxide inhibition of tobacco catalase and ascorbate peroxidase reactive nitrogen species (rns) in plants under physiological and adverse environmental conditions: current view nitro-oxidative stress vs oxidative or nitrosative stress in higher plants (eds) plant parasitic nematodes in subtropical and tropical agriculture the pivotal function of dehydroascorbate reductase in glutathione homeostasis in plants defense responses of thuja orientalis to infestation of anholocyclic species aphid cinara tujafilina maize proteomic responses to separate or overlapping soil drought and twospotted spider mite stresses herbivoria de tetranychus urticae koch (acari: tetranychidae) induz defesa direta em morangueiro? the role of sugars in the regulation of the level of endogenous signaling molecules during defense response of yellow lupine to fusarium oxysporum reactive oxygen species, oxidative signaling and the regulation of photosynthesis the presence of glutathione and glutathione reductase in chloroplasts: a proposed role in ascorbic acid metabolism assessing antioxidant and prooxidant activities of phenolic compounds supersage analysis of the nicotiana attenuata transcriptome after fatty acidamino acid elicitation (fac): identification of early mediators of insect responses variable chlorophyll fluorescence and its use for assessing physiological condition of plant photosynthetic apparatus integrating pests and pathogens into the climate change/food security debate the oxidation of ascorbic acid by hydrogen peroxide. catalysis by ethylenediaminetetraacetato-iron(iii) polyamine action under metal/metalloid stress: regulation of biosynthesis, metabolism, and molecular interactions the ustilago maydis effector pep suppresses plant immunity by inhibition of host peroxidase activity peroxidative responses of leaves in two soybean genotypes injured by twospotted spider mites (acari: tetranychidae) improving the thiobarbituric acid-reactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds oxidative stress and inflammation: what polyphenols can do for us s-nitrosoglutathione reductase-the master regulator of protein s-sitrosation in plant no signaling influence of the erineum strain of colomerus vitis (acari: eriophyidae) on grape (vitis vinifera) defense mechanisms aluminum-induced effects on photosystem ii photochemistry in citrus leaves assessed by the chlorophyll a fluorescence transient the salivary protein repertoire of the polyphagous spider mite tetranychus urticae: a quest for effectors optimized assay for hydrogen peroxide determination in plant tissue using potassium iodide effects of salt stress on photosystem ii efficiency and co assimilation of two syrian barley landraces chlorophyll a fluorescence as a tool to monitor physiological status of plants under abiotic stress conditions carbonylated proteins accumulated as vitality decreases during long-term storage of beech (fagus sylvatica l.) seeds antioxidant enzymes regulation in plants in reference to reactive oxygen species (ros) and reactive nitrogen species (rns) defensive function of herbivore-induced plant volatile emissions in nature impact of plant growth promoting rhizobacteria in the orchestration of lycopersicon esculentum mill. resistance to plant parasitic nematodes: a metabolomic approach to evaluate defense responses under field conditions oxygen and ros in photosynthesis superoxide-driven oxidation of quercetin and a simple sensitive assay for determination of superoxide dismutase ros-mediated inhibition of s-nitrosoglutathione reductase contributes to the activation of antioxidative mechanisms thermal niches of two invasive genotypes of the wheat curl mite aceria tosichella: congruence between physiological and geographical distribution data ascorbate-glutathione pathway as an important player in redox regulation in nematode-infested plants: what we have learned so far glutathione-dependent responses of plants to drought: a review arginase activity in arabidopsis thaliana infected with heterodera schachtii protease activity and phytocystatin expression in arabidopsis thaliana upon heterodera schachtii infection systemic changes in photosynthesis and reactive oxygen species homeostasis in shoots of arabidopsis thaliana infected with the beet cyst nematode heterodera schachtii heterodera schachtii infection affects nitrogen metabolism in arabidopsis thaliana activity profiling of barley vacuolar processing enzymes provides new insights into the plant and cyst nematode interaction carbonyl assays for determination of oxidatively modified proteins chlorophylls and carotenoids: pigments of photosynthetic biomembranes peroxidase. in: bergmeyer hu (ed) methoden der enzymatischen analyse. verlag chemie oxidative stress in pea seedling leaves in response to acyrthosiphon pisum infestation is a blue-red light a good elicitor of phenolic compounds in the family droseraceae? a comparative study intra and extracellular journey of the phytohormone salicylic acid roles of apoplastic peroxidases in plant response to wounding photosynthetic metabolism under stressful growth conditions as a bases for crop breeding and yield improvement phytohormonal signaling in plant responses to aphid feeding the role of heavy metals in plant response to biotic stress oxylipins: structurally diverse metabolites from fatty acid oxidation dual role of metallic trace elements in stress biology-from negative to beneficial impact on plants changes in proteolytic activity and protein carbonylation in shoots of alyssum montanum ecotypes under multi-metal stress ecotype-specific pathways of reactive oxygen species deactivation in facultative metallophyte silene vulgaris (moench) garcke treated with heavy metals heavy metal tolerance in contrasting ecotypes of alyssum montanum hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts chloroplast thioredoxin systems: prospects for improving photosynthesis the influence of aphid-induced plant volatiles on ladybird beetle searching behavior nadph oxidase function as a hydrogen peroxide sensor exogenous polyamines elicit herbivore-induced volatiles in lima bean leaves: involvement of calcium, h o and jasmonic acid salicylic acid-mediated plant defense: recent developments, missing links, and future outlook genome-wide association study in wheat identifies resistance to the cereal cyst nematode heterodera filipjevi the acclimatization strategies of kidney vetch (anthyllis vulneraria l.) to pb toxicity pglox encoding a lipoxygenase contributes to jasmonic acid biosynthesis and ginsenoside production in panax ginseng acclimation of the photosynthetic apparatus and alterations in sugar metabolism in response to inoculation with endophytic fungi arabidopsis glutathionedependent formaldehyde dehydrogenase is an s-nitrosoglutathione reductase response to cadmium in higher plants cross talk between h o and interacting signal molecules under plant stress response reactive oxygen species: re-evaluation of generation, monitoring and role in stresssignaling in phototrophic organisms olfactory responses of two specialist insect predators of spider mites toward plant volatiles from lima bean leaves induced by jasmonic acid and/or methyl salicylate lipoxygenase, trypsin inhibitor, and lectin from soybeans: effects on larval growth of manduca sexta (lepidoptera: sphingidae) the wheat curl mite aceria tosichella (acari: eriophyoidea) is a complex of cryptic lineages with divergent host ranges: evidence from molecular and plant bioassay data spatial and hostrelated variation in prevalence and population density of wheat curl mite (aceria tosichella) cryptic genotypes in agricultural landscapes genetics of lineage diversification and the evolution of host usage in the economically important wheat curl mite, aceria tosichella keifer the interface between wheat and the wheat curl mite, aceria tosichella, the primary vector of globally important viral diseases how to define obligatory anaerobiosis? an evolutionary view on the antioxidant response system and the early stages of the evolution of life on earth abiotic and biotic stress combinations a simple and robust protocol for fast rp-hplc determination of salicylates in foods flavonoids and some other phenolics as substrates of peroxidase: physiological significance of the redox reactions response of dionaea muscipula j. ellis to light stress in in vitro: physiological study mechanisms involved in photosynthetic apparatus protection against lead toxicity can ceylon leadwort (plumbago zeylanica l.) acclimate to lead toxicity?-studies of photosynthetic apparatus efficiency cereal cyst nematodes: importance, distribution, identification, quantification, and control a novel-dehydroascorbate reductase from spinach chloroplasts homologous to plant trypsin inhibitor polyamines and plant disease oxidative stress links response to lead and acyrthosiphon pisum in pisum sativum l acknowledgements as a corresponding author, i express my gratitude conflict of interest the authors declare that they have no conflict of interest.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- -g ux authors: loh, hwei-san; green, brian j; yusibov, vidadi title: using transgenic plants and modified plant viruses for the development of treatments for human diseases date: - - journal: curr opin virol doi: . /j.coviro. . . sha: doc_id: cord_uid: g ux production of proteins in plants for human health applications has become an attractive strategy attributed by their potentials for low-cost production, increased safety due to the lack of human or animal pathogens, scalability and ability to produce complex proteins. a major milestone for plant-based protein production for use in human health was achieved when protalix biotherapeutics produced taliglucerase alfa (elelyso(®)) in suspension cultures of a transgenic carrot cell line for the treatment of patients with gaucher's disease, was approved by the usa food and drug administration in . in this review, we are highlighting various approaches for plant-based production of proteins and recent progress in the development of plant-made therapeutics and biologics for the prevention and treatment of human diseases. hwei-san loh , , brian j green production of proteins in plants for human health applications has become an attractive strategy attributed by their potentials for low-cost production, increased safety due to the lack of human or animal pathogens, scalability and ability to produce complex proteins. a major milestone for plant-based protein production for use in human health was achieved when protalix biotherapeutics produced taliglucerase alfa (elelyso ) in suspension cultures of a transgenic carrot cell line for the treatment of patients with gaucher's disease, was approved by the usa food and drug administration in . in this review, we are highlighting various approaches for plant-based production of proteins and recent progress in the development of plant-made therapeutics and biologics for the prevention and treatment of human diseases. infectious diseases remain as one of the leading causes of mortality and morbidity in developing countries and are exacerbated by the lack of resources and infrastructure to prevent, treat and control diseases. therefore, emerging and re-emerging pathogens have frequently resulted in epidemics in these countries. over the past several decades, production of proteins in plants has been shown to be a promising approach for the manufacture of targets for human health applications. plants, when compared to other production systems, offer some advantages, including ease of scaling and lack of human and animal pathogens [ ] [ ] [ ] (table ) . this review focuses on several approaches that have been used to produce proteins in plants for prophylactic and therapeutic applications to combat human disease conditions. the various approaches for plant-based production of proteins are illustrated in figure . stable nuclear and chloroplast transformations are the two approaches utilized to express heterologous recombinant proteins in plants. agrobacterium-mediated stable transformation has a long history in plant genetic manipulation, and is achieved by stable integration of t-dna into plant nuclear genome [ ] . however, the approach is time consuming, with a lead time ranging from to months and typically has low levels of the target protein expressed [ ] . stable introduction of target genes into chloroplast genome, that is, chloroplast transformation or transplastomics, however, allows for higher levels of target expression as compared to nuclear transformation, largely due to the lack of gene silencing and high gene copy number [ ] , but it is technically difficult, lacks most post-translational modifications and has only been successful in a limited number of plant species. transient expression of target proteins in plants using modified plant viruses or viral vectors integrated into binary vectors delivered via agrobacterium [ , ] is often considered a more robust approach when compared to stable transformation, due to its rapid production capabilities and relatively high protein expression [ ] . the majority of plant viral vectors used to date are based on single-stranded rna viruses, such as tobacco mosaic virus, potato virus x and cowpea mosaic virus (cpmv), which encode for at least three proteins with functions in viral replication (replicase), encapsidation (coat protein) and movement from cell-to-cell (movement protein) [ ]. the initial strategy involved production of recombinant proteins using plant viruses by exploiting their natural ability to infect (full virus) plants. however, this approach generally failed due to instability of viral genome modified by the introduction of large target genes [ ] . this issue was largely resolved by using agrobacterium-mediated gene delivery or agroinfiltration. the target gene can either be directly cloned into an agrobacterium vector or through a modified plant viral vector which has been integrated into an agrobacterium binary plasmid, and delivered into the plant tissues by infiltration with the transformed agrobacterium [ , ] . agroinfiltration allows for high levels of target protein expression with the table general comparison of expression hosts for the production of heterologous proteins for medical and pharmaceutical applications potential for cost-effective production [ , ] . the peak protein expression is typically observed in less than days postinfiltration which is significantly faster when compared to the full virus strategy which requires more than weeks in order to generate a systemic infection for expression. the promise of this platform has been evidenced in numerous successful clinical trials, which demonstrated safety and efficacy of plant-made protein therapeutics and biologics [ ] . for example, in responding to the h n influenza virus pandemic that occurred in , medicago, a canadian company, reported producing the vaccine candidate, hemagglutinin in days in nicotiana benthamiana [ ] . as such, agroinfiltration provides a rapid response capability and is currently the preferred approach for the production of proteins in plants. modified plant viruses for treatment of human diseases loh, green and yusibov detection of serum igg in immunized mice (sc) and fecal iga in immunized mice via oral administration. [ ] hepatitis b virus (hbv) small surface antigen (s-hbsag) evlp vaccine against hbv lactuca sativa (lettuce)/transgenic (nuclear) detection of serum igg in immunized mice via oral administration. [ ] hbv surface antigen (hbsag) suv against hbv solanum tuberosum (potato)/transgenic (nuclear) induction of serum antibodies and stable immunological memory in immunized mice fed with transgenic potato tubers. [ ] human immunodeficiency virus (hiv) gp multi-epitopic envelope protein (c (v ) ) lettuce/transgenic (nuclear) detection of cell-mediated and humoral immunities in immunized mice via oral administration. [ ] hiv gp and gp multi-epitopic envelope proteins (multi-hiv) tobacco/transgenic (chloroplast) detection of antibody and cellular responses as well as specific ifn-g production in immunized mice via oral administration. [ ] hiv- envelope proteins (gag/dgp ) evlps vaccine against hiv- purified ctb-ex increased level of insulin secretion from pancreatic cells. oral feeding of lyophilized ctb-ex lowered blood glucose level in mice. [ ] keys for abbreviations: ade, antibody-dependent enhancement; c h , constant domains of immunoglobulin heavy chain; c l , constant domain of immunoglobulin light chain; ctb, cholera toxin b; cvlp, chimeric virus-like particle; diii, domain iii; dpp, dipeptidyl peptidase; e, envelope; evlp, enveloped virus-like particle; ex, exendin; f, coagulation factor; gaa, acid alpha glucosidase; glp, glucagon like peptide; gp, glycoprotein; ha, hemagglutinin; hi, hemagglutination-inhibition; hc, heavy chain; ig, immunoglobulin; ltb, heat-labile enterotoxin b subunit; im, intramuscular; in, intranasal; ip, intraperitoneal; iv, intravenous; mab, monoclonal antibody; n, nucleocapsid; pa, protective antigen; rtb, ricin toxin b; sag, surface antigen; sc, subcutaneous; scfv, single-chain variable fragment of immunoglobulin; suv, subunit vaccine; vlp, virus-like particle. numerous examples of plant-produced proteins targeting prophylactic and therapeutic applications (subsectioned as vaccines, antibodies and other biopharmaceuticals) in preclinical development are shown in table . several lead candidates have gone through clinical trials (table ) and have been comprehensively reviewed [ , ]. vaccines are highly effective tools for the prevention of infections. over the last three decades, plant-produced antigens targeting various pathogens have been shown to be effective in animal models ( table ) . several of these candidates have progressed into early stage clinical development and were evaluated in phase - human clinical trials ( (table ) . planet biotechnology (hayward, ca) produced the world's first plant-derived clinically tested secretory iga monoclonal antibody which recognizes the surface antigen i/ii of streptococcus mutans (carorx tm ) that predominantly causes dental caries. following the successful demonstration of safety and efficacy in a phase clinical trial, carorx tm has been licensed in europe in a medical device category [ , ] and applied as an oral topical solution to prevent tooth decay. in , the recombinant human growth hormone was the first plant-based biopharmaceutical protein produced in plants [ ] . then over two decades later, the fda in may approved elelyso (human recombinant taliglucerase alfa or glucocerebrosidase), an enzyme produced in genetically engineered carrot cells for treating type gaucher's disease (gd) by protalix biotherapeutics and its partner, pfizer [ ] . gd is a lysosomal storage disorder caused by a hereditary deficiency of the enzyme, glucocerebrosidase (gcd). gd is currently treated by enzyme replacement therapy using this recombinant gcd that is administered intravenously every weeks [ ] . in addition to offering a versatile production platform for numerous plant-made proteins, plant viruses have been engineered to provide medical applications in other ways [ ] . vlps offer advantages over recombinant protein vaccines as they tend to elicit a higher immune response [ ] . virus nanoparticles have also been developed for the targeted delivery for disease treatment and diagnostic purposes. for example, cpmv represents an icosahedral nanoparticle with its capsid surface displaying accessible lysine residues; each of these can be conjugated to various chemical moieties like fluorescent dyes/arrays, polyethylene glycol polymers and subcellular targeting molecules [ , ] . the use of this technology includes engineering for viral resistance the construction of cpmv nanoparticles displaying gastrin-releasing peptide receptors that are overexpressed in human prostate cancers [ ] . another example, cowpea chlorotic mottle virus can stably assemble in vitro and package the rna derived from sindbis virus, a mammalian virus. these hybrid cowpea chlorotic mottle virusbased vlps were shown to protect against rna degradation by cellular nucleases and were able to deliver and release their rna contents within the cytoplasm of mammalian cells. moreover, these hybrid vlps with the fusion of subcellular targeting moieties could be directed toward distinct sites within the cell [ ] and potentially applied as a medical targeted delivery tool. plant viruses have also been engineered to act as adjuvants to elicit an immune response that is more potent and effective. the rodshaped papaya mosaic virus nanoparticles have been engineered to express an influenza epitope on their surface, and mice and ferrets immunized with these recombinant nanoparticles exhibited an increase in robust humoral response to influenza virus infection [ ] . there is growing evidence that plants are capable of making proteins with desired quality to address a range of human health-related issues. plant production platforms for protein therapeutics and biologics, in particular the transient agroinfiltration approach, have demonstrated the ability to be used for broad research and development, as well as commercial needs. it has been extensively discussed that the transient agroinfiltration approach is the ideal platform for fast and scalable production in response to new outbreaks of highly infectious diseases and has been demonstrated under various programs. the success of protalix biotherapeutics in gaining fda approval for the therapeutic enzyme, elelyso for human use was a significant milestone for the plant molecular pharming field. more importantly, the primary benefits of plant-made protein therapeutics and biologics in terms of product safety and potential cost-effectiveness will further contribute to global public health in both developed and developing nations. yao j, weng yq, dickey a, wang yj: plants as factories for human pharmaceuticals: applications and challenges. int j mol sci , : - . this paper illustrates the plant molecular farming or pharming concept in relation to human pharmaceutical applications. several types of plantbased production platforms are described. the challenges such as planttype glycosylations, downstream bioprocesses and biosafety concerns are also discussed. besides, some of the preclinical and clinical studies that have been enlisted in our review paper are elaborated here. the production of recombinant pharmaceutical proteins in plants production of vaccines and therapeutic antibodies for veterinary applications in transgenic plants: an overview comparative evaluation of recombinant protein production in different biofactories: the green perspective the integration of t-dna into plant genome magnifection -a new platform for expressing recombinant vaccines in plants engineering the chloroplast genome for hyperexpression of human therapeutic proteins and vaccine antigens a launch vector for the production of vaccine antigens in plants. influenza other respir viruses production of secretory iga antibodies in plants production of antibodies in plants: status after twenty years the expression of a nopaline synthase -human growth hormone chimaeric gene in transformed tobacco and sunflower callus tissue first plant-made biologic approved plant-based oral delivery of b-glucocerebrosidase as an enzyme replacement therapy for gaucher's disease plant virus expression vector development: new perspectives virus-like particles as a highly efficient vaccine platform: diversity of targets and production systems and advances in clinical development plant viral capsids as nanobuilding blocks: construction of arrays on solid supports cowpea mosaic virus nanoparticles target surface vimentin on cancer cells intravital imaging of human prostate cancer using viral nanoparticles targeted to gastrinreleasing peptide receptors reconstituted plant viral capsids can release genes to mammalian cells improvement of the trivalent inactivated flu vaccine using papmv nanoparticles a plantproduced protective antigen vaccine confers protection in rabbits against a lethal aerosolized challenge with bacillus anthracis ames spores. hum vaccines immunother generation of protective immune response against anthrax by oral immunization with protective antigen plant-based vaccine novel vaccination approach for dengue infection based on recombinant immune complex universal platform expression of an immunogenic ebola immune complex in nicotiana benthamiana expression of an immunogenic ltb-based chimeric protein targeting zaire ebolavirus epitopes from gp in plant cells brief background about ebola virus is mentioned in this paper. development of plant-based vaccine against ebola virus is in fact scarcely reported till date. the authors have demonstrated the successful expression of the chimeric protein, ltb-ebov in plant cells and its immunogenicity in balb/c mice via subcutaneous and oral administrations freeze-drying of plant tissue containing hbv surface antigen for the oral vaccine against hepatitis b study of the immunogenicity of hepatitis b surface antigen synthesized in transgenic potato plants with increased biosafety immunogenic properties of a lettuce-derived c (v ) multiepitopic hiv protein a plantderived multi-hiv antigen induces broad immune responses in orally immunized mice immunological characterization of plant-based hiv- gag/dgp virus-like particles transgenic tobacco expressed hpv -l and lt-b combined immunization induces strong mucosal and systemic immune responses in mice immunization with an hpv- l -based chimeric virus-like particle containing hpv- e and e epitopes elicits long-lasting prophylactic and therapeutic efficacy in an hpv- tumor mice model a plant produced h n trimeric hemagglutinin protects mice from a lethal influenza virus challenge immunogenicity of h n influenza virus-like particles produced in nicotiana benthamiana. hum vaccines immunother the authors have developed the recombinant hemagglutinin from the a/ california/ / strain of h n influenza a virus in the form of enveloped vlps (hac-vlps) in plants. hac-vlps resemble the influenza a virus by morphology expression of h n nucleoprotein in maize seeds and immunogenicity in mice purification and immunogenicity of hemagglutinin from highly pathogenic avian influenza virus h n expressed in nicotiana benthamiana expression of rabies glycoprotein and ricin toxin b chain (rgp-rtb) fusion protein in tomato hairy roots: a step towards oral vaccination for rabies antigen production in plant to tackle infectious diseases flare up: the case of sars a non-glycosylated, plant-produced human monoclonal antibody against anthrax protective antigen protects mice and non-human primates from b. anthracis spore challenge. hum vaccines therapeutic intervention of ebola virus infection in rhesus macaques with the mb- monoclonal antibody cocktail structural and functional characterization of an anti-west nile virus monoclonal antibody and its single-chain variant produced in glycoengineered plants generation and analysis of novel plantderived antibody based therapeutic molecules against west nile virus a plant produced antigen elicits potent immune responses against west nile virus in mice suppression of inhibitor formation against fviii in a murine model of hemophilia a by oral delivery of antigens bioencapsulated in plant cells low cost industrial production of coagulation factor ix bioencapsulated in lettuce cells for oral tolerance induction in hemophilia b oral delivery of acid alpha glucosidase epitopes expressed in plant chloroplasts suppresses antibody formation in treatment of pompe mice the authors have demonstrated the successful expression of acid alpha glucosidase (gaa) in fusion with a transmucosal carrier, cholera toxin b in chloroplasts. by using oral administration of gaa bioencapsulated in plant cells, an induction of oral tolerance and significant suppression of gaa-specific inhibitory antibody (which will jeopardize the enzyme replacement therapy) were evidenced in pompe mice oral delivery of bioencapsulated exendin- expressed in chloroplasts lowers blood glucose level in mice and stimulates insulin secretion in beta-tc cells the authors would like to thank dr stephen streatfield (fhcmb) for editorial assistance. the authors declare no conflict of interest.papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest key: cord- -df w l authors: rosales-mendoza, sergio; márquez-escobar, verónica a.; gonzález-ortega, omar; nieto-gómez, ricardo; arévalo-villalobos, jaime i. title: what does plant-based vaccine technology offer to the fight against covid- ? date: - - journal: vaccines (basel) doi: . /vaccines sha: doc_id: cord_uid: df w l the emergence of new pathogenic viral strains is a constant threat to global health, with the new coronavirus strain covid- as the latest example. covid- , caused by the sars-cov- virus has quickly spread around the globe. this pandemic demands rapid development of drugs and vaccines. plant-based vaccines are a technology with proven viability, which have led to promising results for candidates evaluated at the clinical level, meaning this technology could contribute towards the fight against covid- . herein, a perspective in how plant-based vaccines can be developed against covid- is presented. injectable vaccines could be generated by using transient expression systems, which offer the highest protein yields and are already adopted at the industrial level to produce vlps-vaccines and other biopharmaceuticals under gmpc-processes. stably-transformed plants are another option, but this approach requires more time for the development of antigen-producing lines. nonetheless, this approach offers the possibility of developing oral vaccines in which the plant cell could act as the antigen delivery agent. therefore, this is the most attractive approach in terms of cost, easy delivery, and mucosal immunity induction. the development of multiepitope, rationally-designed vaccines is also discussed regarding the experience gained in expression of chimeric immunogenic proteins in plant systems. the coronaviruses (covs) are enveloped viruses having a positive-sense, single-stranded genomic rna [ ] and are grouped into four genera: α-covs, β-covs, γ-covs, and δ-covs. the ones that affect mammals are αand β-covs, while the other two genera infect birds and could also infect mammals [ ] . lately, the coronavirus has become a remarkable concern for global health after the diagnosis of a cluster of unknown pneumonia patients in december in wuhan, china. the outbreak was associated with workers in the wuhan wholesale seafood market, in which live exotic animals are sold [ ] . the pathogen was named sars-cov- given its similarity ( % of genomic similarity) to sars-cov- , which was responsible for the - severe acute respiratory syndrome epidemic (sars) . until now, the official and accepted name for this new coronavirus strain is covid- virus, an acronym for coronavirus infectious disease [ ] . a systematic genomic analysis has revealed amino acid substitutions between sars-cov- and sars-cov- , which is a starting point to study its functional and pathogenic divergence [ ] . presently, there is no specific treatment for covid- . as an emerging pathogen, several knowledge gaps exist about the sars-cov- virus and the coming months will be critical to refine clinical management and advancement in the validation of possible treatments. a myriad of efforts is ongoing to perform clinical trials and determine the efficacy of preexisting drugs, with hydroxychloroquine and remdesivir as the most promising candidates [ ] . receptor blockers are also under evaluation [ ] , along with the assessment and development of monoclonal antibodies. moreover, transfusions using plasma from individuals recovered from sars-cov- infection (containing neutralizing antibodies) are under exploration with promising findings [ ] . nonetheless, vaccination is the most effective approach to control and ultimately eradicate infectious diseases. since sars-cov- possesses high transmissibility (asymptomatic and presymptomatic virus shedding, which results in a high number of patients with mild symptoms), the development of vaccines is an urgent goal to fight against this pathogen. the straightforward path to generate vaccine candidates is the technology of inactivated vaccines, which can be formulated with sars-cov- virions previously inactivated by chemical or physical treatments. a vaccine based on live-attenuated virus is another possible approach [ ] . for sars-cov- and mers-cov, inactivated vaccine candidates have induced robust humoral responses leading to virus neutralization. alternatively, the construction of a chimeric attenuated virus constitutes an interesting path. for instance, the influenza virus can be used as a scaffold to expose sars-cov- antigens and generate a bivalent vaccine targeting two relevant pathogens causing respiratory diseases [ ] . similarly, adenoviral vectors could be applied in this field [ ] . vaccines based on whole viruses are associated with concerns about antibody-dependent enhancement of viral infection, reactogenicity, and strain reversion to pathogenic forms, among other safety issues [ ] . therefore, the path for vaccine development will require a detailed characterization, not only in terms of efficacy but also safety [ ] . although inactivated vaccines will perhaps be the main avenue for generating the first experimental vaccines, alternative approaches should be explored in parallel, namely the development of subunit vaccines. since the coronavirus spike (s) glycoproteins initiate entry into cells; they are considered the primary target for neutralizing antibodies (figure ). cytotoxic t-lymphocyte (ctl) responses are also of key relevance to protect against viral infections [ ] . under these principles, vaccine development against covid- has been initiated and efforts announced in this field include the development of rna-based vaccines by curevac [ , ] , and an rna vaccine candidate formulated with a novel lipid nanoparticle (lnp) carrying mrna encoding for a full-length, prefusion stabilized s protein [ ] . another candidate (developed by shenzhen geno-immune medical institute [ ] ) consists of a multiepitopic vaccine based on the generation of artificial antigen presenting cells through transduction as a way to express viral antigens and immune modulatory genes to ultimately activate t cells. vaccines , , x of these principles, vaccine development against covid- has been initiated and efforts announced in this field include the development of rna-based vaccines by curevac [ , ] , and an rna vaccine candidate formulated with a novel lipid nanoparticle (lnp) carrying mrna encoding for a fulllength, prefusion stabilized s protein [ ] . another candidate (developed by shenzhen geno-immune medical institute [ ] ) consists of a multiepitopic vaccine based on the generation of artificial antigen presenting cells through transduction as a way to express viral antigens and immune modulatory genes to ultimately activate t cells. figure . structure of the sars-cov- virus. the virus is formed by an envelope membrane associated with the following structural proteins: spike protein (s), which mediates binding to the host cell receptors and considered a critical target for the induction of antibodies capable of neutralizing the virus; hemagglutinin-esterase dimer (he), which acts as a potent mediator of attachment and destruction of sialic acid receptors on the host cell surface; a membrane glycoprotein (m), which is important to generate the virus; and the envelope protein (e), which adheres to the m protein to form the viral envelope. the viral structure also comprises a nucleocapsid protein (n) that, along with the rna genome, produces the nucleocapsid. although the conventional developmental paths for vaccines take several years, the vaccine against hand, foot, and mouth disease (hfmd) that was approved in china; demonstrates how joint efforts can lead to new vaccines that are accepted in relatively short periods after a pathogen emerges [ ] . similarities with the sars-cov- virus constitute a valuable reference for vaccine development, however it should be considered that the genetic variability of sars-cov- seems higher as more than two variants have been described. despite this, the cross protection of sars-cov- and mers experimental vaccines remains to be explored. thus far, there is evidence that some sars-cov- neutralizing antibodies cross react with sars-cov- , however extensive research in this regard is needed. the epitopes conserved among sars-cov- and sars-cov- have been identified and proposed for vaccine development [ ] . the race for generating fundamental knowledge on covid- is leading to rapid advances, which will be useful to refine vaccine design. for instance, walls et al. [ ] , reported cryo-em structures of the ectodomain trimer, which will be of critical importance for designing vaccines and viral entry blockers. they also found evidence of potent inhibition of sars-cov- entry into host cells by using murine polyclonal antibodies against sars-cov- , which suggests that conserved s figure . structure of the sars-cov- virus. the virus is formed by an envelope membrane associated with the following structural proteins: spike protein (s), which mediates binding to the host cell receptors and considered a critical target for the induction of antibodies capable of neutralizing the virus; hemagglutinin-esterase dimer (he), which acts as a potent mediator of attachment and destruction of sialic acid receptors on the host cell surface; a membrane glycoprotein (m), which is important to generate the virus; and the envelope protein (e), which adheres to the m protein to form the viral envelope. the viral structure also comprises a nucleocapsid protein (n) that, along with the rna genome, produces the nucleocapsid. although the conventional developmental paths for vaccines take several years, the vaccine against hand, foot, and mouth disease (hfmd) that was approved in china; demonstrates how joint efforts can lead to new vaccines that are accepted in relatively short periods after a pathogen emerges [ ] . similarities with the sars-cov- virus constitute a valuable reference for vaccine development, however it should be considered that the genetic variability of sars-cov- seems higher as more than two variants have been described. despite this, the cross protection of sars-cov- and mers experimental vaccines remains to be explored. thus far, there is evidence that some sars-cov- neutralizing antibodies cross react with sars-cov- , however extensive research in this regard is needed. the epitopes conserved among sars-cov- and sars-cov- have been identified and proposed for vaccine development [ ] . the race for generating fundamental knowledge on covid- is leading to rapid advances, which will be useful to refine vaccine design. for instance, walls et al. [ ] , reported cryo-em structures of the ectodomain trimer, which will be of critical importance for designing vaccines and viral entry blockers. they also found evidence of potent inhibition of sars-cov- entry into host cells by using murine polyclonal antibodies against sars-cov- , which suggests that conserved s epitopes among these viruses are relevant for immunization and perhaps preexisting immunization models will be useful in the fight against covid- . the characterization of other key targets for drug design has been accelerated (e.g., the protease x-ray structure has been recently described [ ] ). therefore, these recent advances will set the basis to accelerate the development of covid- subunit vaccines taking into consideration the sars-cov- vaccine development, especially the approach targeting the s protein. in parallel with these advances in vaccine design, defining vaccine platforms amenable for rapid and large scale production is required. these must keep in mind that low cost, safe, and easy distribution and delivery are the required attributes to implement broad coverage of vaccination campaigns; especially in the developing countries. genetically engineered plants constitute a consolidated platform for the manufacturing of biopharmaceuticals. plants have been used over the last three decades for this purpose. thus far, a diverse group of biopharmaceuticals has been functionally produced in plant systems that include antibodies, vaccines, growth factors, and cytokines [ ] . remarkably, a recombinant enzyme produced in carrot cells has been already approved by the fda for gaucher's disease treatment [ ] . the main advantages of plant-based platforms include the inability to replicate human pathogens (diminishing the risk of contamination and making the purification process less strident), use of unsophisticated bioreactors, and efficient synthesis of complex proteins (multimeric or glycosylated) [ , ] . the current expression approaches for recombinant proteins using the plant cell as host comprise stably-transformed plants at the nuclear or chloroplast levels and transiently-transformed plants. these expression modalities are summarized in table . the conventional approach for expression of transgenes in plants comprises transgene insertion into the nuclear genome. currently, agrobacterium-mediated transformation is the most popular method to achieve this modification since this bacterium has the ability to transfer large segments of dna with minimal rearrangement at high efficiency with low number of insertions. nonetheless, the transgene is randomly inserted into the genome, which often leads to positional effects that make expression levels unpredictable and interruption of endogenous genes a possibility. another limitation is the induction of silencing mechanisms that hamper productivity. nevertheless, it should be considered that new technologies are emerging to cope with these limitations by providing ways to achieve site-directed insertion through a number of mechanisms [ ] . the n-terminal fragment of the sars-cov- s protein (s ) was expressed in stably transformed tomato and low-nicotine tobacco plants, which induced iga and igg responses in mice. [ ] transient nuclear genome transformation rapid production; high productivity; implemented at the industrial level seed bank cannot be generated; requires purification of the antigen to eliminate toxic compounds from the host and ag-robacteria residues s protein; multiepitope vaccines a chimeric protein of gfp and amino acids - of the sars-cov- s protein (s :gfp) was transiently expressed in tobacco leaves and stably transformed in tobacco and lettuce. no immunization assays were performed the sars-cov- n protein was transiently expressed in nicotiana benthamiana, which induced in mice high levels of igg and igg a and up regulation of ifn-γ and il- in splenocytes. a chimeric protein of gfp and the sars-cov- s protein was transiently expressed in tobacco plants. no immunization tests were performed. the sars-cov- m and n proteins were transiently expressed in n. benthamiana. the n protein was antigenic but immunogenicity was not assessed. [ [ ] [ ] [ ] transplastomic technologies high productivity; multigene expression is possible; improved biosafety since the transgene is inherited maternally; site-specific insertion through recombination; not affected by silencing or position effects complex post-translational modifications are not performed; transformation protocols available for few species and the generation of lines takes long time. a chimeric protein of gfp and amino acids - of the sars-cov- s (s :gfp) was expressed in transplastomic tobacco plants. [ , ] vaccines , , in the case of transplastomic technologies, they typically comprise the site-directed insertion of the foreign dna into the chloroplast genome in an event induced by homologous recombination. the main advantage of this technology is the high protein yield, which is a consequence of the high copy number of the transgene and the fact that silencing events have not been reported thus far, and position effects are avoided due to the site-directed insertion. in addition, transgene confinement is achieved given the maternal inheritance shown by most plant species and several proteins can be produced from a single transformation event through the use of operon-like arrangements [ , ] . for more information regarding transplastomic technologies, readers are referred to reviews published by [ , ] . another approach to express heterologous protein in plants relies on the use of viral-based vectors, which exploit the efficient promoters, utrs, and dna/rna replication mechanisms found in plant viruses. interestingly, the agrobacterium-mediated delivery of viral vectors has consolidated as a highly efficient strategy to achieve rapid production of biopharmaceuticals with yield up to g of protein per kg of fresh plant biomass. the tobamoviruses, potexviruses, tobraviruses, geminiviruses, and comoviruses have served as a basis for the development of transient, efficient expression systems in plants [ ] . this technology, however, demands the purification of the target protein given the presence of toxic compounds (e.g., alkaloids) in the typical host (nicotiana benthamiana), as well as bacterial residues, especially endotoxins. therefore, at present, this technology is applied for the formulation of injectable or nasal vaccines. thus far, some plant-vaccine candidates have entered clinical trials, including candidates against swine influenza, rabies, and hepatitis b [ ] . the most promising candidates are the influenza vaccines developed by medicago inc. that rely on using a non-replicative vector carrying viral regulatory sequences to mediate the transient expression of hemagglutinin (ha) in n. benthamiana, which has led to injectable vaccine candidates [ , ] . overall, these vaccines have been seen as safe and their immunogenic properties have been positively evaluated using in vitro assays with human and mice cells. the trials conducted in volunteers revealed proper immunogenicity with no serious adverse effects (see table ). moreover, a group of respiratory diseases has been targeted through the plant-based production of antigens with promising immunogenicity in preclinical trials [ ] . both homologous and heterologous antigen-specific cd + t cells were elicited. additionally, production of ifn-γ, il- , and/or tnf-α was achieved upon ex vivo antigenic re-stimulation. ha from a/california/ / h n . vlps were evaluated in vitro using human monocyte-derived macrophages. the plant-made vlps were efficiently captured and subjected to endosomal processing and cross-presentation. [ ] ha from a/h n /california/ / (pdmh n ). the inactivated h n vaccine (iiv) was included as a reference vaccine. mice were i.m.-immunized twice. cd + (tnf-α, ifn-γ) and cd + (ifn-γ) t cell responses were higher for the plant-made vaccine than the iiv formulation. the plant-made vlp vaccine elicited stronger and more balanced immune responses than iiv. [ ] ha from a/california/ / (h n ) and a/indonesia/ / (h n ). in vitro assays were performed using mouse and human dcs. mice were immunized by the i.m. route using alhydrogel as an adjuvant. human dcs exposed to plant-made vlps showed high stimulation in terms of secretion of il- , il- , and tnfα and cd expression, along with activation of cd + and cd + t cells. vlps induced accumulation of both cdc s and cdc in the draining lymph nodes of test mice. lymphocytes from mice immunized with plant-made vlps secreted higher concentrations of pro-inflammatory cytokines (including il- , il- , and tnf-α) upon antigenic stimulation. [ ] ha from a/california/ / or a/indonesia/ / strains. in vitro assays were performed using mouse dendritic cells. the plant-made vlps trimmers were morphologically stable over time and interacted with activated antigen-presenting cells similar to the wild type virus. [ ] nonetheless, the ultimate goal for low-cost vaccine development could be achieved by generating oral formulations not requiring purification and being composed of freeze-dried biomass encapsulated in gelatin pills or tablets. under this focus, the goal is to trigger specific immune responses via the gut-associated lymphoid tissues (galt). for this purpose, edible plants lacking toxic metabolites should be used. perhaps the main disadvantage of this technology is the long time required to generate transformed lines of edible crops efficiently expressing the antigen of interest (e.g., rice or corn transgenic lines or transplastomic lines) [ ] . these oral vaccines will overcome the disadvantages of injectable vaccines such as painful administration and the risk associated with invasive delivery routes. in terms of costs, avoiding the requirements of sterile devices and trained personnel represent substantial savings. the fact that plant-based vaccine formulations will not require antigen purification will be, without a doubt, the main factor that will make them low-cost alternatives [ ] , which is necessary to provide wide vaccination coverage in developing and low-income countries. recent promising reports in the viability of formulating oral vaccines include the case of the hepatitis b virus surface antigen [ ] and models of immunotherapy against allergy with promising results [ ] , among other diseases. therefore plant-based vaccines are becoming a reality, although it should be recognized that the progress has been slower than initially expected. this is particularly true for the development of oral vaccines, with the main drawbacks being poor characterization of antigen stability, bioavailability, and reproducibility [ , ] . the currently available expression strategies for foreign proteins in plants allow projecting the generation of several vaccine candidates against covid- in the short term. the expression approach and targeted organelle will depend on the nature of the selected target antigen. in the following section, possible avenues for the development of plant-based vaccines are envisaged (table and figure ). the currently available expression strategies for foreign proteins in plants allow projecting the generation of several vaccine candidates against covid- in the short term. the expression approach and targeted organelle will depend on the nature of the selected target antigen. in the following section, possible avenues for the development of plant-based vaccines are envisaged (table and figure ). developmental paths for plant-based vaccines. vaccine antigens can comprise full length viral proteins or chimeric proteins rationally designed to serve as multiepitopic vaccines. this is based on the selection of t cell and b cell epitopes with the aid of immune bioinformatics tools and experimental data of their protective capacity. current genetic engineering technologies allow expressing target antigens either stably or transiently, which allow exploring distinct immunization approaches. transient expression typically requires purification of the antigen-which is useful for the formulation of injectable vaccines-whereas stable transformation, especially in edible crops, allows implementing oral immunization schemes without purification. combined schemes, using pure antigens for priming by parenteral administration and plant biomass containing the antigen for oral boosting, are likely ideal approaches capable of inducing long-term protection in several compartments, especially in mucous membranes. vaccine antigens can comprise full length viral proteins or chimeric proteins rationally designed to serve as multiepitopic vaccines. this is based on the selection of t cell and b cell epitopes with the aid of immune bioinformatics tools and experimental data of their protective capacity. current genetic engineering technologies allow expressing target antigens either stably or transiently, which allow exploring distinct immunization approaches. transient expression typically requires purification of the antigen-which is useful for the formulation of injectable vaccines-whereas stable transformation, especially in edible crops, allows implementing oral immunization schemes without purification. combined schemes, using pure antigens for priming by parenteral administration and plant biomass containing the antigen for oral boosting, are likely ideal approaches capable of inducing long-term protection in several compartments, especially in mucous membranes. one prominent approach for vaccine design is based on the use of virus-like particles (vlps), which are macromolecular complexes resembling viruses, but lacking their genome. in this way, vlps mimic the native structure of viruses, but are not infective. this avoids the disadvantages of vaccines formulated with attenuated or inactivated viruses that include reactogenicity and reversion to pathogenic forms [ , ] . a myriad of reports on the production of vlps can be found in the literature that comprise the cases of the influenza virus, human papillomavirus, human immunodeficiency virus, foot and mouth disease virus, norwalk virus, rift valley fever virus, and hepatitis b virus [ ] [ ] [ ] [ ] . the precedents of sars-cov- and mers antigens expressed in recombinant systems leading to the formation of vlps constitute important guides for the topic of covid- vaccine development. mortola and roy ( ) produced coronavirus vlps applying baculovirus/insect cells expression (sf cells), which was based on the following structural proteins: s (spike), e (envelope), m (membrane), and n (nucleocapsid) of sars-cov, either individually or simultaneously. simultaneous expression at high levels was achieved for s, e, and m proteins, leading to an efficient vlps assembly and release, evidenced by electron microscopy and immunofluorescence. the authors demonstrated that the formed vlps were similar in morphology to the sars-cov- virions [ ] . another group reported in the same year that m and e proteins were sufficient for the efficient formation of vlps in insect cells [ ] . in , the immunogenicity of sars-cov- vlps was first described by evaluating insect cell-made vlps based on the m, e, and s proteins. electron microscopy demonstrated vlps formation in co-infected insect cells. mice subjected to an immunization scheme consisting of four subcutaneous (s.c.) doses of vlps emulsified with freund's adjuvant showed high antibody titers against sars cov. furthermore, vlps elicited cellular immunity following increased production of ifn-γ and il- [ ] . subsequent in vitro assays using vlps designed with the bat-isolated coronavirus protein s and the sars-cov- proteins e and m; demonstrated ability to stimulate dcs in terms of cytokine induction, evidenced by il- and tnf-alpha production. furthermore, the study indicated that ifn-γ+ and il- + cd + t cells increased in co-culture with dcs pre-exposed to the vlps tested [ ] . given that immunization by mucosal routes is the most pertinent for vaccination, sars-cov- vlps were analyzed in a mouse model based on intraperitoneal or nasal immunization. both routes led to sars-cov- -specific igg responses, igg levels in the groups immunized intraperitoneally were higher. nasal immunization usually results in the induction of secretory iga responses at the genital tract, saliva, and lungs; a type of response that is not efficiently induced by systemic administration. in the mentioned study, iga production at the intestinal tract was higher for intraperitoneal administration when compared to intranasal administration, however only genital tract secretions from the i.n.-immunized group showed neutralization activity [ ] . another approach for the production of vlps consisted in co-expressing the sars-cov- s protein and the m influenza protein in the baculovirus/insect cell expression system. interestingly, the chimeric vlps showed similar size and morphology compared to the wild type sars-cov- . immunogenicity and protective efficacy of these chimeric vlps were tested in a mouse lethal challenge model. complete protection from death was observed in mice vaccinated intramuscularly or intranasally with the chimeric vlps, which contained . µg of the sars-cov- protein s [ ] . in the case of the middle east respiratory syndrome (mers), vlps with proteins s, e, and m were generated using the baculovirus/insect cell expression system by wang et al. ( ) [ ] . electron microscopy and immunoelectron microscopy revealed high structural similarity between the mers-cov vlps and the native virus. rhesus macaques inoculated with mers-cov vlps and aluminum adjuvant showed final virus neutralizing antibody titers reaching : ; inducing t-helper (th ) cell-mediated immunity. in a more recent study, the structural proteins of mers-cov were expressed in silkworm larvae and bm cells for the development of vaccine candidates. however, protein s was not detected in the form of vlps despite the fact that e and m proteins were secreted to the culture supernatant. surfactant treatment and mechanical extrusion using protein s or bm cells expressing structural proteins allowed producing nanovesicles exhibiting protein s with a diameter ranging from to nm as revealed by immuno-tem [ ] . although insect cells possess a protein processing capacity similar to that of higher eukaryotic cells, the protein processing pathways are not totally equivalent [ ] . in terms of n-acetylglycosylation, insect cells have the ability to assemble n-glycans to a nascent polypeptide. however, they have unusual final processing activity mediated by β-n-acetylglucosaminidase (soluble or membrane-bound form), which trims an intermediate product common to mammalian and insect pathways resulting in the insect-specific paucimannose as a final product that avoids the galactosylation process occurring in mammals [ ] . considering that vlps of enveloped viruses have been successfully expressed in plants by expressing the ha protein, it is anticipated that sars-cov- vlps could be assembled by expressing the s protein. in fact, although not published in the literature yet, medicago has described the generation of vlps in plants [ ] . for this purpose, nuclear expression targeting the trans-golgi secretion route is proposed to yield a protein subjected to the glycosylation and secretion machinery that could make possible the production of vlps [ ] . therefore, adoption of these works would be valuable during the development of covid- plant-based vaccines (see the section below). besides vlps resembling the sars-cov- virus, another possible approach is to adopt sars-cov- epitopes and express them in chimeric vlps. in this way, a vlp from an unrelated virus can serve as the scaffold to present the target sars-cov- epitopes. for this purpose, the hepatitis b core protein has been applied as scaffold to display unrelated antigens of some pathogens. in particular, this has been applied at the immunodominant c/e loop region, which allows displaying the target on the spike structures of the vlps [ ] . a recent review revealed that at least vaccine candidates have been developed based on plant viruses covering infectious agents, cancer, and autoimmune disorders [ ] . the glycosylation patterns of proteins that form vlps can impact their immunogenicity and protective capacity. interestingly, glycoengineering strategies have been successfully implemented for plants, which allows diversifying their application as hosts for the production of biopharmaceuticals. this is a relevant aspect considering that plants lack the ability to perform glycosylation, which is a characteristic of mammalian systems. for instance, plants perform beta , -xylosylation, core alpha , -fucosylation, and the addition of a second n-acetylglucosamine (glcnac) to the mannose core. moreover, plant glycans lack β ( , ')-galactose and sialic acid, as well as bi-antennary n-glycans [ ] . in some cases, such as antibody production, these differences on glycosylation can be associated to adverse effects that include generation of immunogenic activity, which can eventually lead to blocking antibodies against the therapeutic antibody. nonetheless, in the case of vaccines these differences could add more immunogenic potential and improve vaccine efficacy [ ] . in any case, it is of interest to comment that glycoengineering has allowed developing knock down n. benthamiana plants for beta , -xylosyltransferase (xylt) and alpha , -fucosyltransferase (fuct) genes, which showed significant reduction in the production of xylosylated and/or core alpha , -fucosylated proteins with no phenotypic alterations [ ] . these lines will be valuable tools to study the impact of glycosylation in the efficacy of covid- vlps, which will allow optimizing the plant-based vaccines. another approach that deserves attention is the development of multiepitopic vaccines, which offers the opportunity of achieving a fine rational vaccine design by selecting epitopes that potentially induce robust and protective immune responses. at the same time, this approach will discard those related to non-protective responses or even those inducing antibody-dependent enhancement of the disease. in this way, a highly efficacious and safe vaccine can be obtained. of special interest are the reports proving that specific s protein epitopes enhance the disease upon a pathogen challenge, which highlights the relevance of recurring to the rational design of vaccines to ensure not only immunoprotection, but safety [ ] . for the case of multiepitope vaccines against infectious agents, several reports have focused on selecting the most promising th, b cell, and t cell epitopes that might allow the rational design of a vaccine inducing robust protective responses, while at the same time avoiding deleterious or non-relevant responses. one key factor that adds importance to the development of multiepitopic vaccines against viral disease is genetic variability. in fact, it has been suggested that the sars-cov- virus evolved into two major types, l and s. the l type (∼ %) predominates versus the s type (∼ %), with the latter being proposed as the ancestral version. the l type is more aggressive than the s type and the analysis performed by tang et al. [ ] suggests that human interference influenced the prevalence of l and s types right after the outbreak appearance. another study has also suggested a rapid evolution of this coronavirus [ ] . therefore, the design of multiepitopic vaccines must adopt the selection of epitopes conserved among the viral variants with the capacity to induce neutralizing humoral responses as critical criterion. it should also be considered that ctl responses are relevant to cope with covid- [ ] . the selected epitopes are the targets to which adaptive immune responses should be induced, but they lack the required complexity to trigger robust immune responses. therefore, a carrier is required to increase antigen complexity and, through adjuvant effects, enhance potency of the induced immune response, making it properly polarized. among these carriers, the b subunit of the cholera toxin and the heat-labile enterotoxin from escherichia coli have been extensively used to carry unrelated antigens. the main advantage of these molecules is their adjuvant effects at the mucosal levels, as well as their ability to efficiently deliver the antigens to the submucosa thanks to a mechanism of translocation mediated by binding to the gm receptor at the epithelial cells in the mucosal surfaces [ ] . once at the submucosa, the antigen is efficiently captured and processed by antigen presenting cells, which subsequently induce robust mucosal and systemic th immune responses at the lymph nodes. the design of ltb/ctb-based chimeras carrying sars-cov- epitopes is an interesting approach that should be explored. proline-containing linkers have been used to properly display unrelated antigens without altering the ability of those carriers to form the pentameric structure responsible for gm binding [ ] . the accumulation of those chimeric proteins in the er is advisable, although toxicity has been reported for some plants accumulating high levels of those recombinant proteins [ ] . given that plant systems have been successfully adopted for the production of multiepitope proteins, the generation of such vaccines against covid- is considered highly viable [ ] [ ] [ ] . besides expressing chimeric proteins, multiple antigen expression can be achieved by transplastomic technologies (operon-like expression) or at the nuclear level using the picornaviral a sequence that allows releasing individual antigens from a single coding gene [ ] . the production of immune complexes (ics) in plants is another approach that renders highly immunogenic agents. ics consist of antigens complexed to antibodies recognizing them, constituting macromolecular entities that are efficiently captured and processed by antigen presenting cells [ ] . this results in the induction of robust humoral and cell-mediated immune responses [ , ] . taking advantage of the machinery of plant cells for protein synthesis and processing, they have been exploited as antibodies and ics factories [ ] . for instance, ics based on the tetanus toxin fragment c fused to a monoclonal antibody were produced in transgenic tobacco plants. these ics were highly immunogenic, inducing immunoprotective effects when administered without accessory adjuvants by the s.c. (subcutaneous) route in mice [ ] . this approach has also been applied to the case of the ag b and acr antigens from mycobacterium tuberculosis and the gp antigen from the ebola virus [ , ] . perhaps the main disadvantage of this approach is the requirement of defined antibodies targeting the antigen, which is not available yet for sars-cov- . however, it should be considered that the cross reactivity of anti-sars-cov- s protein antibodies with the counterpart of sars-cov- has been reported [ ] . the purification of antigens is a laborious and expensive activity as part of the production of injectable vaccines [ ] . one alternative to simplify purification relies on the fusion of elastin-like polypeptides (elp) that possess a unique property named reversible phase transition, which allows precipitating the protein of interest by changing the temperature. this approach is an alternative to the complex/expensive affinity chromatography [ ] that has been applied to develop plant-based vaccines candidates with relevant findings. for instance, the m. tuberculosis antigens ag b and esat- were fused to elp and expressed in transgenic tobacco [ ] , and the plant-made antigens were subcutaneously (s.c.)administered to mice and able to trigger long-lasting humoral immune responses. the elp did not negatively impact the immunogenic properties of the antigens. the haemagglutinin (ha) antigen from the influenza virus has also been expressed under this modality in tobacco, where antigen purification was simplified and the obtained product induced neutralizing antibodies in mice after two s.c. immunizations. elpylation did not alter the immunogenicity of ha [ ] . therefore, these precedents suggest that the elp technology is a possible approach to be explored for the production of antigens against the sars-cov- virus. the common steps to the above-mentioned antigen design will comprise determining antigen yields and antigenic activity, assessing the immunogenic activity in test animals under distinct administration routes, and validating the safety and stability of the vaccine. the latter will be critical to justify the application and get approval to conduct clinical trials. a key precedent for this field is the vaccine candidates already reported for sars-cov- and mers, which are both closely related to sars-cov- . following nuclear expression approaches, the n-terminal fragment of the sars-cov- s protein (s ) was expressed in tomato and low-nicotine tobacco plants using an agrobacterium-mediated method. mice orally immunized with this transgenic tomato revealed significantly increased levels of sars-cov- -specific iga. sera of mice parenterally primed with the transgenic tobacco showed the presence of anti-sars-cov- -igg [ ] . another study focused on expressing a chimeric protein of gfp and amino acids - of the sars-cov- spike protein (s :gfp) by using tobacco leaves subjected to transient expression. after microscopy localization, the fusion protein was observed in the cytosol and the periphery of the nucleus. stable transgenic tobacco and lettuce plants were generated by agrobacterium-mediated transformation. the expression of the chimeric antigen was also achieved in tobacco chloroplasts. however, no immunization assays were performed [ , ] . the sars-cov- nucleocapsid (rn) protein of -aa was transiently expressed in n. benthamiana. yields reached up to µg/g of leaf fresh weight (corresponding to . %- % of the total soluble protein, tsp) at dpi under silencing suppression conditions (p ). mice immunization revealed that three doses led to effective b-cell maturation and differentiation, achieving high levels of igg and igg a. ifn-γ and il- were up-regulated in splenocytes, while the expression of il- and il- was not [ ] . the nucleocapsid protein (n) and the membrane protein (m) were transiently expressed in n. benthamiana. yields reached up to - µg/g fresh leaf weight ( . % tsp) for the n protein, while the m protein yields were . %- . % tsp. the purified n protein reacted with human sera having n-specific antibodies. no immunization assays were performed [ ] . these studies constitute a valuable guide to select the most convenient expression platforms for specific sars-cov- antigens. although most of the vaccines against respiratory diseases are administered by parenteral routes, it should be recognized that immune profiles deserve improvements, especially in terms of immunogenicity and efficacy in the elderly [ ] . mucosal vaccines, especially intranasal-administered vaccines, are a promise for immunization against respiratory diseases given the protections induced in lungs and other mucosal tissues that are critical ports for pathogen entry. critical alternatives for the development of mucosal vaccines include guaranteeing antigen delivery and immunostimulating capacity at the mucosa, while at the same time being minimally reactogenic/toxic. among the immunopotentiator molecules that can be explored are bacterial toxin derivatives, toll-like receptor ligands, and cytokines [ , ] . although some companies have already started the production of plant-based vlps vaccines [ , ] using transient expression systems, these efforts are mainly directed to developing injectable vaccines. therefore, it is imperative to start developing alternatives related to mucosal immunization. for this purpose, generating plants stably expressing sars-cov- antigens will open new avenues for the exploitation of this technology. this approach will result in the production of edible plant material containing sars-cov- antigens that could be used for the development of oral boosting agents. interestingly, this concept has been assessed by some groups with relevant results. in the case of a vaccine targeting the hepatitis b virus, a scheme comprising intramuscular (i.m.) boosting with the pure hbsag antigen followed by double oral boosting with plant material containing hbsag at six week intervals led to a comparable response to that induced by the standard intramuscular (i.m.) vaccination scheme [ ] . moreover, in the case of poliomyelitis vaccination, a plant-based vaccine based on transplastomic plants expressing the vp antigen fused to a carrier has been assessed as oral boosting agents after priming with the inactivated polio vaccine administered subcutaneously. boosting with the plant-made vp -based antigen significantly enhanced vp -igg and vp -iga titers when compared to the response in animals not receiving boosts. the scheme also allowed inducing neutralizing antibodies for the three poliovirus sabin serotypes when two doses of ipv and plant-cell oral boosts were administered, whereas a single dose of ipv resulted in low neutralization potential [ ] . in this way, freeze-dried plant material can be used as a low-cost vaccine rendering thermostability and easy to administer features. similarly, pure antigens produced in plants have been assessed with promising results in schemes where a conventional vaccine is used as a priming agent and the plant-made antigen is administered by the oral route as boosting [ ] . exploring these schemes will be useful to achieve the best immune profile against sars-cov- in terms of safe and long-lasting protective immune responses. in fact, the potential of using prime-boosting schemes through different immunization routes has been proven as an efficacious approach to achieve the desired immune profiles [ ] . the emergence of covid- has led to a global emergency that demands the development of new biologics, especially vaccines, to counteract against this threat. in this scenario, a plant-made vaccine is a viable approach to rapidly respond to this need. the current expression technologies offer relevant paths for developing anti-covid- vaccines. vlps constitute an attractive approach for the development of efficient and safe vaccines, which is associated with high immunogenicity, preservation of the antigenic determinants, and lack of replicative capacity. thus, vlps based on the main sars-cov- structural proteins is an attractive approach for vaccine development against coronavirus infections. the transient expression systems based on deconstructed viral vectors and n. benthamiana as host will allow for immediate exploitation of plants as efficient biofactories of injectable vaccine candidates, which are expected to be implemented and entered into clinical trials in a year. the development of vaccines based on transplastomic lines or edible plant species transformed at the nuclear level and intended to result in oral vaccines (especially boosting agents to provide mucosal immunity) are considered long term goals. however, they have special importance given their low cost and potential to serve as effective boosting agents, which could lead to attractive immune profiles characterized by proper humoral response in the mucosal compartments and long-lasting protection, especially for the elderly. in parallel to these developments, the production of monoclonal antibodies in plants will provide another strategy to generate alternatives to the convalescent plasma transfusion, in which plant-made antibodies will constitute a low cost and safer intravenous treatment for critically ill patients. perhaps the main challenge envisioned for the development of covid- plant-based vaccines will be, as is typical for all vaccines, testing their efficacy in large clinical trials to validate their safety while fulfilling the requirements of regulatory agencies. the fact that there are precedents of a plant-made biopharmaceutical approved for human use and plant-made vaccines against influenza under clinical trials (with promising safety and efficacy) is encouraging. therefore, plant-based vaccines have a realistic potential to contribute to the fight against covid- . as the covid- epidemic advances, the exploitation of plant-made vaccines is a promise to generate low cost, easy to administer, and safe/effective vaccines to fight against this pandemic. the next few months will be critical to envision the real potential of this technology. the authors declare no conflict of interest. emerging coronaviruses: genome structure, replication, and pathogenesis potent binding of novel coronavirus spike protein by a sars coronavirus-specific human monoclonal antibody genome composition and divergence of the novel coronavirus ( -ncov) originating in china the epidemiology and pathogenesis of coronavirus disease (covid- ) outbreak coronavirus covid- global cases by the center for systems science and engineering characteristics of and public health responses to the coronavirus disease outbreak in china transmission routes of -ncov and controls in dental practice taking the right measures to control covid- use of antiviral drugs to reduce covid- transmission angiotensin receptor blockers as tentative sars-cov- therapeutics treatment of critically ill patients with covid- with convalescent plasma combination attenuation offers strategy for live attenuated coronavirus vaccines recombinant live attenuated influenza vaccine viruses carrying cd t-cell epitopes of respiratory syncytial virus protect mice against both pathogens without inflammatory disease adenovirus-based vaccine prevents pneumonia in ferrets challenged with the sars coronavirus and stimulates robust immune responses in macaques pathogenesis of oral type i feline infectious peritonitis virus (fipv) infection: antibody-dependent enhancement infection of cats with type i fipv via the oral route don't rush to deploy covid- vaccines and drugs without sufficient safety guarantees quantification of epitope abundance reveals the effect of direct and cross-presentation on influenza ctl responses advances in rna vaccines for preventive indications: a case study of a vaccine against rabies curevac focuses on the development of mrna-based coronavirus vaccine to protect people worldwide safety and immunogenicity study of -ncov vaccine (mrna- ) to prevent sars-cov- infection safety and immunity of covid- aapc vaccine ev vaccine, a new tool to control outbreaks of hand, foot and mouth disease (hfmd) a sequence homology and bioinformatic approach can predict candidate targets for immune responses to sars-cov- structure, function, and antigenicity of the sars-cov- spike glycoprotein crystal structure of sars-cov- main protease provides a basis for design of improved α-ketoamide inhibitors molecular farming-the slope of enlightenment large-scale production of pharmaceutical proteins in plant cell culture-the protalix experience current status of viral expression systems in plants and perspectives for oral vaccines development mucosal immunology and oral vaccination. in genetically engineered plants as a source of vaccines against widespread diseases-an integrated view emerging genome engineering tools in crop research and breeding severe acute respiratory syndrome (sars) s protein production in plants: development of recombinant vaccine accumulation of recombinant sars-cov spike protein in plant cytosol and chloroplasts indicate potential for development of plant-derived oral vaccines boosted expression of the sars-cov nucleocapsid protein in tobacco and its immunogenicity in mice antigen production in plant to tackle infectious diseases flare up: the case of sars. front vaccination via chloroplast genetics: affordable protein drugs for the prevention and treatment of inherited or infectious human diseases chloroplasts: state of research and practical applications of plastome sequencing production of polyhydroxybutyrate by polycistronic expression of bacterial genes in tobacco plastid plastid-based expression strategie. in genetically engineered plants as a source of vaccines against wide spread diseases-an integrated view when plant virology met agrobacterium: the rise of the deconstructed clones principles of plant-based vaccines techno-economic analysis of a plant-based platform for manufacturing antimicrobial proteins for food safety improving plant transient expression through the rational design of synthetic ' and ' untranslated regions plant-based vaccines against respiratory diseases: current status and future prospects prime-pull vaccination with a plant-derived virus-like particle influenza vaccine elicits a broad immune response and protects aged mice from death and frailty after challenge immunogenicity and safety of a quadrivalent plant-derived virus like particle influenza vaccine candidate-two randomized phase ii clinical trials in to and ≥ years old adults plant-derived virus-like particle vaccines drive cross-presentation of influenza a hemagglutinin peptides by human monocyte-derived macrophages a plant-derived vlp influenza vaccine elicits a balanced immune response even in very old mice with co-morbidities characterization of the innate stimulatory capacity of plant-derived virus-like particles bearing influenza hemagglutinin morphological characterization of a plant-made virus-like particle vaccine bearing influenza virus hemagglutinins by electron microscopy immunological aspects of using plant cells as delivery vehicles for oral vaccines magnifection-a new platform for expressing recombinant vaccines in plants plant lyophilisate carrying s-hbsag as an oral booster vaccine against hbv oral edible plant vaccine containing hypoallergen of american cockroach major allergen per a prevents roach-allergic asthma in a murine model clinical development of plant-produced recombinant pharmaceuticals: vaccines, antibodies and beyond targeting of plant-derived vaccine antigens to immunoresponsive mucosal sites green therapeutic biocapsules: using plant cells to orally deliver biopharmaceuticals plant molecular farming of virus-like nanoparticles as vaccines and reagents highly immunogenic and protective recombinant vaccine candidate expressed in transgenic plants elastin-like polypeptides: a strategic fusion partner for biologics encompassing transition from the trivalent to bivalent oral poliovirus vaccine hitzeroth, i.i. substitution of human papillomavirus type l neutralizing epitopes into l surface loops: the effect on virus-like particle assembly and immunogenicity. front chimaeric rift valley fever virus-like particle vaccine candidate production in nicotiana benthamiana minimally processed crude leaf extracts of nicotiana benthamiana containing recombinant foot and mouth disease virus-like particles are immunogenic in mice efficient assembly and release of sars coronavirus-like particles by a heterologous expression system assembly of human severe acute respiratory syndrome coronavirus-like particles immune responses against severe acute respiratory syndrome coronavirus induced by virus-like particles in mice virus-like particles of sars-like coronavirus formed by membrane proteins from different origins demonstrate stimulating activity in human dendritic cells effect of mucosal and systemic immunization with virus-like particles of severe acute respiratory syndrome coronavirus in mice chimeric severe acute respiratory syndrome coronavirus (sars-cov) s glycoprotein and influenza matrix efficiently form virus-like particles (vlps) that protect mice against challenge with sars-cov mers-cov virus-like particles produced in insect cells induce specific humoural and cellular imminity in rhesus macaques preparation of virus-like particle mimetic nanovesicles displaying the s protein of middle east respiratory syndrome coronavirus using insect cells baculovirus as versatile vectors for protein expression in insect and mammalian cells insect cells contain an unusual, membrane-bound beta-n-acetylglucosaminidase probably involved in the processing of protein n-glycans covid- : medicago's development programs the molecular biology of coronaviruses recombinant expression of tandem-hbc virus-like particles (vlps) recent advances in the use of plant virus-like particles as vaccines n-glycosylation of recombinant pharmaceutical glycoproteins produced in transgenic plants: towards an humanisation of plant n-glycans n-glycosylation of cholera toxin b subunit in nicotiana benthamiana: impacts on host stress response, production yield and vaccine potential production of a monoclonalantibody in plants with a humanized n-glycosylation pattern immunodominant sars coronavirus epitopes in humans elicited both enhancing and neutralizing effects on infection in non-human primates on the origin and continuing evolution of sars-cov- genetic diversity and evolution of sars-cov- virus-specific memory cd t cells provide substantial protection from lethal severe acute respiratory syndrome coronavirus infection edible vaccine protects mice against escherichia coli heat-labile enterotoxin (lt): potatoes expressing a synthetic lt-b gene immunogenic properties of a lettuce-derived c (v ) multiepitopic hiv protein a multi-epitope plant-made chimeric protein (ltbentero) targeting common enteric pathogens is immunogenic in mice an env-derived multi-epitope hiv chimeric protein produced in the moss physcomitrella patens is immunogenic in mice expression of multiple taenia solium immunogens in plant cells through a ribosomal skip mechanism murine fc receptors for igg are redundant in facilitating presentation of immune complex derived antigen to cd + t cells in vivo cellular immune response to the antigen administered as an immune complex in vivo high level production of monoclonal antibodies using an optimized plant expression system plant-derived recombinant immune complexes as self-adjuvanting tb immunogens for mucosal boosting of bcg expression of an immunogenic ebola immune complex in nicotiana benthamiana vaccines elastin-like polypeptides revolutionize protein expression and their biomedical application expression and immunogenicity of the mycobacterial ag b/esat- antigens produced in transgenic plants by elastin-like peptide fusion strategy elpylated haemagglutinins produced in tobacco plants induce potentially neutralizing antibodies against h n viruses in mice use of gfp to investigate expression of plant-derived vaccines a plant-derived multi-hiv antigen induces broad immune responses in orally immunized mice influenza vaccination in older adults: recent innovations and practical applications mucosal vaccines and technology cold chain and virus-free chloroplast-made booster vaccine to confer immunity against different poliovirus serotypes production and immunogenicity of soluble plant-produced hiv- subtype c envelope gp immunogens. front prime-boost vaccine strategy against viral infections: mechanisms and benefits this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -mk mzc z authors: morris, cindy e.; bardin, marc; kinkel, linda l.; moury, benoit; nicot, philippe c.; sands, david c. title: expanding the paradigms of plant pathogen life history and evolution of parasitic fitness beyond agricultural boundaries date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: mk mzc z nan how do pathogens, whether they parasitize plants or animals, acquire virulence to new hosts and resistance to the arms we deploy to control disease? the significance of these questions for microbiology and for society at large can be illustrated by the recent worldwide efforts to track and limit the emergence of human transmissible strains of swine and avian influenza virus and of multidrug-resistant lines of human pathogenic bacteria, and to restrain the spread of ug , a strain of stem rust of wheat. recent research in medical epidemiology has elucidated the impact of pathogen ecology in environmental reservoirs on the evolution of novel or enhanced pathogen virulence. in contrast, the evolution of virulence in plant pathogens has been investigated from a predominantly agro-centric perspective, and has focused overwhelmingly on evolutionary forces related to interactions with the primary plant host. here, we argue that current concepts from the field of medical epidemiology regarding mechanisms that lead to acquisition of novel virulence, biocide resistance, and enhanced pathogenic fitness can serve as an important foundation for novel hypotheses about the evolution of plant pathogens. we present numerous examples of virulence traits in plant pathogenic microorganisms that also have a function in their survival and growth in nonagricultural and nonplant habitats. based on this evidence, we make an appeal to expand concepts of the life history of plant pathogens and the drivers of pathogen evolution beyond the current agro-centric perspective. the classification of diseases in terms of their epidemiology is a useful starting point for a comparison of plant and human pathogens [ ] . in medical epidemiology, anthroponoses are diseases trans-mitted among humans that have no other known reservoirs for multiplication. typhoid fever, smallpox, and certain venereal diseases are examples. zoonoses, such as rabies, lyme disease, severe acute respiratory syndrome (sars), and avian and swine influenzas, are transmitted to humans from living animals. sapronoses are diseases transmitted to humans from environmental reservoirs where the pathogen thrives saprophytically. these habitats include soil, water, and decaying plant and animal matter. examples include legionnaire's disease, cholera, aspergillosis, and the emerging epidemics of melioidosis (burkholderia pseudomallei). human pathogens with saprophytic phases or residing in environmental reservoirs are also referred to as ''environmental pathogens'' [ ] [ ] [ ] [ ] [ ] . studies of virulence factors of human pathogens in environmental reservoirs have begun to reveal the importance of alternate hosts, of dual-use virulence factors, and in general of how environmental habitats can select for traits that confer enhanced fitness as human pathogens. for example, interactions with microbial eukaryotes seem to have led to the acquisition of traits useful for pathogenicity to mammalian cells. numerous environmental pathogens, including cryptococcus neoformans, legionella spp., chlamydophila pneumoniae, mycobacterium avium, listeria monocytogenes, pseudomonas aeruginosa, and francisella tularensis, might have acquired virulence traits via their resistance to predation by amoebae. this resistance, associated with the ability to grow inside the amoebae-which are essentially alternate hosts-has likely led to the selection of traits conferring survival in macrophages [ ] . resistance to macrophages involves the capacity of the bacteria to resist or debilitate the macrophage's phagosomes and to multiply in the cytoplasm. many of the traits essential for virulence to humans likewise seem to play roles in adaption to the environments where the organisms are saprophytes (table ). these traits have dual roles in environmental and parasitic fitness and are thus referred to as ''dualuse traits''. melanins, siderophores, and the capacity to form biofilms are among the frequently cited examples. c. neoformans provides one of the richest examples of dual-use traits. this fungus, frequently found in soils that contain high levels of bird guano and in association with certain plants, causes meningoencephalitis. a nonexhaustive list of its dual-use traits includes capsule formation and production of melanin, laccase, phospholipase, proteases, and ureases [ ] . in the environment these traits contribute to survival and in human hosts they contribute to the capacity of c. neoformans to avoid host resistance mechanisms and to attack host tissue. microbial efflux pumps have also evolved dual uses. these transport systems are used for managing toxic compounds in the environment of the microorganism and can have a broad spectra of activity leading to multidrug resistance among environmental microorganisms [ ] . human activities resulting in the disposal of a wide range of chemical products into the environment, including household cleaners that contain the broad spectrum antimicrobial triclosan, may be inadvertently exacerbating the abundance of multidrug-resistant bacteria [ ] . virulence of environmental pathogens has been described as a set of cards, or a diverse set of attributes acquired as a function of the life history of a pathogen and its adaptation to different environments [ , ] . it is becoming increasingly clear that evolutionary forces outside the context of human-pathogen interactions are responsible for the acquisition and maintenance of some virulence factors [ ] . genomics and phylogenetics are revealing the evolutionary link between, for example, commensal strains of escherichia coli and modern pathogens such as enterohaemorrhagic strains of this species (such as o ). the mechanisms proposed to explain how these commensals have become pathogens are grounded in their ecology and life histories, culminating in the notion of ecological evolution (''eco-evo'') [ ] . the eco-evo approach to understanding the emergence of pathogens gives credence, from the perspective of genomics, to evolutionary and adaptive scenarios that are surmised from a thorough understanding of the ecology and life history of pathogens. at present, epidemiological classifications of plant diseases are based on the interaction of the pathogen and the host (biotrophic or necrotrophic, obligate or facultative), on the number of cycles of propagule production (mono-and polycyclic diseases), on the importance of latency in symptom expression, and on the role of vectors, but there is no formalized equivalent of ''sapronoses''. nevertheless, numerous plant pathogens are present in diverse nonagricultural habitats or survive saprophytically in agricultural contexts. these include a range of bacteria, fungi, and stable viruses (a nonexhaustive list of examples is presented in table ). a striking characteristic of many of the virulence factors of these plant pathogens is that they are linked to-or are in themselves-traits critical to adaptation to the nonplant environment, as will be illustrated below. this provides a compelling reason to adopt a holistic view of the life history and evolution of plant pathogens, to move beyond the traditional borders of agriculture and the presumed ''primary'' plant host. adaptation to biotic and abiotic stresses, within or outside of agricultural habitats, likely plays as important a role in the evolution of parasitic fitness of plant pathogens as it does for human pathogens. as illustrated above, traits that confer fitness in response to biotic and abiotic environmental stress can have dual-use as virulence factors in human pathogens. toxins and toxin transport systems (including efflux pumps, in particular) are among the common adaptations for antagonizing and defending against the co-inhabitants of a habitat. in plant pathogens, the transport systems for toxins and antimicrobials can have broad spectrum activity, leading to resistance to agricultural fungicides and also contributing to virulence [ ] . genes coding for wide spectrum efflux pumps are present in the chromosomes of all living organisms [ ] . the efflux pump bcatrb of botrytis cinerea confers resistance to antimicrobials produced by soil and plant microflora ( , diacetylphloroglucinol and phenazine antibiotics) [ , ] and also to the fungicide fenpiclonil and the plant defensive phytoalexin resveratrol [ ] . the transporter abc from magnaporthe grisea protects the fungus against azole fungicides and the rice phytoalexin sakuranetin [ ] . numerous plant pathogenic bacteria, including erwinia amylovora, dickeya spp. (formerly the multiple biovars of e. chrysanthemi), and agrobacterium tumefaciens, also produce efflux pumps that are involved in their resistance to plant antimicrobials (reviewed by martinez et al. [ ] ). toxins themselves can have a broad spectrum of action. for example, mycotoxins, well known for their human and animal toxicity, have broad spectrum activity and are thought to have evolved as a defense against predators (nematodes) and antagonists (other microorganisms) [ ] . one family of these, the trichothecenes, contributes significantly to the virulence of many gibberella (fusarium) species [ ] . adaptation to biotic stress also implicates systems for the detection or inhibition of arms of aggression used by co-inhabitants. recent work on fungi suggests that systems to detect enzymes that degrade fungal cell walls are also deployed as virulence factors. lysin motifs (lysms) are carbohydratebinding protein modules that have been found in mammalian and plant pathogenic fungi as well as in saprophytes [ ] . bolton et al. [ ] demonstrated that the lysm protein ecp acts as a virulence factor in the plant pathogenic fungus cladosporium fulvum. as virulence factors they may suppress host defenses by sequestrating chitin oligosaccharides that are known to act as elicitors of plant defense responses [ ] and also as activators of host immune responses in mammals [ ] . de jonge and thomma [ ] suggest that these proteins may also have a role in the protection of saprophytic fungi against chitinase-secreting competitor microbes or mycoparasites. protection against abiotic stress can involve molecules that have also become virulence factors. siderophores [ ] [ ] [ ] and various pigments including melanins [ ] are virulence factors in some human pathogens. siderophores contribute to resistance to oxidative stress and sequestering iron when it is rare in the environment. in the plant pathogens alternaria brassicicola, cochliobolus spp., fusarium graminearum [ ] , and m. grisea [ ] , siderophores or their precursors are virulence factors. melanins offer protection from extreme temperatures, uv radiation, and antimicrobials. in the plant pathogens m. grisea and colletotrichum spp., melanins are also virulence factors via their essential role in the formation of tissue-penetration structures such as appressoria [ ] . in many cases, toxins and siderophores are produced by nonribosomal peptide synthase or polyketide synthase pathways. these pathways, widely distributed in the microbial world, are highly adaptable and have given rise to a wide range of compounds with a plethora of activities, including many of pharmaceutical importance [ ] . hc-toxin of cochliobilus carbonum, victorin in c. victoriae, and t-toxin in c. heterostrophus are products of these pathways [ ] . the key virulence factor of streptomyces spp., thaxtomin [ ] , and the multitude of host-specific and nonspecific toxins in pseudomonas syringae pathovars [ ] are also produced by these pathways. the capacity to detect changes in conditions of the abiotic environment has also become part of the virulence factors of some plant pathogens. for example, to detect changes in environmental conditions, organisms exploit two-component histidine kinase complexes. these are key elements of the machinery for signal sensing, allowing bacteria, yeasts, fungi, and plants to adapt to changing environments. in the plant pathogen b. cinerea, one of its multiple histidine kinases, bos , not only mediates osmosensitivity and resistance to fungicides, but is also essential for formation of macroconidia and expression of virulence [ ] . recognition and understanding of the full complexity of the life history of plant pathogens will enhance our capacity to evaluate the diversity and intensity of environmental stresses that microorganisms face and will contribute novel hypotheses concerning the role of environmental stresses in the evolution of pathogenicity. stress is considered to play an important role in adaptive evolution in general, in particular via its effect on mutation rates [ ] . for certain fungi and bacteria, including plant pathogens, stress increases the activity of transposable elements [ ] [ ] [ ] and induces the sos response and other systems involved in the modification or repair of dna [ ] . mutations can target the ensemble of the microbial genome. however, it has been suggested that adaptation of bacteria to multiple stresses can lead, in particular, to the acquisition of virulence factors and to the emergence of pathogenic variants [ ] . adaptation to specific habitats-which involves adapting to a particular ensemble of biotic and abiotic parameters-could also influence the evolution of parasitic fitness. available examples focus on soilborne and rhizosphere microorganisms. the rhizosphere is a dynamic soup whose chemistry changes as plants grow, die, and degrade. chemicals in the rhizosphere are food substrates and means of communication, antagonism, and collaboration among microorganisms, among plants, and between plants and microorganisms. to decompose dead plant material and recycle carbon, microorganisms have developed a range of cell wall-degrading enzymes, without which our planet would be quite encumbered by the accumulation of tissue from dead plants. pectolytic, cellulolytic, and lignolytic enzymes are also well-known pathogenicity factors [ ] [ ] [ ] . to hone the efficiency of these enzymes in planta, pectinolytic fungi are adept at modulating the surrounding ph. alternaria, penicillium, fusarium spp., and sclerotinia sclerotiorum also exploit these ph changes to enhance the action of these enzymes as virulence factors [ ] . streptomyces spp. are considered quintessential soil inhabitants. their ability to degrade biopolymers, including cellulose and chitin, contributes greatly to nutrient cycling, and their vast array of antimicrobials contributes to survival and microbial communication in soil [ ] . some streptomyces species are pathogenic to root crops and to potatoes in particular. a recently discovered virulence factor in streptomyces, a saponinase homologue [ ] , may be the result of adaptation to the rhizosphere. saponins are plant glycosides that contribute to resistance against fungi and insect herbivores. bacteria, and especially gram-positive bacteria, can also be sensitive. saponins are also exuded from the roots of some plant species where they have allelopathic as well as antimicrobial activity [ , ] . key vital functions, housekeeping functions, and basic life cycle processes should also be considered for their potential to give rise to pathogenicity factors. traits fundamental to fitness and survival in general can confer or enhance pathogenic fitness. in plant pathogenic bacteria these include flagella, motility, lipo-and exopolysaccharides, o-antigens, fimbriae, mechanisms for iron acquisition and for quorum sensing, toxin production, cell wall-degrading enzymes, and resistance to oxidative stress [ ] . motility, for example, is essential to dispersal and for attaining new resources. in ralstonia solanacearum it is also essential for early stages of plant invasion and colonization during pathogenesis [ ] . in the fungus aschochyta rabiei, kinesins that are essential for polarized growth and transport of organelles are suspected to be a virulence factor [ ] . an f-box protein of giberrella zeae has been reported to be involved in sexual reproduction and in pathogenicity [ ] . the enzymes that allow fungi to detoxify compounds resulting from plant defense mechanisms are probably also simply means of acquiring nutrients [ ] . for example, detoxification of tomatine in tomatoes by septoria lycopercici and by fusarium oxysporum f. sp. lycopersici is achieved by the deployment of glycosyl hydrolases by these fungi; gaeumannomyces graminis detoxifies avenacins in oats via a beta-glucosidase [ ] . another example of adaptation of basic cellular functions into pathogenicity factors concerns elicitins. elicitins are part of one of the most highly conserved protein families in the phytophthora genus and are widespread throughout phytophthora species. elicitins of p. infestans induce hypersensitivity in plants. recent work from jiang and colleagues [ ] suggests that a primary function of elicitins is the acquisition of sterols from the environment. how can we make sense of the processes that have led to the wide variety of pathogenicity factors in plant pathogens and that continue to drive the evolution of pathogens? bacterial plant pathogens are particularly illustrative of the differences in suites of secretion systems [ , , , ] and of effectors [ , , , , , ] among members of different genera, species, or strains of the same species that attack plants. effectors are proteins secreted by plant pathogens that modulate plant defense reactions, thereby enabling the pathogen to colonize the plant tissues. it is tempting to wonder if the effectors and secretion systems have critical roles in fitness elsewhere other than in association with the host plant. the examples listed above that describe traits that play roles in both environmental fitness and virulence to plants provide a compelling incentive to expand our paradigms concerning the forces that drive evolution of plant pathogenicity. the evolutionary forces that have been described to date for plant pathogens [ ] need to be extended beyond the current agro-centric paradigm. to expand this paradigm we propose that the life cycles and life histories of plant pathogens be reconsidered. studies of pathogen ecology, evolution, and life history should include the full range of habitats and reservoirs these organisms can inhabit. this in turn will permit testing a range of novel hypotheses about the role of ecological contexts-other than direct interaction with host plants-as forces of evolution. in table we propose some such hypotheses. for example, rates of mutation and of transposition of insertion sequences or of transposable elements including phages might be different when a microorganism inhabits nonagricultural habitats (biofilms, lake water, or inert surfaces exposed to uv, for example) than when it colonizes plants. the consequences of these mutations for pathogenicity might in turn be markedly different than for fitness in nonagricultural habitats. likewise, the formation of spores or aggregates that can be released into the air and their survival over long distances might be highly influenced by the nature of the reservoir that the pathogen colonizes, resulting in direct effects of habitat on gene flow. furthermore, the biotic and abiotic stresses endured in nonagricultural habitats might exert positive selection for adaptive survival traits that have dual-use as virulence factors as illustrated in the examples above. these questions are clearly pertinent for pathogens that are not obligate biotrophs. however, the complexity of the biotic and abiotic environment perceived by obligate biotrophs during colonization of plants (powdery mildews on leaf surfaces inhabited by other microorganisms, for example) or during their dissemination (survival in air or in association with vectors) are also likely to exert selection independent of that due to the host plant genotype per se. these are only some of the ways in which environmental parameters other than the host plant are expected to have a marked influence on the diversification of plant pathogens. if nonagricultural environments can foster the evolution of traits that contribute to pathogen virulence, other scenarios are also probable where i) crop plants foster the emergence of traits antagonistic to survival outside of agricultural contexts ii) or nonagricultural environments foster the emergence of traits that are detrimental to pathogen virulence in crops. understanding the prevalence and significance of alternative habitats to pathogen life history is crucial to determining the broad costs of virulence for pathogen fitness. the cost of virulence in terms of fitness in association with plants has been explored extensively for several obligate parasites such as rusts and powdery mildews. work by thrall and burdon [ ] has shown clear fitness tradeoffs between pathogen aggressiveness (capacity to induce intense disease symptoms) and dissemination (via intense spore production). for nonobligate pathogens we do not know the cost of fitness outside of agricultural habitats. the interplay between evolutionary forces and habitat has not been explored for plant pathogens and might be a key feature in the emergence of certain diseases. by expanding our paradigms concerning pathogen life history and the selective forces that drive plant pathogen evolution, we will enhance our understanding of how table . novel hypotheses to be tested concerning the impact of substrates other than host plants on the evolutionary potential of plant pathogens. evolutionary force a novel hypothesis arising from expanded paradigms about the evolution of plant pathogenicity concerning: modifications of the genome. relative to its association with cultivated plant hosts, association of the pathogen with a given nonagricultural substrate leads to: n a significantly greater overall mutation rate. n a greater rate of transposition of insertion sequences or of transposable elements. n more frequent mutations or transpositions that target genes involved in pathogenicity. n a higher probability of acquisition of alien nucleic acids. n genetic exchange with more phylogenetically diverse microbes. effective population size. the effective sub-population size of a pathogen associated with a given nonagricultural (or nonplant) substrate is significantly different from that for sub-populations from cultivated host plants. this could lead to genetic and/or phenotypic differentiation of sub-populations based on substrate of origin. dissemination. the habitats occupied by the plant pathogen influence the mode(s) of dissemination, thereby influencing the distance of dissemination and the spatial and temporal scales of gene flow. mode of reproduction (recombination) genetic recombination. the frequency of recombination (via sexual cycle or other means) varies among strains of plant pathogens as a function of the habitat or substrate. selective pressures and impact on fitness. strains of pathogens adapted to a broad range of habitats have the greatest parasitic fitness. a the evolutionary forces listed here are those that have been considered for plant pathogens in agricultural contexts [ ] . these hypotheses concern pathogens with a marked saprophytic phase or for which nonagricultural or nonplant substrates can be a notable reservoir for survival. reservoirs can include irrigation water, natural waterways and bodies of water, biological vectors (animals, fungi, etc.), abiotic vectors (aerosols, clouds, precipitation), wild plants and weeds, soil, and physical structures in agricultural systems (greenhouse materials, tubing, plastics). doi: . /journal.ppat. .t pathogens survive in the absence of hosts, how and where new pathotypes are likely to emerge, and the significance of natural habitats to agricultural epidemics. insights will come from fundamental research to identify the mechanisms that drive the evolution of pathogenic traits and to explore the ecological significance of pathogenic traits to microbial fitness apart from the plant host. distinguishing the role of adaptation sensu stricto in the emergence of plant pathogenicity relative to that of exaptation [ ] , the useful cooptation of phenotypes that have arisen under natural selection due to forces unrelated to interaction with the primary host plant, will yield critical insight into how plant pathogens evolve independently of agricultural practices. a more complete understanding of the forces that drive plant pathogen evolution will be critical to enhancing and diversifying sustainable disease control strategies, and will improve prediction of the conditions that support the emergence of novel pathogens. emerging human infectious diseases: anthroponoses, zoonoses and sapronoses from outside to inside: environmental microorganisms as human pathogens. a report from the american academy of microbiology accidental virulence, cryptic pathogenesis, martians, lost hosts, and the pathogenicity of environmental microbes biofilm formation and dispersal and the transmission of human pathogens the virulence of human pathogenic fungi: notes from the south of france processes controlling the transmission of bacterial pathogens in the environment microorganisms resistant to free-living amoebae ready made' virulence and 'dual use' virulence factors in pathogenic environmental fungi-the cryptococcus neoformans paradigm functional role of bacterial multidrug efflux pumps in microbial natural ecosystems the biocide triclosan selects stenotrophomonas maltophilia mutants that overproduce the smedef multidrug efflux pump bacterial pathogenomics fungal transporters involved in efflux of natural toxic compounds and fungicides fungal abc transporters and microbial interactions in natural environments involvement of the abc transporter bcatrb and the laccase bclcc in defence of botrytic cinerea against the broad-spectrum antibiotic , -diacetylphloroglucinol the abc transporter bcatrb affects the sensitivity of botrytis cinerea to the phytoalexin resveratol and the fungicide fenpiclonil pathogenicity genes of phytopathogenic fungi fungal lysm effectors: extinguishers of host immunity? the novel cladosporium fulvum lysin motif effector ecp is a virulence factor with orthologues in other fungal species tlr- and il- a in chitin-induced macrophage activation and acute inflammation histoplasma requires sid , a member of an iron-regulated siderophore gene cluster, for host colonization iron metabolism in pathogenic bacteria distinct roles for intra-and extracellular siderophores during aspergillus fumigatus infection color me bad: microbial pigments as virulence factors functional analysis of all nonribosomal peptide synthetases in cochliobolus heterostrophus reveals a factor, nps , involved in virulence and resistance to oxidative stress ferricrocin synthesis in magnaporthe grisea and its role in pathogenicity in rice phylogenetic analysis of condensation domains in nrps sheds light on their functional evolution the phylogeny of plant and animal pathogens in the ascomycota evolution of plant pathogenicity in streptomyces pseudomonas syringae phytotoxins: mode of action, regulation and biosynthesis by peptide and polyketide synthetases a class iii histidine kinase acts as a novel virulence factor in botrytis cinerea stress-induced mutagenisis in bacteria heat shock, copper sulfate and oxidative stress activate the retrotransposon maggy resident in the plant pathogenic fungus magnaporthe grisea ihf is the limiting host factor in transposition of pseudomonas putida transposon tn in stationary phase foxy: an active family of short interspersed nuclear elements from fusarium oxysporum evolution of microbial virulence: the benefits of stress mini review: recent advances in the molecular genetics of plant cell wall-degrading enzymes produced by plant pathogenic fungi advances in molecular genetics of plant-microbe interactions the importance of fungal pectinolytic enzymes in plant invasion, host adaptability and symptom type pathogenic fungi: leading or led by ambient ph? the role of root exudates in rhizosphere interactions with plants and other organisms principles and practices in plant ecology: allelochemical interactions comparative genomics reveals what makes an enterobacterial plant pathogen ralstonia solanacearum needs motility for invasive virulence on tomato towards identifying pathogenic determinants of the chickpea pathogen ascochyta rabiei a novel f-box protein involved in sexual development and pathogenesis in gibberella zeae nutrition acquisition strategies during fungal infection of plants ancient origin of elicitin gene clusters in phytophthora genomes genome sequence of the enterobacterial phytopathogen erwinia carotovora subsp. atroseptica and characterization of virulence factors comparison of the genomes of two xanthomonas pathogens with differing host specificities insights into genome plasticity and pathogenicity of the plant pathogenic bacterium xanthomonas campestris pv. vesicatoria revealed by the complete genome sequence roadmap to new virulence determinants in pseudomonas syringae: insights from comparative genomics and genome organization diverse evolutionary mechanisms shape the type iii effector virulence factor repertoire in the plant pathogen pseudomonas syringae groovy times: filamentous pathogen effectors revealed bacterial phytopathogens and genome science the population genetics of plant pathogens and breeding strategies for durable resistance evolution of virulence in a plant host-pathogen metapopulation exaptation -a missing term in the science of form global impact of vibrio cholerea interactions with chitin virulence and the environment: a novel role for vibrio cholera toxincoregulated pili in biofilm formation on chitin legionella pneimophila -a human pathogen that co-evolved with fresh water protozoa identification of a novel virulence factor in burkholderia cenocepacia h required for efficient slow killing of caenorhabditis elegans transmission of yersinia pestis from an infectious biofilm in the flea vector cloning and characterization of smedef, a novel multidrug efflux pump from stenotrophomonas maltophilia multidrug efflux pumps of gram-negative bacteria acinetobacter baumannii: emergence of a successful pathogen diversity of the burkholderia cepacia complex and implications for risk assessment of biological control strains presence of erwinia chrysanthemi in two major river systems and their alpine sources in australia long distance transport of erwinia carotovora in the atmosphere and surface water presence of erwinia carotovora in surface water in north america soft rot erwinia bacteria in surface and underground waters in southen scotland and in colorado pantoea agglomerans, a plant pathogen causing human disease pantoea agglomerans pvs. gypsophilae and betae, recently evolved pathogens? a surprising niche for the plant pathogen pseudomonas syringae the life history of the plant pathogen pseudomonas syringae is linked to the water cycle applied aspects of rhodococcus genetics rhodococcus fascians in herbaceous perennials versatile persistence pathways for pathogens of animals and plants high diversity of fungi in air particulate matter alternaria spp.: from general saprophyte to specific parasite the broad-scale distribution of microfungi in the windmill islands region, continental antarctica a comparative study of siderophore production by fungi from marine and terrestrial habitats habitat and temporal differences among soil microfungal assemblages in ontario lack of host specialization in aspergillus flavus ecology of the fungus, fusarium: competition general ecology of the fusaria biodiversity of the genus fusarium in saline soil habitats effects of water potential on spore germination and viability of fusarium species metabolites of fusarium the stem canker (blackleg) fungus, leptosphaeria maculans, enters the genomic era insectassociated fungi in soils of field crops and orchards diversity, host, and habitat specificity of oomycete communities in declining reed stands (phragmites australis) of a large freshwater lake penicillium mycobiota in arctic subglacial ice detection of infectious tomato mosaic tobamovirus in fog and clouds detection of tomato mosaic tobamovirus rna in ancient glacial ice detection of infectious tobamovirus in forest soils we thank the three anonymous reviewers for their constructive comments and for the suggestion of additional materials to incorporate into the text. we also thank dr. melodie putnam (oregon state university, united states of america) for useful discussions about the ecology of bacterial plant pathogens. key: cord- - md fq authors: sofo, adriano; sofo, antonino title: converting home spaces into food gardens at the time of covid- quarantine: all the benefits of plants in this difficult and unprecedented period date: - - journal: hum ecol interdiscip j doi: . /s - - - sha: doc_id: cord_uid: md fq people are facing uncertain and difficult times in the face of the covid- pandemic. the benefits of plants (psychological, health, economic, productive) in this period of forced isolation can be of key importance. if many of us have to self-isolate in urban or suburban environments, we need something to do to keep our bodies and minds active and fed. in such a challenging scenario, a vegetable garden in home spaces can bring recreational, health, economic and environmental benefits. regardless of the covid- pandemic, there is untapped potential for this kind of garden to impact environmental outcomes, public awareness, and market trends. home vegetable gardens could provide a small-scale approach to the sustainable use of natural resources, leading towards self-sufficiency, self-regulation, sustainability, and environmental protection. i am a professor of plant biology and soil chemistry in an italian university. i and my family are at the moment practicing "social isolation" at home, and this situation will be probably continue for the unforeseeable future. we are allowed to go outside only for the purchase of food and other basic necessities, and the disposal of rubbish, in a radius of m from home. because of the extreme contagiousness of covid- , unnecessary activity outside is strongly discouraged. within a few days, our daily life was turned upside down and we were forced to change the old habits. right now (april , ), italy is one of the countries hardest hit by positive diagnoses, , deaths, and , recoveries (dipartimento della protezione civile ), but we are not sure if the contagion peak has been reached and how long this pandemic will last. as do most italian citizens, i live in a condominium apartment, where families share common spaces and services, without a garden nor plants. in these conditions, one would think we would have a great deal of time on our hands, but on the contrary, this time is unusable. this is because of a general lack of concentration, also because of the daily bad news, and the significant efforts dedicated to the reorganization of family life. schools, universities, libraries, museums, and theatres are shut down. lessons for schoolchildren and students take place only remotely and, as a university lecturer, i am spending at least - h per day in front of my laptop for lecturing and tutoring my students, many of whom are concerned about the unprecedented terrible and unimaginable situation. during these laptop-hours, i am also in contact with colleagues and friends all over the world, many of whom i hadn't heard in years, all asking how we are. all these things are substitutes for the life i had but i considered myself very lucky and grateful: i can work from my laptop, an opportunity that not everyone has, and i am physically healthy and mentally active. despite these distractions, i had to reduce all this accumulated mental stress, psychological burden and, why not, make myself partly self-sufficient from a food standpoint, while not living in the countryside and having open spaces to cultivate. as a plant and soil biologist, i know that plants are the foundation of a multitude of ecosystem services (primary production, provisioning, supporting, regulating and cultural, etc.) and that an environment rich of plants, much more than we are aware of, can guarantee our physical and mental wellbeing (bratman et al. : russell et al. shwartz et al. ; bratman et al. ) . my apartment is not so big but i have an empty and unutilized -m terrace upstairs and our mediterranean climate is mild in the spring, so, i asked myself, why not convert it into a vegetable garden? besides cultivating and trying to be partially self-sufficient for fruit and vegetables (that are expensive and hard to find now), this could give me psychological benefits. there is an association between home gardening and physical and mental well-being, and this has been demonstrated for various categories of people at risk (grabbe et al. ; john et al. ; quick et al. ; van lier et al. ) . i had a lot of material in my garage, some pipes, shelves, supports, other stuff, and hardware. what i didn't have, i would order online. then, i acted scientifically, calculating the total costs, needed materials and items, plant species to be cultivated, types of soil and pots, production per unit of surface, psychological effects of plants on my family. documenting everything, taking photographs, and updating a personal/laboratory diary were my imperatives. we all, humans, plants, and soil would be part of an experiment, and all this without moving from home. here i present a short paper on my experience, focusing on the benefits of plants (psychological, health, economic, productive) in this period of forced isolation. if many of us have to self-isolate in urban or suburban environments characterized by the lack of spaces and resources, which negatively affects our brain activity (lambert et al. ) , we need something to do to keep our bodies and minds active and fed. i couldn't think of better work than setting up a home vegetable garden. i know that the idea seems, at a first glance, strange and inappropriate -considering the number of current and future problems -but, as a scientist, i felt i had to do it. with people now facing uncertain and difficult times in the face of covid- , i thought i might dedicate this article to the positive action of plants and see how they can help us. i have always been interested in urban permaculture, aimed at producing food in urban areas and promoting energy efficiency and self-production. because it starts from personal initiative and your own home, it is here, more than in all the other sectors of permaculture, that the imagination and the flair of the solutions adopted are surprising. urban permaculture can be concrete and pragmatic, aimed at the production of food in urban areas, replacing ornamental plants with edible species. the selection of plant species to cultivate in outside home spaces should be based primarily on their ability to cope with the harsh conditions of the urban environment, such as high wind and irradiance, lack of organic material and nutrients, and intermittent drought (pavao-zuckerman ). therefore, careful plant selection should be integrated into outside space design (lee et al. ; john et al. ; chaudhary et al. ) . urban habitats are unique and harsh environments for established plant communities, largely because of increased abiotic stresses, such as disturbance, pollution, drought, radiation, heat and microclimate extremes, but also because of the reduction of colonization and modifications in soil microbial diversity (e.g., mycorrhizas or bacteria). another additional difficulty, confirmed by my usual plant dealer, is that there is a rush on vegetable seedlings in garden centers because many more people are now wanting to grow their food. the spread of covid- has caused panic buying at supermarkets, so many families are skipping the supermarket and heading to their local garden center to grow their own and become self-suff cient. therefore, it is often necessary to wait on average one week to get seedlings (starting from the seed is not advisable, as it would take too long). in my small way, i will show you what you could do in your apartment space, on a balcony, a terrace or in a little courtyard. mine is a very practical example of urban permaculture and i hope that could contribute to lightening your stress in this complicated period. from various sites on the web, it is possible to draw countless ideas for designing and managing a small home garden. regardless of space and financial availability, you devise your slice of food independence. the most important thing is to start and be ready to try, without being afraid of making mistakes. a vegetable garden in the city has many advantages. first, you will produce healthy vegetables for yourself. then, there is the psycho-physical well-being that comprises doing a little physical activity and enjoying the satisfaction of seeing your vegetables grow (reed et al. ) . even at the community level, a vegetable garden has its positive sides: a green spot in the cement brightens the view, purifies the air, and cools the environment because of plant transpiration. on an ecological level, an urban garden is a refuge and a shelter for many animals -you cannot imagine how many. but let's proceed step by step. here is my decalogue based on my experience at the moment. for clarity, please note that i describe a garden in a mediterranean environment in the springtime. ) water is the most important issue. on terraces, there are usually rain gutters. instead of wasting rainwater, which moreover is of excellent quality, as it is saltfree, just adapt a tank (in fig. , a -liter resin tank) to the collection of rainwater. the lid, necessary to avoid water evaporation, has been perforated to facilitate the entry of the tube. considering the heat of a terrace in the summer months and the outrageous demand for water for transpiration and evapotranspiration, an additional reserve of water is useful and falls within the perspective of sustainability and saving of natural resources. in a few hours of moderate rain, the tank will fill. if in excess, the accumulated water could be also very useful for cleaning the terrace, which gets dirty when it is transformed into a vegetable garden. in the long run, it might be better to buy a pressure washer to save more water. after the first initial investment (not excessive), you will save a lot of water. other simpler tanks and buckets can also collect water. for aqueduct water, i recommend an irrigation system with an electronic control unit that sends water to the root system of the plants, reduces losses by evaporation, water consumption and costs of the water bill. here, the higher costs for the irrigation system should be taken into account. ) the scaffolding. here too there are various solutions. if we use large pots (a choice that i recommend), they should be raised and not positioned on the ground. this is useful both to better clean the terrace and to not tire you out. the most sustainable choice is the perforated steel or aluminum beams used to construct the shade canopies in the parking areas of shopping malls and supermarkets in europe, and they are inexpensive (fig. ) . by positioning several poles, the structure will be light, stable and will not bend even with the wind because the perforated surface does not offer much resistance. this kind of structure is very useful if you want to train a wild vine (ampelopsis brevipedunculata and parthenocissus quinquefolia) or wisteria (wisteria spp.), or other climbing plants that also have a decorative and shading function. in mediterranean climates, the hot late spring/summer days can raise soil temperature in the pots to even reach - °c and air temperature - °c , so it is advisable to use shade cloth or shading vegetation on the scaffolding to avoid burning roots and shoots of the plants so laboriously cultivated. ) the containers. here you are spoiled for choice but remember that we want to recycle water and save money. we will therefore not buy expensive pots. as simple containers, you can also use inexpensive black/transparent plastic containers that you will drill at the base for drainage, or the wooden boxes from greengrocers (nowadays very rare to find). if the choice falls on the pots, one must be careful and containers without good drainage and/or not transpiring are not recommended. here, it is easy for the roots to suffer from high heat or water stagnation. the best choice would be that of the classic rectangular brown clay pots, but they are heavy, fragile, and expensive (fig. ) . on the other side, the plastic ones are light, not expensive, and long-lasting. instead, we will use common plastic containers or other containers to transport soil, grass clippings, root residues, and compost. if desired, the containers can be built with wooden beams and sheets of various kinds but, considering that it is a terrace and that we must avoid water infiltration and stagnation, better to go with the clay or plastic pots (or a mixture of both). ) what to plant? here too the choice is wide. since the surface, even if you use several pots, is limited, all species that have a too low total fruit/biomass ratio (e.g., legumes) are not recommended. you would risk wasting too much space and having a minimum harvest. in springtime, you can grow different cultivars of lettuce (lactuca sativa), zucchini (cucurbita pepo), onion (allium cepa), chicory (cichorium intybus), rocket (arugula) (eruca sativa), green and red beet (beta vulgaris), fennel (foeniculum vulgare), chive (allium schoenoprasum), carrot (dacus carota), strawberry (fragaria spp.), late cauliflower/broccoli (brassica oleracea) and spinach (spinacia oleracea), early potato (solanum tuberosum), tomato (lycopersicon esculentum), eggplant (solanum melongena) and sweet/spicy pepper (capsicum spp.), and some fresh legumes, such as broad bean (vicia faba), fresh bean (phaseolus vulgaris), and pea (pisum sativum) (fig. ) . tomatoes and fresh legumes need support when they grow, such as wire or bamboo and plastic posts. each season has its particular harvest, so you will follow the rhythms marked by nature, and together you will have vegetables at zero meters. the potting soil should be organic and perhaps mixed with manure (maximum % w/w), with an expanded clay base to avoid water stagnation. it is best to start from seedlings rather than seed. the cost of the seedlings is not high, so starting from seed, especially for some broad-leaved vegetables, it is also sometimes uneconomical and requires too much time and too much care. after the initial investment of soil, remember that the subsequent additions could be produced from compost. if needed, you can carry out a mineral nitrogen fertilization, particularly recommended in spring. ) and lavender (lavandula officinaliswhose dry inflorescences are excellent for perfuming linen), or saplings (these latter better in circular pots placed on the ground), such as olive (olea europaea), pomegranate (punica granatum) and mulberry (morus spp.), or other useful and undemanding plants, such as aloe (aloe spp.whose inner leaf is a strong antioxidant and can be used for healing wounds and burns), citronella (cymbopogon nardusmosquito repellent) and prickly pear (opuntia ficus-indica) (fig. ) . you might put in some ornamental plants but remember that you are in a permaculture regime and therefore you must not have too many demands. ) the compost. another important consideration is the recycling of waste materials. the vegetable biomass of waste, even of a small vegetable garden, can be high and mixed with waste that would end up in the garbage (fruit skins, vegetable residues, eggshells, coffee grounds, tea bags, etc.) it becomes an excellent starting point for the production of compost. you can also add the shredded roots of the plants you want to replace and recover this fig. the containers used hum ecol precious soil. the waste of a vegetable garden has an optimal carbon/nitrogen ratio (around - ), so it is quickly transformed into soil. the soil residues that remain tied to the roots of old plants can be chopped and mixed with the rest, thus providing a natural starter of bacteria and saprophytic fungi. you will notice that your residues will smell first of fruit and vegetables, then of garbage, then of manure, and finally of fresh soil. at that point, compost became humus. the latter has many advantages: it is a fertilizer rich in carbon, nitrogen, and phosphorus, makes the soil soft and improves its structure (it is, therefore, a soil conditioner), keeps more water when it does not rain, contains billions of microorganisms (many of which with antibiotic effects against phytopathogens, others with plant growth-promoting action) and many others. for chopping, you can use an electric shredder that chops everything except very hard material, such as kernels (fig. ) . with the same volume, smaller pieces offer a larger surface area to the microbial attack and therefore composting is faster and more effective. to contain the compost, you can buy a compost bin (there are various sizes but, for a family of four, a -liter one is more than enough) or, with a little patience, you can build it (fig. ). it will surprise you how soon it will fill up and how much less waste you will produce. on warm days, when the bacterial metabolism is high, turning it now and then with a hoe, the compost will be ready in about three months. in this way of recycling you can produce fresh soil in situ and save on the soil buy. ) biodiversity. your home garden is also a cure-all for passing species. in my case, it is not unusual to see plenty of ladybugs (beneficial because they are carnivorous), various spiders, geckos, lizards, bats (attracted by insects), passerines, turtle doves, magpies, and thrushes (fig. ) . unfortunately, since your vegetable garden is free of pesticides (less pollution), it is normal for pests such as mealy cochineals that attack lemon and olive trees, nematodes that affect tomato roots, and finally cabbagewhite butterflies that lay eggs from which voracious leaf-eating caterpillars will appear, to come, but with patience, by hand or with natural remedies, these can be kept at bay. ) personal satisfaction. the home vegetable garden is satisfying because looking at the vegetables and fruit you have cared for gives a pleasant sense of fulfilment. a pod of a pea plant that tries to climb anywhere or the contrast of colors of a red beet leaf can enchant you. you can make yourself a salad, a fresh sauce or turnip greens, au gratin cauliflower, a vegetable soup with pasta, cooking the vegetables you have harvested, or use the sun-dried leaves or seeds of officinal plants as spices or ingredients for your dishes (fig. ) . with a vegetable garden at home, the value of the products is their real worth, because it only takes into account personal work and there are no additional costs, such as fuel for transportation, synthetic fertilizers, plant protection products, marketing, advertising. ) the costs. the terrace garden allows you to save money in the medium-term, even if this is not the only purpose. of course, there is an initial investment (table ) . we can find much of the listed material for free, build something, and find excellent offers on the internet. therefore, the cost of installation of a home vegetable garden is variable, and it could be much lower than the one i calculated in table . besides, you don't have to do all the things i have listed. it would be better to start on a small scale and expand step by step. as the garden will produce kilos of fresh vegetables (we make an average of - kg year ), at an average price of - € kg , you can recover annual management costs in a brief time. the initial costs are not excessive, if you consider the satisfaction of eating home-grown vegetables. ) not all apartments have large terraces or courtyards to devote to the cultivation of vegetables; often the area is limited to a balcony or even a windowsill. even in cases a vegetable garden in home spaces can provide recreation, enhance physical and mental health, and provide economic and environmental benefits (dunnett and qasim ; hartig et al. ; jennings and gaither ) . there are no greenhouse gas emissions, no use of synthetic fertilizers and pesticides, no leaching losses. regardless of practical challenges and the existential difficulties we face, there is untapped potential for home vegetable gardens to influence environmental outcomes, citizens' consciousness, and market trends. it is time to think about a new way of living that starts from daily activities, with a small-scale and bottom-up approach, based on sustainable use of natural resources and promotion of a subsistence economy and bartering, aimed at lasting well-being in generations both in material terms (food and energy) and psychological terms, able to integrate various disciplines (such as agriculture and animal husbandry, forestry, biology, architecture, engineering, but also economics, sociology, philosophy, and psychology), tending towards selfsufficiency, self-regulation, sustainability, and environmental protection. bill mollison, who coined the term "permaculture" in the s, said that "a culture cannot survive long without a sustainable agricultural base and ethics of land use." my heartfelt suggestion is to stay safe at home now and consider that this pandemic could be an opportunity for a whole new -and likely better -world. covid- is the last warning that gives us time to reconsider human behaviorfrom global warming to the ever-increasing intensity and speed of activities, all of which are related to the principle of the capitalistic system. this pause can help us deliberate how all this can be transformed into a sustainable system; maybe it is a big chance! the impacts of nature experience on human cognitive function and mental health the benefits of nature experience: improved affect and cognition urban mycorrhizas: predicting arbuscular mycorrhizal abundance in green roofs covid- italia -monitoraggio della situazione perceived benefits to human wellbeing of urban gardens gardening for the mental well-being of homeless women nature and health approaching environmental health disparities and green spaces: an ecosystem services perspective the potential for mycorrhizae to improve green roof function brains in the city: neurobiological effects of urbanization living roof preference is influenced by plant characteristics and diversity the nature of urban soils and their role in ecological restoration in cities vegetable garden as therapeutic horticulture for patients with chronic pain a repeated measures experiment of green exercise to improve self-esteem in uk school children humans and nature: how knowing and experiencing nature affect well-being enhancing urban biodiversity and its influence on city-dwellers: an experiment home gardening and the health and well-being of adolescents publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -masvn gu authors: soria-guerra, ruth elena; alpuche-solís, angel g.; rosales-mendoza, sergio; moreno-fierros, leticia; bendik, elise m.; martínez-gonzález, luzmila; korban, schuyler s. title: expression of a multi-epitope dpt fusion protein in transplastomic tobacco plants retains both antigenicity and immunogenicity of all three components of the functional oligomer date: - - journal: planta doi: . /s - - - sha: doc_id: cord_uid: masvn gu expression of genes in plant chloroplasts provides an opportunity for enhanced production of target proteins. we report the introduction and expression of a fusion dpt protein containing immunoprotective exotoxin epitopes of corynebacterium diphtheriae, bordetella pertussis, and clostridium tetani in tobacco chloroplasts. using biolistic-mediated transformation, a plant-optimized synthetic dpt gene was successfully transferred to tobacco plastomes. putative transplastomic t plants were identified by pcr, and southern blot analysis confirmed homoplasmy in t progeny. elisa assays demonstrated that the dpt protein retained antigenicity of the three components of the fusion protein. the highest level of expression in these transplastomic plants reached . % of total soluble protein. to assess whether the functional recombinant protein expressed in tobacco plants would induce specific antibodies in test animals, a mice feeding experiment was conducted. for mice orally immunized with freeze-dried transplastomic leaves, production of igg and iga antibodies specific to each toxin were detected in serum and mucosal tissues. simultaneous vaccination against diphtheria, tetanus, and pertussis using a dpt vaccine during infancy and childhood has markedly reduced the incidence of cases and deaths from each of these serious bacterial diseases (cdc ; kalies et al. ; who ) . concerns over the safety of whole-cell pertussis vaccines have prompted the development of acellular vaccines that are less likely to provoke adverse reactions as they contain puriwed antigenic components of the causal bacterium bordetella pertussis. however, current acellular pertussis vaccines must be administered in a series of multiple doses, thereby contributing to their high production costs (cdc ; salmaso et al. ; tan et al. ) . attempts to produce safer, inexpensive, and more eycient dpt vaccines are underway (abomoelak et al. ; aminian et al. ; kamachi et al. ; meriste et al. ) . in the last few years, plants have been genetically engineered to express various recombinant biopharmaceuticals (ma et al. ) . plants can be used as bioreactors for the production of appropriately safe and inexpensive vaccines, and contribute to reduced costs associated with vaccine transportation and storage (giddings et al. ; koya et al. ; la et al. ). plant-based vaccine production has been reported in several plant species, including potato (mason et al. ) , tobacco (liu et al. ; mason et al. ; zhang et al. ), tomato (mcgarvey et al. sandhu et al. ) , lettuce (kapusta et al. ) , carrot (marquet-blouin et al. ; rosales-mendoza et al. ) , and alfalfa (dong et al. ), among others. feeding studies conducted in animals (huang et al. ; rosales-mendoza et al. ; thanavala et al. ) and humans (tacket et al. (tacket et al. , have demonstrated that candidate vaccines produced in plants are evective in eliciting protective immune responses. this vaccine production approach will also have a positive impact on public health measures in developing countries that lack of proper refrigeration systems for storage of traditional vaccines. as plant-based vaccines are administered orally, these vaccines avoid the use of injections, thus eliminating discomfort and more importantly the risk of disease transmission via re-use of contaminated syringes and needles richter et al. ) . plastid transformation provides an alternative strategy for expressing foreign genes in plants and overs several advantages over nuclear plant expression systems (chargelegue et al. ; sala et al. ) . these advantages include higher levels of foreign protein accumulation, site-speciwc integration of transgenes, and transgene containment due to maternal transmission of plastids thus alleviating environmental concerns over gene xow (maliga ; daniell et al. ) . moreover, the destination of a protein inxuences both its stability and modiwcation (drakakaki et al. ) . to date, many antigenic proteins have been expressed in chloroplasts, such as the cholera toxin b subunit (daniell et al. ) , anthrax protective antigen (koya et al. ) , tetanus toxin fragment c (tregoning et al. ) , escherichia coli k wmbrial subunit antigen (garg et al. ) , and the spike (s) protein of the severe acute respiratory syndrome coronavirus (sars-cov) . recently in our laboratory, a fusion protein of the heat labile toxin b subunit (ltb) of the enterotoxigenic escherichia coli along with the heat stable toxin (st) fusion protein (ltb-st) has been expressed in transplastomic tobacco plants, and demonstrated its immunogenic characteristic in tested mice (rosales-mendoza et al. ). previously, we have expressed an antigenic polypeptide containing epitopes of diphtheria, pertussis, and tetanus exotoxins in tomato plants via nuclear transformation ). following analysis of transgenic tomato plants, it has been demonstrated that the dpt transgene is integrated into the genome, transcribed, properly translated, and accumulating at levels of . % of total soluble protein (tsp) ). in addi-tion, we have demonstrated that three doses of mg each of freeze-dried tomato triggers speciwc immune responses in mice (soria-guerra et al., unpublished) . these studies suggest that the dpt could be used as a viable multicomponent dpt subunit vaccine. however, low levels of expression of the dpt protein in tomato plants render this plant-based candidate vaccine less desirable for commercial use as an oral vaccine. in this study, we report on the transfer and expression of the dpt fusion gene, re-designed for expression in tobacco chloroplasts. recovery of transplastomic t tobacco plants accumulating high levels of the recombinant dpt fusion protein is reported. the antigenicity of all three components of the dpt fusion protein is also conwrmed in these transplastomic tobacco plants by elisa assays. following oral feeding of freeze-dried transplastomic tobacco leaves in test mice, immunogenic responses are observed. as previously described, a multi-epitope dpt fusion protein was selected as the target for plastid expression in tobacco plants ). this fusion protein contains six immunoprotective exotoxin epitopes of corynebacterium diphtheriae, bordetella pertussis, and clostridium tetani. a synthetic gene encoding for the dpt fusion protein is designed for optimal expression in tobacco plastome based on codon usage in plants. the -bp sequence includes a ribosome binding site and xbai and xhoi restriction sites at the Ј and Ј ends, respectively. this gene has been synthesized by genscript corp. (piscataway, nj), and no changes were made in the amino acid sequence for the toxin subunits, linkers, and adjuvants, except for the deletion of the sekdel sequence. the two adjuvant-coding sequences of the heavy chain tetanus toxin were added near the c-terminal. for plastid transformation, the plasmid pbic was used; it was derived from the chloroplast transformation vector pkcz (kindly provided by dr. hans-ulrich koop). this vector is designed for integration of foreign genes into an muni restriction site between trnn-guu and trnr-acg in the inverted repeat region of the chloroplast genome (zou et al. ) . several modiwcations have been made to the pkcz vector in order to express the dpt and aada genes as bicistrons under the control of the plastid s rrna promoter (prrn). first, a synthetic plastid s-rrn-promoter (prrn) fused to the Ј-utr of gene from the bacteriophage t (t g ) was prepared using synthetic oligonucleotides. the Ј-utr region of this promoter was ligated to the pkcz vector at restriction sites nhei and bglii of the pkcz-utr. then, the vector pkcz-utr was digested with spei and nhei in order to delete the aada expression cassette, and self-ligated to generate pkcz-utrdel. an aada expression cassette was released from the pkcz vector by smai-ecorv digestion, and cloned downstream of the prrn-utr at afei and pmli sites of pkcz-utrdel to generate the pbic vector. the dpt coding sequence was subcloned into the pbic vector digested with xbai and xhoi restriction enzymes. the resulting plasmid was named pbic-dpt (fig. a) . a positive clone was selected following restriction analysis and sequenced. all cloning and analysis procedures were performed following standard protocols (sambrook et al. ) . dna for plastid transformation was prepared using the qiagen plasmid midi kit (qiagen, valencia, ca). tobacco (nicotiana tabacum cv. petit havana) seedlings were grown aseptically on murashige and skoog (ms) medium for - weeks. dna was coated onto . m gold particles, and bombarded onto fully expanded leaves using the pds- /he (bio-rad, hercules, ca) biolistic delivery system as previously described (daniell et al. ) . following bombardment, leaves were incubated for h in the dark at °c. then, leaves were cut into small sections (» mm £ mm), and cultured with the abaxial side in contact with the rmop medium (svab and maliga ) and containing mg/l spectinomycin. after - weeks, leaves from putative transplastomic shoots on selection medium were cut into small sections ( . cm ), and placed on a selection medium for the next round of selection. following three selection rounds, spectinomycin-resistant shoots were transferred onto fresh ms medium containing mg/l spectinomycin for rooting. whole plants were transferred to soil, acclimatized, and grown in the greenhouse to maturity. after blooming, seeds were collected, subject to surface sterilization and germinated on ms media containing spectinomycin. total dna in t plants was extracted from putative transplastomic and non-transformed tobacco leaves using the dnaeasy™ plant mini kit (qiagen inc., valencia, ca). for pcr analysis, a l reaction mixture containing ng dna, . mm magnesium chloride, . u taq dna polymerase, mm dntps, and m of each of forward and reverse primers was used. the forward primer f ( Ј ggtatgattctaggccaccagagg) and reverse primer r ( Ј gagcggctattcaagatgtgaagc) were used to amplify the dpt gene. primers p ( Ј gctcccccgccgtcgttcaatgaga) and t ( Ј gcatctaagtagtaagcccaccccaagatg) were used to detect integration of the expression cassette into the tobacco plastome. the pcr protocol included an initial denaturation step at °c for min followed by cycles of denaturation, annealing, and extension steps of s at °c, s at °c, and min at °c, respectively. pcr products were analyzed on % agarose gels. pcr-positive lines were transferred to the greenhouse for plant growth and seed production. t seeds were harvested and germinated on a medium containing mg/l spectinomycin. southern blot analysis was performed in t progeny as previously described (sambrook et al. ) . fifty micrograms of total genomic dna from leaves of each transformed and fig. a schematic diagram of the plastid transformation vector harboring the synthesized dpt gene. this gene is under the control of the prrn promoter; moreover, the Ј-utr of phage t gene contains a ggagg ribosome binding site. this gene is inserted at the trnr/trnn insertion site into the tobacco chloroplast genome. rbcl , Ј untranslated region of the rbcl gene; aada, spectinomycin resistance gene. f and r correspond to primers used for the detection of the dpt gene; while, p and t, primers used to detect transplastomic lines. b pcr analysis of plants using f and r primers for the dpt gene. a speciwc . -kb pcr band was detected in putative transplastomic tobacco plants ( - correspond to lines b , c , d , and e ), but it was absent in wild-type tobacco (wt). c t mature plants transferred to soil and grown in the greenhouse exhibiting normal phenotypes. d t seeds germinating on a medium containing mg/l spectinomycin together along with wild-type (wt). e pcr analysis using primers p and t; a kb product is ampliwed from transplastomic plants ( - correspond to lines b and e ) which is absent in the wild-type (wt) e wild-type plants was digested with hindiii, xbai, and saci electrophoresed on a % agarose gel, and blotted onto a hybond n membrane (amersham-pharmacia biotech, piscataway, nj). the dpt fusion gene and a fragment of the trnn region were used as probes, generated using the dig-dna labeling mixture (boehringer mannheim, germany). the probe was hybridized with the membrane and resolved using the cspd substrate for alkaline phosphatase according to the manufacturer's protocol. for western blot analysis, protein samples were separated by sds-page using - % pre-cast polyacrylamide electrophoresis gels (nusep inc., austell, ga). proteins were blotted onto biotrace pvdf membrane using a mini trans blot tm electrophoretic transfer cell (bio-rad). membranes were incubated overnight at °c in ml of either : , dilution of a rabbit anti-tetanus toxin (ab , abcam, cambridge ma) or : , dilution of a goat anti-diphtheria toxin (us biological, swampscott, ma). after washing, membranes were incubated for h in a : , dilution of either an anti-rabbit igg or anti-goat igg conjugated with horseradish peroxidase (sigma a ). the antibody binding signal was detected with a lumi-light western blotting substrate (roche co., mannheim, germany) according to the manufacturer's protocol. total soluble proteins (tsp) were extracted from leaves of transplastomic and non-transformed t plants according to kang et al. ( ) . protein concentration was determined by the bradford protein assay reagent kit (bio-rad). for elisa assays, ng tsps were loaded into a -well microtiter plate (immulux™ hb, dynex technologies, germany) diluted in bicarbonate buver ( mm na co , mm nahco ; ph . ) in a l volume, and incubated overnight at °c. after washing with pbst and blocking with % nonfat dry milk, the plate was incubated with goat anti-diphtheria toxin (us biological, swampscott, ma), mouse anti-bordetella pertussis toxin (abcam, cambridge, ma), or rabbit anti-tetanus toxin (ab , abcam, cambridge ma) antibodies diluted : , ( l per well). as secondary antibodies, anti-goat igg alkaline phosphatase conjugate (sigma a ), anti-mouse igg alkaline phosphatase conjugate (sigma a ), or anti-rabbit igg alkaline phosphatase conjugate (sigma a ) were used. following incubation with l p-nitrophenyl phosphate liquid substrate (sigma n ) per well for min at room temperature, reactions were stopped with n hcl, and optical density was determined at nm using an elisa microplate reader (mrx revelation, dynex technologies). for each assay, a standard curve was included, and a tetanus toxoid (nibsc / ), at levels of . , . , . , and ng, was used for quantiwcation of the tetanus toxoid. male balb/c mice, -to -week-old, (harlan sprague dawley, inc., indianapolis, in) were used for evaluation of the immune response against dpt protein. all animals were handled according to federal regulations for animal experimentation and care (nom- -zoo- , ministry of agriculture, mexico), and approved by the institutional animal care and use committee. mice were immunized via oral route delivery with mg of freeze-dried tobacco powder from e line containing . , . , and g of tobacco-derived diphtheria, tetanus, and pertussis exotoxin epitopes, respectively. positive and negative controls included dpt toxoids [diphtheria toxoid (nibsc / ), tetanus toxoid (nibsc / ), or pertussis toxoid (nibsc / )] and mg of wild-typetobacco material, respectively. each test group contained wve animals for which three oral doses were administered on days , , and . tobacco powder was hydrated in . ml water, and the suspension was administered to test animals via the intragastric route. mice were fasted overnight prior to immunization. the animals were sacriwced on day to collect serum samples and intestinal xuids. serum samples were obtained from blood extracted by cardiac puncture from ether-anesthetized mice. fluids from the gut were collected, and contents of the small and large intestines were xushed out with ml of cold rpmi medium. then, l of mm p-hydroxymercuribenzoate (dissolved in mm tris-base) was added. samples were centrifuged at °c for min at , g, their supernatants were immediately frozen, and stored at ¡ °c until assay. elisa assays of epitope-speciwc antibodies responses in serum and intestinal xuids were determined by an indirect enzyme-linked immunosorbent assay (elisa). briexy, -well plates were coated with either . limit of xocculation (lf)/ml diphtheria toxoid (nibsc / ), . lf/ml (nibsc / ) tetanus toxoid, or . international unit (ui)/ml (nibsc / ) pertussis toxoid, and with l per well of bicarbonate-carbonate buver ( mm na co , mm nahco , ph . ) for h at °c. plates were washed, and then blocked with % nonfat dry milk in pbs ( mm nacl, mm na hpo , mm kh po , ph . ). the serum was diluted : in pbst ( . % v/v tween- in pbs). intestinal xuid samples were diluted : in % nonfat milk dissolved in pbst, added to wells of sensitized plates, and incubated overnight at °c. the following antibodies were used as secondary antibodies: biotinylated goat anti-mouse igg, iga, igg , or igg a (zymed laboratories, san francisco, ca). plates were incubated for h at °c and then washed with pbst. a conjugated horseradish peroxidase-streptavidin was added to each well, and plates were incubated for h at room temperature. after washing, plates were incubated at room temperature with l volume of a substrate solution ( . mg/ml o-phenylenodiamine, . % h o , mm citrate buver, ph . ). following color development ( min), the reaction was stopped with l of . m h so . speciwc antibody levels were expressed as corresponding optical density values, and measured at nm (a ) using a multiskan ascent (thermo electron corporation, waltham, ma) microplate reader. elisa data correspond to the geometric means of n = mice per group and representative of duplicate experiments, and error bars correspond to standard deviations. statistical signiwcance of diverences (p < . ) was determined using analysis of variance. the sequence of the plant-optimized synthetic gene encoding the dpt recombinant polypeptide, containing immunoprotective epitopes of tetanus, pertussis, and diphtheria exotoxins along with coding sequences of two adjuvants of the heavy chain tetanus toxin, was optimized as per codon bias for tobacco genes. following optimization, the gc content was %, and although % of the codons were changed, the original amino acid sequence of the wild-type bacterial genes was maintained. the gene was synthesized by genscript, and both mrna processing and destabilizing motifs were avoided. the dpt gene was inserted into the plastid expression cassette of the pbic vector as described in "materials and methods". the plasmid pbic-dpt utilizes tobacco plastid genome sequences spanning the trnr-acg and trnn-guu regions to target the gene of interest into the chloroplast genome by double homologous recombination. the promoter is derived from the strong - -type plastid rrna operon promoter (prrn) linked to the Ј untranslated region (utr) of the t phage gene (t g ). the selectable marker gene aada provides spectinomycin resistance for selecting stable transformants, and the plastid rbcl Ј untranslated region is involved in mrna stability (fig. a) . the pbic-dpt construct was introduced into tobacco leaf tissues by microprojectile bombardment, and callus was observed on explants after - weeks on selection medium. after approximately months, those plantlets regenerated in the third round of spectinomycin selection were allowed to root. presence of the transgene in t transformed plants was determined by pcr screening using primers speciwc ( f and r) for the dpt transgene. a . -kb pcr-speciwc band was ampliwed in all four putative transplastomic plants, but it was absent in wild-type tobacco plants (fig. b) . pcr-positive plants were transferred to pots containing soil mix, and grown in the greenhouse until maturity (fig. c) . among these four t lines, designated as b , c , d , and e , three plants, including b , c , and e , were fertile and produced seeds. to check for stability of the dpt transgene, seeds from both wild-type (wt) and the three fertile transformed lines were surface-sterilized, and allowed to germinate on a mg/l spectinomycin-containing medium. all wt seeds failed to germinate; while, t seeds of transformed lines e and b readily germinated on the spectinomycin containing medium (fig. d) . seeds of c line become bleached under spectinomycin selection and were deemed non-transformed. pcr reactions were also carried out to assay for presence of the expression cassette using primers xanking the native chloroplast genome, those adjacent to the site of integration ( t), and the prrn promoter ( p) (fig. a) . the detection of a kb pcr product in all four putative transplastomic plants conwrmed presence of the expression cassette in the chloroplast genome (fig. e) . transgene integration and homoplasmy were also conwrmed in t plants, lines b and e by southern blot analysis. total dna from leaves was isolated from nontransformed and putative transplastomic plants and digested with hindiii, xbai and saci. the plastid genome organization of transplastomic tobacco using pbic-dpt and nontransformed tobacco is shown in fig. a and b . when dna was digested with hindiii, a . kb fragment was detected in spectinomycin-resistant plants, while a . kb fragment was detected in non-transformed plants (fig. c) . when dna was digested with xbai, a kb fragment was detected in spectinomycin-resistant lines; while, a kb fragment was detected in non-transformed plants (fig. d) using a diglabeled fragment of the trnn gene. the presence of a . kb hybridizing band in transformed lines when dna was digested with sac i using a dig-labeled dpt probe further conwrmed the integration of the dpt transgene (fig. e) . these results veriwed that the transgene was inserted within the intergenic region between trnn and trnr genes and con-wrmed homoplasmy in both b and e lines. the dpt protein was detected by speciwc antibodies against dpt toxoids using western blotting and elisa assays for tsp extracted from three plants of t transplastomic line e , non-transformed plants, and positive controls (toxoids). a single band of the expected size, kda, was detected in t plants of line e using polyclonal antibodies against either tetanus toxid (fig. a) or anti-diphtheria toxoid (fig. b) . chloroplast-produced dpt protein showed signiwcant high readings for all three elisa assays using polyclonal antibodies against the three dpt toxoids. anti-tetanus toxin assays showed the highest signal, and followed by the antidiphtheria assay (fig. c) . although signals were weak, signiwcant readings were detected for the anti-bordetella pertussis toxin assay (fig. c) . it is likely that the pertussis moiety was either not properly displayed or not as well-displayed as those for the other two toxins. all these wndings suggested that native epitopes of the three bacterial components present in the dpt polypeptide were conserved, and were properly displayed. based on the elisa assays, protein quantiwcation was carried out by comparing absorbance readings of plant samples with known quantities of the tetanus toxoid. the linear standard curve was used to estimate the amount of the dpt protein present in the transgenic lines. the accumulation of the recombinant dpt protein in three plants derived from transplastomic line e was up to . g per mg of leaf tissue, and approximately accounting for . % of tsp (fig. d) . to evaluate immune responses of the recombinant dpt protein produced, we proceeded to feed mice with transplastomic tobacco leaves. mice were inoculated orally with mg of freeze-dried leaves, collected from the transplastomic e line (t-e ), in three doses. mice fed transplastomic tobacco expressing dpt elicited signiwcant serum igg antibody responses to tetanus, diphtheria, and pertussis moieties, and similar to those elicited by the positive dpt(+) group (fig. a) . while, mice fed wild-type tobacco (wt) had the lowest igg responses (p < . ) (fig. a) . signiwcant speciwc iga antibody levels were detected in intestinal tissues (fig. b) , but no signiwcant igg antibody responses were detected (fig. c) . similar high levels of speciwc iga responses were induced in mice immunized with e tobacco leaves. speciwc serum anti-dpt igg (fig. a) and igg a (fig. b) antibodies were elicited in mice immunized with the tobacco-derived dpt. overall, a higher igg than igg a antibody response was detected. this suggested that a humoral immune response, rather than a cellular-mediated response, was predominantly elicited. altogether, the above wndings indicated that oral delivery of tobacco-derived dpt was immunogenic in test animals. the diphtheria, pertussis, and tetanus (dpt) vaccine is widely used for vaccination of infants and children worldwide. although its eycacy is well documented, it is expensive due to the production process which involves both scale-up production and puriwcation of recombinant proteins from three diverent bacteria. in order to reduce side evects, a few attempts have been made to produce a multicomponent recombinant dpt vaccine. boucher et al. ( ) engineered a fusion protein comprising a fragment of the tetanus toxin and the c peptide of the s subunit of the pertussis toxin (barbieri et al. ) . the resulting chimeric protein displayed the protective epitopes of both components. recently, aminian et al. ( ) reported on the expression of a fusion protein comprised of the immunoprotective s fragment of the pertussis toxin, the fulllength nontoxic diphtheria toxin, and the c fragment of the tetanus toxin. the resulting polypeptide appeared to conserve the native epitopes. however, for all these approaches, bacterial fermentation and puriwcation steps were required to purify the recombinant protein, thereby contributing to the high cost of production. to date, several important human diseases have been targeted for plant-based vaccine development that will contribute to a safe as well as inexpensive vaccine production system (korban et al. ) . recently, a novel polypeptide containing the dpt immunoprotective exotoxin epitopes has been designed and used to demonstrate expression of this fusion protein in transgenic tomato plants ). the tomato-derived dpt polypeptide was recognized by antibodies directed against diphtheria, pertussis, and tetanus toxins; however, expression levels in tomato lines were low, t . % tsp . in this study, attempts were made to enhance the expression of the dpt polypeptide in tobacco plants via plastid transformation. a synthetic gene encoding for the dpt polypeptide was designed in order to optimize its expression in tobacco chloroplasts. following microprojectile bombardment with the dpt gene construct and after several cycles of selection, several putative transformants were obtained. pcr analysis of four t plants demonstrated presence of the dpt transgene. moreover, presence of the transgene, speciwc site integration, and homoplasmy were conwrmed in the t progeny. western blots and elisa assays conwrmed that the tobacco-derived dpt protein was recognized by speciwc antibodies against each of diphtheria, pertussis, and tetanus toxins. this also suggested that the three components present in the dpt polypeptide properly displayed the native epitopes. protein quantitation using elisa analysis revealed a dpt protein content of . % of tsp. this dpt level was approximately -fold higher than that detected previously in our transgenic tomato plants derived via nuclear transformation ). using transplastomic technologies, herz et al. ( ) indicated that expression levels of up to - % of tsp could be obtained. in another report, expression of the tetanus fragment c toxin in chloroplast transformed plants reached - % of tsp content (tregoning et al. ) . in this study, the dpt transgene was expressed under the control of the prrn promoter and the Ј-utr t g , reported to mediate high expression in plastids (kuroda and maliga a) . the relatively low dpt content obtained in transplastomic tobacco plants in this study might be attributed to several factors. among these, it is likely that the sequence immediately downstream of the start codon, which has been associated with translation eyciency, might contribute to low levels of expression (kuroda and maliga b) . several reports indicated that the amount of recombinant proteins expressed in chloroplasts was lower than % of tsp (maliga ) . for example, lee et al. ( ) reported levels of . - . % of tsp of the viral capsid protein antigen against epstein-barr virus expressed in tobacco plastids. they suggested that this low level of protein accumulation was likely due to post-transcriptional events and protein stability. herz et al. ( ) reported that expression levels in plastids were very much dependent on the vector, promoter, and translational control element(s), among others. therefore, these various factors might have contributed to the low levels of expression observed in this study. mice feeding studies were conducted using freeze-dried tobacco leaves of transplastomic line e , expressing dpt. results suggested that the recombinant dpt protein induced speciwc systemic and mucosal antibody responses in vaccinated mice. to determine whether evective protective immunity was induced, it will be necessary to assess animal responses and survival to lethal challenge with each of the exotoxins. moreover, future research would involve targeting the transfer of the dpt transgene into edible crops such as lettuce or carrot that have been recently successfully transformed via plastid transformation (kanamoto et al. ; kumar et al. ). in conclusion, we have successfully expressed the multi-epitope dpt polypeptide in transplastomic tobacco plants at levels higher than those reported in transgenic tomato plants. this further demonstrated successful transformation of plastids with a fusion protein of complex oligemeric structures that remain functional and retain their antigenicities. moreover, this candidate plant-based vaccine was immunogenic via oral route delivery in mice. this model system would allow for the development of new experimental dpt subunit vaccines having several advantages, including safe and evective production and delivery, reduced side evects, and low cost of production. humoral and cellular immune responses in mice immunized with recombinant mycobacterium bovis bacillus calmette-guérin producing a pertussis toxin-tetanus toxin hybrid protein expression and puriwcation of a trivalent pertussis toxin-diphtheria toxin-tetanus toxin fusion protein in escherichia coli construction of a diphtheria toxin a fragment-c peptide fusion protein which elicits a neutralizing antibody response against diphtheria toxin and pertussis toxin neutralizing antibodies and immunoprotection against pertussis and tetanus obtained by use of a recombinant pertussis toxin-tetanus toxin fusion protein update to supplementary acip statement. recommendations of the advisory committee on immunization practices (acip) preventing tetanus, diphtheria, and pertussis among adults: use of tetanus toxoid, reduced diphtheria toxoid and acellular pertussis vaccine transgenic plants for vaccine production: expectations and limitations expression of the native cholera toxin b subunit gene and assembly as functional oligomers in transgenic tobacco chloroplasts chloroplast genetic engineering to improve agronomic traits oral immunization with pbsvp transgenic alfalfa protects mice against rotavirus infection intracellular fate of a recombinant protein is tissue dependent chloroplast targeting of fanc, the major antigenic subunit of escherichia coli k wmbriae, in transgenic soybean transgenic plants as factories for biopharmaceuticals development of novel types of plastid transformation vectors and evaluation of factors controlling expression plantderived measles virus hemagglutinin protein induces neutralizing antibodies in mice the use of combination vaccines has improved timeliness of vaccination in children dna vaccine encoding pertussis toxin s subunit induces protection against bordetella pertussis in mice eycient and stable transformation of lactuca sativa l. cv. cisco (lettuce) plastids expression of the b subunit of e. coli heat-labile enterotoxin in the chloroplasts of plants and its characterization a plant-derived edible vaccine against hepatitis b virus foods as production and delivery vehicles for human vaccines plant-based vaccine: mice immunized with chloroplast-derived anthrax protective antigen survive anthrax lethal toxin challenge plastid-expressed betaine aldehyde dehydrogenase gene in carrot cultured cells, roots, and leaves confers enhanced salt tolerance complementarity of the s rrna penultimate stem with sequences downstream of the aug destabilizes the plastid mrnas sequences downstream of the translation initiation codon are important determinants of translation eyciency in chloroplasts edible vaccines: current status and future expression of viral capsid protein antigen against epstein-barr virus in plastids of nicotiana tabacum cv accumulation of recombinant sars-cov spike protein in plant cytosol and chloroplasts indicate potential for development of plant-derived oral vaccines expression of human papillomavirus type l protein in transgenic tobacco plants plant-derived pharmaceuticals-the road forward progress towards commercialization of plastid transformation technology plastid transformation in higher plants neutralizing immunogenicity of transgenic carrot (daucus carota l.)-derived measles virus hemagglutinin expression of hepatitis b surface antigen in transgenic plants expression of norwalk virus capsid protein in transgenic tobacco and protein and its oral immunogenicity in mice expression of the rabies virus glycoprotein in transgenic tomatoes safety and immunogenicity of a primary course and booster dose of a combined diphtheria, tetanus, acellular pertussis, hepatitis b and inactivated poliovirus vaccine production of hepatitis b surface antigen in transgenic plants for oral immunization expression of escherichia coli heat-labile enterotoxin b subunit (ltb) in carrot (daucus carota l.) ingestion of transgenic carrots expressing the escherichia coli heat-labile enterotoxin b subunit protects mice against cholera toxin challenge expression of an escherichia coli antigenic fusion protein comprising the heat labile toxin b subunit and the heat stable toxin and its assembly as a functional oligomer in transplastomic tobacco plants vaccine antigen production in transgenic plants: strategies, gene constructs and perspectives sustained eycacy during the wrst years of life of -component acellular pertussis vaccines administered in infancy: the italian experience molecular cloning: a laboratory manual oral immunization of mice with transgenic tomato fruit expressing respiratory syncytial virus-f protein induces a systemic immune response transgenic tomatoes express an antigenic polypeptide containing epitopes of the diphtheria, pertussis and tetanus exotoxins, encoded by a synthetic gene high-frequency plastid transformation in tobacco by selection for a chimeric aada gene immunogenicity in humans of a recombinant bacterial antigen delivered in a transgenic potato human immune responses to a novel norwalk virus vaccine delivered in transgenic potatoes epidemiology of pertussis immunogenicity of transgenic plant-derived hepatitis b surface antigen expression of tetanus toxin fragment c in tobacco chloroplasts protection against tetanus toxin using a plant-based vaccine informal consultation on the control of pertussis with whole cell and acellular vaccines expression and characterization of helicobacter pylori heat-shock protein a (hspa) protein in transgenic tobacco (nicotiana tabacum) plants the stem-loop region of the tobacco psba 'utr is an important determinant of mrna stability and translation eyciency acknowledgments this research was partially supported by a grant received from the conacyt project , conacyt university of illinois joint research program, and conacyt scholarships and to support graduate studies of ruth soria-guerra and sergio rosales-mendoza, respectively. funding support was also received from the simon f. guggenheim memorial foundation (for ssk). key: cord- -rfu bkag authors: gómez, n.; carrillo, c.; salinas, j.; parra, f.; borca, m. v.; escribano, j. m. title: expression of immunogenic glycoprotein s polypeptides from transmissible gastroenteritis coronavirus in transgenic plants date: - - journal: virology doi: . /viro. . sha: doc_id: cord_uid: rfu bkag abstract the use of transgenic plants as vaccine production systems was described recently. we report on the immunological response elicited by two recombinant versions of the glycoprotein s from the swine-transmissible gastroenteritis coronavirus (tgev) expressed in transgenic plants. arabidoposis plants were genetically transformed with cdnas constructs encoding either the n-terminal domain (amino acid residues – ) or the full-length glycoprotein s of tgev, responsible for the neutralizing antibody induction against the virus, under the control of the cauliflower mosaic virus s (camv s) promoter. genomic dna and mrna analyses of leaf extracts from transformed plants demonstrated the incorporation of the foreign cdna into the arabidopsis genome, as well as their transcription. expression of recombinant polypeptides were observed in most transgenic plants by elisa using specific antibodies. mice immunized with leaf extracts from transgenic plants developed antibodies that reacted specifically with tgev in elisa, immunoprecipitated the virus-induced protein, and neutralized the virus infectivity. from these results, we conclude that transgenic plants expressing glycoprotein s polypeptides may possibly be used as a source of recombinant antigen for vaccine production. swine-transmissible gastroenteritis virus (tgev) is the causative agent of acute diarrhea of newborn piglets that provokes high mortality rates in affected farms. protective immunity against this disease must be developed in pregnant sows to confer passive protection to the piglets through colostrum and milk. neutralizing antibodies against the virus are directed mainly to glycoprotein s (garwes et al., ; jimenez et al., ) , and relevant epitopes in neutralization have been mapped into the n-terminal domain of this protein . four major antigenic sites have been described in glycoprotein s, of which site a is the immunodominant (de diego et al., ; delmas et al., ; sa  nchez et al., ) . glycoprotein s from tgev has been expressed using different vectors with tropism that favored antigenic presentation in the mucosal surfaces (smerdou et al., ; torres et al., ) . these vaccination approaches promoted systemic and mucosal antibody induction and, in the case of adenovirus vector, conferred protection to suckling piglets (torres et al., ) . the development of genetic transformation technology in plants has made possible the expression of foreign genes in different plant species, making reasonable the idea of using plants as bioreactors to produce recombi-nant proteins. the concept of vaccine production in transgenic plants was first introduced by mason et al. in . proteins involved in protective immune response can be produced at a low cost and easily purified from plant extracts for parental inoculation. in addition, oral immunization by edible vaccines produced in transgenic plants could stimulate immune responses at the portal entrance of many pathogens, facilitating the design of large-scale immunization programs. the presence of specific antigens into plants, even at low levels, can raise by the oral route immune reactions comparable to those raised by conventional vaccines (haq et al., ; mason et al., ) . hepatitis b surface antigen (thanavala et al., ) , escherichia coli heat-labile enterotoxin (lt-b) antigen (haq et al., ; tacket et al., ) , norwalk virus capsid protein (mason et al., ) , vp antigen from foot and mouth disease virus (carrillo et al., ) , and cholera toxin b subunit (arakawa et al., ) are the vaccine antigens expressed in transgenic plants and tested for the immune response elicited in immunized animals. additionally, rabies virus glycoprotein was expressed in transgenic tomatoes, but the immune response induced by administration of these plants to animals was not tested (mcgarvey et al., ) . in the present study, we investigated the feasibility of expressing the glycoprotein s from tgev in transgenic plants, as well as the antigenicity and immunogenicity of the plant-derived protein. the s protein is an excellent model for developing oral vaccines against enteric pathogens of mammals because of its immunogenicity and resistance to degradation in the gut. the binary prok i and prok ii recombinant plasmids ( fig. ) , carrying a cdna coding for the n-terminal region or the full-length glycoprotein s respectively, were obtained by subcloning the corresponding sequences from previously obtained constructs. recombinant prok plasmids allow selection of transformants on media containing kanamycin and stable integration into nuclear chromosomal dna from the plant. prok uses the cauliflower mosaic virus s (camw s) promoter for nominally constitutive transcription of the cloned genes. plant transformation with prok i and ii was carried out as described in materials and methods by agrobacterium tumefaciens-mediated transformation. the transgenic plants resistant to the selective medium appeared similar in morphology to the nontransgenic arabidopsis plants. more than different lines of transformants containing each construct were obtained and self-pollinated to obtain f lines. all lines were positive when screened for the presence of the recombinant genes by polymerase chain reaction (pcr) analysis ( fig. a ). most plants harboring recombinant genes showed specific transcription of foreign genes by reverse transcription (rt)-pcr analysis (fig. b ). to rule out the possibility of amplification of contaminant dna sequences present in the rna preparations, we treated the purified rna with ribonuclease before foreign gene amplification by using taq polymerase. no amplified dna fragments were detectable under those conditions, assessing the rna dependence of the reaction (fig. b ). the presence of the recombinant polypeptides in the plants harboring and expressing the foreign genes was investigated in four plants of each construct, selected to be analyzed by elisa and western blotting using an anti-tgev polyclonal serum. results demonstrated that leaf extracts from all selected plants were positive on elisa (fig. ) . however, no specific reaction on western blotting was detected in any of the plant extracts analyzed (data not shown), probably due to the low levels of recombinant protein expression and to the conformational nature of most of the immunodominant epitopes present in this protein. from a titration elisa using different virus dilutions and a monospecific anti-glycoprotein s antibody, we found that ϳ ± g of soluble leaf protein contains a glycoprotein s antigenic mass equivalent to that contained in . g of purified tgev. the percentage of the total soluble protein corresponding to recombinant glycoprotein s polypeptides accumulated in the leaves of arabidopsis transformants could represent . ± . % of the total soluble leaf protein. leaf extracts from transgenic plants expressing the n-terminal (plants ± ) or full-length glycoprotein s (plants ± ) were used to immunize mice. a control mouse was immunized with a leaf extract from a plant transformed with prok plasmid. after three immunization doses, the specificity of mice sera was tested by an elisa using purified tgev as antigen. figure a shows that all sera reacted with the virus showing, as expected, different titers. a kinetic of antibody induction in an immunized mouse (number ) was studied by immunoprecipitation of glycoprotein s induced by tgev in infected st cells. this mouse serum immunoprecipitated specifically the virus protein after two immunizations (fig. b) . finally, sera from all immunized mice were tested in a tgev neutralization assay. both glycoprotein s polypeptides produced in transgenic plants elicited virus-neutralizing antibodies (neutralization indexes of . ± . ; fig. c ). serum from a nonimmunized mouse (not shown) or from the mouse immunized with the plant transformed with prok plasmid did not show virus neutralization activity (fig. c ). in this report, we show that full-length or the globular part (n-terminal domain) of tgev spike protein (glycoprotein s) expressed in transgenic plants retained the antigenic properties and elicited neutralizing antibodies when used to immunize animals. expression in eukaryotic hosts is required for antigenic determinants that are dependent on glycosylation. of the three major antigenic sites defined on glycoprotein s involved in the induction of tgev-neutralizing antibodies, sites a and b are complex, conformational, and glycosylation dependent. site d can be represented by synthetic peptides, although glycosylation has a minor effect on its conformation (gebauer et al., ) . several genetically engineered vaccines using prokaryotic vectors have failed against tgev. glycoprotein s expressed at high levels in escherichia coli and used to inoculate animals did not induce neutralizing antibodies or confer protection in vivo (hu et al., ) . plant cells present differences in protein glycosylation with respect to animal cells that could determine the lose of antigenic determinants in antigens expressed in transgenic plants. glycosylation in plants may differ in the extent of glycosylation, processing, or both of n-linked oligosaccharide side chains (faye et al., ) . furthermore, the complex glycans of plants are often smaller than those of animals, in part due to the absence of sialic acid (faye et al., ) . the only precedent of a glycoprotein expressed in plants for vaccine development is the glycoprotein g of rabies virus (mcgarvey et al., ) . this protein expressed in tomato plants showed a molecular mass ϳ and ϳ kda less than that obtained from virus-infected cells but still larger than the protein size predicted for the unglycosylated polypeptide chain (mcgarvey et al., ) . the molecular mass of glycoprotein s expressed in arabidopsis thaliana could not be determined because we were not able to detect the recombinant protein on western blotting. however, antigenic determinants with strong dependence of glycosylation seem to be preserved because the plant-derived antigens induced neutralizing antibodies in immunized animals, indicating that critical antigenic sites are at least in part correctly glycosylated in plants. this work demonstrates the feasibility of expressing glycoprotein s polypeptides in plants. because the site of insertion of the transferred dna into the cellular chromosomal dna is random, different levels of protein expression in independent transformants are expected. we obtained expression levels similar to that described with equivalent constructs expressing hepatitis b surface antigen or rabies virus glycoprotein (mason et al., ; mcgarvey et al., ) . more recently, expression levels of norwalk virus capsid protein in tobacco have been shown to be higher than the above mentioned antigens (up to . % of total soluble protein; mason et al., ) . we have not found significant differences in foreign antigen plant expression between the two forms of glycoprotein s studied. the use of different promoters, the use of plant-derived leader sequences and signal peptides, and mainly the modification of the codon usage of this protein could improve expression levels in plants. the demonstration that many proteins from pathogens, including some expressed in transgenic plants (haq et al., ; mason et al., ) , are immunogenic when administered orally, encourages the study of other antigens expressed in plants to develop edible vaccines. glycoprotein s from tgev is an interesting model because this protein is resistant, at least when incorporated into the viral particle, to gut degradation. in addition, the protective immune responses against tgev have to be stimulated at the mucosal surfaces to induce secretory and lactogenic immunity (de diego et al., saif and bohl, ; wesley et al., ) . once we have determined the feasibility of expressing immunological active polypeptides from tegv glycoprotein s in plants, studies on the immune response of plant-derived glycoprotein s polypeptides in pigs are necessary. seeds of arabidopsis thaliana (heynh, ecotype columbia) were sown in pots containing a mixture of universal substrate and vermiculite ( : ). to synchronize germination, pots were placed at °c for h in darkness and then transferred to a growth chamber at °c with a -h photoperiod. irrigation was carried out with distilled water and, occasionally, with a mineral nutrient solution (haughn et al., ) . a -pb cdna fragment (nucleotides ± ; fragment i) and a -pb cdna fragment (nucleotides ± ; fragment ii) encoding for the n-terminal and full-length glycoprotein s from tgev purdue strain, respectively, were amplified by rt-pcr from viral rna and cloned into pbacpak plasmid (clontech). the rt primers used were Ј-cccaactatggtaccatcaat aacagc- Ј (complementary primer to nucleotides ± ) and Ј-cgcgggatccttaatggacgtg-cactttttc- Ј (complementary primer to nucleotides ± ). then, the cdna was synthesized by using the primer Ј-gcgcggatccatgaaaaactatttgt-gg- Ј. subsequently, dna fragments i and ii were subcloned in the binary prok plasmid (baulcombe et al., ) under the control of the camw s promoter, yielding the recombinant plasmids prok i and prok ii, respectively (fig. ) . plasmids prok i and prok ii were used for arabidopsis plant transformation as described elsewhere (bechtold et al., ) with slight modifications. a. tumefaciens (c c strain) containing prok i or prok ii plasmids was grown in ml of lb medium con- taining g/ml kanamycin until an od value of was reached. after centrifugation, bacteria were resuspended in ml of . g/l murashige and skoog medium containing g/l -benzilaminopurine and % sucrose. the ± -week-old plants were immersed in the a. tumefaciens suspension by inversion of the pots, and vacuum infiltration was performed in a vacuum chamber at mb for min. infiltrated plants were rinsed with water and placed in the greenhouse until attaining maturity. transgenic t seeds were selected by germination in petri dishes containing gm [ . g/l murashige and skoog, % sucrose, . g/l -(n-morpholino)ethanesulfonic acid (mes), g/l agar, ph . ] and g/ml kanamycin. two-week-old transgenic plants were transplanted into soil and allowed to attain maturity. the plants were self-pollinated to obtain t plants and used for further analysis. the presence of the foreign cdna sequences in generated transgenic arabidopsis was detected by pcr. plant extracts were prepared by macerating leaves (ϳ mg) with pestle and mortar in l of a buffer containing mm tris±hcl, ph . , mm nacl, mm edta, and . % sds. the resulting extract was mixed with l of m ch coona, ph . , and incubated for min at Ϫ °c. then, samples were centrifuged, and the dna contained in the supernatant was precipitated and resuspended in l of te buffer. pcr was performed on . g of dna with a pair of primers that specifically amplify a -bp fragment of the glycoprotein s gene (sense primer, Ј-gcgcggatccatgaa-aaactatttgtgg- Ј; antisense primer, Ј-gcgcgg-tacccgatgtgaagctattg- Ј). glycoprotein s mrna in transgenic plants was analyzed by rt-pcr. total rna from the leaves of transformed plants was isolated using the fast rna kit (bio ) proteins from leaves were obtained by homogenization of leaves in a blender with liquid nitrogen, and the resulting powder was resuspended in buffer ( . g of fresh wt/ml) containing mm mes, ph , mm nacl, mm edta, . % triton x- , . m sucrose, . mm spermine, . mm spermidine, mm dtt, and mm phenylmethylsulfonyl fluoride. the extract was filtered and centrifuged min at , g, and the resulting supernatant was used for glycoprotein s polypeptides expression analyses. elisa plates were coated with l ( g/ml of pbs) of a mixture of two monoclonal antibodies, ac and dh (kindly provided by dr. l. enjuanes, centro nacional de biotechnologõ Âa, csic, spain), recognizing the antigenic sites a and d of the glycoprotein s, respectively . antibodies were incubated for h at °c, and then plates were washed and blocked h at °c with % fetal bovine serum in pbs containing . % tween . after washing the plates, leaf proteins from transgenic plants ( g of total soluble protein per well, diluted in l of pbs, ph ), containing full-length or the n-terminal domain of glycoprotein s, were added to react with the previously adsorbed antibodies in the microtiter elisa plates during h at °c. plates were then washed six times with . % tween in pbs, and l of rabbit anti-s protein, obtained after three immunization doses with the baculovirus-expressed n-terminal fragment of glycoprotein s and diluted at : in pbs containing . % tween , was added per well and left to react for h at °c. plates were washed again six times with pbs±tween buffer, and immunocomplexes were incubated with protein a±peroxidase (sigma) diluted : in pbs±tween for h at °c. finally, plates were washed again, and l of a freshly prepared solution of o-phenylenediamine dihydrochloride (sigma) and h o was added. reactions were stopped with n h so , and the absorbance was measured at nm. balb/c mice (one per arabidopsis plant) were immunized intramuscularly on days , , and with leaf extract in pbs ( g of total protein per animal per injection) in complete freund's adjuvant for the first inoculation and in incomplete adjuvant for the others. mice sera were evaluated for anti-glycoprotein s-specific antibodies by elisa using purified tgev as antigen. coated elisa plates with l of pbs, ph . , containing . g of virus were blocked as described above with % fetal bovine serum, and after washing of the plates six times, sera diluted : in pbs±tween were added ( l per well) and incubated for h at °c. then, plates were washed again to remove unbound antibodies, and goat anti-mouse antibodies ( : ) were added to reveal immunocomplexes. after being washed and developed with o-phenylenediamine dihydrochloride substrate as described above, reaction was stopped with n h so , and plates were read at nm. immunoprecipitation of glycoprotein s by sera from a mouse after different immunization doses was carried out essentially as previously described for mouse antibodies (bullido et al., ) . briefly, st cells infected with tgev (m.o.i. ) were incubated for h, pulse labeled for h with ci/ml of s-methionine ( ci/mmol; amersham international, amersham, england)/ml, and lysed with lysis buffer ( mm tris±hcl, mm nacl, mm edta, % nonidet p- , ph . , mg/ml bovine serum albumin, and mm phenylmethylsulfonyl fluoride). the lysate ( cpm) was incubated with a control mouse serum ( l) for h and precleared with a % (v/v) suspension of protein g±sepharose (pharmacia, sweden) in lysis buffer. the precleared s-labeled cell extract was incubated with mice sera ( l) for h at °c, and immunocomplexes were incubated with % suspension of protein g±sepharose for h with gentle mixing. beads were washed three times with lysis buffer and boiled in sds±electrophoresis buffer. the antigen± antibody complexes were analyzed in . % sds±page. a plaque reduction assay with sera from immunized mice was performed as described previously (jime  nez et al., ) . the neutralization index of each serum was expressed as the log of the ratio of the pfu/ml of virus obtained using a normal serum and that observed in the presence of a given anti-glycoprotein s mouse serum. efficacy of a food plant-based oral cholera toxin b subunit vaccine expression of biologically active viral satellite rna from nuclear genome of transformed plants agrobacterium mediated gene transfer by infiltration of adult arabidopsis thaliana plants monoclonal antibody f / recognizes the ␣ chain of the porcine ␤ integrin involved in adhesion and complement mediated phagocytosis protective immune response to foot-and-mouth disease virus with vp expressed in transgenic plants antigenic structure of e -glycoprotein of transmissible gastroenteritis coronavirus epitope specificity of protective lactogenic immunity against swine transmissible gastroenteritis virus characterization of the iga and subclass igg responses to neutralizing epitopes after infection of pregnant sows with the transmissible gastroenteritis virus or the antigenically related porcine respiratory coronavirus four major antigenic sites of the coronavirus transmissible gastroenteritis virus are located on the amino-terminal half of spike glycoprotein s detection, biosynthesis and some functions of glycans n-linked to plant secreted proteins antigenicity of structural components from porcine transmissible gastroenteritis virus residues involved in the antigenic sites of transmissible gastroenteritis coronavirus s glycoprotein oral immunization with a recombinant bacterial antigen produced in transgenic plants sulfonylurea-resistant mutants of arabidopsis thaliana studies of tgev spike protein gp expressed in e coli and by a tgevvaccinia virus recombinant critical epitopes in transmissible gastroenteritis virus neutralization expression of hepatitis b surface antigen in transgenic plants expression of norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice expression of the rabies virus glycoprotein in transgenic tomatoes passive immunity in transmissible gastroenteritis of swine: immunoglobulin classes of milk antibodies after oral-intranasal inoculation of sows with a live low cell culturepassaged virus antigenic homology among coronaviruses related to transmissible gastroenteritis virus characterization of transmissible gastroenteritis coronavirus s protein expression products in avirulent s. typhimurium ⌬cya ⌬crp: persistence, stability and immune response in swine immunogenicity in humans of a recombinant bacterial antigen delivered in a transgenic potato immunogenicity of transgenic plant-derived hepatitis b surface antigen tropism of human adenovirus type -based vectors in swine and their ability to protect against transmissible gastroenteritis coronavirus induction of antibodies protecting against transmissible gastroenteritis coronavirus (tgev) by recombinant adenovirus expressing tgev spike protein lack of protection in vivo with neutralizing monoclonal antibodies to transmissible gastroenteritis virus we thank j. c. oliveros, p. go  mez puertas, and a. brun for helpful discussions and suggestions and covadonga alonso for critical reading of the manuscript. this work was supported by grant bio ± from comision interministerial de ciencia y tecnologõ Âa of spain and by grant bid /oc-ar pid from secyt-conicet, rep. argentina. key: cord- -dhkz co authors: chamorro, melina fernanda; ladio, ana title: native and exotic plants with edible fleshy fruits utilized in patagonia and their role as sources of local functional foods date: - - journal: bmc complement med ther doi: . /s - - - sha: doc_id: cord_uid: dhkz co background: traditionally part of the human diet, plants with edible fleshy fruits (peff) contain bioactive components that may exert physiological effects beyond nutrition, promoting human health and well-being. focusing on their food-medicine functionality, different ways of using peff were studied in a cross-sectional way using two approaches: a bibliographical survey and an ethnobotanical case study in a rural community of patagonia, argentina. methods: a total of studies were selected for the bibliographical review. the case study was carried out with % of the families inhabiting the rural community of cuyín manzano, using free listing, interviews, and participant observation. in both cases we analyzed species richness and use patterns through the edible consensus and functional consensus indices. local foods, ailments, medicines and drug plants were also registered. results: the review identified peff, the majority of which ( %) were native species, some with the highest use consensus. peff were used in different local foods, but mainly as fresh fruit. of the total, % were used in a functional way, in different medicines. the principal functional native species identified in the review were aristotelia chilensis and berberis microphylla. in the case study peff were in current use ( % were native), and consensus values were similar for native and exotic species. these were used in different local foods, mainly as fresh fruit. only % were recognized for their functional value by inhabitants (mainly as gastrointestinal and respiratory treatments). the species with the highest functional consensus were the exotic sambucus nigra and rosa rubiginosa, followed by the native a. chilensis, ribes magellanicum and b. microphylla. infusions also constituted important local functional foods. conclusions: this survey highlights the importance of studying the different local functional foods to depict the biocultural diversity of a human society. the preparation of different beverages and herbal medicines was relevant, and would be a promising subject to investigate in the future. the living heritage of peff appears to have undergone hybridization processes, such that exotic species play an increasingly significant role. a poor diet, with little consumption of fruit and vegetables, among other characteristics, is known to be strongly associated with mortality through noncommunicable diseases (such as cardiovascular diseases and cancer) [ ] . extra-nutritional substances present in plants have the capacity to reduce cellular oxidative stress, thus lessening the probability of developing these illnesses [ ] [ ] [ ] . the current challenge is to generate policies centered not just on nutrients, but on healthy foods [ ] . species with edible fruits deserve special attention not only for their value per se, but also as local foods. the importance in this study of the variety of ways of ingesting plant-based foods is highlighted by heinrich et al. [ ] . they developed the concept of "local foods" through the study of the mediterranean diet, distinguishing those recipes that are shared in a territory or culture, and which form a fundamental part of local food knowledge [ ] . this represents an interesting field of study in the subject of functional foods, a concept that originated in japan in the s as foods for specified health use (foshu). at the present time, "functional food science in europe" (fufose) proposes that functional food is that which is "satisfactorily demonstrated to affect beneficially one or more target functions in the body, beyond adequate nutritional effects, in a way that is relevant to either an improved state of health and wellbeing and/or reduction of risk of disease" [ ] . from an ethnobotanical perspective, other concepts arise such as "folk functional foods" [ ] , or what we will call here "local functional foods", based on the multifunctional character of food use by local inhabitants [ ] . the study of plant-based local functional foods currently offers an opportunity for ethnopharmacological research, as a wide range of biocompounds is revealed, which is made available to humans in the different recipes used, capable of transforming human gastronomic habits. the study of native and exotic edible plant use in rural and indigenous communities offers an opportunity to appreciate how humans have experimented with the diversity of their plant surroundings in search of wellbeing. as established by etkin and ross [ ] , plant studies in these communities should be approached with understanding of the integral nature of food and health concepts. in their search for this functionality, humans have intentionally selected dual-purpose resources (ediblemedicinal) and have also tended towards diversification of food types and new forms of consumption. consequently, we are interested in showing the multidimensionality and multifunctionality of the peff, considering all their plant parts in an integral way. the study of the potential of patagonian native fruit species is relatively recent. progress has been made from the perspectives of agronomy [ , ] , phytochemistry [ ] [ ] [ ] and nutrition [ , ] . several studies have demonstrated the antioxidant properties of some patagonian fruits and their usefulness in regulating glucose metabolism, thus indicating their nutraceutical value [ ] [ ] [ ] . nevertheless, the diversity of existing species and their local foods has been little described up to now [ ] . one aspect highlighted in the ethnobotanical studies carried out in patagonia is the high proportion of exotic edible plant use in some local communities [ ] [ ] [ ] . for example, in rural areas, the gathering of edible plants seems to be linked to environmental changes, with the incorporation of more exotic fruit-bearing species that grow wild, including invasive plants like sweet briar, elmleaf blackberry and apple (rosa rubiginosa l., rubus ulmifolius schott, malus domestica borkh) [ ] . due to processes of colonization and imposition, since the arrival of europeans the region has tended towards the cultivation of exotic fruits [ ] . in market gardens and fruit farms % of the fruit grown is exotic in origin, such as plums and apples [ ] . this pattern, called the hybridization process [ ] , is prevalent and of great interest when considering local foods. in this study we focus on native and exotic plants with edible fleshy fruits (peff) that grow in patagonia; that is, species that may be wild, cultivated or in an intermediate state of domestication, which bear fruit that is distinguished by its flavor, preferably sweet, and its use principally as a food resource. in general, they have a high proportion of water and sugars that are easily digested and absorbed, and they have long contributed fiber, minerals and vitamins to the diet of local populations [ ] . very little information is available concerning the total number of species involved in this group and how people have used them. in addition, far fewer studies exist that deal with the safety aspect of consumption of peff and their different preparations. understanding the use patterns of peff as part of the local biocultural heritage of patagonia is crucial [ ] . in recent times, as in the rest of the world, rural and urban societies of this region have experienced sociocultural and environmental changes. these changes have led to a reduction in traditional plant use [ ] , and some elements of the flora seem to be in danger [ ] . these alterations will have serious long-term repercussions for peff use, as motivation for the generational transmission processes (oral traditions) is weakened, and consequently, the patrimony for future generations is reduced. in this study we propose a cross-sectional approach which enables exotic and native species richness, local foods, medicines and their use patterns to be evaluated, and which will also help us understand in greater depth, from an ethnobotanical perspective, that diet and health are linked concepts. we will carry out a bibliographical review and a case study of a current rural community in order to synthesize existing information on the functionality of native and exotic peff, and analyze their use patterns and potential. our main questions were: which native and exotic plants from patagonia constitute peff? which species are currently in use? what proportion is used as functional food? how many local foods are involved? this analysis of mixed information also enables us to show the continuity or change in use of functional and nonfunctional peff, shedding light on the existing biological and cultural diversity, and its potential. the bibliographical review involved quali-quantitative analysis of ethnohistorical and ethnobotanical texts published since which mention the use of native and exotic fruits in the patagonian region [ ] . the review was conducted using scopus, science direct and scielo, and google scholar search engine, as well as the library and database of the grupo de etnobiología. the key words and phrases used in the search were: native and exotic edible fruits and patagonia; edible plants and patagonia; medicinal plants and patagonia; patagonian flora and uses; ethnobotany of patagonia; medicinal and alimentary uses and patagonia, and native fruits of patagonia. the search was carried out in spanish and english, and a total of documents were examined. the works selected were mainly primary field studies, but compilations were also included given that each review used independent protocols for species evaluation, according to the author of the work. location of database publications: the articles selected were from studies carried out in argentine rural and urban communities with non-indigenous people, creoles, and people of mapuche-tehuelche-selk'nam ancestry, distributed over an area extending approximately between o and o lat. s. of the articles studied, % dealt with organized indigenous communities, while the remainder were rural and/or urban communities which were pluricultural in character. the phytogeographical heterogeneity of the bibliographical approach led to the inclusion of a high diversity of plants, life forms and botanical families that were used by the majority of the indigenous populations in the region [ , ] . fieldwork location: the fieldwork was carried out in the rural community of cuyín manzano, which is located in nahuel huapi national park, ( ° s and ° w), km from bariloche city. this location lies within the andino norpatagonica biosphere reserve (unesco). the climate in the region is temperate-cold and humid, with a mediterranean precipitation regime, such that rain and snow fall principally in winter (mat . °c). the rural community is located in an ecotonal environment between steppe and forests of austrocedrus chilensis and nothofagus spp. the population is of mestizo origin; some individuals are direct descendants of the mapuche people, while others have mixed ancestry, known locally as criollos (creoles). at present only families live here, due to strong processes of emigration. the economic activity of inhabitants has become more varied with time; they work with livestock, tourism, handicrafts and also as employees in the state school hostel and a private ranch. this community has a longstanding history of settlement in the area, according to archeological records [ ] . our previous studies in this location [ ] [ ] [ ] have shown that this population is very representative of a typical patagonian rural community. considering its low population density at present, the native and mixed ancestry of the people, its immigration and migration process, its contact with the city and the maintenance of its traditions, cuyin manzano reflects shared sociocultural patterns with other rural settlements. in addition, the fieldwork was greatly facilitated due to the close ties of trust we have built up with the people over several years. the information obtained from each source was systematized in a database according to the following criteria: ) the species should be identified taxonomically in the original study; ) it should grow in argentine patagonia; ) native and exotic species were included. native species were defined here as plants whose origin is central or southern argentina, below °southern latitude, and exotic species were those which did not fulfill this condition [ ] ; ) wild, semi-domesticated and cultivated species were included; ) the species were categorized as exclusively edible, and , functional, when the plant had at least one functional citation (edible, with also one or more medicinal uses) in the bibliography. following these criteria, studies and citations were included. each citation corresponds to a species referred to in a publication, with its corresponding ethnobotanical information. the following information was selected: species, common and indigenous names, richness of local foods, medicinal uses, herbal medicines made with peff and parts of the plant used (named plant drugs). the local foods were classified as: fresh fruit (f), jams and sweets (s), non-alcoholic drinks (d), fermented drink called "chicha" (ch), other (ot). the classification of ailments was adapted from molares and ladio [ ] : respiratory (rs), gastrointestinal (gi), urinary (u), pain and inflammation (pi), dermatologic (de), fever (f), obstetric (obs), gynecological (gyn), blood (bl), circulatory and heart (ch), nervous system (ns), cultural syndromes (cs), osteo-articular and muscular (oa), allergies (al), dentistry (den), ophthalmological (op), refreshment (re), others (ot). the different herbal medicines were classified according to method of administration: fruit ingestion (f), infusion (in), decoction (de), bathing, washing and rubbing (ba), gargling (ga), poultices and compresses (ca), creams and ointments (oi), other (ot). the plant drugs were categorized according to the part of the plant used: fruit (f), flowers (fl), leaves (l), branches (b), bark or stalk (s), root (r), the whole plant (pl). fieldwork was carried out according to the code of ethics of the international society of ethnobiology (ise) and the consensus statement on ethnopharmacological field studies [ ] . free listing, semi-structured interviews, in-depth interviews and participant observation were performed with participants [ , ] , prior oral informal consent. if the participant consented to being recorded during the interview, oral consent was also taperecorded. this procedure is supported by national research regulatory agencies and international agreements (argentinian national law n.° , ). a total of % of the population ( households) took part in the study, including several members of each family. the ages of the adult participants ranged from to years. inhabitants were consulted on their knowledge of the peff that grew in the area, and their alimentary and functional uses. the information gathered was reorganized following the categories used for the bibliographical review. the database included citations with information on peff. each citation corresponds to a species referred by each interviewee. the same quali-quantitative analysis [ ] was performed for both approaches. due to the categorical nature of the data, the analyses were principally non-parametric, using the spss package for windows. species richness (s) was calculated in total, according to biogeographical origin, and by botanical family. native and exotic species richness were compared using the binomial test (p < . ) [ ] . whether a native or exotic species was used as edible or functional was evaluated by consensus indices, mainly utilized for use pattern analysis of ethnobotanical data [ ] [ ] [ ] [ ] . these indices enable us to consider the agreement in a community or between diverse authors (and their publications) on the use of a certain species or plant family. the cultural importance index of edible plants (cie) was calculated for the species and for the families: cie = fc/n × , where fc = the frequency of citation of the species or family and n = the number of total publications/informants. we also calculated the cif index for functional species and families, as cif = fc/n × , where fc = the frequency of citation of the functional species or family. the mann whitney test was performed to compare these indexes for native and exotic species (p < . ). the percentage of each category was calculated in relation to the total reported methods of use of all the peff, that is, the total foods. %sc a = s sca / s sca..i × . where %sc a = the percentage of the subcategory a (for instance, drinks); s sca = the richness of species of the subcategory a; and s sca..i = the total of local foods. the use value is an index that indicates the versatility of a species, that is, the diversity of ailments that can be treated by a given species [ , , ] . it was obtained as follows: uvs = uvis/n [ ] , where uvis = the number of different medicinal uses registered by publication/informant for species s. similarly to local foods, the percentage of each category was calculated in relation to the sum of the plant richness for each subcategory. that is, in relation to the total medicines or plant drugs (medicinal parts of the plants). in the field study all native and exotic plant species were mentioned by their common names, and were later identified taxonomically by the authors. the collection of wild and cultivated species with fleshy fruits was performed with the assistance of local dwellers. in addition, field herbaria and photographs were utilized in the interviews to confirm the taxonomic identity of plants. plant identification followed correa [ ] [ ] [ ] plant specimens were placed in the ecotono-ethnobiology group-inibioma-university of comahue herbarium, and will be deposited in the herbarium of the centro regional universitario bariloche (bcru). voucher specimens of all reported species are shown in table . it should be noted that the berberidaceae family was treated according to landrum [ ] . all scientific names were updated using world flora online (www.worldfloraonline.org). a total richness (s) of species was found through the bibliographical review (table , fig. a ). the average richness per study was . species (min: , max: ). richness of native species ( %, species) was notably greater than that of exotic plants ( %, species) (p < . , binomial test). coincidentally, the cie of native peff was higher than for exotic plants (p < . , mann whitney test) (fig. b) . the native species with highest cie were berberis microphylla, ribes magellanicum, fragaria chiloensis, ephedra ochreata, aristotelia chilensis, gaultheria mucronata, berberis darwinii and berberis empetrifolia (fig. a) . the exotic species with highest cie were sambucus nigra, rubus idaeus, prunus cerasus, rosa rubiginosa, ribes aureum and ribes uva-crispa. the peff belonged to botanical families, the main ones being rosaceae ( species), grossulariaceae ( species), anarcardiaceae and berberidaceae ( species each). the families with the highest cie values were berberidaceae, rosaceae and grossulariaceae. in the bibliography, local foods were identified. most publications reported the use of fresh fruits ( % of the total local foods). a wide variety of preparations were found that generally included the addition of sugar, such as jams and jellies ( %). the most significant species used to make jams were b. microphylla, b. darwinii, r. magellanicum, r. cucullatum and f. chiloensis. another important way of taking advantage of peff was the preparation of drinks, mainly as soft drinks. these included non-alcoholic beverages ( %) made from the fruit of different species, such as ephedra triandra, aristotelia chilensis and berberis microphylla. the next most common drink was the fermented "chicha" ( %), which used different peff, amongst which stood out schinus polygama, geoffroea decorticans, r. magellanicum and amomyrtus luma. the production of "chicha" from a. luma mixed with fruits of other species is also mentioned. fifteen percent of local foods includes preparations with cooked, boiled or roasted fruits, as in the case of maihueniopsis darwinii. wines are also prepared, such as those made from a. chiloensis fruit, or liqueurs from b. microphylla or gaultheria poeppigi fruits. similarly, this category includes flours such as those made with condalia microphylla and geoffroea decorticans fruits. of the total number of species registered, the proportion of functional peff was % ( species, table ), similar to the non-functional species ( %, species) (p > . , binomial test). the richness of functional species was composed mainly of native species ( %, p < . , binomial test). it was also found that the cif of native species was significantly higher than that of exotic plants (p < . , mann whitney test). the principal functional species according to the cif values were the native aristotelia chilensis, ribes magellanicum, ephedra ochreata, berberis microphylla, fragaria chiloensis, luma apiculata and amomyrtus luma, and the exotic sambucus nigra, rosa rubiginosa and prunus cerasus ( table ) . the functional peff found belonged to botanical families. among these were anacardiaceae ( species); berberidaceae, ephedraceae and rosaceae with species each, and myrtaceae with species. at botanical family level, elaeocarpaceae, ephedraceae, grossulariaceae, myrtaceae, anacardiaceae, rosaceae, adoxaceae and berberidaceae were families with high cif values. the functional peff registered in the bibliography were used for a wide range of ailments (table ) , mainly gastrointestinal, respiratory, dermatological, gynecological, obstetric, related to the nervous system, heart and circulatory system, fever, pain and inflammation. the most versatile species according to their uv value were aristotelia chilensis, used to treat more than ten ailments, followed by berberis microphylla, luma apiculata, sambucus nigra, fragaria chiloensis and fuchsia magellanica ( table ) . the peff used as herbal medicines totaled (table ) , which mainly included forms that can be ingested, but also some of external use. the most important were two forms of drink: decoctions ( %) and infusions ( %). for example, infusions made of leaves or bark of r. magellanicum were used to "componer la sangre" (depurative) and for digestive ailments. a decoction of b. microphylla bark is also used to bring down a fever, or its fruit is used to combat diarrhea. the direct ingestion of a. chilensis fruit was also used to treat fever and diarrhea. among the methods of external use that stand out were: bathing ( %), for example, using luma apiculata leaves to bathe infected wounds; gargling ( %), with the same species but in this case to treat lesions of the gums. the application of exudates and poultices ( % each) has also been documented, for which the aerial parts of the plant were generally used, such as the exudate of schinus johnstonii leaves used to treat toothache. the exudate of l. apiculata as an anti-inflammatory is also frequently cited in the literature [ , , ] . poultices such as those prepared from dried a. chilensis leaves were used to clean wounds. creams and fresh fruit made up the remaining %. the parts of the plant most frequently used medicinally were the leaves ( %), fruits ( %), bark or stalk ( %) and roots ( %). a total richness of peff was found (table , fig. a) , an average of species being cited per informant (min: , max: ). this richness was divided equally between native ( species) and exotic ( species) plants (p > . , binomial test). in contrast to the findings from the review, in cuyín manzano the cie was the same for native and exotic species (p > . , mann whitney test) (fig. b) . the native species with highest cie were berberis microphylla, fragaria chiloensis and aristotelia chilensis (fig. a) . these were followed by other berberidaceae species, such as the "michay de la costa" (b. empetrifolia) and "michay de cordillera" (b. serratodentata). according to the cie values, some of the most important exotic species were rosa rubiginosa, prunus cerasus, sambucus nigra, prunus avium, prunus domestica, and finally rubus idaeus, which is known throughout the world (fig. a) . the peff in cuyín manzano belonged to eight botanical families. rosaceae contributed most to the list ( the asterisks show the species listed in the iucn red list of threatened species * = least concern, ** = near threatened, *** = vulnerable, **** = endangered, na native, ex exotic, f fruit, s sweets, d drinks, ch chicha, o others, nm not mentioned chamorro and ladio bmc complementary medicine and therapies ( ) : page of species), followed by grosulariaceae ( species) and berberidaceae ( species). however, the families with the highest cie were berberidaceae, rosaceae and elaeocarpaceae. in cuyín manzano local foods with peff were registered. of these, % were consumed as fresh fruits. in general, this method of use implied consumption at the time of gathering, with no storage involved. locals reported consuming these fruits when they were out in the countryside and felt hungry, the main reasons being that they like them a lot (such as the berberis fruits), and they are "good for them". to a lesser extent, the peff were lightly processed, through the addition of sugar or cream. also frequently consumed were sweets ( %), including jams, jellies and syrups made from mature fruit. among other preparations ( %), were the alcoholic beverages, which in this case played a role as social drinks. the most frequently cited of these was the "guindado" made with the fruit of the exotic prunus cerasus, which was served to visitors in the home. non-alcoholic drinks represented % of this category, mainly in the form of infusions, such as those prepared with the fruit of rosa rubiginosa and sambucus nigra, but also in the form of juices. other forms of consumption were also mentioned, such as the addition of fruit to baking products in the home. furthermore, the fruit tended to be used in the form of desserts, as in the case of stewed fruit, where fresh fruit such as plums were boiled in water and sugar. consumption of dried fruit called "orejones" was also registered. it is worthy of note that the preparation of "chicha" from peff was not mentioned here as it was in the review. a total of species ( %) were known to locals for their functional value (table ) ; however, the proportion of edible and/or functional species did not differ statistically (p > . , binomial test). the functional species included native and exotic species. in contrast to the review, in cuyín manzano the cif values were similar for native and exotic species (p > . , mann whitney test). as shown in table , the functional peff with highest cif were the exotic rosa rubiginosa and sambucus nigra, followed by the native aristotelia chilensis, ribes magellanicum, berberis microphylla and ribes cucullatum. the peff in cuyín manzano belonged to botanical families: grossularaciaceae ( species), and rosaceae, berberidaceae, elaeocarpaceae and adoxaceae ( species each). in terms of cif, the families that stood out were: adoxaceae ( %), rosaceae ( %), and to a lesser extent, elaeocarpaceae ( %). the peff in cuyín manzano were used principally to treat respiratory, gastrointestinal and dermatological ailments (table ) . sambucus nigra was one of the most versatile species, followed by rosa rubiginosa, aristotelia chilensis and ribes magellanicum, and finally, berberis microphylla. the inhabitants of cuyín manzano used medicines prepared from peff to treat specific local ailments (table ). these species were mainly used in the form of infusions ( %), utilized generally to treat or prevent respiratory disease. one of the most frequently used infusions was sauco (elderberry) tea, which was drunk at night to "warm up the body", and probably played a preventative role. this infusion was prepared by putting a spoonful of sambucus nigra syrup in a cup, then adding boiling water. the syrup was also consumed alone, without dilution, mainly by children but also by adults suffering cold symptoms ( %). mosqueta (rosehip) tea was used similarly as a recreational drink, to warm up the body and treat illness. fresh fruits were always consumed ( %) in the context of relieving gastrointestinal problems, and decoctions were also prepared ( %). the only external method of application was using creams ( %) made from rosa rubiginosa fruits by women from a nearby community. the medicinal parts of functional peff are mainly the fruits ( %) ( table ) , and to a lesser extent, leaves ( %), the whole plant ( %), followed by flowers, seeds, bark and roots ( % each). finally, five of the medicines were also considered as food by locals: elderberry and/or rosehip infusions, the fresh fruits of maqui and michay, and elderberry syrup, which in total represent % of local foods. the bibliographical analysis revealed a high number of native and exotic peff ( species), providing an overall picture (spanning years and an ample geographical range) that shows the study potential of all these patagonian species. the nutritional role of this plant diversity and the safety of their use is still unknown, but they presumably represent food security for locals. food security is founded on three elements: the availability, access, and utilization of food, elements that were elucidated indirectly in both the bibliographical texts and the testimonies of cuyin manzano inhabitants. it is also important to consider that the conservation status of some of the native species ( species, table ) is challenging, and must be solved by cultivation and/or sustainable management, given that their availability and access are threatened. the need for an integrated study of these aspects must be taken into account in future investigations. moreover, the field study showed that peff on this list continue to be used. although this rural community is probably experiencing a process of dietary transition from smallholder farming-based to industrialized food systems, as well as integration into the global food trade, peff continue to be part of their local heritage. lifestyle changes probably affect food production, plant gathering and consumption, with consequences for the healthfulness of diets, but these species still have an appreciated role in home-produced foods. further field research should be carried out in other patagonian communities, using this tool to survey food security in order to investigate whether local people keep peff use alive in their food traditions. this aspect is of great importance given the processes of abandonment and loss of knowledge associated with plant use observed in cuyín [ ] and numerous other rural communities throughout the world [ ] . our cross-sectional approach coincides in the cultural importance of the three native species with highest consensus: berberis microphylla, fragaria chiloensis and aristotelia chilensis. the michay plant has attractive blue-violet berries with a flavor which is sweet, but sour, and is widely distributed in forest and the patagonian steppe, therefore present also in the most arid zones. the native strawberry has strongly scented fruits, and has been used to obtain fragaria x annanasa, the strawberry which is commercially cultivated throughout the world [ ] . the berries of the maqui plant are acid to the taste, and have been designated a superfood due to their high content of anthocyanins [ , ] . at present these species are being intensively studied as target species [ ] and are attracting much attention for future innovation and development projects [ , ] . the remainder of the list of peff may also be considered by policy makers and researchers in the planning of agricultural and phytochemical research action that will support sustainable diets and local food systems. all this biodiversity is introduced into local gastronomy through a large variety of local foods. remarkably, in both studies the use of fresh fruit is high ( % in the review and % in cuyín manzano). traditional culinary habits could promote better use of nutrients, since a lower level of processing generally leads to greater availability of nutrients such as vitamin c [ ] . in second place, the preparation of preserves and beverages from the fruit indicates strategies that favor preservation over time, storage, and the use of excess fruit, as found in other parts of the world [ , ] . preserves include some forms, such as syrups, which are of great interest due to the compounds that contribute phenolic acids, fiber, and soluble solids [ ] [ ] [ ] . these plant preparations could be also highlighted, as local people could be skillfully managing the plant chemicals during food processing. patagonian communities have experimented extensively with the diversity of peff fruits in the preparation of fermented beverages, mainly in the form of "chicha". this drink is made with fruit and seeds, which in many cases were chewed by the people before being stored for fermentation. this preparation holds great cultural and spiritual value, as it is drunk in traditional mapuche festivities [ ] . nevertheless, in cuyín manzano preparation of these beverages with the peff berries was not identified. although the medicinal use of these preparations was not documented, some studies associate moderate consumption of fermented beverages with prevention of cardiovascular diseases and cancer, due to their polyphenol and alcohol content [ ] . this opens up a wide range of possibilities for the exploration of fermented beverages, taking into account ancient food traditions. these customs are considered key elements for development projects related to food sovereignty [ ] . the peff heritage possesses a great richness of species whose functional value has been well taken advantage of, and which can therefore constitute alternative healthy or phytotherapeutic food. the species taken from the bibliography and listed here are mainly native to patagonia, and represent opportunities for study, for which the consensus values can be a useful guide. in this sense, our findings are in accordance with jennings et al. [ ] as to functional species use. to understand the meaning of their functionality, contextualization of the food-plant spectrum based on both local beliefs and wider structural factors is needed. in our field study we found that dwellers consider that the fruit did them good. from the mapuche worldview, their consumption of wild fruits implies nourishing the earth's energy (called in native mapuzungum "afutum"), energy that cannot be transferred in any other way [ ] . in other words, the classification of a species as functional is inseparably supported by both the local worldview and its chemical and biological attributes. a whole universe of chemical compounds may be related to the health effects of the peff. the medicinal value of the species used as food has been explained in detail by authors such as johns [ , ] , based on the presence of a wide range of phytoconstituents. today we know that food plants have diverse constituents such as polyphenols, in addition to nutritional compounds. these molecules are recognized for their antioxidant properties; they are important radical scavengers through inhibition of prooxidant enzymes and restoration of antioxidant enzymes. however, new mechanisms of action have been proposed to explain their bioactivity, such as interaction at the level of the plasmatic membrane with proteins and phospholipids, and the regulation of signal transduction pathways [ , ] . of the peff, those which have berry-type fruits stand out as functional species, and are known worldwide as healthy foods. berries contain vitamins, minerals and phenolic compounds, and are credited with antioxidant, antiaging, chemoprotective and chemotherapeutic, anti-inflammatory and neuroprotective properties [ , ] . with a strong consensus between our two studies, the key species are: a. chilensis, ribes magellanicum, ephedra ochreata and berberis microphylla. a. chilensis is the species with the highest number of described pharmacological properties, including the ability to attenuate pain and inflammation [ ] . this activity matches the local ailment treatments described in our work. little is known about ribes magellanicum, but the antioxidant potential in vitro of the fruits found in argentine-chilean patagonia has been demonstrated [ ] . unfortunately, ephedra ochreata is one of the leaststudied species in the region. concerns about it eventual toxicity, should be carefully considered, since other ephedra spp. have been demonstrated to exhibit toxicity (https://www.health.harvard.edu/staying-healthy/thedangers-of-the-herb-ephedra). it must be used with great care, given that toxic effects have been reported for other species of ephedra, such as cardiac and psychiatric disorder [ ] [ ] [ ] . future research should focus on e. ochreata in order to test its food safety. the study of other species, such as b. microphylla, have begun only recently, revealing promising antioxidant content and antidiabetogenic activity according to chamorro in vitro and in vivo studies [ ] [ ] [ ] . again, more studies and further analysis is required to relate these chemical and biological findings to the health claims presented here. similar to the peff heritage, the use of functional species seems to be undergoing hybridization processes. this was evident in the rural community we worked in, where the cif values were similar in native and exotic plants. these results show how locals have been using a combination of native and foreign species in edible and functional terms. although this has been happening for a long time in the region, it seems to be more prevalent at the present time [ ] , probably due to changes in food procurement. the use of exotic functional peff in our region may be interpreted as a reflection of the long, strong historical influence of european immigration on the use patterns of this area. various studies carried out in south america have shown that the introduction of eurasian plants led to diversification in the use of medicinal plants [ ] , and many edible fruits form part of the pharmacopeias brought from europe [ ] . shikov et al. [ ] and totelin [ ] suggest that the use of edible fruits as medicine is due to the great influence of the food-medicine conceptions of ancient greece. however, the food-medicine interface had also been described previously, without the use of exotic plants, in patagonian indigenous communities [ ] . we suggest, therefore, that this food-medicine continuum may have been generated independently, and that the knowledge of indigenous and european communities converged at a later date. some of the exotic berries registered in this work have been extensively studied with regard to the biological activity that supports their local functional use. in the case of sambucus nigra, there is in vitro evidence to support the effectiveness of its antibacterial and antiviral properties [ , ] . hawkins et al. [ ] recently found that elderberry fruit can improve respiratory complaints associated with influenza, and so mccarty and dinicolantonio [ ] propose its use (in a dose of - mg) for the treatment of covid- infection. rosa canina, a species closely related to rosa rubiginosa, has been studied with regard to its use in treating skin ailments [ ] . the dermatological use of r. rubiginosa and use of the other functional exotic species were also registered in our field work. this pattern shows how the berry repertoire grows with input from highly reputed exotic plants. with respect to ailments, the review shows that more than ailments have been treated with peff. in cuyín manzano this number is much lower (only five ailments) and is focused on primary health care issues. the broad scope of the review shows the potential of these species, even though the main ailments treated with peff are those most frequently treated by traditional medicine, such as digestive and respiratory complaints. several studies have proposed that this redundancy gives flexibility to the regional herbal medicine, the use of a. chilensis and s. nigra fruits standing out as being the most versatile elements the population has within its reach [ ] . the peff are also important sources of herbal medicines ( in the review and in cuyín manzano) since, as previously shown, different plant parts of the peff are used in an integral way. infusions (and decoctions according to the bibliography) constitute the main method of medicinal use in the region, and this is also, within local foods, the main form of beverage in cuyín manzano. recent research has revealed how infusions of exotic rosa rubiginosa fruit can contribute compounds with antioxidant activity [ ] . the same occurs with the infusion of exotic sambucus nigra fruit, which was shown to contain considerable amounts of polyphenols and anthocyanins, comparable to an ethanolic extract [ ] . infusions are therefore, in general, good vehicles for phenolic compounds, and this is possibly the basis for their functional value in patagonian cultures. according to our analysis of the information gathered from the review, complemented by the fieldwork, we can reinterpret the comprehensiveness of the food-medicine interface in patagonia, outlined in fig. . in the review work we found a notable superposition of use (edible and medicinal) for single native or exotic species (given by the sum of citations of different authors and different study sites). however, in the analysis of each one of the studies included in the review and the fieldwork, we observed that the patagonian communities have not only experimented with native and exotic plants and discovered their medicinal properties, but they have also developed diverse forms of preparation. these make up a wide range of methods of use, but also form part of local food tradition, which considers plants from the perspective of functional, complementary logic, for their general health in their daily lives, local foods and medicines. within this group there are forms of preparation that prove effective as food and medicine (superposition of use forms depending on context), known in the literature as food medicines or healthy foods [ ] . within this group we find the subgroup of the local functional foods. as shown in our field study, this is the case of rosa rubiginosa and sambucus nigra, two exotic berries that are used in the form of infusions and indistinguishable as food or medicine. they are used as a preventative food, which in nutrition is known as a functional food [ ] . these infusions seem to be important local functional foods, given that these water solutions are good vehicles for active compounds found in plants, such as polyphenols [ , ] . finally, the functionality attributed by a culture to its native and exotic species includes superposition at different levels, and may be described in theoretical terms as different categories. however, most important is that they are principally based on the use of the entire plant. in the case of patagonian native and exotic plants with edible fruit, the people seek food, healing, and also prevention ("it warms up the body", "it does you good") through the use of infusions and the ingestion of fresh fruit. previous studies have shown the potential of some native peff, due to their polyphenol content, but little has been studied on the teas prepared with them. more integrative research is required into this use pattern of native and exotic species, their local foods and their potential as local functional foods. argentine patagonia holds great richness of native and exotic peff and the cultures who have lived on these lands have experimented with them as a source of local foods and medicines, generating a food-medicine continuum. the living heritage of patagonian peff appears to have undergone hybridization processes, such that exotic cosmopolitan species play an increasingly significant role. ethnobotanical research can contribute solutions to health problems worldwide by surveying food security and investigating whether local people maintain peff use alive in their food traditions. this should be carried out with locals in a collaborative way, and this synergic effort could provide ideas and opportunities for the sustainable and multipurpose use of plants. the development of functional local foods in any culture could offer contemporary strategies for preserving ecological and cultural diversity, which includes both the tangible and the intangible cultural heritage. a systematic analysis for the global burden of disease study potential synergy of phytochemicals in cancer prevention: mechanism of action in vitro antiproliferative, apoptotic and antioxidant activities of punicalagin, ellagic acid and a total pomegranate tannin extract are enhanced in combination with other polyphenols as found in pomegranate juice how natural dietary antioxidants in fruits, vegetables and legumes promote vascular health global diet and health: old questions, fresh evidence, and new horizons. lancet. the author(s) functional foods from science to health and claims, ilsi europe concise monograph series. brussels: intenational life sciences instiute functional foods or food medicines? on the consumption of wild plant among albanians and southern italians in lucania local food-nutraceuticals: bridging the gap between local knowledge and global needs food as medicine and medicine as food: an adaptative framework for the interperetation of plant utilization among the hausa of northern nigeria shoot growth and development of berberis buxifolia lam fruit growth and chemical properties of ribes magellanicum "parrilla free radical scavenging activity and phenolic content in achenes and thalamus from fragaria chiloensis ssp. chiloensis, f. vesca and f. x ananassa cv anthocyanins in berries of maqui antioxidant activity and phenolic profiles of the wild currant ribes magellanicum from chilean and argentinean patagonia detailed analyses of fresh and dried maqui (aristotelia chilensis (mol.) stuntz) berries and juice fruit mineral contents of six wild species of the north andean patagonia delphinidin-rich maqui berry extract (delphinol®) lowers fasting and postprandial glycemia and insulinemia in prediabetic individuals during oral glucose tolerance tests patagonian ribes and rubus: native fruits with nutraceutical potencial patagonian berries as native food and medicine patagonian berries: an ethnobotanical approach to exploration of their nutraceutical potential non-timber forest product use in two human populations from nw patagonia: a quantitative approach el uso de plantas silvestres comestibles en una población suburbana del noroeste de la patagonia traditional knowledge of edible wild native and exotic plants in the context of cultural change in human populations of arid patagonai ethnobiology and research on global environmental change: what distinctive contribution can we make? ethnobiol conserv horticultural practice and germplasm conservation: a case study in a rural population of the patagonian steppe the concept of hybridization and its contribution to urban ethnobiology frutas: frescas, secas y preservadas ecología e historia natural de la patagonia andina: un cuarto de siglo de investigación en biogeografía, ecología y conservación. buenos aires: fundación de historia natural félix de azara ecología e historia natural de la patagonia andina: un cuarto de siglo de investigación en biogeografía, ecología y conservación. buenos aires: fundación de historia natural félix de azara recife: nupeea plantas del nahuel huapi. catálogo de la flora vascular del parque nacional nahuel huapi, argentina. bariloche: universidad nacional del comahue-red latinoamericana de botánica buenos aires: colecciones científicas del inta las ocupaciones tempranas de la transición pleistoceno-holoceno al holoceno medio en el área boscosa-lacustre . cuartas jornadas de historia de la patagonia. bariloche cultural transmission of ethnobotanical knowledge in a rural community of northwestern patagonia plantas silvestres con órganos subterráneos comestibles: transmisión cultural sobre recursos subutilizados en la patagonia (argentina) ethnoecology of oxalis adenophylla gillies ex hook chemosensory perception and medicinal plants for digestive ailments in a mapuche community in nw patagonia best practice in research: consensus statement on ethnopharmacological field studies -consefs métodos e técnicas na pesquisa etnobiológica y etnoecológic selected guidelines for ethnobotanical reserach: a field manual methods and techniques in ethnobiology and ethnoecology practical nonparametric statics cultural transmission of traditional knowledge in two populations of north-western patagonia ethnobotanical review of the mapuche medicinal flora: use patterns on a regional scale ethnobotanical and phytomedical knowledge in the north-western ligurian alps cultural importance indices: a comparative analysis based on the useful wild plants of southern cantabria (northern spain) the useful plants of tambopata, peru: i. statistical hypotheses tests with a new quantitative technique cultural importance indices: a comparative analysis based on the useful wild plants of southern cantabria (northern spain) evaluating two quantitative ethnobotanical techniques buenos aires: colecciones científicas del inta dicotiledóneas, dialipétalas (salicaceae a cruciferae) dicotiledóneas, dialipétalas (oxalidaceae a cornaceae) revision of berberis (berberidaceae) in chile and adjacent southern argentina. ann of the missouri bot gard plantas indígenas de la argentina con frutos o semillas comestibles breve panorama de las plantas utilizadas por los indios de patagonia y tierra del fuego the chilean strawberry (fragaria chiloensis): over years of domestication underutilized fruits and vegetables as potential novel pigment sources. handbook on natural pigments in food and beverages: industrial applications for improving food color polyphenols and antioxidant activity of calafate (berberis microphylla) fruits and other native berries from southern chile characterization and clonal selection of berberis microphylla g. forst in the chilean patagonia region for natural colorant purposes aristotelia chilensis): morpho-phenological characterization to design highyielding cultivation techniques nutraceuticals and functional foods: whole versus processed foods medicinal and local food plants in the south of alava forest as stronghold of local ecological practice: currently used wild food plants in polesia, northern ukraine effect of processing conditions on phenolic compounds and antioxidant properties of date syru antioxidant effect and characterization of south american prosopis pods syrup the effect of processing on the composition of sea buckthorn juice la chicha en el chile precolombino. first. santiago de chile: mare nostrum wine, beer, alcohol and polyphenols on cardiovascular disease and cancer an ethnobotanical perspective on traditional fermented plant foods and beverages in eastern europe food or medicine? the food-medicine interface in households in sylhet etnoconservacionismo y prácticas locales en patagonia: avances y perspectivas with bitter herbs they shall eat it: chemical ecology and the origins of human diet and medicine the chemical ecology of human ingestive behaviors new insights into the mechanisms of polyphenols beyond antioxidant properties; lessons from the green tea polyphenol, epigallocatechin -gallate polyphenols and human health: prevention of disease and mechanisms of action edible berries: bioactive components and their effect on human health. nutrition berries: improving human health and healthy aging, and promoting quality life-a review efficacy and safety of ephedra and ephedrine for weight loss and athletic performance: a meta-analysis advisories & safety information adverse cardiovascular and central nervous system events associated with dietary supplements containing ephedra alkaloids a polyphenol-rich calafate (berberis microphylla) extract rescues glucose tolerance in mice fed with cafeteria diet berberis species and wild strawberry from the argentinean patagonia a review of the potential of chilean native berries in the treatment of obesity and its related features complejos vegetales comestibles y medicinales en la patagonia argentina : sus componentes y posibles procesos asociados. bol latinoam y del caribe plantas med y aromat edible and healing plants in the ethnobotany of native inhabitants of the amazon and atlantic forest areas of brazil plants used as food and medicine by polish migrants in misionesl traditional and current food use of wild plants listed in the russian pharmacopoeia when foods become remedies in ancient greece: the curious case of garlic and other substances gathering of wild plant foods with medicinal use in a mapuche community of northwest patagonia bioactive properties of sambucus nigra l. as a functional ingredient for food and pharmaceutical industry black elderberry (sambucus nigra) supplementation effectively treats upper respiratory symptoms: a metaanalysis of randomized, controlled clinical trials nutraceuticals have potential for boosting the type interferon response to rna viruses including influenza and coronavirus. prog cardiovasc dis a systematic review on the rosa canina effect and efficacy profile rosa rubiginosa and fraxinus oxycarpa herbal teas: characterization of phytochemical profiles by liquid chromatography-mass spectrometry, and evaluation of the antioxidant activity in vitro antioxidant properties and anthocyanin compositions of elderberry extracts tea and herbal infusions: their antioxidant activity and phenolic profile evaluation of antioxidant and antimutagenic activity of herbal teas from native plants used in traditional medicine in argentina we are deeply grateful to the families from cuyín manzano for their willingness to participate, and for sharing their knowledge. special thanks to mr. antonio bergant, who was not only a great person, but also the best field assistant. the authors acknowledge with appreciation the constructive comments made by the referees andrea pieroni and timothy johns on the earlier version of this paper. we would also like to thank the cultiva (cyted) network. authors' contributions m.f.c.: conducted the review process, the fieldwork, analyzed the data and contributed to the manuscript. a.l.: conceived and designed the research, conducted the review process, analyzed the data and contributed to the manuscript. the author(s) read and approved the final manuscript. this investigation was supported by the consejo nacional de investigaciones científicas y técnicas (conicet) of argentina (pip ), and by centro de investigación y extensión forestal andino patagónico (ciefap). the datasets used and/or analyzed during the current study are available from the corresponding author on request. this study was conducted according to the ethics guidelines of ise code of ethics (http://ethnobiology.net/code-of-ethics/) and nagoya protocol (argentinian national law n.° ). all informants orally confirmed free and informed consent prior to data collection. no other specific additional procedure is mandatory for this kind of study in our country. not applicable. the authors declare that they have no competing interests.received: august accepted: may key: cord- -zkuj oc authors: mason, h. s.; herbst-kralovetz, m. m. title: plant-derived antigens as mucosal vaccines date: - - journal: mucosal vaccines doi: . / _ _ sha: doc_id: cord_uid: zkuj oc during the last two decades, researchers have developed robust systems for recombinant subunit vaccine production in plants. stably and transiently transformed plants have particular advantages that enable immunization of humans and animals via mucosal delivery. the initial goal to immunize orally by ingestion of plant-derived antigens has proven difficult to attain, although many studies have demonstrated antibody production in both humans and animals, and in a few cases, protection against pathogen challenge. substantial hurdles for this strategy are low-antigen content in crudely processed plant material and limited antigen stability in the gut. an alternative is intranasal delivery of purified plant-derived antigens expressed with robust viral vectors, especially virus-like particles. the use of pattern recognition receptor agonists as adjuvants for mucosal delivery of plant-derived antigens can substantially enhance serum and mucosal antibody responses. in this chapter, we briefly review the methods for recombinant protein expression in plants, and describe progress with human and animal vaccines that use mucosal delivery routes. we do not attempt to compile a comprehensive list, but focus on studies that progressed to clinical trials or those that showed strong indications of efficacy in animals. finally, we discuss some regulatory concerns regarding plant-based vaccines. advances in plant biotechnology in the last two decades have facilitated production of recombinant proteins in plants, including subunit vaccine antigens. several good review articles (rybicki ; streatfield ; thanavala et al. ; yusibov and rabindran ) have summarized much of this work and have focused attention on the utility of plants for subunit vaccine expression. during the twenty years since the seminal work that demonstrated the potential of plantderived vaccines (curtiss and cardineau ; mason et al. ) , a large body of literature has demonstrated expression of antigens in many plants, including edible plants like corn, potato, and tomato, as well as tobacco and its relatives. and yet, commercial development of plant-derived vaccine products has been very slow, for a variety of reasons, as discussed previously (rybicki ). one of the biggest obstacles is the reluctance of the pharmaceutical industry to invest in a new technology when the existing seems adequate. however, a steady accumulation of promising data has shown that plant-based expression has great potential, especially if a few hurdles can be overcome. the original and oft-touted goal for plant-derived vaccines was to provide cheap and conveniently delivered oral recombinant subunit vaccines to underdeveloped countries where poor economic conditions limit the application of modern medical science. the hypothesis was that ingestion of edible plant material, either raw or minimally processed, could induce protective immune responses via activation of immune effectors in gut-associated lymphoid tissue (galt) (streatfield ; thanavala et al. ) . oral delivery of bacterial antigens has generated protective immunity against enteric pathogens. however, there is scarce evidence that protection against respiratory or urogenital tract infectious disease can be achieved using the oral vaccine delivery route. digestive acids and proteases that degrade proteins for nutrient absorption are a substantial problem for oral vaccine development. antigens must be relatively resistant to gut proteases, and typically very large antigen doses be administered. encapsulation might better protect immunogens and their delivery into galt (streatfield ) . however, little work in this area has been done with plantderived antigens. another problem for edible vaccines is that accumulation of antigens in transgenic plant tissues is usually rather low (b % of total soluble protein, tsp), creating a requirement for processing of plant material to concentrate the antigen. moreover, the inherent plant-to-plant variability in antigen content requires processing of plant material to produce a well-characterized batch of antigen that can be administered in controlled dosages. thus, plant-derived oral vaccine immunogens will require substantial purification and concentration, and probable delivery in capsules that protect them from proteolytic degradation in the small intestine. recently, plant-based recombinant protein production has been revolutionized by transient expression of antigens in plants using viral vectors (gleba et al. ; yusibov et al. ) . the plant host is usually the tobacco relative nicotiana benthamiana (goodin et al. ) , which is remarkably permissive to many plant viruses. expression levels obtained with viral vectors in n. benthamiana leaves are variable depending on the protein, but are generally ten-fold greater than in stable transformants of the nuclear genome, and frequently in the range of - mg/kg of leaf mass. thus, the prospect of plant-based expression and purification of vaccine antigens, especially virus-like particles (vlp) (huang et al. ), for mucosal delivery is substantially brighter than that obtained using stably integrated transgenes. moreover, the high yields of recombinant antigens that can be obtained using viral vectors may facilitate intranasal delivery, which requires smaller volumes and hence, more concentrated vaccine solutions than the oral delivery route. an ideal mucosal vaccine would induce both antibody-and cell-mediated protection, not only at the relevant mucosal site, but also throughout the body. the most convenient means to achieve mucosal immunity in global health programs is oral delivery. oral vaccination eliminates the possibility of transmission of other infectious diseases by contaminated needles, as well as elimination of pain associated with injections and the need for trained personnel to deliver the vaccines (holmgren and czerkinsky ; lavelle ). however, nasal vaccines are not hampered by the physical and chemical barriers of the gut. nasal vaccination has demonstrated particular potential with regard to induction of broadly disseminated immunity (neutra and kozlowski ; staats et al. ) . in humans, monkeys, and mice, nasal immunization induced antigen-specific mucosal iga responses in salivary glands, upper and lower respiratory tracts, small and large intestines, and most notably male and female reproductive tracts (harandi et al. ; imaoka et al. ; kozlowski et al. ; rudin et al. ; staats et al. ). in addition, the nasal route of immunization can induce cytotoxic t lymphocytes (ctl) in distant mucosal tissues including the female reproductive tract (gallichan and rosenthal ) . in both humans and mice, nasal immunization has produced greater systemic antibody responses than other mucosal immunization routes (kozlowski et al. (kozlowski et al. , staats et al. ) . kunkel and butcher ( ) provided evidence from naïve human vaccine recipients that mucosal immunization can prime the immune system for both mucosal and systemic responses by inducing the expression of both mucosal and systemic homing receptors in responding lymphocytes. thus, delivery of subunit antigens or vlp via the nasal route has excellent prospects as a vaccine strategy. for further reading on vlp vaccines, readers are directed to ''recent advances in microparticle and nanoparticle delivery vehicles for mucosal vaccination'' of this volume. the strategies used for recombinant protein expression in plants are conceptually similar to those used for mammalian, yeast, or other eukaryotic hosts (rybicki ; thanavala et al. ; yusibov and rabindran ) . they include stably integrated transgenes in the nuclear or chloroplast genomes, and transient expression using vectors that are either non-replicating or that utilize plant virus replication elements to amplify the mrna for the target gene. nuclear genes behave in a mendelian fashion, and utilize the typical eukaryotic pathways of protein translation, processing, and subcellular localization. organ-and development stage-specific promoters can be utilized, such that foreign proteins can be directed to accumulate in seeds (nochi et al. ; streatfield et al. ; wu et al. ). expression of antigens in seeds has a particular advantage in regard to protein stability due to drying of the storage tissue during seed development. thus, seeds can be stored at ambient temperatures for months to years with little loss of protein activity. although site-specific recombination of plant nuclear genomes has been studied intensively (hanin and paszkowski ) , it is currently not routine and usually rather inefficient. in most cases, the recombinant construct is delivered as dna using agrobacterium tumefaciens, a plant pathogen that has the ability to transfer genes into the plant cell nucleus (gelvin ) . the foreign dna may be integrated stably into the host nuclear chromosomal dna by non-homologous recombination at random sites, although some work suggests that transcriptionally active sites may be preferentially targeted. the transferred dna may also reside in the nucleus without integration for several days, and can thus act as a template for transcription to effect transient expression. unless the foreign dna is constructed as a viral replicon, it will remain in low-copy numbers, but can be used to evaluate expression of foreign antigens (huang and mason ) . agrobacterium-mediated dna transfer can also be used to deliver viral replicons. dna viruses such as geminiviruses have been used to develop gene amplification systems (huang et al. ). genomes of rna viruses such as tobacco mosaic virus (tmv) can be constructed as cdna fused to a plant promoter, which is transcribed in the nucleus to produce viral rna that then moves to the cytoplasm to establish replication (gleba et al. ) . a method called ''magnifection'' was developed to allow whole-plant inoculation of n. benthamiana by vacuum infiltration with agrobacterium lines containing recombinant tmv replicons that lack the coat protein, resulting in foreign gene expression in all leaves (gleba et al. ) . recombinant viral genomes can also be delivered directly as rna after in vitro transcription from a plasmid by simply abrading the leaf surface and applying a solution of rna (gleba et al. ; yusibov et al. ) . in this case, the replicon needs to contain viral elements that mediate longdistance transport in the plant vasculature so that expression is not restricted only to the site of inoculation. chloroplast transformation is routine in only a few laboratories, but can yield very high-expression levels of some antigens, due to the high gene copy numbers from many chloroplasts per cell and genome molecules per chloroplast (arlen et al. ; singh et al. ). the dna construct is delivered by micro projectile bombardment (gene gun), and site-specific recombination allows targeted gene insertion. one great advantage of chloroplast transgenes is that they are not usually subject to gene silencing mediated by rna interference, which is a common problem with nuclear transgenes. another advantage is that the chloroplast genome is exclusively maternally inherited, making gene containment much easier because pollen grains are devoid of plastids. however, the chloroplast genetic system is a prokaryotic one; thus, some protein processing events, notably glycosylation, will not occur. noroviruses are non-enveloped positive-sense rna members of caliciviridae, and are enteric pathogens that cause gastroenteritis. the capsid protein of norwalk virus (nvcp) can be expressed in insect cells (jiang et al. ) or plants (mason et al. ) , and self-assembles into vlp (rnv) that are orally immunogenic. a clinical study evaluated the antibody responses after ingestion of or doses of g raw potato tubers expressing rnv (tacket et al. ) . although of subjects who ingested the rnv expressing potato developed measurable increases in circulating antibody-secreting cells, the overall response was rather weak. the variable rnv content of the tubers ( - lg per dose) and relatively poor vlp assembly in the potato (effective rnv dose of b lg per dose) were likely limiting factors. however, a detrimental effect of the potato vehicle, perhaps due to poor release of vlp in the gut lumen, cannot be discounted. a clinical trial using orally delivered purified insect cell-derived rnv (i-rnv) produced stronger antibody responses at lg per dose than were obtained with potato (tacket et al. ) , suggesting that purified rnv is a more potent oral immunogen. later, rnv was expressed at higher levels in transgenic tomato fruit using a plant codon-optimized nvcp gene (zhang et al. ) . oral immunogenicity in mice was excellent: doses of . g freeze-dried tomato fruit containing lg nvcp ( lg rnv) induced anti-rnv serum igg and fecal iga in c % of mice, and . g doses generated systemic and mucosal antibodies in all mice. it was also shown that air-dried tomato fruit was at least as active in mice as freezedried tomato, indicating that sophisticated drying technology is not required for rnv in tomato. interestingly, rnv delivered in freeze-dried potato tuber was less orally immunogenic in mice, due to oxidation by phenolic compounds and polyphenol oxidase in potato (zhang et al. ) . tomato is much better in this respect, having low-phenolic content, as well as a high level of ascorbic acid that can act as antioxidant. thus, dried rnv tomato fruit would be worthy of study in humans. in recent years, our group has been experimenting with viral vectors for transient expression of rnv in plants because of the potential for high-level expression and the rapidity of protein production (gleba et al. ; marillonnet et al. ). we used the magnifection system to express nvcp in the tobacco relative n. benthamiana at levels of mg/kg leaf tissue (santi et al. ). further, we developed a vlp purification process that did not require ultracentrifugation but instead utilized ph . precipitation of the major leaf proteins, followed by filtration through a kda membrane to remove unassembled nvcp subunits. in the absence of adjuvant, the tobacco-derived rnv (t-rnv) was orally immunogenic in mice at a lg dose, generating systemic and mucosal anti-rnv antibodies (santi et al. ) . inclusion of cholera toxin adjuvant with t-rnv substantially increased anti-rnv responses. in velasquez et al. studies with mice, we have found that nasal co-delivery of t-rnv and a toll-like receptor (tlr) agonist can produce robust systemic and mucosal antigen-specific antibody responses. we undertook these experiments to evaluate the potential of these vlp to induce distal mucosal iga responses with or without adjuvant. in unpublished studies we examined the tlr agonist, cpg-containing oligodeoxynucleotides (cpg-odn) as adjuvant because it is known to trigger an immunostimulatory cascade that culminates in the maturation, differentiation, and proliferation of multiple cell types, including b cells, and cpg-odn had been used previously in mice by the nasal route (abe et al. ; balmelli et al. ; mccluskie et al. ) . we nasally immunized conscious female balb/c mice with lg purified t-rnv alone or t-rnv with lg type b cpg-odn. nasal immunization of conscious mice with vlp has been shown to target antigen to the nasopharyngeal-associated lymphoid tissue (nalt) but not deeper respiratory tract tissues (balt); whereas nasal immunization of sedated mice is more analogous to intratracheal administration (balmelli et al. ) . we also immunized mice orally with lg t-rnv alone. the rnv-specific igg and igg a antibodies induced in serum were analyzed to determine if immunization had produced a th , th , or mixed th /th immune response. overall, rnv-specific serum igg levels were higher in nasally versus orally immunized mice, with greatest levels achieved by nasal co-delivery of cpg-odn with rnv ( fig. a-c) . within the nasal groups, both anti-rnv igg and igg a in serum were significantly increased in the cpg-odn immunization group relative to controls. this suggests that cpg-odn drives a mixed th /th response with respect to the production of anti-rnv antibodies. in these immunized animals, igg levels had increased by days following a single immunization and continued to rise following booster immunizations at days (intranasal and oral) and (oral only). antibody levels began to plateau by day (fig. a-c) . the orally delivered t-rnv at lg was not very immunogenic; however, nasal immunization with lg t-rnv produced robust immune responses. fecal iga was produced only in the cpg-odn immunization groups (fig. d ). in addition, the levels of rnv-specific iga and igg in vaginal lavages were significantly elevated (p\ . ) in nasal groups given rnv with cpg-odn ( fig. e and f) . to further characterize distal induction of antigen-specific iga, we significantly higher levels of rnv-specific antibody production were observed throughout the time course in mice immunized in with t-rnv and cpg odn compared to t-rnv alone or vehicle alone-immunized mice. prism software from graphpad was used to conduct one-way anova with bonferroni post test (p\ . was considered significant) evaluated iga production at additional mucosal sites, including salivary samples, gastrointestinal lavages, nasal washes, and bronchoalveolar lavages. at each of these distal mucosal sites, iga production was significantly increased relative to controls in the rnv/cpg-odn-immunized mice (fig. a-d) . these results demonstrate that t-rnv co-delivered with tlr agonist by the nasal route can induce robust antigen-specific iga production at distal mucosal sites that would be important for mucosal protection against not only enteric organisms, but also respiratory and sexually-transmitted pathogens. overall, the data demonstrate that immunization with t-rnv by the nasal route is more effective than the oral route for inducing robust rnv-specific systemic igg and mucosal iga antibody responses in upper and lower respiratory (nasal and bronchoalveolar), gastrointestinal (salivary, intestine and fecal), and female reproductive (vaginal) tracts. we are now planning a phase i clinical trial to evaluate systemic and mucosal antibody responses to t-rnv after nasal delivery with a selected adjuvant. fig. induction of antigen-specific iga at distal mucosal sites following intranasal, but not oral administration of tobacco-derived norwalk virus-like particles (t-rnv). female balb/c mice were immunized as described in the fig. . salivary samples (a), gastrointestinal lavages (b), bronchoalvelolar lavages (c), and nasal lavages (d) were collected from mice on day and analyzed for rnv-specific iga production by elisa (presented as gmt). distal mucosal sites contained significantly higher (p\ . ) levels of antigen-specific iga in cpg odn ? t-rnv in vaccinated groups relative to t-rnv alone delivered via in or oral routes chronic infection with hepatitis b virus (hbv) can result in liver cancer, accounting for up to one million deaths per year among approximately million chronic carriers worldwide. hbv is transmitted mainly by blood or through sexual contact, but vertical transmission occurs frequently when hbv-infected mothers give birth. in this regard, a mucosal hbv vaccine may be an improvement over the current intramuscular hepatitis b surface antigen (hbsag) vaccine made in recombinant yeast (mcaleer et al. ) . moreover, an orally delivered plantderived vaccine could improve patient compliance for multiple doses due to the convenience, and it would obviate the need for needles. however, we must take care to avoid interference with current efforts to expand hbv vaccination in the developing world, and move forward with a plant-derived vaccine only if distinct advantages are demonstrated. the perception among plant vaccinologists that a plant-derived hbv vaccine is important is apparent from the large body of work demonstrating expression of hbsag in a variety of plants including potato, lettuce, tomato, tomatillo, corn, and banana (streatfield ). the first publication on plant-based vaccine antigen expression was with hbsag in transgenic tobacco (mason et al. ) . after optimization of hbsag expression in transgenic potato tubers (richter et al. ) , its oral immunogenicity in mice (albeit dependent upon inclusion of ct; kong et al. ) , encouraged a human trial. due to fda concerns that immunologically naïve subjects might experience antigenic tolerance after ingestion of hbsag, the clinical study used hbsag seropositive volunteers who had been previously vaccinated with the yeast recombinant hbsag (thanavala et al. ) . titers of anti-hbsag in serum were increased in / and / volunteers who consumed or doses of transgenic potatoes, respectively, with each dose containing roughly lg hbsag. this finding suggests that oral boosting with plant-derived hbsag could be incorporated as a viable component of immunization programs. oral delivery of hbsag can activate systemic memory cells generated by intramuscular immunization, however, the possibility of priming immunization by hbsag potato ingestion cannot be excluded. an earlier study had shown that presumably naïve human subjects who ingested transgenic lettuce expressing low levels of hbsag underwent seroconversion (kapusta et al. ) . it is interesting to note that neither of these clinical trials employed an adjuvant, which suggests that oral delivery can be substantially optimized in further work. the fear of oral tolerance is one that must be taken seriously; however, very little work has addressed this issue with plant-derived vaccines. one recent paper examined regulatory t cells (tregs) in humanized mice orally immunized with transgenic hbsag tobacco (kostrzak et al. ). t regulatory cells mediate immunological unresponsiveness to self-antigens and are implicated in oral tolerance (sakaguchi et al. ) . the humanized mice were immunized by gavage with different amounts of powdered tobacco leaves, and foxp ? cd ?cd ? tregs were measured in spleen and peripheral lymph nodes (kostrzak et al. ). serum igg and fecal iga were inversely correlated with antigen dose, while the frequency of tregs positively correlated with tobacco dose, leading the authors to conclude that oral tolerance occurred at higher doses. while the data are not strongly conclusive due to the relatively poor antibody response rate among groups, they do suggest that, in the absence of adjuvant, oral plant vaccines may induce tolerance, and that higher-vaccine dosages are not necessarily better. nonetheless, much more work is needed with different antigens, plant vehicles, and dosages in order to make firm conclusions regarding the potential of plant-derived antigens to induce oral tolerance. rabies is a zoonotic disease that is invariably fatal after development of clinical symptoms (nagarajan et al. ) . because most human deaths from rabies occur in developing countries, new technologies like plant-derived recombinant antibodies and vaccines could make a significant impact. yusibov and colleagues (yusibov et al. ) have shown that oral delivery of rabies virus epitopes expressed in spinach is immunogenic in humans. a chimeric peptide containing determinants from rabies virus glycoprotein (g protein) and nucleoprotein (n protein) was fused to the n-terminus of the alfalfa mosaic virus coat protein, and expressed with viral vectors in tobacco and spinach. virus particles were expressed at . mg per g leaf tissue, of which % was recovered in purified form. in mice, intraperitoneal doses of recombinant virus particles (with lg of rabies peptide) generated neutralizing antibodies and protected against lethal rabies virus challenge. thus, a human study was performed using volunteers who had previously received the conventional rabies vaccine (as with the hbv potato vaccine, there were concerns about potential oral tolerance). five subjects were fed g raw recombinant spinach leaves ( . mg of recombinant virus with lg chimeric rabies peptide per dose) a total of times at biweekly intervals, while another five subjects ingested control vector-only spinach. three of the antigen-treated volunteers, but none of the individuals in the control group, showed significant boosting of anti-rabies virus antibody in serum. based on these results, naïve subjects were tested. nine volunteers consumed doses of raw rabies-recombinant spinach leaves ( g per dose) and were fed control spinach. all subjects then received a single dose of commercial rabies vaccine days after the last oral dose. six of the subjects who ate the rabies antigen-containing spinach showed significant increases in serum igg or iga specific for rabies virus (yusibov et al. ) . these results are similar to those obtained with hbsag-expressing potatoes (thanavala et al. ) , and although a greater response rate would be desirable, the studies demonstrate the potential for successful oral delivery of plant-derived vaccines in humans. little further work on rabies vaccines has been reported, but in one study (loza-rubio et al. ) the rabies virus g protein was expressed in transgenic maize seeds at % tsp. mice were fed a single dose of transgenic seeds containing lg of g protein, and challenged days later with a lethal dose of rabies virus ( ld ). the vaccine induced virus-neutralizing antibodies and impressively protected all mice against challenge. these results suggest that maize seeds may be an ideal vehicle for expression and delivery of rabies g protein. measles virus (mv) causes substantial morbidity and mortality worldwide, but developing countries carry the heaviest burden due to the instability of the attenuated virus vaccine and the requirement for inoculation needles (webster et al. ) . moreover, evidence indicates that vaccinated individuals generate less robust and long-lasting antibody titers than individuals who recovered from a natural mv infection (muller et al. ) . thus, boosting with a convenient mucosally delivered mv vaccine would be a great boon to health, especially in resource-poor countries. two groups of researchers have approached this problem using plant-derived vaccines. webster and colleagues expressed a soluble form of the mv hemagglutinin (mv-h) surface protein in stable transgenic tobacco and lettuce (webster et al. (webster et al. , . the mv-h was stable in freeze-dried lettuce leaf stored at room temperature for months, and showed only - % loss after months (webster et al. ) . moreover, the dried mv-h was orally immunogenic in mice after resuspension and delivery by gavage with a crude saponin adjuvant. since no reference standard was available, the amount of mv-h antigen in the lettuce could not be accurately determined. best results were obtained when mice were systemically primed with a mv-h dna vaccine, then orally boosted five times with lettuce expressing mv-h. the mean titers of mv-specific serum igg were boosted tenfold by the oral doses, while control lettuce had no effect. the virus-neutralizing titers were roughly . -fold higher in mv-h lettuce boosted mice than in control lettuce mice. these data indicate the potential for oral boosting with plant-derived mv-h for humans that have been intramuscularly vaccinated but experience waning of anti-mv antibody titer. another group has used a mv polyepitope strategy, expressing four copies of the loop-forming hemagglutinin protective b cell epitope fused to four repeats of the human promiscuous t cell epitope of tetanus toxoid in transgenic carrots (bouche et al. ) . although the vaccine was delivered by the intraperitoneal route and not mucosally, the antigen was immunogenic and generated serum antibodies that neutralized ten different mv strains. this approach might be developed for oral or nasal delivery, with appropriate mucosal adjuvants, in order to produce a vaccine that is more convenient for populations in developing countries. no plant-derived mv vaccines have been tested in humans yet, but the data above show very promising indications that oral boosting could provide substantial benefits. the first plant-derived vaccine immunogen to be tested in humans was the b subunit protein of the enterotoxigenic escherichia coli heat-labile toxin (lt) expressed in transgenic potatoes tacket et al. ). the non-toxic ltb protein mediates binding of the lt holotoxin to epithelial cells, triggering intracellular delivery of the toxic a subunit. the rationale for a mucosal vaccine rationale presumes that oral delivery of ltb will induce local secretion of anti-ltb antibodies in the gut, which will prevent the binding of the holotoxin to enterocytes. data from mouse studies supports this hypothesis, and mice that ingested ltbcontaining potato tubers were partially protected against lt challenge, as indicated by reduced fluid secretion in the small intestine ). the clinical trial showed that ingestion of ltb in potatoes stimulated anti-lt serum igg in of subjects, serum iga in six subjects, and fecal iga in of subjects; and further showed that the antibodies could neutralize lt (tacket et al. ). another clinical trial using transgenic maize germ (embryo of seeds) expressing ltb yielded similar results (tacket et al. ) . the fact that ltb is one of the most orally immunogenic proteins known does not diminish the impact of these findings, which provide clear evidence that the ingestion of crude plant material, in the absence of adjuvant, could potentially stimulate protective antibody responses to enteric pathogens in humans. recently, transgenic rice seeds expressing the structurally and functionally similar b subunit of cholera toxin were developed (nochi et al. ). cholera toxin b subunit (ctb) was expressed in rice seeds using the glub- seed storage protein promoter at up to lg per seed ( . % tsp). it was localized in protein storage bodies, which allowed high stability (immunogenicity after . years at room temperature), and resistance to pepsin treatment. thus, rice and corn appear to be good vehicles for delivery of antigens in the gut. a plant-derived vaccine against enterohemorrhagic e. coli (o :h ) has also been created by expressing part of the bacterial adhesin intimin in cultured transgenic tobacco cells (judge et al. ). the c-terminal domain of intimin (int ) mediates binding to the translocated intimin receptor, as well as hostspecific surface molecules, for bacterial colonization in the gut. oral immunization of mice by ingestion of int expressing tobacco cells after intraperitoneal priming with plant-derived int stimulated specific fecal iga in of mice, and reduced bacterial shedding after challenge with o :h (judge et al. ). however, low expression of int in the transgenic tobacco cells limited the amount of antigen that could be produced. using viral vectors and transient expression, our research team has substantially enhanced int expression in leaves of n. benthamiana (e. topal & h. mason, manuscript in preparation) . further testing in more appropriate hosts, such as cattle, will be needed to assess the efficacy of this and other intimin expressing plant-based vaccines for preventing infection by e. coli :h . plant-derived vaccines for animals are likely to be realized sooner than those for humans, due to more relaxed regulations (rybicki ). in fact, the only licensed plant-derived vaccine is for prevention of newcastle disease virus (ndv) in chickens, comprising recombinant hemagglutinin-neuraminidase protein expressed in cultured transgenic tobacco cells and prepared as an injectable vaccine for chickens (dow agrosciences, www.dowagro.com/animalhealth/resources/faq. htm#faq ). as discussed below, several other plant-derived mucosally delivered vaccines against animal diseases have shown potential. foot-and-mouth disease virus (fmdv) is a very important veterinary pathogen because it is highly infectious in animals and has economically devastating effects on meat and milk production. the fmdv vp capsid protein contains virusneutralizing determinants (wigdorovitz et al. ) . several studies have shown expression of vp in various plant hosts (yusibov and rabindran ) . transgenic vp alfalfa leaves were immunogenic in mice by intraperitoneal or oral delivery (wigdorovitz et al. ) . for oral delivery, mice were fed . g of fresh leaves three times per week for weeks. ten days later serum was obtained. all mice in two separate experiments developed serum antibodies against fmdv particles, at titers ranging up to . moreover, of mice were protected against intraperitoneal challenge with fmdv, as measured by the absence of viremia h later, while none of the control mice were protected. a different strategy fused a vp epitope (amino acids - ) to b-glucuronidase (gus), a stable enzyme that is readily measured using a fluorometric assay (dus santos et al. ) . expression in transgenic alfalfa plants ranged from . to . % of tsp, and crude extracts injected by the intraperitoneal route elicited completely protective immune responses in mice. another fusion protein study used vp amino acids - replacing the n-terminal residues of bamboo mosaic virus coat protein, and the recombinant virus used to infect leaves of chenopdium quinoa (a spinach relative) (yang et al. ). infected leaves produced chimeric virions expressing vp epitopes in its coat protein. although the yield of recombinant protein was not quantified, the data suggest accumulation to * - % tsp (yang et al. ) . intramuscular immunization of pigs with mg of vp virions resulted in the induction of anti-fmdv neutralizing antibodies, vp -specific ifn-c-producing cells, and complete protection against fmdv challenge. the high antigen dose used in these studies indicates robust expression of this viral coat protein fusion. in view of the promising results obtained through oral delivery of plant-derived vp (wigdorovitz et al. ) , it is perhaps surprising that these later fusion protein strategies that yielded improved expression were not tested by the oral route. further investigation is needed in order to evaluate oral delivery with adjuvants, and in animal species that have more economic relevance. transmissible gastroenteritis virus (tgev) is a coronavirus that significantly affects swine production (hammond and nemchinov ). the disease is highly infectious, and the severe diarrhea and vomiting produce high mortality in young piglets. the envelope spike (s) protein is a target for neutralizing antibody, and thus has been expressed in plant systems for oral delivery (howard ; lamphear et al. ) . transgenic corn seed expressing tgev-s protein fed to piglets stimulated antibody production and partially protected animals from viral challenge (howard ) . the truncated soluble s protein product was targeted to the cell wall instead of protein bodies, which allowed accumulation of antigen at mg/kg seed, and a dose of - mg s protein in a single feeding. further studies examined the potential to immunize gilts (young sows) and stimulate anti-s antibody secretion in the milk for protection of suckling piglets (lamphear et al. ) . oral dosing by ingestion of tgev-s corn by gilts on days - and - before farrowing (day ), after having received the modified live tgev vaccine orally at days - and - , stimulated significantly higher tgev-neutralizing activity in serum, colostrum (day ), and milk (day ) compared to a placebo group. antibody isotypes were not determined, but the authors suggest that the milk antibodies later than day two are mostly iga (lamphear et al. ) . protective efficacy was not examined, but the neutralizing titers in milk suggest that suckling piglets would have been protected. however, the milk titers dropped precipitously at day - , suggesting that continued boosting would be necessary to maintain protection. an interesting chimeric antibody against tgev was expressed in plants and tested for its ability to protect piglets after oral delivery (monger et al. ) . these investigators fused an anti-tgev single-chain antibody (scfv) to the ch domain of ige to mediate dimerization. the protein was expressed in cowpea leaves using a cowpea mosaic virus vector, achieving accumulation of * %tsp. this plant-derived antibody (dubbed ''e-small immune protein'' or esip) showed similar tgev-neutralizing activity as the parent antibody a.c or the esip expressed in mammalian cells. moreover, oral gavage in piglets reduced viral titers in lung and gut of piglets challenged with tgev, though less effectively than antibody a.c . these studies illustrate the potential for plant-derived antibodies to provide mucosal protection against enteric viral disease, and deserve further attention for application in veterinary and human medicine. infectious bursal disease virus (ibdv) is highly contagious and deadly in young chickens. it thus has a major economic impact on the poultry industry. ibdv is a member of the birnaviridae with two double-stranded rna genome segments (wu et al. ). the vp viral capsid protein is the immunodominant antigen that generates antiviral neutralizing antibodies. the currently used vaccines are either attenuated or killed viruses, but suffer problems regarding the potential for the live virus vaccine to recombine and generate variants, or poor efficacy in the case of the whole-killed vaccine. one of the more compelling plant-derived vaccine successes to date is recombinant vp delivered orally. the vp was expressed in leaves of arabidopsis thaliana at levels up to . % tsp (wu et al. a) , which is among the highest obtained for a recombinant subunit vaccine in stably transformed plants. leaves of transgenic plants were dried, powdered, and resuspended in water for oral delivery (wu et al. b ). one-week old birds received five oral doses at days intervals (* lg vp per dose), or the live intermediate vaccine bursine- , or priming with bursine- followed by boosting with oral vp . although the bursine- primed birds developed serum anti-vp antibodies earlier, the titers were similar in all three groups after weeks. furthermore, all groups exhibited similar protection ( - %) after challenge with virulent ibdv (wu et al. b) . later, vp was expressed in stable transgenic rice seeds at up to . % tsp, or lg per seed (wu et al. ) . two week old chickens were fed weekly doses of , , or g rice seed, or they received the b live attenuated nasal virus vaccine. anti-vp serum antibodies increased during the weeks following immunization, with the g oral group attaining levels similar to those in the b nasal vaccine group. among the different vp -rice oral groups, antibody levels were positively correlated with the dosage. interestingly, the g oral vp group was better protected against virulent ibdv challenge than the b vaccine group ( / vs. / , respectively). although the bursal lesion score used to calculate efficacy could be considered somewhat subjective, efficacy was correlated with oral antigen dose (wu et al. ) . we have estimated that the g rice seed contained roughly a mg dose vp protein, which is rather high. in this regard, seeds have a distinct advantage over leaves, by enabling a high dose as well as excellent protein stability on long-term storage. actinobacillus pleuropneumoniae is an agent of porcine pleuropneumonia, which is an important swine disease. although the pathogenic mechanism is not fully understood, apx toxins are associated with bacterial virulence (shin et al. ) . the apx i, ii, and iii toxins mediate virulence by inserting into host cell membranes and creating pores; thus they may be good targets for immune intervention in this disease. one group has demonstrated the protective potential of apxia and apxiia proteins as vaccine antigens after oral delivery in mice (lee et al. ; shin et al. shin et al. , . their first study used the yeast saccharomyces cerevisiae for expression of apxiia and oral delivery in mice, showing apxiiaspecific iga production in lung and intestine, as well as partial (b %) and dosedependent protection against challenge with a. pleuropneumoniae. in a second study, stable transgenic tobacco was used to express apxiia at relatively low levels (b . % tsp) (lee et al. ) . mice were gavaged with weekly doses of dried tobacco leaf ( mg or mg dry mass, which we estimate contained or ng apxiia) suspended in pbs, which evoked modest serum igg specific for apxiia in the higher dose group. challenge of the mice by intraperitoneal injection of a. pleuropneumoniae killed all mice in the control nontransgenic tobacco group within h, while % of the higher-dose apxiia tobacco group survived. by comparison, % of mice immunized subcutaneously with recombinant e. coli-expressed apxiia survived (lee et al. ) . although the ultimate fate of the mice beyond h was not reported, it appears that oral delivery of apxiia tobacco afforded some protection. a later oral immunization study using both apxia and apxiia expressed in yeast showed enhanced protective efficacy over either toxin delivered alone, suggesting that multiple toxins should be included in a swine vaccine (shin et al. ). certainly, the levels of antigen expression in plants must be increased in order to facilitate more robust protection by oral delivery. moreover, testing in swine must be performed in order to examine the potential for protection by oral delivery of the apx antigens. plant-based expression of vaccine antigen and oral delivery of crude or minimally processed plant tissues is most promising for antigens expressed in seeds, such as rice or corn. the seed vehicle provides a stable environment for accumulated proteins, thus the need for refrigeration could be obviated or minimized. nonetheless, only a few antigens have been expressed in seeds at sufficient levels to afford protection in animals upon ingestion. due to the variable nature of protein structure and biochemical characteristics, it is difficult to predict which proteins will accumulate well and be correctly folded to display protective epitopes. the generation and screening for optimal expression in stable transgenic plant lines is a time-and labor-intensive process, but in some cases may yield excellent results. regulatory agencies have a substantial concern regarding the use of plants, especially plants used as food for humans, for the production of pharmaceutical proteins and vaccines. this issue was discussed in a recent review (rybicki ). although the world health organization, united states department of agriculture, and the food and drug administration agree that applicable regulatory and good manufacturing practice requirements are in place for plant-derived vaccines, enthusiasm is dimmed by fears that noncompliance could compromise food security. thus, investment of funds for commercial development of vaccines in food crops is likely to suffer. meanwhile, much recent work has focused on the use of non-food plants (tobacco and relatives) for production of vaccine antigens using robust transient expression via plant virus replicons (gleba et al. ; huang et al. ; santi et al. ). these systems provide expression levels high enough to facilitate purification of antigens in good yield and concentrated enough to use for intranasal delivery, or to incorporate into oral delivery formulations. ongoing work to develop mucosal adjuvants will synergize with these efficient plant expression systems to provide more alternatives for vaccination and, ultimately, vaccines tailored for particular pathogens and their hosts. agonists for pattern recognition receptors (e.g. toll-like receptors) show great promise as mucosal adjuvants, and deserve further research and development. nasal vaccination with cpg oligodeoxynucleotide induces protective immunity against non-typeable haemophilus influenzae in the nasopharynx effective plague vaccination via oral delivery of plant cells expressing f -v antigens in chloroplasts nasal immunization of mice with human papillomavirus type virus-like particles elicits neutralizing antibodies in mucosal secretions induction of broadly neutralizing antibodies against measles virus mutants using a polyepitope vaccine strategy oral immunisation by transgenic plants. world intellectual property organization a novel methodology to develop a foot and mouth disease virus (fmdv) peptide-based vaccine in transgenic plants long-term immunity and protection against herpes simplex virus type in the murine female genital tract after mucosal but not systemic immunization agrobacterium-mediated plant transformation: the biology behind the ''gene-jockeying'' tool magnifection-a new platform for expressing recombinant vaccines in plants viral vectors for the expression of proteins in plants nicotiana benthamiana: its history and future as a model for plant-pathogen interactions plant production of veterinary vaccines and therapeutics plant genome modification by homologous recombination a protective role of locally administered immunostimulatory cpg oligodeoxynucleotide in a mouse model of genital herpes infection mucosal immunity and vaccines commercialization of plant-based vaccines from research and development to manufacturing conformational analysis of hepatitis b surface antigen fusions in an agrobacterium-mediated transient expression system a dna replicon system for rapid high-level production of virus-like particles in plants nasal immunization of nonhuman primates with simian immunodeficiency virus p gag and cholera toxin adjuvant induces th /th help for virus-specific immune responses in reproductive tissues expression, self-assembly, and antigenicity of the norwalk virus capsid protein plant cell-based intimin vaccine given orally to mice primed with intimin reduces time of escherichia coli o :h shedding in feces a plant-derived edible vaccine against hepatitis b virus oral immunization with hepatitis b surface antigen expressed in transgenic plants oral administration of low doses of plant-based hbsag induced antigen-specific igas and iggs in mice, without increasing levels of regulatory t cells comparison of the oral, rectal, and vaginal immunization routes for induction of antibodies in rectal and genital tract secretions of women differential induction of mucosal and systemic antibody responses in women after nasal, rectal, or vaginal immunization: influence of the menstrual cycle homeostatic chemokines and the targeting of regional immunity a corn-based delivery system for animal vaccines: an oral transmissible gastroenteritis virus vaccine boosts lactogenic immunity in swine generation of improved mucosal vaccines by induction of innate immunity induction of protective immune responses against the challenge of actinobacillus pleuropneumoniae by the oral administration of transgenic tobacco plant expressing apxiia toxin from the bacteria development of an edible rabies vaccine in maize using the vnukovo strain in planta engineering of viral rna replicons: efficient assembly by recombination of dna modules delivered by agrobacterium expression of hepatitis b surface antigen in transgenic plants expression of norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice edible vaccine protects mice against escherichia coli heat-labile enterotoxin (lt): potatoes expressing a synthetic lt-b gene human hepatitis b vaccine from recombinant yeast intranasal immunization of mice with cpg dna induces strong systemic and mucosal responses that are influenced by other mucosal adjuvants and antigen distribution an antibody derivative expressed from viral vectors passively immunizes pigs against transmissible gastroenteritis virus infection when supplied orally in crude plant extracts immunogenic measles antigens expressed in plants: role as an edible vaccine for adults human monoclonal antibody and vaccine approaches to prevent human rabies mucosal vaccines: the promise and the challenge rice-based mucosal vaccine as a global strategy for cold-chain-and needle-free vaccination production of hepatitis b surface antigen in transgenic plants for oral immunization antibody responses in the lower respiratory tract and male urogenital tract in humans after nasal and oral vaccination with cholera toxin b subunit plant-produced vaccines: promise and reality regulatory t cells and immune tolerance virus-like particles production in green plants an efficient plant viral expression system generating orally immunogenic norwalk virus-like particles induction of antigen-specific immune responses by oral vaccination with saccharomyces cerevisiae expressing actinobacillus pleuropneumoniae apxiia enhancement of protective immune responses by oral vaccination with saccharomyces cerevisiae expressing recombinant actinobacillus pleuropneumoniae apxia or apxiia in mice chloroplast-derived vaccine antigens and biopharmaceuticals: protocols for expression, purification, or oral delivery and functional evaluation intranasal immunization is superior to vaginal, gastric, or rectal immunization for the induction of systemic and mucosal anti-hiv antibody responses oral hepatitis b vaccine candidates produced and delivered in plant material mucosal immunization using recombinant plant-based oral vaccines corn as a production system for human and animal vaccines immunogenicity in humans of a recombinant bacterial antigen delivered in a transgenic potato human immune responses to a novel norwalk virus vaccine delivered in transgenic potatoes humoral, mucosal, and cellular immune responses to oral norwalk virus-like particles in volunteers immunogenicity of recombinant lt-b delivered orally to humans in transgenic corn immunogenicity in humans of an edible vaccine for hepatitis b plant-derived vaccines: a look back at the highlights and a view to the challenges on the road ahead an intranasally delivered toll-like receptor agonist elicits robust systemic and mucosal responses to norwalk virus-like particles the development of a plant-based vaccine for measles measles virus hemagglutinin protein expressed in transgenic lettuce induces neutralising antibodies in mice following mucosal vaccination induction of a protective antibody response to foot and mouth disease virus in mice following oral or parenteral immunization with alfalfa transgenic plants expressing the viral structural protein vp expression of immunogenic vp protein of infectious bursal disease virus in arabidopsis thaliana immunization of chickens with vp protein of infectious bursal disease virus expressed in arabidopsis thaliana oral immunization with transgenic rice seeds expressing vp protein of infectious bursal disease virus induces protective immune responses in chickens induction of protective immunity in swine by recombinant bamboo mosaic virus expressing foot-and-mouth disease virus epitopes recent progress in the development of plant derived vaccines expression in plants and immunogenicity of plant virus-based experimental rabies vaccine the potential of plant virus vectors for vaccine production tomato is a highly effective vehicle for expression and oral immunization with norwalk virus capsid protein key: cord- -imwjoecx authors: lotter-stark, hester c.t.; rybicki, edward p.; chikwamba, rachel k. title: plant made anti-hiv microbicides—a field of opportunity date: - - journal: biotechnology advances doi: . /j.biotechadv. . . sha: doc_id: cord_uid: imwjoecx abstract hiv remains a significant global burden and without an effective vaccine, it is crucial to develop microbicides to halt the initial transmission of the virus. several microbicides have been researched with various levels of success. amongst these, the broadly neutralising antibodies and peptide lectins are promising in that they can immediately act on the virus and have proven efficacious in in vitro and in vivo protection studies. for the purpose of development and access by the relevant population groups, it is crucial that these microbicides be produced at low cost. for the promising protein and peptide candidate molecules, it appears that current production systems are overburdened and expensive to establish and maintain. with recent developments in vector systems for protein expression coupled with downstream protein purification technologies, plants are rapidly gaining credibility as alternative production systems. here we evaluate the advances made in host and vector system development for plant expression as well as the progress made in expressing hiv neutralising antibodies and peptide lectins using plant-based platforms. recent reports show that the number of new hiv infections has declined by % since the peak of the disease almost years ago (unaids, ) . however, worldwide more than million people are still living with the disease (unaids, ) . furthermore, in sub-saharan africa, the most heavily hiv affected region, it is estimated that only . % of the population have been tested for hiv in (unaids, . thus, globally there still exists a huge reservoir of hiv-infected people with the potential to infect millions more. despite commendable research efforts over nearly years, a protective hiv vaccine is still not available. thus, it has become crucial to develop other strategies for disease prevention, such as microbicides that would effectively block the initial transmission of the virus. women comprise % of the hiv infected population and are high risk candidates who are in many cases unable to protect themselves due to domestic violence, cultural and social habits, lack of education and financial security (unaids, ) . due to these difficult socioeconomic conditions a successful microbicide should further lend itself to formulations that can be applied topically or orally in order for women to self-manage the use of it (moscicki, ) . the microbicide development field received a boost with the progress made in caprisa studies where it was demonstrated that a microbicide gel containing % tenofovir, a reverse transcriptase inhibitor, could prevent the risk of hiv infection by % (karim et al., ) . anti-hiv microbicide candidates include surfactants, vaginal milieu protectors, viral entry inhibitors, reverse transcriptase inhibitors and other agents with an unknown mode of action (cutler and justman, ) . surfactants and vaginal milieu protectors were the first generation candidate microbicides (cutler and justman, ) . these were broad acting and failed to produce effective hiv inhibition, even enhancing infection in some instances (van damme et al., ) . of these, n- was the first surfactant that was tested in a clinical trial (garg et al., ) . although no adverse effects were reported for n- in preclinical and phase i clinical trials, genital ulcers, irritation, inflammation and subsequent higher hiv risk were reported from phase iii trials (garg et al., ; moscicki, ) . further surfactant development as microbicides faded under the risk of vaginal damage and inconclusive clinical trials. vaginal milieu protectors stabilise the low mucosal ph. in this class of microbicides acidform (amphora, instead inc., dallas, tx, usa) and buffergel (carbopol p, reprotect, baltimore, md, usa) have been evaluated extensively, displayed good contraceptive properties and were shown to be well tolerated in human subjects . whilst in vitro anti-hiv activity has been reported for acidform, it has only been subjected to safety and acceptability pre-clinical studies (reviewed by cutler and justman, ; http:// www.insteadsciences.com/amphora.htm#results). buffergel failed to show reduction of hiv infectivity when compared to the placebo gel in a study that evaluated its effectiveness in reduction of hiv incidence in a high risk study group (karim et al., ) . it is thus likely that these vaginal milieu protectors will not be effective in preventing hiv transmission in single formulations and will probably be used in combination with other antiviral entities. in fact, carbopol p is being used as the polymer base to formulate gels for the application of reverse transcriptase inhibitors such as tenofovir and uc (garg et al., ) . other strategies to maintain a healthy mucosal environment include the restoration of the microflora population by products such as lactin v and mucocept from osel, santa clara, ca, usa (moscicki, ) . entry inhibitors are a group of microbicides that interact either with viral or host cell structures to prevent attachment, fusion and entry. the first type of entry inhibitors was chemical molecules such as anionic polymers that establish an interaction with the virus based on surface charges (reviewed by cutler and justman, ) . most of these compounds failed to show significant protection in clinical trials, were associated with unwanted side effects and in some instances associated with an enhanced hiv infection risk (pironne et al., ) . subsequent microbicide development focused on more potent specialised molecules such as reverse transcriptase inhibitors, ccr antagonists and viral entry inhibitors. reverse transcriptase inhibitors target viral enzymes (campiani et al., ; cihlar, ) , ccr antagonists compete with the virus for host cell co-receptors (baba, ; schols, schols, , , whilst entry inhibitors bind to viral envelope components to prevent entry of the virus into the cell (balzarini, ; botos and wlodawer, ) . in the later group, antibody and peptide lectins represent a class of molecules that are in advanced stages of development as microbicides. to avoid repeating past failures, newly researched microbicide candidate molecules are currently undergoing strict evaluation in several preclinical test studies using specialised models and formulations (buckheit et al.; , ; doncel and clark, ; mcgowan, ) . rigour is necessary in preclinical testing because clinical trials are complex and expensive (minces and mcgowan, ) and the largest market segment for hiv prophylaxis resides in resource limited countries that can ill afford the development costs. because of these cost hurdles, it is crucial that microbicides be produced with minimum upfront capital outlay so as to facilitate development, testing and ultimate availability of the final product. plants are emerging as cost friendly alternative production systems for a variety of pharmaceuticals. numerous therapeutic proteins have been produced in plant systems (giddings et al., ; ma et al., ) . protein based microbicides, namely, neutralising antibodies and peptide lectins-lend themselves to production in plants (de muynck et al., ; matoba et al., ; o'keefe et al., ; sexton et al., ) . although these microbicides have been extensively studied in terms of their structure and mode of action, their production in plant host expression systems has not been audited to date. in this study, we evaluate the progress made in the expression and development of peptide and antibody candidate microbicides in plants. over the past two decades plants have been extensively investigated as alternative production systems for pharmaceutical proteins. even with careful consideration of existing production systems, plants provide several attractive features that are equivalent or more beneficial (mett et al., ; twyman et al., twyman et al., , . like mammalian and yeast cells, plants possess the cellular machinery which enables them to perform the post translational modifications essential for maturation and sometimes function of proteins. unlike mammalian fermentation systems, plants are not at risk of being contaminated with human pathogens. furthermore compared to mammalian and yeast systems, plant production systems are more easily scaled up; plants can either be propagated in large numbers in designated land plots or in contained greenhouses. maintenance of plants in soil, hydroponic or cell culture is simple and cheap compared to the complex growth media and requirements of yeast and mammalian cell systems (knäblein, ) . furthermore, plants provide a huge biomass in the form of green leafy tissue or as the numerous seeds of certain crops. the latter provides a further advantage of stable storage over longer time periods and high protein content that can be exploited for recombinant protein production (cunha et al., a, b; lau and sun, ) . whilst costs of downstream processing remain as high as that required for purifying proteins made from conventional systems, the burden can be alleviated by maximising production yields and utilising innovative purification strategies to improve product recovery (paul and ma, ) . thus, for plant production systems the upfront investment required for infrastructure is lower, which potentially lowers the barriers to entry by more players or especially players in developing countries. considering the time and effort invested over twenty-years, relatively few plant made pharmaceuticals (pmps) are currently marketed (faye and gomord, ) . the main reasons for this are that the production levels in plants were often too low to be commercially viable, and regulatory approval of pmps was perceived as being difficult to obtain. recombinant proteins were initially expressed in transgenic plants through stable nuclear transformation using agrobacterium-mediated delivery of binary vectors or alternative methods such as biolistic introduction of dna into plant cells (banta and montenegro, ; vianna et al., ) . these are lengthy, labour intensive processes which mostly generated progeny with variable and, for the most part, low target protein accumulation levels. expression of multiple component proteins such as antibodies required numerous crossings and screening of plants in breeding programmes. transient expression with binary vectors was mainly used as a rapid screen to validate the expression potential of a gene and did not generally result in high protein accumulation (gleba et al., ) . whilst viral vectors were useful to produce proteins in plants, these were limited by the insert size or the fidelity of the transcript (pogue et al., ) . furthermore, systemic spread of the virus sometimes resulted in loss of the foreign gene insert and raised concerns of containment. however developments in this arena have resulted in significantly improved state-of-the-art technologies for plant based protein expression. another perceived limitation for pmps in clinical applications is the variation in plant glycan structure compared to that of humans (gomord et al., ) . this shortcoming is also typical of other systems such as yeast and insect cells (mett et al., ) . thus far there has been no clinically significant evidence that plant-specific glycans are inappropriately immunogenic in humans (van der veen et al., ) . another development that has done a lot to allay such fears is the recent fda approval of a plant made therapeutic for gaucher disease; glucocerebrosidase (also known as taliglucerase alfa or elelyso), which is produced in carrot cells, has proven to be not only well tolerated but perhaps even more efficacious and stable-in other words, to be a "biobetter" (aviezer et al., ). however, the pressure remains on plant production systems to deliver therapeutic proteins with a humanised glycan profile. several advances in expression vector systems and plant hosts have addressed some of these limitations to a large extent. to increase protein yield, second generation agrobacterium binary vectors have incorporated various elements to enhance transcription and translation (veluthambi et al., ) . these improved vectors, used in conjunction with transient infiltration, have improved protein expression levels in plants. for instance, transient expression of a human optimised hpv- l capsid protein gene using a specialised binary ptra vector resulted in a yield of more than . g/kg of fresh leaf weight ( % total soluble protein, maclean et al., ) . significant lower levels were obtained when the same protein was transgenically produced in tobacco and potato resulting in the l protein accumulating to . and . % of total soluble protein respectively (biemelt et al., ) . further development in vector systems has seen the merging of viral and binary vector technology to increase yields and address insert size restrictions, retention of target genes and containment issues. icon genetics gmbh (halle, germany) developed a deconstructed viral vector system initially based on tobacco mosaic virus (tmv) in which target genes and different viral vector components are carried on several pro-module vectors (marillonnet et al., ) . in this system the viral coat protein has been removed to eliminate systemic spread. instead agroinfiltration provides the delivery of the modules to the plant cell and limits replication to the infiltrated area. in the cell, high-level expression is facilitated by a rna dependent rna polymerase. a site-specific recombinase facilitates the assembly of the modules into a dna molecule which is transcribed and spliced into a functional transcript. the transcript moves to the cytosol where it is translated into the specified protein. alternatively target signals can be incorporated to direct proteins to specified subcellular compartments (marillonnet et al., ) . this system has been used to accumulate various proteins at levels over g/kg plant material (bendandi et al., ) . one shortcoming of the initial icon system vectors was the inability to co-express more than one protein in the same spatial location (giritch et al., ) . this was problematic with the production of multi-component proteins such as immunoglobulins. a solution to this came by co-expression from two non-competing monopartite viral genomes such as tmv and pvx (giritch et al., ) . alternatively, viral vectors derived from bi-or tri-partite viral rna genomes do not seem to be competing and are able to co-function in the same area. the cowpea mosaic virus (cpmv) is a bipartite viral rna genome from which two types of vector systems were developed. in the full-length system (wild type, wt) the coding sequence for the protein of interest is fused to the c-terminus of the rna- polypeptide which is co-translationally released via a-peptide mediated cleavage . replication is facilitated by the co-expression of rna- . the full-length version allows for local co-expression of two different proteins; however segregation of the co-expressed proteins occurs with systemic movement . a deleted cpmv version of rna- (hypertranslatable, ht) was developed which lacked the ability of systemic spread and was thus able to co-express more than one protein without the occurrence of segregation. moreover the deleted cpmv system obtained higher expression levels than the full-length version (sainsbury et al., , . using this system, protein expression levels exceeded . g/kg protein . several pharmaceutically important molecules have been expressed in plants using vectors based on the ssdna geminivirus bean yellow dwarf virus (beydv) (chen et al., ): huang et al. ( and regnard et al. ( ) have independently developed vector systems based on the dna genome of the virus. the beydv system requires only two viral components for co-expressing heteromeric proteins: these are the long intergenic region (lir) and the short intergenic region (sir) control sequences, and the single alternatively spliced gene for the replication-associated proteins rep/repa. in the system non-competing co-expression can be achieved either from two replicons encoding different proteins, or from a single replicon containing the different proteins. in nicotiana benthamiana, transient expression levels from the beydv vector were -to -fold more for egfp and hiv- p compared to levels obtained using a binary ptra agrobacterium tumefaciens vector (regnard et al., ) . furthermore, expression with beydv resulted in accumulation levels of . g/kg monoclonal antibody against ebola virus gp (mab d ) (huang et al., ) . it is anticipated that the system will be able to simultaneously produce as many as four different protein subunits. several other geminiviruses and also the multicomponent ssdna nanoviruses have been investigated for their potential as expression vectors; their further development could greatly expand the range of plant species that can be used for transient production of recombinant proteins (rybicki and martin, ) . in eukaryotes the n-glycan biosynthesis pathway is conserved for the endoplasmic reticulum (er) (kukuruzinska and lennon, ) . variations between species occur in modifications to glycan structures in processing steps after the protein exits from the er. in plants these variations depend on the protein itself, plant species and plant organ used for expression . unless the plant glycosylated form of the therapeutic protein is more attractive as is the case with the carrot cell produced glucocerebrosidase (shaaltiel et al., ) , it is considered more ideal if plants are able to produce therapeutic proteins that have mammal-like glycans. where glycan structure is not critical to protein function, recombinant proteins without glycan structures are desirable (rodriguez et al., ) . therapeutic proteins that are produced as aglycosylated forms are only feasible if the proteins need to stimulate an inflammatory response or in the case of a recombinantly produced antibody that does not require an effector function since glycan structures are often crucial for this biological function of the protein (jefferis, ). the current gel formulation of hiv neutralising antibodies and peptide lectins (tsai et al., ) suggests that these microbicides will most likely be applied topically. when these are exposed to the mucosal surfaces it will only have a limited interaction with the immune system and thus not cause inflammation. thus for the production of these microbicides in plants the nature of the glycan structures on these microbicides might not be as important as the yield obtained. another means to produce a protein that more closely resembles a humanised glycan profile is to restrict the recombinant protein to the er by using kdel, hdel or sekdel er retention signals (ko et al., ; triguero et al., ) . for regulatory purposes it is generally regarded as "safer" to produce a native version of the protein . however in the case of er retention signals regulatory approval might be less stringent seeing that these sequences are also found in mammalian proteins. of note is that protalix's carrot cell produced glucocerebrosidase, which has just received fda approval, was produced with a storage vacuole targeting signal (shaaltiel et al., ) . another argument for er retention is that for the production of some proteins, er retention is needed to increase the accumulation levels (bortesi et al., ; yang et al., ) . on the other hand er retention can result in the degradation of the protein or low stability and reduced half life of the therapeutic in vivo (ko et al., ; loos et al., ) . studies have also shown that er retention of recombinantly produced proteins is not always successfully achieved, leading to some proteins leaking from the er that are then further processed to contain complex immunogenic plant glycans (floss et al., ; loos et al., ; rademacher et al., ) . subsequently, improved plant hosts have been developed with the aim of humanising the glycan patterns of recombinant proteins. plants such as arabidopsis, tobacco and moss have been generated in which the plant-specific glycosylation genes have been knocked out (koprivova et al., ; schähs et al., ; strasser et al., ) . in these mutants, plant specific α , -fucose and β , -xylose residues are replaced by complex n-acetylglucosamine (gngn) structures. further glycan improvements are made by co-expressing mammal like glycosylation and sialylation enzymes such as β , galactosyltransferase (galt), n-acetylglucosaminyltransferase iii (gntiii), core α , -fucosyltransferase, udp-n-acetylglucosamine epimerase/n-acetylmannosamine kinase (gne), n-acetylneuraminic acid phosphatase synthase (nans), cmp-n-acetylneuraminic acid synthetase (cmas), cmp-n-acetylneuraminic acid transporter (cmp-neu ac) and α , -sialyltransferase (st) in these plant glycosylation knock-out mutants (castilho et al., (castilho et al., , strasser et al., ). resulting proteins not only lack plant-specific glycans but also contain human glycan structures. neutralising antibodies play a very important role in the development of vaccines for passive immunisation and as viral entry inhibitors (reina et al., ) . they are directed against the viral envelope protein and interfere with viral docking and fusion. they thus inhibit the infectivity of the virus and also potentially facilitate viral clearance via their fc related effector functions . several hiv- neutralising antibodies have been isolated from hiv infected individuals (simek et al., ; walker et al., ; wu et al., ) . novel broad acting neutralising antibodies such as vrc , vrc , pg and pg were isolated which displayed a larger breadth and potency than some of the well-known neutralising antibodies (wu et al., ) . however the four well-known hiv- neutralising antibodies, namely, g , e , f and b have been well researched in terms of structure, interaction with the virus, protection in animal models and safety in clinical trials and were produced in various plant platforms. these antibodies have fared well in protecting macaques from systemic or vaginal simian/human immunodeficiency virus (shiv) challenges and are well tolerated in human subjects (armbruster et al., (armbruster et al., , hessell et al., ; mascola et al., mascola et al., , parren et al., ) . furthermore, passive administration of neutralising monoclonal antibodies (mabs) e , f and g reduced viral rebound in established hiv infections (trkola et al., ) . three of these neutralising monoclonal antibodies (mabs)- g , e , f are being assessed as a gel formulated microbicide in a phase i clinical trial for their safety and pharmacokinetic effects . so far vaccination attempts with these antibody epitopes have failed to produce equivalent neutralising antibodies in humans (coëffier et al., ; lenz et al., ; mcgaughey et al., ) . it is thus most likely therefore, that these antibodies will have to be administered passively and that a large production quantity will be required. mammalian cells are currently used for fda-approved therapeutic antibody production . given that the capacity of these traditional fermentor systems will not meet the demand, plants can be employed as alternative manufacturing platforms (knäblein, ) . several antibodies have been successfully produced in plant platforms (de muynck et al., ) . apart from yield, the glycan composition and efficacy will be important criteria for plant made manufacturing of these antibodies. therefore we evaluated the progress of plant production of these neutralising antibodies in light of these criteria. table summarises the expression of these four antibodies in plant systems. it has been reported that g neutralises a and b clade hiv- virus entry by recognition of a manα → man rich epitope on the exterior face of the gp protein (binley et al., ; scanlan et al., ) . monoclonal antibody (mab) g can activate the complement system and display antibody dependent cellular cytotoxicity (adcc) against virus infected cells (trkola et al., ) . passive infusion of g combined with other neutralising antibodies including f , protected macaques from a vaginal and intravenous challenge with shiv (baba et al., ; mascola et al., ) . passively infused g and f were well tolerated in human subjects in a phase-i clinical trial (armbruster et al., ) . clinical trials with g produced in transgenic tobacco have commenced in (paul and ma, ) . mab g is unique in its structure in that it naturally forms a single fab region via domain swapping between the variable regions of the light chain (v l ) and heavy chain (v h ) and between the constant region of the light chain (c l ) and heavy chain (c h ) respectively (calarese et al., ; west et al., ). the g dimer has shown to be more than times more efficient as the monomer in neutralising several hiv- strains in both in vitro (west et al., ) and in vivo assays (luo et al., ) . the production of g has been actively pursued in maize, arabidopsis and tobacco. mab g was produced in the seed endosperm of two different maize lines under control of the rice glutelin (gt- ) promoter. the antibody was produced in hi-ii maize with endoplasmic reticulum (er) retention signals and in the elite maize line m w as a secreted form (ramessar et al., ) . er retained antibody accumulated to μg/g not mentioned both b and b -cv-n fusion were able to bind gp . in a virus neutralisation assay the sexton et al. ( ) in the t generation and μg/g in the t generation whilst the secreted form reached μg/g. identification of the glycan structures showed that the majority of the er retained antibodies contained oligo-mannose type glycans (omt). however a few immunoglobulins contained glycans of the vacuolar type, indicating that er retention was not completely successful. different glycoforms were detected for the secreted g with the majority being single n-acetylglucosamine (glcnac) residues and the rest containing complex type fucose and xylose glycans with a small number also containing omt type glycans. the efficacy of the maize produced antibody was compared with the chinese hamster ovary cells (cho)-produced g derivative. both the er retained and secreted g had a similar antigen binding ability as the cho produced g . however in an hiv neutralisation assay, the er retained and secreted forms were four and threefold more effective than the cho produced equivalent respectively. the increased potency was attributed to the dimerisation and aggregation of the antibody. in arabidopsis, g was expressed in both the leaves (schähs et al., ) and seeds (loos et al., ) of a knockout line (Δxt/ft), that lacked the ability to generate immunogenic plant-specific β , -xylose and α , -fucose glycans (strasser et al., ) . in leaves, expression was driven by the s camv promoter without any er retention signals. the antibody levels varied between . and . % tsp in both the wildtype (wt) and Δxt/ft line (schähs et al., ) . the glycans on the antibody that was produced in the Δxt/ft line were mainly terminal n-acetylglucosamine (gn) residues that lacked plant-specific β , -xylose and α , -fucose residues. a small population of the antibody molecules produced contained omt residues indicating that the processing of all antibodies was incomplete. notably, the binding capacity of the Δxt/ft produced g antibody was similar to the cho produced g . for seed expression in arabidopsis Δxt/ft, expression of g was driven by the β-phaseolin promoter (loos et al., ) . the antibody was expressed with and without er retention signals. there was no significant difference in accumulation levels between secreted and er retained antibody. expression levels peaked around . μg/mg. the n-glycan profile of the purified antibodies revealed that the secreted antibody of the wild type line contained complex n-glycans containing n-acetylglucosamine-xylose-fucose (gngnxf) residues whilst the g produced in the mutant line contained a homogenous n-glycan structure consisting of n-acetylglucosamine (gngn). the majority of the er retrieved antibody of the wild type line contained oligo-mannosidic n-glycans, with a small amount of antibody carrying gngnxf. the efficacy of the seed produced g in an hiv neutralisation assay was slightly inferior to the cho derivative. in n. benthamiana leaves, g was transiently expressed using the full length and the deleted rna- (ht) version of the cpmv vector sainsbury et al., ) . the antibody was expressed in both systems with and without er retention signals. overall higher antibody accumulation was obtained by using the deleted cpmv vector and er retention signals. levels of mg/kg were reported. the glycan analysis of the antibodies showed that er retained forms consisted mainly of oligo-mannose type structures (omt) with a few containing more complex glycans. secreted antibodies contained complex gngnxf, n-acetylglucosamine-mannose-xylose-fucose (gnmxf) with a few omt also present. in vitro evaluation of the binding ability and neutralisation efficacy of the n. benthamiana produced antibody showed that it was equal to the mammalian cell derived g . to further humanise the glycan structures on g , strasser et al. ( ) produced g in a n. benthamiana Δxt/xt galt + mutant line. this n. benthamiana line does not produce plant-specific xylose and fucose glycans but produces partially humanised glycans via the activity of a highly active human derived β , -galactosyltransferase. although no mention was made of the accumulation levels, the g antibody produced in this system was fully galactosylated and was more effective in neutralising hiv- than the cho produced version. mab f displays a broader neutralisation activity than g , inhibiting hiv isolates from clades a, b, d and e (binley et al., ) . it docks onto to the core epitope eldkwa on the lipid embedded membrane proximal exterior region (mper) of gp and potentially interferes with the fusion step of the virus (binley et al., ; de rosny et al., ; franquelim et al., ; muster et al., ) . on its own and in combination with other antibodies including g and e , f displayed the ability to protect against an intravenous, vaginal and oral challenge of shiv in macaques (baba et al., ; hessell et al., ; mascola et al., mascola et al., , . furthermore passive administration of this antibody did not seem to cause immune responses or other adverse effects in hiv infected human participants (armbruster et al., ) . production of f was explored in nicotiana species. the heavy chain (hc) and light chain (lc) were expressed with sekdel retention sequences in tandem under control of the s camv promoter in nicotiana tabacum l. cv bright yellow (by- ) cell cultures (sack et al., ) . accumulation of f reached a maximum of . mg/kg fresh weight and was further enriched by protein-a purification to . mg/kg wet cell weight. no degradation products were observed following purification, however minor impurities were detected. n-glycans were expected to be of the omt, but this was not confirmed by analyses. fc region binding between the by- and cho produced f was equivalent. however, the antigen binding capacity of cho produced f ( %) was slightly superior to the plant derived f ( %). in hiv neutralisation studies the by- produced antibody was threefold less efficient than the cho produced counterpart. this lower potency was attributed either to the presence of impurities, the added sekdel motif or different glycan structures that could have interfered with the antibody access to the epitope. to further enhance the accumulation of f in tobacco, the antibody was expressed as er retained elastin-like polypeptide (elp) fusions (floss et al., ) . the elp peptide has been used to facilitate accumulation of proteins in green leaf tissue (patel et al., ) . four transgenes; hc unfused, hc-elp fused, lc unfused and lc-elp fused were introduced in n. tabacum cv. samsun nn. plants were subsequently crossed resulting in combinations with neither gene carrying the fusion or both hc and lc carrying elp fusions or either the hc or lc fused to elp. prior to crossing the transgenic lines, it was observed that the presence of elp increased the accumulation of the chains with the lc accumulating to higher levels than the hc. in the crossed lines, the lc-elp fusion facilitated a higher accumulation of the unfused hc as well. accumulated total soluble protein (tsp) levels reached . % for the lcelp-hc, . % hcelp-lc, . % for hcelp-lcelp and . % for hclc. the elp fusion eased the purification process of the plant-produced antibodies and did not interfere with the assembly of the antibody. the glycans of the plant-produced f were mainly oligo-mannose type (omt) with lesser amounts of complex glycans consisting of n-acetylglucosamine (gngn), nacetylglucosamine-xylose (gngnx), galactosyl-n-acetylglucosamine-xylose (agnx), n-acetylglucosamine-mannose-xylose (gnmx) and n-acetylglucosamine-xylose-fucose (gngnxf) moieties. f variants were all similar to the cho produced f in their antigen binding capacity. mab e is one of the most broadly neutralising antibodies that are active against several viral isolates of different clades including clade c, which is the most prevalent clade in the heavily affected sub-saharan africa region (binley et al., ; walker et al., ) . both e and vrc were able to neutralise over % of the key hiv subtypes (walker et al., ; wu et al., ) . although vrc is more potent, it uses a different mode of action with the virus than e . vrc interacts with the envelope in a way that resembles the cd -gp interaction (li et al., ) . the e epitope interaction is also somewhat complex; the antibody recognises a linear epitope adjacent to the f epitope on the membrane proximal exterior region (mper) and interacts with the lipids on the cell membrane (franquelim et al., ; zwick et al., ) . whether lipid binding is involved in the broadly neutralising ability of e is still debatable (scherer et al., ; xu et al., ) . thus both antibodies can be used in combination against several hiv isolates. in a phase i clinical trial, it was demonstrated that e can be safely administered to hiv infected participants alone or combination with f and g (armbruster et al., ) . when e was administered intravenously, rhesus macaques were protected from a mucosal challenge with shiv (hessell et al., ) . mab e has been expressed via nuclear transformation in n. benthamiana (strasser et al., ) . it was produced in a wild type (wt), a glycoengineered Δxt/fx mutant line and in a Δxt/fx galt + line that produced an altered version of the human β , -galactosyltransferase. the glycans of the wt produced e contained n-acetylglucosamine-xylose-fucose (gngnxf), n-acetylglucosamine (gngn) for the xt/fx mutant and galactosylated (aa) glycans for the xt/fx galt + line. the later mab form was more potent than the other plant made forms and more efficient than the cho produced derivative in a neutralisation assay, possibly because of the galactosylated glycans that enhance the stability, half-life and functionality of the antibody. mab b can effect hiv neutralisation across different clades from different geographic locations (binley et al., ) . unlike other neutralising antibodies that are restricted to certain conformations of the virus, in vitro studies show that b can bind different conformations of the envelope (eggink et al., ; zhou et al., ) . this antibody has been shown to protect macaques in a vaginal challenge with shiv when administered systemically or topically (parren et al., ; veazy et al., ) . mab b was expressed in the milk of female mice and displayed the same hiv neutralisation ability as the cho cell derived antibody (yu et al., ) . in combination with cd -igg (pro ), b potently inhibited hiv infection of cervical tissue (hu et al., ) . more importantly, in this combination or administered alone, mab b is able to stay associated with the virus that leaves the mucosal environment on migrating cells and prevents subsequent infection of target lymphocytes (hu et al., ; van montfort et al., ) . other neutralising antibodies in the study did not display this property. sexton et al. ( ) produced b and a b -cv-n (cyanovirin) fusion in n. tabacum. cv-n is a cyanobacterium lectin that displays potent anti-hiv activity . plants were generated that expressed b hc, lc or a fusion where cv-n was fused to the b hc. subsequent crosses were performed to generate progeny that expressed both an unfused b ( . μg/ml) as well as b -cv-n fusion ( . μg/ml). the authors demonstrated that both modules of the fusion molecule were functional and the fusion molecule to be more potent than cv-n or b alone in an hiv neutralisation assay. the glycan profile of the plant made proteins was not presented. lectins are proteins of non-immune origin that selectively bind to carbohydrate moieties (goldstein and hayes, ) . these proteins have been isolated from all life forms including bacteria, viruses, algae, mushrooms, nematodes and plants. based on plant lectin information, distinct families have already been described (van damme et al., ) . lectins have been useful for several applications including pest resistance in crop plants (peumans and van damme, ) , therapeutic agents for cancer treatment (liu et al., (liu et al., , and as antiviral microbicide candidates (francois and balzarini, ) . the hiv envelope is heavily populated with mainly high mannose type glycans (doores et al., ; geyer et al., ) . it comes as no surprise that the majority of these anti-hiv lectins show an affinity for mannose moieties (botos and wlodawer, ) . by interacting with the glycan residues on the viral envelope they prevent attachment and fusion. many of these lectins have a broad range of activity against different viral clades of various serotypes and co-receptor dependability. furthermore, some have displayed the potential to inhibit viral capture and dissemination by dc-sign bearing host cells (balzarini et al., ; nabatov et al., ) . the anti-hiv peptide lectins fall into different families with different modes of interaction with mannose glycans. furthermore variations occur in their quaternary structures, efficacy level towards hiv and immune stimulatory effect of human cells (barre et al., ; ziółkowska and wlodawer, ) . anti-hiv lectins have been reviewed extensively with regards to structure and mode of binding (balzarini, ; francois and balzarini, ; ziółkowska and wlodawer, ) . the majority of these lectins are remarkably stable across broad ph ranges and high temperatures. this allows their manipulation in expression, purification, formulation and applications as microbicides. they represent a rich source of proteins that can be developed as anti-hiv microbicides. the first group of anti-hiv lectins was originally isolated from plants (van damme et al., ) . thereafter numerous others have been isolated from other organisms including prokaryotic algae, bacteria, fungi and nematodes bulgheresi et al., ; chiba et al., ; inokoshi et al., ; mori et al., ; zhao et al., ) . recombinant expression of lectins in plants has been applied to some extent to introduce pest resistance in valuable crops or promote rhizosphere symbiotic associations (rovenská and zemek, ; sreevidya et al., ; wang et al., ) . plant lectins which show anti-hiv activity have been isolated directly from their natural source such as the case of galathus nivalus agglutinin (gna) where the bulbs of snowdrop (g. nivalus) contain reasonably high levels of the lectin (van damme et al., ) . on the other hand, hiv inhibiting lectins such as those from prokaryotes and some plants are produced in low quantities and it is thus not feasible for direct isolation from the source (koshte et al., ; . lectins isolated from cyanobacteria, red algae and fungae displayed generally higher potency than most plant lectins and have been extensively researched as topical anti-hiv microbicides. since the proteins seem to occur in low quantities in their native host, recombinant production in alternative systems, such as plants has been explored. as lectins occur naturally in plants, it seems that production of recombinant lectins from other sources will not be problematic. however, it has come to light that plants produce two types of lectins, classical and nucleocytoplasmic (lannoo and van damme, ; van damme et al., ) . classical lectins reside in storage organelles whilst the nucleocytoplasmic lectins occur mainly in the cytoplasm. generally classical lectins are synthesised with signal peptides, are produced in abundance and serve a defence and storage purpose for the plant. nucleocytoplasmic lectins are produced without any signal peptides in small quantities and are thought to play a role in regulatory processes in the plant cell (lannoo and van damme, ) . it is thus evident that in a plant cell there is a clear distinction in signalling and abundance of different lectins with different roles. thus, heterologous production of lectins in plants could have an effect on the viability of the plant cell. the native roles of many of these hiv neutralising lectins have not been resolved and although the majority mainly bind to mannose residues on the viral envelope, one cannot rule out the possibility that other ligands may exist in the plant cell environment which may affect their expression, accumulation or recovery from the plant matrix. thus subcellular targeting may play an important role in the resolution of the optimal compartment for high yield lectin accumulation that is not detrimental for plant cells during heterologous expression. here the progress that has been made with heterologous expression of anti-hiv lectins in plants is briefly reviewed. table highlights the major findings. cyanovirin (cv-n) is an kda protein that was isolated from the blue-green algae nostoc ellipsosporum with an ec value of . nm . cv-n inhibited in vitro fusion of hiv- with target cells as well as subsequent viral spread between virus infected and uninfected cells. it displayed antiviral activity against primary and laboratory modified hiv strains of several clades including m, t and dual tropic viruses . furthermore cv-n inhibits gp binding to ccr or cxcr co-receptor dependent strains (dey et al., ; mori and boyd, ) . antiviral activity of cv-n against hepatitis c (helle et al., ) , ebola (barrientos et al., ; smee et al., ) , shiv , measles and herpes virus (dey et al., ) has also been reported. cv-n has very low homology to other known protein sequences, but contains a sequence motif that is typical to the cv-n type lectin family gustafson et al., ; percudani et al., ; van damme et al., ) . in solution, cv-n exists as a monomer or dimer depending on ph and temperature conditions (barrientos and gronenborn, ; barrientos et al., ) . cv-n interacts with terminal mannose residues of the oligomannose glycan structures of gp (bewley and otero-quintero, ; shenoy et al., ) . the monomer contains two carbohydrate binding domains with different affinities to di-and trimannose respectively (bewley and otero-quintero, ) . although anti-hiv activity has been reported for both monomeric and dimeric forms, it appears that the potency of cv-n depends more on the formation of multisite interactions with glycan residues rather than the affinity and presence of each binding domain (barrientos et al., ; fromme et al., ; kelley et al., ) . in vitro test with host cells and cv-n displayed no or little loss of cell viability as a result of host cell exposure to cv-n. furthermore, in vivo studies with gel-formulated cv-n caused no adverse effects in the test animals (tsai et al., ) . however, more extensive tests showed that cv-n induced the production of chemokines and cytokines and stimulated cell proliferation (huskens et al., ) . this cytotoxicity was however not linked to its carbohydrate-binding property. thus with further development such as mutations or pegylations (zappe et al., ) that could potentially reduce the cytotoxicity of cv-n, the lectin might still be considered as a potential microbicide. cv-n has been used in ground-breaking microbicide development work to pave the way for future development of lectins as viable microbicide molecules. it displayed the potential for lectins to be used as a gel formulated microbicide to protect against vaginal and rectal challenged with hiv (tsai et al., ) . cv-n has been recombinantly produced in commercial bacteria such as streptococcis gordonni (giomarelli et al., ; pozzi et al., ) and lactobacillus jensinii (liu et al., ) . cv-n displayed on the surface of s. gordonni was able to capture hiv virions, whilst if secreted from the bacteria it could bind to gp . recombinant l. jensinii were able to colonise the vagina in mice and secrete full length cv-n. l. jensinii produced cv-n was able to inhibit ccr hiv in vitro in nanomolar concentrations. several fusions of cv-n have been explored with different applications in mind: for example, to form high potency chimaeras cv-n has been fused to the broadly neutralising b antibody (sexton et al., ) and to the linear pi peptide (mcfadden et al., ) . both partners in the fusions were active and the new chimaeras displayed similar stability and higher antiviral activity. cv-n was also fused to a pseudomonas exotoxin a . the chimeric protein potently eliminated hiv infected cells that expressed gp on their surface. the recombinant expression of cv-n in alternative systems has recently been reviewed by xiong et al. ( ) . in brief, initial production and purification in escherichia coli was not optimal, resulting in low levels of cv-n accumulation. further optimisations resulted in high accumulation levels but consisted of a heterogeneous cv-n population of intact, truncated and signal peptide containing cv-n forms. chaperone fusions of cv-n resulted in homogenous cv-n that accumulated to mg/l. expression of cv-n was also pursued in yeast, which only resulted in low yields of non-functional protein. sexton et al. ( ) showed that it is feasible to produce cv-n in tobacco plants as well as hydroponic cultures. the cv-n gene was transformed into n. tabacum and expressed under the s camv constitutive promoter and er targeting signal peptide. cv-n accumulated to ng/mg fresh leaf weight (or . % tsp). hydroponic cultures derived from the transgenic plants secreted cv-n at . μg/ml. crude cv-n extracts from tobacco were able to inhibit hiv infection of tzm-bl cells comparable to that of purified e. coli derived cv-n. cv-n was also produced in tobacco as a fusion with the monoclonal antibody b (sexton et al., ). the fusion accumulated at . μg/ml and was more active than cv-n or b alone. griffithsin (grft) was isolated from the red alga griffithsia (mori et al., ) . its amino acid sequence contains an unknown amino acid at position and codes for a . kda protein that is sequence unrelated to any known protein. in its folded state, the monomer displays the β-prism-i motif found in other lectins such as jacalin, whilst the dimer is formed by a unique domain swopping between two grft molecules that are not typically found in this lectin family . grft displayed broad activity against corona viruses and hiv (o'keefe et al., ) . grft has shown antiviral activity against hiv clades a, b and c that are prevalent in sub-saharan africa, india and the west. it is active against both clinical and laboratory adapted t and m tropic hiv-isolates and inhibits both ccr and crcx orientated strains. of all the prokaryotic anti-hiv lectins, grft is thus far one of the most potent and promising lectins for microbicide development with an ec value as low as . nm (mori et al., ) . the potency of this lectin is attributed to its remarkable dimeric structure that contains six mannose binding sites that are most likely spaced for optimal interaction and subsequent cross linking of the glycans of the gp coat protein (moulaei et al., ; . any successful topically applied microbicide must ultimately be able to function in the mucosal environment. given that the infection rate of hiv is quite rapid, the microbicide should remain stable and efficacious to neutralise hiv immediately on contact. its presence furthermore should not compromise tissue viability or initiate an inflammatory response. in the light of these criteria, preclinical test shows that grft is a good microbicide candidate. grft is virucidal upon contact with the virus and remains stable over several hours prior to or after application (emau et al., ) . grft is stable and functional in cervical lavage fluid over a wide ph range. furthermore grft was not cytotoxic to human and primate cell lines, does not initiate an inflammatory response and did not cause adverse effects in a rabbit vaginal irritation model (emau et al., ; o'keefe et al., ) . it is likely that an effective anti-hiv therapeutic will consist of more than one microbicide to limit the risk of viral resistance. it is thus important that candidate microbicides should be compatible with other microbicides without compromising efficacy and safety of the component molecules. grft was tested in combination with tenofovir (nucleotide reverse transcriptase inhibitor), maraviroc (ccr hiv co-receptor inhibitor) and enfuvirtide (a gp fusion inhibitor) against calde b and c virus isolates to evaluate possible synergistic effects of the lectin in combination with other microbicides (férir et al., ) . when grft was combined with other microbicides the potency of the combination was higher than that of the individual molecules. recombinant production of grft was initially pursued in e. coli. although the lectin accumulated to mg/l, % was irreversibly lost to inclusion bodies . whilst the expression of grft in e. coli illustrated the feasibility of an alternative production system of functional grft, it remains an expensive production platform with high optimisation and maintenance demands. o'keefe et al. ( ) used a tmv based vector system for the transient production of grft in the cytosol of n. benthamiana leaves. grft accumulated to more than g/kg fresh weight and allowed the purification of g grft from . kg processed leaf material. the gp binding potential and efficacy of the plant made grft were similar to e. coli produced and native grft respectively, demonstrating the potential of plant expression approaches as viable alternatives for the production of the lectin for use as a candidate microbicide. actinohivin (ah) is a lectin isolated from the actinomycete longispora abida with a reported ic value of nm (chiba et al., ) . ah harbours a lot of potential to be developed as an anti-hiv microbicide; the lectin inhibits both t and m tropic hiv strains and is particularly potent against c clade viruses (chiba et al., ; matoba et al., ) . furthermore ah exhibits an impressive safety profile; the lectin did not cause proliferation or mitogenic stimulation on host cells (hoorelbeke et al., ) . unlike other prokaryotic lectins, ah binds to clustered high mannose type glycans instead of single moieties (chiba et al., ; tanaka et al., ) . it is thus possible that the cluster binding of ah confers its specificity towards glycan types that are typical of the hiv envelope and is linked to its low mitogenic effects. matoba et al. ( ) investigated a plant based production system for ah. the native gene (chiba et al., ) was expressed using the icon vector system. expression levels between and mg/kg were obtained. when the small ubiquitin-like modifier (sumo) was fused to the n-terminus of ah, the protein levels accumulated to over mg/kg in the apoplast (davis, ) . the plant-produced ah was able to inhibit hiv mediated syncytium formation. the burden of the continuing hiv pandemic together with the lack of an effective vaccine, has spurred the development of several microbicidal candidate molecules to curb hiv transmission. of these, neutralising antibodies and peptide lectins have shown encouraging potency and protection against the virus in in vivo and in vitro studies. plants present a viable option for the cost effective production of protein based candidate microbicides. we reviewed here the progress made with the production of hiv neutralising antibodies and peptide lectins in plant systems. so far four hiv neutralising antibodies- g , b , f and e have been successfully made in plants. expression levels in transgenic plant leaves and seeds and in plant cell cultures were relatively low compared to that obtained with transient expression technologies. for instance, transient infiltration using new generation viral vector technologies such as the deleted cpmv vector resulted in g accumulating to mg/kg. expression and targeting of product to various subcellular locations are generally used to optimise recombinant protein yield. in the case of antibodies, this aspect of expression also influences the glycan structure of the molecule. also, depending on plant organ, subcellular targeting can influence the overall yield and efficacy of the plant made antibody. where er retention of g in arabidopsis seed showed no significant difference in expression levels, it resulted in almost twofold lower levels of g in maize seed. in contrast to maize seed, er retention of g in tobacco leaves improved yields. furthermore er retention does not seem to be entirely optimal, leaving a small fraction of immunoglobulins that will contain immunogenic plant glycans. separating these during downstream processing will only add to production costs. a promising solution lies in the use of a modified host system that is incapable of producing plant glycans but instead adds human type glycans to secreted proteins. both g and e were produced in such a modified n. benthamiana plant. the resulting antibodies contained only human like galactosylated glycan structures. furthermore these galactosylated antibodies were more efficacious than the cho produced equivalent. no mention was made of the accumulation levels of the antibodies in the mutant plant, but the ability to express mab products with a human glycan profile represents a significant step towards the production in plants of fully functional proteins that are likely to be well tolerated. it is difficult to say which factors have the most influence on the efficacy of plant made immunoglobulins. whilst some plant made antibodies were as active as their cho counterparts, others were less effective and others yet up to fourfold more active. impurities in the recovered products and glycan structure might play a role in decreasing activity, whilst aggregation and human-like glycan structures appear to improve activity as was the case for mab g antibody produced in maize and a modified tobacco. furthermore, fusion with other anti-hiv agents also showed an increase in the potency of the antibody against the virus. a few lectins with highly potent anti-hiv activity have been isolated from different biological organisms. in general, these lectins do not occur in large amounts in their natural sources, or these sources are difficult to propagate. thus recombinant expression of the lectins was pursued in other production systems such as bacteria and yeast. with the later systems, lectin production was not optimal resulting in heterogeneous products, non-functional products or products that formed insoluble inclusion bodies. subsequently these lectins were produced in plants with generally improved outcomes with respect to these challenges. three potent anti-hiv lectins-namely, cv-n, grft and ah-have been expressed in tobacco. stable transgenic expression resulted in significantly lower levels of accumulated product relative to transient production of lectins. the highest accumulation level reported thus far for a lectin produced in a plant is for grft. by using a tmv-based viral vector the lectin accumulated in tobacco leaves to more than g/kg protein. the ah lectin also accumulated to a good level of mg/kg using the icon viral vector system. furthermore all the plant-produced lectins reviewed here seem to retain their native efficacy against the virus. subcellular targeting is an important aspect to consider when producing these lectins in plants, since lectin expression may affect plant processes, yields and viability of the plant cell. the lectins reviewed here have either been produced as secreted or cytosolic proteins. it is not clear which compartment suits which lectin since high levels for both a cytosolic (grft) and a secreted lectin (ah) have been reported. thus each lectin that is expressed in a plant system might have to be produced in different cell compartments to evaluate the optimal expression conditions for that lectin. apart from expressing solitary candidate microbicides, plants were also able to produce fusions of lectins and antibodies, either with each other (antibody-lectin fusions) or with entirely different molecules. any administered hiv microbicide will most likely consist of several compounds with a different mode of action to ensure broad maximum activity without the risk of developing resistance. by producing fusion microbicides one combines the neutralisation potential of two molecules in a single production run, with positive implications for lowering cost and increasing efficacy. additionally fusions can stabilise the target protein to ensure higher yields, as in the example of elp fused to anti-hiv antibodies (floss et al., ) . clearly, production of microbicidal candidate molecules offers advantages beyond simple challenges of expression of efficacious molecules. after twenty years of research, plants are on the brink of entering the playing field of protein production platforms for human therapeutics. their progress from potential to actual production platform has been facilitated largely by technical developments in vector systems and plant hosts. for a disease such as hiv, where there is a desperate demand for an effective microbicide, these advances could potentially enable plants to meet the supply gap. although several anti-hiv neutralising antibodies and peptide lectins have been produced in plants, only two have entered clinical trials . mapp is a cocktail of several antibodies produced by the magnicon system in n. benthamiana whilst plant-made g entered clinical trials in . in addition, plant-produced grft has passed pre-clinical studies and is considered safe to be evaluated in clinical trials. advancements in plant-made therapeutics in clinical development and regulatory approval such as the use in advanced broad access trials of carrot cell glucocerebrosidase, provide a new perspective on the potency and utility of pmps. the hope is that as production and purification technology become more standardised in the field, and as more plant-made candidates progress along the preclinical and clinical developmental pipeline, plants will become a source of routinely used, effective therapeutic and preventative biologics. a phase i trial with two human monoclonal antibodies (hmab f , g ) against hiv- passive immunization with the anti-hiv- human monoclonal antibody (hmab) e and the hmab combination e / f / g a plant-derived recombinant human glucocerebrosidase enzyme-a preclinical and phase i investigation recent advances of ccr antagonists human neutralizing monoclonal antibodies of the igg subtype protect against mucosal simian-human immunodeficiency virus infection large-molecular-weight carbohydrate-binding agents as hiv entry inhibitors targeting glycoprotein gp carbohydrate-binding agents efficiently prevent dendritic cell-specific intercellular adhesion molecule- -grabbing nonintegrin (dc-sign)-directed hiv- transmission to t lymphocytes agrobacterium and plant biotechnology structure-function relationship of monocot mannose-binding lectins the domain-swapped dimer of cyanovirin-n contains two sets of oligosaccharide binding sites in solution cyanovirin-n binds to the viral surface glycoprotein, gp , and inhibits infectivity of ebola virus flipping the switch from monomeric to dimeric cv-n has little effect on antiviral activity rapid, high-yield production in plants of individualized idiotype vaccines for non-hodgkin's lymphoma the potent anti-hiv protein cyanovirin-n contains two novel carbohydrate binding sites that selectively bind to man d d and man with nanomolar affinity: implications for binding to the hiv envelope protein gp production of human papillomavirus type virus-like particles in transgenic plants comprehensive cross-clade neutralization analysis of a panel of anti-human immunodeficiency virus type monoclonal antibodies viral and murine interleukin- are correctly processed and retain their biological activity when produced in tobacco proteins that bind high-mannose sugars of the hiv envelope discovery of cyanovirin-n, a novel human immunodeficiency virus-inactivating protein that binds viral surface envelope glycoprotein gp : potential applications to microbicide development development of topical microbicides to prevent the sexual transmission of hiv a new c-type lectin similar to the human immunoreceptor dc-sign mediates symbiont acquisition by a marine nematode antibody domain exchange is an immunological solution to carbohydrate cluster recognition non-nucleoside hiv- reverse transcriptase (rt) inhibitors: past, present, and future perspectives in planta protein sialylation through over-expression of the respective mammalian pathway rapid high yield production of different glycoforms of ebola virus monoclonal antibody geminiviral vectors based on bean yellow dwarf virus for production of vaccine antigens and monoclonal antibodies in plants actinohivin, a novel anti-human immunodeficiency virus protein from an actinomycete, inhibits viral entry to cells by binding high-mannose type sugar chains of gp nucleotide hiv reverse transcriptase inhibitors: tenofovir and beyond antigenicity and immunogenicity of the hiv- gp epitope eldkwa inserted into permissive sites of the male protein accumulation of functional recombinant human coagulation factor ix in transgenic soybean seeds expression of functional recombinant human growth hormone in transgenic soybean seeds vaginal microbicides and the prevention of hiv transmission development of novel vaccines and therapeutics using plant-based expression systems production of antibodies in plants: status after twenty years binding of the f monoclonal antibody to native and fusion-intermediate forms of human immunodeficiency virus type gp : implications for fusion-inducing conformational changes multiple antiviral activities of cyanovirin-n: blocking of human immunodeficiency virus type gp interaction with cd and coreceptor and inhibition of diverse enveloped viruses preclinical evaluation of anti-hiv microbicide products: new models and biomarkers envelope glycans of immunodeficiency virions are almost entirely oligomannose antigens antibodies to hiv- : aiming at the right target griffithsin, a potent hiv entry inhibitor, is an excellent candidate for anti-hiv microbicide success stories in molecular farming-a brief overview synergistic activity profile of griffithsin in combination with tenofovir, maraviroc and enfuvirtide against hiv- clade c gmp issues for recombinant plant-derived pharmaceutical proteins biochemical and functional characterization of anti-hiv antibody-elp fusion proteins from transgenic plants potential of carbohydrate-binding agents as therapeutics against enveloped viruses anti-hiv- antibodies f and e interact differently with lipids to bind their epitopes a monovalent mutant of cyanovirin-n provides insight into the role of multiple interactions with gp for antiviral activity the future of hiv microbicides: challenges and opportunities advances in development, scale-up and manufacturing of microbicide gels, films, and tablets carbohydrates of human immunodeficiency virus. structures of oligosaccharides linked to the envelope glycoprotein transgenic plants as factories for biopharmaceuticals the microbicide cyanovirin-n expressed on the surface of commensal bacterium streptococcus gordonii captures hiv- recombinant production of anti-hiv protein, griffithsin, by auto-induction in a fermentor culture rapid high-yield expression of full-size igg antibodies in plants co-infected with noncompeting viral vectors magnifection-a new platform for expressing recombinant vaccines in plants the lectins: carbohydrate-binding proteins of plants and animals biopharmaceutical production in plants: problems, solutions and opportunities plant-specific glycosylation patterns in the context of therapeutic protein production isolation, primary sequence determination, and disulfide bond structure of cyanovirin-n, an anti-hiv (human immunodeficiency virus) protein from the cyanobacterium nostoc ellipsosporum cyanovirin-n inhibits hepatitis c virus entry by binding to envelope protein glycans fc receptor but not complement binding is important in antibody protection against hiv broadly neutralizing monoclonal antibodies f and e directed against the human immunodeficiency virus type gp membrane-proximal external region protect against mucosal challenge by simian-human immunodeficiency virus shiv ba-l actinohivin, a broadly neutralizing prokaryotic lectin, inhibits hiv- infection by specifically targeting high-mannose-type glycans on the gp envelope blockade of attachment and fusion receptors inhibits hiv- infection of human cervical tissue high-level rapid production of full-size monoclonal antibodies in plants by a single-vector dna replicon system safety concerns for the potential use of cyanovirin-n as a microbicidal anti-hiv agent molecular cloning of actinohivin, a novel anti-hiv protein from an actinomycete, and its expression in escherichia coli recombinant antibody therapeutics: the impact of glycosylation on mechanisms of action effectiveness and safety of tenofovir gel, an antiretroviral microbicide, for the prevention of hiv infection in women safety and effectiveness of buffergel and . % pro gel for the prevention of hiv infection in women engineering an obligate domain-swapped dimer of cyanovirin-n with enhanced anti-hiv activity encyclopedia of molecular cell biology and molecular medicine function and glycosylation of plant-derived antiviral monoclonal antibody n-glycosylation in the moss physcomitrella patens is organized similarly to that in higher plants isolation and characterization of banlec-i, a mannoside-binding lectin from musa paradisiac (banana) protein n-glycosylation: molecular genetics and functional significance nucleocytoplasmic plant lectins plant seeds as bioreactors for recombinant protein production trimeric membrane-anchored gp inhibits hiv membrane fusion cell culture processes for monoclonal antibody production mechanism of neutralization by the broadly neutralizing hiv- monoclonal antibody vrc engineered vaginal lactobacillus strain for mucosal delivery of the human immunodeficiency virus inhibitor cyanovirin-n induction of apoptosis by polygonatum odoratum lectin and its molecular mechanisms in murine fibrosarcoma l cells plant lectins: potential antineoplastic drugs from bench to clinic production of monoclonal antibodies with a controlled n-glycosylation pattern in seeds of arabidopsis thaliana dimeric g as a potent protection against hiv- the production of recombinant pharmaceutical proteins in plants optimization of human papillomavirus type (hpv- ) l expression in plants: comparison of the suitability of different hpv- l gene variants and different cell-compartment localization in planta engineering of viral rna replicons: efficient assembly by recombination of dna modules delivered by agrobacterium protection of macaques against pathogenic simian/human immunodeficiency virus . pd by passive transfer of neutralizing antibodies protection of macaques against vaginal transmission of a pathogenic hiv- /siv chimeric virus by passive infusion of neutralizing antibodies hiv- neutralization profile and plant-based recombinant expression of actinohivin, an env glycan-specific lectin devoid of t-cell mitogenic activity safety and tolerability of buffergel, a novel vaginal microbicide, in women in the united states a recombinant allosteric lectin antagonist of hiv- envelope gp interactions hiv- vaccine development: constrained peptide immunogens show improved binding to the anti-hiv- gp mab public health aspects of hiv/aids in low and middle income countries plants as biofactories advances in the development of microbicides for the prevention of hiv infection cyanovirin-n, a potent human immunodeficiency virus-inactivating protein, blocks both cd -dependent and cd -independent binding of soluble gp (sgp ) to target cells, inhibits scd -induced binding of sgp to cell-associated cxcr , and dissociates bound sgp from target cells construction and enhanced cytotoxicity of a cyanovirin-n-pseudomonas exotoxin conjugate against human immunodeficiency virus-infected cells isolation and characterization of griffithsin, a novel hiv-inactivating protein, from the red alga griffithsia sp microbicides and hiv prevention: lessons from the past, looking to the future a prospective randomized double blind placebo-controlled phase pharmacokinetic and safety study of a vaginal microbicide gel containing three potent broadly neutralizing antibodies ( fs, g , e ) (mabgel) vaginal microbicides: where are we and where are we going? monomerization of viral entry inhibitor griffithsin elucidates the relationship between multivalent binding to carbohydrates and anti-hiv activity a conserved neutralizing epitope on gp of human immunodeficiency virus type c-type lectin mermaid inhibits dendritic cell mediated hiv- transmission to cd + t cells biologically active proteins from natural product extracts scaleable manufacture of hiv- entry inhibitor griffithsin and validation of its safety and efficacy as a topical microbicide component broad-spectrum in vitro activity and in vivo efficacy of the antiviral protein griffithsin against emerging viruses of the family coronaviridae antibody protects macaques against vaginal challenge with a pathogenic r simian/human immunodeficiency virus at serum levels giving complete neutralization in vitro elastin-like polypeptide fusions enhance the accumulation of recombinant proteins in tobacco leaves plant-made pharmaceuticals: leading products and production platforms the anti-hiv cyanovirin-n domain is evolutionarily conserved and occurs as a protein module in eukaryotes lectins as plant defense proteins the rise and fall of polyanionic inhibitors of the human immunodeficiency virus type making an ally from an enemy: plant virology and the new agriculture mucosal delivery of microbicides by recombinant commensal bacteria: expression of the hiv-inactivating protein cyanovirin-n in gram-positive bacteria recombinant antibody g produced in maize endosperm efficiently neutralizes hiv- and contains predominantly single-glcnac n-glycans cost-effective production of a vaginal protein microbicide to prevent hiv transmission high level protein expression in plants through the use of a novel autonomously replicating geminivirus shuttle vector hiv microbicides: state-of-the-art and new perspectives on the development of entry inhibitors transient expression in tobacco leaves of an aglycosylated recombinant antibody against the epidermal growth factor receptor host plant preference of aphids, thrips and spider mites on gna-expressing and control potatoes virus-derived ssdna vectors for the expression of foreign proteins in plants functional analysis of the broadly neutralizing human anti-hiv- antibody f produced in transgenic by- suspension cultures extremely high-level and rapid transient protein production in plants without the use of viral replication expression of multiple proteins using full-length and deleted versions of cowpea mosaic virus rna- rapid transient production in plants by replicating and non-replicating vectors yields high quality functional anti-hiv antibody the broadly neutralizing anti-human immunodeficiency virus type antibody g recognizes a cluster of mannose residues on the outer face of gp production of a monoclonal antibody in plants with a humanized n-glycosylation pattern aromatic residues at the edge of the antibody combining site facilitate viral glycoprotein recognition through membrane interactions hiv coreceptor cxcr antagonists hiv co-receptor inhibitors as novel class of anti-hiv drugs transgenic plant production of cyanovirin-n, an hiv microbicide design, expression, and characterization of a multivalent, combination hiv microbicide production of glucocerebrosidase with terminal mannose glycans for enzyme replacement therapy of gaucher's disease using a plant cell system selective interactions of the human immunodeficiency virus-inactivating protein cyanovirin-n with high-mannose oligosaccharides on gp and other glycoproteins human immunodeficiency virus type elite neutralizers: individuals with broad and potent neutralizing activity identified by using a high-throughput neutralization assay together with an analytical selection algorithm treatment of influenza a (h n ) virus infections in mice and ferrets with cyanovirin-n expression of the legume symbiotic lectin genes psl and gs promotes rhizobial colonization of roots in rice generation of arabidopsis thaliana plants with complex n-glycans lacking β , -linked xylose and core α , -linked fucose improved virus neutralization by plant-produced anti-hiv antibodies with a homogeneous , -galactosylated n-glycan profile mechanism by which the lectin actinohivin blocks hiv infection of target cells plant-derived mouse igg monoclonal antibody fused to kdel endoplasmic reticulum-retention signal is n-glycosylated homogeneously throughout the plant with mostly high-mannose-type n-glycans human monoclonal antibody g defines a distinctive neutralization epitope on the gp glycoprotein of human immunodeficiency virus type delay of hiv- rebound after cessation of antiretroviral therapy through passive transfer of human neutralizing antibodies cyanovirin-n gel as a topical microbicide prevents rectal transmission of shiv . p in macaques molecular farming in plants: host systems and expression technology transgenic plants in the biopharmaceutical market the production of vaccines and therapeutic antibodies in plants report on the global hiv- /aids epidemic isolation and characterization of a lectin with exclusive specificity towards mannose from snowdrop (galanthus nivalis) bulbs plant lectins: a composite of several distinct families of structurally and evolutionary related proteins with diverse biological roles effectiveness of col- , a nonoxynol- vaginal gel, on hiv- transmission in female sex workers: a randomised controlled trial cytoplasmic/nuclear plant lectins: a new story poor biologic activity of cross-reactive ige directed to carbohydrate determinants of glycoproteins efficient capture of antibody neutralized hiv- by cells expressing dc-sign and transfer to cd + t lymphocytes prevention of virus transmission to macaque monkeys by a vaginally applied monoclonal antibody to hiv- gp the current status of plant transformation technologies a minimal dna cassette as a vector for genetic transformation of soybean (glycine max) broad and potent neutralizing antibodies from an african donor reveal a new hiv- vaccine target enhancement of resistance to aphids by introducing the snowdrop lectin gene gna into maize plants design and expression of a dimeric form of human immunodeficiency virus type antibody g with increased neutralization potency rational design of envelope identifies broadly neutralizing human monoclonal antibodies to hiv- the antiviral protein cyanovirin-n: the current state of its production and applications interactions between lipids and human anti-hiv antibody e can be reduced without ablating neutralizing activity high yield recombinant silk-like protein production in transgenic plants through protein targeting neutralization of hiv by milk expressed antibody. poster presented at federation of pegylation of cyanovirin-n, an entry inhibitor of hiv a novel lectin with highly potent antiproliferative and hiv- reverse transcriptase inhibitory activities from the edible wild mushroom russula delica structural definition of a conserved neutralization epitope on hiv- gp structural studies of algal lectins with anti-hiv activity domain-swapped structure of the potent antiviral protein griffithsin and its mode of carbohydrate binding broadly neutralizing antibodies targeted to the membrane-proximal external region of human immunodeficiency virus type glycoprotein gp this research forms part of a phd study that is funded by the council for scientific and industrial research (csir), biosciences, pretoria, south africa. key: cord- -k gs ohp authors: makhzoum, abdullah; benyammi, roukia; moustafa, khaled; trémouillaux-guiller, jocelyne title: recent advances on host plants and expression cassettes' structure and function in plant molecular pharming date: - - journal: biodrugs doi: . /s - - - sha: doc_id: cord_uid: k gs ohp plant molecular pharming is a promising system to produce important recombinant proteins such as therapeutic antibodies, pharmaceuticals, enzymes, growth factors, and vaccines. the system provides an interesting alternative method to the direct extraction of proteins from inappropriate source material while offering the possibility to overcome problems related to product safety and source availability. multiple factors including plant hosts, genes of interest, expression vector cassettes, and extraction and purification techniques play important roles in the plant molecular pharming. plant species, as a biosynthesis platform, are a crucial factor in achieving high yields of recombinant protein in plant. the choice of recombinant gene and its expression strategy is also of great importance in ensuring a high amount of the recombinant proteins. many studies have been conducted to improve expression, accumulation, and purification of the recombinant protein from molecular pharming systems. re-engineered vectors and expression cassettes are also pivotal tools in enhancing gene expression at the transcription and translation level, and increasing protein accumulation, stability, retention and targeting of specific organelles. in this review, we report recent advances and strategies of plant molecular pharming while focusing on the choice of plant hosts and the role of some molecular pharming elements and approaches: promoters, codon optimization, signal sequences, and peptides used for upstream design, purification and downstream processing. a plant molecular pharming system has been thoroughly explored over the last two decades. molecular pharming has had positive impacts on the production of pharmaceutically important proteins. it has proven that some pharmaceuticals displaying advantageous clinical properties over their native counterparts could be produced in efficient ways by molecular pharming technologies. as molecular pharming platforms, plants are excellent biofactories for the production of drugs, antibodies, and vaccines in various host systems such as whole transgenic plants, cell suspension culture, hairy roots, and hydroponic culture [ ] [ ] [ ] . however, based on the biochemical pathway features, plant species are different in their ability to express and accumulate recombinant proteins. various plant species have already been investigated for their relevance to produce bioactive molecules in an artificial way in plants. table summarizes some human antibodies and vaccines produced in different plant species and eukaryotic algae. thanks to important advantages over other prokaryotic or eukaryotic systems, plants have gained great importance in molecular pharming because they offer ( ) reduced contamination risks, ( ) reduced costs, ( ) scaling-up possibilities [ ] [ ] [ ] , and ( ) synthesizing of large and complex protein compounds while retaining their activities (post-translational modifications) [ ] . there is, however, a major drawback to a plant molecular system regarding low expression and accumulation levels of some foreign proteins. to overcome this shortcoming, several studies have been conducted to improve the different aspects that allow increasing the yield of the recombinant proteins in plants. some of these studies have focused on the choice of plant hosts, others on the bioengineering strategies of the target proteins and the expression cassettes with their components such as promoters, terminal sequences, epitope tags, and signal peptides. these aspects must be taken into account when we attempt to maximize the expression and yield of the targeted proteins to increase their production. intensified efforts have also been done to improve heterologous gene structures and codon optimization. here, we review these aspects and report recent advances in the improvements of plant molecular pharming to increase protein yield and accumulation based on upstream and downstream processing studies and empirical essays. the choice of suitable plants for molecular pharming technology is an essential factor for the success of the plant molecular pharming approach. the choice of host plant depends on a broad range of criteria including the nature of potato [ ] hbsag-env, gag epitopes of hiv- vaccine tomato hepatitis [ ] norwalk virus capsid protein (nvcp) vaccine potato gastroenteritis phase i [ ] major capsid protein vp vaccine potato severe viral diarrhea preclinical [ ] hbsag hepatitis b surface antigen, hsv herpes simplex virus, icam intercellular adhesion molecule, lt-b heat labile enterotoxin b subunit, mab monoclonal antibody, tgev transmissible gastroenteritis virus the protein, ability for transformation and regeneration, post-translational modifications, scale-up of production and maintenance costs, span of production cycles, and the downstream processing requirements. a wide range of plant crops have thus been tested for molecular pharming purposes, including leafy crops, cereal and legume seeds, oil crops, plant cell suspensions, hairy roots, and microalgae. leafy crops are helpful in terms of biomass yield and high soluble protein levels [ ] . additionally, leaf harvesting does not need flowering and thus significantly reduces contamination through pollen or seed dispersal [ ] . however, there is a major problem of instability of the expressed proteins in leaves due to proteolytic degradation with aging of the leaves. in fact, the instability of proteins present in leaf cells, and also in cells of the other plant tissues, may start during the translation of the foreign proteins, which hold a natural tendency towards a structural heterogeneity in a heterologous environment [ ] . despite this, one of the major causes of protein instability inside the leaf cells is the presence of numerous proteolytic vacuoles in their cytoplasm. in fact, the mature leaves possess very large extra cytoplasmic vacuolar compartments containing numerous active proteolytic enzymes that are involved in the degradation of native and foreign proteins, notably after harvesting or during downstream processing (extraction/ purification from freshly collected leaves). one main lifesupporting function of the vacuolar compartment is protein breakdown. indeed, numerous vacuoles are major sites of cellular proteolysis and contribute largely to amino acid recycling in the cell, in vivo [ ] . to avoid this hurdle, the leaves must be processed immediately at the farm or transported as dried or frozen material [ ] . moreover, protein expression in plant aerial parts could affect the growth and development of the host plant. tobacco is the most suitable leafy crop for many reasons such as high biomass yield, well-established technology for gene transfer and expression, year-round growth and harvesting, and the existence of large-scale infrastructure for processing. furthermore, tobacco has little risk in contaminating either food or feed chains because it is a non-food or non-feed crop. although many tobacco cultivars contain high levels of toxic alkaloids and phenolic substances [ ] , these compounds can be removed during the purification process. the use of transgenic tobacco chloroplasts as an alternative bioreactor presents important advantages including high transgene-copy number and high level of accumulated proteins with reduced toxicity for the host plant [ ] . many recombinant proteins have been produced at high levels in tobacco chloroplasts. for example, oey et al. [ ] obtained a proteinaceous antibiotic by transformation of the tobacco chloroplast by up to % of the total soluble proteins (tsp), which is the highest recombinant protein accumulation accomplished so far in plants. more recently, a proinsulin has been expressed in tobacco and lettuce chloroplasts with reduced cost and facility for oral delivery [ ] . alternative leafy crops such as alfalfa, lettuce, and soybean are also being investigated as suitable hosts for molecular pharming [ ] . alfalfa and soybean produce lower amounts of leaf biomass than tobacco, but they have a major advantage of using atmospheric nitrogen through symbiotic relations, resulting in reduced fertilizer inputs [ ] . alfalfa is particularly useful because it can be harvested up to nine times a year and has high leaf protein content (up to mg total protein per gram in fresh weight). medicago inc. company (http://www.medicago. com) has invested in the development of transformation methods for the production of recombinant proteins in alfalfa leaves in order to improve the yield of antibodies with human-like n-glycans [ ] . lettuce is also being investigated as a production host for edible recombinant vaccines and it has been used in a series of clinical trials for vaccine production against hepatitis b [ ] , pig edema disease [ ] , pneumonic plague [ ] , cholera [ ] , severe acute respiratory syndrome coronavirus [ ] , and porcine epidemic diarrhea viruses [ ] . one disadvantage, however, of the chloroplast transgenic system is that plant plastids do not ensure some of the important post-translational modifications, such as glycosylation. the latter is required for the function of many human proteins; the only organelle that can achieve this process is the golgi apparatus. however, there are differences between the models of human and plant glycosylation, and these differences can cause an immune response in humans [ ] . therefore, plastid-based expression technology is limited to the production of non-glycosylated products [ ] . seeds of cereals and legumes offer many advantages, in particular the long stability of their expressed proteins because seeds have an appropriate biochemical and physiologic environment such as dormancy and low water content that promote stable protein accumulation [ ] . indeed, the lost water by desiccation changes the ph ambient, consequently decreasing the activity of proteases inside seed cells and protecting the recombinant protein contents. hence, the protein storage in seed endosperm allows long-term storage for at least years at room temperature with no detectable loss of protein activity [ ] . cereal seeds also lack the phenolic substances that are present in tobacco leaves, increasing the efficiency of downstream processing [ ] . the high seed protein content, ranging from to %, is translated into high expression for several seed-targeted recombinant proteins [ ] [ ] [ ] [ ] . however, the overall yields of recombinant proteins in seed crops are much lower than in tobacco. additionally, the transgenic plants must pass through a flowering cycle to produce seeds; therefore, there is a possibility of contamination that may occur by pollen transfer [ , ] . maize, rice, barley, and wheat are the most important platforms for seed recombinant protein production. generally, maize seeds are excellent bioreactors because their kernel sizes are large (with % of endosperm), which is the principal site of protein accumulation [ , ] . maize also has several advantages such as having the highest biomass yield among food crops, and ease of transformation and scaling-up [ ] . the foremost disadvantage of using maize as a bioreactor is that it is a cross-pollinating plant, and thus there is substantial risk of contamination of maize crops by their transgenic counterparts [ ] . similar to maize, rice is easy to transform and to scale-up with a selfpollinating trait, reducing the probability of horizontal gene flow [ ] . recent studies report that rice has the potential to offer an oral delivery system for vaccine antigens and therapeutic proteins [ , ] . like rice, barley is a selfpollinating crop and could be considered a better choice than maize [ ] . however, barley is less widely grown than maize and harder to transform [ , ] . on the other hand, wheat shows the lowest yields per unit biomass and it is still under development and improving transformation efficiency [ ] . legume seeds are also an interesting production system because they have exceptionally high protein content ( - %) , which could produce high yields of recombinant proteins too. soybean seeds offer the advantage of a low risk of contamination by pollen because soybean is largely self-pollinating, with a low production cost. furthermore, soybean has been explored for its efficacy in expressing human growth hormone [ ] and humanized antibody against herpes simplex virus [ ] . similar to soybean, peas present comparable advantages. however, only a few studies have reported on the use of pea seeds as a platform for commercial production of recombinant proteins. transgenic pea seeds are used as bioreactors for the production of a single-chain fv fragment (scfv) antibody [ ] . on the other hand, saalbach et al. [ ] have reported a high level of expression of scfv antibody with % of the total seed protein. because of their inherently low associated proteolytic activity and simplified protein isolation via oil body separations, oleosin-fusion is an emerging approach as a promising system for recombinant protein production. the target recombinant protein is produced as a fusion with oleosin, accumulates in seed oil bodies, and can be purified using simple extraction and centrifugation procedures followed by the release of the target protein by proteolytic cleavage [ , ] . however, oleosin expression levels are still not high enough for economical production. safflower seeds are also considered advantageous for their high protein yield, low acreage, and self-pollination [ ] . plant cell suspension culture has been recognized as a promising alternative biosynthetic platform for valuable proteins, and combines the qualities of whole-plant systems with those of microbial fermentation and mammalian cell culture [ , ] . suspension cells in particular increase safety compared with mammalian cell systems which can harbor human pathogens. additionally, it is well-suited for product isolation when it is secreted into the culture medium. the first plant-produced commercial therapeutic protein for human infusion, glucocerebrosidase, was produced in carrot cells [ ] , though other recombinant proteins have previously been produced in plants for therapeutic purposes [ , ] . insertion into the plant nuclear genome of the t-dna segment, carried on a voluminous and extrachromosomal ri-plasmid (ri for root-inducing) present in the phytopathogen gram-negative soil bacterium, agrobacterium rhizogenes [ , ] , leads to the emergence of neoplastic adventitious roots, referred to as ''hairy root syndrome'' [ , ] . the tumorous roots, resulting from an a. rhizogenes-mediated transformation, can be grown indefinitely in vitro and have become a viable alternative production platform for heterologous proteins, as well as for other plant metabolites naturally biosynthesized in wild roots [ ] . similar to suspension cells, hairy roots can be axenically cultured in controlled and contained environments for the production of high-value pharmaceutical proteins. in addition, the possible extracellular secretion or rhizosecretion of expressed proteins from hairy root cultures [ ] offers a simplified method for the recovery of foreign proteins from a low-cost process. the advantages of rapidly growing hairy roots over suspension cells include long-term genotype and phenotype stability, efficient productivity, and good growth on hormone-free media [ ] . the swiss biotechnology company rootec (http://www. rootec.com) is currently focusing on the commercialization of plant-derived products with hairy roots grown in mist bioreactors. hairy roots appear to be an attractive in vitro expression system with several advantages compared with field-grown plants and suspension cultured cells. other systems, such as eukaryotic algae and higher aquatic plants (e.g., duckweeds) have been envisaged for heterologous protein expression. the main advantages of these platform types are fast growth, easy harvest, and high protein contents (up to % dry weight) [ ] . moreover, these eukaryotic systems are able to produce glycosylated proteins contrary to the microbial cultures and their production costs are lower than that of the mammalian cells [ ] . biolex, inc. (http://www.biolex.com) is working to develop a duckweed-based expression system to produce recombinant pharmaceuticals and proteins. the leading product of biolex, a hepatitis c treatment, is a controlledrelease interferon (ifn)-a b that is currently on the market [ ] . monoclonal antibodies (mabs), i.e., anti-cd mab and anti-cd mab, represent another range of products that are actually in preclinical development [ ] . microalgae also offer potential production systems of recombinant proteins because of their ease of genetic transformation, rapid growth, short life cycles, and cost effectiveness [ ] . currently, most of the work is achieved with one green unicellular alga, chlamydomonas reinhardtii [ ] . c. reinhardtii taken as a recombinant protein expression system combines, at once, the traditional bacterial fermentation or mammalian cell-culture methods. these microalgae offer post-translational modification systems that are not present in bacteria and essential for the biological activity of many therapeutic proteins [ ] . furthermore, c. reinhardtii has a rapid growth rate with a doubling time of h under optimal growth conditions and supports easy nuclear and chloroplast genome transformation. therefore, only the chloroplast is considered a realistic production system for a high level of recombinant protein accumulation [ , ] . however, as mentioned earlier, the principal limitation of chloroplast recombinant protein production is that the expressed proteins lack posttranslational modifications such as glycosylation [ ] . despite this limitation, functional recombinant proteins, including antibodies and growth factors [ ] , vaccines [ ] , and autoantigens [ ] have been expressed in the chloroplast of c. reinhardtii. plant molecular pharming technology has attracted more attention in the corporate world and more international companies are specializing in this field. table provides the names and web addresses of biotech companies working on molecular pharming to develop relevant plant platforms. the high costs of the production of drugs in transgenic plants can be minimized by increasing the expression levels of the target molecules. to improve protein expression levels, it is necessary to understand the functional gene structure and all genetic and epigenetic parts in spatio-temporal expression context. the prerequisite for making the system effective in molecular pharming is to improve the expression level based on the expression cassette structures to enhance the transcription and translation of recombinant proteins. to optimize the transcription of recombinant proteins, some approaches have been envisaged with a focus on promoter elements, translation and stability of the transcripts, leader sequences and exogenous or endogenous signal peptides. tandem repeats of recombinant proteins or peptides and rna interference (rnai) technology to suppress the proteinase activity have also been attempted in order to increase the yield of proteins of interest. gene promoter is an essential element in genetic engineering for plant molecular pharming. promoter can have ubiquitous expression ( s promoter) or spatiotemporal expression features as deacetylvindoline -o-acetyl transferase gene promoter from the medicinal plant catharanthus roseus [ ] . for its strength and efficiency to express recombinant genes in living cells, the s promoter is widely used for driving the expression of various recombinant genes in the molecular pharming industry [ , [ ] [ ] [ ] . the human growth factor placental lactogen (hpl) is an example transcribed by s promoter in transgenic tobacco cells [ ] . the s promoter is used as single or multiple copies to increase gene transcription efficiency. it is used, for example, as a part of the vector prjc-htf to express the human serum transferrin (htf) in transgenic tobacco [ ] , and in arabidopsis protoplasts and maize callus to study the localization of the escherichia coli heatlabile enterotoxin b subunit (lt-b) [ ] . dual s promoter is used as a part of the pzp vector to increase the expression levels of the surface antigen (sag ) [from to ] open reading frame (orf) antigen of toxoplasma gondii in tobacco leaves [ ] . although s promoter is a good choice for molecular pharming systems, it has moderate expression levels in some seed crops expressing recombinant proteins due to the lack of cis-acting regulatory elements specific for promoters of mature seeds' genes and the elements on which specific transcription factors bind to activate or repress the transcription. moreover, s promoter is not as efficient in monocots as in dicots. for this reason, monocot seed-adapted promoters have to be used instead of s promoter. as such, the fatty acid elongase (fad) was employed as a seed-specific promoter to express a chimeric protein in canola seeds [ ] . the maize kda c-zein promoter with c-zein signal peptide was also used as a part of recombinant gene cassettes to drive the gene expression of the cholera toxin b subunit (ct-b) in maize seeds [ , ] . another example of seed-specific promoters is the barley horde in promoter ( . kb), which is employed to express and produce an active recombinant human hematopoietic growth factor flt in pb gl- in barley plants [ ] . on the other hand, it was demonstrated that extending the length of the globulin promoter from . to kb or using three tandem repeats of the . kb globulin promoter was a successful strategy to increase the expression level of the hepatitis b surface antigen (hbsag) in maize seeds. the obtained yield of this antigen shifted from . to . % of the tsp [ ] . on the other hand, inducible promoters are a powerful approach to restricting the gene expression at the spatiotemporal level and expressing the recombinant genes when and where they are needed. for example, the rice gene promoter a-amy , which is strongly inducible by sugar depletion, was successfully employed to produce the mouse granulocyte-macrophage colony-stimulating factor (mgm-csf) in rice suspension culture with a final yield up to % of total secreted proteins [ ] the microalgae c. reinhardtii has also employed for production of recombinant proteins based either on a nuclear or a chloroplast gene expression systems. for nuclear one, a few promoters have been used such as the promoters of the photosystem i complex, ribulose bisphosphate carboxylase, heat shock protein a genes while for chloroplast gene expression systems, other genes promoter were exploited as the rbcl (ribulose bisphosphate), atpa (alpha subunit of adenosine triphos-phatase), psbd (photosystem ii d ) and psba (photosys-tem ii psba) promoters are considered a good choice. these promoters have been found to be more efficient when they are used with their -and -untranslated regions ( utr and utr) [ ] . chloroplast-based systems have the advantage of avoiding the nuclear gene silencing and methylation with enhanced increasing of the level of transgene expression. tobacco chloroplast is envisioned as a relevant host to produce native and synthetic insulin-like growth factor (igf)- (igf- s), while employing its specific regulatory elements including utr and utr regions and s rrna (ribosomal rna) promoter as part of the expression cassette. the chloroplast recombinant protein is fully functional based on its ability to stimulate the growth and proliferation of the human hu- cells [ ] . another example of using chloroplast as a model to produce human recombinant protein is the production of the ifn-a gene in tobacco chloroplasts [ ] . in this study, the chloroplast rbcl light-inducible promoter was able to produce a yield up to , pg/g fresh weight [ ] . table summarizes some promoters and other elements in the expression cassettes used in molecular pharming systems. in addition to the importance of promoter architecture for gene expression in molecular pharming, other strategies based on using specific peptides at n-and c-termini have been employed to enhance the transcript level of recombinant proteins. these peptides are recognizable by the host system and can increase the level of transcription and translation of recombinant proteins with enhanced stability and protection against protease degradation by targeting recombinant protein in the secretory pathway to specific organelles [ ] . the cereal a-amylase signal peptide is largely used in seed-specific vectors, e.g., to produce the mouse mgm-csf in a gateway vector under the control of the a-amylase gene promoter [ ] . replacing the native signal with a-amylase signal peptide did not show significant effect on the expression and accumulation of the protein in transgenic tobacco and potato [ ] . the signal peptide from the s seed storage protein was successfully used to direct the human recombinant proteins into a seed secretory pathway in arabidopsis, petunia, and tobacco [ , ] . another signal peptide, the ap osmotin, is used to target the surface-expressed antigen (sag ) and granule dense (gra ) to the apoplast of tobacco leaves [ ] . the bacterial lt-b from e. coli can drive the localization of recombinant proteins to the secretory pathway in arabidopsis protoplasts and maize [ ] . the sequence kdel (lys-asp-glu-leu), encoding for endoplasmic reticulum (er) retention signal, was used to express the mouse (mgm-csf) into tobacco transgenic plants [ ] . kozak consensus peptide plays an important role in eukaryotic gene expression system [ ] . kozak and kdel (vacuole retention peptides) are largely exploited to enhance the expression, stability, and retention of recombinant protein [ ] . ma and co-workers [ ] showed that pleiotropic cytokine interleukin (il)- expression level under the control of s promoter in transgenic tobacco and potato was increased when using constructs containing the er sekdel (ser-glu-lys-asp-glu-leu) in c-terminal. hdel (his-asp-glu-leu) retention peptide was also shown to be crucial for er targeting of the igg-hdel fusion into transgenic tobacco plants [ ] . the histidine tag (his-tag) is widely used for targeting and facilitating the purification of recombinant proteins [ , ] . the zz linker from staphylococcus aureus is also used as an epitope tag in chloroplast expression cassette to facilitate the purification process of synthetic and native igf- [ ] . other specific signals and peptides are used for their suitability to host plants and their positive roles in transcription and/or translation of the recombinant proteins. table summarizes some of these specific signals and their usage as parts of the expression cassettes. re-engineering recombinant genes could generate active proteins with increased expression levels. the possibility utr -untranslated region, camv cauliflower mosaic virus, fw fresh weight, gad glutamic acid decarboxylase, hdel his-asp-glu-leu, hgad human glutamic acid decarboxylase , hfix human coagulation factor ix, hgm-csf human granulocyte-macrophage colonystimulating factor, hpv- human papillomavirus- , il- interleukin- , kdel lys-asp-glu-leu, mgm-csf mouse granulocyte-macrophage colony-stimulating factor, nls nuclear localization signal, rnai rna interference, sp storage protein, tsp total soluble protein of making a fusion between a few genes using linkers to assemble amino acids or orfs in a chimeric structure is a successful strategy in molecular pharming. using the optimal codon to match the endogen proprieties of the host plant can significantly increase the level of transcription and translation of recombinant proteins. amani et al. [ ] , for example, employed linkers to create a chimeric structure of three virulence factors: espa, intimin, and tir receptor from e. coli o :h . the designed vaccine was able to reduce bacterial infection and colonization in mice. another example was constructed from two heavy-and light-chain sequences of lo-bm antibody (a therapeutic igg) and expressed in tobacco bright yellow (by)- cells [ ] . the two chains were fused in tandem or inverted in a convergent orientation to be expressed as part of an expression cassette under the control of pma promoter with two s enhancer sequences. in this work, the tandem construct had a much higher level of expression than the inverted one in tobacco suspension cells and transgenic tobacco plants [ ] . a number of chimeric proteins have been constructed from multiple hiv proteins to be evaluated as hiv vaccines. targets for hiv vaccines included the different categories of hiv proteins: structural proteins (ex. gag, env, glycoprotein [gp] , gp , gp ); enzymes (pol); and regulatory proteins (nef, tat, rev, vpr). more information about potential vaccines reported as candidate plant-based vaccines for human is reviewed by soria-guerra et al. [ ] . protein accumulation could also be increased by duplicating a small peptide to form a large multimeric protein. for example, ten sequential tandem repeats of the small peptide glucagon-like peptide- (glp- ) were combined into one large synthetic gene that was introduced to tobacco [ ] . western blot analysis revealed that the multimerized glp- protein accumulated up to . % of the tsp in transgenic tobacco leaves. additionally, cerovska et al. [ ] constructed a chimeric version of an epitope driven from the human papillomavirus type l (amino acids from to ), fused to the coat protein of the potato virus x (pvx cp). then, the chimeric construction tamavidin /fungal avidin-like protein that binds biotin with high affinity, a versatile affinity tag psp sba-tamavidin (sba-s-tm ) fusion tobacco by- cells mg/l highly purified ( - % purity) on the microbeads [ ] thrombin protease/cleavage site ptz human interferon-a tobacco chloroplasts/ , pg/g fw [ ] basb barley amylase signal peptide, ct-b cholera toxin b subunit, er endoplasmic reticulum, fw fresh weight, gfp green fluorescence protein, hbsag hepatitis b surface antigen, hfbi hydrophobin, hgh human growth hormone, orf open reading frame, sba soybean agglutinin, tsp total soluble protein was transiently expressed in transgenic tobacco with a resulting yield of the fused protein of mg/kg of the fresh leaf tissue. codon optimization of the surface antigen, sag - from toxoplasma gondii (protozoan parasite), was investigated in transgenic tobacco leaves [ ] . the synthetic orf of sag was designed by overlapping oligonucleotides strategy with codon optimized by er signal peptide. the synthetic sag was then introduced into the plant binary vector, pzp , and expressed in tobacco leaves. it was found that the tobacco leaves transformed with unmodified sag gene accumulated protein five-to tenfold more than the leaves transformed with a codon-optimized sag gene [ ] . this may be due to the er localization signal that allowed high accumulation levels of the native sag . while this study shows a negative impact of the codon optimization on the expression of synthetic, optimized gene, daniell et al. [ ] found that igf- s was expressed, and fully functional, in tobacco chloroplasts at similar levels of the native igf protein. in fact, in a comparison study between the expression levels of the native igf- and an igf- s, the authors found that, after optimization of the igf- gene, the yield of the optimized igf- was . % of tsp, while the native igf- was . % of tsp. moreover, the expression of the igf- s was detected by western blots in e. coli, while no native igf- protein was observed, suggesting that the translation machinery in chloroplast is more flexible than in e. coli, with regard to codon optimization and usage [ ] . in plant molecular pharming, linkers are important elements for producing multimeric proteins, and introducing multiple and more complex traits. linkers are short peptide sequences composed of flexible residues such as glycine and serine between adjacent domains to ensure that these domains do not sterically interfere with each other. linkers are important elements assembling few proteins, orfs, or peptides together without interfering with the structure of fused units. linkers have no negative effects on the activity and stability of the assembled parts in the new structure. variable short peptides have been exploited as linkers in genetic engineering. for example, the linker eaaak was used to design a new synthetic gene encoding the carboxy-terminal fragment of intimin, the middle region of tir receptor and the carboxy-terminal part of the espa factor in e. coli [ , ] . the new synthetic gene was translated into transgenic canola and tobacco plants with a yield up to . - . % tsp in transgenic canola seeds and . - . % tsp in transgenic tobacco. another example is the ag simple alanine-glycine linker (ag linker), consisting of six amino acids with three ag repeats (agagag). this linker was used as a part of the expression cassette in plm , plm , and plm vectors and resulted in a positive shift of yield from undetectable to . % extracted from maize kernels [ ] . as a part of a cleavable polyprotein precursor with native kex p-like protease activity, the kex p linker (igkrgk) ef (amino acids leu/glu and glu/phe) was also successfully used to fuse red fluorescent protein (dsred) or human cytokine (gmcsf) to the green fluorescent protein (gfp) into the viral vector ttosa - spekcd and the two fusions were expressed in tobacco cells (referred to as rkg and gkg cells). the expression levels of rkg and gkg were . and . % of tsp, respectively [ ] . to test the effect of the length of linker on the accumulation of recombinant proteins, two different lengths were compared by plchova et al. [ ] . the first link was four amino acids in length (gpgp) and the second amino acids in length (sgggg) . both of them were employed to link different mutagenized versions of the human papillomavirus e oncoprotein with pvx cp in c-and n-terminal [ ] . the resulting fusions were then expressed in e. coli and tobacco (nicotiana benthamiana), but no significant difference was observed between the two lengths of linker on the accumulating protein constructs. however, the n-terminus fusions resulted in twofold higher protein yield than the c-terminus fusion (approximately . mg/ml culture medium against . mg/ml, respectively). more information about linkers and the choice of linkers are provided by soria-guerra [ ] and crasto and feng [ ] . the gateway recombination system is a relatively recent powerful tool used in molecular cloning and gene expression investigations. based on specific recombination sequences, the gateway system allows cloning and transferring of dna fragments in a high-throughput manner between different expression vectors while maintaining the orientation of the cloned reading frame [ ] . the system is based on the insertion of a dna fragment, or a complete orf, between two flanking recombination sequences in a specific plasmid vector to create an ''entry clone''. once an entry clone is created, the cloned dna could be sub-cloned to any gateway-designed expression vector, independent of the vector function or the host background [ ] . due to its advantage as a high-throughput cloning tool, gateway technology is suitable for creating 'molecular archives' containing the whole predicted orfs, orfeome, of an organism [ ] . different orfeome projects have thus been generated using gateway technology for protein expression and functional analysis, such as the yeast orfeome [ ] and the human orfeomes [ ] . the gateway orfeomes also allow straightforward expression of native or n-terminal and/or c-terminal fusion proteins [ ] . the use of gateway technology in molecular pharming is emerging at great pace. a recent study support this trend and reports the suitability of gateway-based vectors to transform plant chloroplast [ ] . the author of this study converted a standard transformation vector into a gateway destination vector in which they cloned the gfp associated with a ribosome binding site, t g . then, they transformed the tobacco chloroplast by the biolistic method, and found that gfp was successfully integrated into the plastid genome and resulted in an accumulation level of gfp protein up to % of the tsp. pcr testing and confocal laser scanning microscopy confirmed the presence of gfp in the chloroplast, suggesting that gateway system is suitable for plant pharming in this organelle. in another recent study, buntru et al. [ ] report that multigene constructs carrying seven transgenes, up to kb size, were successfully transformed into tobacco cells using gateway technology and were stably inherited for at least two generations. one of the implications of the findings in this study is the availability of a powerful and efficient tool for multigenes transformation that may facilitate the genetic engineering for the production of pharmaceutical compounds at an industrial scale. kagale et al. [ ] have also constructed a series of tobacco mosaic virus (tmv) compatible with gateway technology and intended for orfeome exploitation with the facility of n-or c-terminal fusions with a wide range of epitope tags. similarly, liu et al. [ ] have recently constructed a gateway-compatible binary t-dna destination vector to produce the mgm-csf in rice suspension cell culture under the control of the rice a-amy promoter with its signal peptide. a binary gateway vector, pk wg , has been successfully used to produce the human glutamic acid decarboxylase (gad ), an autoantigen implicated in type diabetes mellitus, in transgenic tobacco with protein accumulation up to . % of tsp, but retaining its autoantigenecity [ ] . morandini et al. [ ] also investigated the use of gateway vectors such as the pphasgw for seedspecific transgene expression system. the pphasgw vector was successfully employed to express a few human recombinant proteins such as a chimeric version of gad / , an isoform of the glutamic acid decarboxylase ( kda), a murine anti-inflammatory cytokine il- and proinsulin. these proteins were produced in arabidopsis and tobacco under the control of the specific seeds promoter phaseolin and resulted in a high yield of the chimeric gad / in arabidopsis seeds ( . % of tsp) while il- and proinsulin reached up to . and . % of tsp, respectively [ ] . the pphasgw vector was also a good choice to express a few other constructs harboring recombinant single-chain variable fragments to the crystallizing fragment of the antibody (scfv-fc) of two antiviral mabs ( g and ha ) in arabidopsis transgenic seeds and leaves under the control of the seed-specific bphaseolin promoter from phaseolus vulgaris; the maximal expression levels ranged from . to . mg recombinant protein per gram dry seed weight [ ] . more information about gateway-compatible plants can be found elsewhere [ , ] . . rna interference, gene silencing and suppressing of the proteases activity in molecular pharming the limitations to producing a high yield of recombinant proteins are sometimes related to the plant host system and its metabolism. this obstacle can be overcome by inhibiting some of the major metabolic activities in the plant host. for example, the production and accumulation of the recombinant human granulocyte colony-stimulating factor was significantly increased in transgenic rice suspension culture by an rnai approach designed to suppress the cysteine proteinase gene expression or by the inhibition of proteinase [ , ] . the inactivation of atp/adp transporters in potato tubers by rnai methodology increased the accumulation level of human recombinant mab, scfv l g , in the tubers up to twofold, with % increase in tuber biomass and % more soluble protein than wild-type tubers [ ] . in rice, it was found that the co-transformation of cell suspension culture with recombinant human granulocyte-macrophage colony-stimulating factor (rhgm-csf) and a serine proteinase inhibitor ii gene resulted in a reduction of the protease activity and an increase of the yield of the recombinant rhgm-csf by twofold compared with cells expressing rhgm-csf only [ ] . the strategy of a gene silencing suppressor was applied on a few systems to enhance the gene expression and recombinant protein accumulation. for example, adding a suppressor of gene silencing can have a positive role on gene expression such as the derivatives of peaq-ht vector, which was used to express transiently the human papillomavirus under the control of s promoter in agroinfiltrated n. benthamiana leaves [ ] . the anthrax receptor fusion protein co-expressed with viral gene silencing suppressors in agro-infiltrated tobacco leaves showed a tenfold increase in the recombinant protein compared with the absence of suppression, particularly with p suppressor [ ] . rice seeds are an efficient model to produce pharmaceuticals for human health, particularly when storage proteins ( kda prolamin and glutelin) have been silenced by the rnai technology, so the yield of the human growth hormone polypeptides reached the level of lg/g dry weight [ ] . protease activities, in turn, can be exploited to increase the level of recombinant proteins. the protease activities were investigated to express multiple secretory proteins in fusions between red fluorescent protein (dsred), or human cytokine (gmcsf), and downstream gfp as a single transgene by using a cleavable polyprotein precursors containing kex p linkers such as leagg(igkrgk) ef [ ] . using protease inhibitors in a specific manner demonstrated its potential to increase the recombinant protein yield. the transient expression of tomato cathepsin d, inhibitors of a and s proteinases, led to improvement of the murine diagnostic antibody c - accumulation and protection. it altered the a /s protease activities in the targeted specific cell compartment (leaf apoplast) in n. benthamiana and increased the protein content by % [ ] . pharmaceutical proteins glycosylation represents the most important post-translational modification that corresponds to covalent linkage of an oligosaccharide side chain to the natural or biopharmaceutical proteins [ ] . shared by all eukaryotic organisms, this fundamental mechanism significantly modulates folding, activity, yield, and immunogenicity of therapeutic glycoproteins [ ] . nevertheless, there are interkingdom differences in the glycosylation patterns, particularly in the n-glycoform design. n-glycosylation starts in the er and continues in the golgi apparatus with the addition of a( , )-fucose and b( , )-xylose residues to n-glycans in plants and with the addition of a( , )-fucose moieties, glucose, and acid sialic residues to n-glycans of glycoproteins in mammals [ ] . plant-specific xyloglucan is likely associated with allergenicity disorders limiting the use of therapeutic glycosylated proteins by parenteral administration. to overcome such hurdles, humanization of plant-made glycoproteins must be achieved. in recent years, promising strategies of glycoengineering have made humanizing plant n-glycolysation possible [ ] , either by inactivation of the host plant's endogenous glycosyltransferases (a , -fucosyltransferases and b , xylosyltransferase) or de novo expression of heterologous glycosyltransferases (b ( , ) galactosyltransferase and sialyltransferase), similar to those of mammals [ ] . besides, proteins resident of the er lumen possess high-man (mannose)-type n-glycans with structures common to plants and mammals [ ] . thereby, the addition of the h/kdel sequence at the c-terminal end of a secreted protein makes its retention in the er possible and its glycosylation is limited to only high mannose-type n-glycans [ ] . another non-immunogenic glycosylation approach, based on rna-interference, modulates the levels of xylosyl and fucosyl glycans and prevents the addition of undesirable sugar residues to biotherapeutic proteins [ ] . many genetic, upstream and downstream aspects of the molecular pharming technology must be taken into account when we attempt to maximize the expression and translation of the recombinant proteins of interest. various plants species, organs, tissues, cells, and subcellular organelles have been extensively studied to optimize the yield of pharmaceutical recombinant proteins with more or less successful results. genetic transformation and regeneration systems are still not feasible or are unavailable for other plant species. this fact narrows the spectra of plant hosts available to explore new approaches and tools to enhance the potential of drug design, discovery, and production. the design of expression cassette in molecular pharming has profound effects on gene expression and protein accumulation. it is thus of high interest to elucidate the mechanisms underlying the transcription and translation process of the recombinant proteins. in this review, we attempted to bring into focus the involvement of some transcription and translation constituents. although a limited number of studies report the applications of gateway vectors in molecular pharming studies, these vectors facilitate the cloning and the expression in a short time in comparison with classical cloning techniques. epigenetic rnai or gene silencing suppressors can also have significant impacts on the whole plant metabolism and the expression and accumulation of heterologous proteins. the obstacle of plant glycosylation features can be overcome by humanization of plant-made glycoproteins. the plant-made pharmaceuticals industry will flourish in the near future when it is able to provide a high quality and quantity of pharmaceutical products at low costs and with basic infrastructure. it is currently in dire need of a standard regulation system to avoid all environmental and healthcare system troubles (biosafety and regulatory issues). the recent advancements and progress in molecular biology and genomic studies and molecular cloning techniques will certainly give more effective expression and purification systems for the plant molecular pharming platform. molecular farming for new drugs and vaccines. current perspectives on the production of pharmaceuticals in transgenic plants hairy root research: recent scenario and exciting prospectscommentary development of rhizosecretion as a production system for recombinant proteins from hydroponic cultivated tobacco plants and human health in the twenty-first century molecular farming in plants: host systems and expression technology two decades of plant-based candidate vaccines: a review of the chimeric protein approaches molecular pharming in cereal crops protein bodyinducing fusions for high-level production and purification of recombinant proteins in plants approaches to achieve high-level heterologous protein production in plants protein dynamics and proteolysis in plant vacuoles microalgae as bioreactors for production of pharmaceutical proteins comprehensive biotechnology optimization of elastin-like polypeptide fusions for expression and purification of recombinant proteins in plants a novel expression platform for the production of diabetes-associated autoantigen human glutamic acid decarboxylase (hgad ) exhaustion of the chloroplast protein synthesis capacity by massive expression of a highly stable protein antibiotic low-cost production of proinsulin in tobacco and lettuce chloroplasts for injectable or oral delivery of functional insulin and c-peptide expression of an immunogenic f -v fusion protein in lettuce as a plantbased vaccine against plague advances in plant molecular farming transient co-expression for fast and high-yield production of antibodies with human-like n-glycans in plants transgenic lettuce seedlings carrying hepatitis b virus antigen hbsag production of double repeated b subunit of shiga toxin e at high levels in transgenic lettuce plants as vaccine material for porcine edema disease expression of a cholera toxin b subunit in transgenic lettuce (lactuca sativa l.) using agrobacterium-mediated transformation system accumulation of recombinant sars-cov spike protein in plant cytosol and chloroplasts indicate potential for development of plant-derived oral vaccines expression of a cholera toxin b subunit-neutralizing epitope of the porcine epidemic diarrhea virus fusion gene in transgenic lettuce (lactuca sativa l.) biopharmaceutical production in plants: problems, solutions and opportunities chloroplast biotechnology, genomics and evolution: current status, challenges and future directions seed-based expression systems for plant molecular farming production and localization of recombinant pharmaceuticals in transgenic seeds commercial production of avidin from transgenic maize: characterization of transformant, production, processing, extraction and purification the production of recombinant proteins in transgenic barley grains maize (zea mays)-derived bovine trypsin: characterization of the first large-scale, commercial protein product from transgenic plants biopharming to increase bioactive peptides in rice seed plant seeds as bioreactors for recombinant protein production maize plants: an ideal production platform for effective and safe molecular pharming a bacterial signal peptide is functional in plants and directs proteins to the secretory pathway molecular farming on the rise-gmo regulators still walking a tightrope endosperm tissue is good production platform for artificial recombinant proteins in transgenic rice recent advances in barley transformation barley genomics: an overview advances and remaining challenges in the transformation of barley and wheat host limits to accurate human growth hormone production in multiple plant systems second-generation hiv microbicides: continued development of griffithsin transgenic pea seeds as bioreactors for the production of a single-chain fv fragment (scfv) antibody used in cancer diagnosis and therapy high-level expression of a single-chain fv fragment (scfv) antibody in transgenic pea seeds oleosins and oil bodies in seeds and other organs bioreactor engineering for recombinant protein production in plant cell suspension cultures a novel plant cell bioproduction platform for high-yield secretion of recombinant proteins production of glucocerebrosidase with terminal mannose glycans for enzyme replacement therapy of gaucher's disease using a plant cell system transgenic plants for therapeutic proteins: linking upstream and downstream strategies production of human lysosomal proteins in plant-based expression systems transgenic hairy roots: recent trends and applications hairy root: plasmid encodes virulence traits in agrobacterium rhizogenes dna from agrobacterium rhizogenes in transferred to and expressed in axenic hairy root plant tissues agrobacterium rhizogenes inserts t-dna into the genomes of the host plant root cells rhizosecretion of recombinant proteins from plant hairy roots the duckweeds: a valuable plant for biomanufacturing spirodela (duckweed) as an alternative production system for pharmaceuticals: a case study, aprotinin novel controlled-release lemna-derived ifn-alpha b (locteron): pharmacokinetics, pharmacodynamics, and tolerability in a phase i clinical trial glycan optimization of a human monoclonal antibody in the aquatic plant lemna minor the microalga chlamydomonas reinhardtii as a platform for the production of human protein therapeutics micro-algae come of age as platform for recombinant protein production recent developments in the production of human therapeutic proteins in eukaryotic algae green factory: plants as bioproduction platforms for recombinant proteins production of therapeutic proteins in algae, analysis of expression of seven human proteins in the chloroplast of chlamydomonas reinhardtii microalgal vaccines functional analysis of the dat gene promoter using transient catharanthus roseus and stable nicotiana tabacum transformation systems identification of dna sequences required for activity of the cauliflower mosaic virus s promoter expression of a soybean b-conclycinin gene under the control of the cauliflower mosaic virus s and s promoters in transformed petunia tissues molecular farming of pharmaceutical proteins tobacco as biofactory for biologically active hpl production: a human hormone with potential applications in type- diabetes plant-derived recombinant human serum transferrin demonstrates multiple functions effect of codon optimization and subcellular targeting on toxoplasma gondii antigen sag expression in tobacco leaves to use in subcutaneous and oral immunization in mice immunogenicity of a plant-derived edible chimeric espa, intimin and tir of escherichia coli o :h in mice expression of the cholera toxin b subunit (ct-b) in maize seeds and a combined mucosal treatment against cholera and traveler's diarrhea barley as a green factory for the production of functional flt ligand production of highly concentrated, heat-stable hepatitis b surface antigen in maize production of mouse granulocyte-macrophage colony-stimulating factor by gateway technology and transgenic rice cell culture chlamydomonas reinhardtii as a viable platform for the production of recombinant proteins: current status and perspectives optimization of codon composition and regulatory elements for expression of human insulin like growth factor- in transgenic chloroplasts and evaluation of structural identity and function synthesis and expression of recombinant interferon alpha- gene in tobacco chloroplasts, a non-edible plant production of biologically active human interleukin- in transgenic tobacco and potato expression of antibody fragments with a controlled n-glycosylation pattern and induction of endoplasmic reticulum-derived vesicles in seeds of arabidopsis recombinant mouse granulocyte-macrophage colony-stimulating factor is glycosylated in transgenic tobacco and maintains its biological activity considerations for extraction of monoclonal antibodies targeted to different subcellular compartments in transgenic tobacco plants the development, characterization, and demonstration of a novel strategy for purification of recombinant proteins expressed in plants different subcellular localization and glycosylation for a functional antibody expressed in nicotiana tabacum plants and suspension cells a proficient approach to the production of therapeutic glucagon-like peptide- (glp- ) in transgenic plants transient expression of human papillomavirus type l epitope fused to n-and c-terminus of coat protein of potato virus x in plants in silico analysis of chimeric espa, eae and tir fragments of escherichia coli o :h for oral immunogenic applications coordinate expression of multiple proteins in plant cells by exploiting endogenous kex p-like protease activity expression of human papillomavirus e ggg oncoprotein on n-and c-terminus of potato virus x coat protein in bacterial and plant cells linker: a program to generate linker sequences for fusion proteins dna cloning using in vitro site-specific recombination gateway recombinational cloning: application to the cloning of large numbers of open reading frames or orfeomes orfeome cloning and global analysis of protein localization in the fission yeast schizosaccharomyces pombe from orfeome to biology: a functional genomics pipeline constructing orfeome resources with removable termination codons a novel chloroplast transformation vector compatible with the gateway recombination cloning technology delivery of multiple transgenes to plant cells by an improved version of multiround gateway technology tmv-gate vectors: gateway compatible tobacco mosaic virus based expression vectors for functional analysis of proteins recombinant human gad accumulates to high levels in transgenic tobacco plants when expressed as an enzymatically inactive mutant non-food/feed seeds as biofactories for the high-yield production of recombinant pharmaceuticals gateway-compatible vectors for plant functional genomics and proteomics gateway technology: a universal technology to clone dna sequences for functional analysis and expression in multiple systems improvement of recombinant hgm-csf production by suppression of cysteine proteinase gene expression using rna interference in a transgenic rice culture coexpression of proteinase inhibitor enhances recombinant human granulocyte-macrophage colony stimulating factor production in transgenic rice cell suspension culture the development of a high-yield recombinant protein bioreactor through rnai induced knockdown of atp/adp transporter in solanum tuberosum comparative analysis of recombinant human papillomavirus l production in plants by a variety of expression systems and purification methods transient co-expression of post-transcriptional gene silencing suppressors for increased in planta expression of a recombinant anthrax receptor fusion protein production of human growth hormone in transgenic rice seeds: co-introduction of rna interference cassette for suppressing the gene expression of endogenous storage proteins a protease activity-depleted environment for heterologous proteins migrating towards the leaf cell apoplast from planta to pharma with glycosylation in the toolbox pharming and transgenic plants down-regulated expression of plant-specific glycoepitopes in alfalfa plant-specific glycosylation patterns in the context of therapeutic protein production is the drought over for pharming? rice-based mucosal vaccine as a global strategy for coldchain-and needle-free vaccination analysis of a cholera toxin b subunit (ctb) and human mucin (muc ) conjugate protein in a muc -tolerant mouse model plant-derived vaccines against diarrheal diseases plant molecular farming: opportunities and challenges regulatory issues for plant-made pharmaceuticals and vaccines clinical trials fuel the promise of plant-derived vaccines immunogenicity of a novel, bivalent, plant-based oral vaccine against hepatitis b and human immunodeficiency viruses human immune responses to a novel norwalk virus vaccine delivered in transgenic potatoes expression of rotavirus capsid protein vp in transgenic potato and its oral immunogenicity in mice accumulation of functional recombinant human coagulation factor ix in transgenic soybean seeds high-level expression of human interferon alpha- b in transgenic carrot (daucus carota l.) plants expression of the major mugwort pollen allergen art v in tobacco plants and cell cultures: problems and perspectives for allergen production in plants production of the -kda fragment of plasmodium falciparum merozoite surface protein , a leading malaria vaccine antigen, in arabidopsis thaliana seeds influence of elastin-like peptide fusions on the quantity and quality of a tobacco-derived human immunodeficiency virus-neutralizing antibody hydrophobin fusions for high-level transient protein expression and purification in nicotiana benthamiana bioseparation of recombinant proteins from plant extract with hydrophobin fusion technology tamavidin, a versatile affinity tag for protein purification and immobilization acknowledgments abdullah makhzoum thanks prof. mark bernards for his helpful to publish the manuscript. no sources of funding were used to assist in the preparation of this review. the authors have no conflicts of interest that are directly relevant to the content of this review. key: cord- -rtac hh authors: bhatia, saurabh; dahiya, randhir title: chapter edible vaccines date: - - journal: modern applications of plant biotechnology in pharmaceutical sciences doi: . /b - - - - . - sha: doc_id: cord_uid: rtac hh abstract vaccines are considered primary tools for health intervention in both humans and animals. vaccines can be used more widely, especially in developing countries, if their cost of production can be reduced and they can be preserved without refrigeration. in developing countries certain limitations, like vaccine affordability, the need for “cold chains” from the producer to the site of use of the vaccine, and the dependence on injection, are barriers to health care services. plant-derived vaccines do not face such limitations. research under way is dedicated to solving these limitations by finding ways to produce oral (edible) vaccines from transgenic plants. plant-derived vaccines offer increased safety, envisage low-cost programs for mass vaccination, and propose a wider use of vaccination for veterinary use. with the advent of modern molecular biology techniques in the s, new strategies were developed for the production of vaccines. these vaccines are comprised of proteins derived from pathogenic viruses, bacteria, or parasites (generally proteins are produced not by the pathogens themselves, but by expression of the gene encoding the protein in a "surrogate organism") [ ] . in the last decade, it was found that green plants can also be used as the "surrogate production organism" to produce antigens of human pathogens (including hb-sag). these proteins can elicit priming and also can boost the immune response in humans when administered orally. in addition, unlike almost all other cell lines used for production of vaccines, components of plant cells have always been an important part of the normal human diet. plants, therefore, offer significant new opportunities for making safe and effective oral vaccines [ ] . the introduction of selected desired genes into plants and then inducing these altered plants to produce the encoded proteins is the primary condition for the development of edible vaccines. this process is known as transformation and altered plants are called transgenic plants. selection of important epitope region(s) from the pathogen of interest is the one of the key factors that determines the success of potential edible vaccines. edible vaccine development has been challenged by low expression levels of foreign proteins in transgenic plants. reported expression rates range from . % to % total soluble protein (tsp), which can render edible vaccine proteins less immunogenic. selection of strong plant-specific super promoters to improve expression levels is another key factor that can determine the success of edible vaccines [ ] . after transformation of tobacco, great efforts have been made to develop efficient methods for genetic transformation and optimizing expression of foreign genes in plants. the techniques used to introduce foreign genes into plants have been extended to major crops, vegetables, and ornamental and medicinal plants. various foreign proteins including serum albumin, human a-interferon, human erythroprotein, and murine igg and iga immunoglobulins have been successfully expressed in plants. in recent years, several attempts have been made to produce various antigens and antibodies in plants. antigens or antibodies expressed in plants can be administered orally as any edible part of the plant, or by the parenteral route (such as intramuscular or intravenous injection) after isolation and purification from the plant tissue. the edible part of the plant to be used as a vaccine is fed raw to experimental animals or humans to prevent possible denaturation during cooking, and avoid cumbersome purification protocols [ ] . while agrobacterium-mediated transformation still remains the method of choice for dicots, a general method, the biolistics method, of transformation of plants, including monocots, has come into existence. other strategies for expression of foreign genes in plants include use of strong and organ-specific plant promoters, targeting of the protein into endoplasmic reticulum (er) by incorporating er-targeting and er-retention signals, creation of an optimized translation start site context, as well as alteration of codons to suit the expression of prokaryotic genes in a plant. for the production of edible vaccines or antibodies, it is desirable to select a plant whose products can be consumed raw to avoid degradation during cooking. thus, plants like tomato, banana, and cucumber are generally the plants of choice. while expression of a gene into the genome allows maintenance of the material in the form of seeds, some virus-based vectors can also be used to express the gene transiently to develop the products in a short period ( fig. . ). this may have the additional advantage of allowing expression of the product at a very high level; not always attainable in transgenic systems [ ] . plus-sense, single-stranded plant rna viruses have been proposed as an effective alternative to produce vaccine antigens in plants. in this technique, the epitope of interest is engineered into a plant virus, usually within the coat protein gene. infection of a susceptible non-gm-plant results in intracellular production and accumulation of the epitope. the epitope sequence, as well as the viral genome, never become integrated into the plant genome and hence are only expressed by the generation of infected cells. a recombinant cowpea mosaic virus has shown to elicit protective immunity in mink when engineered to express the antigenic epitope against mink enteritis virus. recombinant alfalfa mosaic virus (almv) has enabled expression of significant quantities of rabies virus and hiv epitopes upon integration of their respective coding sequence into the almv coat protein. the extra sequences were found to protrude from the virion surface without interfering with virus assembly. recombinant almv coat protein molecules have also demonstrated the ability to assemble into particles containing three different epitopes from hiv and rabies. this demonstrates the ability of plant viruses to produce multicomponent vaccines [ ] . claimed advantages of transient viral expression of transgenes over transgenic plants are: lesser time for cloning of the foreign gene in the viral genome as compared to time required for transformation of plants; the ease at which antigen production can be scaled up; and the wide host range of plant viruses, which allows use of multiple plant species as biofactories. each single antigen expressed in plants must be verified by animal studies and western blots, and quantified by enzyme-linked immunosorbent assay (elisa). most pathogens enter at mucosal surfaces lining the digestive, respiratory, and urinoproductive tracts, which are collectively the largest immunologically active tissue in the body. the mucosal immune system is the first line of defense and the most effective site for vaccination against pathogens. nasal and oral vaccines are most effective for mucosal infections. the goal of oral vaccine is to stimulate both mucosal and humoral immunity against pathogens. edible vaccines when taken orally undergo mastication, and degradation of plant cells occurs in the intestine due to the action of digestive enzymes. peyer's patches are an enriched source of iga producing plasma cells and have the potential to populate mucosal tissue and serve as mucosal immune effector site. the breakdown of edible vaccine occurs near peyer's patches, which consist of - lymphoid nodules on the outer surface of the intestine and also contain follicles from which the germinal center develops after antigenic stimulation. these follicles act as a site for the penetration of antigens in intestinal epithelium. the antigen then comes in contact with m-cells. m-cells express class- major histocompatibility complex molecules, and antigens transported across the mucous membranes by m-cells can activate b-cells within these lymphoid follicles. the activated b-cells leave the lymphoid follicles and migrate to diffuse mucosal associated lymphoid tissue where they differentiate into plasma cells that secrete the iga class of antibodies. these iga antibodies are transported across the epithelial cells into secretions of the lumen where they can interact with antigens present in the lumen (figure . ). • edible vaccines are effective as a delivery vehicle for immunization because adjuvants that enhance the immune response are not required. • edible vaccine can elicit mucosal immunity, which is not observed in traditional vaccines. • edible vaccines are also cost effective in availability, storage, preparation, production, and transportation. vaccines produced by biotechnological methods are stable at room temperature, unlike traditional vaccine, which needs cold chain storage, which multiplies the yearly cost to preserve vaccines. moreover, the seeds of transgenic plants could be dried as there is less moisture content in seeds and the plants with oil or their aqueous extracts possess more storage opportunities. manufacturing cost is low as there is no need for special premises to manufacture them. edible vaccine can be easily produced at mass level in comparison to an animal system. • edible vaccines are well tolerated, as they do not require administration by injection unlike traditional vaccines. thus, there is also a reduced need for medical personnel and risk of contamination is low. the feasibility of oral administration compared to injection is also an advantage. • plant-derived vaccines could be the source for new vaccines combining numerous antigens. these multicomponent vaccines are called second generation vaccines as they allow for several antigens to approach m-cells simultaneously. • edible vaccines are subunit preparations, do not involve attenuated pathogens, and improve the safety of individuals as compared to traditional vaccine since there is no possibility of proteins reforming into infectious organisms. • the separation and purification of vaccines from plant materials is very easy and pathogenic contamination from animal cells can be effectively prevented. • there is the possibility of development of immunotolerance to the vaccine protein or peptide. • consistency of dosage form differs from plant to plant and generation to generation. • protein content varies from plant to plant and generation to generation. • ripeness also affects the proteins that are present in form of antigens in the fruits. • limitations of methods for standardization of plant material/product. • stability of vaccine differs from plant to plant. • some food cannot be eaten raw (e.g., potato) and needs to be cooked, which will denature or weaken the protein present in it. • variable conditions for edible vaccine are also a major problem. potatoes containing vaccine can be stored at °c for longer time, while tomato does not last long at this temperature. thus, these vaccines need to be properly stored to avoid infection through microbial spoilage. • another concern regarding edible vaccine is the need for proper distinguishing characters to identify between "vaccine fruit" and "normal fruit" to avoid maladministration of vaccine, which could lead to tolerance. • the glycosylation pattern of plants and humans is different, which could affect the functions of vaccines. the first report of the production of edible vaccine (a surface protein from streptococcus) in tobacco, at . % of total leaf protein level, appeared in in the form of a patent application published under the international patent cooperation treaty. subsequently, a number of attempts were made to express various antigens in plants (table . ). since acute watery diarrhea is caused by enterotoxigenic escherichia coli and vibrio cholerae that colonize the small intestine and produce one or more enterotoxin, an attempt was made to produce edible vaccine by expressing heat-labile enterotoxin (lt-b) in tobacco. transgenic potatoes were created and grown by charles arntzen hugh s. mason and their colleagues at the boyce thompson institute for plant research, an affiliate of cornell university. transgenic potatoes containing enterotoxin stimulated strong immune responses in animals. an edible vaccine could stimulate an immune response in humans. volunteers ate bite-sized pieces of raw potato that had been genetically engineered to produce part of the toxin secreted by the e. coli bacterium, which causes diarrhea. ten of the volunteers ( %) who ingested the transgenic potatoes had four-fold rises in serum antibodies at some point after immunization, and six of the ( %) developed four-fold rises in intestinal antibodies. the potatoes were well tolerated and no one experienced serious adverse side effects (table . ). tomatoes serve as an ideal candidate for this hiv antigen because unlike other transgenic plants that carry the protein, tomatoes are edible and immune to any thermal process, which helps retain its healing capabilities. even more importantly, compared to bananas tomatoes were found to grow at a high rate of success in russia. for vaccines or subunit vaccinations, bananas seem to be the desired vector. the advantage of bananas is that they can be eaten raw as compared to potatoes or rice, which need to be cooked, and bananas can also be consumed in a pure form. research is leaning toward the use of bananas as the vector since most third world countries, which would benefit most from edible vaccines, are in tropical climates that are suitable for growing bananas. egyptian scientists have genetically engineered maize plants to produce a protein used to make the hepatitis-b virus vaccine. a team of researchers led by hania el-itriby, director of cairo's agricultural genetic engineering research institute, developed genetically modified (gm) maize plants that produce the protein known as hbsag, which elicits an immune response against the hepatitis-b virus and could be used as a vaccine (table . ). when predominant t-cell epitope peptides, which were derived from japanese cedar pollen allergens, were specifically expressed in rice seed and delivered to the mucosal immune system, the development of an allergic immune response of the allergen-specific th cell was suppressed. furthermore, not only were specific ige production and release of histamine from mast cells suppressed, but the inflammatory symptoms of pollinosis, such as sneezing, were also suppressed. these results suggest the feasibility of using an oral immunotherapy agent derived from transgenic plants that accumulate t-cell epitope peptides of allergens for allergy treatment (table . ). when the seed expression system is used as a platform for foreign protein production, substantial amounts of recombinant proteins can be accumulated, because the seed is a natural storage organ for accumulating the starch, protein, and oil required for seedling growth. also, artificial peptides or proteins accumulate in seed, which is in remarkable contrast with other tissues. plant-derived vaccines are free from animal pathogen contaminants. furthermore, plant dna is not known to interact with animal dna and plant viral recombinants does not invade mammalian cells. further safety of plant-derived vaccines can be achieved by following similar regulations established for traditional vaccines. nevertheless, the present concern over the use of gm plants is now affecting research in this important field, especially in europe. one of the fears is that gm pollen may outcross with sexually compatible plants (related crops or weeds) and affect biodiversity. in order to address this alarm, several pollen containment approaches have been developed. these are essentially based on the exploitation of different forms of male sterility (suicide genes, infertility barriers, apomixis). an alternative way of solving the problem is engineering vaccines into the chloroplast dna (cpdna), which is not transmitted to the sexual progeny through the pollen grains. an additional safety feature would be the recognition of gm plants that produce vaccines by the addition of genes encoding colored plant pigments. it is important to recognize that plants that produce vaccines are medicinal plants and should be grown, processed, and regulated as pharmaceutical products. in the majority of earlier papers, level of antigen accumulation in the plant organ was in the order of . - . % of total soluble protein, while the more recent developments on cpdna integration promises to increase this value to % or more. at the latter value, land requirements for industrial plant-derived vaccine production will be in the order of a few thousand square meters. this will definitely enable vaccine-producing plants to be set apart from field grown crop plants and offer added safety when engineered plant viruses are used for transient antigen expression. a further point of public concern in gm plants is the presence of antibiotic resistance genes (used as selective marker in most transgenic plants). approaches have now been developed to generate gm plants (with both nuclear or cpdna integration) that do not carry these genes. edible vaccines edible vaccines: current status and future transgenic plants as factories for biopharmaceuticals using transgenic plants as bioreactors to produce edible vaccines edible vaccines and antibody producing plants medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants expression of chimeric hcv peptide in transgenic tobacco plants infected with recombinant alfalfa mosaic virus for development of a plantderived vaccine against hcv a review of oral vaccination with transgenic vegetables edible vaccines: a concept comes of age food as production and delivery vehicles for human vaccine key: cord- - gxkwi n authors: farrar, ashley j.; farrar, francisca c. title: clinical aromatherapy date: - - journal: nurs clin north am doi: . /j.cnur. . . sha: doc_id: cord_uid: gxkwi n clinical aromatherapy is an alternative medicine therapy that can be beneficial in the inpatient or outpatient setting for symptom management for pain, nausea, general well-being, anxiety, depression, stress, and insomnia. it is beneficial for preoperative anxiety, oncology, palliative care, hospice, and end of life. essential oils can be dangerous and toxic, with some being flammable, causing skin dermatitis, being phototoxic with risk of a chemical burn, or causing oral toxicity or death. the article investigates history, supporting theories, guidelines, plant sources, safety, pathophysiologic responses, and clinical nursing aromatherapy. recommendations for developing a best practice clinical nursing aromatherapy program are provided. conditions. this article investigates the use of clinical aromatherapy. credibility is seen in the historical evolution and nursing theorist support. aromatherapy regulation of guidelines, plant sources for aromatic oils, and safe use of essential oils in symptom management in clinical aromatherapy is reviewed. suggestions are recommended for a best practice model for clinical aromatherapy. aromatherapy is a fast-growing complementary therapy worldwide. according to the national institutes of health national center for complementary and integrative health, americans spend more than $ . billion annually on this therapy. it is predicted the global market will grow in spending to $ trillion by . aromatherapy also is called integrative medicine. it is especially important for frontline nurses to understand the difference between alternative therapy and integrative therapy. in alternative medicine, the therapy works as an addition to conventional medical treatment, whereas integrative therapy is solo and replaces any conventional medical care. the national institutes of health national center for complementary and integrative health developed categories for these therapies-mind-body therapy, biologically based practices, manipulative and body-based practices, energy medicine, and whole medical systems, such as ayurvedic medicine and traditional chinese medicine. , nursing health care aromatherapy falls into the category of mind-body therapy. nursing health care uses essential oils to complement therapeutic interventions, decrease anxiety. it is expected that the plant-based essential oil applications can be measured, such as a preanxiety symptoms, interventions with essential oil, and postanxiety symptoms. the outcome from the administration of essential oil can be measured with a pre anxiety level and post level of anxiety to determine if the essential oil is effective. , aromatherapy has been used for thousands of years. hippocrates, father of modern medicine, advocated the use of aromatherapy due to his belief that aromatic baths and scented massage were key to good health. essential oils leaders emerged, supporting aromatherapy as a credible therapy for mind, body, and spirit. table summarizes major historical timelines of countries and cultural influences, validating aromatherapy as medical, clinical, and holistic. historical evolution of medical, clinical, and holistic uses of essential oils is embraced by major nursing theorists. their theoretic frameworks and concepts reflect the use of clinical aromatherapy as a patient-centered and holistic approach for balancing physical health, spiritual needs and well-being. the theorists' embracement confirms health care aromatherapy is a credible alternative method ( table ) . the food and drug administration (fda) of the united states guidelines classify essential oils for aromatherapy as cosmetics, because they are not drugs for treating or prevention of a disease. therefore, aromatherapy essential oils are not regulated by the fda. the us consumer product safety commission (cpsc) monitors unsafe products. the cspc enforces federal laws to protect consumers against unreasonable injury and death from products. the following are examples of how these federal organizations monitor essential oils. clinical aromatherapy . aromatherapy waterless vaporizers and diffusers were recalled due to a defective heater causing a fire multiple times with consumers. the cpsc had the authority to cease the sale of the products and refund consumers. . the fda protects consumers from false claims and mislabeled products that mislead the public. surveillance found quinessence aromatherapy ltd posted on their website advertisement that essential oils protect against and cure coronavirus disease- . . this false statement triggered a fda letter warning to the owner to take the information off their website within hours and cease the sale of essential oil for prevention and cure of covid- . these hours included developing a plan to be approved by the covid- task force. the company was located in europe with essential oils sold in the united states. the owner ignored the warning. due to the fraudulent statement describing essential oils as a curing drug for covid - , a second joint letter was sent to the owner by the fda, cpsc, the secretary of health and human services, and the u.s. public health services ordered the owner of the company to immediately take down the website and cease the sell of essential oils as a curing drug. the company's website was also put on the federal surveillance website list. administrators were ordered to cease sale and remove from the web site immediately. this incidence of a false claim has brought awareness that currently essential oils are complementary therapy and do not treat and/or prevent a disease. sellers need to be aware of descriptions of aromatherapy and consumers need to know that aromatherapy is complementary. essential oils are used every day for their aromatic scents-for example, perfumes, candles, essential oil plug-ins, scented aerosol sprays for the home, fabric softeners farrar & farrar for clothes, hair shampoos, and spices to add flavor to food. essential oils also are used in over-the-counter herbs and added to medications to add a pleasant flavor to bitter medications. these aromatic essential oils are growing in popularity, with nurses needing to learn about essential oils, their benefits, and safety measures. essential oils come from seeds, stems, leaves, needles, petals, flowers, rinds and fruits, woods and resins, roots and rhizomes, and grasses. oil is extracted from the plant by distillation by steam or mechanical cold press. cher kaufman, a certified aromatherapist, wrote a book with a series of chapters on plant sources for aromatic essential oils-seeds, petals and flowers, rinds and fruits, woods and resins, roots and rhizomes, and grass. the following is a summary of the plant sources of each category, with examples that could be significant to health care nurses. three common examples of essential oils that come from seeds from plants are uses for this oil include an anti-inflammatory, antibacterial, antifungal, antispasmodic, detoxifier, and digestive and for relieving gas. there are common examples of essential oils derived from stems, leaves, and needles. . cistus (cistus ladanifer) is from the plant family cistaceae. this essential oil comes from stems, twigs, dried leaves, and dried flowers. uses for this oil include as a cictrisant or for cell regeneration; as an antibacterial, anti-infectious, antimicrobial, astringent, and antiviral agent; as an immunity booster and regulator; as a tonic and support for parasympathetic and central nervous systems; and for wound healing. . eucalyptus is a tree from the plant family myrtaceae. it also is referred to by many names, such eucalyptus oil, blue gum oil, blue mallee oil, and gully gum oil. the leaves and twigs are used for burns, wounds, nasal congestion, lowering blood glucose, nasal congestion, and asthma and as a tick repellent. it also is used in medications and supplements. . laurel (laurus nobilis) is from the plant family lauraceae. this aromatic evergreen scrub is known for its aromatic dark green, glossy leaves. dried and fresh leaves oil is used as an analgesic, antibacterial, antimicrobial, antiseptic, antispasmodic, and antiviral; for boosting the immune system and calming the nervous system; and as an expectorant and fungicide. . patchouli (pogostemon cablin) comes from the plant family lamiaceae that is commonly called the mint or dead needle busy herb. oil from leaves are used as an antidepressant, anti-inflammatory, antimicrobial, antiviral, aphrodisiac, astringent, deodorant, and digestive; for relieving gas soothing the nervous system; and as a stimulant and tonic. . peppermint (mentha x piperita l) comes from the plant family lamiacae in the mint family. peppermint essential oil is a common flavoring agent in pharmaceuticals, soaps, cosmetics, food, and beverages. this essential is used as an analgesic, antibacterial, anti-inflammatory, antispasmodic, antimicrobial, decongestive, digestive, and expectorant and relieves coughs. . pine (pinus sylvestris)-pinus edulis is from the plant family lamiaceae and from the mint family. pine essential oil is derived from the needles on the pine tree. the scent is known for the uplifting and positive impact on the mood. it is known for treatment of postsurgery nausea and vomiting. essential pine oil is used as an analgesic, antibacterial, antibiotic, anti-infectious, anti-inflammatory, antifungal, and antimicrobial agent; assisting in opening lungs and air pathways; as an expectorant; and for soothing nerves. . rosemary (rosmarinus officinalis) is from the plant family lamiaceae. this aromatic evergreen shrub's essential oil is derived from leaves, flowers, and stems. this essential oil is known for folk medicine, flavoring food, and herbal tea. rosemary has been known as a sacred oil. uses for this essential oil are as an analgesic, anti-inflammatory, anti-infectious, antiseptic, and antispasmodic agent; for breaking up mucus; as a cognitive stimulant, decongestant, expectorant, muscle relaxant (cineole), stimulant, and tonic; and for wound healing (verbenone). there are common essential oils derived from petal and flowers. is an herbaceous perennial in the plant family lamiaceae with a history of petal and flowers used as an herb. the essential oil of clary sage is used in perfumes and muscatel flavoring in wines and liqueur. this essential oil is used as an antidepressant, antifungal, anti-inflammatory, antispasmodic, and aphrodisiac and for calming the nervous system, relaxing the uterus, and stimulating the blood flow. is in the plant family asteraceae and is a common name for several daisy-like flowers. chamomile essential oil from flowers is used in herbal tea and is a popular night herbal tea due to the sedative affect. this essential oil is used for support for the nervous system, inflammation, insomnia, menstrual issues, headaches, and skin concerns. . geranium (pelargonium x asperum) and rose (pelargonium graveolent)-this essential oil comes from the plant family geraniaceae. this perennial plant has a sweet floral scent with uses in high-end perfumes and skin products with essentials oils resulting in young radiant skin. essential oil from the flowers are used for reducing anxiety, as a sedative, for stimulating relaxation, as aids in symptoms from menstruation, as an anti-inflammatory, and for supporting healthy lymph drainage. . jasmine (jasminum sambac; jasminum grandiflorum)-this essential oil is from the plant family oleaceae. jasmine is a genus of shrubs and vines in the olive family. flowers of this bushy strong-scented perennial plant are used for scent and in tea as a base for green and white teas. as an essential oil, jasmine is used as an antidepressant and aphrodisiac, for calming the nervous system, and as a sexual tonic and stimulant. . lavender (lavandula angustifolia) -this essential oil is in the plant family of lamiaceae and is a bushy strong-scented perennial plant. lavender is a popular house dé cor and frequently used with dried flowers as a complement in weddings. the popular scent is used in balms, salves, and cosmetics. as an essential oil, lavender is used as analgesic, anti-inflammatory, antifungal, and antispasmodic; for calming farrar & farrar the nervous system, lowering blood pressure, and reducing anxiety and sensations of pain; as a sedative; and for wound healing. . neroli (citrus aurantium var. amara)-this essential oil is in the plant family rutaceae and is from the bitter orange tree. this essential oil from flowers has a rich floral scent and is known as orange blossom oil. neroli is used in scented products, such as perfumes and lotions. this essential oil is used as an antidepressant, antifungal, anti-inflammatory, antimicrobial, antioxidant, antiparasitic, antiseptic, and aphrodisiac; for calming; and as a digestive, nervous system stimulant, sedative, and tonic. . rose (rosa damascena; r damascena var. alba)-this essential is from the plant family lamiaceae and is a flowering shrub known as a rosebush. rose oil is a powerful rich sweet smell. it is used commonly in perfumery. this essential oil is used as an antibacterial, antidepressant, anti-infectious, anti-inflammatory, antiseptic, antiviral, aphrodisiac, and astringent agent; for calming the nervous system and reducing anxiety; as a sedative; as a sexual, general, and uterine tonic; and for wound healing. . ylang-ylang (cananga odorata)-this essential oil is from the plant family annonaceae, or custard apple family. this tropical flower is a yellow-shaped flower that grows on the cananga tree. oil from ylang-ylang is used in cologne, lotion, food flavoring, and soap. this essential oil elevates the mood. ylang-ylang essential oil is used as an antidepressant, anti-inflammatory, antiparasitic, antispasmodic, and aphrodisiac; for calming the nervous system and lowering blood pressure; and as a sexual tonic. rinds and fruits is from the plant family rutaceae. this yellow or green fruit is a hybrid of lemon and bitter orange and has a bitter taste that is more than grapefruit but less than a lemon. the essential oil from the peel or zest of the fruit can cause photosensitivity, with sun exposure causing damage to sun-exposed skin. the essential oil has a citrus fruit smell, with uses in oil perfumes, cosmetics, and scenting food. this essential oil is used as an air purifier, antibacterial, antidepressant, antifungal, anti-inflammatory, and antiviral; for calming; as a deodorant; for digestive regulating (undereating or overeating); for reducing anxiety; as a sedative and tonic; and for wound healing. . lemon (citrus limonum)-this essential oil is fruit from a small evergreen tree. this oil is from the rutaceae plant family, with the peel of the fruit and pulp used in culinary and noncultural from lemon essential oil, lemon pie for culinary to cleaning products. the distinct sour taste of lemon is a popular essential oil. the essential oil from lemon is used as an antibacterial, anticoagulant, antidepressant, antiinfectious, anti-inflammatory, antiseptic, antiviral, astringent, antioxidant, and antimicrobial agent; as a digestive stimulant, immunity booster, and lymphatic; and for reducing anxiety. . mandarin (citrus reticulata) -this essential oil is from the rutaceae plant family. this small citrus tree grows mandarin oranges that are smaller than oranges. a hybrid of the mandarin orange is the tangerine. the mandarin essential oil from peel and rind is sweeter and can be dried for seasoning and used in various food. this essential oil is used as an analgesic, antidepressant, antiseptic, central nervous system tonic, deodorant, digestive tonic, and immunity booster; for reducing reduces anxiety and fevers; and as a sedative. . sweet orange (citrus sinensis)-this essential oil is from the plant family rutaceae. this sweet citrusy greenish orange fruit oil is from the peel and zest. this oil is used in top perfumes. the leaves are photosensitive but not the fruit. the sweet orange essential oil is used as an analgesic, antidepressant, antibacterial antifungal, antiseptic, antiviral, deodorant, and digestive tonic; for reducing anxiety; as a sedative; for soothing the nervous system; and as a stimulant. cone. this essential oil is from the plant family cupressaceae, derived from conifers, and often is used as a spice. the essential oil is used as an analgesic, antiseptic, antiseborrheic, anti-inflammatory, antifungal, antiviral, decongestant, and detoxifier and for increasing circulation and reducing fever. woods and resins . cedarwood (cedrus atlanticia)-cedarwood is from the plant family pinaceae and the needles, leaves, bark, and wood are for extracting the essential oil. the evergreen conifers have a soothing woodsy scent. the essential oil is used as an antifungal, antiseptic, and astringent; for breaking up mucus; and as a calmative, insect repellent, lymphatic decongestant, and general tonic. . frankincense (boswellia carteri)-this essential oil is in the plant family of burseraceae and is from a boswellia tree. resin that is a hardened gumlike material is used in aromatic incense and perfumes. the essential oil is used as an analgesic, antibacterial, antidepressant, anti-infectious, antimicrobial, and astringent agent; for immunity tonic; for reducing anxiety; as a sedative; and for soothing the nervous system and wound healing. . sandalwood (santalum album)-this essential oil is from the plant family santalaceae. the oil is extracted from wood, heartwood of the trunk, and sawdust. the essential oil from sandalwood is used in medications, skin beauty treatment, incense sticks, perfumes, mouthwashes, deodorants, and antiseptics. as an essential oil, it is used as an antibacterial, antidepressant, anti-inflammatory, antimicrobial, antiviral, aphrodisiac, and sedative; for soothing the soothes nervous system; and as a general tonic. roots and rhizomes is distilled from the rhizome or underground stem of a root of the herb zingiber. ginger also is known as the oil of empowerment for the feeling of confidence. ginger root oil is a frequently used spice. in addition, this dried and ugly root is used as an analgesic, antibacterial, antispasmodic, digestive support, immunity harmonizer, and rubefacient. . vetiver (vetiveria zizanoides) is derived from the aromatic roots and also called khus oil. it is derived from the vetiver plant that is a clumpy, green grass that can grow feet or more. this essential oil is used as an antiseptic, antispasmodic, antiinflammatory, digestive stimulant, immunity booster, and sedative, and for skin support and soothing the nervous system. is an essential oil that comes from the leaves and stalk of the lemongrass plant. this grassy plant is used in cooking and herbal tea. the oil from the grass has a lemony powerful scent and is bright or pale yellow. this essential oil is used as an analgesic, antidepressant, antiviral, immunity booster, and general tonic. . palmarosa (cymbopogon martinii var. motia) is an essential oil that comes from a tall herbaceous grass and can be called indian geranium or rose oil. the oil has a sweet citrus lemony scent that has a yellow color. farrar & farrar there are basic methods for administration of essential oils. nursing commonly uses topical skin application of essential oil for administration. if a facility has an integrative medicine department, massage therapy usually includes an essential oil. the following is an overview of the methods by which essential oils are absorbed. . topical application with skin absorption of the essential oil. examples include massage, scented bath, cosmetics, and perfumes. when essential oil in aromatherapy is inhaled, molecules activate the olfactory, respiratory, gastrointestinal, and/or integumentary systems based on the pathway of activation. these molecules are capable of releasing neurotransmitters, such as endorphins, to trigger a sense of well-being and an analgesic effect. , there are common pathways triggering a pathophysiologic response to aromatherapy molecules. the most common pathway is inhalation, such as by a diffuser. activation of olfactory stimulation produces immediate change in parameters for blood pressure, pulse rate, muscle tension, pupil dilation, body temperature, and blood flow. , the following summarizes this pathway: the olfactory stimulation by aromatherapy travels via nostrils to the olfactory bulb. the stimulus then travels to the brain for processing, where the amygdala triggers an emotional response and the hippocampus retrieves and/or forms memories. the limbic system interacts with the cerebral cortex, activating thoughts and feelings. the inhaled aromatherapy molecules travel to the upper respiratory tract and then to the lower respiratory tract. molecules than travel to the pulmonary blood vessels to the blood stream then to organs and tissues. , in summary, the inhaled aromatherapy molecules affect mind, body, and spirit. the second common pathway is through the skin, such as by a massage, in which molecules are absorbed through the skin. the pathway is summarized: the molecules travel to the upper respiratory track and then the lower respiratory tract. molecules then travel to the pulmonary blood vessels, to the blood stream, and then to organs and tissues. , the skin pathway can activate olfactory stimulation and also activates application of scented oil to the skin pathway triggering a mental and physiological response. the skin pathway absorption of essential oils can reduce a patient's perceived stress, enhance healing, and increase communication. coming home from a long challenging day of work to the aromatic smell of a favorite essential oil can immediately decrease the stress from a busy and challenging day. relaxing in a scented uplifting bubble bath can make someone feel like a new person. on the flip side, aromatic essential oils can be toxic, causing chemical burns and even death. aromatherapy is growing in usage and can be extremely dangerous if not used safely because of a knowledge deficit. two ethical principles apply to nurses when administering essential oils. the first is beneficence, in which the nurse takes positive steps to prevent harm. the second ethical principle is nonmaleficence, which means having an obligation to do no harm to a patient. legal consequences could result if harm to a patient occurs due to negligence from administering of aromatherapy. therefore, the bottom line is the need for nurses to learn about aromatherapy essential oils and their potential harms, such as poison and lethal complication. the following case reports portray safety considerations, complications, and interventions. tf is a -year-old man who lives in phoenix, arizona; he is an advocate of essential oils and frequently uses them for anxiety and to promote sleep. on his day off, tf has several errands, including his monthly supply of essential oils. his first errand was to purchase essential oils. during his last errand, tf heard his name being called by an old friend. tf sat down to visit with his friend for a few minutes; the visit lasted minutes. when tf returned to his car, he found black smoke in the car and a large burnt hole in his backseat where his purchased essential oils were placed. tf called the police to file a report. critical analysis revealed the large supply of essential oils were stored in the back seat. tf left the oils in the car for minutes with the outside temperature of f, with the potential increase in the temperature in the car increasing to f. the essential oils caught on fire, resulting in the smoke and burnt hole in the backseat of the car. intervention for this unsafe use of essential oil is education that these oils are flammable and need to be stored in a cool dark place in the original bottle, which is colored to prevent direct sunlight penetrating to the essential oil. unsafe storage by leaving essential oils in a hot car can cause a combustion reaction, triggering flames and a fire. cf, a -year-old woman, was admitted for inpatient treatment of sepsis from an acute urinary infection. at night she can become agitated and screamed that snakes were crawling up her wall. the provider ordered aromatherapy and increased lighting in the room. turning on a bed alarm and increased rounding also were ordered. the nurse brought the aromatherapy essential oil to the room and left to get a steam diffuser. when the nurse returned, cf appeared drunk. the nurse found the bottle open on the bedside table. the nurse called for assistance. critical analysis revealed the nurse left the bottle of essential oil on the nightstand unattended when she left the room. the patient was able to open the bottle and drink a small amount of the essential oil, causing the drunken behavior. intervention for essential oil left attended with a confused elderly patient was administration of milk to dilute the essential oil. the provider was called for further orders and farrar & farrar an incident report was completed. the elderly and children are vulnerable to adverse effects from inappropriate use of essential oils. early recognition is appearing drunk. essential oils should be locked in a container in a hospital and kept away from elders and children. an essential oil bottle should not be left unattended, especially with this confused patient with delusions. the diffuser with the essential oil needs to be prepared outside of the patient's room. allergic contact dermatitis and primary contact dermatitis ta, a -year-old woman, works at a massage therapist and is extremely popular. bookings must be in advance because her schedule stays full. each day she works hours to hours, with mostly . -hour massages. she uses a lotion with an aromatic essential oil. after months, she developed a bright bred rash on her hands and lower arms. she used a steroid cream on the rash without resolving the rash. over the next month, the rash got worse. ta scheduled an appointment with a dermatologist. critical analysis evaluated allergic contact dermatitis versus primary contact dermatitis. in allergic contact dermatitis, the allergy occurs over a period whereas primary contact dermatitis occurs the first time the essential oil is used. in allergic contact dermatitis, the symptoms are a bright red rash that worsens with time whereas the primary contact dermatitis a presents as a red wheal or burn. , intervention was based on treating allergic contact dermatitis based on symptomatology and length of time. patch testing revealed the specific essential oil to stop using, allowing her to continue as a massage therapist. if ta had primary contact dermatitis, the red wheal or burn area from the toxic oil would be diluted with vegetable oil or milk then washed with unscented soap. essential oil phototoxicity aj is a -year-old woman who loves the sun. she lives in an apartment with a swimming pool. the average summer temperature is f. on weekends she can be found at the swimming pool for hours per weekend day. aj's pool relaxing is hours to hours per day. she sets an alarm hourly to turn from back to stomach. aj says the sunrays lift her up and gives her a beautiful brown tan with sunscreen oil. due to the shutdown of her state due to covid- and stores closed, aj decided to shop online for home delivery of essential oils. aj found a web site with a sale on essential oils that had a pop-up advertisement declaring breaking news that essential oils prevent and cure the covid- virus. aj purchased several citrus fruit essential oils. aj applied a mandarin essential oil to her neck and chest to ward off the covid- virus. aj left the pool early because of a burning sensation on her neck and chest. she took a shower and noticed several burned areas. the next day, the red burned areas turned to a brown skin damage appearance unlike any sunburn she ever experienced. aj scheduled a dermatologist appointment due to the discoloration and discomfort not resolving. , , critical analysis revealed that aj was scammed by a fraudulent online statement to sell essential oils in a pandemic covid- fearful time. the mandarin essential oil was not diluted when applied to the skin, increasing the risk for dermal toxicity. the pure mandarin essential oil was phototoxic and inflected damage to the skin, resulting in dark pigmented skin that could be permanent. , dermal toxicity also occurred with the essential oil not being diluted. the regular sunburn resulted in redness and blisters on her skin. aj scheduled an urgent dermatology appointment. she used an over-the-counter steroid cream for pain relief. the intervention to stop using the phototoxic essential oil and seek a specialist, which aj did, with scheduling the dermatology appointment. the essential oil label clinical aromatherapy always should be read for safety instructions. if a photosensitivity essential oil is used, wait a minimum of hours before exposure to sun ultraviolet radiation. aj should consult a qualified aromatherapist who has training for aromatherapy, not a seller of essential oils, to prevent harm from essential oils. a registry database for trained aromatherapists who have passed the core level of aromatherapy examination can be found at thttps://www.aromatherapycouncil.org.uk/about_us essential oils always should be diluted in a carrier oil. a carrier oil prevents irritation to the skin and side effects of the essential oil. examples of carrier oils include coconut oil, coconut oil, aloe vera gel, unscented lotion, vegetable oil, and avocado oil. because oil and water do not mix, milk is used to remove the oil and calm the skin followed by washing of the skin by unscented soap. phototoxic essential oil contains constituents that triggers a chemical process that changes the skin dna, making the skin susceptible to sun ultraviolet radiation. this chemical change in the skin is called photosensitivity and the primary constituent is confurocoumarins that causes phototoxic reaction. exposure of the applied photosensitivity essential oil to ultraviolet radiation from the sun inflicts skin damage with darkly pigmented skin that can be permanent due to the long period of exposure to sun ultraviolet radiation. it is extremely important to determine if an essential oil is phototoxic. aj should find this warning on the label of the essential oil. essential oils are not regulated by the fda but the fda monitors web sites for fraudulent postings. aj should report the online site and harm to her skin to the fda for investigation. oral toxicity lm, a -pounds -year-old woman who lived with her mother, was told by an essential oil seller that she heard essential oils could prevent and cure covid- . lm was terrified that she could contract the virus with a resurgence of covid- later in the year. lm was extremely excited about this information and asked which essential oil she should use orally to protect herself from this deadly pandemic covid- virus. the seller recommended a safe dose for eucalyptus essential oil twice weekly orally. after a week lm decided to increase the oral dose to ward off the covid- virus. she decided to drink half of an oz glass of eucalyptus essential oil. within minutes she experienced burning in her throat, mouth, and stomach. lm yelled for her mother to come quickly. the mother found the daughter vomiting, staggering, and with slurred speech. the mother found the eucalyptus essential oil bottle and a glass indicating she had drunk eucalyptus essential oil. the mother called with the dispatcher sending an ambulance and notifying the poison control center for eucalyptus poisoning. critical analysis of oral toxicity of eucalyptus revealed lm had drunk an unsafe dose causing poisoning with central nervous system depression and a chemical burn in her mouth, throat, and stomach. lm was admitted to the intensive care unit. intervention was police investigation of the fraudulent information that eucalyptus could prevent and cure covid- information by the seller of the essential oil with unintentional poisoning lm. a qualified aromatherapist that is certified or completed aromatherapy curriculum. don't take advise from a seller of essential oils without expertise. seek consultation for safe use of essential oils. even with a safe oral dose of eucalyptus can cause harm. safe use of essential oils is to not take essential oils internally. it is extremely important that induced vomiting is not done for this toxicity. oral toxicity symptoms are rapid decline with complaint of burning in the mouth and throat and abdominal pain. central nervous symptoms are ataxia and respiratory depression, and, with a higher dose, possible nasal intubation is needed for mechanical ventilation and deep coma. death can occur with a toxic dose. , eye safety mf accidently splashed essential oil in an eye when she was preparing essential oil for a diffuser. her eye was burning and painful and her vision blurred in the eye. critical analysis reveals essential oils are toxic to eyes and can result in a chemical burn. the eye should be rapidly irrigated with milk or a vegetable oil carrier. a washcloth or cotton ball can help with the irrigation. after treatment flush the eye with water. do not flush the eye with water initially due to oil and water not mixing. clinical nursing aromatherapy is patient symptom management with measuring the outcome in a clinical setting. when aromatherapy is ordered by a provider for symptom management, a nurse needs to perform a history assessment, obtain vital signs, identify the symptom, educate the patient, measure symptom management, evaluate the effectiveness, and document the plan of care. , , , the following is an overview of clinical management of the essential oil: allergy-inhalant, skin, food, and medication allergy or sensitivity. consider the need for a patch test. chronic conditions-assess condition that could be impacted by aromatherapy, such as plant source triggering asthma attack or cancer that is fed by estrogen, with a few essential oils having estrogenic activity. obtain vital signs-assess if there is a problem proceeding with essential oil administration. symptoms needing to be managed-such as anxiety, depression, insomnia, nausea, and pain educate the patient about the essential oil, procedure, safety, symptom management, patient-centered selection of the essential oil, and consent for implementation. outcome measurement of symptom relief-select a tool for measuring the symptom, such as pain. the pain tool could be measurement of pain from to or visual picture rating of pain; or, a nonverbal patient's pain could be measured with a visual picture range, and pain in a patient unable to communicate could be measured with physiologic changes, such as vital signs, guarding of the area, and facial grimaces from discomfort. after selection of the pain measurement tool, rate the presymptom range, and post-implementation, measure at end of post symptom score for a change in outcome findings. evaluate the effectiveness of the essential oil on the symptom. the outcome goals are decrease in the symptom and increased well-being and quality of life. patient-centered symptom management and presence of a nurse could increase patient satisfaction. document the procedure and incorporate into the plan of care. examples of clinical conditions and settings that can benefit in the management of symptoms in the inpatient and outpatient settings are pain, nausea and vomiting, preoperative anxiety, critical care, general well-being, anxiety, depression, stress, insomnia, respiratory, dementia, oncology, palliative care, hospice, and end of life. , , , aromatherapy is used as an alternative medicine and complement to traditional care. aromatherapy is rising in popularity and is a cost-effective symptom manager. the following are suggested steps for guidance and triggers for brainstorming to develop a customized patient-centered symptom management program using essential oils in an inpatient or outpatient setting. . buy-in from major stakeholders. develop a committee that includes interprofessional members. input from all stakeholders, including frontline nurses, needs to be embraced; and, commit, by a recorded vote, to proceeding with the aromatherapy program. . develop a policy and procedure manual. search the literature for best practice aromatherapy models. if possible, contact the facility for assistance with the startup of the program. for example, a best practice model is at the mayo clinic in phoenix arizona. this research and education facility uses aromatherapy for alternative medicine and has integrative medicine. . upon approval, establish guidelines for safe and effective implementation, including infection control, safe storage, and disposal of the chemical oil. . identify common symptoms that could occur in the facility, such as pain, anxiety, depression, nausea, and insomnia. . identify nursing considerations, such as assessment, chronic illness, administration, and safety. . identify preoutcome and postoutcome measurements and best tools for measurement. for example, anxiety is a symptom: identify a pretest and post-test to measure anxiety that is a short tool. . identify and educate aromatherapy champions to lead the new program by supervising and mentoring nurses, for example, a classroom course for hospital nurses and a certified clinical aromatherapy practitioner course. . evaluate the data from the pre-intervention and post-intervention of aromatherapy. interpret the findings and refine if needed. . data analysis to justify aromatherapy is an effective intervention for symptom management. . seek a provider standing order for aromatherapy for sustainability. , summary aromatic scents and oils used in clinical aromatherapy can be beneficial for symptom management such as pain, nausea, vomiting, anxiety, depression, stress, insomnia, agitation with dementia, cancer pain, and end of life symptoms, clinical aromatherapy has been found beneficial in the inpatient and outpatient settings especially critical care, oncology, palliative care, hospice, and surgical. on the flip side, aromatic essential oils can be dangerous and toxic due to certain oils being flammable, causing skin dermatitis, or being phototoxic, with risks of chemical burn, oral toxicity, and even death. therefore, it is important that nurses learn about essential oils. if a facility has a clinical aromatherapy program, it is critical that frontline nurses be educated with a classroom course on essential oils. champions need to be selected for a clinical aromatherapy practitioner course. these certified aromatherapists can lead the program, serve as consultants, and mentor nurses. essential oils in hospitals: the ethics, safety, cost and application of clinical aromatherapy antimicrobial properties of plant essential oils against human pathogens and their mode of action: an updated review. evidence based complementary and alternative medicine alternative, or integrative health: what's in a name? aromatherapy and nursing: historical and theoretical conception aromatherapy with essential oils (pdq)-health professional version. beethesda,md: national cancer institute complementary and alternative therapies in nursing clinical aromatherapy essential oils in healthcare national association of holistic aromatherapists. what is aroma therapy? available at consumer product safety commission. regulations, laws & standards. available at chapter . seeds. in: nature's essential oils: aromatic alchemy for well-being stems, leaves, & needles in nature's essential oils: aromatic alchemy for well-being petals & flowers. in: nature's essential oils: aromatic alchemy for well-being rinds & fruits in nature's essential oils: aromatic alchemy for well-being woods & resins. in: nature's essential oils: aromatic alchemy for well-being roots & rhizomes. in: nature's essential oils: aromatic alchemy for well-being grass. in: nature's essential oils: aromatic alchemy for well-being clinical aromatherapy essential oils in healthcare clinical aromatherapy essential oils in healthcare clinical aromatherapy essential oils in healthcare key: cord- -df luh v authors: dos santos-silva, carlos andré; zupin, luisa; oliveira-lima, marx; vilela, lívia maria batista; bezerra-neto, joão pacifico; ferreira-neto, josé ribamar; ferreira, josé diogo cavalcanti; de oliveira-silva, roberta lane; pires, carolline de jesús; aburjaile, flavia figueira; de oliveira, marianne firmino; kido, ederson akio; crovella, sergio; benko-iseppon, ana maria title: plant antimicrobial peptides: state of the art, in silico prediction and perspectives in the omics era date: - - journal: bioinform biol insights doi: . / sha: doc_id: cord_uid: df luh v even before the perception or interaction with pathogens, plants rely on constitutively guardian molecules, often specific to tissue or stage, with further expression after contact with the pathogen. these guardians include small molecules as antimicrobial peptides (amps), generally cysteine-rich, functioning to prevent pathogen establishment. some of these amps are shared among eukaryotes (eg, defensins and cyclotides), others are plant specific (eg, snakins), while some are specific to certain plant families (such as heveins). when compared with other organisms, plants tend to present a higher amount of amp isoforms due to gene duplications or polyploidy, an occurrence possibly also associated with the sessile habit of plants, which prevents them from evading biotic and environmental stresses. therefore, plants arise as a rich resource for new amps. as these molecules are difficult to retrieve from databases using simple sequence alignments, a description of their characteristics and in silico (bioinformatics) approaches used to retrieve them is provided, considering resources and databases available. the possibilities and applications based on tools versus database approaches are considerable and have been so far underestimated. proteins and peptides play different roles depending on their amino acids (aa) constitution, which may vary from tens to thousands residues. peptides are conventionally understood as having less than aa. proteins, on the contrary, would be any molecule presenting higher amino acid content and bothproteins and peptides-present a plethora of variations in plants. despite that, plant proteomes have been much more studied than peptidomes. it is well-known that the biochemical machinery necessary for the synthesis and metabolism of peptides is present in every living organism. from the variations of this machinery, a wide structural and functional diversity of peptides was generated, justifying the growing interest in their study. in eukaryotes, peptides are prevalent in intercellular communication, performing as hormones, growth factors, and neuropeptides, but they are also present in the defense system. besides plants and animals, several pathogenic microorganisms, peptides can serve as classical virulence factors, which disrupt the epithelial barrier, damage cells, and activate or modulate host immune responses. an example of this performance is represented by candidalysin, a fungal cytolytic peptide toxin found in the pathogenic fungus candida albicans that damages epithelial membranes, triggers a response signaling pathway, and activates epithelial immunity. there are also reports of defense-related fungal peptides. for example, the copsin, a peptide-based fungal antibiotic recently identified in the fungus coprinopsis cinerea kills bacteria by inhibiting their cell wall synthesis. regarding bacterial peptides, certain species from the gastrointestinal microbial community can release low-molecular-weight peptides, able to trigger immune responses. there are additionally peptides that act like bacterial "hormones" that allow bacterial communities to organize multicellular behavior such as biofilm formation. some peptides are known for their medical importance, as defensins that bioinformatics and biology insights present antibacterial, antiviral, and antifungal activities. for example, human alpha-and beta-defensins present in the saliva may potentially impede virus replication, including sars-cov- , besides other roles as protection against intestinal inflammation (colitis). considering the roles of plant peptides, they can also be multifunctional, and have been classified into main categories (supplementary figure s ) : ( ) peptides with no bioactivity, primarily resulting from the degradation of proteins by proteolytic enzymes, aiming at their recycling, and ( ) bioactive peptides, which are encrypted in the structure of the parent proteins and are released mainly by enzymatic processes. the first group is innocuous regarding signaling, regulatory functions, and bioactivity. so far, it has been reported that some of them may play a significant role in nitrogen mobilization across cellular membranes. the second group of bioactive peptides has a substantial impact on the plant cell physiology. some peptides of this group can act in the plant growth regulation (through cell-to-cell signaling), endurance against pathogens and pests by acting as toxins or elicitors, or even detoxification of heavy metals by ion-sequestration. comprising bioactive peptides, additional subcategorization has been proposed regarding their function. tavormina et al figure s ) based on the type of precursor: • • derived from functional precursors: originated from a functional precursor protein; • • derived from nonfunctional precursors: originated from a longer precursor that has no known biological function (as a preprotein, proprotein, or preproprotein); • • not derived from a precursor protein: some sorfs (small open read frames; usually < codons) are considered to represent a potential new source of functional peptides (known as "short peptides encoded by sorfs"). a more intuitive classification of bioactive peptides was further proposed by farrokhi et al receptors in leaves. another example is the pls (polaris) peptide that acts during early embryogenesis but later activates auxin synthesis, also affecting cytokine synthesis and ethylene response. regarding the second group, it includes peptides with signaling roles in plant defense, comprising at least subgroups, including syst (systemin) (supplementary figure s ). the syst peptides were identified in solanaceae members, like tomato and potato (acting on the signaling response to herbivory). the syst leads to the production of a plant protease inhibitor that suppresses insect's proteases. stratmann suggested that in plants, systs act to stimulate the jasmonic acid signaling cascade within vascular tissues to induce a systemic wound response. • • defense peptides or antimicrobial peptides (amps): to be fitted into this class, a plant peptide must fulfill some specific biochemical and genetic prerequisites. regarding biochemical features, in vitro antimicrobial activity is required. concerning the genetic condition, the gene encoding the peptide should be inducted in the presence of infectious agents. in practice, this last requirement is not ever fulfilled as some amps are tissue-specific and are considered as part of the plant innate immunity, while other isoforms of the same class appear induced after pathogen inoculation. plant amps are the central focus of the present review, comprising information on their structural features (at genomic, gene, and protein levels), resources, and bioinformatic tools available, besides the proposition of an annotation routine. their biotechnological potential is also highlighted in the generation of both transgenic plants resistant to pathogens, and new drugs or bioactive compounds. antimicrobial peptides are ubiquitous host defense weapons against microbial pathogens. the overall plant amp characterization regards the following variables ( figure ): electrical charge, hydrophilicity, secondary and -dimensional ( d) structures, and the abundance or spatial pattern of cysteine residues. these features are primarily related to their defensive role(s) as membrane-active antifungal, antibacterial, or antiviral peptides. regarding the nucleotide sequence, plant amps are hypervariable and this genetic variability is considered crucial to provide diversity and the ability to recognize different targets. for their charges, amps can be classified as cationic or anionic ( figure ). most plant amps have positive charges, which is a fundamental feature for the interaction with the membrane lipids of pathogens. concerning hydrophilicity, amps are generally amphipathic, that is, they exhibit molecular conformation with both hydrophilic and hydrophobic domains. silva et al with respect to their d structure, amps can be either linear or cyclic ( figure ). some linear amps adopt an amphipathic α-helical conformation, whereas non-α-helical linear peptides generally show or predominant amino acids. in turn, cyclic amps, including cysteine-containing peptides, can be divided into subgroups based on the presence of a single or multiple disulfide bonds. a peculiar feature of these peptides is a cationic and amphipathic character, what improves their functioning as membrane-permeabilizing agents. considering the secondary structures, amps may exhibit α-helices, β-chains, β-pleated sheets, and loops ( figure ). wang classified plant amps into families (α, β, αβ, and non-αβ), based on the protein classification of murzin et al, with some modifications. antimicrobial peptides of the "α" family present α-helical structures, whereas amps from the "β" family contains β-sheet structures usually stabilized by disulfide bonds. , some plant amps show an α-hairpinin motif formed by antiparallel α-helices that are stabilized by disulfide bridges. such amps present a higher resistance to enzymatic, chemical, or thermal degradation. antimicrobial peptides from the "αβ" family having both "α" and "β" structures are also stabilized by disulfide bridges. an example of amp presenting "αβ" structures are defensins, usually with a cysteine-stabilized αβ motif (csαβ), an α-helix, and a triplestranded antiparallel β sheet stabilized mostly by disulfide bonds. finally, amps that do not belong to the "αβ" group exhibit no clearly defined "α" or "β" structures. plant amps are also classified into families considering protein sequence similarity, cysteine motifs, and distinctive patterns of disulfide bonds, which determine the folding of the tertiary structure. therefore, plant amps are commonly grouped as thionins, defensins, heveins, knottins (linear and cyclic), lipid transfer proteins (ltp), snakins, and cyclotides. , these amp categories will be detailed in the next sections, together with other groups here considered (impatienlike, macadamia [β-barrelins], puroindoline (pin), and thaumatin-like protein [tlp]) and the recently described αhairpinin amps. the description includes comments on their structure, pattern for regular expression (regex) analysis (when available), functions, tissue-specificity, and scientific data availability. thionins are composed by to amino acid residues with a molecular weight around kda, considering the mature peptide. they are synthesized with a signal peptide together with the mature thionin and the so-called acidic domain. to date, there is no experimental information available about possible functions of the acidic domain, even though it is clearly not dispensable as shown by the high conservation of the cysteine residues. the thionin superfamily comprises distinct groups of plant peptides α/β-thionins and γ-thionins with distinguished structural features. the α/β thionins have homologous amino acid sequences and similar structures. besides, they are rich in arginine, lysine, and cysteine. in turn, γ-thionins have a greater similarity with defensins, and some authors classify them within this group. however, compared with the defensins, they present a longer conserved amino acid sequence. regarding the cysteine motif, it can be divided into subgroups, one with residues connected by disulfide bonds called c and the other with residues connected by disulfide bonds called c. the general designation of thionins has been proposed as a family of homologous peptides that includes purothionins. the first plant thionin was isolated in from wheat flour and labeled as purothionin. since then, homologues from various taxa have been also identified, like bioinformatics and biology insights viscotoxins (viscum album) and crambins (crambe abyssinica). they have also been isolated from different plant tissues like seeds, leaves, and roots. , thionins have been tested for different elicitors: grampositive , or gram-negative bacteria, , yeast, , insect larvae, nematode, and inhibitory proteinase. thionins are hydrophobic in nature, interact with hydrophobic residues, and lyse bacteria cell membrane. their toxicity is due to an electrostatic interaction with the negatively charged membrane phospholipids, followed by either pore formation or a specific interaction with a membrane. it has been reported that they are able to inhibit other enzymes possibly through covalent attachment mediated by the formation of disulfide bonds, as previously observed for other thionin/enzyme combinations. thionin representatives with known d structures determined by x-ray crystallography are crambin (pdb id: crn), α and β-purothionins (pdb id: phn and bhp), β-hordothionin (pdb id: wuw), and viscotoxin-a (pdb id: okh). the first to be determined was the mixed form of crambin. , it showed a distinct capital Γ shape with the n terminus forming the first strand in a βsheet. the architecture of this sheet is additionally strengthened by disulfide bonds. after a short stretch of extended conformation, there is a helix-turn-helix motif. in crambin, there is a single disulfide involved in stabilizing the helix-tohelix contacts. at the center of this motif, there is a crucial arg that forms hydrogen bonds to tie together the first strand, the first helix, and the c terminus. the first plant defensins were isolated from wheat and barley grains, initially called γ-hordothionins. due to some similarities in cysteine content and molecular weight, they were classified as γ-thionins. later, the term "γ-thionin" was replaced by "defensin" based on the higher number of primary and tertiary structures of these proteins and also on their antifungal activities more related to insect and mammalian defensins than to plant thionins. plant defensins belong to a diverse protein superfamily called cis-defensin and exhibit cationic charge, consisting of to aa with to disulfide bonds. , plant defensins share similar tertiary structures and typically exhibit a triple-stranded antiparallel β sheet, enveloped by an α-helix and confined by intramolecular disulfide bonds (figure a ). this motif is called cysteine-stabilized αβ (csαβ). the csαβ defensins were classified into groups based on their sequence, structure, and functional similarity. defensins are known for their antimicrobial activity at low micromolar concentrations against gram-positive and gramnegative bacteria, fungi, viruses, and protozoa. in addition, they present protein inhibition, insecticidal, and antiproliferative activity, acting as an ion-channel blocker, being also associated with the inhibition of pathogen protein synthesis. instead, plant defensins act in the regulation of signal transduction pathways and induce inflammatory processes, in addition to wound healing, proliferation control, and chemotaxis. in general, plant defensins do not present high toxicity to human cells, having in vivo efficacy records, with relevant therapeutic potential, and can be applied in treatments associated with traditional medicine. cools et al reported that a peptide derived from a plant defensin (hsafp ) acted synergistically with caspofungin (an antimycotic) (in vivo and in vitro) against the formation of candida albicans biofilm on polystyrene and catheter substrate, indicating that the hsafp variant presented a strong antifungal potential in the proposed treatment. other biotechnological applications of defensins are described, as in the case of ecgdf , which was isolated from a legume (erythrina crista-galli), heterologously expressed in escherichia coli and purified. ecgdf inhibited the growth of various plant and human pathogens (such as candida albicans and aspergillus niger and the plant pathogens clavibacter michiganensis ssp. michiganensis, penicillium expansum, botrytis cinerea and alternaria alternate). due to these features, ecgdf is a candidate for the development of antimicrobial products for both agriculture and medicine. non-specific lipid transfer proteins (ns-ltps) were first isolated from potato tubers and are actually identified in diverse terrestrial plant species. they comprise a large gene family, are abundantly expressed in most tissues, but absent in most basal plant groups as chlorophyte and charophyte green algae. they generally include an n-terminal signal peptide that directs the protein to the apoplastic space. some ltps have a c-terminal sequence that allows their post-translational modification with a glycosylphosphatidylinositol molecule, facilitating the integration of ltp on the extracellular side of the plasma membrane. the ns-ltps are small proteins which were thus named because of their function of transferring lipids between the different membranes carrying lipids (non-specifically, the list includes phospholipids, fatty acids, their acylcoas, or sterols). they consist of approximately aa and are relatively larger in size than other amps, such as defensins. depending on their sizes, ltps may be classified into subfamilies: ltp and ltp , with relative molecular weight of and kda, respectively. , the limited sequence conservation turned this classification inadequate. thus, a modified and expanded classification system was proposed, presenting main types (ltp , ltp , ltpc, ltpd, and ltpg) and additional types with a smaller number of members (ltpe, ltpf, ltph, ltpj, and ltpk). the new classification system is not based on molecular size but rather on ( ) the position of a conserved intron, ( ) the identity of the amino acid sequence, and figure s ) . although this latter classification system is the most recent, the conventional classification of ltp and ltp types has been maintained by most working groups. lipid transfer protein nomenclature has been confusing and without consistent guidelines or standards. there are several examples where specific ltps receive different names in different scientific articles. the lack of a robust terminology sometimes turns it quite difficult, extremely time-consuming, and frustrating to compare ltps with different roles/functions. therefore, an additional nomenclature was also proposed by salminen et al, naming ltps as follows: atltp . , osltp . , hvltpc , ppltpd , and taltpg , with the first letters indicating the plant species (eg, at = arabidopsis thaliana, pp = physcomitrella patens); ltp , ltp , and ltpc indicating the type; while the last digit (here - ) regards the specific number given to each gene or protein within a given ltp type. for the sake of clarity, the authors recommend the inclusion of a point between the type specification (ltp and ltp ) and the gene number. for ltpc, ltpd, ltpg, and other types of ltp defined with a letter, the punctuation mark was not recommended. this latter classification system is currently recommended as it comprises several features of ltps and is more robust than the previous classification systems. lipid transfer proteins are small cysteine-rich proteins, having to helices in their tertiary structure ( figure b ), which is stabilized by several hydrogen bonds. such a folding gives ltps a hydrophobic cavity to bind the lipids through hydrophobic interactions. this structure is stabilized by disulfide bridges formed by conserved cysteines, similar to defensins, although bound by cysteines in different positions. the disulfide bridges promote ltp folding into a very compact structure, which is extremely stable at different temperatures and denaturing agents. [ ] [ ] [ ] these foldings provide a different specificity of lipid binding at the ltp binding site, where the ltp structure is relatively more flexible and present a lower lipid specificity when compared with ltp . the first d structure of an ltp was established for taltp . based on d and d data of h-nmr, purified from wheat (triticum aestivum) seeds in aqueous solution. , currently, several d structures of ltps have been determined, either by nuclear magnetic resonance (nmr) or x-ray crystallography; in their free, unbound form or in a complex with ligands. the heveins were first identified in in the rubber-tree (hevea brasiliensis), but its sequence was determined later, whereas a similarity was detected to the chitin-binding domain of an agglutinin isolated from urtica dioica (l.) with cysteine residues forming a typical cys motif. the primary structure of the hevein consists of to aa, positively charged, with abundant glycine ( ) and cysteine ( - ) residues, and aromatic residues. , the chitin-binding domain is a determinant component in the identification of hevein-like peptides whose binding site is represented by the amino acid sequence sxfgy/sxygy, where x regards any amino acid. , most heveins have a coil-β -β -coil-β structure that occurs by variations with the secondary structural motif in the presence of turns in long coils in the β chain. antiparallel β chains form the central β sheet of the hevein motif with long coils stabilized by disulfide bonds ( figure c ). although the presence of chitin has not been identified in plants, there are chitin-like structures present in proteins that exhibit a strong affinity to this polysaccharide isolated from different plant sources. the presence of aromatic amino acids in the chitinbinding domain favors chitin binding by providing stability to the hydrophobic group c-h and the π electron system through van der waals forces, as well as the hydrogen bonds between serine and n-acetylglucosamine (glcnac) present in the chitin structure. , this domain is commonly found in chitinases of classes i to v, in addition to other plant antimicrobial proteins, such as lectins and pr- (pathogenesis-related protein ) members. , it may also occur in other proteins that bind to polysaccharide chitin, such as the antimicrobial proteins ac-amp and ac-amp of amaranthus caudatus (amaranthaceae) seeds which are homologous to hevein but lack the c-terminal glycosylated region. plant chitinases (class i) have the hevein-like domains, called hlds. due to the similar structural epitopes between chitinases and heveins, they are responsible for the cross reactive syndrome (latex-fruit syndrome). , among the several classes of proteins mentioned, the proteins with a high degree of similarity to hevein are chitinases i and iv. chitinases are known to play an essential role in plant defense against pathogens, also inhibiting in vitro fungal growth, especially when combined with β- , -glucanases. it also interferes with the growth of hyphae, resulting in abnormal ramification, delay, and swelling in their stretching. however, it has been shown that heveins have a higher inhibitory potential than chitinases and that their antifungal effect is not related only to the presence of chitinases ; pn-amp and pn-amp amps with hevein domains have potent antifungal activities against a broad spectrum of fungi, including those without chitin in their cell walls. , modes of action of chitinases usually include degradation and disruption of the fungal cell wall and plasma membrane due to its hydrolytic action, causing extravasation of plasma particles. , therefore, heveins have good antifungal activity, and only a few are active against bacteria, most of them with low activity. another role of hevein chitinases regards the antagonistic effect in triggering the aggregation of rubber particles in the latex extraction process in rubber trees. unlike heveins, other chitinases inhibit rubber particle aggregation. however, its action in conjunction with other proteins (β- , -glucanase) increases the effect of β- , -glucanase on rubber particle aggregation. a study by shi et al found that the interaction of the protein network related to the antipathogenic activity released by lutoids (lysosomal microvacuole in latex) is essential in closing laticiferous cells (cells that produce and store latex), not only providing a physical barrier, but a biochemical barrier used by laticiferous cells affected by pathogen invasion. knottins are part of the cysteine-rich peptides (crps) superfamily, sharing the cysteine-knot motif and therefore resembling other families as defensins, heveins, and cyclotides. their structure was initially identified by crystallography of carboxypeptides isolated from potato, showing the cysteine-knot motif with aa and cysteine residues. they are also called "cysteine-knot peptides," "inhibitor cysteine-knot peptides," or even "cysteine-knot miniproteins" because their mature peptide presents less than aa, forming interconnected disulfide bonds in the cysteine-knot motif, characterizing a particular scaffold. this conformation confers thermal stability at high temperatures. for example, the cysteine-stabilized β-sheet (csb) motif derived from knottins presents stability at approximately °c with only disulfide bonds. the knottins may have linear or cyclic conformation. however, both exhibit connectivity between the cysteines at positions - c, - c, and - c, forming a ring at the last bridge ( figure d ). knottins have different functions, such as signaling molecules, response against biotic and abiotic stresses, root growth, symbiotic interactions as well as antimicrobial activity against bacteria, fungi, virus, and insecticidal activity, among others. knottins antimicrobial activity has been attributed to the action of functional components of the plasma membrane, leading to alterations of lipids, ion flux, and exposed charge. the accumulation of peptides on the surface of the membrane results in the weakening of the pathogen membrane, resulting in transient and toroidal perforations. in the course of a large-scale survey to identify novel amps from australian plants, , an amp with no sequence homology was purified. its complementary dna (cdna) was cloned from macadamia integrifolia (proteaceae) seeds, containing the complete peptide coding region. the peptide was named miamp , being highly basic with an estimated isoelectric point (pi) of and a mass of kda. the miamp is aa long, including a aa signal peptide in the n-terminal region, bound to a aa mature region with cysteine residues. its d structure was determined using nmr spectroscopy, revealing a unique conformation among plant amps, with beta-strands arranged in greek key motifs, forming a greek key beta-barrel ( figure e ). due to its particularities, miamp was classified as a new structural family of plant amps, and the name β-barrelins was proposed for this class. this structural fold resembles a superfamily of proteins called γ-crystallin-like characterized by the precursors βγ-crystallin. this family includes amps from other organisms, for example, wmkt, a toxin produced by the wild yeast williopsis mraki. the miamp exhibited in vitro antimicrobial activity against various phytopathogenic fungi, oomycetes, and grampositive bacteria with a concentration range of . to μm generally required for a % growth inhibition (ic ). in addition, the transient expression of miamp in canola (brassica napus) provided resistance against blackleg disease caused by the fungus leptosphaeria maculans, turning miamp potentially useful for genetic engineering aiming at disease resistance in crop plants. there are few scientific publications with macadamia-like peptides, maybe because they prevail in primitive plant groups (eg, lycophytes, gymnosperms to early angiosperms as amborella and papaver), being apparently absent in derived angiosperms (eg, asteridae, including brassicaceae as arabidopsis thaliana). on the contrary, they have been identified in some monocots (as zantedeschia, zea, and sorghum). in fact, peptides similar to miamp appear to play a role in the defense against pathogens in gymnosperms, including species of economic importance (as pinus and picea) thus deserving attention for their biotechnological potential. four closely related amps (ib-amp , ib-amp , ib-amp , and ib-amp ) were isolated from seeds of impatiens balsamina (balsaminaceae) with antimicrobial activity against a variety of fungi and bacteria, and low toxicity to human cells in culture. these amps are the smallest isolated from plants to date, consisting of only aa in length. the ib-amps are highly basic and contain cysteine residues that form disulfide bonds. interestingly, they have no significant homology with other amps available in public databases. sequencing of cdnas isolated from i. balsamina revealed that all peptides are encoded within a single transcript. concerning the predicted precursor of ib-amp protein, it consists of a pre-peptide followed by mature peptide domains, each one of them flanked by propeptide domains ranging from to aa in length (supplementary figure s ) . this primary structure with repeated domains of alternating basic peptides and acid propeptide domains has, to date, not been reported in other plant species. patel et al conducted an experiment to purify ib-amp from seeds of impatiens balsamina. after purification, this peptide had its secondary structure tested by circular dichroism (cd). the results revealed a peptide that may include a β-turn but do not show evidences for either helical or β-sheet structure over a range of temperature and ph. structural information from d h-nmr was obtained in the form of proton-proton internuclear distances inferred from nuclear overhauser enhancements (noes) and dihedral angle restraints from spinspin coupling constants, which were used for distance geometry calculations. owing to the difficulty in obtaining the correct disulfide connectivity by chemical methods, the authors had built and performed separate calculations: ( ) a model with no disulfides; ( ) another with predicted disulfide bonds; and ( ) a model with alternative connectivity disulfide, as assigned from the nuclear overhauser effect spectroscopy (noesy) nmr spectra. as a result, hydrophilic patches were observed at opposite ends and opposite sides of the models, whereas in between them a large hydrophobic patch was identified. however, the study did not conclude which of the models would be the most likely representative of ib-amp , reporting only that cysteines are necessary for maintaining the structure. based on the experiment performed by patel et al, the present work built different models: model : without disulfide bonds, and the other models with different disulfide connections-model : nmr prediction by patel et al -cys; -cys and -cys; -cys, and model : disulfide bond partner prediction by dianna -cys; -cys and -cys; -cys. calculations have shown that although the peptide is small, the cysteines constrain part of it to adopt a well-defined main chain conformation. from residue to (except ), the main chain is well-defined, whereas residues to in the n-terminal region present few restrictions and appear to be more flexible (supplementary figure s ) . analyzing the rmsd (root mean square deviation), we observed that all the models lost the initial conformation and, among them, model was the most stable. models and showed a similar pattern (supplementary figure s ) , as in the models of patel et al, although model was the most flexible. little is known about impatiens-like amps mode of action. lee et al investigated the antifungal mechanism of ib-amp noting that when oxidized (bound by disulfide bridges), there occurs a -fold increase in antifungal activity against aspergillus flavus and candida albicans, as compared with reduced ib-amp (without disulfide bridges). confocal microscopy analyses have shown that ib-amp can either bind to the cell bioinformatics and biology insights surface or penetrate cell membranes, indicating an antifungal activity by inhibiting a distinct cellular process, rather than ion channel or membrane pore formation. fan et al reported the ib-amp antimicrobial activity dependent of β-sheet configuration to enable insertion into the lipid membrane, thus killing the bacteria through a non-lytic mechanism. current approaches aim to make changes in ib-amp to improve its antimicrobial activity. as an example, synthetic variants of ib-amp were fully active against yeasts and fungi, where the replacement of amino acid residues by arginine or tryptophan improved more than twice the antifungal activity. another study involving amp modification generated a synthetic peptide without the disulfide bridges (ie, a linear analog of ib-amp ), which showed an antimicrobial specificity . to . times higher than the wild-type ib-amp . puroindoline puroindolines are small basic proteins that contain a single domain rich in tryptophan. these proteins were isolated from wheat endosperm, have a molecular mass around kda, and a calculated isoelectric point higher than . at least main isoforms (called pin-a and pin-b) are known, which are encoded by pina-d and pinb-d genes, respectively. these genes share . % identical coding regions but exhibit only % identity in the ′ untranslated region. both pin-a and pin-b contain a structure with conserved cysteine residues and a tertiary structure similar to ltps, consisting of α-helices separated by loops of varying lengths, with the tertiary structure joined by disulfide bonds, of which identical to ns-ltps. the conformation of the pin isoforms was studied by infrared and raman spectroscopy. both pin-a and pin-b have similar secondary structures comprising approximately % helices, % β-sheets, and % non-ordered structures at ph . it has been proposed that the folding of both pins is highly dependent on the ph of the medium. the reduction of the disulfide bridges results in a decrease of pins solubility in water and to an increment of the β-sheet content by about % at the expense of the α-helix content. no high-resolution structure for any of the pin isoforms is available, bringing challenges to understanding the function of their hydrophobic regions, with some evidence coming only from partially homolog peptides. however, wilkinson et al proposed a theoretical model for several sequences of this amp. puroindolines are proposed to be functional components of wheat grain hardness loci, control core texture, besides antifungal activity. [ ] [ ] [ ] [ ] although the biological function of pins is unknown, their involvement in lipid binding has been proposed. while ltps bind to hydrophobic molecules in a large cavity, pins interact only with lipid aggregates, that is, micelles or liposomes, through a single stretch of tryptophan residues. this stretch of tryptophan residues is especially significant in the main form, pin-a (wrwwkwwk), while it is truncated in the smaller form, pin-b (wptwwk). [ ] [ ] [ ] puroindolines form protein aggregates in the presence of membrane lipids, and the organization of such aggregates is controlled by the lipid structure. in the absence of lipids, these proteins may aggregate, but there is no accurate information on the relationship between aggregation and interaction with lipids. the antimicrobial activity of pins is targeted to cell membranes. charnet et al indicated that pin is capable of forming ion channels in artificial and biological membranes that exhibit some selectivity over monovalent cations. the stress and ca + ions modulate the formation and/or opening of channels. puroindolines may also be membranotoxins, which may play a role in the plant defense mechanism against microbial pathogens. morris reported that the pin-a and pin-b act through similar but somewhat different modes, which may involve "membrane binding, membrane disruption and ion channel formation" or "intracellular nucleic acid binding and metabolic disruption." natural and synthetic mutants have allowed the identification of pins as key elements for antimicrobial activity. snakins are crps first identified in potato (solanum tuberosum). , due to their sequence similarity to gasa (gibberellic acid stimulated in arabidopsis) proteins, the snakins were classified as members of the snakin/gasa family. the genes that encode these peptides have ( ) a signal sequence of approximately aa, ( ) a variable region, and ( ) a mature peptide of approximately residues, with highly conserved cysteine residues. these cysteine residues maintain the d structure of the peptide through disulfide bonds, besides providing stability to the molecule when the plant is under stress , , , (figure f; supplementary figure s ). snakins may be expressed in different parts of the plant, like stem, leaves, flowers, seeds, and roots, - both constitutive or induced by biotic or abiotic stresses. in vitro activity was observed against a variety of fungi, bacteria, and nematodes, acting as a destabilizer of the plasma membrane. , , moreover, they were reported as essential agents in biological processes such as cell division, elongation, cell growth, flowering, embryogenesis, and signaling pathways. [ ] [ ] [ ] [ ] alpha-hairpinins as reported by nolde et al, alpha-hairpin emerged as a new amp with unusual motif configuration. these peptides prevail in plants and their structure was resolved based on nmr data obtained from the ecamp- peptide isolated from barnyard grass seeds (echinoa crus-galli). some α-hairpinins comprise trypsin inhibitors with helical hairpin structure and this group silva et al was recently proposed as a new plant amp family. similar to other amps, the amino acid sequences of α-hairpinins are variable. they share the conserved cysteine motif (cx cx - cx c) that form a helix-loop-helix fold and may have disulfide bridges c -c and c -c . its structural stability is maintained by forming hydrogen bonds, so that the side chains have a relatively stable spatial orientation. as reviewed by slavokhotova et al, members of alphahairpin family have been described in both mono and dicot groups, including species as echinochloa crus-galli and zea mays (both poaceae, monocot), fagopyrum esculentum (polygonaceae, eudicot), and stellaria media (caryophyllaceae, eudicot). several transcripts with α-hairpinin motif exhibit similarities to snakin/gasa genes and are sometimes positioned within this family. although the α-hairpinins structure has been published, its mechanism of action is still not resolved ( figure j , pdb id: l r). however, studies indicate they present a potential dna binding capacity. the term cyclotide was created at the end of the past century to designate a family of plant peptides with approximately aa in size and a structural motif called cyclic cysteine knot (cck). this motif is composed by a head-to-tail cyclization that is stabilized by a knotted arrangement of disulfide bridges, with conserved cysteines, connected as follows: c - , c - , c - . cyclotides are generally divided into subfamilies, mӧbius and bracelets, based on structural aspects. in addition to ccks, loops (between c - and c - ) have high similarity between both subfamilies, while the other loops (between c - and c - ) exhibit some conservation within the subfamilies , (supplementary figure s ) . to date, several cyclotides were identified in eudicot families such as rubiaceae, violaceae, fabaceae, and solanaceae, in addition to some monocots of poaceae family. in general, cyclotides may act in defense against a range of agents like insects, helminths, or mollusks. in addition, they can also act as ecbolic (inducer of uterus contractions), antibacterial, anti-hiv, and anticancer factors. all these characteristics added to the stability conferred by the cck motif turn these peptides into excellent candidates for drug development. , thaumatin-like protein thaumatins or tlps belong to the pr- (pathogen-related protein) family and received this name due to its first isolation from the fruit of thaumatococcus daniellii (maranthaceae) from west africa. thaumatin-like proteins are abundant in the plant kingdom, being found in angiosperms, gymnosperms, and bryophytes, being also identified in other organisms, including fungi, , insects, and nematodes. thaumatin-like proteins are known for their antifungal activity, either by permeating fungal membranes or by binding and hydrolyzing β- , -glucans. , in addition, they may act by inhibiting fungal enzymes, such as xylanases, α-amylases, or trypsin. besides, the expression of tlps is regulated in response to some stress factors, such as drought, injuries, freezing, and infection by fungi , viruses, and bacteria. as to the tlp structure, this protein presents characteristic thaumatin signature (ps ): , most of the tlps have molecular mass ranging from to kda, possessing conserved cysteine residues (supplementary figure s ) involved in the formation of disulfide bonds, which help in the stability of the molecule, allowing a correct folding even under extreme conditions of temperature and ph. thaumatin-like proteins also contain a signal peptide at the n-terminal, which is responsible for targeting the mature protein to a particular secretory pathway. the tertiary structure presents distinct domains, which are conserved and form the central cleft, responsible for the enzymatic activity of the protein, being located between domains i and ii. this central cleft may be of an acidic, neutral, or basic nature depending on the binding of the different linkers/receptors. all plant tlps with antifungal activity have an acidic cleft known as motif reddd due to highly conserved amino acid residues (arginine, glutamic acid, and aspartic acid; supplementary figure s ), being very relevant for specific receptor binding and antifungal activity. , , crystallized structures were determined for some plant tlps, such as thaumatin (figure g ), zeamatin ( figure h ), tobacco pr- d and osmotin, the cherry allergen pruav , and banana allergen ba-tlp, among other tlps. some tlps are known as small tlps (stlps) due to the deletion of peptides in one of their domains, culminating in the absence of the typical central cleft. these stlps exhibit only -conserved cysteine residues, forming disulfide bonds, resulting in a molecular weight of approximately to kda. they have been described in monocots, conifers, and fungi, so far. , , other tlps exhibit an extracellular tlp domain and an intracellular kinase domain, being known as pr k (pr -like receptor kinases) and are present in both monocots and dicots. for example, arabidopsis contains pr k genes, while rice has only . with the rapid growth in the number of available sequences, it is unfeasible to handle such amount of data manually. thus, amp sequences (as well as their biological information) have been deposited in large general databases, such as uniprot and trembl, which contain sequences of multiple origins. , in this sense, the construction of databases that deal specifically with amps was an important step to organize the data. during the past decade, several databases were built to support the deposition, consultation, and mining of amps. thus, these databases can be classified into groups: general and specific. the specific databases can be divided into subgroups: those containing only specific group (defensins or cyclotides) and those containing data from a supergroup of peptides (plant, animal, or cyclic peptides) (supplementary table ). in general, both types of databases share some characteristics such as the way that the data are available or the tools to analyze amps. the collection of antimicrobial peptides (campr ) is a database that comprises experimentally validated peptides, sequences experimentally deduced and still those with patent data, besides putative data based on similarity. [ ] [ ] [ ] the current version includes structures and signatures specific to families of prokaryotic and eukaryotic amps. the platform also includes some tools for amp prediction. the antimicrobial peptide database (apd) collects mature amps from natural sources, ranging from protozoa to bacteria, archaea, fungi, plants, and animals, including humans. amps encoded by genes that undergo post-translational modifications are also part of the scope, besides some peptides synthesized by multienzyme systems. the apd provides interactive interfaces for peptide research, prediction, and design, statistical data for a specific group, or for all peptides available in the database. the lamp (database linking antimicrobial peptides) comprises natural and synthetic amps, which can be separated into groups: experimentally validated, predicted, and patented. their data were primarily collected from the scientific literature, including uniprot and other amp-related databases. the database of antimicrobial activity and structure of peptides (dbaasp) contains information about amps from different origins (synthetic or non-synthetic) and complexity levels (monomers and dimers) that were retrieved from pubmed using the following keywords: antimicrobial, antibacterial, antifungal, antiviral, antitumor, anticancer, and antiparasitic peptides. this database is manually curated and provides information about peptides that have specific targets validated experimentally. it also includes information on chemical structure, post-translational modifications, modifications in the n/c terminal amino acids, antimicrobial activities, cell target and experimental conditions in which a given activity was observed, besides information about the hemolytic and cytotoxic activities of the peptides. due to the diversity of amps and the need to accommodate the most representative subclasses, several databases were established, focusing on specific types, sources, or features. there are several ways to classify amps, and they can range from biological sources such as bacterial amps (bacteriocins), plants, animals, and so on; biological activity: antibacterial, antiviral, antifungal, and insecticide; and based on molecular properties, pattern of covalent bonds, d structure and molecular targets. , the "defensins knowledgebase" is a database with manual curation and focused exclusively on defensins. this database contains information about sequence, structure, and activity, with a web-based interface providing access to information and enabling text-based search. in addition, the site presents information on patents, grants, laboratories, researchers, clinical studies, and commercial entities. , the cybase is a database dedicated to the study of sequences and d structures of cyclized proteins and their synthetic variants, including tools for the analysis of mass spectral fingerprints of cyclic peptides, also assisting in the discovery of new circular proteins. the phytamp is a database designed to be solely dedicated to plant amps based on information collected from the uniprot database and from the scientific literature through pubmed. plantpepdb is a database with manual curation of plantderived peptides, mostly experimentally validated at the protein level. it includes data on the physical-chemical properties and tertiary structure of amps, also useful to identify their therapeutic potential. different search options for simple and advanced compositing are provided for users to perform dynamic search and retrieve the desired data. overall, plantpepdb is the first database that comprises detailed analysis and comprehensive information on phyto-peptides from a wide functional range. biological data banks (dbs) are organized collections of data of diverse nature that can be retrieved using different inputs. the management of this information is done through various software and hardware resources, whose retrieval and organization can be performed in a quick and efficient way. considering biological data, information can be classified into ( ) primary (sequences), ( ) secondary (structure, expression, metabolic pathways, types of drugs, etc), and ( ) specialized, for example, containing information on a species or on a class of protein. within this third group, some references to amps can be mentioned, such as campr and apd that compile sequence data and structure retrieved from diverse sources, and also the defensin knowledgebase and the cybase which are dedicated to specific classes of peptides (defensins and cyclotides, respectively), in addition to phytamp, a specific database of plant amps (supplementary table ). the first step to infer the function of a given sequence (annotation) is to retrieve it in databases. for this purpose, approaches have been used mostly: ( ) local alignments, especially by using basic local alignment search tool (blast) and fasta ; by searching for specific patterns using ( ) regex or ( ) hidden markov model (hmm). the first approach has been widely used, since most of the information is available in databases as sequences, together with tools to align them, whereas the blast is the primary tool for doing so. this tool splits the sequence into small pieces (words), comparing it with the database. however, this approach has a limitation. small motifs may not be significantly aligned as they comprise small portions of the sequences that can be smaller than % of the total size. , due to the high variability of amps, only few highly conserved sequences can be identified using this type of inference. to reduce the effects of local alignment limitations, other strategies based on the search for specific patterns were introduced, such as regex (supplementary table ) and hmm. the regex is a precise way of describing a pattern in a string where each regex position must be set, although ambiguous characters (or wildcards) can also be used. for example, if we want to find a match for both amino acid sequences caiessk and waiesk, we can use the following expression: [cw]aies{ , }k, this expression would find a pattern starting with the letter "c" or "w," followed by an "a," an "i," and an "e," or "s," and ending with a "k." the hmms are well-known for their effectiveness in modeling the correlations between adjacent symbols, domains, or events, and they have been extensively used in various fields of biological analysis, including pairwise and multiple sequence alignment, base-calling, gene prediction, modeling dna sequencing errors, protein secondary structure prediction, noncoding rna (ncrna) identification, protein and rna structural alignments, acceleration of rna folding and alignment, fast noncoding rna annotation, and many others. using hmm, a statistic profile is included in the model, which is calculated from a sequence alignment, and a score is determined site-to-site, with conserved and variable positions defined a priori. , predicting antimicrobial activity the design of new amps led to the development of methods for the discovery of new peptides, thus allowing new experiments to be done by researchers. in this sense, the new challenge lies in the construction of new prediction models capable of discovering peptides with desired activities. the apd db has established a prediction interface based on some parameters defined by the entire set of peptides available in this database. these values are calculated from natural amps to consider features like length, net charge, hydrophobicity, amino acid composition, and so on. if we take as an example the net load, the amps deposited in the apd range from - to + . this is the first parameter incorporated into the prediction algorithm. however, most amps have a net load ranging from - to + , which then becomes the alternative prediction condition. therefore, the same method is applied to the remaining parameters. the prediction in apd is performed in main steps. first, the sequence parameters will be calculated and compared. if defined as an amp, the peptide can then be classified into groups: ( ) rich in given amino acids, ( ) stabilized by disulfide, and ( ) linear bridges. finally, sequence alignments will be conducted to find peptides of higher similarity. , , the advent of machine learning (ml) methods has promoted new possibilities for drug discovery. in ml inferences, both a positive and a negative dataset are usually required to train the predictive models. the positive data, in this case, regard preferably experimentally validated amps that can be collected in databases, whereas negative data are randomly selected protein sequences that do not have amp characteristics. , machine learning methods based on support vector machine (svm), random forest (rf), and neural networks (nn) have been the most widely used. svm is a specific type of supervised method of ml, aiming to classify data points by maximizing the margin between classes in a high-dimensional space. , random forest is a non-parametric tree-based approach that combines the ideas of adaptive neighbors with bagging for efficient adaptive data inference. neural networks is an information processing paradigm inspired by how a biological nerve system process information. it is composed of highly interconnected processing elements (neurons or nodes) working together to solve specific problems. [ ] [ ] [ ] evaluating proteomic data regarding the use of amps in peptide therapeutics, as an alternative to antimicrobial treatment, new efficient and specific antimicrobials are demanded. as aforementioned, amps are naturally occurring across all classes of life, presenting high active potential as therapeutic agents against various kinds of bacteria. the identification of novel amps in databases is primarily dependent on knowing about specific amps together with a sufficient sequence similarity. however, orthologs may be divergent in sequence, mainly because they are under strong positive selection for variation in many taxa, leading to remarkably lower similarity, even in closely related species. in this scenario, where alignment tools present limited use, strategy to identify amps is related to proteomic approaches. proteins and peptides are biomolecules responsible for various biochemical events in living organisms, from formation and composition to regulation and functioning. thus, understanding of the expression, function, and regulation of the proteins encoded by an organism is fundamental, leading to the so-called "proteomic era." the term "proteome" was first used by marc wilkins in and it represents the set of proteins encoded by the genome of a biological system (cell, tissue, organ, biological fluid, or organism) at a specific time under certain conditions. protein extraction, purification, and identification methods have significantly advanced our capacity to elucidate many biological questions using proteomic approaches. , due to the wide diversity of proteomic analysis, methods makes the choice of the correct approach dependent on the type of material and compounds to be analyzed. , two main tools are used to isolate proteins: ( ) the -dimensional electrophoresis ( -de) associated with mass spectrometry (ms) and ( ) liquid chromatography associated with ms, each one with its own limitations. [ ] [ ] [ ] obtaining native proteins is a challenge in proteomics or peptidomics, due to high protein complexity in samples, as the occurrence of post-translational modifications. alternative strategies applied to extraction, purification, biochemical, and functional analyses of these molecules have been proposed, favoring access to structural and functional information of hard-to-reach proteins and peptides. based on d gel, al akeel et al evaluated spots obtained from seeds of foeniculum vulgare (apiaceae) aiming at proteomic analyses and isolation of small peptides. extracted proteins were subjected to kda dialysis, and separation was carried out by deae-ion exchange chromatography while further proteins were identified by d gel electrophoresis. one of its spots showed high antibacterial activity against pseudomonas aeruginosa, pointing to promising antibacterial effects, but requiring further research to authenticate the role of the anticipated proteins. for amps, de is challenging due to the low concentration of the peptide molecules captured by this approach, their small sizes, and their ionic features (strongly cationic). in addition, the limited number of available specific databases and high variability turn their identification through proteolysis techniques and mass spectrometry, matrix-assisted laser desorption/ionization (maldi-ms) difficult. in addition, the partial hydrophobicity characteristics and surface charges facilitate peptide molecular associations, making analysis difficult by any known proteomic approaches. in addition, peptides are most often cleaved from larger precursors by various releasing or processing enzymes. furthermore, profiles generated do not represent integral proteome, as de has limitations to detect proteins with low concentration, values of extreme molecular masses, pis, and hydrophobic proteins, including those of membranes. due to these limitations, multidimensional liquid chromatography-high-performance liquid chromatography (mdlc-hplc) has been successfully employed as an alternative to d gels. techniques and equipments for the newly developed separation and detection of proteins and peptides, such as nano-hplc and multidimensional hplc, have improved proteomics evaluation. molecular mass values obtained are used in computational searches in which they are compared with in silico digestion results of proteins in databases. in silico approaches, usually by the action of trypsin as a proteolytic agent, may generate a set of unique peptides whose masses are determined by ms. , these methodologies are widely adopted for large-scale identification of peptide from ms/ms spectra. theoretical spectra are generated using fragmentation patterns known for specific series of amino acids. the first widely used search engines in database searching were sequest and mascot (matrix science, boston, ma; www.matrixscience.com). they rank peptide matches based on a cross-correlation to match the hypothetical spectra to the experimental one. mascot is widely used for peptidomics and proteomics analysis, including amp identification in many organisms, or to evaluate the antibacterial efficacy of new amps. evaluating new amp against multidrug-resistant (mdr) salmonella enterica, tsai et al used d gel electrophoresis and liquid chromatography-electrospray ionization-quadrupole-time-offlight tandem ms to determine the protein profiles. the protein identification was performed using the mascot with trypsin as cutting enzyme, whereas ncbi nr protein was set as a reference database. the methodology used in this study indicated that the novel amp might serve as a potential candidate for drug development against mdr strains, confirming the usability of mascot. in a similar way, umadevi et al described the amp profile of black pepper (piper nigrum l.) and their expression on phytophthora infection using label-free quantitative proteomics strategy. for protein/peptide identification, ms/ms data were searched against the apd database using an in-house mascot server, established full tryptic peptides with a maximum of missed cleavage sites and carbamidomethyl on cysteine, besides an oxidized methionine included as variable modifications. the apd database was used for amp signature identification, together with phytamp and campr . to enrich the characterization parameters, isoelectric point, aliphatic index, and grand average of hydropathy were also used (gravy) (using protparam tool), besides the net charge from phytamp database. based on label-free proteomics strategy, they established for the first time the black pepper peptidomics associated with the innate immunity against phytophthora, evidencing the usability of proteomics/ peptidomics data for amp characterization in any taxa, including plant amps, aiming the exploitation of these peptides as next-generation molecules against pathogens. other tools use database searching algorithms, such as x!tandem, open mass spectrometry search algorithm (omssa), probid, radars, and so on. these search engines are based on database search but use different scoring schemes to determine the top hit for a peptide match. general information on database search engines, their algorithms, and scoring schemes were reviewed by nesvizhskii et al. despite its efficient ability to identify peptides, database searching presents several drawbacks, like false positive identifications due to overly noisy spectra and lower quality peptides score (related to the short size of peptides). so, the identification is strongly influenced by the amount of protein in the sample, the degree of post-translational modification, the quality of automatic searches, and the presence of the protein in the databases. , in this scenario, the knowledge about the genome from a specific organism is important to allow the identification of the exact pattern of a given peptide. if an organism has no sequenced genome, it is not searchable using these methods. , once the sequences are obtained, bioinformatic tools can be used to predict peptides structure and estimate bioactive peptides. more recently, an interactive and free web software platform, mixprotool, was developed, aiming to process multigroup proteomics data sets. this tool is compiled in r (www.r-project. org), providing integrated data analysis workflow for quality control assessment, statistics, gene ontology enrichment, and other facilities. the mixprotool is compatible with identification and quantification outputs from other programs, such as maxquant and mascot, where results may be visualized as vector graphs and tables for further analysis, in contrast to existing softwares, such as giapronto. according to the authors, the web tool can be conveniently operated, even by users without bioinformatics expertise, and it is beneficial for mining the most relevant features among different samples. the central tenet of structural biology is that structure determines function. for proteins, it is often said the "function follows form" and "form defines function." therefore, to understand protein function in detail at the molecular level, it is mandatory to know its tertiary structure. experimental techniques for determining structures, such as x-ray crystallography, nmr, electron paramagnetic resonance, and electron microscopy, require significant effort and investments. all methods mentioned have their own limitations, and the gap between the number of known proteins and the number of known structures is still substantial. thus, there is a need for computational framework methods to predict protein structures based on the knowledge of the sequence. in addition, in recent years, there has been impressive progress in the development of algorithms for protein folding that may aid in the prediction of protein structures from amino acid sequence information. historically, the prediction of a protein structure has been classified into categories: comparative modeling, threading, and ab initio. the first approaches construct protein models by aligning the query sequences with already solved model structures. if the models are absent in the protein data bank (pdb), the models must be constructed from scratch, that is, by ab initio modeling, considered the most challenging way to predict protein structures. in the case of comparative modeling methods, when inserting a target sequence, the programs identify evolutionarily related models of solved structures based on their sequence or profile comparison, thus constructing structure models supported by these previously resolved models. this approach comprises main steps: ( ) fold assignment, which identifies similarity between the target and the structure of the solved model; ( ) alignment of the target sequence to the model; ( ) generation of a model based on alignment with the chosen template; and ( ) analysis of errors considering the generated model. there are several servers and computer models that automate the comparative modeling process, with swiss-model and modeler figuring as the most used. , although automation makes comparative modeling accessible to experts and beginners, some adjustments are still needed in most cases to maximize model accuracy, especially in the case of more complex proteins. therefore, some caution must be taken regarding the generated models, considering the resolution and quality of the model used, as well as homology between the model and the protein of interest. threading modeling methods are based on the observation that known protein structures appear to comprise a limited set of stable folds, and those similarity elements are often found in evolutionarily distant or unrelated proteins. the most used servers based on this approach are muster, sparks-x, raptorx, prosa-web, and most notably the i-tasser. in some cases, the incorporation of structural information to combine the sequence used in the search with possible models allows the detection of similarity in the fold, even in the absence of an explicit evolutionary relation. the prediction of structures from known protein models is, at first sight, a more straightforward task than the prediction of protein structures from available sequences. therefore, when no solved model is available, another approach is recommended, namely, the ab initio modeling. this method is intended to predict the structure only from the sequence information, without any direct assistance from previously known structures. the ab initio modeling aims to predict the best model, based on the minimum energy for a potential energy function by sampling the potential energy surface using various searchable information. , such approaches turn it challenging to produce high-resolution modeling, essential for determining the native protein folding and its biochemical interpretation. on the contrary, later resolved structures and comparisons with previously predicted proteins point to a higher successful modeling generated by ab initio methods than those generated by pure energy minimization methods, classical or even pure methods. among the most used servers and programs for ab initio modeling, we highlight the rosetta, quark, and touchstone ii. the accuracy of the models calculated by many of these methods is evaluated by cameo (continuous automated model evaluation) and by casp (critical assessment of protein structure prediction). probably the first reasonably accurate ab initio model was built in casp . since then, sustained progress was achieved in ab initio prediction, but mainly for small proteins ( residues or less). in casp , for the first time, a novel -residue protein with a sequence identity with known structures lower than % was constructed with high precision for sequences of this size. in casp , a significant improvement was reported in areas: contact prediction, free modeling, template-based modeling, and estimating the accuracy of models. the authors report that this improvement is due to the accuracy of modeling and alignment methods, as well as increased data availability for both sequence and structure. due to the number of amps deposited in the pdb (to date approximately structures), comparative modeling is the most used. however, when it comes to de novo peptide design, the most recommended choice would be ab initio or a hybrid approach that uses more than modeling method. after the generation of a model, the amp stability should be evaluated using molecular dynamics (md). molecular dynamics comprises the application of computational simulations that predict the changes in the positions and velocities of the constituent atoms of a system under given time and condition. this calculation is done through a classical approximation of empirical parameters, called "force field." if, on one hand, this approximation makes the dynamics of a system containing thousands of atoms numerically accessible, it obviously limits the nature of the processes that can be observed during the simulations. no quantum effect is visualized in a md simulation; just as no chemical bond is broken, no interactions occur between orbitals, resonance, polarization, or charge transfer effects. however, the molecules go beyond a static system. thus, md is a computational technique that can be used for predicting or refining structures, dynamics of molecular complexes, drug development, and action of molecular biological systems. molecular dynamics simulation is widely used for protein research, aiming to extract information about the physical properties of individual proteins. the results of such simulations are then compared with experimental results. as these experiments are generally carried out in solvents, it is necessary to simulate molecular systems of protein in water. these simulations have a variety of applications, such as determining the folding of a structure to a native structure and analyzing the dynamic stability of this structure. the use of md to simulate protein folding processes is one of the most challenging applications and should be relatively long (in the order of microseconds to milliseconds) to allow observing a single fold event. in addition, the force field used must correctly describe the relative energies of a wide variety of shapes, including unfolding and poorly folded shapes that may occur during the simulation. the considerable application potential led to the implementation of md simulation in many software packages, including gromacs, - amber, namd, charmm, lammps, and desmond. in addition to the above mentioned, there are other simulation types available, such as the monte carlo method, stochastic dynamics, and brownian dynamics. in the last decades, md simulation has become a standard tool in theoretical studies of large biomolecular systems, including dna or proteins, in environments with near realistic solvents. indeed, simulations have proven valuable in deciphering functional mechanisms of proteins and other biomolecules, in uncovering the structural basis for disease, and in the design and optimization of small molecules, peptides, and proteins. historically, the computational complexity of this type of computation has been extremely high, and much research has focused on algorithms to achieve unique simulations that are as long or as large as possible. the interplay between a given pathogen (eg, virus, bacteria, fungus) must be studied through a holistic approach. hostpathogen relationships are very complex and occur at diverse conceivable levels, including the cellular/molecular level of both, pathogen and host, under given environmental conditions. a most approximate understanding of these interactions at every level is the ultimate goal of "systems biology" (sb). it comprises a holistic approach, integrating distinct disciplines, as biology, computer science, engineering, bioinformatics, physics, and others to predict how a given system behaves under given conditions and what is the role of its parts. systems biology stands out because it is capable of correlating omics data for the understanding of plant-pathogen interaction. the construction of a plant-pathogen interaction network includes the reconstruction of metabolic pathways of these organisms, identification of the degree of pathogenicity, besides the expression of genes and proteins from both plant and pathogen. the networks can be classified into types: ( ) regulatory; ( ) metabolic; ( ) protein-protein interaction; ( ) signaling and regulatory; and ( ) signaling, regulatory, and metabolic. each of these networks can be plotted according to computational approaches. also, further studies are required to contemplate the construction of evolutionary in silico models and the characterization of these molecular targets in vitro. , studies of protein-protein interactions to understand the regulatory process are essential and new computational methods are necessary for this purpose with more optimized algorithms, also to remove potential false positives. thus, in-depth studies on the orientation of molecules and their linkages to the formation of a stable complex are of great importance for understanding plant-pathogen studies and also to develop new drugs. the understanding of the regulatory principles by which protein receptors recognize, interact, and associate with molecular substrates or inhibitors is of paramount importance to generate new therapeutic strategies. in modern drug discovery, docking plays an important role in predicting the orientation of the binder when it is attached to a protein receptor or enzyme, using forms and electrostatic interactions, van der walls, coulombic, and hydrogen bond as parameters to quantify or predict a given interaction. , molecular docking aims at exploring the predominant mode(s) of binding of a molecule (protein or ligand) when it binds to a protein with a known d structure based on a scoring function that has main functions: the first is to determine the binding mode and the binding site of a protein, the second is to predict the absolute binding affinity between protein and ligand (or other protein) in lead optimization, and the third is virtual screening, which can identify potential drug leads for a given protein target by searching a large ligand or protein in database. protein-protein interactions are essential for cellular and immune function. in many cases, due to the absence of an experimentally determined structure of the complex, these interactions must be modeled to obtain an understanding about their structure and molecular basis. few studies on plant-pathogen interactions include docking approaches and most studies focus on drug development for medical purposes. drug research based on structure is a powerful technique for the rapid identification of small molecules against the d structure of available macromolecular targets, usually by x-ray crystallography, nmr structures, or homology models. due to abundant information on protein sequences and structures, the structural information on specific proteins and their interactions have become crucial for current pharmacological research. even in the absence of knowledge about the binding site and limited backbone movements, a variety of algorithms have been developed for docking over the past decades. although the zdock, the rdock, and the hex have provided results with high coupling precision, the complexes provided are not very useful for designing inhibitors for protein interfaces due to constraints on rigid body docking. in this context, more flexible approaches have been developed which generally examine very limited conformations compared with rigid body methods. these docking methods predict that binding is more likely to occur in broad surface regions and then defines the sites in complex structures of high affinity. the best example is the haddock software, which has been successful in solving a large number of precise models for protein-protein complexes. a good example of its use is the study of the complex formed between plectasin, a member of the innate immune system, and a precursor lipid of bacterial cell wall ii. the study identified the residues involved in the binding site between the proteins, providing valuable information for planning new antibiotics. however, the absolute energies associated with intermolecular interaction are not estimated with satisfactory accuracy by the current algorithms. some significant issues as solvent effects, entropic effects, and receptor flexibility still need to be addressed. however, some methods, such as moe-dock, gold, glide, flexx, and surflex which deal with lateral chain flexibility, have proven to be effective and adequate in most cases. realistic interactions between small molecules and receptors still depend on experimental wet-lab validation. , despite the current difficulties, there is a growing interest in the mechanisms and prediction of small molecules such as peptides, as they bind to proteins in a highly selective and conserved manner, being promising as new medicinal and biological agents. while both "small molecule docking methods" and "custom protocols" can be used, short peptides are challenging targets because of their high torsional flexibility. proteinpeptide docking is generally more challenging than those related to other small molecules, and a variety of methods have been applied so far. however, few of these approaches have been published in a way that can be reproduced with ease. [ ] [ ] [ ] although it is difficult to use peptide docking, a recent focus of basic and pharmacological research has used computational tools with modified peptides to predict the selective disruption of proteinprotein interactions. these studies are based on the involvement of some critical amino acid residues that contribute most to the binding affinity of a given interaction, also called hot-spots. , despite the number of docking programs, existing algorithms still demand improvements. however, approaches are being developed to improve all issues related to punctuation, protein flexibility, interaction with plain water, among other issues. in this context, the capri (critical assessment of predicted interactions) is a community that provides a quality assessment of different docking approaches. it started in and since then has aided the development and improvement of the methodologies applied for docking. an evaluation was carried out for capri in , resulting in an improvement in the integration of different modeling tools with docking procedures, as well as the use of more sophisticated evolutionary information to classify models. however, adequate modeling of conformational flexibility in interacting proteins remains an essential demand with a crucial need for improvement. different docking programs are currently available, and new alternatives continue to appear. some of these alternatives will disappear, just as others will become the top choices among field users. molecular docking technique is not often used for amps, due to its standard mechanism of action based on the classical association with the external membrane of the pathogen. despite that, some amps have the ability to bind other proteins and/or enzymes, a feature still scarcely studied. in such cases, molecular docking can be useful. an example of success is the study performed by melo et al, where they showed the specific binding of a trypsin to a cowpea (vigna unguiculata) thionine, revealing that this interaction occurs in a canonical manner with lys , located in an extended exposed loop. therefore, further application of docking may bring new evidences about the antimicrobial mechanisms revealing other molecular targets of interest. it is clear that the combination of data bank information with bioinformatic tools (especially those allowing the identification of patterns, rather than sequence order) is able to revolutionize the identification of amps and prediction of their activity. the data may come from genomic, transcriptomic, or proteomic databases, or a combination of different information sources (eg, genomic and transcriptomics, transcriptomics and proteomics). supplementary figure s brings a schematic flowchart describing the steps for mining, annotation, and structural/ functional analysis of amps, in addition to some wet-lab analyses that can be integrated to assess/confirm candidate amps. similar bioinformatic approaches have been actually used to identify potential peptide candidates with anti-sars-cov- activity, especially those potentially able to interact with the spike protein and proteases involved in viral penetration. , as emphasized, plant amps show greater diversity and abundance, when compared with other kingdoms. it can be speculated that plants shelter many yet undescribed amp classes, given their vast abundance and isoform diversity. the genomic and peptidic structure of amps can be variable, with few key residues conserved, which turns their identification, classification, and comparison challenging even in the omics age. nevertheless, advances in the generation of new bioinformatics tools and specialized databases have led to new and more efficient approaches for both the identification of primary sequences and molecular modeling, besides the analysis of the stability of the generated models. despite the large availability of omics data and bioinformatics tools, most new plant peptides have been discovered by wet-lab approaches regarding single candidates. high throughput in silico methods have the potential to transform this scenario, revealing many new candidates, including some new or "non-canonical" peptides. it may be also speculated that a myriad of new peptides may exist considering even smaller peptides, still less considered and more difficult to identify. finally, in silico approaches shall in future studies be mandatory to define the design of wet-lab studies, turning the identification more efficient and requiring reasonably less time to track, identify, and confirm new candidate amps. considering the actual pandemic scenario of covid- , plant amps may be regarded as an important source of antiviral drug candidates, especially considering that some amp categories present not only antiviral effects but also a wide spectrum antimicrobial activity, act as anti-inflammatory, and also induce the immune response. of higher education personnel, biocomputational program), cnpq (brazilian national council for scientific and technological development), and facepe (fundação de amparo à ciência e tecnologia de pernambuco) for fellowships. the project is supported by the interreg italia-slovenia, ise-emh / and rc / from irccs burlo garofolo/ italian ministry of health cass performed the literature review and whore the manuscript. lz, mol, lmbv, jpbn, jrfn, jdcf, rlos, cjp, ffa, and mfo wrote specific chapters, eak and sc critically revised the text and included relevant suggestions. ambi conceived the review, wrote the introduction and concluding remarks, besides critically revising the manuscript. all authors have read the manuscript and agree to its content. supplemental material for this article is available online. prediction of protein function from protein sequence and structure plant peptides in defense and signaling plant bioactive peptides: an expanding class of signaling molecules candidalysin is a fungal peptide toxin critical for mucosal infection copsin, a novel peptide-based fungal antibiotic interfering with the peptidoglycan synthesis innate and specific gut-associated immunity and microbial interference the wide world of ribosomally encoded bacterial peptides oral saliva and covid- human β-defensin mediated immune modulation as treatment for experimental colitis plant peptides and peptidomics nucleic acids and proteins in plants i the plant peptidome: an expanding repertoire of structural features and biological functions a small peptide modulates stomatal control via abscisic acid in long-distance signalling interaction of pls and pin and hormonal crosstalk in arabidopsis root development peptide signals for plant defense display a more universal role protease inhibitors in plants: genes for improving defenses against insects and pathogens long distance run in the wound response-jasmonic acid is pulling ahead rodríguez-palenzuéla p. plant defense peptides overview on plant antimicrobial peptides ethnobotanical bioprospection of candidates for potential antimicrobial drugs from brazilian plants: state of art and perspectives conopeptide characterization and classifications: an analysis using conoserver adaptive hydrophobic and hydrophilic interactions of mussel foot proteins with organic thin films cathelicidins, multifunctional peptides of the innate immunity antimicrobial peptides: discovery, design and novel therapeutic strategies scop: a structural classification of proteins database for the investigation of sequences and structures antimicrobial peptides from plants cyclotides insert into lipid bilayers to form membrane pores and destabilize the membrane through hydrophobic and phosphoethanolamine-specific interactions analysis of two novel classes of plant antifungal proteins from radish (raphanus sativus l.) seeds antifungal plant defensins: mechanisms of action and production h-nmr studies on the structure of a new thionin from barley endosperm: structure of a new thionin antimicrobial peptides from plants arabidopsis thionin-like genes are involved in resistance against the beet-cyst nematode (heterodera schachtii) host defense peptides and their potential as therapeutic agents plant thionins-the structural perspective plant antimicrobial peptides de smet i. plant peptides-taking them to the next level antimicrobial peptides from plants and their mode of action the inhibitory effect of a protamine from wheat flour on the fermentation of wheat mashes characterization and analysis of thionin genes thionin genes specifically expressed in barley leaves antimicrobial peptides as effective tools for enhanced disease resistance in plants identification of a cowpea γ-thionin with bactericidal activity novel thionins from black seed (nigella sativa l.) demonstrate antimicrobial activity synthetic and structural studies on pyrularia pubera thionin: a single-residue mutation enhances activity against gram-negative bacteria antimicrobial activity of γ-thionin-like soybean se in e. coli and tobacco plants inhibition of trypsin by cowpea thionin: characterization, molecular modeling, and docking toxicity of purothionin and its homologues to the tobacco hornworm, manduca sexta (l.) (lepidoptera:sphingidae) studies on purothionin by chemical modifications full-matrix refinement of the protein crambin at . Å and k γ-purothionins: amino acid sequence of two polypeptides of a new family of thionins from wheat endosperm primary structure and inhibition of protein synthesis in eukaryotic cell-free system of a novel thionin, gammahordothionin, from barley endosperm plant defensins: novel antimicrobial peptides as components of the host defense system the evolution, function and mechanisms of action for plant defensins plant γ-thionins: novel insights on the mechanism of action of a multi-functional class of defense proteins disulfide bridges in defensins comparative analysis of the antimicrobial activities of plant defensin-like and ultrashort peptides against food-spoiling bacteria isolation, purification, and characterization of a stable defensin-like antifungal peptide from trigonella foenum-graecum (fenugreek) seeds antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? plant defensins-prospects for the biological functions and biotechnological properties defensins and paneth cells in inflammatory bowel disease plant defensins: types, mechanism of action and prospects of genetic engineering for enhanced disease resistance in plants benko-iseppon am, cecchetto g. gene isolation and structural characterization of a legume tree defensin with a broad spectrum of antimicrobial activity recent advances in the chemistry and biochemistry of plant lipids evolutionary history of the non-specific lipid transfer proteins lipid transfer proteins: classification, nomenclature, structure, and function lipid-transfer proteins in plants purification and characterization of a small ( . kda) putative lipid transfer protein from maize seeds surprisingly high stability of barley lipid transfer protein, ltp , towards denaturant, heat and proteases structural stability and surface activity of sunflower s albumins and nonspecific lipid transfer protein involvement of gpi-anchored lipid transfer proteins in the development of seed coats and pollen in arabidopsis thaliana two-and three-dimensional proton nmr studies of a wheat phospholipid transfer protein: sequential resonance assignments and secondary structure three-dimensional structure in solution of a wheat lipid-transfer protein from multidimensional h-nmr data. a new folding for lipid carriers an unusual lectin from stinging nettle (urtica dioica) rhizomes hevein-like antimicrobial peptides of plants structural basis for chitin recognition by defense proteins: glcnac residues are bound in a multivalent fashion by extended binding sites in hevein domains ginkgotides: proline-rich hevein-like peptides from gymnosperm ginkgo biloba structure and function of chitin-binding proteins structural features of plant chitinases and chitin-binding proteins a novel antifungal peptide from leaves of the weed stellaria media l antimicrobial peptides from amaranthus caudatus seeds with sequence homology to the cysteine/glycinerich domain of chitin-binding proteins overview of plant chitinases identified as food allergens the latex-fruit syndrome the n-terminal cysteine-rich domain of tobacco class i chitinase is essential for chitin binding but not for catalytic or antifungal activity a chitin-binding lectin from stinging nettle rhizomes with antifungal properties biochemical and molecular characterization of three barley seed proteins with antifungal properties hevein: an antifungalprotein from rubber-tree (hevea brasiliensis) latex two hevein homologs isolated from the seed of pharbitis nil l. exhibit potent antifungal activity comparative proteomics of primary and secondary lutoids reveals that chitinase and glucanase play a crucial combined role in rubber particle aggregation in hevea brasiliensis the formation and accumulation of protein-networks by physical interactions in the rapid occlusion of laticifer cells in rubber tree undergoing successive mechanical wounding plant cystineknot peptides: pharmacological perspectives: plant cystine-knot proteins in pharmacology refined crystal structure of the potato inhibitor complex of carboxypeptidase a at . Å resolution squash inhibitors: from structural motifs to macrocyclic knottins small signaling peptides in arabidopsis development: how cells communicate over a short distance use of scots pine seedling roots as an experimental model to investigate gene expression during interaction with the conifer pathogen heterobasidion annosum (p-type) tying the knot: the cystine signature and molecular-recognition processes of the vascular endothelial growth factor family of angiogenic cytokines a cactus-derived toxin-like cystine knot peptide with selective antimicrobial activity circular proteins from plants and fungi circulins a b. novel human immunodeficiency virus (hiv)-inhibitory macrocyclic peptides from the tropical tree chassalia parvifolia isolation, solution structure, and insecticidal activity of kalata b , a circular protein with a twist: do möbius strips exist in nature? purification, characterisation and cdna cloning of an antimicrobial peptide from macadamia integrifolia miamp , a novel protein from macadamia integrifolia adopts a greek key β-barrel fold unique amongst plant antimicrobial proteins peptides of the innate immune system of plants. part ii. biosynthesis, biological functions, and possible practical applications nmr structure of the streptomyces metalloproteinase inhibitor, smpi, isolated from streptomyces nigrescens tk- : another example of an ancestral βγ-crystallin precursor structure ancestral beta gamma-crystallin precursor structure in a yeast killer toxin enhanced quantitative resistance to leptosphaeria maculans conferred by expression of a novel antimicrobial peptide in canola (brassica napus l.) primitive defence: the miamp antimicrobial peptide family a novel family of small cysteine-rich antimicrobial peptides from seed of impatiens balsamina is derived from a single precursor protein structural studies of impatiens balsamina antimicrobial protein (ib-amp ) antifungal mechanism of a cysteine-rich antimicrobial peptide, ib-amp , from impatiens balsamina against candida albicans antimicrobial peptide hybrid fluorescent protein based sensor array discriminate ten most frequent clinic isolates ib-amp insertion causes surface rearrangement in the phospholipid bilayer of biomembranes: implications from quartz-crystal microbalance with dissipation antifungal activity of synthetic peptides derived from impatiens balsamina antimicrobial peptides ib-amp and ib-amp antimicrobial specificity and mechanism of action of disulfide-removed linear analogs of the plant-derived cys-rich antimicrobial peptide ib-amp triticum aestivum puroindolines, two basic cystine-rich seed proteins: cdna sequence analysis and developmental gene expression determination of the secondary structure and conformation of puroindolines by infrared and raman spectroscopy sequence diversity and identification of novel puroindoline and grain softness protein alleles in elymus, agropyron and related species puroindolines: their role in grain hardness and plant defence molecular genetics of puroindolines and related genes: allelic diversity in wheat and other grasses isolation, characterization and antimicrobial activity at diverse dilution of wheat puroindoline protein the wheat puroindoline genes confer fungal resistance in transgenic corn: the puroindolines confer corn slb resistance mini review: structure, biological and technological functions of lipid transfer proteins and indolines, the major lipid binding proteins from cereal kernels puroindolines: the molecular genetic basis of wheat grain hardness plant lipid binding proteins: properties and applications puroindolines form ion channels in biological membranes the antimicrobial properties of the puroindolines, a review snakin- , a peptide from potato that is active against plant pathogens snakin- , an antimicrobial peptide from potato whose gene is locally induced by wounding and responds to pathogen infection snakin: structure, roles and applications of a plant antimicrobial peptide the new casn gene belonging to the snakin family induces resistance against root-knot nematode infection in pepper radiation damage and racemic protein crystallography reveal the unique structure of the gasa/snakin protein superfamily gasa , a regulator of flowering time and stem growth in arabidopsis thaliana isolation and characterization of the tissue and development-specific potato snakin- promoter inducible by temperature and wounding the gibberellic acid stimulatedlike gene family in maize and its role in lateral root development analysis of expressed sequence tags (ests) from avocado seed (persea americana var. drymifolia) reveals abundant expression of the gene encoding the antimicrobial peptide snakin increased tolerance to wheat powdery mildew by heterologous constitutive expression of the solanum chacoense snakin- gene recombinant production of snakin- (an antimicrobial peptide from tomato) in e. coli and analysis of its bioactivity geg participates in the regulation of cell and organ shape during corolla and carpel development in gerbera hybrida two osgasr genes, rice gast homologue genes that are abundant in proliferating tissues, show different expression patterns in developing panicles gasa , one of the -member arabidopsis gasa family of small polypeptides, regulates flowering and seed development identification of novel genes potentially involved in somatic embryogenesis in chicory (cichorium intybus l.) disulfide-stabilized helical hairpin structure and activity of a novel antifungal peptide ecamp from seeds of barnyard grass (echinochloa crus-galli) buckwheat trypsin inhibitor with helical hairpin structure belongs to a new family of plant defence peptides novel antifungal αhairpinin peptide from stellaria media seeds: structure, biosynthesis, gene structure and evolution design, synthesis and docking of linear and hairpin-like alpha helix mimetics based on alkoxylated oligobenzamide defense peptide repertoire of stellaria media predicted by high throughput next generation sequencing influence of cysteine and tryptophan substitution on dna-binding activity on maize α-hairpinin antimicrobial peptide plant cyclotides: a unique family of cyclic and knotted proteins that defines the cyclic cystine knot structural motif plants defense-related cyclic peptides: diversity, structure and applications discovery, structure, function, and applications of cyclotides: circular proteins from plants cyclotide evolution: insights from the analyses of their precursor sequences, structures and distribution in violets (viola) isolation of oxytocic peptides from oldenlandia affinis by solvent extraction of tetraphenylborate complexes and chromatography on sephadex lh- fractionation protocol for the isolation of polypeptides from plant biomass discovery of cyclotides in the fabaceae plant family provides new insights into the cyclization, evolution, and distribution of circular proteins cyclotides associate with leaf vasculature and are the products of a novel precursor in petunia (solanaceae) discovery and characterization of novel cyclotides originated from chimeric precursors consisting of albumin- chain a and cyclotide domains in the fabaceae family the cyclotide cycloviolacin o from viola odorata has potent bactericidal activity against gram-negative bacteria cyclotides: a novel type of cytotoxic agents potential therapeutic applications of the cyclotides and related cystine knot mini-proteins disulfide-rich macrocyclic peptides as templates in drug design the superfamily of thaumatinlike proteins: its origin, evolution, and expression towards biological function plant thaumatin-like proteins: function, evolution and biotechnological applications some fungi express beta- , -glucanases similar to thaumatin-like proteins lentinula edodes tlg encodes a thaumatin-like protein that is involved in lentinan degradation and fruiting body senescence plant stress proteins of the thaumatin-like family discovered in animals plant pathogenesis-related proteins: molecular mechanisms of gene expression and protein function the crystal structure of the antifungal protein zeamatin, a member of the thaumatin-like, pr- protein family several thaumatin-like proteins bind to β- , -glucans some thaumatin-like proteins hydrolyse polymeric beta- , -glucans tlxi, a novel type of xylanase inhibitor from wheat (triticum aestivum) belonging to the thaumatin family zeamatin inhibits trypsin and alpha-amylase activities drought-inducible-but aba-independent-thaumatin-like protein from carrot (daucus carota l.) ethylene-responsive genes are differentially regulated during abscission, organ senescence and wounding in peach (prunus persica) antifreeze proteins in winter rye are similar to pathogenesis-related proteins differential gene expression in arachis diogoi upon interaction with peanut late leaf spot pathogen, phaeoisariopsis personata and characterization of a pathogen induced cyclophilin transcriptome and metabolite profiling of the infection cycle of zymoseptoria tritici on wheat reveals a biphasic interaction with plant immunity involving differential pathogen chromosomal contributions and a variation on the hemibiotrophic lifestyle definition a classification of plant food allergens molecular, biochemical and structural characterization of osmotin-like protein from black nightshade (solanum nigrum) molecular characterization of a novel soybean gene encoding a neutral pr- protein induced by high-salt stress thaumatin-like proteins-a new family of pollen and fruit allergens biochemical and structural characterization of tlxi, the triticum aestivum l resolution of the structure of the allergenic and antifungal banana fruit thaumatin-like protein at . -Å crystal structure of tobacco pr- d protein at . Å resolution reveals a conserved acidic cleft structure in antifungal thaumatin-like proteins crystal structure of osmotin, a plant antifungal protein crystal structure of a sweet tasting protein thaumatin i, at · Å resolution crystallization and preliminary structure determination of the plant identification of conidialenriched transcripts in aspergillus nidulans using suppression subtractive hybridization analysis of the aspergillus nidulans thaumatin-like ceta gene and evidence for transcriptional repression of pyr expression in the ceta-disrupted strain the pr k receptor protein kinase from arabidopsis thaliana is structurally related to a family of plant defense proteins uniprot: the universal protein knowledgebase discovering new in silico tools for antimicrobial peptide prediction computational tools for exploring sequence databases as a resource for antimicrobial peptides camp: a useful resource for research on antimicrobial peptides camp: collection of sequences and structures of antimicrobial peptides campr : a database on sequences, structures and signatures of antimicrobial peptides apd : the antimicrobial peptide database as a tool for research and education dbaasp: database of antimicrobial activity and structure of peptides new trends in peptide-based anti-biofilm strategies: a review of recent achievements and bioinformatic approaches a large-scale structural classification of antimicrobial peptides defensins knowledgebase: a manually curated database and information source focused on the defensins family of antimicrobial peptides computational resources and tools for antimicrobial peptides cybase: a database of cyclic protein sequence and structure phytamp: a database dedicated to antimicrobial plant peptides plantpepdb: a manually curated plant peptide database bdbms-a database management system for biological data bioinformatics: a way forward to explore "plant omics basic local alignment search tool rapid and sensitive sequence comparison with fastp and fasta comparative analysis of the quality of a global algorithm and a local algorithm for alignment of two sequences programming techniques: regular expression search algorithm profile hidden markov models hidden markov models and their applications in biological sequence analysis what are the ideal properties for functional food peptides with antihypertensive effect? a computational peptidology approach computational peptidology c-pamp: large scale analysis and database construction containing high scoring computationally predicted antimicrobial peptides for all the available plant species predstp: a highly accurate svm based model to predict sequential cystine stabilized peptides assigning biological function using hidden signatures in cystine-stabilized peptide sequences random forests and adaptive nearest neighbors multiple incremental decremental learning of support vector machines evolutionary artificial neural networks: a review novel peptideprotein assay for identification of antimicrobial peptides by fluorescence quenching a reverse search for antimicrobial peptides in ciona intestinalis: identification of a gene family expressed in hemocytes and evaluation of activity positive selection drives a correlation between non-synonymous/synonymous divergence and functional divergence progress with proteome projects: why all proteins expressed by a genome should be identified and how to do it proteomic tools for biomedicine compatibility of plant protein extraction methods with mass spectrometry for proteome analysis proteomic profiling for target identification of biologically active small molecules using d dige proteomics technologies and challenges separomics applied to the proteomics and peptidomics of low-abundance proteins: choice of methods and challenges-a review mining the active proteome in plant science and biotechnology screening, purification and characterization of anionic antimicrobial proteins from foeniculum vulgare peptidomics coming of age: a review of contributions from a bioinformatics angle d-lc/ms techniques for the identification of proteins in highly complex mixtures hplc techniques for proteomics analysis-a short overview of latest developments bioinformatics in proteomics computational methods for protein identification from mass spectrometry data de novo sequencing methods in proteomics a fast sequest cross correlation algorithm probability-based protein identification by searching sequence databases using mass spectrometry data novel antimicrobial peptides with promising activity against multidrug resistant salmonella enterica serovar choleraesuis and its stress response mechanism proteomics assisted profiling of antimicrobial peptide signatures from black pepper apd: the antimicrobial peptide database protein identification and analysis tools on the expasy server tandem: matching proteins with tandem mass spectra open mass spectrometry search algorithm probid: a probabilistic algorithm to identify peptides through sequence database searching using tandem mass spectral data radars, a bioinformatics solution that automates proteome mass spectral analysis, optimises protein identification, and archives data in a relational database analysis and validation of proteomic data generated by tandem mass spectrometry improved method for proteome mapping of the liver by -de maldi-tof ms bioinformatics-coupled molecular approaches for unravelling potential antimicrobial peptides coding genes in brazilian native and crop plant species prediction of bioactive peptides from chlorella sorokiniana proteins using proteomic techniques in combination with bioinformatics analyses graphical interpretation and analysis of proteins and their ontologies (giapronto): a one-click graph visualization software for proteomics data sets principles, challenges and advances in ab initio protein structure prediction practically useful: what the rosetta protein modeling suite can do for you. biochemistry critical assessment of methods of protein structure prediction (casp)-round xii template-based protein structure modeling using the raptorx web server comparative protein structure modeling of genes and genomes swiss-model: modelling protein tertiary and quaternary structure using evolutionary information comparative protein structure modeling using mod-eller: comparative protein structure modeling using modeller muster: improving protein sequence profile-profile alignments by using multiple sources of structure information improving protein fold recognition and template-based modeling by employing probabilistic-based matching between predicted one-dimensional structural properties of query and corresponding native properties of templates prosa-web: interactive web service for the recognition of errors in three-dimensional structures of proteins i-tasser server for protein d structure prediction touchstone ii: a new approach to ab initio protein structure prediction ab initio modeling of small proteins by iterative tasser simulations integration of quark and i-tasser for ab initio protein structure prediction in casp : ab initio structure prediction in casp critical assessment of methods of protein structure prediction: progress and new directions in round xi: progress in casp xi in silico optimization of a guava antimicrobial peptide enables combinatorial exploration for peptide design high-resolution comparative modeling with rosettacm developing a molecular dynamics force field for both folded and disordered protein states challenges in protein-folding simulations molecular dynamics simulations of biomolecules relaxation mode analysis for molecular dynamics simulations of proteins gromacs : algorithms for highly efficient, load-balanced, and scalable molecular simulation : a high-throughput and highly parallel open source molecular simulation toolkit gromacs: high performance molecular simulations through multi-level parallelism from laptops to supercomputers the amber biomolecular simulation programs scalable molecular dynamics with namd charmm: a program for macromolecular energy, minimization, and dynamics calculations fast parallel algorithms for short-range molecular dynamics scalable algorithms for molecular dynamics simulations on commodity clusters molecular dynamics simulation for all targeting antibiotic tolerance, pathogen by pathogen measuring and mapping the global burden of antimicrobial resistance university of texas medical branch at galveston antibiotic drugs targeting bacterial rnas antimicrobial drugs in fighting against antimicrobial resistance protein-ligand docking: current status and future challenges peptide docking and structurebased characterization of peptide binding: from knowledge to know-how software for molecular docking: a review bacterial multidrug efflux pumps: mechanisms, physiology and pharmacological exploitations zdock server: interactive docking prediction of protein-protein complexes and symmetric multimers the haddock . web server: user-friendly integrative modeling of biomolecular complexes rdock: a fast, versatile and open source program for docking ligands to proteins and nucleic acids protein docking using case-based reasoning principles of flexible protein-protein docking plectasin, a fungal defensin, targets the bacterial cell wall precursor lipid ii variability in docking success rates due to dataset preparation development and validation of a genetic algorithm for flexible docking extra precision glide: docking and scoring incorporating a model of hydrophobic enclosure for protein−ligand complexes protein-ligand docking: current status and future challenges surflex-dock: docking benchmarks and real-world application docking small peptides remains a great challenge: an assessment using autodock vina advances in the prediction of protein-peptide binding affinities: implications for peptide-based drug discovery: protein-peptide binding affinities recent work in the development and application of protein-peptide docking peptide docking and structurebased characterization of peptide binding: from knowledge to know-how protein-ligand docking in the new millennium-a retrospective of years in the field unal eb, gursoy a, erman b. vital: viterbi algorithm for de novo peptide design modeling protein-protein and proteinpeptide complexes: capri th edition docking, scoring, and affinity prediction in capri potential chimeric peptides to block the sars-cov- spike receptor-binding domain peptide-like and small-molecule inhibitors against covid- the authors are very grateful to capes (coordination for the improvement key: cord- - rbbe c authors: mukherjee, pulok k. title: antiviral evaluation of herbal drugs date: - - journal: quality control and evaluation of herbal drugs doi: . /b - - - - . - sha: doc_id: cord_uid: rbbe c the viral infection and resistance to the existing antiviral drugs are alarming, which is a serious public health concern. medicinal plants are valuable resources for treatment of viral infections and can be used for the management of infections like herpes simplex virus (hsv), human immunodeficiency virus (hiv), influenza, etc. the antiviral screening of plant extracts should be highly selective, specific, and sensitive for bioactivity guided isolation of the active compounds from the plant extracts. the antiviral screening system should be validated for accuracy, reproducibility, simplicity, and cost effectiveness. this chapter highlights on various aspects for screening and evaluation of antiviral natural components including factors affecting antiviral in vivo studies, host cells, organisms, and culture media followed by different virus-specific assays for antiviral screening of natural products. although vaccines have been very successful in controlling many viral diseases, some diseases are likely to be controlled only by antiviral chemotherapy. the concept of antiviral drugs has only been accepted slowly, partly because of the toxicity of many of the earlier antiviral agents. in contrast to the development of antibiotics, attempts to develop antiviral drugs have indeed met a variety of problems. being strictly dependent on cellular metabolic processes, viruses possess only limited intrinsic enzyme systems and building blocks that may serve as specific targets for a drug. moreover, contrary to a bacteriostatic compound, an effective antiviral drug should not only display considerable specificity in its antiviral action, but should also irreversibly block viral synthesis in order to stop cell suicide due to the viral infection and restore normal cell synthesis (vanden berghe et al., ) . in addition to this inhibition, the antiviral agent must have a broad spectrum of activity, favorable pharmacodynamic properties, and not be immunosuppressive. in the ideal situation, the antiviral drug checks the infection while the immune system prepares to destroy the last virus particles (munro et al., ) . this point is critical for those immune-compromised by illness (aids, cancer) or drug therapy (transplants, cancer). a frequent cause of death in these instances is from viral infections, so that adjuvant antiviral chemotherapy is vital in these circumstances (shannon and schabel, ) . many viral infectious diseases still cause high mortality. although antiviral chemotherapy has shown outstanding progress, antiviral agents are still required. the emergence of drug-resistant viruses during treatment raises a potential problem for effective therapy. furthermore, new viral pathogens may be discovered. biologically active substances of plant origin have long been known as viral inhibitors. these antiviral compounds may be extracted from sources, such as higher plants, which have, for various reasons, been explored considerably less than the traditional ones. the first clinically useful antiviral drug was methylisatin-thiosemicarbazone (methisazone), which was active against pox viruses, especially smallpox virus. methisazone ( mg/kg) was found to inhibit variola virus in mice and was later used successfully to treat cases of eczema vaccinatum and to treat and prevent smallpox. kaufman et al. ( ) , an ophthalmologist, successfully treated a herpes eye infection with an antineoplastic drug, idoxuridine. at the same time, a group of chemists at the du pont chemical company in the united states investigated the antiviral activity of a molecule called amantadine. it was active against influenza virus type a. in rapid succession, further nucleoside analogs, cytosine arabinoside, trifluorothymidine, and adenine arabinoside, were found to inhibit herpes virus. in the s, the antiherpes activity of a new compound, -( -hydroxyethoxymethyl) guanine (acyclovir), was detected by j. bauer at the wellcome research labs. within a decade, the same group was to discover azidothymidine (zidovudine), the first effective inhibitor of the newly emerged hiv. the alkaloid from the australian horse chestnut (castanospermine australe), isolated in the s, was also found to be active against hiv. a research program to detect and isolate antiviral compounds from higher plants is best carried out by a multidisciplinary team, consisting of at least a pharmacognosist and a virologist. the antiviral screening system should meet all requirements of any good assay, including validity, lack of ambiguity, accuracy, reproducibility, simplicity, and reasonable cost. moreover, because we are dealing with plant extracts, the antiviral screen should be highly selective, specific, and sensitive; it is advisable to discriminate a true antiviral activity from a viricidal one at this stage. because most of the aforementioned requirements are better met by in vitro testing, we not only prefer in vitro screening of the plant extracts, but also the use of the same bioassay to guide the isolation of the antivirally active compounds from the plant extracts. the antiviral activity of the pure compounds then has to be confirmed in a later stage by in vivo assays (leven et al., ; vanden berghe et al., ) . one of the possible strategies for finding new antiinfective drugs may involve the search for compounds with chemotherapeutic activities supplementary to, and structures widely different from, those in current use. these compounds could be extracted from sources that have, for various reasons, been explored considerably less than the traditional microorganisms, including, among others, higher plants (mitscher and rao, ) . considering the enormous number and the amazing structural diversity of the currently available antimicrobially and antivirally active plant constituents, one might hope that promising systemic and/or locally acting antiinfective agents might be discovered in the plant kingdom (vanden berghe et al., ; vlientinck et al., ) . the increase of drug-resistant viruses in treatment raises an important issue for effective treatment. moreover, new viral pathogens might be found. along these lines, there is a strong requirement for promptly accessible antiviral medications at moderate costs with minimal side effects. henceforth, traditional medicines ought to be investigated as novel antiviral agents, as many of these ancient medicaments, containing different plant metabolites, have potent antiviral activities (chattopadhyay and khan, ) . the design of new antiviral drugs potentially focuses on the structural dynamics and replication cycles of the various infections causing viruses. a suitable example is the invention of acyclovir, which hinders certain proteins of herpes infections responsible for the triggering of disease. ethnomedicines are a vast repository of structural diversities and extensive bioactivities that can serve as a huge source of potential antiviral drugs. a significant number of medicinal plants from ayurveda and the traditional chinese system of medicine serve as potential remedies to decrease the severity of illness caused by viruses (chattopadhyay et al., ; khan et al., ; jadhav et al., ) . research on the antiviral potentials of plants was first started in , and out of plants were found to be effective against influenza (chattopadhyay and naik, ) . numerous screening studies have been conducted in the last few years to determine the antiviral efficacy of medicinal plants using in vitro and in vivo assays. a few out of a british colombian medicinal plants showed antiviral efficacy against respiratory syncytial virus (rsv), corona virus, influenza virus, and herpes simplex virus (hsv) (mccutcheon et al., ) , while the marine algae spirulina showed antimutagenic, immunomodulatory, and antiviral activities (chamorro et al., ) . interestingly, cyanovirin n, an -kda protein of blue green algae, inactivates hiv- by binding with its glycoprotein (clercq , while sulfated polysaccharides of seaweeds and algae showed anti-hiv and anti-hsv activities (schaeffer and krylov, ) . the plant kingdom is highly diverse and ranges from unicellular microscopic plants to long lived, huge trees. to screen each and every plant or their individual parts for the identification of antiviral components is a huge task. several examples of plants having antiviral properties and newly identified active compounds from them are reported in various journals. one of the examples that can be cited here is cyanovirin n (cv-n), an -kda protein isolated from the cyanobacterium nostoc ellipsosporum, which exhibits the property of inhibiting hiv- infection and also possesses broad-spectrum activity. the phytochemical, podophyllotoxin, isolated from the aqueous extract of podophyllum peltatum l., inhibited hsv type (hsv- ) (bedows and hatfield, ) . the acetone extract of another plant, phyllanthus urinaria, also suppressed hsv- and hsv- (yang et al., ) . bessong et al. ( ) reported a comparison of various plants and their individual parts (stem, leaves, roots, and so forth.) in repressing viral reverse transcriptase (rt) and integrase, the two basic enzymes in hiv disease. after comparing all the extracts and fractions of the various plants, it was found that the n-butanol fraction of bridelia micrantha (hochst) exhibited the highest anti-rt activity. it has also been reported that the aqueous extract from the roots of carissa edulis (forssk.) vahl, a plant grown in kenya, displayed noteworthy activity against hsv for both wild type and resistant strains (tolo et al., ) . polyphenol-rich extract from the medicinal plant geranium sanguineum l. has been reported to show a strong antiinfluenza virus activity, as well as antioxidant and radical scavenging capacities (sokmen et al., ) hepatitis a, b, c, d, and e viruses are the leading causes for the prevalence of viral hepatitis and liver inflammation. despite the fact that presentation to any of these infections prompts intense disease, in any case, types b, c, and d are unique in causing chronic infection. plants belonging to the genus phyllanthus of the euphorbiaceae family were extensively used as a traditional remedy against these infections. clinical investigations were additionally intended to look at the inhibitory effects of different species of phyllanthus, that is, p. amarus (l.), p. niruri (l.), and p. urinaria (l.) (wang et al., ) . the screening of different chinese medicinal herbs led to the identification of two potent plant extracts against duck hepatitis b virus, namely, ardisia chinensis and pithecellobium clypearia . similarly, this also led to the identification of oenanthe javanica (blume) dc flavones (ojf). they acted as a strong inhibitor of hbsag and hbeag secretion (involved in viral pathogenesis) in . . cells and also reduced dhbv-dna levels in the hbv-infected duck model (wang et al., ) . because of the strong prevalence of hcv infection in poor countries, screening for the identification of anti-hcv potentials from medicinal plants are still ongoing. according to hussein et al. ( ) various plant extracts, such as methanol extracts acacia nilotica l. willd ex delile, boswellia carterii, embelia schimperi, quercus infectoria, trachyspermum ammi l., and aqueous extracts of piper cubeba l., q. infectoria, and syzygium aromaticum l., were found to possess significant inhibitory activity against hsv. combination therapy for treating diseases is an age-old practice of traditional medicine in which several plants are mixed together to develop an effective formulation for a particular disease. such combination therapies have also been tried for the inhibition of viral hepatitis. as an example, a chinese herbal medicine, prepared by liquid fermentation broth of ganoderma lucidum supplemented with an aqueous extract of radix sophorae flavescentis, was potent against hepatitis b virus activity in vitro and in vivo. viral infections are a matter of great concern for this planet. plants having broad-spectrum activity against many viruses could be evaluated for isolation and identification of molecules for treating viral infections. as an example, glycyrrhizin, a bioactive component of licorice (glycyrrhiza uralensis fisch), and lycorine, isolated from lycoris radiata l., showed strong anti-sars-cov activity, and was initially used for treating other indications (li et al., a, b) . a variety of herbal preparations have shown potentials for inhibiting viruses that cause serious infections among humans, such as measles viruses (olila et al., ) , human rotaviruses (hrv) (husson et al., ; takahashi et al., ) , respiratory syncytial virus (rsv), human rhinoviruses (glatthaar-saalmuller et al., ) , the coxsackie group of viruses (evstropov et al., ; su et al., ) , neurotropic sindbis virus (nsv) (paredes et al., ) , and various strains of polio virus (andrighetti-frohner et al., ; melo et al., ) . one such illustration is the atomic investigation of the heated water concentrates of stevia rebaudiana l., which blocked a section of different irresistible serotypes of hrv into permissive cells by an anionic polysaccharide having a molecular weight of with uronic acid as a noteworthy sugar constituent (takahashi et al., ) . thus, an alkaloid concentrate of haemanthus albiflos globules repressed rna amalgamation of hrv spread in ma- cells (husson et al., ) . in contrast to the many publications on antibacterial and antifungal screening of plant extracts that have appeared in the last decades, far fewer antiviral screening studies of plant extracts have been reported. this is due chiefly to the complexity of the different techniques involved in such research, which consequently requires the know-how and dedication of a multidisciplinary team. nevertheless, many antiviral agents have been isolated from natural sources and partly or completely characterized. from these studies, several substances have emerged as true antivirals having a good chemotherapeutic index based on the viability of infected cells and on in vivo tests. thus, different -methoxy flavones and synthetic derivatives have shown to be promising leads for the development of antirhinovirus drugs (van hoof et al., ; vlientinck et al., ) , whereas the spanonin, glycyrrhizic acid, was found to be highly active in vitro against herpes simplex (pompei et al., ) , varizella-zoster (baba and shijeta, ) , and human immunodeficiency viruses (ito et al., ) . whether these compounds have any clinical potential, that is, in the therapy of the corresponding viral diseases, remains to be determined. there, in vivo bioavailability and other pharmacokinetic parameters are subjects of future study. because of the problems of drug resistance stated earlier and of side effects, the pharmaceutical industry is looking forward toward natural products (mainly medicinal plant extracts) as a source of possible antiviral agents. approximately medicinal plant species have been recorded globally to treat a myriad of inflictions and diseases. polyphenols, alkaloids, flavonoids, saponins, quinones, terpenes, proanthocyanidins, lignins, tannins, polysaccharides, steroids, thiosulfonates, and coumarins are prominent bioactive phytochemicals that have been observed to combat viral infections, as they are harmless to the systems of the human body. some selected anthraquinones and anthraquinone derivatives are noted for their dammarane saponins, ginsenoside rb (grb ), and chikusetsusaponin (chi-iii) have been found to possess antiviral activity against hsv-i using an in vitro plaque elimination assay. chi-iii prevented plaque formation at half the concentration of grb (fukushima et al., (hudson and towers, ) . the inhibitory effect of ferulic acid and isoferulic acid on murine interleukin- production in response to influenza virus infections in vitro has been reported (hirabayashi et al., ) and the effect of isoferulic acid was found to be greater than that of ferulic acid. hayashi et al. ( ) found a direct inhibitory activity of the steam distillate prepared from fresh plants of houttuynia cordata (saururaceae) against hsv- , influenza virus, and hiv- , without showing cytotoxicity, but not against poliovirus and coxsackie virus. montanha et al. ( a) evaluated the action of a series of aporphine alkaloids against hsv- in cell cultures. on the basis of viral titer reduction, six alkaloids were found to be active. ellagitannins isolated from phyllanthus myrtifolius and p. urinaria (euphorbiaceae) showed activity against epstein-barr virus dna polymerase. flavonoidal constituents, such as biflavonoids and robustaflavones, exhibited strong inhibitory effects against influenza a and b viruses. the antiviral potential of the flavonoids of chamaesyce thymifolia has been reported; they showed high cytotoxicity on hep- cells and moderate inhibitory activity against hsv- and bovine viral diarrhea virus (amaral et al., ) . sotanaphun et al. ( ) isolated sclerocarpic acid from the stem bark of glyptopetalum sclerocarpum, which exhibited antiviral activity against herpes simplex virus types and . rhamnan sulfate, a natural sulfated polysaccharide isolated from monostroma latissimum, showed potent inhibitory effects on the virus replication of hsv- , hcmv, and hiv- in vitro . matsuse et al. ( ) tested aqueous and methanolic extracts of panamanian medicinal plants for anti-hiv effects. seven of these were found to be moderate inhibitors of hiv-protease enzyme. serkedjieva and ivancheva ( ) investigated the inhibitory effect of five extracts from the bulgarian medicinal plant g. sanguineum on herpes simplex virus types and . yoosook et al. ( ) evaluated the anti-hsv- activities of barleria lupulina and clinacanthus nutans. the results suggested a therapeutic potential against hsv- for b. lupulina, but not for c. nutans. the antiviral activities of various water and methanol soluble substances isolated from g. lucidum against hsv types and , influenza a virus, and vesicular stomatitis virus were studied using cytopathic effect inhibition assay and plaque reduction assay (eo et al., ) . kudi and myint ( ) investigated the antiviral activity of medicinal plant extracts against poliovirus, astrovirus, hsv, and parvovirus. most of the extracts showed activity against more than one virus at a dose rate of between and μg/ μl (eo et al., ) . bourne et al. ( ) assessed plant products in vitro by plaque reduction assay to determine their activity against a commonly transmitted pathogen, herpes simplex virus type . docherty et al. ( ) found that resveratrol, a phytoalexin, inhibited hsv- and hsv- replication in a dose-dependent reversible manner. anani et al. ( ) prepared methanol extracts from medicinal plants of togo and analyzed them for antiviral and antibiotic activities. ten of the showed significant antiviral activity against one or another of the test viruses (herpes simplex, sindbis, and poliovirus). hudson et al. ( a, b) evaluated ethanolic extracts of plants, endemic to madagascar, in order to determine the potential of malagasy plants as sources of antiviral activities. nine of the extracts had significant activity against hsv, whereas only four were active against the sindbis virus. a bioactive flavonoid, "baicalein," isolated from the chinese medicinal plant scutellaria baicalensis georgi showed antiviral properties using the high-speed countercurrent chromatography (hsccc) technique (li and chen, ) . many other substances, including flavonoids, phenolics, tannins, triterpenes, and alkaloids, interfere with host cell replication at their antivirally active concentrations or only exhibit extracellular viricidal activities. some of these viricides, including flavonoids and tannins present in foodstuffs, might be important, because they can inhibit virus replication of picorna-, rota-, and arena viruses in the gastrointestinal tract of humans and animals. artemisia capillaris was found to possess an active , -dimethylesculetine having potent cytotoxic potential. in the fruits of schisandra chinensis (schizandraceae) used in oriental medicine, lignans were identified, some of which, such as wuweizisu c and gomisine n, are very active. the same method threw light on the mechanism of the antihepatotoxic action of such well-known compounds as glycyrrhizin and its intestinal metabolites, which are protective against the first stages preceding hepatic lesions. other tests of this type are used to track down active substances. from taxus baccata, potier's group isolated new analogs of taxol, a terpenic compound displaying very good antileukemic and antitumor activities. taxol promotes the polymerization of tubulin into microtubules and inhibits their depolymerization. the choice among various fractions obtained by extraction was guided by the antitubulin activity in an in vitro test. many substances that are present in only trace quantities and are difficult to purify have been studied chemically; for example, the demonstration of xylose-bearing derivatives is new in this series of compounds. regarding structure activity relationships, in vitro cytotoxicity tests have shown that the activity is mainly related to the presence of a complex ester function in the compound. a list of plant extracts and their phytoconstituents that have antiviral potentials are listed in table . . in recent years, a lot of development has taken place regarding the identification of antiviral molecules from plant sources and the molecular mechanistic approach. compounds, such as spiroketalenol ether derivatives isolated from rhizome extract of tanacetum vulgare, have been reported to block virus entry and also arrest the synthesis of hsv- gc and hsv- gg glycoproteins (fernandes et al., ) . samarangenin b from the roots of limonium sinense exhibited inhibition of hsv- α gene expression (kuo et al., ) , whereas oxyresveratrol from artocarpus lakoocha plant was found to inhibit the early and late phases of viral replication of hsv- and hsv- , respectively (chuanasa et al., ) . also, pterocarnin a compound isolated from pterocarya stenoptera inhibited hsv- from binding and penetrating to the host cells (cos et al., ) . the structures of some of the potential phytoconstituents having significant antiviral activity are depicted in fig. . . many of the antiviral drugs now known have been discovered by random search in the laboratory. most labs use a biological test system in which new molecules are added to tissue culture cells at a range of concentrations (e.g., - μg/ ml); the drug-treated cells (and untreated cells as control) are then infected with a known multiplicity of infective virus particles. thousands of compounds can inhibit viral replication in a cell culture. in general, the more complex the regulatory mechanisms of a virus, the easier it is to find molecules that inhibit it. a broad estimate of the ratios of the activity of antiviral compounds in a cell culture, animal models, and humans is : : . during the evaluation of antiviral agents, many test conditions, such as the cell culture system, virus strain, virus challenge dose, virus input multiplicity of infection, and time of harvesting, can affect or even alter the test results. to test the inhibitory activity of a new antiviral agent, it is first necessary to select the host cell system(s) in which the virus replication can be measured. viruses vary considerably in their ability to replicate in cultured cells. many viruses can cause cpe while some of them can form plaques. others can produce some specialized functions, such as hemagglutination, hemadsorption, or cell transformation. virus replication in cell cultures can also be detected by the presence of viral products, namely, viral dna, rna, or polypeptides. the antiviral tests selected may be based on: (a) inhibition of the virus-induced cytopathic effect (cpe) in which the % effective dose (ed ) of the antiviral agent is expressed as the concentration that inhibits cpe in half of the quadruplicate cultures. (b) plaque reduction assay in which the dose of the drug required to reduce the plaque number by %, that is, ed is calculated. (c) virus yield reduction assay in which the drug concentration required to reduce % ( log reduction), or % ( log reduction) of the virus yield as compared with the virus control (infected cultures without drug) are determined from the dose-response curves and are expressed as ed or ed of the antiviral agent. (d) assay systems based on the measurement of specialized functions and viral products; a number of viruses do not produce plaques nor do they cause cpe readily, but they may be quantified by certain specialized functions based on their unique properties, for example, hemagglutination and hemadsorption tests used to study the antiviral activity against myxoviruses and elisa, used to determine the extent of virus replication and, thus, obtain a measure of the inhibitory effect of various antiviral agents on virus replication, etc. (hu and hsiung, ). colorimetric assays quantify cell viability through enzyme-mediated biochemical reactions owing to ingress of certain dyes inside living cells. mosmann, borenfreund, and puerner first advocated the application of tetrazolium (mtt) and neutral red (nr) assay, respectively, to quantitate cell viability and the cytotoxicity to cells in vitro. parida et al. ( ) compared the efficacy of two colorimetric assays (mtt and neutral red) to determine viral infectivity in microculture virus titration employing polio virus type- and dengue virus type- . mtt assay, also known as tetrazolium assay, has been exploited extensively to reveal the protective efficacy of therapeutic agents and plant extracts against cancer, hiv- , hsv, polio virus type , den- virus, and to the determination of neutralizing antibody levels to hiv and respiratory syncytial virus. mtt assay using microculture virus titration (mcvt) was applied for the determination of infectivity titers of influenza viruses and was found to be compatible with the well-established procedure of egg infectivity assay. unlike mtt, neutral red dye uptake assay has not been substantially exploited in virologic research. nr dye assay was earlier performed for the study of the antiviral efficacy of basil leaves extract against polio virus type . mtt, a tetrazolium salt, is a yellow-colored dye [ -( , -dimethylthiazol- -yl)- , -diphenyltetrazolium bromide] which gets cleared by mitochondrial succinate dehydrogenase enzyme into a blue-colored formazan in active cells. this product does not form crystals when it interacts with isopropyl alcohol and thus can be accurately measured. there is no need to harvest the viable cells, wherein the cell viability can be directly measured by a spectrophotometer (parida et al., ) . the assay procedure has been discussed in section . . . a colorimetric assay based on the cleavage of the tetrazolium salt wst- has been developed for human cytomegalovirus (hcmv) antiviral susceptibility testing and adapted to a microtiter plate format. bedard et al. ( ) developed a colorimetric assay based on the cleavage of the tetrazolium salt wst- for human cytomegalovirus antiviral susceptibility testing. the response of different cell cultures to a given antiviral agent, including the drug metabolism and toxicity, among other factors, may vary greatly. to perform antiviral testing against a particular virus infection, it is necessary to obtain a suitable host cell system for that virus infection. the same antiviral agent may behave very differently in different cell culture systems, although the same virus strain is used. (ii) virus strain and passage history the variability of sensitivity to a given antiviral agent has been noted among different strains of hsv and cmv. drugresistant strains have emerged to some antiviral agents, especially in the herpes virus group. the passage history of a virus strain may also affect its sensitivity to some antiviral agents. moi can influence substantially the evaluation of antiviral activity by the plaque reduction method or the virus yield reduction assay. high moi will decrease the sensitivity of the virus to an antiviral agent. this may also contribute to the disparity among antiviral evaluation results, even when the virus moi is kept constant (hu and hsiung, ) . ( the activities of several known antiviral phytochemicals are profoundly affected by the presence of serum components. for example, the terthiophene, α-terthienyl (α-t) was inhibited in a concentration-dependent manner by serum. in the case of the carboxylic acid derivative of α-t, the compound appeared to have no antiviral activity at all in the presence of serum, yet in its absence this compound was as effective as α-t. in contrast, the complex anthraquinone hypericin required a small amount of serum for maximal antiviral activity. the reactions were also strongly affected by the order of incubation of the components: virus, compound, serum, and light (hudson and towers, ) . the viruses to be selected for initial evaluation of plant extracts are obviously of major importance. they must be chosen to represent the different groups of viruses according to their morphology and various multiplication mechanisms and a range of virus diseases for which chemical control would be useful. besides the need for control, also the prevalence of the viral diseases and the resulting projection of the market potential, which are determined by the antigenic abundance of the causative viruses and the problems this represents for vaccine control, are important selection criteria (grunert, ) . in vitro methods are therefore more appropriate, because they allow simultaneous screening of a battery of viruses. in vivo screening of extracts against a broad array of viruses, in contrast, is not only very expensive but also extremely laborious. in vitro antiviral bioassays utilize thinly confluent monolayers of tissue culture cells with sufficient susceptibility to the infecting viruses that a visibly cytopathogenic effect (cpe) occurs, for example, rounding up. shrinking or detaching of cells from the monolayer can be produced and readily observed microscopically. a monolayer of cells consists of animal or human cells, such as chick embryo, rabbit, or green monkey kidney cells (vero cells), or human skin fibroblasts and carcinoma cells (hela cells), grown in a culture medium. such continuous cell lines used in virology are mostly "transformed" cells that can be maintained for an indefinite number of generations. the host cells require an appropriate tissue culture medium in which they can survive for at least week without having to renew the medium. renewal of the medium causes changes in intra-and extracellular products and alters the virus concentration. the medium must have a stable ph during the whole incubation time and may contain only a small amount of serum, because blood products tend to absorb many compounds. mostly, a defined synthetic medium, supplemented by some type of serum (such as fetal bovine, calf, or horse), a buffer on sometimes bacterial and fungal inhibitory antibiotics, is used. according to experience, vero cells, which allow the growth of many human viruses with visible cpe, grown in the medium described by hronovosky et al. ( ) , and slightly modified by lowering the pyruvate concentration, are most suitable for antiviral screening of plant extracts (van den berghe et al., ) . many combinations of test viruses are possible, but a battery of six viruses seems to be quite acceptable. virus types and strains may vary in sensitivity, but have to be selected as a function of their ability to multiply in the same tissue culture when cell culture models are used as screening systems. in this way, an objective comparison of antiviral activities is possible, whereas toxicity tests are minimized. moreover, virus multiplication must cause a visible cpe within a reasonable period of time, preferably within a week after infection. which are expressed as the number of infectious units per volume. an infectious unit is defined as the smallest amount of virus capable of producing a reaction after virus inoculation and can be carried out by two generally applied methods, namely, the plaque test (pt) and the % endpoint titration technique (eptt). in the plaque test, monolayers of cells grown in plastic or glass petri dishes are inoculated with dilutions of a viral suspension. after adsorption of infectious virus particles to the host cell, the monolayers are overlaid with an agarosecontaining medium so that the newly formed virus particles are localized by the solid agar over layer. these newly formed infectious particles spread from the initially infected cell to the adjacent cells and develop well-circumscribed foci of cellular degeneration. these areas of dead cells are called "plaques" and are visualized by staining the cell monolayer with a vital dye, such as neutral red. the plaques may also be detected microscopically by determining the multinucleated cells (e.g., measles) or by immunofluorescence. the concentrations of viral suspensions measured by counting the plaques are expressed as plaque-forming units per ml (pfu ml − ). in the endpoint titration technique, the concentration of infectivity is measured by determining the highest dilution of the suspension, which produces cpe in % of the cell cultures inoculated. this dilution is called the % tissue culture dose endpoint (tcd ). dilutions are therefore made in a maintenance medium and a certain volume of each of them ( . - . ml) is added to four or more tube cultures. the proportion of positive cultures is registered for each dilution and the precise dilution at which % of the inoculated tube cultures are infected is calculated. at this dilution, the inoculum contains, on average, one tcd or one tissue culture (infectious) dose for % of the tissue culture tubes. the influence of a plant extract on virus multiplication can be determined as a viricidal or an antiviral activity. the viricidal activity is measured by titration of the residual infectious virus after incubation of the plant extract with a virus suspension of at least tcd ml − . on the other hand, the antiviral activity is determined by comparing the virus titers of infected cells, which have been cultured with a maintenance medium containing plant extracts or test substances and a maintenance medium without test material (colegate and molyneux, ) . in contrast with antibacterial screening, no solvents, other than physiological buffer solutions, should ideally be used in the in vitro antiviral screening of plant extracts because the samples have to be added to tissue culture cells. it has been observed that many nonpolar plant extracts, prepared and evaporated, are reasonably soluble in dimethyl sulfoxide (dmso), especially if little or no water is present in the sample and the dissolving sample is heated on a water bath. viricidal and antiviral determinations may then be carried out on test solutions containing not more than % and % dmso, respectively. therefore, dissolved samples of nonpolar plant extracts in dmso are added dropwise to the maintenance medium in a ratio of : or : under stirring. as already mentioned, the maintenance medium may contain antibiotics, such as penicillin g ( μg ml − ), neomycin ( μg ml − ), and amphotericin b ( μg ml − ), in order to avoid sterilization of the test solutions. any contamination by bacteria or fungi would indeed ruin the in vitro antiviral bioassay (colegate and molyneux, ) . there are various methods for validation of antiviral activity. the major techniques have been highlighted in the preceding sections and in fig. . . most currently used antiseptics and disinfectants kill pathogenic bacteria and fungi at °c within min when present in a concentration of about . % ( -log titer reduction). because it has been noticed that most of these preparations fail to kill all pathogenic viruses under these circumstances, a method has been developed for testing the in vitro viricidal effect of plant extracts, as will be described in the following section. thoroughly mix the preincubated ( °c) plant extracts, dissolved in a physiological buffer, or their twofold dilutions (e.g., / to / ), with the same volume of a preincubated ( °c) virus suspension of, for example, pfu ml − or tcd ml − in physiological buffer. incubate the mixture at °c for min. stop the incubation by the addition of a fold volume of ice-cold maintenance medium and filter the mixture immediately through a . μm filter to eliminate all possible precipitate. then, filter the ice-cold filtrate through a . μm filter to concentrate residual virus on the filter and separate the virus from possibly cytotoxic plant components, which pass the filter. remove the residual virus from the filter with maintenance medium, supplement with % serum ( ml), sonicate in an ice-bath for s, and titrate in -fold dilutions at °c by plaque formation or in microtiter plates according to the eptt. carry out a virus control in a physiological buffer containing no plant extract simultaneously. an essential step of this methodology is the separation of all cytotoxic plant components from the residual virus, which has to be measured at °c. cytotoxic substances have a greater influence on the activity of an extracellular virus at °c than at °c. this step, however, can be omitted when the plant extracts to be tested are not toxic to the host cells under the conditions of the evaluation procedure (colegate and molyneux, ) . the eptt technique is performed on preemptied confluent monolayers of vero or other cells, grown in the holes of microtiter plates, which are infected with serial -fold dilutions of a virus suspension ( μl). starting with monolayers containing cells per hole and a virus suspension of, for example, tcid ml − or pfu ml − , infect the first monolayers of cells with a multiplicity of infection (moi). the antiviral activity is expressed as the virus titer reduction at the maximal nontoxic dose (mntd) of the test substance, that is, the highest concentration (μg ml − ) that does not affect the monolayers under the antiviral test condition. viral titer reduction factors, that is, the ratio of the viral titer reduction in the absence (virus control) and presence of the mntd of the test sample of × to × indicate a pronounced antiviral activity and are suitable as selection criteria for further investigation of plant extracts (colegate and molyneux, ) . the eptt is more suitable for testing complex samples, such as plant extracts, for many reasons. (i) first, the concentration of many compounds in the extract remains constant, and consequently the proportion of toxic versus active compounds does not change. (ii) second, the exact duration of the antiviral action can be determined by using the eptt, because the action starts from the moment the extract is added to the cells. (iii) third, the eptt using serial diluted extracts deals with a dynamic process, because the cells are subsequently infected with different moi. (iv) this system allows the correlation of all possible moi values in the same microtiter plate with decreasing amounts of plant extracts, so that the noncytotoxic concentration of plant extracts can be determined. at the same time, a correlation between extract toxicity and antiviral activity according to the corresponding moi can be determined in the same microtiter plate. (v) it can be stated as a general rule that the detected antiviral activity should be stable in at least two subsequent dilutions of nontoxic concentration of the extract; otherwise the activity is directly correlated with the toxicity of the extract or is only viricidal. moreover, a true antiviral product has to protect the cells, which have been infected with low virus dilutions (starting from . pfu per cell onward). (vi) finally, it should be stressed that all possible steps of virus manipulation are to be completed before the plant extracts are added. this means that an antiviral product tcd ml − ), under nontoxic conditions, must act on virus replication steps after uncoating. when the cells are completely protected only in the lower moi ( . tcd ml − ), replication processes before uncoating may be involved. an important aspect of the inhibition of viral cytopathic effect (cpe) is the determination of tcid ( % tissue culture infective dose). after harvesting, dilute the virus stock -fold. add . ml of each dilution in wells each of a -well microtiter plate. add . ml of cell suspension of , cells/well, incubate at °c with % co atmosphere, and observe for vial cpe on alternate days. take a final reading on the fifth day and calculate tcid as per the method of reed and muench ( ) , from which tcid can be further calculated from the log value. an antiviral drug will inhibit the cpe of a virus. therefore, for detecting an antiviral agent, the amount of inhibition of cpe of a virus can be observed microscopically (kenny et al., ) . for this purpose, trypsinise the monolayer and allow to seed in -well microtiter plates. after a -h incubation, wash the monolayer in each well and add different test drug dilutions and incubate. after h, wash the cell culture and inoculate with . ml of tcid , tcid , and tcid of the virus suspensions in different wells. incubate them for h at °c in an incubator for the adsorption of the virus onto the cells. after incubation, remove excess virus suspension by washing with rpmi. add . ml of selected concentration of the test compound and keep both virus and cell control wells with . ml of rpmi containing % sheep serum. observe the plates under a microscope every h until the virus control shows % cpe and tabulate the results (hu and hsiung, ) . trypsinize the cell monolayer, allow to seed in a -well microtiter plate and incubate for h at °c with % co atmosphere. remove the medium, wash the cell monolayer with rpmi without serum, and add . ml of different virus suspensions in different wells containing the cell layer and incubate for h for virus adsorption; wash off the excess virus suspension after adsorption. to each well, add the selected concentration of the test drug diluted in rpmi containing % sheep serum. to the virus control and cell control, rpmi is added ( % serum) and incubated for h. freeze the plates at − °c and thaw at room temperature a couple of times to liberate the associated virus. determine the virus titer by the end point dilution method and express as tcid (cinatl et al., ) . trypsinize the monolayer culture and allow to seed in a -well microtiter plate. after a -h incubation, wash the monolayer in each well and add different test drug dilutions and incubate. after h, wash the cell culture and inoculate with . ml of tcid , tcid , and tcid of the virus suspensions in different wells. incubate for h at °c in a co incubator for adsorption of the virus onto the cell. after incubation, remove excess virus suspension by washing with rpmi without serum. add . ml of the selected concentrations of the test compound and keep both the virus and cell control wells with . ml of rpmi containing % sheep serum. incubate the plates at °c for h. after h, discard the old media from the cell cultures and add μl of mg/ml of mtt ( -( , -dimethylthiazol- -yl)- , -diphenyltetrazolium bromide) to each well and incubate for h. after h of incubation, discard the mtt and add μl of isopropyl alcohol to each culture and set aside for min. record the absorbance using an elisa plate reader at nm. in this experiment, the effect of the test drug on mitochondrial synthesis due to viral infection is studied. the "plaque" is a confined region of contaminated cells formed by multiplying virus particles. the plaques are revealed either as regions of dead/decimated cells recognized by cell stains or as zones of polluted cells by immunostaining. the blended cell monolayer is infected with a log dilution of viral plaque-forming unit (pfu) in the absence or presence of the test drug and permitted to adsorb ( h at °c in % co ); afterward, the cells should be washed twice with prewarmed tcid cpe at dilution next above cpe at dilution nex % % % t t above cpe at dilution next below % % % § © · minimum essential medium (mem). the overlaid drug dilutions are arranged in the overlay medium on the contaminated culture, without the test drug. then, ml of carboxy methyl cellulose is added to ml of the medium; the plates are incubated for - days, then settled with % formalin or % formaldehyde for min. the cells are stained with methylene blue ( ml/well) or % gem violet (w/v), washed, and dried to check the plaques (dark areas) by low-power amplification of a binocular microscope. the antiviral impact should be measured as the percentage inhibition of plaque formation: the concentration of the test drug required to exert % of virus inhibition (ic or ec ), as compared with the infection control, is evaluated from the graphical plot as dose-response curves by regression analysis . viruses, for example, influenza, can agglutinate rbcs due to surface ha proteins, which can be analyzed visually by blending viral dilutions with rbc. this can be utilized to inspect the inhibitory impact of any medication on ha. the -overlay serially diluted ( - x) test drug is used alongside the diluted viral stocks ( : to : ). this dilution ( ml/well) is added to drug-containing wells. it should be preincubated for min and mixed with rbc ( / in pbs) sample solution. here, up to a specific dilution, the viral particles possibly lose their capacity to agglutinate rbcs, which shows a linkage of the drug with the viral ha. known quantities of virus (moi - ) are used for infecting both untreated or drug-treated cells and allowed to adsorb for - min. the unabsorbed virus particles are washed and fresh media is added to incubate for - h. then, cells are washed with pbs, fixed with paraformaldehyde ( %- %), and permeabilized with acetone or triton x- . the cells are again washed and blocked with % bovine serum albumin (bsa) in pbs for min. then, the cells are incubated with mouse or rabbit antibody against a specific viral protein for - h at °c. after that, the cells are subjected to repeated washing and incubated with a fluorescent-tagged secondary antibody for h and washed again. after washing, the cells are visualized under a fluorescent microscope and compared with the fluorescence of untreated and drug-treated cells. on the other hand, for quantitation, the cells are trypsinized after treatment and fixed with % paraformaldehyde. the cells are then washed, permeabilized, and labeled with a fluorescent-tagged antibody, followed by propidium iodide (pi: mg/ml in pbs). the cells are then counted in a fluorescent-activated cell counter to quantitate the fluorescence percentage. known quantities of virus (moi - ) are used for infecting both untreated and drug-treated cells, adsorbed for h, washed, and incubated ( - days) for evaluation of the inhibition of the virus-induced cytopathic effect (cpe). the virus stock is centrifuged after freeze thawing and diluted for elisa. each well of a plate coated with a virus-specific antibody is mixed with ml of controls or test drug and incubated at room temperature ( h) with horseradish peroxidase conjugate, alkaline phosphatase, or b-d-galactosidase-labeled virus-specific antibody. the wells are washed five times and the substrate ( ml) is added and incubated in the dark for min. the reaction is stopped by adding % h so solution and the absorbance is read at nm. the -well plates are seeded with a quadruplicate cell monolayer having a log dilution of the test drug followed by infection with the virus. after - h of incubation at °c, the monolayers are fixed with . % glutaraldehyde and examined for virus-specific protein(s) on the cell surface. the elisa should be performed with a monoclonal antibody to the specific protein of the corresponding virus and protein and the od (optical density) is measured at nm. the concentration is calculated by % reduction in od values (ec ) from extrapolating graphical plots followed by the determination of si (ratio of cc :ec ) in which the results are expressed as a percentage of virusinfected cells (virus control) . the test drug and the virus ( pfu/ml) are mixed to incubate for h at °c. then, immediately dilute the virus-drug mixture to -fold (final inoculums pfu/well) with media containing % fbs to get a subtherapeutic concentration of mean number of plaques in control mean number of plaques in sample m ean number of plaque in control the test drug. following that, mix the monolayer, with the virus inoculums seeded using a -well plate. alternately, add the virus-test drug mix diluted to -fold, with no incubation period, with the respective cells for infection. allow to adsorb for h at °c, discard the diluted inoculums, and wash the cells with pbs. pour an overlay medium (with % fbs), and incubate at °c for h, followed by plaque reduction assay. count the viral plaque numbers obtained from infections set in the presence of the test drug and compare it with the control. viral attachment to the cell surface is carried out at °c, as it permits binding, but stops viral entry, by elisa. briefly, incubate susceptible cells ( × cells/well) in -well plates and allow growth overnight. the cell monolayers are cooled at °c. the cells are infected with the virus (moi ) using heparin in presence of the test drug for h at °c as a control. after washing the wells with ice-cold pbs, fix with prechilled % paraformaldehyde in pbs for h on ice, blocked with % bsa at °c. the samples are incubated at °c for h with a primary antibody in pbst pbs with . % tween along with % bsa. the wells are washed twice with pbst, % bsa, and again only with pbst twice, at -min intervals on a shaker. this is mixed with secondary antibody in pbst with % bsa and incubated at °c for h. the reaction is developed with a , ′, , ′-tetramethyl-benzidine two component microwell peroxidase substrate for min; the reaction is stopped with m phosphoric acid. the absorbance is measured at nm, and the values are expressed as the fold change of absorbance relative to the mock infection control (lin et al., ) . the cell monolayers are cooled and grown in -well plates at °c for h. subsequently, infect the prechilled cells with hsv- ( pfu/well) and incubate for h at °c to allow for viral adsorption. incubate the infected cell monolayers in the presence of test drug or heparin ( mg/ml) for an additional min at °c to facilitate hsv- penetration. the extracellular virus is inactivated by citrate buffer (ph . ) for min, and the cells are washed with pbs followed by overlay with dmem containing % fbs. after h of incubation at °c, the viral plaques are stained and counted (lin et al., ) . add the plated cells ( . × cells/well for a -well plate) grown overnight at % confluence ( ml) with virus dilution and deae dextran at a final concentration of mg/ml. after adsorption ( h at °c in co incubator), place the plates in a rocker to prevent the cells from drying and add fresh medium ( - ml) containing the test drug to each well and incubate for - h in % co at °c for subconfluent growth. after removing the media, add fixing solution ( - ml) to each well and incubate for min at room temperature (β-galactosidase activity decreases dramatically if the fixing solution is left for > min). then, discard the fixing solution, wash the cells twice with pbs, stain, and incubate at °c for min. finally, stain the plates to count the number of blue syncytia and express the titration values as the number of stained cells multiplied by the viral dilution . this assay is applicable for hiv- , simian immunodeficiency virus (siv), and simian-hiv and is carried out in tzm-bl cells as it reveal the reduction in tat-induced luciferase (luc) reporter gene expression after a single round of virus infection. place the tzm-bl cells ( × /well) in a -well plate and incubate overnight. in a separate vial, mix the hiv- nl . (moi . ) virion with the test drug or vehicle for h at °c, then add to tzm-bl cells and incubate for h. wash the cells (with cold pbs), and add fresh media with the test drug to culture for h, using untreated hiv- -infected cells (negative) and azidothymidine (azt)-treated cells (positive) as controls. wash the cells twice with pbs, lyse with x lysis buffer, and add the supernatant with the substrate, which should then be analyzed for luciferase activity in an optiplate using a fluorimeter. the results are expressed as percentage inhibition: and the percent inhibition is calculated by subtracting the above value from (wan et al., ) . luminescence in the experimental group test drug or azt lu / m minescence of infected cells without the drug u (b) cem-green fluorescent protein (gpt) cell-based assay cem-gfp is a stable t-cell line-containing a plasmid encoding gfp and is suitable for hiv- nl . (moi . ) culture. for postinfection, incubate the cells ( . × /well) with the test drug up to days, using azt and solvents (used to prepare the test drug) as control(s). lyse the virus-infected cells with x promega cell lysis buffer ( ml), and transfer to a culture plate to read the absorbance at nm (excitation) and nm (emission) by a fluorimeter. the results can be expressed as percentage inhibition: with the he percent inhibition calculated by subtracting the above value from . place hep ad cell suspension ( × viable cells/ml of hep ad seeding medium) in a -well microtiter plate, and incubate at °c for days. discard the medium and wash the cell monolayers with warm ( °c) dpbs. to the proper wells, add μl of hepad assay medium that contains either test or control compounds at the desired concentrations. also include wells with hep ad assay medium alone as "virus only" controls. incubate at °c for days. on day , wash the cells once with warm dpbs and add fresh medium containing the appropriate compound to the wells. after h, transfer the supernatants to v-bottomed -well plates and remove cellular debris by centrifugation ( min, rpm at °c). transfer μl of the clarified supernatants to new v-bottomed plates and store at − °c for quantification of hbv dna. thaw the supernatants that were collected and add μl of x denaturation solution to each well and mix. incubate at °c for min. cut the nylon membrane to size and prepare it for blotting by wetting it first with distilled water and then x hybridization buffer, ssc (saline sodium-citrate). dot-blot the denatured supernatants on to the nylon membrane as directed by the manufacturer. wash the blot with μl of neutralization solution followed by μl of x ssc. remove the blot, rinse it briefly in x ssc, and then crosslink the dna to the nylon filter by uv irradiation. prehybridize the blot at °c for h in μl of hybridization solution. prepare a p-labeled probe by random priming using a portion of the hbv genome as a template. purify the probe through a clontech chroma spin column. denature the probe by boiling for min and add it immediately to the hybridization solution. hybridize the nylon filter overnight at °c. wash the nylon filters twice with ml of x ssc, . % sds (sodium dodecyl sulfate) at room temperature for min and twice with ml of . x ssc, . % sds at °c for min. expose the nylon filters to a molecular imager screen for h and scan on a phosphor imager to obtain the data. to determine the percent inhibition of hbv replication, subtract the background value (counts of radiation detected from the nylon filter itself) from all control and experimental values. divide the average values of the experimental wells (cells treated with test compounds) by the average value for the "virus only" control (cells not treated with compound or tetracycline during the experiment) and multiply this number by (king and ladner, ) . the above mentioned in vitro studies are listed in table . . drug development programs include preclinical screening of immense quantities of chemicals for specific and nonspecific cytotoxicity against numerous sorts of cells, which is imperative to show the potential therapeutic target and safety evaluation. the screening of plant extracts or pure compounds for investigating their antiviral properties can be more significant with cytotoxicity measures (meyer et al., ) . it is essential for an investigational item to establish the antiviral activity at concentrations that can be accomplished in vivo without inducing toxicity to cells. moreover, in a cell culture display, antiviral activity of an investigational item can be the aftereffect of host cell death after exposure to the item. cytotoxicity tests utilize a series of increasing concentrations of the antiviral product to determine what concentration results in the death of % of the host cells. this value is referred to as the median cellular cytotoxicity concentration (cc or ctc or ccic ). the relative effectiveness of the investigational product in inhibiting viral replication compared with inducing cell death is defined as the therapeutic or selectivity index (cc value/ec value). it is desirable to have a high therapeutic index giving maximum antiviral activity with minimal cell toxicity. according to us fda guidelines, it is recommended to determine cc values in both stationary and dividing cells from multiple relevant human cell types and tissues to establish the potential for cell cycle, species, or tissue-specific toxicities. studies determining cytotoxicity and therapeutic indexes should be conducted before the initiation of phase clinical studies. there are a number of advantages for in vitro testing using cell cultures, which include gfp fluorescence in the experimental group fluorescence in / i infected cells without the test drug × analysis of species specificity, feasibility of using only small amounts of test substances, and facility to do mechanistic studies (guidance for industry, ). after confirming the cytotoxic concentration, the drug concentrations are selected for antiviral studies based on the percentage viability of cells and are used to study the antiviral activity by cpe inhibition assay, virus yield assay, followed by mtt assay. any compound that is cytotoxic to cells inhibits the cell proliferation and kills the cells. trypan blue is a dye that is capable of penetrating dead cells; therefore, the dead cells take up the blue stain whereas the viable cells do not. this method gives an exact number of dead and viable cells (strober, ) . protein content is widely used for estimating total cellular material and can be used in growth experiments. the colorimetric method of estimating protein is more sensitive. the cell pellets are treated with % cold trichloroacetic acid to remove amino acid pools and dissolved in alkaline cupric sulfate and folin ciocalteau phenolic reagent. folin's reagent and cupric sulfate together react with amino acid to give a blue color and this color intensity is proportional to the protein concentration, which can be measured colorimetrically (maya et al., ) . to proceed with the same technique, the cells from the wells were trypsinized using μl trypsin and transferred into eppendorf tubes and centrifuged at rpm for min to obtain pellets. the cell pellets are dissolved in naoh and diluted , tzm-bl cell-based assay (hiv), cem-green fluorescent protein cell-based assay (hiv), hep ad assay (hbv), immunofluorescence assay, enzyme-linked immunosorbent assay (elisa) • reduction or inhibition of virus-specific polypeptides synthesis in infected cell cultures, e.g., viral nucleic acids, determination of the uptake of radioactive isotope labeled precursors or viral genome copy numbers other assays for validation of antiviral activity • virus inactivation assay, virus adsorption assay, virus attachment, and penetration assay to . n. the test drug is to be added to μl of protein sample, mixed, and left for min. to this, μl of test reagent is added with constant mixing and left for min in incubator. the absorbance was read at nm using an elisa reader (bio-rad). the color development was correlated with the cell number as follows: the cytotoxic concentration found by dye exclusion techniques gives superficial data. the selected concentrations from a trypan blue dye exclusion study are used further for estimating proteins. mtt ( -( , -dimethylthiazol- yl)- , diphenyl tetrazolium bromide) in live cells enters the cells and enzyme succinate dehydrogenase present in mitochondria reduces it to formazan blue product. the color intensity is directly proportional to the number of live cells. to perform this process, the plates were seeded with hep- cells at , cells/well. they are incubated for h. after h, the medium is discarded and drug concentrations were added and incubated for h. then, μl of mg/ml of mtt is to be added and incubated for h and then μl of isopropyl alcohol is added and absorbance is read at nm in an elisa plate reader (bio-rad). the results are tabulated and percentage growth inhibition is calculated using the following formula: the concentrations of the test drug used in the previous experiments can be further confirmed by studying the mitochondrial synthesis by mtt assay. the formazan blue color formation is directly proportional to the number of viable cells and therefore the absorbance is to be read at nm. in this test, brine shrimp (artemia salina) eggs are hatched in artificial sea water ( g/l of sea salt). the brine shrimp test (bst) bioassay experiment is performed according to the procedure described by meyer et al. ( ) . generally, samples of the test drugs for the experiment are prepared in methanol solution, which acts as control vehicle. after h of incubation, brine shrimps are transferred to each sample vial using a pasteur pipette and artificial sea water is added to make ml. sample vials are previously prepared by dissolving specific concentrations of test drugs with different dilutions. the solvent is then evaporated overnight. survivors are counted after h and the lc values, with % confidence intervals are determined using probit analysis, as described by finney ( ) . control vials are prepared using methanol only. three replicates are prepared for each concentration of the test drugs. control disks are prepared using only methanol. replicates are prepared for each dose level. to begin the bioassay, brine shrimp eggs are hatched in a shallow rectangular dish ( × cm ) under the same conditions described in the literature except that natural instead of artificial seawater is used. ten shrimps are selected and transferred into each sample vial by means of a -cm disposable pasteur pipette and the final volume in each vial is adjusted to ml using natural seawater. a drop of dry yeast suspension ( mg in ml seawater) is added as food to each vial. the vials are maintained under illumination. survivors are counted with the aid of a stereomicroscope, after , , and h, and the deaths at each dose level and control are determined. no deaths are usually observed to occur in the control after h. the brine shrimp test (bst) represents a rapid, inexpensive, and simple bioassay for testing plant extract lethality, which, in most cases, correlates reasonably well with cytotoxic properties (mclaughlin, ) . following the procedure of meyer et al. ( ) , the lethality of the test drugs/plant extracts to brine shrimp is determined. the lethal concentrations of test drugs/plant extract resulting in % mortality of the brine shrimp (lc ) and % confidence intervals are determined from the and -h counts and the dose-response data are transformed into a straight line by means of a trend line fit linear regression analysis; the lc is derived from the best-fit line obtained. caffeine (lc = μg/ml) (meyer et al., ) is used as a positive control and methanol ( μl) as a solvent and a negative control in the bioassay experiments. the current scenario of viral diseases is lethal and there is an upsurge in new viral diseases and resistance to existing viral infections worldwide. the currently accessible antivirals, however effective, are exorbitant and past the reach of a majority of individuals. along these lines, the advancement of safe, effective, and low-cost antiviral medications, for example, rt inhibitors, is among the top priorities, as many viral infections are not yet treatable and have high death rates. for the past few years, substantial work has been carried out regarding the effectiveness of medicinal plants on hiv infection (premanathan et al., ; calabrese et al., ; asres et al., ) and an increasing popularity of over-the-counter plant products containing orthodox drugs has been observed. the main focus is to lower the adverse effects associated with viral infections and an inclination toward synergistic interactions of multiple molecules present in plant extracts. be that as it may, because mostly pharmacological mechanisms of the combinations are not studied, antagonistic impacts or remedial disappointments have been seen (chan et al., ) . a prerequisite that should be considered significant for medicinal plants is to identify and standardize the method of extract preparation, the suitable season for collecting plant material, and the details of its administration (chattopadhyay et al., ) . as a lot of plant extracts and subsequent formulations have shown significant outcomes, it seems to be rational to endorse the idea of the study of medicinal plants as a quest to find potential antivirals. antiviral investigation on the flavonoids of chamaesyce thymifolia investigation of medicinal plants of togo for antiviral and antimicrobial activities in vitro virucidal activity of selected anthraquinones and anthraquinone derivatives antiviral evaluation of plants from brazilian atlantic tropical forest antiviral activity against human immunodeficiency virus type (hiv- ) and type (hiv- ) of ethnobotanically selected ethiopian medicinal plants suppression of hepatitis c virus by the flavonoid quercetin is mediated by inhibition of ns protease activity anti-herpes virus activities of bioactive fraction and isolated pure constituent of mallotus peltatus: an ethnomedicine from andaman islands the mannose-specific plant lectins from cymbidium hybrid and epipactis helleborine and the (n-acetylglucosamine) n -specific plant lactin from urtica dioica are potent and selective inhibitors of human immunodeficiency virus and cytomegalovirus replication in vitro -cinnamoyl- , -dihydroxymeliacarpin is a natural bioactive compound with antiviral and nuclear factor-kappa b modulating properties a high throughput colorimetric cell proliferation assay for the identification of human cytomegalovirus inhibitors an investigation of the antiviral activity of podophyllum peltatum in vitro anti-hiv- activities of kaempferol and kaempferol- -o-glucoside isolated from securigera securidaca further screening of venda medicinal plants for activity against hiv type reverse transcriptase and integrase the natural compound silvestrolis a potent inhibitor of ebola virus replication plant products as topical microbicide candidates: assessment of in vitro and in vivo activity against herpes simplex virus type a phase i trial of andrographolide in hiv positive patients and normal volunteers comparative study of propofol versus midazolam in the sedation of critically ill patients: results of a prospective, randomized, multicenter trial survey for the presence and distribution of human herpesvirus in healthy brain ethnomedicines and ethnomedicinal phytophores against herpes viruses antivirals of ethnomedicinal origin: structure-activity relationship and scope dose dependent therapeutic antiinfectives from ethnomedicines of bay islands recent advancements for the evaluation of anti-viral activities of natural products validation of antiviral potential of herbal ethnomedicine inhibitory effects of quercetin -rhamnoside on influenza a virus replication anti-herpes simplex virus (hsv- ) activity of oxyresveratrol derived from thai medicinal plant: mechanism of action and therapeutic efficacy on cutaneous hsv- infection in mice chemistry, biological activity, and chemotherapeutic potential of betulinic acid for the prevention and treatment of cancer and hiv infection antiviral effects of -diazo- -oxo- -nor-leucine on replication of herpes simplex type- novel compounds in preclinical/early clinical development for the treatment of hiv infections molyneux bioactive natural products plant substances as antiviral agents: an update potential antiviral lignans from the roots of saururus chinensis with activity against epstein-barr virus lytic replication drug screening for autophagy inhibitors based on the dissociation of beclin -bcl complex using bifc technique and mechanism of eugenol on anti-influenza a virus activity structure and in vitro antiviral activity of triterpenoid saponins from calendula arvensis antiviral activity of chlorogenic acid against influenza a (h n /h n ) virus and its inhibition of neuraminidase resveratrol inhibition of herpes simplex virus replication antiviral activities of various water and methanol soluble substances isolated from ganoderma lucidum sennoside a, derived from the traditional chinese medicine plant rheum l., is a new dual hiv- inhibitor effective on hiv- replication anti-enterovirus and immunostimulating activity of the polyphenol complex extracted from pethaphylloides fruticosa (l.) o. schwarz honokiol, a lignanbiphenol derived from the magnolia tree, inhibits dengue virus type infection screening of brazilian plants for antiviral activity against animal herpes viruses probit analysis antiviral effects of glycyrrhiza species biotransformation of ursolic acid by syncephalastrum racemosum cgmcc . and anti-hcv activity anti-aids agents . betulinic acid and platanic acid as anti-hiv principles from syzygium claviflorum, and the anti-hiv activity of structurally related triterpenoids antiviral activity of dammarane saponins against herpes simplex virus type activity of melaleuca alternifolia (tea tree) oil on influenza virus a/pr : study on the mechanism of action antiviral activity of an extract derived from roots of eleutherococcus senticosus search for antiviral agents antiviral product development-conducting and submitting virology studies to the agency. u.s. department of health and human services, food and drug administration michellamines d-f, new hiv-inhibitory dimeric naphthylisoquinoline alkaloids, and korupensamine e, a new antimalarial monomer, from ancistrocladus korupensis virucidal effects of the steam distillate from houttuynia cordata and its components on hsv- , influenza virus, and hiv inhibitory effect of ferulic acid and isoferalic acid on murine interleukin- production in response to influenza virus infections in vitro and in vivo a modified plaque method for arboviruses on plastic panels evaluation of new antiviral agents: i, in vitro perspectives anti-viral effect of a compound isolated from liriope platyphylla against hepatitis b virus in vitro isolation of the anthropogenic compound fluoranthene in a screening of chinese medicinal plants for antiviral compounds further investigations on the antiviral activities of medicinal plants of togo antiviral activities in plants endemic to madagascar inhibitory effects of sudanese medicinal plant extracts on hepatitis c virus (hcv) protease study of antiviral action of total alkaloids from haemanthus albiflos inhibitory effect of glycyrrhizin on the in vitro infectivity and cytopathic activity of the human immunodeficiency virus antiviral potential of selected indian medicinal (ayurvedic) plants against herpes simplex virus and . n anti-hbv active constituents from piper longum the flavonoid ellagic acid from a medicinal herb inhibits host immune tolerance induced by the hepatitis b virus-e antigen comparison of specific antiviral agents in herpes simplex keratitis in vitro and in vivo anti picorna virus activity of some phenoxypyridine carbonitriles extracts and molecules from medicinal plants against herpes simplex viruses hep ad assay a high-throughput, cell-based screen for the evaluation of compounds against hepatitis b virus samarangenin b from limonium sinense suppress herpes simplex virus type replication in vero cells by regulation of viral macromolecular synthesis antiviral activities against hsv- , hcmv, and hiv- of rhamnan sulfate from monostroma latissimum antiviral effects of black raspberry (rubus coreanus) seed and its gallic acid against influenza virus infection in vitro antiviral activities of chinese medicinal herbs against duck hepatitis b virus plant antiviral agents iii. isolation of alkaloids from clivia miniata regel (amaryllidaceae) isolation and purification of baicalein, wogonin and oroxylin a from the medicinal plant scutellaria baicalensis by high-speed counter-current chromatography antiviral activity and mode of action of caffeoylquinic acids from schefflera heptaphylla (l) identification of natural compounds with antiviral activities against sarsassociated coronavirus mechanism of action of glycyrrhizic acid in inhibition of epstein-barr virus replication in vitro hydrolyzable tannins (chebulagic acid and punicalagin) target viral glycoproteine glycosaminoglycan interactions to inhibit herpes simplex virus entry and cell-to-cell spread saikosaponin b is a naturally occurring terpenoid that efficiently inhibits hepatitis c virus entry ethnopharmacology in overdrive: the remarkable anti-hiv activity of artemisia annua the alkaloids. vol antiviral potential of curcumin a search for anti-viral properties in panamian medicinal plants, the effects on hiv and its essential enzymes interaction of filacial proteins on growth regulation of normal lung epithelial cells in vitro antiviral screening of british columbian medicinal plants crown-gall tumors in potato discs and brine shrimp lethality: two simple bioassays for higher plant screening and fractionation the in vitro antivirial activity of an aliphatic nitro compound from heteropteris aphrodisiaca brine shrimp: a convenient general bioassay for active plant constituents natural products and drug development. munksgaard anti-herpes virus activity of apporphine alkaloids anti-herpes virus activities of achyranthes aspera: an indian ethnomedicine, and its triterpene acid bio organic marine chemistry pedilanthus tithymaloides inhibits hsv infection by modulating nf-κb signalling screening extracts of zanthoxylum chalybeum and warburgia ugandensis for activity against measles virus (swartz and edmonston strains) in vitro biological activity of guatteria cardoniana fractions comparison of two colorimetric assays to determine viral infectivity in microculture virus titration characteristic of alkylated chalcones from angelica keiskei on influenza virus neuraminidase inhibition glycyrrhizic acid inhibits virus growth and inactivates virus particles in vitro anti-human immunodeficiency virus activity of polysaccharide from rhizophora mucronata poir maslinic acid, a natural triterpenoid compound from olea europaea, protects cortical neurons against oxygen-glucose deprivation induced injury in vitro study of the antiviral activity of some b-carboline alkaloids a simple method of estimating fifty percent endpoints anti-hiv activity of extracts and compounds from algae and cyanobacteria antiviral flavonoid from pterocaulon sphacelatum, an australian aboriginal medicine in vitro antiviral activity of the anthraquinone chrysophanic acid against poliovirus antiherpes virus activity of extracts from the medicinal plant geranium sanguineum l antiviral agents as adjuncts in cancer chemotherapy effects of phyllanthus plant extracts on duck hepatitis b virus in vitro and in vivo antiviral activity of carnosic acid against respiratory syncytial virus in vitro antioxidant activity of polyphenol extracts with antiviral properties from geranium sanguineum l a new antiviral and antimicrobial sesquiterpene from glyptopetalum sclerocarpum trypan blue exclusion test of cell viability antiviral activity and constituent of ardisia chinensis benth against coxsackie b virus analysis of anti-rotavirus activity of extract from stevia rebaudiana proanthocyanidin from blueberry leaves suppresses expression of subgenomic hepatitis c virus rna anti-viral activity of the extracts of a kenyan medicinal plant carissa edulis against herpes simplex virus screening of higher plants for biological activities. ii. antiviral activity plant products as potential antiviral agents plant antiviral agents: v. -methoxyflavones as potent inhibitors of viral-induced block of cell synthesis in vitro antiviral activity of plant extracts from asteraceae medicinal plants current organic chemistry, natural product chemistry issue plant flavonoids in biology and medicine ii: biochemical, cellular and medicinal properties inhibition of hepatitis c virus replication by chalepin and pseudane ix isolated from ruta angustifolia leaves fangchinoline inhibits human immunodeficiency virus type replication by interfering with gp proteolytic processing herbs of the genus phyllanthus in the treatment of chronic hepatitis b: observations with three preparations from different geographic sites effect of oenanthe javanica flavone on human and duck hepatitis b virus infection antiviral effect of cimicifugin from cimicifuga foetida against human respiratory syncytial virus three new secoiridoids, swermacrolactones a-c and anti-hepatitis b virus activity from swertia macrosperma lignans with antihepatitis b virus activities from phyllanthus niruri l antiviral activity of polymethoxylated flavones from guangchenpi, the edible and medicinal pericarps of citrus reticulata 'chachi the in vitro activity of geraniin and , , , -tetra-o-galloyl-beta-d-glucose isolated from phyllanthus urinaria against herpes simplex virus type and type infection the protective effect of -deoxysappanchalcone on in vitro influenza virusinduced apoptosis and inflammation mechanism of inhibition of hiv- infection in vitro by purified extracts of prunella vulgaris evaluation of anti-hsv- activities of barleria lupulina and clinacanthus nutans novel antiviral activity of baicalein against dengue virus a dihydro-pyrido-indole potently inhibits hsv- infection by interfering the viral immediate early transcriptional events comparative inhibitory effects of suramin and other selected compounds on the infectivity and replication of human t-cell lymphotropic virus (htlv-iii)/lymphadenopathy-associated virus (lav) discovery of cyanovirin-n, a novel human immunodeficiency virus-inactivating protein that binds viral surface envelope glycoprotein gp : potential applications to microbicide development emerging anti-hiv drugs agar diffusion method for detection and bioassay of antiviral antibiotics anti-herpes virus activity of aporphine alkaloids antiviral potentials of medicinal plants evaluation of anti-infective potential of a tribal folklore odina wodier roxb against some selected microbes and herpes simplex virus associated with skin infection sensitive and rapid assay on mt- cells for the detection of antiviral compounds against the aids virus a rapid and simple colorimetric test for the study of anti-hiv agents plant-derived leading compounds for chemotherapy of human immunodeficiency virus (hiv) infection resistance of human immunodeficiency virus type to the high-mannose binding agents cyanovirin n and concanavalin a key: cord- - su m authors: alam, aatif; jiang, linda; kittleson, gregory a.; steadman, kenneth d.; nandi, somen; fuqua, joshua l.; palmer, kenneth e.; tusé, daniel; mcdonald, karen a. title: technoeconomic modeling of plant-based griffithsin manufacturing date: - - journal: front bioeng biotechnol doi: . /fbioe. . sha: doc_id: cord_uid: su m griffithsin is a marine algal lectin that exhibits broad-spectrum antiviral activity by binding oligomannose glycans on viral envelope glycoproteins, including those found in hiv- , hsv- , sars, hcv and other enveloped viruses. an efficient, scalable and cost-effective manufacturing process for griffithsin is essential for the adoption of this drug in human antiviral prophylaxis and therapy, particularly in cost-sensitive indications such as topical microbicides for hiv- prevention. the production of certain classes of recombinant biologics in plants can offer scalability, cost and environmental impact advantages over traditional biomanufacturing platforms. previously, we showed the technical viability of producing recombinant griffithsin in plants. in this study, we conducted a technoeconomic analysis (tea) of plant-produced griffithsin manufactured at commercial launch volumes for use in hiv microbicides. data derived from multiple non-sequential manufacturing batches conducted at pilot scale and existing facility designs were used to build a technoeconomic model using superpro designer(®) modeling software. with an assumed commercial launch volume of kg griffithsin/year for . million doses of griffithsin microbicide at mg/dose, a transient vector expression yield of . g griffithsin/kg leaf biomass, recovery efficiency of %, and purity of > %, we calculated a manufacturing cost for the drug substance of $ . /dose and estimated a bulk product cost of $ . /dose assuming a % net fee for a contract manufacturing organization (cmo). this is the first report modeling the manufacturing economics of griffithsin. the process analyzed is readily scalable and subject to efficiency improvements and could provide the needed market volumes of the lectin within an acceptable range of costs, even for cost-constrained products such as microbicides. the manufacturing process was also assessed for environmental, health and safety impact and found to have a highly favorable environmental output index with negligible risks to health and safety. the results of this study help validate the plant-based manufacturing platform and should assist in selecting preferred indications for griffithsin as a novel drug. griffithsin is a high-mannose binding lectin found natively in the marine red alga griffithsia (mori et al., ) . the protein is composed of amino acids and its monomer has a mass of approximately kda. griffithsin forms a homodimer with six binding pockets with high affinity for mannose, a common sugar found at the terminal end of oligosaccharides on the surface of many enveloped viruses. the protein is thought to inhibit the entry of enveloped viruses into host cells as well as viral maturation and transmission events by binding to oligosaccharides on the viral envelope surface. native griffithsin and its analogs are the most potent hiv- entry inhibitors yet described, with ec values in the picomolar range (mori et al., ; o'keefe et al., ) . griffithsin also effectively inhibits transmission of hsv- (nixon et al., ) , hcv (meuleman et al., ) , sars-cov (o'keefe et al., ) , ebola (barton et al., ) , and possibly other viruses yet to be studied. importantly, griffithsin appears devoid of cellular toxicity that is associated with other lectins. o'keefe et al. conducted studies with explants of macaque and rabbit vaginal tissues ex vivo and showed that griffithsin did not induce changes in the levels of cytokines or chemokines, nor did it alter lymphocyte levels in human cervical tissue nor elicit inflammatory responses in rabbit tissue (o'keefe et al., ) . the combination of extremely wide viral target range and demonstrated preclinical safety makes griffithsin potentially useful as a prophylactic and/or therapeutic in multiple and diverse antiviral indications. the potential indications for griffithsin as a human prophylactic or therapeutic include its use as an active pharmaceutical ingredient (api) in vaginal and rectal microbicides. in spite of the value shown by pre-exposure prophylaxis (prep) drugs to prevent hiv transmission, issues of cost, side effects, the potential for development of viral resistance through chronic use of antiretrovirals (arv) as prevention modalities, and access to prep drugs by underresourced populations remain. these unmet needs could be met by the availability of affordable, safe and effective "on demand" antivirals, especially with griffithsin as the api and its potential to control co-transmitted viruses such as hiv- , hsv- and hcv during intercourse. adoption of griffithsin as a new biologic drug, especially in cost-constrained products such as microbicides, is predicated on the feasibility of a scalable manufacturing process that can supply market-relevant volumes of the api at an acceptable cost of goods sold (cogs). previously, we showed that recombinant griffithsin can be expressed and isolated with high efficiency using transient gene expression in green plants (fuqua et al., a,b) . although the process described can be further optimized, the achieved pilot-scale expression yields of > . g griffithsin per kg of fresh (hydrated) green biomass ("fresh weight"; fw), recovery efficiencies of - % overall, and griffithsin purity of > % of total soluble protein (tsp) are already impressive. in this study, we developed a technoeconomic model for griffithsin manufacturing using a plant-based system with the goal of estimating api manufacturing cost and determined the factors that have the greatest impact on cogs. the output of our study should serve as a basis for additional process improvements, selection of a commercial-scale manufacturer, and should assist in the identification of future product targets for cost-sensitive markets such as prophylactic microbicides as well as those for less cost-constrained therapeutic indications. technoeconomic modeling was performed with the widely used superpro designer modeling software (intelligen, inc., scotch plains, nj, usa). the main analysis in this study was conducted using data available from pilot-scale manufacturing of griffithsin in nicotiana benthamiana plants using tobacco mosaic virus (tmv)-induced transient gene expression, and assuming that manufacturing would take place in an existing and fully equipped state-of-the-art plant-based biomanufacturing facility. modeling costs based on existing resources of a contract manufacturing organization (cmo) instead of a "greenfield" build of a new facility was seen as the most likely scenario for launch of a new product. our reasoning was that dedicated infrastructure could be built subsequently depending on market demand for the drug. as a result, we did not estimate capital equipment or total capital investment costs, and neglected depreciation, insurance, local taxes and factory expenses in the manufacturing operating cost analysis as these investments would have been made by the cmo. our analysis assumed a % net profit margin/fee (sood et al., ) assessed by the cmo and this figure was added to the production cost of the product to arrive at the final total product cost. in addition to the technoeconomic analysis, an environmental health and safety assessment (ehsa) of the designed process was conducted using the method described by biwer and heinzle ( ) to evaluate the environmental, health and safety impact of griffithsin manufacturing using the plant-based system, with the goal of assessing the sustainability of the process. the technoeconomic modeling for this study was performed using superpro designer ("superpro"), version . (intelligen, inc., scotch plains, nj; http://www.intelligen.com/), a software tool for process simulation and flowsheet development that performs mass and energy balances, equipment sizing, batch scheduling/debottlenecking, capital investment and operating cost analysis, and profitability analysis. this software has been used to estimate cost of goods in a variety of process industries including pharmaceuticals produced by fermentation (ernst et al., ) and plant-made pharmaceuticals (evangelista et al., ; zapalac and mcdonald, ; tusé et al., ; nandi et al., ) . it is particularly useful at the early, conceptual plant design stage where detailed engineering designs are not available or warranted. superpro was chosen because it has built-in process models and an equipment cost database for typical unit operations used in the biotechnology industry, such as bioreactors, tangential flow ultrafiltration and diafiltration, chromatography, grinding or homogenization, and centrifugation. there are some specific unit operations and processes used in this study that are currently not included in superpro, such as indoor plant cultivation, transplantation, plant harvesting and screw press/disintegrator. such unit operations were addressed through the "generic box" feature of the application. unless otherwise noted, the maintenance costs of major equipment, unit operation-specific labor requirements and costs (e.g., operators, supervisors), pure components, stock mixtures, heat transfer agents, power and consumables (e.g., filter membranes, chromatography resins) used in the analysis were determined using the superpro built-in equipment cost model and default databanks. additional case study specific design parameters were selected based on experimental data from journal articles, patent literature, the authors' laboratories, interviews with scientists and technologists conducting the work cited, technical specification sheets or correlations, heuristics, or assumptions commonly used in the biotechnology and/or agricultural industry. process flow and unit operations were derived from published methods and unpublished results obtained by the authors and collaborators who have participated in the development and scale-up of the process described and in the development of griffithsin products. on the basis of this information, the superpro software was used to select and size equipment for each of the unit operations to achieve the desired production target ( kg of purified griffithsin/year), simulate the operations by performing material and energy balances, and specify and schedule all operations taking place within each piece of equipment to calculate material inputs and outputs and process times. costs for raw materials, utilities, consumables, labor, laboratory qa/qc, waste disposal and equipment maintenance were then used to determine annual operating costs, and per-unit mass or per-dose costs ($/kg or $/dose). the main case study model was based on an existing plantbased manufacturing facility, operating in batch mode, and excluded new capital investments and other facility dependent costs, except for equipment maintenance costs, which were included. for the downstream portion of the griffithsin manufacturing process, an annual available operating time of , h ( days, -h operation, or % available operating time per year) for the facility was used with indoor-grown nicotiana benthamiana plants. operating time was based on holtz et al. ( ) for a similar facility, which was designed with overlapping utility capacity and in which the largest single utility unit can be down for maintenance and/or repairs and the utility loads can be maintained with redundant (spare) equipment. likewise, per nandi et al. ( ) it was assumed that the plants would be grown continuously throughout the year ( , h, or days, -h operation, or % available operating time per year). land costs, upfront r&d, upfront royalties, and regulatory/certification costs were neglected in the model as these costs can vary widely. griffithsin protein for this modeling study was produced in nicotiana benthamiana host plants. this host is preferred for indoor protein manufacturing due to its metabolic versatility, permissiveness to the propagation of various viral replicons, and high expression yields achievable with a wide range of targets, as reviewed by pogue et al. ( ), de muynck et al. ( , thomas et al. ( ) , gleba et al. ( ) , and others. griffithsin protein can be produced in plants in a number of ways. these include (a) stable expression in recombinant plants; (b) inducible expression in transgenic plants; (c) transient expression induced directly by tobacco mosaic virus (tmv) replicons; or (d) via agrobacterial vectors introduced into the plants via vacuumassisted, or surfactant-assisted, infiltration (gleba et al., ) . relative to stable transgenic plants, the advantages of speed of prototyping, manufacturing flexibility, and ease of indoor scale-up are clearly differentiating features of transient systems and explain why this approach has been widely adopted in the manufacture of many plant-made pharmaceuticals (gleba et al., ) . in our base-case analysis, we modeled expression of griffithsin using tmv induction described in fuqua et al. ( b) and results from pilot-scale manufacturing runs because these batches provided the most extensive and complete data set; however, this process has been corroborated in additional manufacturing runs at pilot-scale or larger. nicotiana benthamiana host plants are generated from seed and propagated indoors under controlled environmental conditions until sufficient biomass is obtained for inoculation with the tmv vector carrying the griffithsin gene. the process is summarized as follows. an n. benthamiana master seed bank is generated from seeds obtained from the u.s. department of agriculture (usda) repository. for biomanufacturing, seeds from the tw- line are obtained in bulk and stored securely. the master seed bank is qualified for germination rate (> %), freedom from disease, and genetic uniformity, and stored in sealed containers under temperature-controlled conditions ( ± • c). if the seed batch passes release tests, it becomes the production seed batch and is used in the designated production run ("working seed lot"). seedlings are allowed to grow for days under controlled environmental conditions ( ± • c and % rh per holtz et al., ) . at this stage, the seedlings are transplanted to accommodate their larger size and moved to another growth room to await inoculation, as described in the following sections. the expression vector is constructed as described in o' keefe et al. ( ) . briefly, a synthetic cdna (genbank no. fj ) encoding the -amino acid griffithsin amino acid sequence is cloned into a tmv-based expression vector. in posttranslational processing in planta, griffithsin's amino-terminal methionine is cleaved and the n-terminal serine is acetylated. the construct containing the tmv vector backbone and griffithsin gene insert are built into a plasmid that is propagated in the e. coli host strain dh b (fuqua et al., b) and constitutes a master plasmid bank. the master plasmid bank is maintained in stocks at − • c and is checked periodically for stability and insert fidelity. excision via t polymerase produces free tmv transcript, which constitutes a working transcript batch used to inoculate n. benthamiana plants days after sowing and generate a tmv virion inoculum batch days post infection, which is checked for conformance to quality control criteria (e.g., infectivity, message fidelity, bioburden, stability) per fuqua et al. ( a) . the tmv inoculum is then applied to host plants to initiate expression. the tmv inoculant is applied to the -day-old plant host production batch by high-pressure spray with an abrasive (diatomaceous earth), to introduce the virus into plant tissue. once the tmv vector gains access to plant tissue, the virion decapsidates and the genomic rna encodes for a polymerase/replicase to multiply the message. as described in shivprasad et al. ( ) and pogue et al. ( ) , subgenomic promoters also drive expression of a movement protein (mp) to translocate the transcript throughout the plant, and a coat protein (cp) that encapsidates the rna and reconstructs the virion that then self-propagates throughout the plant. simultaneously, a subgenomic promoter (tmv u ) also drives expression of the griffithsin gene, which is translated into griffithsin protein. plants at this stage are therefore induced to synthesize the api. using this expression method, griffithsin concentration in planta reaches a maximum without further increase typically days post inoculation (optimized internally based on the amount of inoculum used). at this stage, the plants are ready for api extraction. the api extraction procedure modeled is per holtz et al. ( ) except that a : ratio of biomass:buffer is used. briefly, the aerial parts of the plants (i.e., leaves, stems) containing accumulated griffithsin are mechanically inverted and cut with a mechanical cutter. the harvested biomass is collected in baskets for transport to the extraction suite, to initiate downstream processing. the harvested biomass fresh weight (fw) is determined to calculate the volume of extraction buffer to be added, typically at a rate of kg biomass fw: l buffer mix ( mm sodium acetate, mm sodium chloride, mm ascorbic acid, mm sodium metabisulfite). the ph is adjusted to . and the mixture is heated to • c for min to help precipitate major host plant proteins. the heated mixture is passively cooled and filtered ( . µm cellulose filter) to yield a crude extract. the crude extract is stirred overnight at • c in the presence of bentonite and mgcl . this procedure helps remove tmv coat protein (cp), which at this step represents the largest protein impurity in the extract. the suspension is filtered ( . µm cellulose filter) to remove aggregated tmv cp, yielding a clarified and partially purified api-containing solution and is then sterilefiltered ( . µm polyethersulfone filter). in-process controls are applied throughout downstream processing unit operations to determine reagent volumes and assess yield and quality at key steps. the partially purified extract is subjected to capto r mmc multi-modal chromatography (ge healthcare) per fuqua et al. ( b) using a -step pbs gradient ( % and % phosphate buffered saline [ mm nacl, . mm kcl, mm nah po , mm kh po ]) at ph . . the purified product consists of the drug solution in pbs, which is considered the drug substance (ds). the ds is release-tested per specification and is typically > % pure (fuqua et al., b) . the ds solution is typically bulk-packaged in inert bottles with screw cap closures per usp class vi guidelines. because container options vary, the final packaging step was not included in the model. results used for modeling purposes were averages from non-sequential manufacturing runs at pilot scale conducted at kentucky bioprocessing llc ("kbp, " owensboro, ky, usa), the first of which was described in fuqua et al. ( b) . these results have been corroborated by additional production runs since. under the conditions described, . g griffithsin is expressed per kg plant biomass fw. overall recovery efficiency by the method described is typically ≥ %, or ≥ . g griffithsin/kg fw biomass. to adequately meet the projected initial annual market demand for a rectal microbicidal formulation in the united states, approximately . million doses of griffithsin api at mg/dose would be needed. this translates into a production rate of kg of purified griffithsin api per year. the manufacturing facility to produce the required kg of api per year was assumed to segregate production operations into two broad categories; namely, upstream production and downstream recovery and purification. to accommodate a large number of plants, the facility uses a vertical (layered) cultivation design with integrated irrigation and runoff collection system. each rack is compatible with an integrated transportation infrastructure to move each tray to the next phase of the growth cycle. the upstream portion of the facility houses unit operations for n. benthamiana propagation, inoculation with tmv vector, and griffithsin protein expression and accumulation. these processes begin with seeding and end when the biomass is taken to harvest. the downstream portion of the facility begins at harvest and continues through purification of the griffithsin ds. upstream processing is assumed compliant with good agricultural practices (gap), whereas downstream processing is subject to fda current good manufacturing practice (cgmp). the general layout of the upstream growth rooms was adapted from holtz et al. ( ) , and includes one germination chamber for seeds, one pre-inoculation room for biomass growth, and an isolated post-inoculation chamber where n. benthamiana inoculated with tmv expresses and accumulates griffithsin. all plant growth was modeled to occur indoors using a vertical rack system with hydroponic irrigation. plants are arrayed in equally sized trays under light-emitting diode (led) light systems tuned to the optimized photosynthetic absorbance spectrum of n. benthamiana (composite blue/red spectrum: % ± nm wavelength/ % ± nm wavelength; holtz et al., ) and are continuously illuminated. the plants are rooted in rock wool cubes held in the trays by polystyrene foam floats and perfused with a nutrient solution (the components of which are listed in supplementary table in supplementary material). hydroponic irrigation is on a -h cycle and is accomplished via nutrient film technique (holtz et al., ) . we modeled a hydroponic system because the nutrient solution is recycled; hence, water is conserved, and fertilizer runoff is reduced although not eliminated. the mass of nutrient solution taken up by the plants, the cost of the nutrient solution per liter, and the mass of residual nutrient solution that goes to the wastewater treatment system are shown in supplementary table in supplementary material. to ensure consistency of the nutrient solution, all water was assumed to be treated by reverse osmosis (ro) with solution-monitoring for proper ph and dissolved solids content. a semi-quantitative environmental health and safety assessment was conducted by determining the hazardousness and mass of input materials used in the described upstream and downstream manufacturing operations as well as the hazardousness and mass of waste products generated. the method is referred to as "semi-quantitative" because the amounts of input and output components are quantified from superpro, but the "hazardousness" of each component is determined from the properties of the component (e.g., thermophysical properties, material safety data sheets, national fire protection association (nfpa) ratings, etc.) per three qualitative classifications followed by assignment of a numerical value based on the classification (see biwer and heinzle, ) . the three phases of plant growth (i.e., seed germination, seedling growth and development pre-inoculation, and post-inoculation maturation) require a total batch time of days in the upstream portion of the facility. due to the protracted and continuous nature of plant cultivation, the upstream portion of the facility contains multiple concurrent batches staggered at different stages of growth. when one batch graduates to the next step of production (every . days), the trays containing the batch's biomass are cycled out and the corresponding rack space is immediately filled with a new rotation of trays. we divided the -day growth period into concurrent batch periods, with one batch ready to enter downstream purification every . days. table is a summary of the number of plants, trays and batches that comprise the upstream facility at any given moment. for model building, batch schedules were calculated under the initial assumption of / operation for days per year. plant uptake of nutrients and growth were assumed to be linear reaching g fw per plant at viral inoculation and then increasing in mass to reach g fw per plant at harvest. a % failure rate of tmv inoculation was assumed (pogue et al., ) . the griffithsin expression rate was fixed at . g/kg fw the results generated by the software for the upstream operations are shown in figure , with scheduling shown in the equipment occupancy chart in figure . the following descriptions elaborate on the schema presented in each figure. griffithsin recovery and purification was modeled as a batch process in a facility with an available operating time of days a year for h a day and days a week. in each year, there are batches total to produce kg of purified griffithsin api. since the recovery and purification process only takes . days, the downstream facility has a significant down time of . days between batches. overall, each batch requires . days from seed planting to formulating the final product, with days upstream and . days downstream. in figure , the upstream processes are dictated by concurrent batches (represented by generic boxes) with each batch being . days apart from each other. a batch basis of . days was chosen to decrease equipment idle time and thereby increase downstream equipment utilization efficiency. despite the . -day batch period and a -day operating year, in the model the batch time upstream was reduced to approximately days and the operating year was increased to days to reach the desired batches per year. this was done because superpro reproduces uniform results for each year. the goal of the upstream process operations is to produce sufficient biomass to enable isolation of kg griffithsin per annum. the modeling results show that each batch would produce kg of biomass containing g of griffithsin, assuming an expression yield . g api/kg fw biomass (fuqua et al., b) . because induction was modeled using infection with recombinant tmv vector, the three main phases in upstream are germination, pre-inoculation, and postinoculation. the duration of the phases in the model are days, days, and days, respectively. each batch of n. benthamiana plants goes through a germination phase of days and the germination room is designed with a capacity to grow the , plants necessary to reach the production goal. this step of the process uses germination trays, each holding about plants, distributed among batches in the germination room. after days post germination, the n. benthamiana plants are transplanted to a lower density to enable further growth. thus, seedlings from one germination tray are transplanted into three grow trays (with plants per grow tray), meaning that there are three times the number of trays in pre-and post-inoculation, individually, than in germination. the plant density is plants per m in the germination trays and plants per m after transplantation. in practice, during transplantation each plant will spend only a few minutes away from its growth environment to minimize transplant shock and undue stress. in the model, the overall time was overestimated to be h to accommodate other necessary procedures, such as moving the plants back to the tray stacks. the transplanted trays are relocated to pre-inoculation rooms that are designed to accommodate the increased area from transplanting for ∼ days. the pre-inoculation room contains batch, each containing trays with plants per tray. recombinant tmv for inoculation is produced in and isolated from n. benthamiana. the plant growth model is the same as the rest of n. benthamiana plants. by using infected plants and the purification model defined by leberman ( ) , mg of pure tmv per gram of infected plant material can be recovered (leberman, ; bruckman et al., ) . each batch is equivalent to , plants distributed on trays. less than microgram of tmv virion is needed to inoculate each plant (pogue et al., ) . thus, approximately . mg of tmv is needed per batch and the necessary amount of tmv to inoculate a batch can be produced from a single n. benthamiana plant. multiple batches of tmv solution can be made simultaneously and stored at − • c (fuqua et al., b) . tmv production can be done at lab scale and equipment, labor and material costs are negligible (∼$ , ) compared to the overall cost of plant maintenance. the isolated tmv is incorporated in diatomaceous earth buffer solution at a concentration of micrograms per . ml of diatomaceous earth buffer solution, which contains % by volume diatomaceous earth and % by volume of sodium/potassium-based buffer (pogue et al., ) . the selected inoculation volume of . ml is a safe middle value from the range suggested in the literature (e.g., - ml, pogue et al., ). in the model, the estimated mixing and transfer time for the solution is h, which starts at the beginning of post-inoculation, so the plants and solution enter the same stage together. a forklift is used to transport the plants into the inoculation room. the plants are inoculated with the diatomaceous earth buffer solution described above with a high velocity spray. inoculation machines are often custom made and consist of a conveyor traveling through an enclosed cylinder equipped with high pressure spray nozzles aimed at the plants' aerial structures. once the inoculation is complete, the trays are conveyed to the post-inoculation growth room, which is similar in design to the pre-inoculation growth room; the main difference being its size. the post-inoculation room contains batches at any given time for a total of trays with plants per tray. the scheduling of batches is summarized in the equipment occupancy chart in figure . as shown, seeding, germination, transplant, pre-inoculation, inoculation, and post-inoculation occur sequentially, and the batches are staggered by . days. the downstream unit operations developed in superpro are shown in figure , with scheduling summarized in the equipment occupancy chart shown in figure . the following descriptions elaborate on the schema presented in each figure. at the end of each . day growing rotation cycle upstream, one batch of n. benthamiana plants is ready to be transferred to downstream processing. this is done by placing each tray of plants onto a conveyor system which leads them to the first phase of downstream operations. the matured plants are first harvested for the green biomass from which the majority ( %) of griffithsin can be recovered with a single extraction. additional griffithsin could be recovered from fibrous material by reprocessing (o'keefe et al., ) and from roots (which are not harvested); however, reprocessing was not included in this model. the automated harvester processes the kilograms of biomass at a rate of kilograms of biomass per hour. with an operational buffer time of h, this process is thus expected to take h. as the biomass is processed by the harvester, it is directly fed into a shredder which further comminutes the biomass to improve griffithsin recovery. the shredder operates at a capacity of kg of harvested biomass per hour for . h. the shredded biomass is then mixed with an extraction buffer in a buffer addition tank. for every kilogram of plant material, l of extraction buffer is added. thus, for kg of n. benthamiana in a batch, approximately l of extraction buffer are added. the resultant solid-liquid mixture has a total volume of about , l and is sent through a screw press, which is represented as a generic box in the model. the screw press separates the solidliquid slurry leaving a main process fluid stream of plant extract and a waste stream of biomass. the extract solution contains griffithsin as well as the host and viral protein impurities. a loss about % of the original starting griffithsin was modeled assuming it to be non-liberated from the homogenized biomass. the removal of the biomass leaves a main process stream that contains about l ( kg/batch). to facilitate the aggregation of proteinaceous impurities, the extract solution is transferred into a mixing tank and heated to • c for min. the mixture is passively cooled and simultaneously transferred out of the tank and fed into the first . µm plate-and-frame filter. the extract solution is filter-pressed at - psig to remove the aggregated protein impurities. filtering has a process time of h and requires a filter area of m to handle the kg/batch of the process stream. at this stage, the process loses a further % of the griffithsin but removes all the rubisco (ribulose- , bisphosphate carboxylase/oxygenase) and % of the tmv coat protein impurities. the filtrate from this step is transferred to a second mixing and storage tank, mixed with bentonite clay and magnesium chloride, and stored at • c for a -h period. this stage is the bottleneck operation for the downstream process. after the -h incubation, the solution is filtered through a second . µm filter press and a . µm inline sterilizing filter. these operations remove the remaining protein impurities leaving a griffithsin extract with greater than % purity but at the cost of losing % of the griffithsin. the second plate-and-frame filter has a filter area of about m and will process all of the extract in h. there is approximately g of griffithsin per batch at the end of the filtration phase. following the filtrations steps, the griffithsin extract solution is collected in a storage tank and further purified using an axichrom column with capto mmc resin to remove residual color and potential non-proteinaceous impurities. to accommodate the g of griffithsin in solution, . l of mmc bed resin is needed at a mg/ml binding capacity (per product specification sheet). the order of the operations for this chromatography step are: equilibrate, load, wash, elute, and regenerate. in total, chromatography requires h with the load step taking the longest, at h, because approximately l of solution are processed. chromatography is necessary to decolorize the extract at the expense of losing % of the griffithsin, giving a remaining griffithsin mass of g per batch. the l of eluant process fluid is sent through a viral clearance filter and transferred into a pool/storage tank. subsequently, the extract is sent through an ultrafiltration/diafiltration cycle to remove salts introduced in the chromatography column. after ultrafiltration, the product is transferred into a storage tank to be mixed with the final formulation components. the concentrated griffithsin is diluted to give a concentration of g/l griffithsin in mm na hpo , . mm kh po , . mm kcl and mm nacl at ph . . the final volume of the ds is l per batch. as shown by figure , each batch in the downstream requires h of process time which includes all sip and cip operations. as batches move from the upstream portion of the facility every . days, the remaining time left over in the downstream is set as slack time in the model that may be dedicated toward repair, maintenance, etc. the assumptions and results developed in superpro were used to calculate the economics of the process described. table shows the total operating costs segregated individually for upstream and downstream components. figure displays process category cost contributions graphically, including percentages of total costs. in upstream operations, the largest cost components are utilities ($ , ) and labor ($ , ), representing % and % of total upstream costs, respectively. in downstream operations, labor-dependent costs ($ , ) are the highest contributors at % of total downstream costs, followed by consumables ($ , ) at % of total downstream costs. overall, the upstream component represents nearly % of the total griffithsin production cost, which is calculated as just over $ /g protein. for a microbicide dose of mg, the per-dose manufacturing cost is $ . , excluding any cmo fee. an environmental health and safety assessment was also conducted for this case study following the method of biwer and heinzle ( ) and the results are found in supplementary tables - in supplementary materials. overall, the process uses chemicals that are not harmful to people or the environment, as can be seen by the low magnitude of input and output environmental factor values (typically less than . on a - scale) in supplementary table . the biggest causes for concern (based on the environmental indices) are tmv in the residual biomass, and sodium hydroxide and phosphoric acid used in clean-in-place operations, if released to the environment; however we included costs for a thermal or chemical deactivation step for the tmv-contaminated biomass and ph neutralization for the acid and base cleaning agents which would eliminate the environmental impact of these components. it should also be noted that the upstream nutrient compounds can be more efficiently recycled to increase nutrient utilization by the plants and reduce water/soil impact. waste compounds in the downstream process are disposed of through wastewater and biowaste treatment. an aggregate disposal cost of $ . per liter of non-tmv-contaminated aqueous streams and $ . per kg of biowaste is assigned in superpro for expenses related to wastewater disposal and thermal/chemical deactivation of biowaste streams. compounds introduced during or after the post-inoculation step in the upstream facility are considered as biowaste since they may contain tmv. this includes spent nutrient solution in the post-inoculation step and retentate streams from plate-andframe and dead-end sterilizing filtration skids. disposal of tmv-contaminated materials poses low environmental risk. there is extensive industrial experience in disposing of tmvcontaminated materials, which can be rendered non-infective by treatment with bleach, heat or detergents, diluted and disposed of as municipal waste (pogue et al., ) . the facility modeled can annually produce kg of the potent antiviral griffithsin for use in microbicide products. the host used in our modeling was nicotiana benthamiana. this species was selected because of its aforementioned productivity, but also because our previous report on technoeconomic modeling of nicotiana-produced therapeutic and industrial products nandi et al., ) prefaces the work reported herein. in addition, the use of nicotiana for production of clinical trial materials is also familiar to fda and other regulatory agencies, thus facilitating nicotiana's acceptance in regulation-compliant manufacturing (streatfield and howard, ; mccormick et al., ; bendandi et al., ; tusé, ; gleba et al., ) . the api is manufactured in the host nicotiana benthamiana using tobacco mosaic virus (tmv) as the expression vector. the upstream plant growth and griffithsin production operations are adapted from the facility layout detailed by figure | upstream and downstream cost contributions by process category (units in $ ). holtz et al. ( ) . over , plants are housed in vertically stacked hydroponic grow racks, fitted with high-efficiency led lights. the environment is controlled and monitored for compliance with good agricultural practices (gap). each batch of , plants grows over the course of days and yields a total of kilograms (fresh weight) of biomass. ninety-five batches are seeded and grown annually, with one batch reaching harvest every . days. the downstream griffithsin extraction and purification process is scaled up from the pilot industrial scale process presented by fuqua et al. ( b) . an expression rate of . grams of griffithsin per kilogram of biomass (fresh weight) and a downstream recovery of % were used in the base case and give a combined yield of . grams of griffithsin per kilogram of harvested biomass. sterile filtration and cip/sip systems facilitate compliance with cgmp guidelines. downstream processing commences upon the completion of an upstream batch and takes . h. the stable final formulation is > % griffithsin as the api with negligible endotoxin levels. in the model, the upstream costs account for nearly % of the total cost of griffithsin production. containing both upstream and downstream losses of the protein could significantly reduce cogs. approximately % of the protein api is non-liberated from the homogenized biomass (reprocessing was not modeled) and % is lost during downstream polishing steps. based on the data and assumptions employed in the current analysis, the unit production cost of griffithsin is estimated to be $ . per dose ( milligram). the model was based on published designs for a commercialscale facility and pilot-scale data on griffithsin production adapted to the facility described. this type of modeling is useful for determining ranges of api selling price, production capacity and expression level requirements for commercial supply and profitability. in this study we modeled the manufacturing of griffithsin through a contract manufacturing organization instead of a greenfield build of a new facility because we assumed that that would be the most prudent approach to launching a new product. if the product manufactured using the process modeled is used directly as a vaginal rinse or rectal enema, the additional costs post manufacturing would include transportation, storage, insurance, distribution, marketing, etc., none of which were modeled in this manufacturer-level analysis. if the drug substance produced via the process analyzed is further formulated (e.g., as the api in gels, suppositories, or condom additives), or used as a component of another device (e.g., vaginal ring), those costs and other product-specific costs would be additive and were also excluded from our manufacturer-level analysis. the cost of goods calculated by the current model reflects the manufacturer's cost of production. we are less certain about the wholesale price of the drug because there is no standard "offthe-shelf " profit margin that can be added to toll manufacturing cost to arrive at a standardized answer. often scale up to commercial launch volumes of a product requires additional process development and optimization, validation batches, etc., which lead to negotiated transfer prices depending on volume, duration of engagement, license fees, export duties, and other factors, all of which would impact the cost of bulk griffithsin. nevertheless, for this discussion we assumed a manufacturer's fee of % of cogs for a total production cost of bulk griffithsin drug substance of $ . /dose. additive formulation, storage, distribution, insurance, marketing, sales margins and other costs could lead to a consumer-level use cost of $ - /dose (i.e., ∼ to -times the production cost and < to times the price of a male condom, which varies widely depending on material, features and quantities purchased). this technoeconomic analysis emphasized griffithsin's use in microbicides because such products arguably represent the most price-constrained applications of this new drug. we cannot define the target retail price of a griffithsin microbicide; there is no market reference price for microbicides since no commercial microbicides yet exist. for perspective, the user cost of a griffithsin microbicide can be benchmarked against pre-exposure prophylaxis (prep) with traditional male condoms and prep with microbicides containing antiretroviral (arv) drugs as a newer alternative. analyses have been conducted on the cost of prevention modalities and the cost savings to the healthcare system enabled by preventing hiv transmission, with prevention being far more cost effective than treatment in most scenarios (e.g., pretorius et al., ) . walensky et al. ( ) conducted an analysis of the cost-effectiveness of a tenofovirbased prep microbicide in south african women. in their cost modeling of a vaginal gel, they multiplied the product cost of $ . /dose times (product must be applied twice, pre-and postintercourse) and by . (average sex acts per woman-month) to arrive at a product use cost of approximately $ /womanmonth. however, the price of the microbicide gel used in the study was assumed and region-adjusted and hence pricing in other countries may be different. terris-prestholt et al. ( ) estimated tenofovir gel prices of $ . - . per dose, provided that the gel was used in combination with a condom ($ . - . each; planned parenthood, ), from which an additive cost of use (single-use condom plus double-dose microbicide gel) of $ -$ /person-month can be derived. assuming the same average use rate ( . applications/month) of a griffithsincontaining microbicide applied singly without a condom and priced at $ . -$ . per dose, the cost of use would be $ -<$ /person-month. whether a higher cost of use discourages adoption of griffithsin-based microbicides by men and women remains to be shown. a market study by darroch and frost ( ) of the alan guttmacher institute consisted of detailed interviews of a cross-section of , sexually active women aged - in the continental united states. their statistically rigorous survey identified levels and predictors of women's concerns about stds (including hiv transmission) and interest in microbicides, as well as their preferences regarding method characteristics and likelihood of usage versus price of product, with survey sample results extrapolated to the national level. the results showed that of the estimated . million women aged - interested in microbicides and concerned about stds, including hiv, . million ( %) would still be interested in the method even if it were not % effective, and . million ( %) would remain interested even if the microbicide did not protect against stds other than hiv. the same study found that women's predicted use of a microbicide was affected by price, but interest was still high at $ per application, or roughly up to -times the average price of a male condom. the survey concluded that more than seven million sexually active women in the usa would be interested in a vaginal microbicide even if the product only protected against hiv, was only - % effective and cost them $ per application (darroch and frost, ) . that conclusion was arrived at in ; the $ per application cost back then would be $ . in . one can conclude from these results that there is interest in effective yet inexpensive, self-administered hiv and std prevention modalities even if such products might cost more than conventional prevention methods. the darroch and frost analysis was conducted nearly years ago, and the interviews were limited to women practicing vaginal intercourse. to our knowledge, a more recent study linking likelihood of product use and price sensitivity has not been conducted, or at least not reported, to include other populations of potential microbicide users such as heterosexual couples practicing anal sex or gay men practicing unprotected rectal intercourse. nevertheless, the study established an initial price point and price sensitivity for potential users of microbicides in the usa. griffithsin has a broader spectrum of antiviral activity than hiv-specific prep agents, including activity against hsv- and hcv, which are co-transmitted with hiv- (meuleman et al., ; nixon et al., ) . hence, griffithsin might command a higher price due to its broader antiviral activity and its potential to obviate prevention and treatment costs for co-transmitted viruses. in the usa, the cost of the oral prep drug truvada (emtricitabine and tenofovir disoproxil fumarate) ranges from $ , to over $ , per month (https://www.goodrx.com/ truvada) for the uninsured, but treatment is typically covered by insurance with user co-payments of $ -$ per month. so even if a griffithsin-containing microbicide sold for $ per application (e.g., $ per -use pack), a user of packs per month would pay $ for the microbicide, which is in the range of prep, with the potential added benefit of controlling co-transmitted viruses. consumers in wealthier economies might be receptive to microbicides costing $ - or even more per dose; however, consumers in lesser-developed economies might find $ - /dose to be prohibitive. hence, absent subsidies, there exists a continuing need to lower cogs for apis such as griffithsin. we can conclude that a cogs of <$ . /dose of griffithsin ds as determined in this study, and an estimated user cost of $ - /dose, might enable at least some simpler formulations of the drug (e.g., rinses or enemas) to be economically marketed. for more complex formulations and delivery systems, or for higher doses of the drug, lower cogs for bulk griffithsin would be desirable. the environmental assessment of the plant-based production of griffithsin indicates low impact, particularly if the plant nutrient solutions are recycled in a hydroponic system and if waste streams containing tmv are treated in a biowaste heat or chemical treatment process. the assessment method used, although semi-quantitative, utilizes mass input and output stream data generated by superpro, along with independent assessment of compound toxicity and/or environmental impact (for example using material safety data sheet information), and allows comparison between alternative production strategies, process configurations or chemical components used in the manufacturing process. our low environmental impact assessment for plant-based manufacturing should compare favorably with fermentationbased approaches to producing griffithsin (giomarelli et al., ) . in the latter, the complexities of purification suggest less efficient utilization of materials and higher disposal volumes, although a side-by-side environmental analysis between the two platforms was not conducted in this study. upstream, griffithsin expression rates were based on empirical findings using tmv whole virion as the expression vector, which can achieve typically . - . g griffithsin/kg plant biomass (fuqua et al., a ). an average pilot-scale expression rate of . g/kg was used in our model (fuqua et al., b) . although this expression level is quite good for tmv, higher griffithsin expression levels can be achieved with different technology. for example, nomad bioscience gmbh (halle, germany) has achieved griffithsin expression in n. benthamiana exceeding . g griffithsin/kg fw biomass using nomadic tm agrobacterial vectors applied to plants either through vacuum infiltration or agrospray (hahn et al., ) , albeit these results were obtained in small-scale studies. the utilization of such an induction process instead of tmv virions could further improve process economics. for example, even with the same recovery efficiency of % assumed in the current model, the output of griffithsin at the higher expression level would be . g api/kg plant material, instead of the current . g/kg; this represents more than . times the modeled output of protein per kg biomass. under such conditions, the costliest parts of the current process, namely biomass production and upstream procedures, would be lowered by the reduced biomass needs to produce the required kg/year of api. although a full analysis of the cost of agrobacterial inoculation for griffithsin production needs to be conducted, we know from similar analyses (e.g., nandi et al., ) that economics can be favorably impacted by higher expression efficiencies. we can therefore envision that by using a more efficient induction process the per-dose production cost could be less than the current $ . . still other gene expression methods can be considered, including using transgenic plants expressing griffithsin either in constitutive or inducible systems (werner et al., ; gleba et al., ) , which could also lead to higher api accumulation in host plant biomass and potentially lower cogs . increasing expression yield upstream might shift costs to downstream operations to handle process streams with higher concentrations of api. definition of the comparative cost benefits of these improvements relative to the current process modeled awaits a subsequent evaluation. from a process standpoint, improvements in the efficiency of lighting technologies and/or incorporating solar panels would reduce upstream utilities costs, one of the major contributors to the upstream operating costs. improving hydroponic nutrient utilization through recycling and minimizing runoff in the simulation model will reduce raw material costs as well as aqueous waste disposal costs, thereby reducing the cogs. in the downstream portion of the process consumables play a major role, particularly dead-end filters and plate-andframe filters; if these could be replaced with tangential flow filtration systems that utilize reusable, cleanable ceramic filters, downstream operating costs could be further reduced. at the time of this writing, such systems were being considered and their impact on griffithsin cogs will be the subject of a future analysis. aa, lj, gk, ks, sn, and km contributed to the conception and design of the study. aa, lj, gk, and ks conducted initial modeling calculations, provided information for supplementary tables - , organized preliminary results and prepared an initial report on the findings. dt, jf, and km provided additional data inputs and further refined the scope of the model. km developed the final superpro model and tea results. dt wrote initial and final drafts of the manuscript, including the cost-of-use analysis and consumer price sensitivity discussion, with primary editorial input from jf, kp and km. kp provided critical reading of the manuscript. all authors contributed to manuscript revision, read and approved the submitted version. the participation of aa, lj, gk, ks, sn, and km in this project was supported by the university of california, davis; however, the model developed, results presented, and outcomes of this study are the personal views of independent authors. the participation of dt, jf, and kp was supported in part by us niaid u program project grant no. u ai and funds from the helmsley charitable trust. activity of and effect of subcutaneous treatment with the broad-spectrum antiviral lectin griffithsin in two laboratory rodent models rapid, high-yield production in plants of individualized idiotype vaccines for non-hodgkin's lymphoma environmental assessment in early process development biodistribution, pharmacokinetics, and blood compatibility of native and pegylated tobacco mosaic virus nano-rods and -spheres in mice women's interest in vaginal microbicides production of antibodies in plants: status after twenty years process simulation for recombinant protein production: cost estimation and sensitivity analysis for heparinase i expressed in escherichia coli process and economic evaluation of the extraction and purification of recombinant beta-glucuronidase from transgenic corn bulk production of the antiviral lectin griffithsin improving the large scale purification of the hiv microbicide, griffithsin recombinant production of anti-hiv protein, griffithsin, by auto-induction in a fermentor culture plant viral vectors for delivery by agrobacterium a novel and fully scalable agrobacterium spray-based process for manufacturing cellulases and other cost-sensitive proteins in plants commercial-scale biotherapeutics manufacturing facility for plant-made pharmaceuticals the isolation of plant viruses by means of "simple" coacervates. virology plant-produced idiotype vaccines for the treatment of non-hodgkin's lymphoma: safety and immunogenicity in a phase i clinical study griffithsin has antiviral activity against hepatitis c virus isolation and characterization of griffithsin, a novel hiv-inactivating protein, from the red alga griffithsia sp techno-economic analysis of a transient plantbased platform for monoclonal antibody production griffithsin protects mice from genital herpes by preventing cell-to-cell spread broad-spectrum in vitro activity and in vivo efficacy of the antiviral protein griffithsin against emerging viruses of the family coronaviridae scaleable manufacture of hiv- entry inhibitor griffithsin and validation of its safety and efficacy as a topical microbicide component how do i get condoms? how much do condoms cost making an ally from an enemy: plant virology and the new agriculture production of pharmaceutical-grade recombinant aprotinin and a monoclonal antibody product using plant-based transient expression systems evaluating the cost-effectiveness of pre-exposure prophylaxis (prep) and its impact on hiv- transmission in south africa heterologous sequences greatly affect foreign gene expression in tobacco mosaic virus-based vectors the flow of money through the pharmaceutical distribution system plant-based vaccines cost-effectiveness of tenofovir gel in urban south africa: model projections of hiv impact and threshold product prices evolution of plant-made pharmaceuticals safety of plant-made pharmaceuticals: product development and regulatory considerations based on case studies of two autologous human cancer vaccines manufacturing economics of plantmade biologics: case studies in therapeutic and industrial enzymes the cost-effectiveness of pre-exposure prophylaxis for hiv infection in south african women high-level recombinant protein expression in transgenic plants by using a double-inducible viral vector economic and environmental assessment of the purification of alpha- -antitrypsin from transgenic plant cell suspension cultures the authors thank trena tusé of intrucept biomedicine llc for critical reading of the manuscript and for document formatting in compliance with editorial guidelines.superpro designer is a trademark of intelligen inc. bpg, capto, and sepharose are trademarks of ge healthcare limited. sartopore is a trademark of sartorius-stedim biotech gmbh. truvada is a trademark of gilead sciences, inc. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fbioe. . /full#supplementary-material key: cord- -hcf gsv authors: lin, k.h.; chen, l.f.o.; li, s.d.; lo, h.f. title: comparative proteomic analysis of cauliflower under high temperature and flooding stresses date: - - journal: sci hortic (amsterdam) doi: . /j.scienta. . . sha: doc_id: cord_uid: hcf gsv high-temperature and waterlogging are major abiotic stresses that affect the yield and quality of cauliflower. cauliflower cultivars ‘h ’ and ‘h ’ are tolerant to high temperature and flooding, respectively; however, ‘h ’ is sensitive to both stresses. the objectives of this study were to identify the proteins that were differentially regulated and the physiological changes that occurred during different time periods in ‘h ’, ‘h ’, and ‘h ’ when responding to treatments of flooding, °c, and both stresses combined. changes in the leaf proteome were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (maldi-tof-ms) and identified by mascot peptide mass fingerprint (pmf) and database searching. stress treatments caused significant reductions in electrolyte leakage, chlorophyll fluorescence fv/fm, chlorophyll content, and water potential as stress times were prolonged. by the comparative proteomic analysis, protein peaks that were differentially expressed in response to combination treatments at , , and h, ( in ‘h ’, in ‘h ’, and in ‘h ’) were identified, of which were cultivar specific. differentially regulated proteins predominantly functioned in photosynthesis and to a lesser extent in energy metabolism, cellular homeostasis, transcription and translation, signal transduction, and protein biosynthesis. this is the first report that utilizes proteomics to discover changes in the protein expression profile of cauliflower in response to heat and flooding. cauliflower (brassica oleracea var. botrytis), a member of the brassicaceae family, is an economically and nutritionally important cole crop. the optimum mean temperature range for growing cauliflower is - • c. high temperatures cause cauliflower to form uneven and loose heads, puffy buds, yellow eyes and leaves in the head, narrow leaves, reduced leaf growth, delayed initiation of heading, and increased petiole-to-blade ratio, all of which lower the quality of or even make the heads unmarketable (wurr et al., ; nowbuth and pearson, ) . however, heat-tolerant cultivars are able to form heads at mean temperatures higher than • c. to expand the area of food production, crops are grown under stressful environments that likely lead to lower yields. the main contributing factors in the reduction of yield and quality in these areas are variations in climatic conditions such as flooding caused by rains (lin et al., ) . heavy rainstorms and standing water can leave soils saturated for days before draining, making waterlogging a problem in many parts of the world. air pockets in the soil become filled with water during saturation, thus creating hypoxic conditions followed by anoxia. flooding causes oxygen starvation, which is a consequence of the relatively slow diffusion of gases in water and from oxygen consumption by plant roots (takeshi and julia, ) . rapid leaf chlorosis is seen when cauliflower is subjected to waterlogging (shih et al., ) . flooding from large rainfall events is a major risk to fresh-market cauliflower production in taiwan, and most cauliflower cultivars are unable to tolerate flooding during typhoon-caused heavy summer rains. waterlogging and increasing temperatures associated with global warming are a growing concern, as they limit plant growth and productivity, especially in temperate species. over the past decade in the tropics and subtropics, there have been considerable increases in production because of the availability of new, tropically adapted cultivars, resulting in increased farmer incomes. in the field, the co-occurrence of several abiotic stresses rather than individual stresses are most damaging to crop production (mittler, ) . many physiological changes occur during high temperature and flood stressing that result in increased heat and flood http://dx.doi.org/ . /j.scienta. . . - /© elsevier b.v. all rights reserved. (hf) tolerance. the physiological mechanisms of hf tolerance are extensively studied in various plant species; however, our current understanding of the molecular biology of acquired tolerances to high temperature and waterlogging are still limited, and relatively little is known about proteins that are critical for controlling these dual mechanisms (wahid et al., ) . understanding these processes requires the identification and analysis of major proteins that underlie stress-regulatory networks. plant adaptations to environmental stresses depend on the activation of cascades of molecular networks involved in stress perception, signal transduction, and the expression of stress-related proteins. knowledge of hf-responsive proteins is critical for further understanding of the molecular mechanisms of stress tolerance. plants respond to stress in part by modulating protein regulation, which eventually leads to restoration of cellular homeostasis, neutralization of toxins, and recovery of growth. however, there are no reports on the effect of hf stresses on the functioning of cauliflower. proteins are the direct effectors of the response of plants to stress. they not only include enzymes that mediate changes in the levels of metabolites but also serve as components of the transcription and translation machinery. in recent years, methods for analyzing the proteome have advanced considerably, and together with emerging sequence information in crops, plant proteomics has become increasingly useful for understanding gene functioning and networks in response to environmental stimuli. proteomics is a powerful tool for the quantitative analyses of different biochemical pathways including plant stress-related responses. comparative proteomic analyses of plants subjected to specific stress conditions allow the exploration of various defense-related mechanisms. for instance, proteomics approaches for the comparative analysis of protein abundance between untreated and stress-treated or tolerant and intolerant rice plants have greatly facilitated the study of plant cellular stress responses (komatsu et al., ) . therefore, identifying novel proteins and studying their differential display patterns in response to hf stresses will provide the molecular and physiological bases for improving tolerance to hf by cauliflowers. understanding the basis of hf stress signaling and tolerance mechanisms in cauliflowers is required for engineering local cauliflower genotypes that are more tolerant to heat and flood stressing. this goal can be achieved by deciphering the physiological and proteomic responses of cauliflower genotypes, heat-tolerant 'h ', flood-tolerant 'h ', and hf-sensitive 'h ' to flood stress at • c. the objective of this study was to identify the leaf proteins that are differentially regulated in response to hf stress using a pro-teomelab pf- d aided by improved databases. our hypothesis was that both hf stresses trigger plant responses that result in quantifiable changes in the proteins of 'h ', 'h ', and 'h '. proteomic characterization and functional analysis should facilitate a better understanding of the hf response mechanisms in brassica so that effective strategies for the genetic improvement of hf-tolerant plant cultivars can be established. to the best of our knowledge, there are no published reports addressing the identification of hfresponsive proteins in brassica using a proteomic approach. . . plant materials, culturing, and heat-and flood-stress treatments seeds of cauliflower (b. oleracea var. botrytis) 'h ', 'h ', and 'h ' were obtained from chin-long seed co. (tainan, taiwan). 'h ' is a heat-tolerant cultivar used especially in warm-subtropical regions such as southern taiwan, where average day temperatures reach as high as • c during the summer (june-august). 'h ' is a popular cultivar grown in taiwan and is flood-tolerant during the rainy season, particularly in rain-fed lowlands. 'h ' is a heat-and flood-sensitive cultivar, and mostly grown during winter in taiwan due to its cold tolerance. seeds were sterilized with . % (v/v) sodium hypochlorite, rinsed with distilled-deionized (dd) h o, and sown in a commercial potting soil mixture, and germinated seedlings were transplanted into . -cm diameter plastic pots and raised in a growth chamber under mol m − s − light with a h photoperiod and / • c day/night temperatures at a relative humidity (rh) of %. plants were watered three times a week and fertilizer ( : : , n:p:k) applied once a week to maintain optimal growth for days before the imposition of stress treatments. pots containing 'h ' and 'h ' plants were subjected to four treatments: non-flooding at • c (nfc, as control), flooding at • c (fc, control temperature), non-flooding at • c (nfh, high temperature), and flooding at • c (fh), for periods of , , , , , , and h in four growth chambers having a h photoperiod at mol m − s − radiation and % rh (lin et al., ) . in the case of flooding treatments, pots were randomly placed in cm × cm × cm plastic buckets and subjected to flooding by filling the buckets with tap water to cm above the soil surface. pots were removed from the buckets at different times following flooding, and plants were removed and their leaves from each plant clipped, frozen in liquid nitrogen, and stored at − • c in an ultrafreezer until used. three replicates of each time period for the four treatments were randomly placed in a growth chamber. the experiment was performed twice independently in a randomized design for growth environment, sampling day, and physiological analyses. . . determination of electrolyte leakage (el), chlorophyll fluorescence (cf), chlorophyll content (cc), and water potential (wp) cell membrane stability was estimated by measuring leaf ion leakage according to the method of huang and guo ( ) . leaves were excised and immersed in ml of distilled water in test tubes overnight at room temperature. the initial conductivity of the water was determined using a conductivity meter (model cdm , radiometer, cedex, france). tubes were placed in boiling water for min and then cooled to room temperature, and conductivity was again determined. the relative el (%) was calculated as the ratio of conductivity before boiling to that after boiling. cf components were quantified with a portable modulated fluorometer (mini-pam photosynthesis yield analyzer, walz, effeltrich, germany). the measurement of variable fluorescence to (fv)/maximum fluorescence level in light-adapted leaves (fv/fm) was previously described (lin et al., ) . relative cc per unit leaf area was determined using a spad (soil plant analysis development) analyzer (spad- chlorophyll meter, konica minolta, tokyo, japan). wp (in bars) was measured on the third leaf from the top of each plant using a pressure chamber (plant water system, skye skpm , tokyo, japan) (sairam et al., ) . the data shown in tables - represent the means of at least two independent sets of experiments with similar results. measurements of physiological parameters were analyzed by a three-factor completely randomized anova that compared cultivars, treatments, and time periods. for significant values, means were separated by a least significant difference (lsd) test at p ≤ . using pc sas . (sas institute, cary, nc, usa). because the negative effects of flood stressing on hf-sensitive 'h ' at • c were observed after h of stress treatments (see section ), 'h ', 'h ', and 'h ' plant leaves subjected to hf conditions for , , and h were used for subsequent proteomics analysis. proteins were extracted according to a previously published method (yan et al., ) with some modifications. briefly, two grams of plant leaves were ground in liquid nitrogen and crude protein extracts solubilized in ml of extraction buffer containing . % sds (sodium dodecyl sulfate), % ␤-mercaptoethanol, % glycerol, . % polyvinylpyrrolidone, and mm tris-hcl, ph . after min of incubation at • c, samples were centrifuged for min at , × g at • c; these two steps were done twice. the extraction buffer was further replaced by ml of start buffer ( mm tris-hcl, ph ) with a pd- column (ge healthcare) according to manufacturer (proteomelab pf- d kit, beckman coulter) protocols. protein concentrations in the samples were determined using the bradford assay (bio-rad, hercules, ca, usa) and bovine serum albumin (bsa) was used to generate a standard curve. a g aliquot of the total protein sample was passed through a . m filter before injection into the chromatography column. the high performance chromatofocusing (hpcf) column was treated according to manufacturer instructions (proteomelab pf- d protein fractionation system, beckman coulter, fullerton, ca, usa). briefly, the column was washed with volumes of water at a flow rate of . ml/min for min and then equilibrated with volumes of start buffer for min at . ml/min. after equilibration, each sample was introduced with a manual injector into the column and absorbance of the column effluent was monitored at nm. in the first dimension, proteins were bound to a strong anion exchanger and eluted with a continuously decreasing ph from . to . . fractions were collected at ph intervals of . in a deepwell plate. proteins eluted in the gradient were then separated in the second dimension using high performance reversed phase (hprp) chromatography. fractions were separated from the second dimension with an rp-hplc column using two solvents: . % trifluroracetic acid (tfa) in hplc water (solvent a) and . % tfa in acetonitrile (acn) (solvent b) (lee et al., ) . separation was performed at • c with a flow rate of . ml/min and protein fractions were detected by uv absorbance at nm. equilibration was achieved with solvent a for min followed by solvent b for min prior to each injection. from the selected first dimension fractions, . ml were injected, run for min, and the column eluted with a linear gradient of - % solvent b for min. thereafter, solvent b was continued for min, followed by re-equilibration with % solvent a for min (irar et al., ) . fractions from the second dimension were analyzed with karat tm version . (beckman coulter). protein extraction and two-dimensional gel electrophoresis ( -de) were carried out on three independent technical replicas of the bulk samples. proteins were in-gel digested with trypsin in the automatic investigator progest robot of genomic solutions. briefly, excised gel bands were washed sequentially with mm ammonium bicarbonate (nh hco ) buffer and acn. proteins were reduced and alkylated, respectively, by treatment with mm dithiothreitol (dtt) solution for h at • c and treatment with a mm solution of iodine acetamide. after sequential washings with buffer and acn, proteins were digested overnight at • c with g/ml of trypsin. tryptic peptides were extracted from the gel matrix with % formic acid and acn, and extracts were then pooled and dried in a vacuum centrifuge. digested peptide solutions were desalted with zip-tip pipet tips (nikkyo technos, tokyo, japan) and redissolved in l of . % tfa in % acn according to manufacturer instructions. proteins excised from two-dimensional gels were analyzed in matrix-assisted laser desorption ionization time-of-flight (maldi-tof, proteomics analyzer, applied biosystems) mass spectrometers. a l aliquot was mixed with the same volume of a matrix solution ( mg/ml ␣-cyano- -hydroxycinnamic acid in . % tfa in % acn) and spotted on a maldi plate. ms spectra were acquired in the positive reflector mode, with shots per spectrum being accumulated. three major peaks were selected for further characterization by ms/ms analysis. ms spectra were acquired using collision-induced dissociation (cid) with atmospheric air as the collision gas, the ms kv positive mode being used. the peak lists for ms/ms spectra from same-replicate samples were merged into a single file prior to database searching. the pkl and mgf files were searched against the swiss-prot database using mascot (http://www.matrixscience.com; london, uk). search parameters were set as follows: missed cleavage, fixed modification, peptide charge ± , and variable modifications were carbamidomethyl of cysteine and oxidation of methionine. trypsin was specified as the proteolytic enzyme. peptide tolerance and ms/ms mass tolerance were ppm and . da, respectively. peptide mass fingerprinting (pmf) match confidence was based on the mowse score and confirmed by accurate overlapping of matched peptides with mass spectrum major peaks. scores greater than (p < . ) were considered positive. peaks with multiple proteins detected by ms were not considered. only significant hits, as defined by mascot probability analysis (p < . ), were accepted. characteristic physiological responses of plants to different temperature and waterlogging treatments were evaluated. table illustrates the comparison of fv/fm values under four treatments at seven different times in leaves of three genotypes of cauliflower. levels of fv/fm in all plants progressively decreased as flood stress and heat stress (nfh and fh) durations were extended. fv/fm in 'h ' plants showed significantly lower values after h with table identification of differentially expressed proteins found in cauliflower 'h ' plants by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (maldi-tof ms) in comparison to combination treatments for and h. peak ( table lists the different points in time that waterlogging and high temperature treatments were monitored by measuring the changes in wp. when different treatments across time were compared, wp with stress treatments was significantly lower (more negative) than those with nfc treatment from to h for all genotypes, indicating that high temperature and/or flooding induced a decrease in leaf water level, subsequently affecting leaf wp. the trends in wp differed in all stressing treatments from to h in all plants. thus, the wp of the different genotypes responded differently to specific stresses. the lowest value in wp (− . mpa) was detected at h with exposure to nfh treatment in 'h '. proteome alterations in the three genotypes in response to short-term exposures to flood and high-temperature conditions were compared relative to treatments and controls. the proteome-lab system uses two-dimensional liquid chromatography based on high-performance chromatofocusing in the first dimension followed by high-resolution reversed-phase chromatography in the second dimension, and has become available for sample fractionation and more resolution at extreme ph values (soldi et al., ; pirondini et al., ) . protein peaks were identified and their expression patterns analyzed. protein fractions were separated according to isoelectric point (pi) and hydrophobicity and detected in a ph range of . - . by proteomelab pf- d. proteins were analyzed by maldi-tof-ms and identified via pmf and database searching by their calculated molecular weight, pi score, and percent coverage, and their database accession numbers and alteration values among stress treatments are represented in tables - . changes in the protein levels of 'h ' exposed to fh stresses at and h and their differentially expressed protein peaks are listed in table . compared to nfc control treatment, two protein peaks ( of phosphoglucosamine mutase and of rotidine -phosphate decarboxylase) under fc treatment for h were up-regulated (supplementary fig. s a) . furthermore, the expression level of s-adenosylmethionine synthetase (peak ) was increased during high-temperature stressing over h (supplementary fig. s b ). two identified peaks, of kda calcium-binding protein and of acetyl-coenzyme a carboxylase carboxyl transferase subunit beta, were also up-regulated when 'h ' plants were treated with fc for h in comparison to nfc treatment at h (supplementary fig. s c) . nine protein expression patterns were detected in 'h ' plants under high-temperature and flood stress at h, and the dynamic changes in the level of each protein are displayed in table . compared to nfc treatment at h, nfh treatment for h showed that the abundances of peaks (phosphoserine aminotransferase), (imidazole glycerol phosphate synthase subunit hisf), table identification of differentially expressed proteins found in cauliflower 'h ' plants by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (maldi-tof ms) in comparison to combination treatments for and h. peak ( (phosphoribosyl formyl glycin amidine synthase ), and (probably hydrogenase nickel incorporation protein hypa) were up-regulated (supplementary fig. s a) . moreover, the abundance of peak (putative holliday junction resolvase) in 'h ' plants was also up-regulated during h of fc treatment as compared to nfc treatment at h (supplementary fig. s b ). the last comparison includes four proteins (peaks , , , and , namely trna-methyltransferase, arginyl-trna synthetase, elongation factor g, and methionyl-trna formyltransferase, respectively) whose abundances were consistently up-regulated during flood stressing for h at • c. interestingly, all peaks were up-regulated after all treatments for h and h in both 'h ' (table ) and 'h ' (table ) plants. the identified proteins showed altered expressions by upregulation or down-regulation in 'h ' plants at the h and h time points when combination treatments were compared (table ) . only six protein peaks ( , , , , , and ; supplementary fig. s ) from comparative proteomic analyses between fh and nfc for h were down-regulated, all others being up-regulated. there were peaks found in fh vs nfc for h, with the remaining peaks being among other comparisons, including fh vs nfh, fc vs nfc, nfh vs nfc, nfh vs fc, and fh at h vs nfc at h. the carbohydrate metabolism related protein, fructose- , -bisphosphatase (peak ), was down-regulated under fh treatment compared to nfc treatment. nevertheless, the photosynthetic-related proteins ribulose bisphosphate carboxylase (rubisco) small chain (peak ) and large chain (peak ) were increased in abundance in 'h ' under flooding for h. additionally, dna mismatch repair protein muts was found in peak with down-regulation (fh vs nfc) and peak with up-regulation (fc vs nfc) in protein abundance through a homologue search. differentially expressed proteins identified in 'h ' plants at h and h under stressing are shown in table . the protein abundances of eight peaks ( , , , , , , , and of ribose import atp-binding protein, phenylalanyl-trna synthetase, rubisco large subunit, gmp synthase, atp synthase subunit, and phosphoribosylformyl glycinamidine synthase) were consistently down-regulated during the fh condition when compared to nfc treatment for h, but the other peaks were up-regulated in protein abundance. mitochondrial inner membrane protease (peak ) and rubisco small subunit (peaks and ) were up-regulated in 'h ' upon fc treatment compared to nfc after h ( supplementary fig. s a ). peaks and , respectively identified as uncharacterized . kda and sec cytosolic factor, were increased under heat stress at h ( supplementary fig. s b) . meanwhile, rubisco small subunit (peak ) was also increased in 'h ' under fh at h as compared to nfc at h ( supplementary fig. s c) . notably, the majority of peaks found in 'h ' at h represented rubisco down-regulation (peaks , , and ) and up-regulation (peaks , , , , , , , , , and ) in protein abundance. because flood and heat often co-occur in stress-prone environments, this study compared the combined effects of flood and heat with those of the single stresses on plant physiology. in general, the changes in plant physiology were greater under the combined treatment than under heating or flooding alone. the differences in responses to flood and heat in fv/fm, cc, el, and wp seen between cultivars suggested that the three genotypes have unique mechanisms for coping with environmental stresses. the heat-tolerant 'h ' genotype showed significantly higher fv/fm, cc, and wp values (tables , and ) and lower el% (table ) than the sensitive 'h ' genotype when heated for h under nfh treatment, which reflects its tolerant nature. the exposure to flooding for at least h under fc treatment provoked greater reductions in fv/fm and cc values in 'h ' plants than 'h ' plants (tables and ). in addition, flooding also significantly decreased the el in 'h ' plants relative to 'h ' plants after - h ( table ), indicating that 'h ' is flood-tolerant. similar losses of fv/fm, cc, and wp and an increase in el were observed in 'h ' under flooding and high temperature stresses (fh), but responded at different levels over time (tables - ) . table identification of differentially expressed proteins found in cauliflower 'h ' plants by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (maldi-tof ms) in comparison to combination treatments for and h. peak ( a soil water potential of approximately − mpa was used in greenhouse experiments to simulate field situations under severe stress conditions (ashoub et al., ) . the leaf wp of 'h ' plants under high-temperature treatment was significantly lower than in 'h ' and 'h ' plants after h, indicating that 'h ' plants suffered from heat exposure. a lower osmotic potential is responsible for maintaining turgor when water loss occurs due to high temperature. thus, high electrical conductivity due to a high concentration of electrolytes in the leaf sap of 'h ' plants should lead to alterations in membrane permeability and a reduced ability to retain solutes and water during high temperature and waterlogging. however, the ability of 'h ' and 'h ' plants to tolerate heat and flood stress, respectively, strongly depends on adjustments in water balance and ionic leakage. a decline in wp and an increase in el during heat and flood stress were associated with leaf water deficits as well as increases in ionic leakage. at the physiological level, the many effects of flooding and heat stresses indicate the importance of protecting plants from oxidative damage caused by the overproduction of ros that is elicited by increased ion leakage (kangasjarvi et al., ) . the chlorophyll fluorescence emission parameter, fv/fm, which is widely used as a proxy for the maximum quantum efficiency of ps ii photochemistry, was correlated with flooding and high temperature tolerance. in healthy leaves, the fv/fm value is close to . , which is a typical value for uninhibited plants. a lower table identification of differentially expressed proteins found in cauliflower 'h ' plants by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (maldi-tof ms) in comparison to combination treatments for and h. peak ( value indicates that some proportion of the psii reaction centers are damaged, which is often observed in plants under stress (camejo et al., ) . flooding and high temperatures inhibit photosynthetic co fixation and damages photosynthetic electron transport at the site of psii where there exists a very sensitive photosynthesis apparatus. this reduction in the co assimilation rate observed in 'h ' plants was generated by effects on the calvin cycle and also on psii functioning. exposure of 'h ' plants to flooding and high temperature may lead to reductions in net photosynthetic rate, stomatal closure, and cell activities that damage photosynthetic membranes (jiang and huang, ) . these results suggested that the chlorophyll fluorescence parameters were stress specific and were not expressed solely in response to an increasing excess of photon energy. chloroplast development in 'h ' plants may be particularly sensitive to high temperature and flooding. alternatively, the pigments of the sensitive cultivar might have been destroyed because of a high sensitivity to oxidative stress. 'h ' plants suffered greater cc losses than 'h ' and 'h ' plants after - h of exposure. flooding and heat may induce stomatal closure and consequently reduce cc levels and wp as well. typically, the amount of cc is reduced by stress. this is consistent with our observations that chlorophyll loss was more pronounced in waterlog-and heat-sensitive 'h ' plants in accordance with the more pronounced and increased visible symptoms of leaf injury. leaf curling or folding were the initial and most obvious changes observed. most 'h ' leaves progressively became necrotic, epinastic, or wilted over the course of time; however, most 'h ' and 'h ' leaves visually appeared to be green and healthy after h of flooding and high temperature (photos not shown). along with visual symptoms, reduced cc could be used to monitor flooding and heat damage in green or senescent leaves. attempts have been made to breed for increased flooding tolerance and modify brassica cultivation or management practices to avoid injury from flooding and heat. environmental stresses represent the most limiting conditions for horticultural productivity and plant exploitation worldwide. important factors among them are water and temperature. flooding and high temperature are major abiotic stresses resulting in serious problems for the growth and yield of flood-and heat-sensitive plants. it is necessary to identify the physiological characteristics that reflect their complex underlying genetic make-up. these easily measured physiological biomarkers could be used as generic tools to develop a reliable method for selecting cauliflower cultivars having flood-and heat-stress resistance. tables - demonstrate that the chlorophyll losses in 'h ' plants were in concert with increasing el accumulations after h of flooding and heat treatments, which is an index of oxidative damages to cell constituents in general. this suggests that fv/fm and cc can be effectively used as reporter signals in screening for flooding and high temperature tolerances. these easily measured physiological biomarkers could be used as generic tools for developing a reliable method to select 'h ' and 'h ' cultivars having flooding and heat-stress resistances. these findings are important for farming in high-temperature areas and wetlands or other areas subject to short and intense rainfall events. proteomics techniques are becoming indispensable for understanding how plants adapt to abiotic stresses, and have promoted the identification of numerous stress-related proteins and the pathways in which they function (kosová et al., ) . changes in protein accumulation under stress are directly related to the physiological phenotypic responses of plants to stress. the proteomic responses to high-temperature or flood stresses are known for several plants, including rice (lin et al., ) , wheat (majoul et al., ) , barley (süle et al., ) , soybean (komatsu et al., ) , radish (zhang et al., ) , arabidopsis thaliana (palmblad et al., ) , agave americana (shakeel et al., ) , and populus euphratica (ferreira et al., ) . in the present study, we analyzed physiological and proteomics data in different genotypes of cauliflower under conditions of high-temperature and waterlogging stresses over different time periods. approximately peaks were detected at the tested time points, whereas peaks were successfully observed to be differentially regulated ( down-regulated, up-regulated) and expressed at a minimum of one time point. among them, , , and proteins were identified in 'h ', 'h ', and 'h ' plants, respectively, indicating that they were regulated in a genotype-specific manner ( supplementary fig. s ). rubisco small and large chains, kda calcium-binding protein, molybdenum cofactor biosynthesis protein a, and phosphoserine aminotransferase were observed in both 'h ' and 'h ' plants. phosphoribosylformyl glycinamidine synthase and arginyl-trna synthetase were both found in 'h ' and 'h ' plants ( supplementary fig. s ). moreover, in 'h ' plants, peaks and were both identified as dna mismatch repair protein muts, while and were identified as phosphoglucosamine mutase (tables and ). these sister peaks may represent close homologues, but this could not be resolved based on mass spectrometric data and might instead be isoforms resulting from differential post-translational modifications (rollins et al., ) . as stress times increased, additional responsive proteins continued to be identified. for instance, (table ) and (table ) peaks were observed in 'h ' under stress treatments for h and h, respectively. in this study, some of the proteins identified were well characterized in terms of response to stressing, while others were not. the biological functions of differentially regulated proteins include roles in photosynthesis, energy metabolism, cellular homeostasis, response to stimuli, transcription and translation, protein biosynthesis, mediation of signal transduction, and other functions. for survival, plants must respond to flood and heat stresses in a different manner from regulating protein expressions for biochemical and physiological adaptations. photosynthesis is one of the systems that is most sensitive to high-temperature stress. changes in environmental temperatures are primarily reflected in photosynthesis, triggering a response that is aimed toward the best possible performance under new conditions. for this, a balance is sought between the energy of absorbed light, carbon assimilation, and consumption by metabolic sinks. several studies have shown that high-temperature stress can significantly inhibit the rate of photosynthesis (yan et al., ; salvucci and crafts-brandner, ) . in our study, rubisco units (small and large chain) related to photosynthesis were differentially expressed and regulated by combination treatments and among genotypes. for example, the up-regulation of rubisco was detected in 'h ' (peaks and , table ) and 'h ' (peaks (peaks , (peaks , (peaks , , , (peaks , . however, the level of expression of the rubisco large subunit was down-regulated in 'h ' during fh treatment for h (peaks , , and , table ). rubisco proteins resist high-temperature and flood stresses by inhibiting photosynthesis and other nonessential metabolic processes. in contrast, to increase energy metabolism, the expression of proteins related to redox homeostasis and response to stimuli were up-regulated, thereby maintaining physiological balance during stress. rubisco catalyzes the first major step of carbon fixation in photosynthesis. affinity of rubisco for co decreases with increasing temperature (yan et al., ) . the diminished capacity of rubisco and its low affinity for co suggest that carbon fixation or assimilation was highly inhibited when 'h ' plants were exposed to stress, especially under the high temperature and flooding stresses ( table ) that lead to reduced rates of photosynthetic co assimilation. photorespiration serves as an energy sink, preventing the over-reduction of the photosynthetic electron transport chain and photoinhibition. lee et al. ( ) reported that high temperature over h significantly inhibits the activity and quantity of rubisco subunits in rice seedlings. the effects of drought stress on rubisco represent reductions in rubisco activity (parry et al., ) . exposure to salt stress brings about a reduction in the overall growth and productivity of plants by disturbing the function of vital components of photosynthesis, such as psii and rubisco (ghaffaria et al., ) . consequently, a strong reduction in fv/fm value (table ) and cc content ( table ) while maintaining photosynthesis under stress after h was seen in all plants. some researchers report its up-regulation (ge et al., ; budak et al., ) , whereas others find downregulation (gaoa et al., ) or even both (guoa et al., ) in response to abiotic stresses. as a result, an up-regulation in rubisco levels may also indicate an increase in the photorespiration rate. molecular chaperones, including heat shock proteins (hsps), are key components of innate immunity in plants. they have major roles in protein folding and signal-transduction networks, cellcycle control, protein degradation, and protein trafficking (wang et al., ) . hsp chaperone pathways require energy in the form of atp hydrolysis in order to function. the atp-dependent clp proteases (hsp ) represent a class of hsp. in addition to their function as molecular chaperones, they function in protein disaggregation and protein degradation when removal of potentially harmful polypeptides arising from misfolding, denaturation, or aggregation are important for the maintenance of cellular homeostasis. the mechanism for rescuing proteins from aggregation is proposed to include the cooperation hsp (kregel, ; sarkar et al., ) . hsps are typically induced when cells are exposed to various types of environmental stresses. the synthesis of hsps under high temperatures is reported to be related to thermotolerance in plants, and plants with a decreased expression of hsps show compromised tolerance to acquired thermo-tolerance (charng et al., ) . hsps are closely related to the acquisition of heat resistance in plants. choi et al. ( ) identify several high molecular weight hsps whose expression was up-regulated significantly in pyropia tenera under high-temperature stress for h and h. furthermore, lee et al. ( ) report that several hsps are expressed in rice leaves during heat stress. high temperature might increase the potential risk of protein misfolding, and an active protein quality control system inside cells plays an important role in plant tolerance to heat stress. hsps are also increased by drought stress in the sugar beet (hajheidari et al., ) , wheat (demirevska et al., ) , and sugarcane (jangpromma et al., ) . in our study, peak of atp-dependent clp protease proteolytic subunit was up-regulated in response to heat stress for h in 'h ' plants (table ) . this suggests that the increased level of hsp under high-temperature stress allowed the heat-tolerant 'h ' to produce more energy through atp hydrolysis and the degradation or reactivation of damaged proteins and prevented protein misfolding, resulting in reestablishing normal protein conformations and cellular homeostasis. s-adenosylmethionine (sam) synthetase catalyzes the synthesis of sam that serves as a methyl group donor in transmethylation of proteins, nucleic acids, and a precursor in the biosynthesis of polyamines, biotin, and nicotianamine in plants (roeder et al., ) . sam in shoots underwent increased protein synthesis in a heat-tolerant barley cultivar and showed a reduced trend in an abiotic stress-susceptible cultivar (süle et al., ) . in our study, the expression of sam synthetase was significantly up-regulated (peak , table ) in heat stressed 'h ' over h. therefore, it is most likely that an increased amount of sam synthetase (as a result of the increased formation of sam methyl donors) in 'h ' contributes to its stronger heat tolerance compared to controls. in addition, peak of adenylate kinase (adk) was observed in 'h ' under heat stress for h (table ) . adk catalyzes the salvage synthesis of adenine monophosphate from adenosine and atp (wang et al., ) . heat and flood treatments markedly increased the abundance of two elongation factors (efs): ( ) elongation factor g was increased in 'h ' (peak ) under flooding at h ( table ) and ( ) transcription elongation factor grea was increased in 'h ' (peak ) under fh at h ( table ). the elongation of polypeptide chains during translation is a conserved process among prokaryotes and eukaryotes. efs are the critical regulators of protein synthesis, which is influenced by high temperature and drought stress in arabidopsis (rizhsky et al., ) and rapeseed (mohammadi et al., ) . regulation of the transcriptional and translational machinery is considered to be an important component of cellular stress response. the increased expression of ef grea in 'h ' and ef g in 'h ' likely led to increased protein synthesis, which served to reduce the effects of treatment stress. plants under stress can respond by sensing and transferring stress signals through signal transduction networks. the response to stress is likely mediated through signaling pathways that regulate the expression levels of a range of proteins (hirayama and shinozaki, ) . in our study, gtp-binding protein lepa (peak , table ) involved in signal transduction was identified. the up-regulation of the lepa protein in response to flooding at • c might reflect the role of this protein in cell signaling under stress. ca + -binding protein is mainly resident in the endoplasmic reticulum where it serves as a calcium modulator and chaperone of newly synthesized glycoproteins (nam et al., ) . the latter have functions in plant growth and development as well as biotic and abiotic stress responses. aghaei et al. ( ) indicated that ca + -binding protein was up-regulated by salt stress in potato leaves. salinity induces ca + accumulation in the cytosol that starts the signaling pathway. the modulation of intracellular ca + levels is regulated by calcium-binding proteins, which, after activation, induce specific kinases (capriotti et al., ) . a kda calcium-binding protein was identified as an up-regulated peak ( ) under flooding (table ) , suggesting the involvement of intracellular calcium homeostasis and signal transduction in 'h ' during waterlogging. atp synthase is a large multi-subunit transmembrane enzyme complex. the catalytic site for atp synthesis is localized primarily on the beta subunit. atp synthase is known to be involved in energy metabolism and was found in 'h ' plants upon fh stressing for h (table ) . down-regulation of the catalytic subunit of atp synthase subunit beta (peak ) suggested that the energy production systems of 'h ' were highly affected by heat and flood stresses. huseynova et al. ( ) also found that the atp synthase complex was less accumulated in drought-sensitive wheat 'giymatli- / ' than in drought-tolerant azamatli- under water stress. in addition, impaired atp synthesis under drought is regarded as a major constraint to photosynthesis (flexas and medrano, ) . fructose- , -bisphosphatase (fbpase) is involved in carbohydrate metabolism during gluconeogenesis, and also an important enzyme in the calvin cycle that plays a crucial role in plant responses to some abiotic stimuli (fan et al., ) . the downregulation of fbpase in 'h ' under fh at h (peak , table ) indicated that gluconeogenesis was inhibited under stressing. an increase in ribosomal protein is a candidate for initiating a translation site, and the phosphorylation of ribosomal proteins is important in the regulation of protein synthesis (fatehi et al., ) . the s ribosomal subunit catalyzes the peptidyl transfer reaction of mrna-directed protein biosynthesis (sobhanian et al., ) . up-regulation of the s ribosomal protein l p (# ) was observed under high temperature stress at h in 'h ' (table ) , indicating the resistance of 'h ' plants to the inhibitory effect of high temperature on protein biosynthesis. in addition, pentatricopeptide repeat (ppr) proteins are characterized by tandem arrays of degenerate amino acid motifs (o'toole et al., ) and are thought to be rna binding proteins involved in posttranscriptional processes in mitochondria and chloroplasts (schmitz-linneweber et al., ) . meanwhile, ppr proteins are also essential for rna editing in chloroplasts (emi et al., ) . peak (pentatricopeptide repeat containing protein) of 'h ' was up-regulated under fh within h ( table ), suggesting that 'h ' was able to increase rna maturation and protein function in chloroplasts under fh. taken together, genetic differences are supported by the high number of proteins differentially regulated between genotypes. our data suggest that the early response of cauliflower to flooding and high temperature might be an important stress adaptation for survival following not only hypoxia and heat, but also direct damage to cells by flooding and high temperature. all of the identified proteins likely work cooperatively to reestablish cellular homeostasis under stress. the long term goal of our work is to help breed a competitively higher flood-and heat-tolerant cauliflower to be grown in lowlands during summer. the identification of the unique stress-responsive proteins will allow further dissection of the genetic basis of this transgressive performance in offspring. our results not only provide information for selecting lines having better tolerance to waterlogging and heat stresses, but also provide a basis for understanding cauliflower metabolic pathways and their cross-talk under stress. further verification of the correlative stress-responsive protein by gene expression analyses of these stressed-responsive genes, such as rubisco, efs, hsp , fbpase, adk, atp synthase, sam synthetase, ppr protein, gtp-binding protein lepa, ca + -binding protein, and ribosomal protein l p, would facilitate our understanding of the heat-and flood-response mechanism in cauliflowers. this study provides information on mechanisms underlying the contrasting responses to hf stresses. different genotypes showed differing physiological responses to combinations of stress treatments. all dynamics of the peaks were analyzed across all treatments, and a comparative analysis identified stressresponsive proteins in cauliflower under short-term stressing. the majority of these stressed-responsive proteins were genotype specific. these identified proteins emerged as key participants in stress tolerance. most of the changes in the expression levels of these proteins in response to stressing were involved in photosynthesis. 'h ' plants resisted high-temperature stressing by inhibiting photosynthesis and other unnecessary metabolic processes. energy metabolism was increased and up-regulation of the expression of proteins related to cellular homeostasis, as well as those that respond to stimuli, served to maintain physiological balance during stress. concurrently, the expression levels of proteins related to transcription, translation, and signal transduction were also up-regulated to promote survival under hf stressing. the genetic variations identified in the proteomes, plant growth, and photosynthetic performance in response to flood and heat represent stress adaption mechanisms to be exploited in future cauliflower breeding efforts. proteome analysis of potato under salt stress comparative analysis of barley leaf proteome as affected by drought stress proteome changes in wild and modern wheat leaves upon drought stress by two-dimensional electrophoresis and nanolc-esi-ms/ms changes in photosynthetic parameters and antioxidant activities following heat-shock treatment in tomato plants proteomic study of a tolerant genotype of durum wheat under salt-stress conditions arabidopsis hsa , a novel heat shock protein, is essential for acquired thermotolerance during long recovery after acclimation transcriptome sequencing and comparative analysis of the gametophyte thalli of pyropia tenera under normal and high temperature conditions drought-induced leaf protein alterations in sensitive and tolerant wheat varieties a pentatricopeptide repeat protein is essential for rna editing in chloroplasts cloning and molecular characterization of fructose- , -bisphosphate aldolase gene regulated by high-salinity and drought in sesuvium portulacastrum the proteome response of salt-resistant and salt-sensitive barley genotypes to longterm salinity stress proteome profiling of populus euphratica oliv. upon heat stress drought-inhibition of photosynthesis in c plants: stomatal and non-stomatal limitations revisited proteome analysis of wheat leaf under salt stress by two-dimensional difference gel electrophoresis ( d-dige) comparative proteomic analysis of grain development in two spring wheat varieties under drought stress physiology and proteome responses of two contrasting rice mutants and their wild type parent under salt stress conditions at the vegetative stage comparative proteomic analysis of salt response proteins in seedling roots of two wheat varieties proteome analysis of sugar beet leaves under drought stress research on plant abiotic stress responses in the post-genome era: past, present and future responses of antioxidative system to chilling stress in two rice cultivars differing in sensitivity structural-functional state of thylakoid membranes of wheat genotypes under water stress proteomic analysis of wheat embryos with -de and liquid-phase chromatography (proteomelab pf- d) -a wider perspective of the proteome a proteomics analysis of drought stress-responsive proteins as biomarker for drought-tolerant sugarcane cultivars drought and heat stress injury to cool season turfgrasses in relation to antioxidant metabolism and lipid peroxidation diverse roles for chloroplast stromal and thylakoid-bound ascorbate peroxidases in plant stress responses rice proteomics: a step toward functional analysis of the rice genome proteomic and biochemical analysis of the cotyledon and root of flooding stressed soybean plants plant proteome changes under abiotic stress -contribution of proteomics studies to understanding plant stress response heat shock proteins: modifying factors in physiological stress responses and acquired thermo tolerance a proteomic approach in analyzing heat-responsive proteins in rice leaves application of a peptide-based pf d plateform for quantitative proteomics in disease biomarker discovery proteomic analysis of the expression of proteins related to rice quality during caryopsis development and the effect of high temperature on expression chilling stress and chilling tolerance of sweet potato as sensed by chlorophyll fluorescence identification of flooding-response genes in eggplant roots by suppression subtractive hybridization proteomic analysis of the effect of heat stress on hexaploid wheat grain: characterization of heatresponsive proteins from nonprolamins fraction abiotic stress, the field environment and stress combination comparative proteome analysis of drought-sensitive and drought-tolerant rapeseed roots and their hybrid f line under drought stress comparative proteomic analysis of early salt stress-responsive proteins in roots of snrk transgenic rice the effect of temperature and shade on curd initiation in temperature and tropical cauliflower on the expansion of the pentatricopeptide repeat gene family in plants heat-shock response in arabidopsis thaliana explored by multiplexed quantitative proteomics using differential metabolic labeling rubisco activity: effects of drought stress a -d liquid-phase chromatography for proteomic analysis in plant tissues when defense pathways collide: the response of arabidopsis to a combination of drought and heat stress sam levels, gene expression of sam synthetase, methionine synthase and acc oxidase, and ethylene emission from n. suaveolens flowers leaf proteome alterations in the context of physiological and morphological responses to drought and heat stress in barley (hordeum vulgare l.) role of antioxidant systems in wheat genotypes tolerance to water stress inhibition of photosynthesis by heat stress: the activation state of rubisco as a limiting factor in photosynthesis heat shock proteins: molecules with assorted functions a pentatricopeptide pepeat protein facilitates the trans-splicing of the maize chloroplast rps pre-mrna proteomic and transcriptomic analyses of agave americana in response to heat stress physiological index for tolerance to high temperature and waterlogging in cauliflower proteome analysis of soybean leaves, hypocotyls and roots under salt stress proteome profile of human urine with two-dimensional liquid phase fractionation proteomic analysis of small heat shock protein isoforms in barley shoots plant responses to hypoxia -is survival a balancing act? heat tolerance in plants: an overview role of plant heat-shock proteins and molecular chaperones in the abiotic stress response proteomic alternations of brassica napus root in response to boron deficiency investigating trends in vegetable crop response to increasing temperature associated with climate change proteomic profile analysis of pyropia haitanensis in response to high-temperature stress proteomic analysis of heat stress response in leaves of radish (raphanus sativus l.) this research was supported by grants (nsc , - -b- - -my ) from national science council, taiwan, roc. supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ . /j.scienta. . . . key: cord- - e tb o authors: halewood, michael; jamora, nelissa; noriega, isabel lopez; anglin, noelle l.; wenzl, peter; payne, thomas; ndjiondjop, marie-noelle; guarino, luigi; kumar, p. lava; yazbek, mariana; muchugi, alice; azevedo, vania; tchamba, marimagne; jones, chris s.; venuprasad, ramaiah; roux, nicolas; rojas, edwin; lusty, charlotte title: germplasm acquisition and distribution by cgiar genebanks date: - - journal: plants (basel) doi: . /plants sha: doc_id: cord_uid: e tb o the international collections of plant genetic resources for food and agriculture (pgrfa) hosted by cgiar centers are important components of the united nations food and agriculture organization’s global system of conservation and use of pgrfa. they also play an important supportive role in realizing target . of the sustainable development goals. this paper analyzes cgiar genebanks’ trends in acquiring and distributing pgrfa over the last years, with a particular focus on the last decade. the paper highlights a number of factors influencing the centers’ acquisition of new pgrfa to include in the international collections, including increased capacity to analyze gaps in those collections and precisely target new collecting missions, availability of financial resources, and the state of international and national access and benefit-sharing laws and phytosanitary regulations. factors contributing to centers’ distributions of pgrfa included the extent of accession-level information, users’ capacity to identify the materials they want, and policies. the genebanks’ rates of both acquisition and distribution increased over the last decade. the paper ends on a cautionary note concerning the potential of unresolved tensions regarding access and benefit sharing and digital genomic sequence information to undermine international cooperation to conserve and use pgrfa. treaty's governing body and the fao commission on genetic resources for food and agriculture (cgrfa), this had not been done according to calendar years, so could not be used for this study. these data were combined with pre-existing data on annual acquisitions and distributions by the centers covering the period of - (from the study referenced above). genebank managers were also asked to respond to a survey (see appendix a) providing reflections on trends in acquisitions and distributions from to , contributing factors, and experiences acquiring (or attempting to acquire) new materials during the last five years. staff from the centers' genebanks, the crop trust, and the genebank platform policy module subsequently worked together in group teleconferences to review and synthesize key findings from the assembled data and survey. the respondents/informants provided expert knowledge concerning genebanks' performance targets and quality management standards, methods for identifying and prioritizing gaps in collections to be addressed through new collecting expeditions, centers' efforts to ensure healthy, quarantine organism-free genetic materials, and international crop conservation strategies. literature reviews were conducted to gain insights into the experiences of genebanks outside the cgiar. to validate data and key findings, several consultations with cgiar genebank managers and staff were conducted. there was a dramatic rise in acquisitions of new pgrfa by the genebanks between and , compared to the previous years, though still lower than peaks reached in the s and s, as illustrated in figures and below. the increase over the last years reached its height in , when the genebanks received almost , samples of distinct pgrfa to include as new accessions in the international collections. of course, not all of the materials that the genebanks receive are ultimately accessioned. if materials received are not viable, or are redundant with respect to the materials already in the collection, they are not included in the collections. for the convenience of using a short form, the tables and figures presented below make reference to "accessions"; in fact, the data refer to materials received with the intention of including them as accessions, assuming they are viable and not redundant. in , the number of newly acquired materials by the centers to include in the international collections dropped back down to the lower levels that characterized the mid- s to . plants , , x for peer review of experiences acquiring (or attempting to acquire) new materials during the last five years. staff from the centers' genebanks, the crop trust, and the genebank platform policy module subsequently worked together in group teleconferences to review and synthesize key findings from the assembled data and survey. the respondents/informants provided expert knowledge concerning genebanks' performance targets and quality management standards, methods for identifying and prioritizing gaps in collections to be addressed through new collecting expeditions, centers' efforts to ensure healthy, quarantine organism-free genetic materials, and international crop conservation strategies. literature reviews were conducted to gain insights into the experiences of genebanks outside the cgiar. to validate data and key findings, several consultations with cgiar genebank managers and staff were conducted. there was a dramatic rise in acquisitions of new pgrfa by the genebanks between and , compared to the previous years, though still lower than peaks reached in the s and s, as illustrated in figures and below. the increase over the last years reached its height in , when the genebanks received almost , samples of distinct pgrfa to include as new accessions in the international collections. of course, not all of the materials that the genebanks receive are ultimately accessioned. if materials received are not viable, or are redundant with respect to the materials already in the collection, they are not included in the collections. for the convenience of using a short form, the tables and figures presented below make reference to "accessions"; in fact, the data refer to materials received with the intention of including them as accessions, assuming they are viable and not redundant. in , the number of newly acquired materials by the centers to include in the international collections dropped back down to the lower levels that characterized the mid- s to . in total, over the course of ten years, from to inclusive, the cgiar genebanks acquired , samples of distinct pgrfa to include in their article collections. approximately % of the materials acquired by the genebanks came from providers in different countries. the remaining % came from the centers' own breeding programs, as discussed below. a total of % of those countries are developing countries or countries with economies in transition as defined in the international monetary fund's world economic outlook database (october , accessible at: https://www.imf.org/external/pubs/ft/weo/ / /weodata/groups.htm). approximately % of the distribute the material concerned with the smta. in this context, it is interesting to note that of the countries from which the materials were made available are not currently contracting parties to the plant treaty, yet they were still willing to provide the materials, and have them subsequently redistributed by the cgiar centers, under the conditions established by the plant treaty. we did not analyzed whether some other countries which are now plant treaty members made materials available before becoming members. approximately one third of the pgrfa acquired by the genebanks from providers in countries between and were associated with a project coordinated by the crop trust from to called "securing the biological basis of agriculture" (hereinafter referred to as the "regeneration project"), funded by the bill and melinda gates foundation and the grains research and development corporation. that project provided financial and technical support for organizations around the world to regenerate unique ex situ pgrfa that were at risk of being lost, to send a copy of the regenerated materials to an internationally recognized genebank, to send a copy for safety back-up in the svalbard global seed vault, and to make the materials available through the multilateral system of access and benefit sharing. activities with national partners in countries resulted in the regeneration of approximately , threatened accessions, of which more than half were duplicated in cgiar genebanks with permission to make them available through the multilateral system. as of , another samples of unique accessions collected from countries were sent to cgiar center genebanks (icarda, icrisat, irri, iita, cip) by the millennium seed bank (msb), associated with the project called "adapting agriculture to climate change: collecting, protecting and preparing crop wild relatives" (hereinafter the "cwr project") coordinated by the crop trust from to [ ]. the cwr project, with funding from the norwegian government, provided financial and technical support for project partners to target and collect wild species related in total, over the course of ten years, from to inclusive, the cgiar genebanks acquired , samples of distinct pgrfa to include in their article collections. approximately % of the materials acquired by the genebanks came from providers in different countries. the remaining % came from the centers' own breeding programs, as discussed below. a total of % of those countries are developing countries or countries with economies in transition as defined in the international monetary fund's world economic outlook database (october , accessible at: https://www.imf.org/external/pubs/ft/weo/ / /weodata/ groups.htm). approximately % of the materials from countries came from new collecting expeditions; the other % was material that was already in ex situ conditions prior to being sent to the centers. all of the materials from providers in countries were either received under the standard material transfer agreement (smta) adopted for exchanges of materials under the plant treaty's multilateral system (which allows the center to conserve, use, and pass on the materials with the same smta) or under other agreements whereby the providers gave the centers permission to subsequently distribute the material concerned with the smta. in this context, it is interesting to note that of the countries from which the materials were made available are not currently contracting parties to the plant treaty, yet they were still willing to provide the materials, and have them subsequently redistributed by the cgiar centers, under the conditions established by the plant treaty. we did not analyzed whether some other countries which are now plant treaty members made materials available before becoming members. approximately one third of the pgrfa acquired by the genebanks from providers in countries between and were associated with a project coordinated by the crop trust from to called "securing the biological basis of agriculture" (hereinafter referred to as the "regeneration project"), funded by the bill and melinda gates foundation and the grains research and development corporation. that project provided financial and technical support for organizations around the world to regenerate unique ex situ pgrfa that were at risk of being lost, to send a copy of the regenerated materials to an internationally recognized genebank, to send a copy for safety back-up in the svalbard global seed vault, and to make the materials available through the multilateral system of access and benefit sharing. activities with national partners in countries resulted in the regeneration plants , , of of approximately , threatened accessions, of which more than half were duplicated in cgiar genebanks with permission to make them available through the multilateral system. as of , another samples of unique accessions collected from countries were sent to cgiar center genebanks (icarda, icrisat, irri, iita, cip) by the millennium seed bank (msb) , associated with the project called "adapting agriculture to climate change: collecting, protecting and preparing crop wild relatives" (hereinafter the "cwr project") coordinated by the crop trust from to [ ]. the cwr project, with funding from the norwegian government, provided financial and technical support for project partners to target and collect wild species related to crops, to create a safety back-up, and make collected material available through the multilateral system. it is critically important to have safety duplication for pgrfa accessions hosted by organizations that can ensure the required conditions for long-term storage. details concerning the centers, crops, providing countries, and related programs are set out in table below. as mentioned above, approximately % of the materials acquired by the genebanks between and came from the centers' own plant breeding programs, mostly from the cimmyt wheat breeding program. every genebank has a policy and process, both of which are periodically reviewed, for strategically acquiring materials from breeders' collections for incorporation into the genebank for long-term conservation with the express aim of ensuring that incoming materials are likely either to represent a highly demanded material or diversity that is not already contained in the collection. the availability of funds-both for providers and for the centers as recipients-was one of the most frequently mentioned factors by the genebank managers affecting the ability of cgiar centers to acquire new materials to include in the genebanks. the centers representatives confirm that in many of the instances where the genebanks were able to acquire new materials, it was critically important to be able to provide financial and technical support for providers' activities related to the collection, regeneration, phytosanitary cleaning and inspection, and shipping of accessions. in some cases, the centers provided this support, in other instances-most notably, the regeneration and cwr projects-the support came from other organizations. generally speaking, the financial costs of preparing and sending materials are not particularly high. costs arise because of the lack of capacity and means to multiply, dry, test, and clean seed that is either collected or conserved and is only available in small quantities with unknown viability. the transaction costs are greatly higher when vegetatively propagated germplasms, including roots, tubers, bananas, or trees, are involved because the movement across international borders involves stringent phytosanitary restrictions that demand expensive disease cleaning and testing upon both shipping and receipt along with extensive periods of time in quarantine based on the risk assessed by the national plant protection organizations (nppo). in such cases, the centers need to make substantial investments in strengthening partners' capacity to test and clean materials. once materials arrive at the center, they need to be processed through post-entry quarantine, health tested, and cleaned of infectious diseases, multiplied, tested for viability, and dried before being introduced into the collection and made available for distribution. in the case of clonally propagated crops, the steps can be very expensive and time-consuming, taking generally four years or longer for a new accession to become available (n. l. anglin pers comm). post-entry quarantine (peq) procedures such as growing the first generation in a quarantine greenhouse-a requirement for all newly acquired materials by the genebanks-are also expensive. occasionally, the nppo requires new acquisitions to remain in peq from four months up to two years to assess risks. direct costs for new collecting missions are also relatively modest. of course, the costs per samples of materials collected greatly differs by crop type, wild or cultivated form, and geographical location. for example, similar projects, with similar budgets, working with national agricultural research system (nars) partners to conduct collecting missions resulted in very different numbers of acquisitions; samples of landraces and wild relatives and forages were gathered in tajikistan and lebanon for the same cost as samples of bananas collected in papua new guinea, samoa, and the cook islands. the per accession costs will differ between seed and clonal crop collections by an order of magnitude more when taking into account the costs of incorporating the materials into the collections. in addition to direct costs associated with the management and transfer of biological materials, there are transaction costs associated with getting requisite permissions to provide/access materials from both ex situ and in situ conditions. transactional costs associated with these activities are often substantially increased in situations where the national policies and laws are unclear, or non-existent. in such cases, it can take extended rounds of communications over long periods of time with many different levels of national r&d partners, lawyers, and competent authorities before final decisions to provide materials can be made, or, as sometimes occurs, no final determination is ever communicated. unlike some national genebanks or networks of collections [ ] , cgiar does not have a centralized service that takes responsibility to help centers comply with requisite processes for organizing collecting missions/partnerships, and obtaining requisite permissions. indeed, along with availability of funds, the cgiar genebank managers emphasized that "restrictive or unclear laws or policies" were leading variables influencing their ability to acquire new pgrfa to include in the international collections. they note that the plant treaty's multilateral system has contributed stability and a sound legal basis for providing and receiving germplasm, and that their ability to acquire materials through new collecting is evidence of cooperation/coordination between national authorities responsible for implementing the plant treaty and those responsible for regulating access to genetic resources outside the multilateral system (including those implementing the cbd or nagoya protocol). however, the centers are concerned that unresolved disputes concerning the enhancement of the plant treaty's multilateral system of access and benefit sharing, and digital sequence information (dsi) in particular (both under the plant treaty and the nagoya protocol), are holding back some countries (and providers within countries) from making more pgrfa available through the multilateral system. if international tensions over these issues remain unresolved for too long, they could further undermine the centers' ability to access, generate, use, and distribute pgrfa and associated information. the center genebanks confirm that, over time, they tend to obtain materials to include in their collections from the same set of countries or subregions, where they have established connections. by corollary, there are some countries and subregions from which they rarely, if ever, obtain materials. a number of the genebanks confirm that they rarely make overtures to organizations and or countries which have strongly signaled in the past that they are unwilling to make new materials available. their general perception is that, despite the coming into force of the nagoya protocol and the existence of the plant treaty's multilateral system of access and benefit sharing, some of these same countries have not substantially altered their approach to making materials available upon request for inclusion in the international genebanks' collections. the initial stimulus, and subsequent financial and technical support for pgrfa providers, from these internationally coordinated projects were clearly critically important factors contributing to the extraordinary increase in pgrfa that was made available for the cgiar centers to include in the international collections and thus the multilateral system. the centers genebanks' representatives also report that the strength of the centers' long-term relationships with providers and provider countries is equally important. most materials are made available to centers as part of projects with providers. centers rarely obtain new materials to include in the genebank as a result of "cold-calling" would-be providers with simple requests for materials and no other form of engagement. out of appreciation of these factors, the centers most recent collective efforts under the cgiar genebank platform (suspended temporarily due to the covid- pandemic) to catalyze and support new collecting expeditions involve support for "two-way flows" of germplasm from the genebanks to the providers, identified on the basis of a jointly conducted analysis of potentially useful germplasm to respond to local needs, and from the providers to the genebanks, and financial and technical support for institutional capacity building for partner organizations in the country concerned. this is consistent with practices of other organizations seeking to acquire pgrfa to include in public genebanks through new collecting activities [ ] . of course, these factors affecting the ability of the genebanks to acquire new materials need to be considered within the broader context of what additional pgrfa needs to be collected, backed up, and conserved as part of the global system, either by cgiar genebanks or other organizations hosting globally available pgrfa collections. methods for conducting gap analyses and strategies for coordination with other organizations are considered below. from to , cgiar centers acquired pgrfa through collecting expeditions from countries. collecting in six of those countries was supported by the cwr project. during the same period of time, the center for genetic resources in the netherlands (cgn, with , accessions) received materials through collecting expeditions in five countries, the leibniz institute of plant genetics and crop plant research (ipk, with , accessions) received materials from collecting in six countries, and the national plant germplasm system of the united states department of agriculture (npgs-usda, with , accessions) organized collecting expeditions in at least foreign countries [ ] [ ] [ ] [ ] [ ] [ ] [ ] . accessions from collecting expeditions in other countries account for % of cgn total germplasm acquisitions [ ] . at the beginning of the century, such materials represented % of material acquired by npgs-usda [ ] . in the last decade, collecting expeditions have contributed % of all the acquisitions by cgiar genebanks. the fact that, relative to the cumulative size of their collections, the cgiar centers have engaged in relatively fewer collecting activities during this period than these other organizations may be attributable to a combination of the following factors. the diversity of some cgiar mandate crops is relatively well represented in ex situ collections when compared to the crops that ipk, cgn, and npgs-usda have been prioritizing in their collecting activities, i.e., vegetables (e.g., lettuce, allium, brassica, chicory, spinach, asparagus, and carrot), fruit and nut trees (e.g., apple, pear, pomegranate, pistachio, walnut, and hazelnut), berries (fragaria, rubus, and ribes), and temperate grasses (poa, festuca, agrostis, koeleria, and puccinellia). ipk, cgn, and npgs-usda have been acquiring most of their new materials from the transcaucasia and central asia regions and europe [ , ] , which are arguably more open to allowing new collecting missions than other countries and regions in the world. ipk, cgn, and npgs-usda may also have had more, and more regular, financial resources to dedicate to supporting new collecting activities. we acknowledge that we are only scratching the surface of potential comparisons between cgiar and other genebanks around the world; we hope to be able to deepen such analyses in the future. over the course of the last years, the cgiar genebanks have distributed on average , samples of germplasm per year around the world. while there are significant fluctuations in centers' distributions from year to year, the overall rate of centers' distribution from to is higher than the previous decade at , samples per year ( to annual average), as portrayed in figure above. comparing the total distributions between time periods - and - , four centers increased their annual distributions in the latter half of the last decade, four remained generally the same, and three decreased. there is considerable fluctuation, from year to year, in the ratio of materials the cgiar genebanks send to recipients within the cgiar (mainly breeders) and to recipients outside the cgiar, as can be seen in figure . since , the centers genebanks have been distributing proportionately more materials to recipients outside the cgiar. some centers do not have crop breeding programs (e.g., bioversity, ilri), so almost all of their distributions are to recipients outside the cgiar. in , the centers and crops with a high proportion of materials distributed to breeders (within their own center or in other centers within the cgiar) are africarice (rice); icrisat (pigeon pea, chickpea); icarda (grasspea, barley); cip (sweet potato); irri (rice); iita (cassava); and cimmyt (wheat). between and , approximately % of pgrfa samples distributed by the genebanks to recipients outside the cgiar were to a combination of nars partners, national genebanks, advanced research institutes (aris), and universities (see figure for more details). the proportion of samples distributed to farmers, farmer organizations, and non-governmental organizations (ngos) during this time is still relatively small ( %), approximately the same rate as noted earlier for [ ] . cip and icraf were the two centers whose proportionate distribution of materials to farmers and ngos was largest, between and inclusive, as seen in figure b . much of the material distributed by cip during this period was part of their repatriation program in which desired potato landraces are matched to farmers' descriptions and returned to the farmers that have lost them due to normal attrition, environmental impacts (drought, hail, diseases), or other reasons, to help maintain and/or increase their diversity in farms. cip also gives material to farmers in the repatriation program to support their needs in responding to climate change or to help improve yield. in terms of absolute numbers, the largest provider of germplasm materials was cimmyt, distributing % of the materials cumulatively distributed by the genebanks between and , followed by irri and icrisat, distributing approximately % and %, respectively. (a) all centers between and , approximately % of pgrfa samples distributed by the genebanks to recipients outside the cgiar were to a combination of nars partners, national genebanks, advanced research institutes (aris), and universities (see figure for more details). the proportion of samples distributed to farmers, farmer organizations, and non-governmental organizations (ngos) during this time is still relatively small ( %), approximately the same rate as noted earlier for [ ] . cip and icraf were the two centers whose proportionate distribution of materials to farmers and ngos was largest, between and inclusive, as seen in figure b . much of the material distributed by cip during this period was part of their repatriation program in which desired potato landraces are matched to farmers' descriptions and returned to the farmers that have lost them due to normal attrition, environmental impacts (drought, hail, diseases), or other reasons, to help maintain and/or increase their diversity in farms. cip also gives material to farmers in the repatriation program to support their needs in responding to climate change or to help improve yield. in terms of absolute numbers, the largest provider of germplasm materials was cimmyt, distributing % of the materials cumulatively distributed by the genebanks between and , followed by irri and icrisat, distributing approximately % and %, respectively. between and , approximately % of pgrfa samples distributed by the genebanks to recipients outside the cgiar were to a combination of nars partners, national genebanks, advanced research institutes (aris), and universities (see figure for more details). the proportion of samples distributed to farmers, farmer organizations, and non-governmental organizations (ngos) during this time is still relatively small ( %), approximately the same rate as noted earlier for [ ] . cip and icraf were the two centers whose proportionate distribution of materials to farmers and ngos was largest, between and inclusive, as seen in figure b . much of the material distributed by cip during this period was part of their repatriation program in which desired potato landraces are matched to farmers' descriptions and returned to the farmers that have lost them due to normal attrition, environmental impacts (drought, hail, diseases), or other reasons, to help maintain and/or increase their diversity in farms. cip also gives material to farmers in the repatriation program to support their needs in responding to climate change or to help improve yield. in terms of absolute numbers, the largest provider of germplasm materials was cimmyt, distributing % of the materials cumulatively distributed by the genebanks between and , followed by irri and icrisat, distributing approximately % and %, respectively. (a) all centers landraces are the most frequently requested materials ( % between and ), followed by breeding materials ( %), and wild relatives ( %) (see figure for more details). landraces are the most frequently requested materials ( % between and ), followed by breeding materials ( %), and wild relatives ( %) (see figure for more details). landraces are the most frequently requested materials ( % between and ), followed by breeding materials ( %), and wild relatives ( %) (see figure for more details). the countries which received the highest numbers of samples from cgiar genebanks, between and , are set out in table . these data do not include transfers within or between cciar centers. four of these countries are not contracting parties to the plant treaty. the genebanks' annual distribution is a reflection of demand from users, often driven by the needs of the research and development projects in which they are involved, and by the profile or visibility of the genebanks among research and development, and non-governmental organizations in different countries. the center hosting the genebank is frequently involved in the research in some way. here, we provide some examples of projects with which distributed material was associated. the overall spike in distributions in is largely attributable to the internal cimmyt transfers of , wheat accessions as part of cimmyt's seeds of discovery (seed) project to cimmyt researchers and breeders characterizing maize and wheat genetic diversity for use in breeding programs. cimmyt's high number of distributions in also includes wheat accessions to recipients in iran and turkey as part of those countries' restoration efforts. in , icraf distributed considerably more tree germplasm than in recent years as a result of requests from organizations involved in the mega project "regreening africa" that is being implemented in east and west africa. between and , the icarda genebank's rate of distribution to recipients outside the cgiar increased - % over the previous five-year period, reflecting a combination of the center's progress getting its genebank's operations back "up to speed" after the disruptions associated with having to relocate from syria and a surge of new interest in the genebank's materials as a result of publicity generated when the center retrieved materials from the svalbard global seed vault. a big increase in the icrisat genebank's distributions in and was associated with supporting a consortium of indian organizations conducting chickpea and pigeon pea multilocation trials. in , icrisat genebank's distributions were higher than usual in part because it responded to a request from italian organizations for core collections of cereals (sorghum, pearl millet, finger, kodo, proso, barnyard, little, and foxtail millets) as part of a program to reproduce local varieties and develop new crops adaptable to italian local conditions. the iita genebank's increased rate of distributions in was associated with the requests for cowpea and bambara groundnut germplasm from nigerian universities to support their research programs on those crops. the incidence of a newly emerging rare disease can also lead to a need to screen a large number of accessions for resistance [ , ] . the genebank managers confirm that one of the most important factors affecting demand for pgrfa is the quality and relevance of the accession-level information that the centers compile about the materials in their collections, a finding consistent with the relevant literature [ , ] . accession-level information helps users make informed decisions about what materials from the collections are potentially most useful for their specific purposes. it also makes it possible for the genebank to make more targeted selections of materials in response to their requests since often users do not know where to begin in choosing an appropriate accession for their needs. the genebanks highlighted the importance of trait-specific data including nutritional qualities, biotic and abiotic stresses, agronomic performance, genetic sequence information linked to traits or to provide information about relationships among accessions, and geographic information about place of collection, including climate conditions and soil type. irri reports there was a significant increase in the number of requests for genetic stocks of rice accessions, particularly between and , after the full genome sequences were made publicly available. the genebanks have minted digital object identifiers under the plant treaty's global information system (glis-dois) for almost all of the accessions in the center-hosted international collections, with a long-term view towards helping this process and linking publications and data back to accessions. while such information is absolutely necessary to generate interest in, and demand for, materials in the genebanks, it also permits users to make more targeted requests for a narrower range of materials with each request. otherwise, users must consider thousands of accessions from which to choose. thus, documenting the traits and uniqueness of each accession helps create a path for utilization of the germplasm. further, creating cores or subsets of accessions, where small groups of accessions are defined by the genebank to help narrow down the search for specific traits, has had an influence on demand and could be scaled up significantly. africarice's genebank has used molecular markers to create core and mini-core sets that represent the maximum possible genetic variation contained in the african rice whole collection [ ] . as a result, there has been a significant increase in requests for the mini-core set for use in rice genetic and breeding studies, and gene discovery (m.n. ndjiondjop pers.com). the centers' genebank managers note that as they have been able to increase the quantity of accession-level information, requests are indeed becoming more informed and better targeted. scientists working with genebanks both outside [ , ] and with cgiar are developing methods to assist users to identify useful materials from ex situ collections, including the icarda-led focused identification of germplasm strategy (figs) [ , ] . in , iita developed a figs population of drought-and heat-tolerant cowpea. this significantly increased the requests to over samples distributed to recipients' countries for research purposes (t. marimagne, pers.com) the supply of pgrfa from the genebanks also depends upon the ready availability of a sufficient stock of pest and disease-free materials with legal certainty concerning the conditions under which the materials can be provided and received. costs associated with multiplying, assuring plant health, and distributing samples of crops that are clonally propagated or have recalcitrant seeds are much higher than for crops of orthodox seed behavior. under the framework of the cgiar genebank crp ( - ) and the cgiar genebank platform ( - ), the crop trust, in cooperation with the centers, has developed performance targets and a monitoring system to assess the availability, safety duplication, documentation, and quality management of collections. ultimately, the target is to have % (of accessions in the collections) immediately available and % safety-duplicated at two locations (for seed collections only). as of the end of , % of all materials in the cgiar genebank were immediately available, % of the seed collection was secured in safety duplication at two levels, and % was duplicated at the svalbard global seed vault. a total of % of the clonal crop collection was safety-duplicated in the form of cryopreservation or in vitro cultures in at least one location. the covid- pandemic has highlighted the strategic importance of cryopreserving clonal crops. in vitro cultures require continuous monitoring and upkeep by genebank staff in personam. if scientists' access to these collections is limited as a result of governmental policies restricting movement, some accessions in those collections could deteriorate and be lost. if those accessions are not safety duplicated somewhere else, or if the safety duplications are in the form of in vitro collections that are similarly vulnerable to the same risk, then unique materials and potentially unique varieties no longer found in farmers' fields could disappear if they are not secured in cryopreservation. cryopreserved back-up collections of these materials would address this risk. the cgiar genebanks report that, on occasion, materials they send, and materials they are meant to receive, are held up for long periods of time due to the implementation of phytosanitary regulations, occasionally to the point where materials die before they arrive at their destination. the agreements between the cgiar centers and the plant treaty's governing body (signed in ) have created legal certainty concerning the status of the collections and the conditions under which they can be distributed. this is reflected in the fact that almost all transfers of pgrfa from the genebanks are under the plant treaty's smta (with the exception of materials sent for service agreements, or restoration or direct use by farmers in cultivation, as per the opinions of the ad hoc technical advisory committee on the multilateral system and smta) [ ] . just as the centers received materials under the smta from providers in countries that are not contracting parties, the centers' genebanks also distributed pgrfa, using the smta, to recipients in thirteen countries that are not plant treaty contracting parties (between and inclusive). despite these benefits to the centers operating under the plant treaty's framework, the genebanks note with concern that some large seed companies, some universities, and one national agricultural research organization are unwilling to receive materials under the smta, which makes it impossible for the centers to distribute materials from the genebank (and also most of the materials from the breeding programs) to them. other genebanks noted that their ability to distribute materials from the international collections was being constrained by policies of the country in which they are located. most of the international collections hosted by the cgiar centers originated as working collections to support research and breeding programs of international and national public agricultural research organizations. their subsequent growth depended partly on taking advantage of opportunities to acquire pgrfa from a wide range of sources in an ad hoc manner, for example, being offered material: previously assembled by other organizations [ ]; • as a result of interactions with scientists from national programs; • as part of the international network of base collections organized by the international board for plant genetic resources (ibpgr) [ ] ; • by the centers' own breeders. the collections grew in opportunistic "fits and starts" without resources or tools to systematically analyze their structure and coverage vis-à-vis what exists in situ, or in other collections around the world. consequently, it can be a challenge for genebank managers to be certain that newly acquired materials are duplicates of materials they already have, or the same as materials that are conserved and made internationally available by other genebanks, or that they are truly unique. characterization, documentation, and some cross-referencing are key to resolving this issue, but still duplicates or near duplicates are likely to be abundant within and among genebanks in recent years, the centers' genebanks have started to take advantage of modern molecular tools such as genotyping to characterize the genetic structure of the collections and to identify genetic differences among and within accessions, for example, potato [ ] [ ] [ ] , sweet potato [ , ] , cassava [ ] , forage grasses [ ] [ ] [ ] , mexican wheat landraces [ ] , and african rice [ ] irri generated whole genome sequences of three thousand accessions in its collection [ ] . while still constrained by resource limitations, molecular-level characterization has unprecedented potential to identify redundancies not only within collections, but also across collections, both inside and outside the cgiar along with identifying potential misclassifications, introgressions, levels of domestication, genetic origins, and putative hybrids. however, it is important to note that even with these data, it is often difficult to determine an adequate cut-off threshold for calling a material a duplicate or too genetically similar for incorporation into the collections. the cost of generating raw sequence data has dropped precipitously, but the expertise and computing power necessary to analyze the data are still expensive and time-consuming, especially when dealing with whole genomes instead of reduced-representation sequencing approaches for genotyping. there are also other biological complications to molecular data alone for resolving duplication issues. however, the cgiar genebank platform has been piloting training programs with scientists from national agricultural research organizations to use genotypic information to analyze within and among accession genetic diversity for a range of crops. all cgiar genebanks now have acquisition policies in place and current processes are based on a more critical assessment of whether new materials add diversity to the collections or respond to a specific need or mandate. this is particularly important for clonal collections where, resources permitting, the cgiar genebanks can use genotyping to help confirm if a sample of newly acquired material is unique (to the collection) before undertaking expensive procedures to test, clean, and reproduce the materials for introduction into the collection as new accessions [ ] . centers working with clonal crops have also developed tools to use genetic sequence information to test for the presence of viruses and bacteria, overcoming costly delays for centers acquiring and distributing clonal pgrfa [ ] [ ] [ ] . under the framework of the cgiar genebank platform, the centers and the crop trust have developed three methods for analyzing gaps in the coverage of their own collections. first, so-called "diversity trees" have been constructed for crops. the trees are developed using published literature and expert knowledge to categorize the diversity held in each crop gene pool into known variety or genotype groups or wild species, which allows the mapping of accessions into the groups and the quantitative representation (or not) of the gene pool by the collection [ , , ] . second, spatial analyses have been undertaken using a method to assess the ecogeographic gaps and coverage of current cgiar crop collections. the method, which works best for collections with a high percentage of available information on the latitude-longitude of the origin of accessions, looks for relationships between geographic patterns in crop distribution with the genetic structuring, and uses these relationships to build distribution models for crop landraces [ ] . third, a method for trait-based gap analyses focuses on the analysis of the distribution of adaptive priority traits in relation to the environment using machine learning to make predictions; it works best where landraces have been associated with an environment for long enough for their traits to become associated with their environment, and presupposes well-characterized collections. figure includes a preliminarily indication of the coverage of cultivated gene pools in the cgiar genebank collections through the use of these tools and illustrates clearly that the cultivated diversity of some crops is well represented while others are considerably less. in addition to the three strategies listed above, some genebanks are utilizing collection-wide genotyping in order to make key decisions on any new acquisitions. this strategy is only effective for genebanks that have collected genotyping data on their entire ex situ collection with a particular marker system. after which, any new material being considered for acquisition can be genotyped with the same marker system (gbs, snp, dartseq, etc.) to aid in decision making. the resulting fingerprints of material being considered for acquisition are subsequently compared to the entire germplasm collection to gain genetic insights. phylogenetic results and genetic distance measures produced from the genotyping data can clearly show which samples are unique and which are redundant to the existing germplasm collection. once these data are available, decisions can be made on which material to introduce into the genebank, usually under the framework of maximizing genetic diversity and not introducing material that is genetically similar. this is especially a useful strategy in clonal genebanks in which introduction and virus cleaning are expensive and time-consuming. in addition to the three strategies listed above, some genebanks are utilizing collection-wide genotyping in order to make key decisions on any new acquisitions. this strategy is only effective for genebanks that have collected genotyping data on their entire ex situ collection with a particular marker system. after which, any new material being considered for acquisition can be genotyped with the same marker system (gbs, snp, dartseq, etc.) to aid in decision making. the resulting fingerprints of material being considered for acquisition are subsequently compared to the entire germplasm collection to gain genetic insights. phylogenetic results and genetic distance measures produced from the genotyping data can clearly show which samples are unique and which are redundant to the existing germplasm collection. once these data are available, decisions can be made on which material to introduce into the genebank, usually under the framework of maximizing genetic diversity and not introducing material that is genetically similar. this is especially a useful strategy in clonal genebanks in which introduction and virus cleaning are expensive and timeconsuming. the genebank platform has recently initiated communications with national agricultural research organizations and national plant treaty focal points in countries to use combinations of these tools as part of an effort to identify complementary holdings and gaps in collections, to identify priority areas for collecting (with the understanding that collected materials would be made available through the plant treaty's multilateral system). at the same time, they undertook to work together to identify potentially useful materials from the international collections to test in the countries concerned. while covid- has had an effect on the preparations for this work, the ambition remains to support the collecting of pgrfa in target countries where there are significant gaps in ex situ conservation. the cgiar centers do not have the objective to host complete collections of the diversity of their mandate crops. rather, they seek to coordinate efforts with other collections to ensure the broadest possible coverage, long-term conservation, and availability of materials, in the global system as a whole. the centers will pursue this approach, and the use of the tools and methods described the genebank platform has recently initiated communications with national agricultural research organizations and national plant treaty focal points in countries to use combinations of these tools as part of an effort to identify complementary holdings and gaps in collections, to identify priority areas for collecting (with the understanding that collected materials would be made available through the plant treaty's multilateral system). at the same time, they undertook to work together to identify potentially useful materials from the international collections to test in the countries concerned. while covid- has had an effect on the preparations for this work, the ambition remains to support the collecting of pgrfa in target countries where there are significant gaps in ex situ conservation. the cgiar centers do not have the objective to host complete collections of the diversity of their mandate crops. rather, they seek to coordinate efforts with other collections to ensure the broadest possible coverage, long-term conservation, and availability of materials, in the global system as a whole. the centers will pursue this approach, and the use of the tools and methods described above, in the process of defining collecting priorities and revising the current set of crop conservation strategies (available online at https://www.croptrust.org/resources/#ex-situ-conservation-strategies). the centers (and the crop trust) do not organize collecting expeditions on their own. they work in collaboration with nars partners who take responsibility for conducting the collecting, obtaining requisite permissions in compliance with national laws and regulations, and first depositing samples in their own national genebanks before transferring them to the cgiar genebanks. maintaining the health of the germplasm in the international collections is critically important to ensure against the international spread of quarantine pests and diseases. in , the germplasm health units (ghus) of the centers hosting pgrfa collections collectively facilitated exchanges of materials with countries. altogether , samples of , accessions were analyzed by performing , diagnostics tests, and , samples were rejected to be replaced with healthy materials from other batches or to be subject to phytosanitary treatments to eliminate the pests concerned [ ] . most of the pests detected are target-specific species. viral pathogens are most frequently intercepted in legume seeds and in clonal crops. centers only distribute germplasm that is free of quarantine organisms. untested and unclean materials are not distributed until establishment of the pest-free stocks. as of , nearly % of the germplasm conserved in cgiar genebanks had been tested for quarantine pests and about % of the collection was available for immediate distribution. the international plant protection convention (ippc) is essential for promoting and harmonizing countries' phytosanitary regulations, and for facilitating international movement of plant genetic resources that are free of quarantine pests and diseases. however, the system still can and should be further tailored to address the particularities of the international movement and uses of plant genetic resources. under the framework of the ippc, contracting parties occasionally adopt international standards for phytosanitary measures (ispms) that are tailored for different subject matters that could be a potential carrier of quarantine pests and diseases over international borders (e.g., farm equipment, commercial seed). to date, approximately ispms have been adopted. unfortunately, no ispm for the regulation of international movement of plant germplasm from genebanks has been developed. the ispm- on integrated measures for plants for planting and the ispm- on international movement of seeds partially address some of the issues, but insufficiently. as a result, the national plant quarantine facilities of many countries either develop and follow their own norms or follow those prescribed through ispms dealing with commercial seed (or plantlets), which are not appropriate for plant germplasm shipped to and from genebanks. the commercial seed ispms-which anticipate tons of seed in a single shipment-prescribe testing protocols that deplete most of the very small quantities of seed transferred to and from genebanks, or create long delays which are unnecessary to address risks associated with genebank germplasm. these delays can result in materials dying in transit before they arrive at their intended destinations, or they arrive so late that they miss an entire planting season, thereby contributing to delays or cessation of planned research and or plant breeding activities. while phytosanitary issues are most challenging for recalcitrant seed or clonal crops, the genebanks report similar challenges with seed crops. to address this situation, cgiar germplasm health experts are working with partners from other organizations to develop a draft protocol for a comprehensive phytosanitary compliance assurance procedure that will demonstrate the best procedures in use for germplasm production and health assurance, while maintaining transparency in risk assessment and mitigation strategies to get nppo accreditation as trusted to fast-track germplasm distribution. the initiative is referred to as the cgiar greenpass phytosanitary protocol (greenpass). if the concept is endorsed at the level of the ippc's commission on phytosanitary measures, nppos could justify eliminating redundant checks by cutting some steps or reducing the processing time for material from greenpass-accredited facilities. during the late s, the genebanks received mounting criticism that materials stored in genebanks were rarely used. this was a concern because necessary investments in genebanks are difficult to obtain if potential investors do not appreciate the value of crop diversity, including the multitude of services and benefits it can provide. several studies followed in the early s which contradicted the viewpoint that genebanks were "unused" [ , ] and a large body of research documented the high rates of return from the genetic improvement of crops for yield, yield stability, quality, nutritional composition, resource use efficiency, and resistance to pests and diseases [ ] [ ] [ ] [ ] [ ] [ ] . most of the economic benefits have been generated from farm productivity gains which can be attributed to research and breeding programs by publicly funded institutions, such as the cgiar, and society and consumers have especially benefited from lower food prices. since then, however, the research on the value of international collections and the genetic diversity held in the cgiar genebanks has not kept pace. recently, the cgiar genebank platform and the crop trust supported the establishment of a community of practice on genebank impacts to revive the interest in applied economics research in this area. the work resulted in several papers which have made important contributions to earlier research on genebank valuation [ ] . for example, bernal-galeano et al. ( ) [ ] estimated a gross benefit from the "victoria" potato variety in uganda at usd . billion dollars, which exceeds the annual operational cost of the cip genebank several times over. villanueva et al. ( ) [ ] estimated that a % increase in the genetic contribution of irri genebank accessions to an improved rice variety grown by rice farmers in east india is associated with a yield increase of %. this current set of studies attests to the value of genebanks in at least two ways. first, they contribute to a better understanding of the role, function, and value of genebanks, in the light of climate change and evolving food security challenges. several authors were able to trace the ancestry of varieties currently adopted by farmers to specific accessions in the genebanks and apportion economic gains by drawing from information on pedigrees. second, the studies highlight the importance of long-term conservation and safety duplication of unique and diverse genetic materials, for the potential unknown use of future generations. as with gollin ( ) [ ] , the findings support the need to refocus global conservation strategies on the efficient management of genetic resources, including on acquisition and conservation priorities. the findings presented above concerning the influence of political tensions, restrictive policies, and legal uncertainty are similar to the conclusion of the previous study [ ] of factors affecting centers' acquisitions between and . on the positive side, there is evidence that the plant treaty's multilateral system of access and benefit sharing is contributing positively to the willingness of many countries, national genebanks, and other providers to make pgrfa available and to safety-duplicate material in the cgiar center-hosted international collections. perhaps the most significant evidence (which is so obvious it is often overlooked) is that all of the material in the international collections ultimately came from countries and, to date, countries and the eu have voluntarily ratified the plant treaty, which invites the centers to manage those collections under the plant treaty framework, and to make those materials available under the smta. as of november , at least , accessions maintained in cgiar genebanks (approximately % of the collections) were originally obtained from countries that were not plant treaty contracting parties including, but not limited to, the following countries: mexico, china, nigeria, colombia, thailand, russia, vietnam, azerbaijan, republic of south africa, uzbekistan, and kazakhstan. it is partly for this reason that the cgiar centers feel duty bound to continue making materials available under the smta to recipients in countries that are not plant treaty members. some contracting parties, particularly those who have been criticized in recent years for not making more pgrfa directly available through the multilateral system, complain that there is insufficient recognition of the fact that much of the genetic resources that the cgiar centers' genebanks distribute through the multilateral system (with the exception of accessioned breeders materials) originally came from them. information about the country sources of materials (provenance) held in the center-hosted international collections is available through genesys, and centers have published papers highlighting the origins of materials in the collection [ , ] . however, more can and should be done-through more popular, less expert-oriented mechanisms-to celebrate countries' contributions to the international collections and the multilateral system. indeed, during the plant treaty governing body meeting in october , cgiar undertook to work with the plant treaty secretariat to publicize this information more broadly. more recent evidence of the positive influence of the plant treaty on providers' willingness to include materials in the international collections is the surge of materials received by the centers' genebanks between and . all of those materials were either provided under the smta or with permission for centers to subsequently make those materials available under the smta. it seems likely that many of those providers would not have been willing (or permitted) to provide materials for inclusion in the international collections in the absence of the plant treaty, the multilateral system, and the smta. indeed, some genebanks have been informed by national partners that while those partners can provide pgrfa of annex materials, they cannot provide pgrfa of non-annex materials, because their national rules only apply to materials formally included within the scope of the multilateral system. the fact that the centers were able to acquire materials-particularly materials through new collecting expeditions-is evidence that other regulatory frameworks, apart from the plant treaty's multilateral system, are also contributing to the willingness/ability of providers to provide access to those materials. a lot of in situ pgrfa are not automatically included in the multilateral system, so collection must be approved by a competent authority subject to national law (or in the absence of a law, some other standard observed by the authority) to allow the material to be collected, deposited in the national genebank, and later sent under the smta to the cgiar genebanks. (in this context, it is relevant to note that some countries have explicitly adopted the policy of not wanting to regulate access in this manner). the cgiar genebanks never collect on their own. they work in partnership with national organizations who manage the interface with national authorities, logistics, and the actual collecting. transfer within a country of material from one abs system to another requires coordination and cooperation between the competent authorities and stakeholders. it is certainly the case that the existence of the smta (because it is standard) made it possible for the centers to process agreements to receive and manage those same materials. it would have been impossible for the centers to negotiate unique transfer agreements with sui generis benefit sharing, dispute resolution, scope of use, and other conditions in each case, and then to put systems in place to manage materials under a plethora of different conditions. it is important to consider the significance of the fact that most of the materials made available to the genebanks between and were associated with the regeneration and cwr projects. first, it highlights the fact that conserving and providing pgrfa require financial and technical support and coordination, and in the absence of such support, many potential actors in the global system are unable to play their anticipated roles including securing the unique materials in their collections through safety duplication. in this context, it is important to emphasize that the funds and/or other non-monetary benefits provided to project partners in the regeneration and cwr projects were modest, designed to subsidize/cover the costs of regeneration and or collecting, health testing and shipping, and in some cases training to do those things and for the centers to receive the materials. extra funding is needed to support collecting new pgrfa. those costs, transaction costs in particular, would decrease if countries had well-defined systems for processing requests for germplasm, and clear signals from competent authorities that such requests were acceptable to consider. it is interesting to note that the usda has a dedicated plant exploration program managed by a plant exchange office that is responsible for organizing collecting expeditions, reaching out to national competent authorities on behalf of the genebanks included within the national system [ ] . there is no such service or function within the cgiar. second, the regeneration and cwr projects highlight the continued importance, and need for, scientifically informed priority setting and international coordination to generate a shared sense of purpose and to motivate the wide range of actors, spread across the world, who ended up being engaged in those projects. the rounds of projects supported by the plant treaty's benefit-sharing fund (bsf) have played a similar role, catalyzing interest and engagement of a broad range of actors operating from the levels of individual farms to international organization, and providing financial and technical support for project participants to, among other things, collect, multiply, health-test, and share pgrfa through the multilateral system. it would be interesting, but it is beyond the scope of this paper, to identify the range of materials included in the multilateral system in general as a result of the bsf projects. this is not to say that all of these internationally coordinated projects worked perfectly, or were immune from criticisms, or that many contracting parties, organizations, and people involved in the conservation and use of pgrfa do not have alternative visions or priorities. the point is that while the plant treaty's multilateral system of access and benefit sharing provides an internationally sanctioned, clearly defined legal platform of exchanging plant genetic resources, it is clear that additional financial and technical support, and inspiring visions for dispersed actors to work together, are also necessary to take advantage of the multilateral system. other actors (including potentially the plant treaty's own governing body, or, more realistically, a specialist group it establishes) could develop a vision and internationally coordinated program that similarly stimulate and support activities that take advantage of the multilateral system to access, improve, and share pgrfa, and safeguard threatened pgrfa in furtherance of sustainable development goals (e.g., pooling and evaluating a genetically diverse range of pgrfa for traits adapted to local climate changes). meanwhile, the cgiar centers' genebanks continue to distribute hundreds of thousands of pgrfa samples under the plant treaty's smta. indeed, in the decade between and (inclusive), the centers' genebanks distributed % more pgrfa samples than they did over the previous decade. these numbers attest to the utility of the multilateral system and increased reliance upon it as a means of accessing genetic materials. the plant treaty's positive impact on the availability of pgrfa more generally (i.e., without involvement of the center's genebanks) is further evidenced by the fact that, since , sixteen additional countries became members of the plant treaty. among them, is the usa, with the result that approximately , additional pgrfa accessions are available through the multilateral system of access and benefit sharing. in addition, during the same period, one additional international organization, the international center for biosaline agriculture (icba), agreed to make pgrfa available under the terms of and conditions of the plant treaty. on the negative side, however, there is also evidence that a number of plant treaty contracting parties continue to be reluctant to implement the multilateral system. overall, national level implementation of the multilateral system is still relatively low. this is perhaps most clearly manifested in the fact that only out of countries that are contracting parties have confirmed what pgrfa within their borders are actually included in the multilateral system. (see "material available in the multilateral system" on the plant treaty website at http://www.fao.org/plant-treaty/areas-of-work/themultilateral-system/collections/en/). this may not be a "black letter law" obligation under the treaty framework, but it is a commonly acknowledged prerequisite for the multilateral system to practically function [ ] . furthermore, some of the countries that have provided notice have confirmed only a fraction of the collections maintained in their national genebanks or other national public organizations. there are even fewer notices to the governing body concerning pgrfa that are voluntarily included by natural and legal persons. few countries have reported to the governing body that they have adopted new policies or administrative measures to implement the multilateral system. on one hand, it is not required by the plant treaty that a country develop new policies or laws to implement the multilateral system; on the other hand, many countries report that in the absence of new policy instruments approved by the national government that clarify who has the right to provide materials under the multilateral system, they are unable to do so [ ] . in such cases, the absence of new policy measures does indeed reflect a lack of national level implementation. the relatively slow rate of national level implementation by a number of countries can be partially accounted for by the fact that developing country contracting parties are dissatisfied by the fact that, to date, there has been only one payment to the plant treaty's benefit-sharing fund by a commercial user of materials from the multilateral system. this dissatisfaction contributed to the fifth session of the governing body in launching a process to enhance the functioning of the multilateral system. that process continued until , when it was suspended, with no new agreement reached, and high levels of unresolved political tension between contracting parties. international tension and disagreement concerning access and benefit-sharing issues in other fora have also spilled over into meetings under the plant treaty framework. the nagoya protocol came into force in . it was designed to address (mostly developing) countries' concerns that the convention on biological diversity (cbd) did not sufficiently promote its benefit-sharing objective. however, by , the conference of the parties to the cbd in mexico was overtaken by tensions over benefit sharing from commercial use of dsi, which is not addressed by the nagoya protocol (or at least not in a way that the international community agrees upon). since then, the issue has dominated the agendas of the cbd, plant treaty, fao cgrfa, and others. under these circumstances, some parties who are not content with levels of monetary benefit sharing from the use of pgrfa and/or genomic sequence information may be reluctant to make more materials available through the multilateral system by depositing them in the international collections hosted by the cgiar centers until there is some resolution to the dsi and abs issue. the previous study [ ] of factors affecting the centers' germplasm acquisitions from to adopted an implicitly millennial framework, looking forward to a new era when international tensions over access and benefit sharing were resolved, and national laws implementing international agreements provided requisite legal certainty for pgrfa providers and recipients alike. in , it seemed reasonable to expect that this state of affairs could be achieved within a few years. since that time, tensions over access and benefit sharing have increased, and the possibility of arriving at a set of final international agreement(s) on the issue appears to have receded still further into the distance. over the course of the decade - , the cgiar center genebanks received a surge of pgrfa from providers around the world, with permission to make those materials available through the plant treaty's multilateral system of access and benefit sharing. most of those newly deposited materials were associated with an internationally coordinated project that provided financial and technical support for the providers to regenerate and safety-duplicate unique, at-risk pgrfa in ex situ collections around the world. the regeneration and cwr projects, the bsf project cycles, and the cgiar genebanks' experiences over the last ten years highlight the critical importance of internationally coordinated projects to motivate and instill otherwise dispersed and disconnected actors with a sufficiently shared sense of purpose, to make materials available through the multilateral system. those projects and experiences also underscore the importance of providing modest levels of financial support to cover both the providers' and the recipient genebanks' costs associated with collecting, multiplying, health-testing, sending, and receiving pgrfa, and of providing technical support/training to providers/partners. the number of collecting expeditions organized by cgiar centers in this period was higher than in the previous decade, but lower than some national genebanks which play an important role in the global system of conservation, sustainable use, and exchange of plant genetic resources. increasingly sophisticated and globally coordinated gap analyses are making it possible to identify where gaps in collections exist. gap analyses presented in this paper highlight that ex situ collections managed by the cgiar represent well the cultivated gene pool of some crops, while there are significant gaps for other crops. given the need to keep costs to a minimum, and the possibility of sharing responsibilities with other global system actors, cgiar centers must redouble their efforts to address the results of gap analyses in concert with other organizations that host internationally available collections. while financial support, scientific leadership, and international coordination are indispensable, so too are supportive policies. the cgiar genebanks' experiences highlight the importance of supportive policies, and conversely, the negative impacts of restrictive or unclear policies and laws, on their ability to acquire new materials to include in the international collections, and to distribute those materials to recipients around the world. in retrospect, it is perhaps surprising that over the same period of time, the international community launched and attempted to renegotiate the multilateral system of access and benefit sharing-with all the uncertainty that attends an international negotiation-and the centers' genebanks enjoyed a surge of new materials being made available to include in the international collections. during the same period of time, they continued to distribute an extraordinary diversity of pgrfa to recipients around the world (even more than in the previous decade), a fact which reflects the persistent need/demand for access to those materials for agricultural research and development, the deep rooted nature of the cgiar collections within the global system, and the positive influence of the centers' article agreements with the governing body of the plant treaty. however, with the suspension of the process to enhance the multilateral system, and widespread tensions concerning the governance of digital genomic sequence information, international disagreement over access and benefit sharing is becoming still more geopolitically polarized. there is a significant risk that this increased polarization could further undermine the willingness of a range of actors to make materials available through the multilateral system in general, and to the cgiar genebanks in particular. the centers highlight the importance of resolving those tensions to "head off" unintended potential negative impacts on the cgiar's mission, the global system, and the sdgs. rapid loss of biodiversity, climate change, the covid- pandemic, rising populations, depleted soils, and a range of other challenges make the conservation, availability, and use of pgrfa more important than ever. it is essential that the plant treaty (and nagoya protocol and ippc) is implemented in ways that support all actors in the global system to fulfill their roles. q . which of the following factors have affected the your genebanks' overall rate of acquisition of pgrfa from sources outside the cgiar? answer choices most relevant diversity is already collected no space left in the genebank for additional accessions no money for additional collecting missions no capacity/money to evaluate or characterize what is already in the genebank, so there is no point adding more restrictive or unclear policies or laws make it difficult to get permission to access new pgrfa unwillingness of countries to allow collecting missions or to share their ex situ materials provider's country cannot issue a certificate in compliance that satisfies the phytosanitary rules of the country hosting the my genebank other (please specify) q . please add additional information to further clarify your answer from above. if you chose more than one factor, please rank them in terms of their importance. answer choices number of requests for materials a general tendency towards more targeted requests (i.e., requestors are more specific about the range of materials they are seeking, so you end up sending less material per request) your responses to requests are more targeted (i.e., you spend more time in the past, with more information about your collection, determining which particular materials are best suited to the needs of the requestor, so you end up sending less material per request) you do not have sufficient resources to regenerate enough material to send materials in response to all requests requested materials do not meet requisite phytosanitary standards restrictive policies or laws or conditions make it difficult for the genebank to distribute pgrfa status of information available about materials in the genebank (e.g., characterization, evaluation, subsets, etc.) other (please specify) q . please add additional information to further clarify your answer from above. if you chose more than one factor, please rank them in terms of their impact (either positive or negative) on your distributions. q . are you sometimes asked for pgrfa that you do not have? if 'yes', please provide a brief description of the materials concerned and why they are being asked for. q . please describe the circumstances that you believe contributed to particularly steep spikes or dips in your genebank's rate of acquisition of pgrfa in any year or years between - (e.g., internationally coordinated projects, organizations looking to transfer collections, joint projects with national research organizations, etc.) q . please describe the circumstances that you believe contributed to particularly steep spikes or dips in your genebank's rate of distribution of pgrfa in any year or years between - (e.g., sources of demand linked to particular projects in particular countries, etc.) q . how many requests for pgrfa to include in your collection(s) has your genebank made to organizations outside the cgiar in the last years? (for materials in in situ and ex situ conditions) answer choices - - - other (please indicate number) answer choices (a) how many were explicitly rejected? (b) how many were ignored (i.e., simply no answer)? (c) how many were accepted, but materials are not yet acquired by your center? (d) how many were accepted and the materials have actually been acquired by your center? if (a) to (d) do not describe what happened, please describe the outcome q . was there a difference in the kinds of responses you received depending on whether you were seeking to acquire material from in situ conditions (therefore involving new collecting missions) or materials that were already in ex situ collections? please explain. q . was there a difference in the kinds of responses you received depending on the types of organizations to whom you addressed your request? please explain. q . would you have preferred to make more requests to acquire more pgrfa over the last years? q . are you concerned that unresolved international negotiations concerning digital genomic sequence information (dsi), and/or the suspension of negotiations to enhance the plant treaty's multilateral system of access and benefit-sharing, could, in the future, have a negative impact on the following: cgiar centers' genebanks ability to access to pgrfa to include in international collections? cgiar centers' ability to access, generate, use and share digital genomic sequence information (dsi)? centers' management of their article collections (including distributing pgrfa and related information)? willingness of some organizations to enter into partnership with cgiar centers? if you answered 'yes' to any of the above, please explain q . if you already have evidence of negative impacts as a result of the unresolved issues regarding dsi and the multilateral system enhancement, please provide details here. changing rates of acquisition of plant genetic resources by international gene banks: setting the scene to monitor an impact of the international treaty an overview of the u.s. national plant germplasm system's exploration program genebank operation in the arena of access and benefit-sharing policies collecting in central asia and the caucasus: u.s. national plant germplasm system plant explorations recent npgs coordinated expeditions in the trans-caucasus region to collect wild relatives of temperate fruit and nut crops report of the plant exchange office to the plant germplasm operations committee, the regional technical advisory committees, and the crop germplasm committees summary annual reports of the project acquisition of plant genetic resources through domestic and international plant explorations and associated capacity-building partnerships accessions conserved and available at the leibniz institute of plant genetics and crop plant research (ipk) originating at collecting expeditions organized by ipk, or with ipk participation from collecting missions from the dutch national genebank cgn: a general introduction; ncare: baq'a access to genes: linkages between genebanks and farmers' seed systems large scale germplasm screening for identification of novel rice blast resistance sources. front screening of wheat genotypes for leaf rust resistance along with grain yield plant genetic resources for food and agriculture: opportunities and challenges emerging from the science and information technology revolution seed banks and molecular maps: unlocking genetic potential from the wild genetic variation and population structure of oryza glaberrima and development of a mini-core collection using dartseq. front development and evaluation of a core subset of the usda rice germplasm collection combining focused identification of germplasm and core collection strategies to identify genebank accessions for central european soybean breeding predictive characterization of icarda genebank barley accessions using figs and machine learning the figs (focused identification of germplasm strategy) approach identifies traits related to drought adaptation in vicia faba genetic resources opinions and advice of the ad hoc technical advisory committee on the multilateral system and the standard material transfer agreement are the old international board for plant genetic resources (ibpgr) base collections available through the plant treaty's multilateral system of access and benefit sharing? a review the origins and adaptation of european potatoes reconstructed from historical genomes genetic identity in genebanks: application of the solcap k snp array in fingerprinting and diversity analysis in the global in trust potato collection structural genome analysis in cultivated potato taxa reconciling conflicting phylogenies in the origin of sweet potato and dispersal to polynesia a taxonomic monograph of ipomoea integrated across phylogenetic scales a global overview of cassava genetic diversity molecular markers as a tool for germplasm acquisition to enhance the genetic diversity of a napier grass (cenchrus purpureus syn author correction: genotyping by sequencing provides new insights into the diversity of napier grass (cenchrus purpureus) and reveals variation in genome-wide ld patterns between collections genotyping-by-sequencing reveals population structure and genetic diversity of a buffelgrass unlocking the genetic diversity of creole wheats genomic variation in diverse accessions of asian cultivated rice high throughput sequencing for plant virus detection and discovery complete viral genome sequence and discovery of novel viruses by deep sequencing of small rnas: a generic method for diagnosis, discovery and sequencing of viruses rapid and specific detection of yam mosaic virus by reverse-transcription recombinase polymerase amplification optimization of the composition of crop collections for ex situ conservation a gap analysis modelling framework to prioritize collecting for ex situ conservation of crop landraces demand for genetic resources and the u.s. national plant germplasm system van den houwe, i. the impact of the musa international transit centre-review of its services and cost-effectiveness, and recommendations for rationalization of its operations securing crop diversity for sustainable development genetic resources, international organizations, and improvement in rice varieties searching an ex situ collection of wheat genetic resources valuing pre-commercial genetic resources: a maximum entropy approach the distribution of benefits from public international germplasm banks: the case of beans in latin america saving seeds: the economics of conserving crop. genetic resources ex situ in the future harvest centres of the cgiar valuing genebanks. food secur andean potato diversity conserved in the international potato center genebank helps develop agriculture in uganda: the example the contribution of the international rice genebank to varietal improvement and crop productivity in eastern india transferring diversity of goat grass to farmers' fields through the development of synthetic hexaploid wheat the contribution of the ciat genebank to the development of iron-biofortified bean varieties and well-being of farm households in rwanda use and benefits of tree germplasm from the world agroforestry genebank for smallholder farmers in kenya dynamic conservation of genetic resources: rematriation of the maize landrace jala the tale of taro leaf blight: a global effort to safeguard the genetic diversity of taro in the pacific conserving genetic resources for agriculture: economic implications of emerging science decision-making tool for national implementation of the plant. treaty's multilateral system of access and benefit-sharing key: cord- -z bxsjug authors: martin, r. r.; tzanetakis, i. e. title: pathogen-tested planting material date: - - journal: encyclopedia of agriculture and food systems doi: . /b - - - - . -x sha: doc_id: cord_uid: z bxsjug abstract certification programs have been developed to provide plant material that meets a predetermined level of plant health. the primary objectives of these programs are to limit pathogen incidence in plant material in order to minimize losses by growers and prevent movement of harmful pests and pathogens that may harm the environment. for many fruit and nut crops, orchards are expected to remain productive for years or decades; thus, starting with plants of high health status is essential. the components of certification programs in terms of plant health will be outlined, along with the benefits of harmonizing these programs where possible to facilitate plant movement without increasing trade in plant pathogens. buffer zone an area surrounding or adjacent to an area for production of plants in a certification scheme designed to minimize the probability of spread of the target pathogens, pollen, or seed into or out of the block, to meet phytosanitary or other control measures as defined in a certification standard. certification standard comprehensive process established and authorized by a regulatory entity for the production of plants free of regulated pathogens, with predefined trueness-to-type or genetic purity. these regulations also define plant production, plant identification and labeling, and quality assurance requirements. generation or g-level it indicates the relationship of plants in a certification scheme to the original pathogentested plant material at the top of the scheme. regulations developed by certifying agencies specify the conditions under which each generation (g) level must be maintained in order to meet the standard. heat therapy a method used to eliminate viruses in which plants are grown at - c for - weeks. after this meristem tips ( . - . mm) are removed and used to regenerate plants. this results in the production of plants that are often free of targeted pathogens. once grown in tissue culture, rooted and planted in soil, the plants are retested for the targeted pathogens to determine their health status. index procedure to determine whether a given plant is infected by a targeted pathogen. it involves the transfer of a bud, scion, sap, etc. from one plant to one or more indicator plants sensitive to the virus. international standard for phytosanitary measures it is an international standard adopted by the conference of the meristem-tip (tissue) culture a pathogen-elimination technique whereby tissue pieces (cells) are separated from an organism and cultured in a sterile growth media apart from that organism. one of the preferred pathogenelimination methods for plants is 'microshoot tip culture,' which is effective for eliminating most viral, bacterial, and fungal contaminants. in this technique a meristem tip of less than . - . mm is extracted from the shoot and placed in sterile tissue culture growth media; the meristem then grows to a complete plant. phytosanitary measure any legislation, regulation, or official procedure with the purpose to prevent the introduction or spread of pests or diseases. large-scale production and globalization of agriculture has resulted in a narrow germplasm base for all major food crops being grown over vast areas, which increases the risk of epidemics that could impact food security on a broad scale (strange and scott, ) . in addition, the majority of the production of these crops is in areas far from their centers of origin and subjected to many plant pathogens that they did not coevolve with. cassava is an excellent example of this, as its center of origin is in south america, but african cassava mosaic virus, which causes a devastating disease in africa, is not present in south america (fauquet and fargette, ; thresh et al., ) . most of the production of these crops is isolated from new strains of pathogens as they evolve at centers of origin and are at risk of severe disease outbreaks should new strains of a pathogen be distributed with planting material. this is also true for most of the fruit, vegetable, and ornamental species that are grown commercially. concomitant with the globalization of crop plants is an increased risk of globalization of plant pathogens that can lead to serious epidemics. the classic example of such movement of a pathogen is phytophthora infestans, the causal agent of potato late blight in the mid-nineteenth century, where its impact on human suffering and migration is well documented (schumann, ) . yet, years later, new strains of this pathogen have emerged and continue to cause serious plant disease problems (vleeshouwers et al., ) . there are examples of outbreaks of all types of pathogens (fungi, bacteria, nematodes, and viruses) that have resulted from movement of plant pathogens with the movement of plant propagation material. to protect agricultural production from introduced pathogens, two types of phytosanitary systems have been employed. plant quarantine is generally used at the national level to restrict introduction of plant pathogens that are not present in a country or have limited distribution and active programs to eradicate or delimit the pathogen. there are also domestic quarantines that restrict the movement of plant material within a country to address pests and pathogens that have limited distribution or may have been deregulated as a federal quarantine pest. plum pox virus (ppv), golden nematode, light brown apple moth, ralstonia solanacearum race biovar , and phytophthora ramorum are examples of quarantine pathogens and pests in the united states. certification programs are national, state, or provincial programs used to produce planting materials, vegetative or seed, that are free of or have less than some predetermined threshold of various pathogens (waterworth, ) . these programs primarily target domestic pathogens for plants at the g -g ( figure ) level of certification programs. these programs aim to ensure that the planted crop has a pathogen(s) level low enough to minimize the risk of severe crop loss, increasing the chance of harvesting an acceptable crop. the pathogen tolerances allowed in certified plants depends on the pathogen, crop, and agroecosystem where the crop is grown. the primary goal of quarantine and certification programs is to facilitate movement of plants without increasing trade of plant pathogens. the objective of all plant certification programs is to provide a supply of healthy plants, which is accomplished through adherence to best management practices (bmps) that are science based together with a defined level of testing to monitor the effectiveness of the program. the application of bmps to the production system combined with audit testing has proven to be a very useful means of producing plants of high health status. certification programs should be reevaluated periodically to take into account new pathogens or vectors that have been reported in the region, or other changes that affect the pathogen, vector, host, or environment that may impact the integrity of a certification scheme. bmps require that a hazard analysis and critical control points evaluation of the entire certification system be completed. this process is used to: ( ) identify weak points and greatest risk factors to the system in terms of introduction of pathogens or pests, and ( ) establish inspection, testing and mitigation procedures, and record-keeping requirements (parke and grünwald, ) . once the weak points are identified, management practices are designed to prevent intrusion of pests and pathogens. mesh size on a screenhouse might be adjusted to account for the primary virus vectors in the region to protect a g block. for example, if pathogens in a region are vectored by aphids the screen size might be larger than in another region with pathogens vectored by thrips. vector management in field blocks of certified plants will be adjusted based on the vectors and pathogens present in the region. knowledge of the phenology of vectors is also important, because times of peak vector movement present the greatest risk of pathogen intrusion into the system; thus, vector control measures may well change during the year to take into account the risk of vector migration into a nursery. knowing which of the targeted pathogens are most prevalent in a region and how they are vectored are important considerations when developing bmps for a nursery (martin and tzanetakis, in addition to certification programs that are designed for mass production of planting materials of high health status for plant, food, and fiber production, there are extensive quarantine programs that have been developed in many countries to reduce the risk of introduction of exotic pests. though not the focus of this article, quarantine goes hand-in-hand with certification to provide an integrated system to protect agriculture and environment from plant pests and pathogens, thus its relation to certification will be touched on briefly. the rationale for a brief overview of quarantine is that many certification programs focus on domestic or endemic pathogens, with the assumption that quarantine pathogens are detected and eliminated at the stage where plants enter the country and need not be included in the domestic certification program. quarantine programs are operated primarily at the national level and deal with pathogens that are not known to occur in the country or are present at a very restricted level and have active programs targeted to eradicate or prevent further spread of the pest or pathogen (foster and hadidi, ; reed and foster, ; anonymous, ) . as examples, plant material of berry crops, citrus, grape, potato, sweet potato, pome, and stone fruit, etc. coming into the united states under quarantine are tested for exotic pathogens before they are released for propagation, but they could be released if found to be infected only with viruses already endemic in the country. in some cases plants may be held in quarantine for - years while graft indexing on indicators is carried out. to be effective, quarantine programs are dependent on the public understanding the risks associated with introducing exotic pathogens into natural and agricultural ecosystems. public in this sense includes anyone who may wish to transport plant material from a foreign country to their homeland, including hobbyist interested in a new ornamental or food crop to add to their garden or plant collection, plant breeders interested in new potential germplasm, plant pathologists interested in adding to their pathogen collection for scientific study, germplasm curators attempting to broaden the diversity of their collections, or growers interested in getting a head start on a new cultivar developed in a foreign country. also, an effective quarantine program needs to have an effective and efficient mechanism to introduce plant material into the country. this combination of an educated public that understands the risks of introducing foreign pathogens and pests, together with an efficient system to bring plant material through approved quarantine and testing facilities will reduce the temptation by individuals to do 'suitcase' imports that could threaten local environments and agriculture. the introduction of ppv into pennsylvania highlights the benefits of quarantine systems to exclude exotic pathogens. plum pox is a devastating disease first identified in the early s in bulgaria that has since spread through much of europe and the mediterranean countries, where it is the most serious disease of stone fruits. it was detected for the first time in the western hemisphere in chile in (herrera et al., ) and is now considered to be widespread there and has since been detected in argentina. ppv was detected in a localized area of pennsylvania in the united states in (levy et al., ) and an eradication program was implemented immediately. after years, the eradication effort was deemed successful, but the cost was in excess of $ million us dollars to eliminate the pathogen from a relatively small geographic area (welliver, ) . thus, quarantine programs can be an effective and economical way to reduce international movement of plant pathogens and pests to protect local agriculture and native flora. the international plant protection convention (ippc) is an international agreement on plant health to which countries are signatories. the secretariat of the ippc is provided by the food and agricultural organization of the united nations. the ippc has the mission to protect cultivated and wild plants by preventing the introduction and spread of pests and pathogens. they develop standards (international standards for phytosanitary measures (ispms)) that are recognized by participating countries and provide for science-based standards for the safe movement of plants and plant products. as an example, ismp no. (anonymous, b) provides recognized standard treatments to eliminate plant pathogens and pests, including fumigation, cold treatment, heat treatment, and irradiation; and ismp no. (anonymous, ) provides agreed on sampling levels that participating countries use when shipping plants or plant products internationally to protect against movement of quarantine pests and pathogens. the ippc develops standards for range of issues related to plant protection. once the standards are approved by the ippc member countries, they become an ismp. certification programs have been developed for many vegetatively propagated food crops over the past years hadidi et al., ) . the first certification programs were developed in the netherlands and germany in the early s for potato production when they realized that leaf crinkling, rolling, and mosaics were transmitted through the tubers to the next generation and that by selecting the most vigorous and healthy-looking plants for propagation, tuber production was improved greatly. they developed a program for plant inspection and production, which was adopted in the united states and other countries long before it was known that many of the diseases were caused by viruses. in the united states and canada the first seed potato production programs were established in (slack and singh, ) . visual inspections still play a major part in seed potato certification programs with inspections carried out early and midlate in the growing season to observe foliar symptoms, at harvest for observing tubers symptoms, and a postharvest inspection of plants grown from tubers to look for late-season infections (frost et al., ) . visual inspections are part of all plant certification programs. trained inspectors also watch for other problems, such as herbicide damage, cultivar mixtures or trueness-to-type issues, physiological disorders, etc. during visual inspections, thousands of plants can be observed in a relatively short time. in most programs there is also laboratory testing that is carried out on each tuber or 'seed' lot to identify viruses that may be present and to monitor for symptomless pathogens. as laboratory-based diagnostics are improved in terms of sensitivity, specificity, and costs, more programs are incorporating their use to improve the quality of the program. certification programs have been developed for a wide range of crops . testing methods including mechanical transmission to herbaceous hosts, immunological and molecular techniques (enzyme-linked immunocapture assay (elisa), polymerase chain reaction (pcr), etc.), isolation of fungi or bacteria, etc. that are used for detection of various pathogens are similar in all programs. with woody crops there is often the need to do graft transmission onto indicator plants to test for uncharacterized graft-transmissible agents (converse, ; hadidi et al., ) . there are many definitions for certification programs. north american plant protection organization (nappo) defines a certification program as: "a domestic program consisting of maintenance, multiplication, distribution, and production of plant materials intended for release either domestically or for export, under an officially sponsored certificate attesting to the status of the material" (lanterman et al., ) . many certification programs are based on a published standard that defines site selection and preparation, isolation distances from plants of the same species and other vegetation, number of inspections, record keeping on plant traceability so that tracebacks or traceforwards can be done if a problem should arise, a pest and disease management plan, records of all pest management activities, the conditions and protocols to be followed during plant or seed production, and types and amount of testing that needs to be done at each level in the propagation cycle. the selection of plant material that is trueto-type is an essential first step. the selected plant(s) is then subjected to pathogen testing using a range of laboratory and biological indexing. if infected with any of the targeted pathogens listed in the standard, the plants are subjected to 'cleanup.' after 'cleanup' the plants are retested to ensure the pathogen(s) have been eliminated. once determined to be free of targeted pathogens and true-to-type, this plant can be designated as a g (figure ) plant that enters into the certification program. in many cases these plants are maintained in protected culture (screenhouse) and become the source plants that are propagated in certification programs. there is an effort in the united states to develop auditbased certification programs that rely on bmps outlined in the certification standard (thompson, ) . an audit-based program relies on compliance monitoring and requires a reasonable level of trust between the regulatory agency (inspectors) and nursery managers for the certification systems to function. the audit or inspection assesses the degree of compliance of the nursery to the certification standard, so that plants can be certified to meet that standard. an auditor or inspector designated by the certifying agency is responsible for the audit process. all records of the nursery can be reviewed by the auditor (thompson, ) . with such a system in place, record keeping by the nursery becomes even more critical as the inspector's decision will be based on the records reviewed, visual inspection of the nursery, and some limited testing. the goal is to have a systems approach that uses science bmps for nursery production of plant materials (parke and grünwald, ) . the use of pathogen-tested planting material is the first and arguably the most important step for the control of many systemic plant pathogens. most effort in this area has focused on viruses, viroids, systemic bacteria, and phytoplasmas because there are no postinfection treatments that can be used in plant or food production systems to rid plants of these pathogens. many important systemic pathogens are not transmitted through the seed or transmitted to less than % of the seedlings, or transmitted as seed coat contaminants that can be controlled with various seed treatment methods (ling, ; liu et al., ) . in this case, seedlings free of the targeted pathogen(s) can be identified and then grown in isolation to produce seed free of these pathogens or with an incidence below some predetermined threshold. with seed coat contaminants the seed can be treated to inactivate pathogens on the seed surface (ling, ) . in the case of vegetatively propagated crops it is often necessary to eliminate a specific pathogen, usually through thermal-, cryo-, or chemotherapy combined with meristem-tip culture to produce plants free of the targeted pathogens (mink et al., ; laimer and barba, ) . the plants regenerated from such meristems are then thoroughly tested, and a single plant free of pathogens of interest is the starting point for massive vegetative propagation. in the case of seed or vegetatively propagated plants, the plants are propagated under a defined set of conditions described in a certification standard. certification standards often have tolerance levels of some small percentage of infection, defined requirements for the site where the crop (seed or vegetative) is grown, required field inspection(s) during the production cycle that include cleanliness in terms of weeds, other crops that may serve as contaminants in seed lots or as hosts for targeted pathogens and freedom from pathogen vectors. tolerances for pathogens depend on the rate of pathogen spread for annual crops, but tolerances are much lower for perennial crops. crops that are only grown for a single year or a few years in production fields, such as most seed crops and potatoes, strawberries, sweet potatoes etc., may have higher tolerances than most of the fruit and nut crops, such as citrus, tree fruits, grapevines, or berries, which are expected to be productive for many years or decades in the same field. to set tolerances for various pathogens requires sampling and testing, and a major concern is how many samples to test. the number of samples that need to be tested per lot to achieve a confidence level (i.e., % or %) that a pathogen is not present is outlined in ispm no. (anonymous, ) . in some countries, certification schemes are managed at the national level and in others at the state or provincial level. plant pathogens are recognized as major constraints to agriculture production worldwide. most fruit and nut crops; major food crops such as potato, cassava, sweet potatoes; and many ornamentals are vegetatively propagated, using cuttings, tubers, or rhizomes. more recently, tissue culture propagation has become a major component of mass propagation for many of these crops. elaborate protocols are used to eliminate targeted pathogens from one or a few infected plants to provide a source of plant material free of these pathogens (mink et al., ; laimer and barba, ) . plants entering a certification program are often advanced selections, cultivars, or varieties developed in breeding programs. in some cases breeders work closely with the cleanup programs and get plants into the testing and cleanup phase before cultivar release, in an effort to have certified plants available at the time of release. another source of material entering certification programs are 'heritage' cultivarscultivars that have not been used for many years but are maintained at germplasm repositories. as requests for these cultivars have increased, there is a need to confirm that they meet current certification standards before commercial nurseries are willing to add them to their production systems. in the united states, there is an effort to have new cultivars coming into the country, as well as those developed in-country go through one of the 'clean plant centers' that have been developed over the past years. increased funding for these programs at the federal level since has provided for capacity building and the ability to process materials in a more timely manner to meet the needs for food production. these plants are tested for target pathogens before they enter into a g block, then a certification program and made available for production. in many cases, the g block is maintained by federal, provincial, or state government agencies, or some type of government/private entity, though recently private companies are maintaining their own proprietary g germplasm. these plants are tested on a predetermined schedule to monitor for reinfection, in addition, these blocks are retested if a new virus is discovered in the crop that may have been missed with the testing procedures used previously. producing g plants . candidate plant (advanced selections, heritage, new, or imported cultivars) arrive at clean plant center, (time ¼ , t¼ ). . testing program to determine the health status of the plants with respect to targeted pathogens listed in the certification standard, grafting for many fruit and nut crops. this process can take up to three years for crops, such as grapevines or fruit trees, but less time for other crops (t ¼ . - years). if no grafting is required this can be completed in several weeks; this is the case for some crops, such as potatoes. . if negative for all targeted pathogens, the plant enters the g block. if infected, the plants are put through therapy treatment ( - months), meristem-tip cultured and whole plants regenerated ( - months). . these plants are then retested and, if 'clean,' enter the g block; but if still positive they go through the therapy again (t¼ . - years). this is repeated until 'clean' plants are obtained. each cycle of testing can take up to - years for crops like grapevines or fruit trees, - years for strawberry or rubus. in most cases protocols have been developed such that there is a reasonable likelihood of having 'clean' plants after the initial cycle of therapy. however, if three cycles of cleanup were needed to get plants free of targeted pathogens this can easily require - years, depending on the crop. there is a misconception among many growers and researchers that 'tissue culture' propagated is synonymous with virus free. this is not the case. to eliminate viruses it is necessary to regenerate plants from meristematic tip, generally less than . mm and often in combination with some type of therapy (thermal, cryo, or chemo) before removing meristems (mink et al., ) . some viruses move into the meristematic tissue quite effectively, and a combination of thermal therapy or chemotherapy are required to get meristematic tissue free of the virus (chen and sherwood, ) . in the past these were referred to as 'heat stable' viruses and were 'difficult' to eliminate using thermal therapy and meristem-tip culture, whereas 'heat labile' viruses were much easier to eliminate. one now know that many of the 'heat labile' viruses are phloemassociated viruses, such as luteoviruses and closteroviruses, and the reason they are 'heat labile' is that the phloem tissue is not differentiated in the meristematic dome. thus, the virus does not move readily into the meristem. this is the case for the grapevine leafroll-associated viruses, which are in the closteroviridae family. in raspberry, raspberry leaf mottle and raspberry leaf spot were considered 'heat labile' as they were relatively easy to eliminate. these two diseases are caused by strains of raspberry leaf mottle virus, which is a closterovirus and phloem limited. raspberry bushy dwarf virus (rbdv) was considered a 'heat stable' virus because it is difficult to eliminate using thermal therapy and meristem-tip culture. rbdv infects most cell types and is not restricted to phloem tissue, thus, it likely moves into the meristematic dome much sooner than phloem-restricted viruses. the ease of obtaining meristems free of a virus in combination with heat therapy is not related to the heat stability of the virus, but rather how rapidly or effectively it can invade the meristematic tissues. the 'cleanedup' g plants are maintained under clearly defined conditions to minimize the risk of reinfection and should be free of all targeted pathogens outlined in the certification standard. in many cases the g plants are maintained in protected culture, such as in a screenhouse. the g plants are then sold to private nurseries for mass propagation under a set of conditions outlined in a certification program that is managed by a regulatory agency. the goal is to have a systemsbased approach that addresses risks of reinfection and pathogen spread during plant propagation. there are multiple cycles of plant increase for vegetatively propagated crops and unfortunately a wide range of terms have been used to identify each cycle (figure ). in , the nappo (rsmp no. ; anonymous, ) suggested the use of a simple terminology for the cycles of vegetative propagation. g plants are the plants that have tested negative for all targeted pathogens outlined in the certification scheme. g plants propagated by tissue culture or by traditional vegetative propagation methods become g plants, and multiple cycles of tissue culture can be carried out and still retain g status. the use of tissue-culture propagation and the number of propagation cycles allowed in tissue culture should be defined in the certification standard and will vary depending on the crop and certifying agency. g plants are derived from g or g plants. g plants are grown as potted plants that are propagated from g , g , or g plants and grown at another location. most existing programs use the various terminologies shown in figure , but follow this basic scheme of scale up in stages - to produce plants that are sold to growers. with the application of tissue culture in some propagation schemes, nurseries are able to sell g or g plants to growers, which should translate into higher health-status plants being used by the industry. however, for many crops conventional vegetative propagation is still the primary means of plant multiplication that is used. there are several reasons for this: ( ) costs for conventional propagation of crops, such as strawberry, where a - fold increase can be obtained in field production each year, is relatively inexpensive to produce millions of plants; ( ) the agency that regulates the certification program may prohibit or limit the number of cycles of tissue culture as a means of propagation due to concerns about somatic mutations; ( ) grafted plants may use a rootstock that is generated from seed, which is inexpensive. in such cases, the rootstock may be seed-propagated and the scions by tissue culture or by conventional vegetative propagation methods. during propagation it is important that care is taken to prevent contamination of tools with viruses that are readily mechanically transmitted. tools are sterilized during tissue-culture propagation to maintain sterile conditions during transfers of tissue-culture plants. it is also necessary in conventional propagation to prevent spread of viruses during the cutting of tubers, rhizomes, taking cuttings, etc. (lewandowski et al., ) . contamination using cutting tools is much more likely in herbaceous crops, such as potatoes, than in woody crops like grapevines, tree fruits, nut crops, etc. there are efforts among regional plant protection organizations (rppos) to harmonize quarantine standards between the member countries. there are nine rppos (asian and pacific ppo; caribbean plant protection commission; comite de sanidad vegetal del cono sur; comunidad andina; european and mediterranean ppo (eppo); interafrican phytosanitary council; near east ppo; north american ppo; organismo internacional regional de sanidad agropecuaria and pacific ppo (roy, ) ). there are also efforts at harmonizing certification standards across some of the rppos, such as for fruit trees and grapevines in nappo countries, and the eppo countries have adopted certification schemes for crops (roy, ; eppo website) . these rppo-developed standards are often a minimum standard that is required, but member countries can require a more stringent standard internally. although this is happening at the international level, there are many cases where the harmonization of certification programs within countries has not happened. in countries where these programs are regulated at the province or state level, there are often significant differences between the certification standards. for example, in the united states some states require that the g plants for some crops be maintained within protected culture (screenhouse) to minimize vector transmission of pathogens, whereas other states do not require that same level of protection. also, the type and level of testing required at each level in the certification scheme can vary between states. there is an effort underway to harmonize certification standards for some of the fruit crops across the united states. as these programs are developed, they are looking at certification schemes in other countries and rppos with the goal of harmonizing standards on a broader scale if possible. for seed propagated crops, seed is often increased in a country other than where the seed will be planted for food or fiber production. to speed up increase of seed for commercial production many companies grow seed in the northern and southern hemisphere to get two cycles of increase in a single year. as a result, seed is moved between countries on a frequent basis and are potentially exposed to quarantine or certification pests of concern in the country where the final crop is grown. also, because seed has a long shelf life, it may be moved to several countries before it is planted, or a seed lot may be subdivided and shipped to multiple countries. many countries have phytosanitary requirements for the movement of seed though requirements for field inspections, sampling, and testing can vary and cause problems if trying to move the seed internationally. the ippc's ispm no. provides information on agreed on treatments for a range of regulated plants pathogens and pests (anonymous, a) . the preparation of a phytosanitary certificate is done by the exporting country but must meet the requirements of the importing country.thus, if a seed lot is being shipped to multiple countries the documentation can become very complicated. ippc has also developed ispm no. , which covers the reexport of the seed to a third country, where the phytosanitary requirements would have to be met by the original exporting country (anonymous, ) . for these reasons, harmonized standards could greatly facilitate seed trade to meet the needs of the increasing globalization of agriculture. there are a number of agencies for seed testing and accreditation of certification schemes. the testing and accreditation by these agencies are recognized by various countries or groups of countries and provide a mechanism to harmonize seed certification standards for international movement of seed among a group of countries. the association of official seed analysts, association of official seed certifying agencies, international seed trade association (ista), national seed health system, oecd seed schemes (us), society of commercial seed technologists are all involved in seed testing and are recognized by multiple countries. ista testing and accreditation is recognized by more than countries. nappo regional standards for phytosanitary measures no. provides information on the movement of seed between countries in north america (anonymous, ) . for seed production, the emphasis is on eliminating pathogens from the elite germplasm that can be transmitted through seed or contaminate the seed surface, including viruses, bacteria, and fungi. in many cases, elite germplasm of seed crops is produced and maintained by private companies. with seed certification, genetic purity is often as great a concern as pathogen contamination. there are multiple levels in certification programs for seed crops, similar to those used for vegetatively propagated crops. the seed crops would clearly fit under the g terminology shown in figure , and in this case g would stand for generation in the true sense of the word. the four common levels in seed certification (aosca) include: breeder seed (seed controlled by the plant breeder, or the institution or company where the breeder works), foundation seed (propagated from breeder seed under conditions that retain genetic purity, identity, and health status), registered seed (propagated from breeder or foundation seed under conditions that maintain genetic purity, identity, and health status), and certified seed (progeny of breeder, foundation or registered seed and grown under conditions that maintain genetic purity, identity, and health status). in addition to genetic purity, identity, and health status, seed certification also requires information on percentage germination, date of germination test, and percentage contamination with other seed. for pathogens that are transmitted internally in the seed, it is often possible to grow out seed and identify plants free of the pathogen because this type of transmission is rarely % efficient (liu et al., ; mink, ) . the exceptions to this are the cryptic viruses, which are seed transmitted at %. these viruses are not known to cause disease and are not considered in quarantine or certification programs. heat treatments for eradication of embryo infection by virus have not been successful without a loss in seed viability (maury et al., ) . for seed contaminants, various types of seed treatment have been used to eliminate or reduce pathogen level below set tolerances. dry heat at c for h, followed by c for h, and finally c for h was very effective at controlling very stable viruses, such as tobamoviruses (tobacco mosaic virus, tomato mosaic virus, and cucumber green mottle mosaic virus, lee, ) and potexviruses (pepino mosaic virus, ling, ) as well as for a wide range of fungal and bacterial pathogens with little or no effect on seed germination (lee, ) . soaking tomato seed for min in a % sodium hypochlorite solution (dilution of commercial bleach, depends on the percentage active ingredient in the bleach product) containing . % triton-x- as a wetting agent completely inactivates potexviruses (pepino mosaic virus; ling, ) on the seed surface with little or no impact on seed germination. hydrochloric acid or trisodium phosphate were not as effective as bleach in eliminating virus from seed coats (ling, ) . with woody plants that involve graft transmission assays for detection of viruses, grafted plants may need to be observed for two or more years for symptoms before the plant is determined to be free of the pathogen. these potentially long intervals for detection using biological indexing has prompted most quarantine facilities to adopt laboratory-based testing where possible (rowhani et al., ; martin et al., ; , including: electron microscopy, elisa, immunospecific electron microscopy, nucleic-acid-based techniques such as pcr or reverse transcription -pcr (rt-pcr) combined with gel electrophoresis and sequencing of the any amplicons obtained, double-stranded ribonucleic acid (dsrna) detection; and mechanical transmission to herbaceous hosts (reed and foster, ; miller et al., ). however, with widely used tests currently available, there is still a need for biological indexing onto woody indicators for some exotic pathogens. for many of the crops there are various laboratory tests available for the major pathogens of concern. thus, in some cases, once all laboratory tests have been completed and found negative, plants are released to the importer on a provisional basis while the biological indexing is completed. this is done with an agreement that in case the tests are positive, the plant will be destroyed. this process allows the importer to begin multiplication of a new cultivar, which in some cases can reduce the time to get the materials to production fields by - years. the plants must be maintained and propagated under quarantine-approved conditions until all testing is completed. the currently used assays for virus detection (elisa, pcr, and q-pcr) are great for detecting a virus in a large number of samples, such as in surveys or ecological or epidemiology studies. however, for quarantine and certification programs a method that was capable of detecting all viruses in a single test would be ideal, rather than doing individual tests for each virus known to infect the host. in some crops, this means - tests per plant (berries or grapevines). recent work with macroarrays has shown great promise for detecting the most common viruses in grapevine, with viruses detected in a single test (thompson et al., ) . potentially, within the next five years as 'deep' or 'nexgen' sequencing becomes more widely adapted (studholme et al., ; kreuze et al., ) , and universal plant microarrays are perfected (hammond, ) they could replace indexing. this would require extensive validation of these technologies to ensure they are as good or better than current methods. the advantage of these technologies is that they have the potential to provide information on any virus in a plant without any a priori knowledge of the virus, in contrast to other laboratory techniques that detect known viruses (kreuze et al., ; al rwahnih et al., ; kashif et al., ; thekke-veetil et al., ; seguin et al., ; hammond, ; esteban et al., ) . wang et al. ( ) demonstrated the feasibility and utility of the microarray technology to identify and characterize new viruses. they developed microarrays containing oligonucleotides that represented conserved sequences of all fully sequenced human respiratory viruses, which at the time represented a few hundred viruses. using this array they identified a novel coronavirus and showed that it caused severe acute respiratory syndrome, a newly emerging disease at the time (wang et al., ) . nexgen sequencing has a huge impact on many aspects of biology and is used in virus discovery and is being investigated for virus diagnostics. sequencing of total nucleic acids in plants has led to the identification of multiple viruses and viroids in single plants (adams et al., ; al rwahnih et al., kreuze et al., ; sequin et al., ) and offers the potential to certify plants as virus-free rather than virus-tested. obtaining the 'virome' of a plant provides much information on the range of viruses and their diversity within a single plant. however, work with grapevines has shown that many of the viruses were related to mycoviruses rather than plant viruses (al rwahnih et al., ; coetzee et al., ) . this leads to questions on the significance of these viruses in plant health. the new technology will allow for rapid discovery of many new viruses; unfortunately, characterizing the biological significance of these viruses will take much longer. mycoviruses in grapevines may actually be modifying endophytes and indirectly impacting the health status of grapevines. thus, eventually how viruses and other microorganisms impact the whole plant (understanding the microbiome) may be important in certification and quarantine, but we are not there yet. although it is good to know what is in the plant, this technology is resulting in the identification of many new viruses for which there is no biological information. for the next decade it is likely that the biological significance will lag behind the discovery of microorganisms in plants, and it is important that for certification and quarantine programs, any changes are made in response to the biology of these organisms rather than their presence. it seems reasonable that new plant viruses related to known plant pathogenic viruses should be considered as the highest priority for evaluating their biological significance and determining if they should be part of quarantine and certification regulations. certification standards vary widely among crops and regulatory authorities. the viruses included in a certification program can vary from country to country or state to state. an excellent example is grapevines where certification in italy includes more viruses than programs in germany or france; or grapevine certification in washington state is different from california. some of these differences are due to environmental considerations, where infection by a pathogen may cause severe disease in one setting, such as crown gall in grapevine in new york state, but be latent or symptomless under different environmental conditions, such as crown gall in grapevine in california. attempts to harmonize certification schemes across boundaries to facilitate trade of plants without increasing trade in plant pathogens will require certification programs to account for disease expression by pathogens in areas where the certified plants may be sold, rather than only where the nursery stock is produced. in the united states, where certification programs are regulated by individual states, there are efforts underway in multiple crops (blueberry, grapevines, hops, rubus, strawberry, and fruit trees) to develop a single certification standard that all states with programs for that crop would adopt. if successful, this in essence would provide a national program for certification of these crops. as this process is developing, there is an ongoing communication with trading partners to harmonize these standards as much as possible with their certification programs to facilitate international trade. next generation sequencing and metagenomic analysis: a universal diagnostic tool in plant virology deep sequencing analysis of rnas from a grapevine showing syrah decline symptoms reveals a multiple virus infection that includes a novel virus deep sequencing evidence from single grapevine plants reveals a virome dominated by mycoviruses rspm no. , guidelines for international movement of pome and fruit trees in a nappo member country phytosanitary principles for the protection of plants and the application of phytosanitary measures in international trade ispm no. , phytosanitary treatments for regulated pests glossary of phytosanitary terms ispm no. , international standards for phytosanitary measures ispm no. . phytosanitary certificates rspm no. , phytosanitary guidelines for the movement of seed evaluation of tip culture, thermotherapy and chemotherapy for elimination of peanut mottle virus from arachis hypogeae deep sequencing analysis of viruses infecting grapevines: virome of a vineyard virus diseases of small fruits. u.s. department of agriculture agriculture handbook no. a diagnostic oligonucleotide microarray for simultaneous detection of grapevine viruses african cassava mosaic virus − etiology, epidemiology, and control plant virus disease control integrated control of potato pathogens through seed potato certification and provision of clean seed potatoes virus and virus-like diseases of pome and stone fruits plant virus disease control universal plant virus microarrays, broad spectrum pcr assays, and other tools for virus detection and identification survey of sharka disease (plum pox virus) on stone fruit trees in chile detection of viruses in sweetpotatoes from honduras and guatemala augmented by deep-sequencing of small-rnas complete viral genome sequence and discovery of novel viruses by deep sequencing of small rnas: a generic method for diagnosis, discovery and sequencing of viruses elimination of systemic pathogens by thermotherapy, tissue culture, or in vitro micrografting disease control through crop certification − woody plants advances in seed treatments for horticultural crops first report of plum pox virus (sharka disease) in prunus persica in the united states surprising results from a search for effective disinfectants for tobacco mosaic virus − contaminated tools effectiveness of chemo-and thermotherapeutic treatments on pepino mosaic virus in tomato seed pollen and seed transmission of cucumber green mottle mosaic virus in cucumber viruses and virus diseases of rubus new and emerging viruses of blueberry and cranberry high risk strawberry viruses by region in the united states and canada: implications for certification, nurseries and fruit production seed certification for viruses plant disease diagnostic capabilities and networks pollen-and seed-transmitted virses and viroids heat treatment of perennial plants to eliminate phytoplasmas, viruses, and viroids while maintaining plant survival a systems approach for the management of pests and pathogens of nursery crops exclusion of pome and stone fruit viruses, viroids, and phytoplasmas by certification and quarantine pathogen testing and certification of vitis and prunus species control measures of pome and stone fruit viruses, viroids, and phytoplasmas: role of international organizations plant diseases: their biology and social impact de novo reconstruction of consensus master genomes of plant rna and dna viruses from sirnas control of viruses affecting potatoes through seed potato certification programs plant disease: a threat to global food security application of high-throughput dna sequencing in phytopathology molecular characterization and population structure of blackberry vein banding associated virus, a new ampelovirus associated with blackberry yellow vein disease certified − feasibility of audit-based certification to prevent invasive plant pests in the horticultural industry profiling viral infections in grapevine using a randomly primed reverse transcription-polymerase chain reaction/macroarray multiplex platform the control of african cassava mosaic virus disease: phytosanitation and/or resistance? understanding and exploiting late blight resistance in the age of effectors microarray-based detection and genotyping of viral pathogens viral discovery and sequence recovery using dna microarrays certification for plant viruses − an overview plum pox virus case study: the eradication road is paved with gold key: cord- -pyfc jde authors: lico, chiara; chen, qiang; santi, luca title: viral vectors for production of recombinant proteins in plants date: - - journal: j cell physiol doi: . /jcp. sha: doc_id: cord_uid: pyfc jde global demand for recombinant proteins has steadily accelerated for the last years. these recombinant proteins have a wide range of important applications, including vaccines and therapeutics for human and animal health, industrial enzymes, new materials and components of novel nano‐particles for various applications. the majority of recombinant proteins are produced by traditional biological “factories,” that is, predominantly mammalian and microbial cell cultures along with yeast and insect cells. however, these traditional technologies cannot satisfy the increasing market demand due to prohibitive capital investment requirements. during the last two decades, plants have been under intensive investigation to provide an alternative system for cost‐effective, highly scalable, and safe production of recombinant proteins. although the genetic engineering of plant viral vectors for heterologous gene expression can be dated back to the early s, recent understanding of plant virology and technical progress in molecular biology have allowed for significant improvements and fine tuning of these vectors. these breakthroughs enable the flourishing of a variety of new viral‐based expression systems and their wide application by academic and industry groups. in this review, we describe the principal plant viral‐based production strategies and the latest plant viral expression systems, with a particular focus on the variety of proteins produced and their applications. we will summarize the recent progress in the downstream processing of plant materials for efficient extraction and purification of recombinant proteins. j. cell. physiol. : – , . © wiley‐liss, inc. recombinant dna technology was initially used to express proteins that were difficult to produce in their native organisms. increasing efforts, however, have been focused on designing new molecules with more desirable characteristics and/or functionality. pharmaceuticals and industrial enzymes were the first recombinant biotech products on the world market and biopharmaceuticals were the majority of commercialized recombinant proteins (pavlou and reichert, ) . many protein-based drugs, similar to traditional small molecule pharmaceuticals, function as antagonists by binding to and thereby inhibiting the activity of their target, such as an enzyme or a receptor. classical protein antagonists include full monoclonal antibodies (mabs), their single-chain derivatives (scfv) and mab-fusion proteins. recent research programs have also focused on non-antibody antagonists that consist of a scaffold protein displaying the inserted affinity peptide (walsh, ) . recombinant dna technology also provided an excellent alternative for developing safer vaccines. subunit vaccines are based on immunodominant protein components of a pathogen, but do not contain its genetic material. consequently they cannot replicate, cause disease, or introduce pathogens into non-endemic regions. viral coat proteins are exceptional subunit vaccine candidates and in some cases are able to form virus-like particles (vlps) when expressed in heterologous systems. in fact, the only recombinant subunit vaccines presently available are based on vlps. they are highly immunogenic and able to induce both humoral and cellular responses (chackerian, ) . in addition to the pharmaceutical industry, many other fields are also relying intensely on recombinant proteins. areas as diverse as agro-food technology, chemistry, detergent production, bioremediation, biosensoring, petroleum, and paper industries all receive significant contribution from applications of recombinant proteins. for example, increasing needs for a diversity of food processing enzymes, for example, amylase, lipase, xylanase, pullulanase and pectin modifying enzymes, demand a substantial involvement of recombinant protein technology (olempska-beer et al., ) . in the coming years, there will be a significant increase in demand for high quality recombinant proteins. in response, biological systems used for the production of proteins must be scalable, cost-effective, safe and flexible enough to meet market requirements. current systems rely on ''bio-factories,'' that is, mammalian, insect, yeast, and microbial cell cultures. the majority of the recombinant proteins are currently produced in escherichia coli or mammalian cells with a few exceptions of yeast or insect cells (yin et al., ) . all of these bio-factories are based on fermentation technology of suspension cells in bioreactors, which requires an enormous upfront capital investment and, thereby, severely constrains their scalability. the use of plants as production systems for recombinant proteins has been actively investigated over the last two decades. plants are attractive as protein factories because they can produce large volumes of products efficiently and sustainably and, under certain conditions, can have significant advantages in decreasing manufacturing costs (hood et al., ; giddings, ) . plant systems are far less likely to harbor microbes pathogenic to humans than mammalian cells or whole transgenic animal systems. in addition, one of the major advantages of plants is that they possess an endomembrane system and secretory pathway that are similar to mammalian cells (vitale and pedrazzini, ) . thus, proteins are generally efficiently assembled with appropriate post-translational modifications. these cost, scale, and safety advantages make plant-made pharmaceuticals very promising for both commercial pharmaceutical production and for manufacturing products destined for the developing world. three approaches are commonly used to express heterologous proteins in plants: ( ) stable transformation of the nuclear genome, ( ) stable transformation of the chloroplastic genome, and ( ) viral transient transformation. in stable transformation technology, an expression cassette harboring the exogenous gene of interest is integrated into the nuclear or plastid genome of plant cells, which results in the acquired character to be stably inherited over generations. these lines can be propagated vegetatively or by seed and thus, readily scaled up for protein production. stable nuclear transformation is often achieved using agrobacterium tumefaciens, which delivers fragments of dna into the plant cell nucleus at random positions. alternatively, the ''biolistic'' method (microprojectile bombardment) can be used for plant hosts that are difficult to transform by agrobacterium (hansen and wright, ) . due to their double membrane structure, chloroplast transformation can be achieved only by bombardment with tungsten or gold particles coated with fragments of dna. stable transformation of the chloroplast offers several distinct advantages in areas of transgene targeting, product yield, and regulatory compliance. since the chloroplast genome allows for homologous recombination, transgenes can be precisely targeted to a specific locus of the genome to avoiding positional effect or accidental gene knock-outs. each plant cell has several chloroplasts and each of these contains many circular genomes. therefore, the shear copy number of the transgene enables high-level expression of the target recombinant protein. since the chloroplast is inherited maternally, this technology reduces the risk of potential transgene escape by pollen dissemination. similar to bacterial cells, however, the chloroplast is unable to perform typical eukaryotic posttranslational modifications, such as glycosylation. as a result, this technology cannot be used to produce proteins when such modifications are essential for their function (bock, ) . the third strategy relies on replicating plant viruses. these viruses are small, can be easily manipulated, and their infection process is relatively simple. the above features make viral vectors an attractive alternative to stable transgenic systems for the expression of foreign proteins in plants. in this strategy, the gene of interest is inserted among viral replicating elements, episomically amplified, and subsequently translated in the plant cell cytosol. most of the transient expression systems are based on non-food, non-feed plants like tobacco, therefore, requiring further purification prior to application. production of recombinant proteins with stably or transiently transformed plants has been performed both in green-houses and in field. neither practice requires large investments in hardware or culture media, thus making scale-up more economical than fermentation cultures. the possibility of producing recombinant protein agents on an agricultural scale by ''molecular farming'' is extremely attractive. although the use of plants as expression systems for recombinant proteins was first conceived for oral delivery of antigenic proteins (walmsley and arntzen, ) , plant-made recombinant proteins have also been purified and used in many different applications. an enormous number of proteins different in size, structure, origin and biological function have been successfully expressed in stably transformed monocot and dicot plants such as maize (chikwamba et al., ) , rice (nochi et al., ) , wheat (brereton et al., ) , potato (youm et al., ) , tomato (huang et al., ) , tobacco (watson et al., ) , lettuce (sun et al., ) , alfalfa , lupin (smart et al., ) , carrots (marquet-blouin et al., ) , barley (joensuu et al., ) , soybean (moravec et al., ) , and thale cress (carrillo et al., ) . many antigenic monomeric proteins from viruses (webster et al., ) and prokaryotes (alvarez et al., ) have been produced and in a few cases, their immunogenic properties have been successfully evaluated in human volunteers during phase i clinical studies (tacket et al., (tacket et al., , thanavala et al., ) . complex oligomeric mabs were first expressed in stable transgenic plants by hiatt et al. ( ) . since then, the number and type of plant-expressed antibodies, such as secretory antibodies, antibody fragments and, more recently, immune complexes, have increased steadily. several commercial candidates of mabs have been developed, with five mabs under clinical evaluation, and two having reached phase ii clinical studies . in addition to pharmaceuticals, recombinant proteins for a variety of applications have also been expressed in transgenic plants. examples include alpha-amylase enzyme (pen et al., ) , the protein brazzein for commercial sweeteners (lamphear et al., ) , and the fungal enzyme manganese peroxidase (clough et al., ) . moreover the chicken avidin diagnostic protein (hood et al., ) and the bovine trypsin enzyme (woodard et al., ) from transgenic maize, and the human anti-microbic proteins lysozyme and lactoferrin (humphrey et al., ) from transgenic rice have been commercially produced and are currently marketed by sigma chemical company (st. louis, mo). the major challenges facing transgenic plant technology lay in increasing the quantity of protein, the optimization of expression systems, and improvement of downstream processing. in this review, we will focus on transient production strategies using plant viral expression systems, with a particular focus on the variety of proteins produced, and their applications. we will also discuss the latest developments in the downstream processing of plant materials for efficient extraction and purification of recombinant proteins. the history of genetic engineering and applied virology are intimately connected; the first recombinant molecule assembled was a chimeric sv containing genes from the bacteriophage l (jackson et al., ) . the unique properties of viruses such as ease of manipulation, high level amplification, site specific recombination, strong infectivity, enhanced translation and compact and repetitive morphological structure have enabled their broad application, from basic research to product development, including the generation of robust expression systems. viruses with a variety of host ranges such as bacteriophages, mammalian retroviruses, invertebrate infecting baculoviruses, and plant viruses have been genetically modified to express heterologous proteins. from the discovery of viruses in (tobacco mosaic virus, tmv) (bos, ) , to the first demonstration of rnas role in virus replication by turnip yellow mosaic virus (tymv) (matthews, ) , to the very recent discovery of gene silencing and its implication in host response to infection, gene regulation and transgene expression (baulcombe, ; lu et al., ; waterhouse and helliwell, ) , plant virology has played a crucial role in the understanding of the most fundamental concepts of modern biology. in addition, plant viral elements such as promoters, terminators, translational enhancers and various cis-regulatory sequences have been extensively used in plant biotechnology. plant viruses have been used to introduce foreign genes in plants since the early s and technical advances in molecular biology and plant virology have allowed the generation of many improved expression systems. these recent systems provide many advantages including rapid, high-level transgene expression, and, in the case of movement defective systems, better transgene containment due to the lack of vertical and horizontal gene transfer. the earlier plant virus expression systems were based on the cauliflower mosaic virus (camv) of the caulimoviridae family, the only plant viruses with a double stranded dna genome. however, limited packaging capacity and the restricted amount of viral dna that can be removed without affecting essential functions hindered the application of these initial expression systems. fortunately, technical advances in molecular biology such as generating cdna from an rna template have enabled us to expand our search for expression systems into single stranded rna viruses, the most represented types of plant viruses in nature. another technical breakthrough was the use of agrobacterium tumefaciens to promote viral infection (grimsley et al., ; turpen et al., ) . generally, viral infection can be initiated by mechanical inoculation of infectious viral particles on leaves or alternatively by transfection of nucleic acids. the use of agrobacterium tumefaciens, known as ''agroinfection,'' allows the direct and efficient targeting of cdna to the plant cell nucleus (fig. ) . the cdna constructs in the plant expression cassette would result in an infectious, autonomously replicating nucleic acid after host cell nuclear transcription and processing. moreover agroinfection allowed the use of viruses that in nature are not mechanically transmissible, but instead need a special insect vector to initiate the infection process. these viral vectors are commonly divided into gene substitution vectors, gene insertion vectors, modular or deconstructed vector systems and peptide display vectors. gene substitution or replacement vectors are based on the exchange of an endogenous viral sequence with a heterologous gene of interest. the first successful expression of a foreign gene ever achieved in plants was obtained using a substitution vector based on the camv. usually, the coat protein (cp) gene is the viral coding sequence of choice to be replaced. however, with a few exceptions, the cp is essential for cell to cell and/or systemic movement and infection. however, caution has to be taken if leaves of the whole plants are intended to be infected. for example, a cp replacement vector was developed using the tomato bushy stunt virus (tbsv). in this case the cp is not essential for systemic movement of the virus in certain species of nicotiana (scholthof et al., ) and it was indeed demonstrated that the insertion of a heterologous gene supports a systemic infection (scholthof et al., ) . in tmv-based substitution vectors, where the cp is necessary for systemic infection, a chloramphenicol acetyl transferase (cat)-cp gene replacement vector generated only local lesions and cat activity was confined on the inoculated leaf (takamatsu et al., ) . in another recently developed tmv-based gene substitution vector the cp gene was exchanged with the gene of interest fused to a thermostable carrier molecule, the beta- , - , -glucanase enzyme (lichenase) of clostridium thermocellum, to facilitate target expression, stability and purification. the heat tolerant property of the lichenase ( c) allows easy target proteins purification by heat treatment which precipitates up to % of contaminating plant proteins. the protein of interest, fused at the n-or c-terminus of lichenase, or inserted in the central loop of an engineered version of the enzyme, is protected from the degradation and thus purified. gene insertion vectors consist of complete functional viruses with the addition of an extra open reading frame (orf) for the target protein. they are capable of cell to cell and systemic movement and infection. viruses with both spherical and rod shaped virions have been investigated. viruses with rod-shaped particles have a better potential due to fewer constraints on the amount of nucleic acid inserted. however, there is still a limit on the genome size of the chimeric rod-shaped virion based vectors. it has been proven that chimeric vectors with genome size beyond certain limits resulted in unsustained virion assembly. two of the most famous gene insertion vectors have been derived from tmv and potato virus x (pvx). both viruses have a single stranded positive rna genome. they use subgenomic promoters and consequently subgenomic rnas to express some of their orfs and have a helical virion symmetry which results in rod-shaped particles. a chimeric tmv was constructed with the cat gene inserted between the movement protein (mp) gene and the cp gene; the expression was regulated by an additional copy of the subgenomic promoter of the cp gene that, as a result, was duplicated in the hybrid vector. the vector was able to replicate, to form subgenomic rnas, to assemble correctly, and to produce reporter gene activity. nevertheless the duplication of the subgenomic promoter sustained homologous recombination causing instability and the consequent loss of the exogenous gene (dawson et al., ) . this problem was solved by using a subgenomic cp promoter derived from a different virus belonging to the tobamovirus genus to prevent homologous recombination. similar problems and solutions of recombination between duplicated subgenomic promoters were reported in a recent insertion vector system based on the grapevine virus a (gva) for n. benthamiana (haviv et al., ) . analogous to tmv, a pvx-based vector was generated using the duplicated promoter of the cp gene. however in this case the vector was stable and able to retain the additional coding sequence (chapman et al., ) . nevertheless, recently it has been demonstrated that the size of the inserted gene of interest can play a crucial role in the stability of the modified pvx expression vector, with a positive correlation between the elimination rate of the gene of interest and its length (avesani et al., ) . recombination can involve homologous cp promoter sequences but also other mechanisms mediated by both the host plant and the virus (avesani et al., ) . another well established system is based on cowpea mosaic virus (cpmv) the type member of comoviridae whose bipartite genome is constituted by single stranded positive rna molecules. rna- encodes for proteins involved in replication while rna- carries the genetic information for the movement and structural proteins. the system is very versatile; it can be utilized in a totally transient fashion, co-infiltrating wild-type plants with two separate c-dna constructs representing rna- to sustain amplification and rna- harboring the gene of interest, or it can be utilized combining transgenic plants with virus-mediated transient expression. in this case the rna- is used for stable transformation and the rna- , necessary to prime the active replication of rna- and of the heterologous gene, is supplemented by agro-infiltration. it is worth noticing that a deleted version of rna- , unable to produce infectious clones, it has also been utilized to prevent possible environmental containment concerns (liu et al., ; sainsbury et al., ) . in conclusion it is possible to obtain an inducible expression system if rna- is supplemented by agroinfiltration and a constitutive stable expression system if it is supplemented by crossing with an rna- stable transgenic line (canizares et al., ) . modular or deconstructed vectors are a new generation of viral expression systems. their development was driven by the understanding that: ( ) not all viral components are essential or beneficial for an expression vector and ( ) viruses can be broken down into different genomic elements that would still operate together in the infection process as wild-type multipartite genome viruses. moreover, agroinfection provided the technical possibility to co-deliver multiple different components. hence in modular systems, viral components are separated into distinct portions and inserted into binary vectors contained in an agrobacterium strain. the strains are mixed together and co-infiltrated into plant leaves. this strategy allows the replicative portion, the so called replicon, to be reduced in size, down to minimal to accommodate the insertion of transgene, while other viral components necessary for the vector function can be provided in trans during the infection process. at present, one of the most famous and broadly used vector is the deconstructed system based on tmv and developed by icon genetics (halle, germany), recently acquired by bayer innovation gmbh. the vector has been engineered to divide the tmv genome into two major cdna modules: a module which contains the viral rna dependent rna polymerase and the mp, and a module that carries the gene of interest and the untranslated region (utr) of the virus essential for the efficient replication and amplification of the vector. in this particular case, viral functions are not complemented in trans but the two modules actually assemble together in vivo by a site specific recombinase delivered by a third agrobacterium cell line. furthermore, different modules carry different organelle targeting signals which, fuse in frame with the gene of interest after recombination and nuclear processing, allow a single construct to be combined pair wise with various targeting elements in separate constructs. this feature greatly facilitates the experimental procedure to test optimal expression in terms of accumulation in different sub-cellular compartments. an additional attribute of the vector, that permitted a dramatic enhancement of expression levels, was the introduction of several introns in the coding sequences of the module (marillonnet et al., . levels of expression are impressive and can reach up to mg of recombinant protein per gram of fresh weight (fw). the system was extensively tested with different genes, coding for antibodies and antibody-derivatives, interferons, growth hormones, bacterial and viral antigens, adjuvants and enzymes , confirming each time its versatility and robustness. another interesting modular system was developed using the bean yellow dwarf virus (beydv) of the geminiviridae family. geminivirus are single stranded circular dna viruses known to replicate through the rolling circle process, promoting high level of genome amplification in the nucleus of the infected cell. the beydv based system is depleted of the mp and cp gene and consequently the expression of the protein of interest is confined to the inoculation area. the basic version involves two modules, one containing the gene of interest inserted between the beydv long and short intragenic regions, lir and sir respectively. both intragenic regions are necessary to sustain rolling circle replication. the gene of interest is driven by a strong plant specific constitutive promoter. the second module is responsible for the expression of the rep protein which catalyzes various aspects of the rolling circle replication. initially, the system was used as an expression vector in tobacco plant cell culture (mor et al., ) but its use has recently been extended to whole leaves with agroinfection. the modular nature of this system also permits the co-expression of a variety of sequences of different viral or non-viral origin that are beneficial for replicon amplification and transgene expression. for example, genes of post-transcriptional gene silencing (ptgs) inhibiting proteins can be co-expressed to prevent ptgs and hence enhance target protein accumulation. display viral vectors have been extensively used to provide a molecular scaffolding for different kinds of peptides to be fused with the viral cp and, therefore, to be exposed and displayed on the surface of chimeric virus particles (cvps). coat protein genes of several plant viruses were genetically modified to support fusions with coding sequences of heterologous peptides in specific regions known to be well exposed on the virion surface (johnson et al., ; porta and lomonossoff, ) . therefore, the virus and the plant host would provide the expression system to produce large quantities of the chimeric fusion protein. clearly this is only feasible for well characterized viruses in terms of assembly, movement strategies and structure. fusions must be compatible with the normal assembly and viral fitness, and must avoid any possible steric hindrance or interference with virus movement. defining peptide features for their correct display on cvp is of particular interest among studies in this area (bendahmane et al., ; porta et al., ; lico et al., ) . two strategies have been developed to overcome assembly problems for long or difficult peptides. one strategy is to modify the terminus of the cp gene so that the cp stop codon would function in a leaky fashion. this would cause most of the cp produced to be unfused, while a small portion would be fused to the peptide displayed on the surface (hamamoto et al., ; sugiyama et al., ; borovsky et al., ) . the other strategy employs the a peptide of the small foot and mouth disease virus (fmdv). this sequence, inserted between the epitope coding sequence and the terminus of the cp gene, produces a ribosomal skip during the translational procedure (donnelly et al., ; de felipe et al., ) , with the result that only a small portion of the translational products will consist in a fusion between the heterologous sequence and the cp (fig. ) . the use of these strategies, however, opens the question of the precise dose of the antigen in the vaccine formulation. many plant viruses have been used to display different kinds of peptides. the most widely used viruses for this purpose are the cpmv, the tmv and the pvx. extensive research regarding the symmetry, shape and structure at atomic level for these viruses have allowed the precise definition of specific sites for peptide insertion to ensure the successful display of fusion peptides (klug and caspar, ; lomonossoff and johnson, ; baratova et al., a,b; johnson et al., ; parker et al., ) . major efforts are now directed to the development of new viral vector typologies in terms of both improvement of the expression strategies and virus choice. host plants have also been extended to a wider variety of species including herbaceous plants and cucurbitaceous family. besides the viruses mentioned above, other viruses have also been investigated as useful vectors, such as tomato mosaic virus (tomv) (dohi et al., ) ; maize streak virus (msv) (palmer and rybicki, ) ; bean pod mottle virus (bpmv) (zhang and ghabrial, ) ; beet necrotic yellow vein virus (bnyvv) (schmidlin et al., ) ; tomato golden mosaic virus (tgmv) (hayes et al., ) ; potato virus a (pva) (kelloniemi et al., ) ; zucchini yellow mosaic virus (zymv) (hsu et al., ) ; cucumber mosaic virus (cmv) (nuzzaci et al., ) ; and cucumber green mottle mosaic virus (cgmmv) (ooi et al., ) . antibodies are key molecules of the vertebrates' immune system. they are responsible for the recognition and binding of target antigens with high affinity and specificity. monoclonal antibodies (mabs) are used in a wide range of applications, including diagnosis, prevention and treatment of many diseases. for example, they are being successfully utilized as ''targeting vectors'' to direct drugs, radioisotopes and other therapeutic molecules to their target cells or tissues, as immunomodulators in pathology-related functions, as well as diagnostic tools to identify and localize molecular alterations. in addition to their pharmaceutical applications, mabs can also be used to modulate plant traits, generate disease or pest resistant crops (tavladoraki et al., ; nolke et al., ; peschen et al., ; villani et al., ) , and to study plant metabolic pathways (nolke et al., ) . antibodies are complex glycoproteins consisting of four polypeptides, linked by disulfide bridges and non-covalent bonds. despite their complexity, mabs and their derivatives can be easily expressed in heterologous systems including plants . however, mabs produced by heterologous systems may not share the precise structural and functional properties of the native molecules. for example, mabs produced by a bacterial expression system will lack post-translational modifications including glycosylation. non-mammalian eukaryotic expression systems could fail to use the appropriate glycans groups resulting in different glycosylation patterns. yeasts, for example, tend to hyperglycosylate (harashima, ; malissard et al., ) ; while plants preferentially introduce xylose and fucose residues and fail to use galactose and syalic acid (gomord and faye, ) . although proper glycosylation is required for functions of many mabs, it is to be noted that different glycosylation patterns do not always lead to loss of function in vitro or in vivo, or cause side-effects in humans (chargelegue et al., ) . different strategies have been adopted to circumvent these limitations such as targeting mabs to the endoplasmic reticulum, or abolishing glycosylation sites by mutagenesis to avoid inappropriate glycosylated products. in fact, it has been reported that the absence of glycans in some cases did not interfere with correct assembly and activity (rodriguez et al., ; nuttall et al., ) . in addition a fascinating approach that requires sophisticated genetic and metabolic engineering resides in the inactivation of endogenous glycosyltransferases and/or expression of heterologous mammalian specific enzymes in the host plant system (saint-jore- dupas et al., ) . complete antibodies and their derivatives have been produced with stable plant transformation technologies . thanks to the recent improvements of viral-based vectors, mabs have been produced with transient expression systems to quickly achieve much higher production levels along with other complex proteins. single chain fragments, that preserve antigen-recognition elements of the full length immunoglobulin, are often more effective as drugs or diagnostic tools than mabs due to their increased ability to penetrate target tissues, reduced immunogenicity, and more rapid clearance. a chimeric gene insertion tmv based vector was used for the expression of a human scfv for treatment of non-hodgkin's lymphoma (nhl) (mccormick et al., (mccormick et al., , . the plant-derived scfv generated an antibody response in vaccinated animals, and was an effective vaccine in a murine nhl tumor challenge model. the tmv strategy provides the speed and versatility required for the development of a patient specific treatment and this anti-idiotype vaccine is currently undergoing clinical trials for safety and immunogenicity evaluation in humans. another example is a scfv derivative (small immune protein, sip) to treat transmissible gastroenteritis virus (tgev), a porcine coronavirus. the sip was expressed by a pvx gene insertion vector and by a cpmv based display vector using the a expression strategy. the aim was to provide passive protection against enteric infections upon oral delivery in crude plant extracts . the sip folded correctly and preserved the ability to bind and neutralize tgev in tissue cultures. moreover, it provided in vivo protection against challenge with tgev in piglets vaccinated orally with crude extracts of scfv-expressing tobacco plants. the same researchers also expressed a full length tgev-specific iga by co-infecting plants with two separate pvx insertion vectors for the light chain (lc) and the heavy chain (hc) respectively . the iga was correctly assembled in plant tissue and provided in vivo protection against tgev in piglets after oral administration. a previous work (verch et al., ) also indicated the possibility to produce a full size mab in plants through the co-infection of two modified tmv based viral vectors. these results clearly indicated the possibility to obtain functional mabs through plant expression systems. nonetheless, expression of heterooligomeric proteins such as mabs might be inefficient due to the co-delivery of viral vectors built on the same virus backbone. in fact, this feature may result in early segregation and subsequent preferential amplification of one of the vectors in one cell. this problem has been recently overcome by utilizing two sets of vectors derived from non-competing tmv and pvx to express the two different mab chains . tmv and pvx may interact with different host proteins to sustain their replication and movement without interfering with each other. as a result, neither of the two vectors gains a replicative advantage over the other, allowing efficient co-expression of hc and lc in the same cells. the level of expression by each vector is independent within the same cell and can reach up to . mg/g fw of full assembled mab in few weeks. this strategy represents a useful platform for rapid large-scale manufacturing of mabs and other hetero-oligomeric proteins especially in situations requiring rapid responses such as pandemic threats and bioterrorism events. a vaccine is an antigenic preparation used to establish immunity to a disease. vaccination has been one of the greatest revolutions in medical science and has dramatically improved the quality and length of life expectancy. although vaccines are the most cost effective form of disease protection in healthcare, their cost is still too excessive for many people in the world, especially in developing countries. vaccines can be therapeutic, to overcome an infection already established, or prophylactic, to prevent a future infection. the aim of vaccination strategies is to obtain efficient and safe vaccine formulations to induce a long lasting immunity against various pathogens. to achieve this goal, the vaccine component should generate not only a neutralizing antibody response, a long-term memory b cells stimulation, but also a t cell mediated immunity to eliminate infected cells which represent the pathogen reservoirs. recombinant subunit vaccines, based on a single protein (antigen) or a single peptide (epitope) derived from the pathogen, are particularly attractive strategies when compared to classic inactivated, attenuated or live recombinant vaccines. recombinant subunit vaccines offer potentially equivalent efficacy but are much safer and in some cases easier to produce. plants offer unique advantages for the production of subunit vaccines in terms of scale, speed, costs, yield, and safety. the first work that describes the expression of the hepatitis b surface antigen in stable transgenic tobacco in marked the beginning for developing low cost vaccine formulations in plants (mason et al., ) . researchers focused on expressing their antigens in edible plant tissues as oral vaccines, opening a new prospect for vaccine delivery (mor et al., ) . since then, many research groups have adopted this system and carried out studies in the areas of improving target protein expression levels, analyzing the administration route and schedule, choosing the appropriate animal models, and exploring the use of possible adjuvants. the first report in this area described the expression of the fmdv structural protein vp with a tmv-based gene insertion vector (wigdorovitz et al., ) . a yield of approximately . mg/g fw was achieved and subsequently the antigen was administered to mice without further purification. all mice immunized intraperitoneally developed a protective immune response against experimental challenge with virulent fmdv. a great number of antigens have been produced in plants with different immunologic results (streatfield and howard, ) . altogether, these works demonstrated that plant-derived antigens retained their antigenicity and were able to induce active protective humoral and cell-mediated immune responses. the development of plant derived vaccines for human and veterinary uses against viral pathologies, tumors, allergies, bacterial infections, and diabetes-associated autoantigens has been extensively investigated (table ) . yusibov et al., expressed the e oncoprotein from human papilloma virus (hpv) in n. benthamiana plants with a gene replacement vector. the antigen was subsequently purified from infected leaves using the thermotolerance properties of lichenase (massa et al., ) . mice, immunized subcutaneously with the plant-produced e , showed a specific antibody and cytotoxic t-cell response. the dose also protected the animals against challenge with e tumor cells. moreover, the recombinant antigen was able to prevent tumor development when administered after virus challenge, supporting the ultimate goal to produce an anti-tumor vaccine with both therapeutic and prophylactic uses. a fundamental revolution within the last few years came from the use of modular deconstructed systems. a recent work using the magnicon tmv based system is worth noting. santi et al. ( ) focused on the production of antigens for agents of biological warfare, for example, the yersinia pestis bacterium, the causative agent of plague. in this work the protective efficacy of the fraction capsular antigen (f ) and the low calcium response virulent antigen (v) expressed at levels up to mg/g fw in n. benthamiana leaves was evaluated. guinea pigs, immunized with purified antigens, showed antigen-specific serum igg titers and, more importantly, % of animals were protected from an aerosolized challenge of virulent y. pestis. in a subsequent work mett et al. ( ) expressed the same y. pestis f and v antigens in n. benthamiana plants with an average yield of . mg/g fw with their lichenase optimized vector. the purified proteins were administered subcutaneously to non-human primates, cynomolgus macaques, producing antigen specific igg and iga responses which resulted in complete protection against lethal challenge with y. pestis. recent work show a particular interest on emerging and re-emerging diseases, as well as agents of biological warfare, and have extended plant-derived vaccine development to smallpox (golovkin et al., ) , anthrax , dengue virus (saejung et al., ) , and avian influenza a virus (nemchinov and natilla, ) . altogether, these data collectively show the establishment of a portfolio of well characterized and novel, improved strategies for the expression of effective vaccines in plants. journal of cellular physiology determined by the amino acid sequence, or conformational, determined by neighboring amino acids only in the tridimensional tertiary structure. an epitope alone could be poorly immunogenic and often possess a short half-life in the serum (lien and lowman, ) . as a result, extensive efforts are being directed to the development of new delivery strategies to increase the immunogenicity and half-life of epitope vaccines (purcell et al., ) . plant viral display vectors have the potential to play an important role in such strategic development. plant virus particles can structurally function as a scaffold to support, stabilize and display epitopic peptides. for example, a target epitope can be genetically fused to the cp protein. the chimeric virion will be formed by the cp-epitope fusion, representing the vaccine by itself. as a result, plant viruses can be used as a carrier to stabilize epitopes and to present them correctly to the immune system. strategies such as the use of the amber leaky stop codon and a peptide (described earlier) have been adopted successfully to display peptides (turpen et al., ; marconi et al., ) , as well as for full-length protein fusions (o'brien et al., ) . in addition, new strategies involving biotinylation of the capsid and the subsequent binding of streptavidin-conjugated target protein have been developed for displaying long sequences. the canine oral papillomavirus l protein, displayed on the tmv surface with this strategy, was significantly more immunogenic in animal models compared to the uncoupled antigen . with this strategy, it is also possible to combine helper epitopes and peptides known to facilitate cellular uptake or other additional t-cell targets to avoid the use of adjuvants. coexpression of melanoma-associated cytotoxic t lymphocytes (ctl) epitopes p e and trp on a tmv scaffold stimulated effective tumor protection from challenge and showed a significant survival improvement over the single peptides alone (mccormick et al., a) . the capability of cvps in inducing an antibody response specifically to the displayed epitope upon intranasal, intraperitoneal or oral administration, have been extensively demonstrated in different animal models (table ) . other data show cvps ability to elicit a strong specific neutralizing immune response in the absence of adjuvants (yusibov et al., ; brennan et al., ; marusic et al., ) . oral delivery of spinach leaves infected with cvps elicited a strong antibody response to the displayed rabies virus peptide in mice and human volunteers (yusibov et al., ) , indicating a potential use of this system as a supplementary oral booster for rabies vaccinations. alfalfa mosaic virus (amv)-derived cvps were able to elicit t-and b-cell responses, in non-human primates (yusibov et al., ) . chimeric tmv particles was shown to be able to directly interact with and stimulate mammalian antigen presenting cells to induce a strong cellular anti-tumor immune response (mccormick et al., b) . studies of body distribution for orally administered cvps also suggested their application as nanocapsules in novel drug delivery systems (rae et al., ; smith et al., ) . applications of peptide-displaying cvps extend far beyond the area of vaccine development. of particular interest are a killer peptide to confer broad-spectrum resistance to phytopathogens (donini et al., ) , a metal-binding peptide converting cvps into an artificial metal-adsorbing sink for metal tolerance and phytoremediation (shingu et al., ) , and a mosquito hormone-derived decapeptide as a larvicide to protect plants against agricultural insect pests and to control vector mosquitoes (borovsky et al., ) . finally werner et al. ( ) fused a fragment of protein a ( aa) to the tmv cp c-terminus via a -aa linker. this chimeric nanoparticle allowed a simple purification of mabs with % recovery yield and product purity of greater than %. this technology provides an inexpensive self-assembling matrix that could be used as industrial immuno-adsorbent for antibody purification. due to their simple macromolecular organization, assembly capabilities, structure stability, easy scalability and facile purification, plant viruses can offer a cheap source of biopolymers and nanoparticles. well characterized viruses with stable, highly ordered and repetitive structures, such as tmv and cpmv, are of particular interests. for example, cpmv, cowpea chlorotic mottle virus (ccmv) and other icosahedral viruses have been used as nucleation cages for the mineralization of materials (douglas and young, ) . another promising application is in noninvasive in vivo vascular imaging techniques (manchester and singh, ) . cpmv for example has been fluorescently labeled to visualize blood flow for periods of at least h to identify vessels and to monitor tumor neovascularization (lewis et al., ) . moreover, cpmv can be modified to generate new surface properties for developing novel biomaterials, electrochemical biosensors, or nanoelectronic devices (wang et al., ; chatterji et al., ; steinmetz et al., ) . rod-shaped viruses, like tmv, have been extensively used in the synthesis of a variety of metals nanowires, magnetic materials and semiconductors. tmv can be used as organic templates for the controlled deposition of gold, silver and platinum to prepare -d arrays (dujardin et al., ) . self-assembled, modified cp monomers of tmv were also employed for the construction of photovoltaic components in a novel light-harvesting system (miller et al., ) . cvps can be also incorporated in liquid crystal systems or used to generate thin films and fibers (flynn et al., ) . these examples clearly demonstrate the broad application and emerging potential of plant viruses in nanotechnology fields. over the last decade, plant based production of pharmaceuticals has made remarkable progress as the expression of a diverse set of proteins has been demonstrated, several proteins have moved into clinical testing, and a plant-derived veterinary vaccine has been approved. recent developments in transient expression systems and production in a controlled green house environment are directly addressing the issues of low expression levels and under developed regulatory standards for plant made pharmaceuticals (pmps). in spite of this progress, barriers remain that prevent the broad adoption of this technology platform. one of these is the lack of translational research to bridge the gap between bench discoveries and their corresponding clinical products. in fact, many product failures during development are ultimately caused by problems of transition from laboratory prototype to industrial product, as stated by the fda critical path report (http://www.fda.gov/oc/initiatives/criticalpath/). product industrialization programs are routinely delayed or derailed by inadequate efforts in downstream manufacturing, scale-up development, and quality control. therefore, as tremendous progress has been realized in recombinant protein expression, research focus has gradually been shifted to improvements in downstream processes including extraction, purification and recovery of the final products. downstream processing is fundamentally important to the commercial viability of the specific pmps and the pmp technology in general. an optimized downstream process will not only provide the additional cost-saving measures for the overall product cost, it will also enable the large-scale production of the products to meet the market demands. in addition, downstream process development is an essential step in compliance with fda's current good manufacture practice (cgmp) regulations. the general downstream processing steps for extraction and purification of pharmaceutical proteins are similar to those of other systems. these steps include harvest and fractionation of the tissue containing the targeted protein, extraction of target protein into designed buffer, clarification of cell debris and particles from the extract, purification of the target protein, and vialing of the purified products into the proper container in the desired formulation buffer. the unique structure and biochemistry of plant cells and tissues present different challenges and opportunities for each of the above processing steps. in addition, the choice of host plant species, tissue and subcellular organelle targeting of the product will have profound impact on the strategy and outcome of downstream processing efforts. since this review concerns primarily viral vector based transient production of pmps, our discussion will focus on the downstream bioprocessing of proteins targeted to the cytoplasm of leafy plants. pmp production with seeds has been extensively reviewed by others (menkhaus et al., ; nikolov and woodard, ) . the first step of biomaterial processing is extraction and purification from tissue with a high concentration of the target recombinant protein. the selective harvest of the target tissue will reduce the volume of initial biomaterial, and in turn, reduce the unnecessary need to eliminate the extra host proteins introduced by the inclusion of non-target tissue, and the total operational cost. the selectivity will also allow the avoidance of tissues which are hard to process, prime to introducing environmental contaminants (e.g., roots), or rich in other undesirable molecules such as proteases, alkaloids, and phenolics. for transient expression with viral vectors, leaves are primarily the target tissue for protein accumulation. currently most of the biomaterial production with these vectors is accomplished in greens houses with a controlled environment. for example, - days after inoculation, n. benthamiana leaves will be selectively harvested away from roots and stems (werner et al., ) . for small scale preparation, some researchers perform further fractionation of leaves by manually separating the central vein from the rest of leaves. for largescale production, this kind of operation becomes impractical and whole leaves are processed thoroughly in the next step. if the biomaterial is produced in open fields, extra steps have to be performed to remove the diverse array of environmental contaminants. rinsing and light washing of plants, before harvesting the target tissue, is a common practice for this purpose. regardless of where the biomaterial is generated, it is important to lower the bioburden and other contaminants as much as possible at this step. this measure will prevent microorganisms and other contaminants from entering purification feed streams and thus, greatly simplify the subsequent purification process and ensure the regulatory compliance of the final product. one of critical factors in choosing plant expression platforms is the protein stability in the targeted tissue. with all the advantages of the viral based transient expression system, recombinant proteins generally do have less long-term stability in leaves due to higher water content in contrast to the seedbased stable transformed expression systems (doran, ) . however, some proteins did show relative long-term stability in leaves of up to days at room temperature (fiedler et al., ) or even longer ( weeks) in dry alfalfa tissue (khoudi et al., ) . depending on the nature of the target protein, some of them need to be further processed as fresh leaf tissue, while others can be stored up to months as frozen tissue without losing their recovery and/or biological activity (chen, unpublished work). the possibility of storing tissue at a cold temperature for certain proteins offers and expands the flexibility of this production platform. the major goal of this processing step is to release the target protein into a liquid buffer solution from the plant tissue. to achieve this goal in a leaf-based transient expression system, leaf tissue is first ground in the presence of a desired extraction buffer to break the tissue and cells. the tissue homogenate will then be further pressed to release the protein into the aqueous buffer. the crude extract will be clarified to separate the protein-containing buffer from plant debris, insoluble proteins (see below) and other particulate matters. in comparison to other production hosts, plants produce more solid debris (up to % of totally weight of the biomaterial (menkhaus et al., ) ). the larger and denser solids make it impossible to achieve effective clarification by filtration methods. as a result, continuous centrifugation remains the most effective and scalable technique for plant extract clarification. it should be noted that it is possible to extract small recombinant proteins (< kda) that are targeted through the endomembrane system to the apoplast without breaking the plant cells. for example, some of the smaller proteins can be released directly into the extraction buffer by simply rinsing the tissue or by a specialized centrifugation technique (lohaus et al., ) . the purification process for this class of proteins can be greatly simplified with the result of a reduction in host protein contaminants. the choice of extraction buffer has to be carefully determined based on the properties (e.g., pi, size, hydrophobicity, and stability) of the target protein and the major contaminating host molecules. for example, the major contaminating protein in plant leaves is ribulose , bisphosphate carboxylase-oxygenase (rubisco). correspondingly, the choice of low ph buffers (ph . ) should be encouraged to keep rubisco insoluble and prevent it being extracted into the aqueous phase. at laboratory bench scale, protease inhibitors and antioxidants are routinely added to the extraction buffer to counter the denaturation, degradation and structural modification by proteases and phenolic compounds. due to regulatory restraints and low-cost requirement, these molecules should only be included when deemed absolutely necessary for large-scale production. both chromatographic and non-chromatographic methods have been employed to purify plant-derived pharmaceutical proteins. like proteins from other production hosts, purification strategies are formulated for each individual protein based on its solubility, size, pi, charge, hydrophobicity, and affinity to specific ligands and the parallel characteristics of host proteins. for mab-based pmps, while protein a or g-based chromatography provides a superb and convenient step (langone, ) , much effort is needed to eliminate plant host molecules which cause resin fouling or/and interfere with the binding of the target protein to protein a resin, and thus, reduce its capacity. non-chromatographic scalable processes such as aqueous two-phase partitioning systems (atps) are being developed to address this key issue (platis and labrou, ) . for other non-mab-based vaccines and therapeutics, a purification scheme has to be developed individually which is based on multiple steps of conventional chromatographic methods (menkhaus et al., ) . this time-consuming and challenging process calls for the need to develop more ''universal'' or versatile purification methods. the employment of affinity tags, particularly the tandem affinity tag purification (tap) strategy, provides possibilities for such versatile solution (lichty et al., ; arnau et al., ; tagwerker et al., ) . however, the search for both efficient and precise proteases for tag removal still presents serious challenges. in addition, the inclusion of proteases in the manufacturing process further complicates product purification, as well as raises cost and regulatory concerns (feeney et al., ; kenig et al., ) . several non-enzymatic affinity tag removal techniques have been explored (rais-beghdadi et al., ; wood et al., ) , but still require further optimization before becoming practical for product manufacturing. in addition to native proteins, carbohydrates and other host molecules, unique plant endogenous molecules such as phenolics needed to be removed during purification. phenolics tend to modify proteins by forming complexes, thus impeding purification if not removed earlier during purification (kusnadi et al., ; menkhaus et al., ) . some plants, such as tobacco, also produce a high-level of toxic alkaloids that have to be eliminated from final product . regarding agroinfection, the potential elevated-level of endotoxin (lipopolysaccharide, lps) from a gram-negative bacterium is another concern. specific purification strategies to lower the lps to acceptable levels must be incorporated into the overall scheme to ensure the safety of pmps produced using this platform (magalhaes et al., ) . the overall purification design has to be robust, scalable, cost-effective and compliant to cgmp regulations. ideally, no more than three chromatographic steps should be included to obtain a highly purified product. otherwise, recovery will drop to an impractical level. whenever possible, resins widely accepted by standard pharmaceutical industry should be considered first during process-development to minimize future regulatory expenditure. the requirement for an independent quality management system (qms) to govern the manufacture of pharmaceuticals also applies to the pmps. in addition to cgmp compliance during downstream processing, analytical tests for releasing the final purified products have to address plant specific contaminants in addition to the standard required assays. even though plants do not contain animal viruses and other infectious agents, it must be validated that lps, phenolics, and alkaloids, as well as herbicides and insecticides have been adequately removed from the final product. a significant increase in demand for high quality recombinant proteins is already on the horizon. new biological systems for the production of these proteins must be developed to meet market demands. plant expression systems based on viral vectors have the greatest potential to provide such technology. once optimized and implemented at a commercial scale, these expression systems should create a technology platform to produce recombinant proteins with scalability, speed, efficiency, cost-effectiveness and safety. use of virus vectors for the expression in plants of active full-length and single chain anti-coronavirus antibodies plant-made subunit vaccine against pneumonic and bubonic plague is orally immunogenic in mice current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins stability of potato virus x expression vectors is related to insert size: implications for replication models and risk assessment the organization of potato virus x coat proteins in virus particles studied by tritium planigraphy and model building the topography of the surface of potato virus x: tritium planigraphy and immunological analysis fast forward genetics based on virus-induced gene silencing display of epitopes on the surface of tobacco mosaic virus: impact of charge and isoelectric point of the epitope on virus-host interactions plastid biotechnology: prospects for herbicide and insect resistance expression of aedes trypsin-modulating oostatic factor on the virion of tmv: a potential larvicide beijerinck's work on tobacco mosaic virus: historical context and legacy chimeric plant virus particles administered nasally or orally induce systemic and mucosal immune responses in mice single chain antibody fragments for ocular use produced at high levels in a commercial wheat variety a bipartite system for the constitutive and inducible expression of high levels of foreign proteins in plants protective immune response to foot-and-mouth disease virus with vp expressed in transgenic plants virus-like particles: flexible platforms for vaccine development potato virus x as a vector for gene expression in plants a murine monoclonal antibody produced in transgenic plants with plant-specific glycans is not immunogenic in mice new addresses on an addressable virus nanoblock; uniquely reactive lys residues on cowpea mosaic virus immunogenicity of a subunit vaccine against bacillus anthracis localization of a bacterial protein in starch granules of transgenic maize kernels manganese peroxidase from the white-rot fungus phanerochaete chrysosporium is enzymatically active and accumulates to high levels in transgenic maize seed plant-derived vaccine protects target animals against a viral disease a tobacco mosaic virus-hybrid expresses and loses an added gene co-translational, intraribosomal cleavage of polypeptides by the foot-and-mouth disease virus a peptide inducible virus-mediated expression of a foreign protein in suspension-cultured plant cells production of an engineered killer peptide in nicotiana benthamiana by using a potato virus x expression system analysis of the aphthovirus a/ b polyprotein 'cleavage' mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal 'skip foreign protein degradation and instability in plants and plant tissue cultures superexpression of tuberculosis antigens in plant leaves host-guest encapsulation of materials by assembled virus protein cages organization of metallic nanoparticles using tobacco mosaic virus templates novel protein purification system utilizing an n-terminal fusion protein and a caspase- cleavable linker development of an antigen presentation system based on plum pox potyvirus protection of rabbits against rabbit hemorrhagic disease virus by immunization with the vp protein expressed in plants with a potyvirus-based vector optimization of scfv antibody production in transgenic plants production of antibodies in plants and their use for global health viruses as vehicles for growth, organization and assembly of materials plant-derived human papillomavirus e oncoprotein induces immune response and specific tumor protection transgenic plants as protein factories rapid high-yield expression of full-size igg antibodies in plants coinfected with noncompeting viral vectors magnifection-a new platform for expressing recombinant vaccines in plants smallpox subunit vaccine produced in planta confers protection in mice posttranslational modification of therapeutic proteins in plants agroinfection,'' an alternative route for viral infection of plants by using the ti plasmid a new tobacco mosaic virus vector and its use for the systemic production of angiotensin-i-converting enzyme inhibitor in transgenic tobacco and tomato recent advances in the transformation of plants heterologous protein production by yeast host-vector systems engineering the genome of grapevine virus a into a vector for expression of proteins in herbaceous plants stability and expression of bacterial genes in replicating geminivirus vectors in plants production of antibodies in transgenic plants molecular farming of industrial proteins from transgenic maize oral administration of a mite allergen expressed by zucchini yellow mosaic virus in cucurbit species downregulates allergeninduced airway inflammation and ige synthesis expression of functional recombinant human lysozyme in transgenic rice cell culture viruslike particle expression and assembly in plants: hepatitis b and norwalk viruses expression of avian reovirus sigma c protein in transgenic plants rice expressing lactoferrin and lysozyme has antibiotic-like properties when fed to chicks biochemical method for inserting new genetic information into dna of simian virus : circular sv dna molecules containing lambda phage genes and the galactose operon of escherichia coli presentation of a foreign peptide on the surface of tomato bushy stunt virus glycosylated f (k ) fimbrial adhesin faeg expressed in barley endosperm induces etec-neutralizing antibodies in mice presentation of heterologous peptides on plant viruses: genetics, structure, and function plant based hiv- vaccine candidate: tat protein produced in spinach a potyvirus-based gene vector allows producing active human s-comt and animal gfp, but not human sorcin, in vector-infected plants influence of the protein oligomericity on final yield after affinity tag removal in purification of recombinant proteins production of a diagnostic monoclonal antibody in perennial alfalfa plants the structure of small viruses processing of transgenic corn seed and its effect on the recovery of recombinant beta-glucuronidase expression of the sweet protein brazzein in maize for production of a new commercial sweetener protein a of staphylococcus aureus and related immunoglobulin receptors produced by streptococci and pneumonococci viral nanoparticles as tools for intravital vascular imaging comparison of affinity tags for protein purification peptide display on potato virus x: molecular features of the coat protein-fused peptide affecting cell-to-cell and phloem movement of chimeric virus particles therapeutic peptides cowpea mosaic virus-based systems for the production of antigens and antibodies in plants is the infiltration-centrifugation technique appropriate for the isolation of apoplastic fluid? a critical evaluation with different plant species the synthesis and structure of comovirus capsids virus-induced gene silencing in plants antibody processing and engineering in plants, and new strategies for vaccine production methods of endotoxin removal from biological preparations: a review recombinant soluble beta- , -galactosyltransferases expressed in saccharomyces cerevisiae. purification, characterization and comparison with human enzyme virus-based nanoparticles (vnps): platform technologies for diagnostic imaging in planta production of two peptides of the classical swine fever virus (csfv) e glycoprotein fused to the coat protein of potato virus x in planta engineering of viral rna replicons: efficient assembly by recombination of dna modules delivered by agrobacterium systemic agrobacterium tumefaciens-mediated transfection of viral replicons for efficient transient expression in plants neutralizing immunogenicity of transgenic carrot (daucus carota l.)-derived measles virus hemagglutinin chimeric plant virus particles as immunogens for inducing murine and human immune responses against human immunodeficiency virus type expression of hepatitis b surface antigen in transgenic plants anti-cancer activity of plant-produced hpv e vaccine roy markham: pioneer in plant pathology rapid production of specific vaccines for lymphoma by expression of the tumorderived single-chain fv epitopes in tobacco plants individualized human scfv vaccines produced in plants: humoral anti-idiotype responses in vaccinated mice confirm relevance to the tumor ig tmv-peptide fusion vaccines induce cell-mediated immune responses and tumor protection in two murine models chemical conjugate tmv-peptide bivalent fusion vaccines improve cellular immunity and tumor protection considerations for the recovery of recombinant proteins from plants a plant-produced plague vaccine candidate confers protection to monkeys self-assembling light-harvesting systems from synthetically modified tobacco mosaic virus coat proteins an antibody derivative expressed from viral vectors passively immunizes pigs against transmissible gastroenteritis virus infection when supplied orally in crude plant extracts perspective: edible vaccines-a concept coming of age geminivirus vectors for high-level expression of foreign proteins in plant cells production of escherichia coli heat labile toxin (lt) b subunit in soybean seed and analysis of its immunogenicity as an oral vaccine a launch vector for the production of vaccine antigens in plants cucumber mosaic virus as carrier of a hepatitis c virus-derived epitope transient expression of the ectodomain of matrix protein (m e) of avian influenza a virus in plants development of a plant-derived subunit vaccine candidate against hepatitis c virus downstream processing of recombinant proteins from transgenic feedstock from the cover: ricebased mucosal vaccine as a global strategy for cold-chain-and needle-free vaccination antibody-based pathogen resistance in plants immunomodulation of polyamine biosynthesis in tobacco plants has a significant impact on polyamine levels and generates a dwarf phenotype a functional antibody lacking n-linked glycans is efficiently folded, assembled and secreted by tobacco mesophyll protoplasts cucumber mosaic virus as a presentation system for a double hepatitis c virus-derived epitope rotavirus vp expressed by pvx vectors in nicotiana benthamiana coats pvx rods and also assembles into viruslike particles food-processing enzymes from recombinant microorganisms-a review the full-length clone of cucumber green mottle mosaic virus and its application as an expression system for hepatitis b surface antigen investigation of the potential of maize streak virus to act as an infectious gene vector in maize plants surface features of potato virus x from fiber diffraction recombinant protein therapeutics-success rates, market trends and values to direct screening for high-level expression of an introduced alpha-amylase gene in plants bovine herpes virus gd protein produced in plants using a recombinant tobacco mosaic virus (tmv) vector possesses authentic antigenicity fusion proteins comprising a fusarium-specific antibody linked to antifungal peptides protect plants against a fungal pathogen inactivation and purification of cowpea mosaic virus-like particles displaying peptide antigens from bacillus anthracis development of an aqueous two-phase partitioning system for fractionating therapeutic proteins from tobacco extract scope for using plant viruses to present epitopes from animal pathogens development of cowpea mosaic virus as a high-yielding system for the presentation of foreign peptides cowpea mosaic virus-based chimaeras. effects of inserted peptides on the phenotype, host range, and transmissibility of the modified viruses more than one reason to rethink the use of peptides in vaccine design systemic trafficking of plant virus nanoparticles in mice via the oral route purification of recombinant proteins by chemical removal of the affinity tag transient expression in tobacco leaves of an aglycosylated recombinant antibody against the epidermal growth factor receptor production of dengue envelope domain iii in plant using tmv-based vector system expression of multiple proteins using full-length and deleted versions of cowpea mosaic virus rna- from planta to pharma with glycosylation in the toolbox protection conferred by recombinant yersinia pestis antigens produced by a rapid and highly scalable plant expression system use of a beet necrotic yellow vein virus rna- -derived replicon as a new tool for gene expression the capsid protein gene of tomato bushy stunt virus is dispensable for systemic movement and can be replaced for localized expression of foreign genes conferring cadmium resistance to mature tobacco plants through metal-adsorbing particles of tomato mosaic virus vector a plant-based allergy vaccine suppresses experimental asthma via an ifn-gamma and cd þcd rblow t celldependent mechanism modified tobacco mosaic virus particles as scaffolds for display of protein antigens for vaccine applications assembly of trans-encapsidated recombinant viral vectors engineered from tobacco mosaic virus and semliki forest virus and their evaluation as immunogens immunization with a chimeric tobacco mosaic virus containing an epitope of outer membrane protein f of pseudomonas aeruginosa provides protection against challenge with p. aeruginosa plant viral capsids as nanobuilding blocks: construction of arrays on solid supports plant-based vaccines systemic production of foreign peptides on the particle surface of tobacco mosaic virus functional expression of the taste-modifying protein, miraculin, in transgenic lettuce immunogenicity in humans of a recombinant bacterial antigen delivered in a transgenic potato human immune responses to a novel norwalk virus vaccine delivered in transgenic potatoes a tandem affinity tag for two-step purification under fully denaturing conditions: application in ubiquitin profiling and protein complex identification combined with in vivo cross-linking expression of bacterial chloramphenicol acetyltransferase gene in tobacco plants mediated by tmv-rna transgenic plants expressing a functional single-chain fv antibody are specifically protected from virus attack immunogenicity in humans of an edible vaccine for hepatitis b transfection of whole plants from wounds inoculated with agrobacterium tumefaciens containing cdna of tobacco mosaic virus malarial epitopes expressed on the surface of recombinant tobacco mosaic virus molecular farming in plants: host systems and expression technology expression and assembly of a full-length monoclonal antibody in plants using a plant virus vector immunization with a plant-produced colorectal cancer antigen immunomodulation of cucumber mosaic virus infection by intrabodies selected in vitro from a stable singleframework phage display library recombinant pharmaceuticals from plants: the plant endomembrane system as bioreactor plants for delivery of edible vaccines biopharmaceutical benchmarks natural supramolecular building blocks. wild-type cowpea mosaic virus exploring plant genomes by rna-induced gene silencing expression of bacillus anthracis protective antigen in transgenic chloroplasts of tobacco, a non-food/feed crop measles virus hemagglutinin protein expressed in transgenic lettuce induces neutralising antibodies in mice following mucosal vaccination immunoabsorbent nanoparticles based on a tobamovirus displaying protein a protection of mice against challenge with foot and mouth disease virus (fmdv) by immunization with foliar extracts from plants infected with recombinant tobacco mosaic virus expressing the fmdv structural protein vp a genetic system yields self-cleaving inteins for bioseparations maize (zea mays)-derived bovine trypsin: characterization of the first large-scale, commercial protein product from transgenic plants select what you need: a comparative evaluation of the advantages and limitations of frequently used expression systems for foreign genes oral immunogenicity of potato-derived hbsag middle protein in balb/c mice antigens produced in plants by infection with chimeric plant viruses immunize against rabies virus and hiv- expression in plants and immunogenicity of plant virusbased experimental rabies vaccine peptide-based candidate vaccine against respiratory syncytial virus development of bean pod mottle virus-based vectors for stable protein expression and sequence-specific virus-induced gene silencing in soybean in planta expression of hiv- p protein using an rna plant virus-based expression vector key: cord- -s sxi uj authors: rubio, luis; galipienso, luis; ferriol, inmaculada title: detection of plant viruses and disease management: relevance of genetic diversity and evolution date: - - journal: front plant sci doi: . /fpls. . sha: doc_id: cord_uid: s sxi uj plant viruses cause considerable economic losses and are a threat for sustainable agriculture. the frequent emergence of new viral diseases is mainly due to international trade, climate change, and the ability of viruses for rapid evolution. disease control is based on two strategies: i) immunization (genetic resistance obtained by plant breeding, plant transformation, cross-protection, or others), and ii) prophylaxis to restrain virus dispersion (using quarantine, certification, removal of infected plants, control of natural vectors, or other procedures). disease management relies strongly on a fast and accurate identification of the causal agent. for known viruses, diagnosis consists in assigning a virus infecting a plant sample to a group of viruses sharing common characteristics, which is usually referred to as species. however, the specificity of diagnosis can also reach higher taxonomic levels, as genus or family, or lower levels, as strain or variant. diagnostic procedures must be optimized for accuracy by detecting the maximum number of members within the group (sensitivity as the true positive rate) and distinguishing them from outgroup viruses (specificity as the true negative rate). this requires information on the genetic relationships within-group and with members of other groups. the influence of the genetic diversity of virus populations in diagnosis and disease management is well documented, but information on how to integrate the genetic diversity in the detection methods is still scarce. here we review the techniques used for plant virus diagnosis and disease control, including characteristics such as accuracy, detection level, multiplexing, quantification, portability, and designability. the effect of genetic diversity and evolution of plant viruses in the design and performance of some detection and disease control techniques are also discussed. high-throughput or next-generation sequencing provides broad-spectrum and accurate identification of viruses enabling multiplex detection, quantification, and the discovery of new viruses. likely, this technique will be the future standard in diagnostics as its cost will be dropping and becoming more affordable. introduction viral diseases are a major threat to sustainable and productive agriculture worldwide, resulting in losses of several billion dollars every year (mumford et al., ) . the highest impact occurs with emerging diseases, defined by a rapid increase in disease incidence, geographical range, and/or pathogenicity. the main factors driving virus emergence are: i) the agricultural systems based on monocrops with low genetic diversity and high plant density, which are more vulnerable to pathogens and pests; ii) world trade of plant material (germplasm and live plants) that moves viruses, hosts, and vectors to new regions and environments; iii) the climate change affecting the distribution area of hosts and vectors; and iv) the ability of viruses for rapid evolution and adaptation (anderson et al., ; jones, ; elena et al., ) . presently, curing plants once they have been infected by a virus is not feasible, unlike bacteria or fungi that can be treated with antibacterial or antifungal agents, respectively. so, disease management relies on preventing viruses from entering plants, or getting plants resistant to viral infection, using multiple strategies that must be developed specifically for each virus, host, and environment (pathosystem). specific tools for virus diagnostics and identification are pivotal to set up and evaluate disease management. here, the current state and progress of procedures used for virus detection are reviewed, discussing important features such as their sensitivity, specificity, versatility, portability, capacity for multiplexing, and virus quantification and designability. this review also includes basic concepts of genetic diversity and evolution of plant viruses and how they must be considered to improve detection. finally, the main strategies for disease control are described, showing both the more suitable detection methods and how genetic diversity and evolution of virus populations can affect the efficiency and durability of some control strategies. this review follows a pragmatic approach aimed to guide plant pathologists to design and apply more accurate detection procedures for a more efficient management of viral diseases. viruses have a great potential for high genetic variability due to their rapid replication and generation of large populations. viruses with rna genomes, comprising most plant viruses, and viroids have the highest mutation rate of any group of replicons, since rna polymerases lack a proofreading activity (domingo et al., ; drake and holland, ; gago et al., ) . the mutation rate is so high that replication from a single rna molecule gives rise to a population of mutant sequences (haplotypes or variants) grouped around a master sequence, termed quasispecies (holland et al., ; moya et al., ) . populations of closely related viral or viroidal variants in individual plants have been reported (ambroś et al., ; kong et al., ; gandıá et al., ) . viral populations in individual plants can be even more complex, since mixed infections with different virus species (juaŕez et al., ) or divergent variants of the same virus species (rubio et al., ; goḿez et al., ) are frequent as a consequence of successive inoculations by vectors (e.g., insects). for example, a survey of seven tomato viruses in sicily, italy showed that most plants ( . %) presented multiple infections, whereas . % were infected with a single virus, and only . % were free of these viruses ( table ) . synergistic interactions between different viruses and viroids in mixed infection can lead to increased virulence (symptoms and/or viral accumulation) or even new diseases (wang et al., ; wintermantel, ; murphy and bowen, ; untiveros et al., ; syller, ; moreno and loṕez-moya, ) . mixed infections of two viruses also enable recombination, which, in addition to mutation, is another source of genetic variation and emergence of new viruses. recombinants have been described between different species of plant viruses (padidam et al., ; chare and holmes, ; codoñer and elena, ; davino et al., ) or divergent viral strains lian et al., ; ferriol et al., ) . recombination seems a frequent event coupled to virus replication (froissart et al., ; sztuba-solinska et al., ) , so that populations of different recombinants have been found in individual plants ( figure a ) (vives et al., ; weng et al., ) . recombination in rna viruses is considered as a mechanism for rapid removal of many deleterious mutations produced during replication and regeneration of functional genomes (moya et al., ) . the genetic variation produced by mutation and recombination is restricted and structured by the other three evolutionary forces: natural selection, genetic drift, and gene flow. natural selection is a directional process by which the less fit virus variants will decrease their frequency in the population (negative or purifying selection) as a result of functional restrictions necessary for replication, movement between plant cells, transmission by vectors, and specific interactions between virus and host or virus and vector (power, ; schneider and roossinck, ; chare and holmes, ) . positive or adaptive selection consists in the frequency increase of the fittest variants carrying genetic changes required to become adapted to new hosts and/or vectors (agudelo-romero et al., ; ohshima et al., ; peña et al., ) . genetic drift consists of stochastic changes in the frequencies of genomic variants in a finite population due to random sampling occurred during reproduction (moya et al., ) . the effect is a reduction of genetic variability and fixation of selectively neutral variants that is more evident after a rapid reduction of the population size by population bottlenecks or founder events, which can occur in different steps of the virus life cycle, such as virus movement between plant cells and transmission by vectors (sacristań et al., ; ali et al., ; betancourt et al., ; ali and roossinck, ) . figure b shows genetic changes of citrus tristeza virus (ctv) isolates, revealed by single-strand conformation polymorphism analysis (explained below), after host change or vector transmission. finally, gene flow (migration) among viral populations from distinct geographic areas is another factor shaping the genetic structure and variation, so that high migration rates favor genetic uniformity between populations decreasing the global genetic diversity (moya et al., ) . the rapid evolution of plant viruses implies that epidemiological and evolutionary processes interplay, and they must be considered together to understand and prevent viral emergence. in the last decades, rapid and specific serological (enzyme-linked immunosorbent assay, elisa) and molecular techniques (molecular hybridization and dna amplification) for the detection of plant viruses have been developed. elisa is based on specific binding of viral proteins with antibodies (clark and adams, ) , and molecular hybridization, on binding viral nucleic acids with sequence-specific dna or rna probes, due to their sequence complementarity (hull and al-hakim, ) . these binding events are visualized by attached markers based on fluorescent dyes, enzymes producing colorimetric or chemiluminescent reactions, radioactivity, or others. detection methods based on dna amplification can be classified into two types: polymerase chain reaction (pcr) and isothermal amplification. pcr makes millions of dna copies of a specific region of the viral genome that are usually visualized by electrophoresis or by hybridization with fluorescent probes. pcr can use as template genomic dna, or complementary dna obtained after reverse transcription (rt) of viral rna. amplification occurs in three steps: i) denaturation by heating at °c to °c to separate the double-stranded dna (dsdna) template into single strands; ii) annealing by cooling at °c to °c to allow the primers (two short dna sequences of - nt) to bind the start and end of the target dna; iii) extension by heating at °c to °c, in which a thermostable dna polymerase synthesizes new dna strands starting from the primers. these steps are repeated for to cycles, so the newly synthesized dna segments serve as template in next cycles (mullis and faloona, ) . the pcr product is visualized by electrophoresis, and it can be further characterized by sanger sequencing (first-generation sequencing), enabling a more precise identification by comparison with known sequences from databases like genbank (see below). also, this approach is used to genotype virus populations, evaluate their genetic diversity, and study their evolution (see below). realtime quantitative pcr (qpcr) is a variant of this technique that monitors the reaction progress by detecting a fluorescent reporter that binds to the dsdna or is released from sequence-specific probes of to nt. this pcr variant can be used to quantify nucleic acids (see below). isothermal amplification can be achieved by different approaches (olmos et al., ) : i) helicase dependent amplification (had) uses a helicase to separate the strands of dsdna, allowing primer binding and extension by dna polymerase at a constant temperature of about °c. ii) recombinase polymerase amplification (rpa) uses a recombinase which forms a complex with primers to initiate amplification at a temperature between °c and °c. iii) nucleic acid sequence-based amplification method (nasba) uses a modified primer with the bacteriophage t promoter region that attaches to the rna template. reverse transcriptase and rnase h are used to synthesize complementary ds dna, and a t rna polymerase to synthesize complementary rna strands resulting in amplification. iv) loop-mediated isothermal amplification (lamp) is based on auto cycling and high dna strand displacement activity mediated by bst polymerase from geobacillus stearothermophilus, under isothermal conditions at °c to °c (panno et al., ) . recently, new tools for molecular diagnosis have been developed based on prokaryotic clustered regularly interspaced short palindromic repeats (crispr) immunity system, widely applied for genome editing (chertow, ) . the advent of high-throughput sequencing (hts) technologies, also known as next-generation sequencing, has led to a revolution in plant virus diagnosis (maree et al., ; villamor et al., ) . hts does not require any previous knowledge of viral sequences and can sequence millions or billions of dna molecules in parallel, enabling the detection of all viruses present in a plant (virome), including those still unknown (roossinck, ) . hts allowed elucidating the elusive etiology of some diseases (vives et al., ; he et al., ) , but often it is not possible to find a direct association between the disease and a particular virus (tomasěchováet al., ) among those detected in the infected plant. in this case, the diagnostic must be established by fulfilling koch's postulates, or at least by finding a tight association between the disease and the presence of a certain virus in field surveys (mumo et al., ) . hts can be divided into two types. second-generation sequencing is based on the preparation of random libraries of dna fragments when dna is used as starting material, or of cdna obtained by retrotranscription of the rna with random primers or oligodt. these libraries are clonally amplified, bond to synthetic dna adapters and sequenced in parallel. this produces a large number of short sequence reads ( - nt) that are assembled by connecting overlapping sequence reads according to nucleotide identity by informatic analysis, e.g., geneious package (www.geneious.com). several platforms for second-generation sequencing have been developed by different companies such as roche , illumina, solid and ion torrent (bleidorn, ; goodwin et al., ; heather and chain, ; villamor et al., ) . in addition to detection of new plant viruses and viroids, hts is being used for studies on epidemiology, synergistic interactions between viruses, and genetic diversity and evolutionary mechanisms of virus populations (kehoe et al., ; hadidi et al., ; pecman et al., ; roossinck, ; tan et al., ; xu et al., ) . (vives et al., ) . above is a partial representation of the genomic map of ctv with boxes corresponding to genes. red and green lines indicate sequence types with about % nucleotide identity between them. the relative frequency of each sequence type is indicated. (b) genetic variation of the ctv isolate t after host change and the ctv isolate f after a transmission event by the aphid aphis gossypii (d' urso et al., ) . genetic variants are showed as electrophoretic bands after single-strand conformation polymorphism (sscp) analysis. third-generation sequencing is based on sequencing single molecules in real-time without the need for clonal amplification, thus shortening dna preparation time and giving long reads of several kilobases (goodwin et al., ; van dijk et al., ) . long reads are more appropriate for genome sequencing, genotyping, and detecting recombination. however, third-generation sequencing needs further improvement since error rates are still much higher than in second-generation sequencing. several techniques are being developed by different companies such as single-molecule real-time (smrt) and nanopore sequencing. smrt sequencing uses a flow cell with millions of individual picolitre wells with transparent bottoms (zero-mode waveguides) with a dna polymerase fixed. incorporation on each single-molecule template per well is continuously visualized with a laser and camera system that records the color and duration of emitted light, as the labeled nucleotide momentarily pauses during incorporation at the bottom of the wells. nanopore sequencing is based on translocating the dna or rna through a nanopore (in membrane proteins or synthetic materials such as silicon nitride and aluminum oxide), where an ionic current pass by setting a voltage. the dna passing through the nanopore changes the current depending on the shape, size and length of the dna sequence. nanopore sequencing has several advantages such as the relatively low cost compared to other hts technologies, high mobility due to the sequencer small size and rapid sample processing, without the need for reverse transcription for rna viruses (kiselev et al., ) . this technology has been recently used to detect some plant viruses, such as plum pox virus (ppv) and tomato yellow leaf curl virus (tylcv), and discover new plant viruses (bronzato badial et al., ; chalupowicz et al., ; naito et al., ) . detection procedures must be optimized for accuracy, measured as sensitivity and specificity, which are the statistical measures of performance of binary classification tests (sharma et al., ) . sensitivity measures the proportion of actual positives which are classified as such (probability of true positives) and specificity measures the proportion of negatives which are correctly identified (probability of true negatives). other measures of accuracy are positive predictive value, defined as the proportion of positive samples correctly diagnosed, and negative predictive value, or proportion of samples with negative results correctly diagnosed ( table ) . however, the predictive values depend on the infection prevalence in the samples tested and do not apply universally (olmos et al., ) . low virus titer can limit sensitivity, producing false negatives when the virus concentration is under the technique detection threshold. usually, the molecular techniques are more sensitive than the serological ones. conventional pcr is much more sensitive than molecular hybridization. some modalities of pcr are even more sensitive, such as qpcr and nested pcr (this uses two successive runs of pcr with a second primer pair to amplify a secondary target within the product of the first run). lamp exhibits a sensitivity in the order of qpcr and is less affected than pcr by inhibitors (phenols, tannins, and complex polysaccharides), which are often a cause of false negatives. paradoxically, the high sensitivity of the amplification techniques can be a problem, as contamination of reagents and instruments with amplicons from previous samples and cross-contamination between samples can produce false positives reducing specificity. an important factor affecting the accuracy of the serological and molecular detection methods is the genetic variability within each virus species and the genetic relationships with other virus species. since these methods are based on specific binding (protein with antibody or nucleic acids with probes or primers), some dissimilar virus variants can fail to react giving false negatives. for example, universal detection of ppv by elisa with monoclonal antibodies failed for some ppv isolates (sheveleva et al., ) . on the other hand, false positives by cross-reactions of antibodies with related viruses have been described for some viruses, e.g., arabis mosaic virus (armv) (frison and stace-smith, ) . unlike antibody production, primers and probes are better suited to be optimized by considering the genetic variability. however, genetic variation of viruses is often neglected, and accuracy is tested just with samples from local surveys harboring genetically similar virus isolates. thus, some detection protocols can fail when applied with the same reagents (probes or primers) in other geographical areas or after the emergence of divergent variants, e.g., pepino mosaic virus (pepmv) and apple chlorotic leafspot virus (aclsv) (mansilla et al., ; spiegel et al., ) . to design accurate probes or primers for a given virus, the first step is to get a picture of the genetic variation and structure by gathering as many nucleotide sequences as possible from isolates of that virus and from genetically related viruses. sequences of specific genomic regions or complete genomes can be determined from purified or cloned pcr products or by hts from viral samples and retrieved from databases like genbank (https://www.ncbi.nlm.nih.gov/). the genetic diversity and structure can be estimated easily with the mega x software (kumar et al., ) , after alignment with the algorithm clustalw (higgins et al., ) implemented in mega. nucleotide diversity is the mean distance (proportion of nucleotide differences) between sequence pairs and can be considered as a measure of the genetic variation within a virus population. in this case, p-distance should be used instead of nucleotide substitution models as this analysis is aimed to know the actual genetic differences for application in diagnostic and not the evolutionary changes that occurred. the genetic structure can be visualized with phylogenetic trees, which can be inferred with different methods, such as neighbor-joining, maximum likelihood and maximum parsimony. as an illustration, figure shows the nucleotide diversity and the phylogenetic relationships of randomly selected worldwide isolates of cucumber green mottle mosaic virus (cgmmv) and grapevine leafroll-associated virus (glrav- ). the nucleotide diversity of glrav- is higher, so it is more challenging to develop an accurate detection method for glrav- than for cgmmv. since the viral population of glrav- is structured in eight groups or clades, at least one isolate per clade should be considered to develop detection and disease control procedures for this virus. for universal detection of a virus by pcr, primers should be designed from short sequence stretches with conserved nucleotide positions among all available sequences and they should be degenerated to cover possible genetic variation not found in the sequences analyzed (explained in detection levels). for example, detection of pepmv by rt-pcr (mansilla et al., ) failed for new pepmv isolates and universal detection was only achieved after designing primers targeting conserved sequence stretches among pepmv isolates (ling et al., ) . for molecular hybridization, different genomic regions should be considered for probe design since the genetic diversity can vary widely along the genome due to different selective pressures or recombination. for example, broad bean wilt virus (bbwv- ), with a bipartite single-stranded rna (ssrna) genome, showed the lowest nucleotide diversity in the ′ terminal sequence of rna . thus, only a dna probe binding this genomic region allowed universal detection of bbwv- by molecular hybridization (ferrer et al., ) . hts enables accurate and unbiased identification of viruses unlike the other techniques (elisa, molecular hybridization or amplification) requiring a specific binding that can fail to detect some genetic variants. hts generates hundreds of megabases to gigabases of nucleotide sequence reads in a single run providing a good sensitivity (santala and valkonen, ). however, cross-contamination can produce false positives, so it is necessary validation with other techniques, such as pcr (maree et al., ) . detection and identification of viruses are based on assigning a virus from a plant sample to a group of viruses sharing common characteristics. in most cases, the level of detection is the virus species, but it can also be set for higher taxonomic units such as genus or family, or lower units like strain (virus variants with distinctive biological or molecular characteristics). serological techniques usually detect viruses to the species level and, in some cases, they allow discrimination between virus strains (serotypes) using monoclonal antibodies (permar et al., ; myrta et al., ; sheveleva et al., ) . molecular hybridization has been used mostly to detect virus species (supplementary table s ), but the detection level can be modified to a certain extent by using different probes and hybridization conditions. the stability of the hybrid complexes depends on the probe length and g-c content, the probe type (dna or rna), and the number of global or local mismatches (nucleotide distance) between target and probe. therefore, the distribution of nucleotide variation along the virus genome should be considered to modulate the level of detection and to test for accuracy. regarding the hybridization conditions, a more selective detection can be attained by using more stringent conditions (higher incubation temperature, lower salt concentration or adding denaturing agents like formamide), that reduce the number of mismatches permitted to occur. thus, probes from variable genomic regions with stringent conditions have been used to discriminate between virus strains or isolates (narvaéz et al., ; ferrer et al., ) . designing probes complementary to regions conserved within taxonomic units higher than species is challenging given the high nucleotide variation among species. to our knowledge, only two cases have been reported: i) a single rna probe derived from the ′ untranslated of bbwv- was able to hybridize with other members of the genus fabavirus . this genomic region contains several perfect or near-perfect repeats of ten nucleotides that allow hybridization despite the low nucleotide identity between these virus species. ii) a polyprobe with seven conserved motifs of the genus potyvirus allowed detection of viruses of this genus by hybridizing at low stringency conditions . pcr techniques are the most versatile and primers have been designed for different detection levels from families and genera (supplementary table s ) to strains and genetic variants (ruiz-ruiz et al., b; debreczeni et al., ) , whereas isothermal amplification has been limited to the species level (supplementary table s ). obtaining primers specific for genera or families is more challenging than for virus species given the increasing nucleotide diversity of higher taxons. primer design requires searching for conserved nucleotide positions among the members of the genus or family, which usually correspond to sequence motifs with relevant biological functions, and therefore, subjected to strong negative selection. the conserved positions can occur at the nucleotide level due to structural constraints, codon usage, or at sites where regulatory proteins bind (koonin, ; adams and antoniw, ; watters et al., ) , but most are at amino acid level. for example, primers for the subfamily comovirinae (composed of the genera comovirus, fabavirus and nepovirus) were designed based on amino acid motifs of the rna-dependent rna polymerase: (t/v)ygddn(v/l) and tseg(y/f)p (koonin, ; maliogka et al., ) . to design primers detecting the members of a genus or family, at least one sequence per each virus species should be used for alignment, preferably a codon-based or amino acid alignment. since the genetic code is redundant, the primer must be degenerate, that is, composed of a mixture of almost identical primers differing in some positions and covering all possible nucleotide combinations for that protein sequence. the degeneracy level should be reduced as much as possible due to its negative effect in sensitivity (only a small proportion of the primers would bind the target) and specificity (some primers can bind to nontarget sequences). several approaches can be used, such as i) limiting the degenerate sites to the last to nucleotides from the ′ terminus, which are critical for pcr amplification, whereas some mispairings at the ′ terminus are allowed; ii) choosing low degeneracy (one-or two-fold) codons, particularly at the ′ terminus; and iii) using modified nucleotides such as inosine (i) for four-fold degenerate sites that can base-pair with the four normal nucleotides: a, c, g and t. as an illustration, figure shows conserved amino acid positions in the genus fabavirus, which can be used for a hypothetical universal detection of viruses within this genus. the pcr products obtained with conserved primers for a genus or family can be further purified and sequenced to identify the viral species or discover new ones (zheng et al., ) . in plants infected with two or more species of the same genus or family, it is necessary to clone the pcr products and sequence individual clones to identify each virus species. in some cases, the pcr products obtained with conserved primers are of distinct size for each virus species and can be easily discriminated by electrophoresis (james and upton, ; ferrer et al., ) . tools to detect small genetic variations are also necessary given the great potential of viruses to generate high genetic and biological variation (genetic variants can display different properties, such as host range, virulence and resistancebreakdown). several techniques based on pcr or using pcr products as templates have been used for genotyping: i) randomly amplified polymorphic dna (rapd)-pcr uses a single short primer with an arbitrary nucleotide sequence ( - nucleotides) to produce different random segments depending on the target amplified, which are visualized by electrophoresis. this technique does not require knowledge of the target dna sequence, but its reproducibility is low and has been applied only for few plant viruses (williams et al., ; de araujo et al., ) ; ii) restriction fragment length polymorphism (rflp) analysis is based on digestion of the pcr products with restriction enzymes and electrophoretic separation of the resulting restriction fragments according to their length, revealing sequence differences within the restriction sites. this technique has been used to differentiate isolates of some plant viruses, such as prunus necrotic ringspot virus (pnrsv), tylcv and ctv (gillings et al., ; hammond et al., ; font et al., ) ; iii) single-strand conformation polymorphism (sscp) analysis is based on electrophoresis of denatured dsdna in non-denaturing gels so migration of single-stranded dna depends on its conformation determined by its nucleotide sequence and the electrophoretic conditions. this technique is very resolutive and can detect small differences, even of a single nucleotide, but it is very sensitive to minute changes in the electrophoretic conditions hindering reproducibility. the main advantage of sscp analysis is the ability to detect different genetic variants (visualized as electrophoretic bands) within a sample (plant), allowing to assess within-isolate genetic variation rapidly. this technique followed by sequencing of the different haplotypes detected has been used to evaluate the genetic variation of some plant viruses, such as cucumber mosaic virus (cmv), citrus psorosis virus (cpsv) and ctv (rubio et al., ; rubio et al., ; vives et al., ; lin et al., ; martıń et al., ) . iv) real-time qpcr has been used to differentiate virus strains by high resolution melting dna curve analysis with sybr green or by using taqman ™ fluorescent probes specific for each strain (varga and james, ; ruiz-ruiz et al., ; bester et al., ) . hts techniques are the most powerful and versatile since the nucleotide sequences can be used not only to estimate the genetic variation and structure of virus populations but also to identify and ascribe a virus sample to different taxonomic levels or discover new virus species, genera or families (kreuze et al., ; wu et al., ; pecman et al., ; verdin et al., ) , according to its nucleotide or amino acid identity with known sequences in databases (genbank) or the presence of sequence motifs. this task can be easily carried out with the algorithm blast (https:// www.ncbi.nlm.nih.gov/blast/), which compares the query sequence with all sequences from databases and find those that are more similar. recombination can produce biological and genomic variants of a virus that can be very similar in one genome region and very divergent in other. thus, the complete genome or different regions of it should be analyzed for detection and identification of recombinant variants. recombination can be detected by comparing nucleotide identity and/or phylogenetic relationships along the genome, which can be performed with different procedures implemented in the package rdp (martin et al., ) . for example, identification of ctv strains requires different sets of primers (roy et al., ) as recombination has played an important role in shaping ctv genome (martıń et al., ) . hts can be useful to detect recombination since fulllength or almost full-length viral genomes are sequenced rapidly, in a single analysis (akinyemi et al., ; silva et al., ) , unlike genome walking that requires successive steps of pcr, sanger sequencing and primer design. third-generation sequencing of single molecules seems more appropriate to identify recombination (viehweger et al., ) than secondgeneration sequencing methods that can produce recombinant artifacts, as genomic sequences are assembled from short sequences. nevertheless, it is convenient to confirm the recombinants by pcr followed by sanger sequencing. procedures to detect and identify various viruses or virus strains in a single assay simultaneously reduce time and cost of the analysis (see pallaś et al., for a comprehensive review), and are especially suitable for evaluating mixed infections in individual plants. the detection of individual viruses in a sample is mainly based on three approaches: i) spatial separation of detection sites (wells or spots); ii) separation of distinctly sized amplicons by electrophoresis; and iii) using a different label for each virus (dincer et al., ) . multiplex pcr or rt-pcr is the amplification of multiple targets simultaneously in a single pcr by using several primer pairs specific for each target. development of a multiplex pcr or rt-pcr assay is often complex since primers must comply with several conditions: i) similar melting temperatures (similar length and g-c content) so that all primers can function under the same pcr conditions; ii) compatibility, avoiding cross-binding and competition; iii) similar sensitivity; and iv) flanking genomic regions of different sizes so that the amplicons of each target can be separated and visualized by gel electrophoresis. the last constriction can be avoided by using primers labeled with different color fluorescent dyes or coupling the pcr with hybridization with specific probes . multiplex pcr or rt-pcr have been used to identify: i) the main viruses infecting a particular crop, such as tomato, tobacco, legumes, potato, ornamentals, cucumber and olive (bariana et al., ; bertolini et al., ; dai et al., ; panno et al., ; ge et al., ; ali et al., ; pallaś et al., ) ; ii) viruses from the same genus (uga and tsuda, ; hu et al., ; panno et al., ) ; and iii) different strains of a viral species (hammond et al., ; nie and singh, ; huang et al., ; alfaro-fernańdez et al., ; bester et al., ) . multiplex real-time qpcr with taqman probes labeled with different fluorescent dyes have been used to identify viruses from the same crop (abrahamian et al., ; loṕez-fabuel et al., ; malandraki et al., ) , and strains or isolates from the same viral species (varga and james, ; debreczeni et al., ) . the main problem of multiplex pcr is that only a limited number of targets can be amplified simultaneously since the more primers are used, the higher is the probability of incompatibility between some of them. also, there is a limitation in the number of products of different sizes that can be resolved by electrophoresis or the number of fluorescent dyes that can be used (boonham et al., ) . multiplex lamp has also been developed for the simultaneous detection of some plant viruses (zhang j. et al., ; wilisiani et al., ) . molecular hybridization with cocktails of probes or polyprobes (probes linked in tandem) has been used to detect different viruses affecting a crop (sańchez-navarro et al., ; saade et al., ; herranz et al., ; aparicio et al., ; minutillo et al., ) , although further analyses are necessary to identify each virus. microarray analyses can screen many samples simultaneously. they can be based on serology, but most are based on molecular hybridization (boonham et al., ; boonham et al., ; charlermroj et al., ) . capture probes corresponding to different viruses and/or genomic regions are attached to a solid support (usually glass) and the sample to be examined is fluorescently labeled so the identity of the virus or viruses present in the sample is determined by the fluorescent positions on the array. capture probes can be produced from pcr products ( - bp in length) or synthetic oligonucleotides ( - nucleotides in length). probe design must consider probe length, melting temperature, gc content, secondary structure caused by self-annealing and hybridization free energy since they affect sensitivity and specificity. short oligonucleotides ( - bases) are better to discriminate small sequence differences but exhibit reduced sensitivity. oligoprobes can be designed for different detection levels (genus, species and strain) by choosing conserved or variable sequence stretches. sensitivity and specificity can be improved controlling the hybridization temperature and buffer composition (boonham et al., ) . microarrays have been used to detect: i) viruses infecting a particular crop, such as tomato, cucurbits, potato and grapevine (bystricka et al., ; engel et al., ; sip et al., ; tiberini et al., ; tiberini and barba, ) ; ii) different viral species of a genus (wei et al., ); and iii) isolates or variants of the same virus (deyong et al., ; pasquini et al., ) . microarrays have been improved to detect hundreds of plant viruses, including genus-specific oligoprobes (zhang et al., ; nicolaisen, ; nam et al., ) . microsphere technology, like luminex xmap, can detect up to targets in a single sample. it is based on microspheres (beads) coated with specific antibodies or oligonucleotides, which capture respectively viruses or pcr products obtained from their genetic material. the beads have been dyed into spectrally distinct sets, or "regions," allowing them to be individually identified. after binding, the target is labeled by specific conjugated antibodies or probes which will give a fluorescent signal. the color code of the bead in combination with the fluorescent signal identifies a unique combination (boonham et al., ). luminex xmap system has been used to detect viruses infecting a crop (lim et al., ) , viruses belonging to a genus (van brunschot et al., ; bald-blume et al., a) , and strains or variants within a virus species (bald-blume et al., b) . hts is the most powerful technique for multiplex detection as it can identify and discover an unlimited number of viruses and virus variants within a plant (jones et al., ) . estimating the amount of a specific virus provides more precise information than just determining the presence or absence of that virus. elisa and molecular hybridization can be used for rough quantification of viral particles or nucleic acids based on the signal intensity (rubio et al., a; rohrman et al., ) . real-time qpcr is a very accurate procedure to estimate virus titer with a wide dynamic range and great sensitivity. the principle is to monitor in each cycle the increase of fluorescence. the cycle at which amplification is observed (cycle threshold, ct) is related to the inverse log of the quantity of target being amplified (boonham et al., ) . real-time qpcr or rt-qpcr has been developed for several plant viruses (mumford et al., ; picóet al., ; loṕez et al., ; ling et al., ; hongyun et al., ; ruiz-ruiz et al., a; debreczeni et al., ; ferriol et al., ; sharma and dasgupta, ; mackenzie et al., ; herrera-vaśquez et al., ) . it has been applied to evaluate some disease control methods such as i) study interactions between viruses in mixed infections (mortimer- jones et al., ; abrahamian et al., ) , which can be used for control based on cross protection (ruiz-ruiz et al., b; hanssen et al., ) ; ii) estimation of correlation between virus accumulation and transmission by insect vectors (olmos et al., ; rotenberg et al., ; ferriol et al., ; debreczeni et al., ) , which can be used for epidemiological studies and disease control strategies based on restricting the dispersion of viruses by vectors; iii) evaluation of the resistance level to virus accumulation in plant breeding programs (gil-salas et al., ; galipienso et al., ; soler et al., ) ; and iv) estimation of fitness in competition and evolutionary experiments (carrasco et al., ; peña et al., ) which can be used for evolutionary and epidemiological studies, as well as for evaluation of resistance durability. hts can be used for a relative quantification based on the number of reads for the same sequence, but it is still too expensive for these applications. other important features to be considered in the detection techniques are the costs, throughput screening (number of samples analyzed simultaneously) and easiness, not only during the application but also during the design or development ( table ) . sample processing is a critical step and affects the rapidity, easiness and throughput of the detection process. molecular hybridization and pcr techniques require purification of total rna or dna from plants to remove substances inhibiting the detection process (yielding false negatives) or producing a background signal (yielding false positives). inhibition of pcr can be avoided or minimized by diluting the extracts or by immunocapture (olmos et al., ) . hts also requires purification of dna or rna from plants. libraries can be enriched in viral sequences by using as starting material preparations from which host nucleic acids have been removed by subtractive hybridization or using purified viral particles, double-stranded rnas (dsrnas) preparations, which are enriched in replicative intermediates of rna viruses, or small rnas resulting from rna silencing that is a plant response to virus infection (kreuze et al., ; singh et al., ; kesanakurti et al., ; pecman et al., ; czotter et al., ) . extracts obtained by just grinding plant tissue in buffer can be used for elisa and lamp. for some viruses, tissue-prints made by cutting leaf petioles or rolled leaf blades transversely and gently pressing the fresh cut onto nitrocellulose membranes have been analyzed directly with elisa or molecular hybridization (narvaéz et al., ; rubio et al., b; ferrer et al., ) . an alternative to passive incubation of probes with the targets immobilized onto membranes in solution is the flow-through hybridization, based on directing a probe flow towards the targets immobilized on the membrane, reducing the hybridization time from hours to a few minutes . on-site detection of plant viruses is an interesting feature allowing a prompt response. presently, several techniques are commercially available (donoso and valenzuela, ) . lateral flow assay (lfa) consists of a chromatographic test strip where crude plant extracts are dropped and move capillarily. the virus is detected when a stained band appears by binding virions with labeled antibodies or viral nucleic acids with labeled dna or rna probes (drygin et al., ; zhang et al., ; koczula and gallotta, ) . this procedure takes only about to min. lfa has been used for multiplex detection of potato viruses (safenkova et al., ) and relative quantification (rohrman et al., ) . rpa and lamp isothermal amplification can be performed with crude plant extracts in portable hot blocks and results can be displayed with a portable fluorescence reader or a lateral flow strip so the whole process can take about min. (zhang et al., ; wilisiani et al., ) . oxford nanopore technologies has developed minion, a portable nanopore sequencing device, that can be used for the detection of plant viruses in the field (boykin et al., ; filloux et al., ; shaffer, ) , but it is still too expensive for most routine uses. the ability to develop rapidly new assays is very important given the continuous emergence of new plant viruses. the production of antisera for the serological techniques is lengthy, unpredictable and costly (boonham et al., ) . in contrast, the setup of molecular detection methods is a directed process that is cheap, fast and versatile, enabling to address different detection levels and consider the genetic variability of virus populations. primers and oligoprobes are synthesized and commercialized at a low cost. they can be easily designed with many available software algorithms, such as prime (http://bioinfo.ut.ee/ primer - . . /) and primer express (thermofisher) for pcr, or primer explorer (https://primerexplorer.jp/e/), lamp designer (optigene) and lava (torres et al., ) for lamp. eradication or control of virus diseases is difficult given the complex and dynamic nature of virus epidemics and the great evolvability of viruses (acosta-leal et al., ; elena et al., ) . for efficient and durable control, it is necessary to consider the genetic diversity and evolution of virus populations and have specific, fast and reliable diagnostic tools. disease management in agriculture is based on two approaches: immunization to get resistant plants to viral infections and prophylactic measures to restrain virus dispersion. introgression of resistance genes from cultivated or wild species into susceptible related crops by backcrossing (plant breeding) is the most widely-used immunization method. there are two types: i) active resistance driven by resistance proteins, encoded by dominant alleles, which recognize specifically a sequence or conformational pattern of a virus gene (avirulence determinant, avr) and induces death of the infected cells (hypersensitive response), precluding virus movement to adjacent cells and systemic infection (de ronde et al., ; peiróet al., ) ; and ii) passive resistance, conferred by resistance recessive alleles encoding host factors critical for viral infection, mostly eukaryotic translation initiation factors (eif) e and g, and their isoforms (truniger and aranda, ; hashimoto et al., ) . plant breeders usually aim at complete resistance, in which the virus cannot establish a systemic infection. elisa and molecular hybridization have been used to test germplasm and cultivars for resistance since a good number of plants can be analyzed simultaneously (rubio et al., a; soler et al., ) . when a complete resistance is not possible, breeding for relative or partial resistance (reduction of virus accumulation) or tolerance (reduction of virus damage without affecting virus multiplication) can be a good alternative. the most precise technique to evaluate the level of relative resistance is real-time qpcr (gil-salas et al., ; galipienso et al., ; soler et al., ) . time-course assays can be used to evaluate relative resistance (that can be measured by elisa or molecular hybridization) and relative tolerance (measured by observation of symptoms as a proxy of damage). the levels of resistance or tolerance can be estimated as the probability of no infection or no symptoms, respectively, by survival analyses (kaplan and meier, ) . however, breeding resistant cultivars is unsuitable for many crops and viruses because of the scarcity of resistance genes found in genetically compatible relatives. an alternative may be to change the specificity or range of known resistance genes by artificial evolution so they can confer resistance to novel viruses or virus strains. this approach is based on generating large populations of random mutants from a resistance gene by pcr with a high error rate, followed by the selection of those variants showing the desired resistance properties. resistance is evaluated by agrobacterium-mediated transient co-expression of each resistance gene mutant and the avr from the challenge virus in nicotiana benthamiana leaves so the resistance response is observed as a necrotic lesion. this approach has been used to broaden the resistance specificity of the potato gene rx (farnham and baulcombe, ; harris et al., ) , but its use has not become widespread since most mutants are nonfunctional and screening is time-consuming. genome editing, like the crispr-cas system, could be used to implement in crops the resistance genes obtained by artificial evolution and to mutagenize directly host genes involved in recessive resistance to prevent interaction with viruses (piron et al., ; chandrasekaran et al., ; pyott et al., ) . however, the mutagenized plant genes could be involved in important biological functions, so the mutations may also have unexpected adverse effects in plant development or physiology. resistance can be ineffective for some virus variants or be overcome by i) interaction with other viruses in mixed infections (desbiez et al., ; garcıá-cano et al., ) , ii) positive selection of punctual mutations (weber et al., ; hebrard et al., ; lópez et al., ) or iii) recombination or reassortment events (qiu and moyer, ; miras et al., ) . plant genes conferring dominant resistance usually target viral protein domains whose function is essential for the virus biology (replication, movement, transmission) and are under strong negative selection. thus, it is difficult for the virus to fix the mutations producing resistance breakdown. in some cases, overcoming the resistance implemented in a cultivar by plant breeding involves a tradeoff leading to a loss of the virus fitness in non-resistant hosts, thus limiting the cases of emergence and spread of resistance-breaking isolates in the field (garcıá-arenal and mcdonald, ) . polygenic resistance is more durable, but resistance implemented in most breeding programs is monogenic because its introgression in the crops is easier (palloix et al., ) . understanding the molecular, evolutionary and epidemiological factors involved in the emergence of resistance-breaking virus isolates is progressing (garcıá-arenal and mcdonald, ; elena et al., ) , but predicting the durability of new resistances remains elusive and it needs to be tested in the field. plant breeders are keen on the host genetic variability, but often they neglect the virus genetic variability, which should be considered when new resistance genes are tested to minimize the possibility of resistance breakdown. multiplex real-time qpcr is well-suited to evaluate the effect of mixed infections in overcoming resistance. detection of virus variants with punctual mutations leading to resistance breakdown would be a valuable tool to monitor the dispersion of these variants. however, this has proved to be a difficult task as in most cases resistance breakdown occurs by only one nucleotide substitution (producing one amino acid change). the presence of other neutral substitutions around the critical mutation hinders the design of molecular markers for resistance breakdown. real-time qpcr with taqman probes has been developed to detect single nucleotide polymorphisms associated with resistance breakdown for beet necrotic yellow vein virus (bnyvv) and tomato spotted wilt virus (tswv) (acosta- leal and rush, ; di rienzo et al., ) ;. however, there is no guarantee that these techniques are universal for all isolates of each virus species, since other polymorphic sites nearby can affect the detection process or have epistatic interactions affecting resistance. another strategy to obtain resistant plants is based on the rna silencing mechanism. rna silencing is a regulatory mechanism of gene expression in eukaryotes and a natural antiviral defense mechanism. the host rna silencing machinery targets dsrna that arise from replicative viral intermediates or secondary structures in the genomic rna due to internal complementarity, which are detected by rnases (dicer-like proteins) and cleaved into small rna duplexes (sirna or mirnas) of - nucleotides (nt) in length. one of the two strands of the small rnas is recruited to the rna-induced silencing complex (risc) that subsequently cleaves cognate viral rnas in a sequence-specific manner. these cleaved rnas are recognized by rna dependent rna polymerase (rdr), which amplifies the dsrna molecules, thus contributing to the amplification of the host defense mechanism that results in effective inhibition of local and systemic viral infection (kaldis et al., ) . to counteract this defensive mechanism, many viruses encode rna silencing suppressors (voinnet et al., ) , which can act in different steps of the silencing pathway, either by binding sirna duplex or by directly interacting with key components of the rna silencing machinery. some synergetic interactions between coinfecting viruses (increasing viral accumulation or symptoms) are caused by the cumulative effect of the silencing suppressors of both viruses (syller, ) . resistance can be obtained by plant transformation, introducing into plants dna constructs to produce viral dsrnas or ssrna with some degree of secondary structure to trigger rna silencing. since rna silencing requires a certain nucleotide identity between the targeted virus and the transgene, it is necessary to evaluate the nucleotide variation of the virus population as explained above. the best technique to evaluate the efficiency of the rna silencing-based resistances is realtime qpcr. silencing resistance breakdown can occur by mutation and selection (de haan et al., ) or by mixed infection with other viruses (syller, ) . transgenic plants with multiple constructs from different viral genomes (from the same species and/or different species) can be used to minimize the risk of resistance breakdown (bucher et al., ; duan et al., ) . another strategy is using a transgene mimicking the secondary structure of endogenous mirna precursors (involved in plant gene expression and development) to express artificial mirnas (amirnas) targeting viral sequences (niu et al., ; qu et al., ; liu et al., ) . the main advantage is that the short sequence of amirnas makes it easier to find conserved sequences that are more difficult to overcome (they are usually under strong negative selective pressure) and can be used for broad range targets (genera and families). however, the amirna resistance can be also overcome by mutation and selection (simoń-mateo and garcıá, ; lin et al., ; lafforgue et al., ) or interaction with co-infecting viruses (pacheco et al., ; martıńez et al., ) . a strategy to obtain more durable resistances is to express multiple amirnas to target different regions within a single viral genome (fahim et al., ; kung et al., ; lafforgue et al., ; kis et al., ) . synthetic trans-acting small interfering rnas (syn-tasirnas) is another class of artificial small rnas engineered in plants, which are especially suited to target multiple sites within a viral genome or different unrelated viruses (carbonell et al., ; chen et al., ; . the durability of these resistances can be evaluated by experimental evolution based on successive passages of the virus in the resistant plants. resistance-breakdown can be detected by qpcr as an increase in virus accumulation (carrasco et al., ; peña et al., ) . the mutations fixed after the passages can be detected by pcr followed by cloning and sanger sequencing or hts. identification of the mutations leading to resistance breakdown is possible if infectious clones exist, so that each mutation can be tested for the increase of virus accumulation. an alternative to transgenic plants is the exogeneous application of in vitro-produced dsrnas from viral sequences onto plants (tenllado and dıáz-ruiz, ; kaldis et al., ; niehl et al., ) . the efficacy of this technique of immunization can be improved by high-pressure spraying plants (dalakouras et al., ) , using cell-penetrating peptides (numata et al., ) or clay nanoparticles stabilizing dsrnas (mitter et al., ) . another immunization method is cross-protection based on inoculating mild or attenuated viral strains to protect plants against severe strains of the same virus. cross-protection has been applied to several viruses and crops, such as pepmv in tomato and ctv in citrus crops (pechinger et al., ) . the mechanism of crossprotection is poorly understood and several models have been proposed: prevention of virus entry into cells; competition for host factors for replication, interference with disassembly, translation or replication; and induction of rna silencing leading to sequence-specific degradation of the superinfecting virus (ziebell and carr, ; folimonova, ) . it has been suggested that crossprotection in some viruses might be an active virus-controlled function involving virus-coded proteins (folimonova, ) . a recent model proposed that cross-protection is a mechanism that prevents the virus progeny to replicate in the cells to minimize mutation rate and collaterally targets highly homologous superinfecting viruses that are indistinguishable from progeny viruses . to apply cross-protection it is necessary to evaluate the genetic and biological variability of the local virus population and search for mild isolates genetically close to the severe ones (hanssen et al., ) . mild or attenuated strains can also be generated by thermal treatment, random mutagenesis by using chemicals as nitrous acids and selection or directed mutagenesis in viral rna silencing suppressors (ziebell and carr, ) . cross-protection assays can be evaluated by real-time qpcr with probes specific for each virus variant (ruiz-ruiz et al., b; hanssen et al., ) and sscp analysis (sambade et al., ) . however, the protection exerted by the mild isolate can be overcome and even a more severe disease can emerge by transmission to a different host species, interaction with other viruses in mixed infections, or recombination between divergent strains or viruses (fulton, ) . therefore cross-protection should be used only for devastating diseases when other protection measures fail, and the process should be monitored closely. quarantine (control of borders) and sanitary certification of virusfree germplasm (seeds or asexual propagative tissues) are the first measures to prevent the introduction and emergence of new viruses in a geographical area. virus detection should be based on sensitive and broad-spectrum methods, since discarding healthy material is preferable to the risk of spreading new diseases. on-site detection devices can be useful to make decisions quickly, thus preventing importation and exportation delays. hts is the most powerful detection procedure since it can detect all the viruses (known and unknown) present in a plant in an unbiased way. its use in quarantine and clean plant programs is increasing as it is becoming more economically affordable (villamor et al., ) . phytosanitary certificates should be based on propagative material free from only harmful viruses, since plants can harbor many viruses (maree et al., ) . since epidemiology and evolution are coupled in viruses, phylogeographic studies, comparing genetic variation in space and time, can provide useful information on the introduction sites of new viruses and the dispersal paths (goḿez et al., ; davino et al., ) . as an illustration, figure shows the migration paths of one of the strains in the first introduction of ctv in sicily, italy , which were inferred by bayesian phylogeographic analysis with the program beast v . . (drummond and rambaut, ) . phylogenetic analyses showed that these sicilian ctv isolates were genetically close to ctv isolates from mainland italy and california. sanitation, that is, removing viruses from valuable cultivars, is necessary when no healthy plants are available. virus-free plants are usually produced by thermotherapy, chemotherapy, electrotherapy, and tissue culture alone or combined (naik and buckseth, ) . thermotherapy could inactivate viruses by viral rna breakage, viral particle disruption or coat protein rupture, viral replicase inactivation, virus movement inhibition and/or translation reduction (hull, ) . chemotherapy is based on antiviral drugs to inhibit or disrupt specific steps of the virus life cycle, e.g., nucleoside analogs inhibiting replication and protease inhibitors preventing protein processing. antiviral drugs are costly and are used only to regenerate healthy mother plants for vegetative propagation or seed production (panattoni et al., ) . the sanitated plants must be evaluated and confirmed to be virus-free with very sensitive techniques such as real-time qpcr. nucleoside analogs can also be used to increase the already high mutation rate of rna viruses, so that the excess of mutations would lead to a loss of genetic information and virus extinction (lethal mutagenesis or error catastrophe). this approach has been assayed recently with a plant virus, tobacco mosaic virus (tmv), resulting in a loss of viral infectivity (dıáz- martıńez et al., ). an agronomical practice to limit virus dispersal consists of removing virus-infected plants from crops or weeds acting as inoculum source. this requires rapid and specific detection techniques able to analyze many samples from the field, such as elisa and molecular hybridization, especially using tissue-prints rubio et al., b) . roguing is effective only if the virus incidence is low after a recent introduction or in isolated areas. other practices consist of interrupting the virus transmission chain. many seed-borne viruses (carried on the seed coat) can be removed with chemical disinfectants such as sodium hypochlorite, trisodium phosphate, hydrochloric acid and ozone, whereas some seed-transmitted viruses (infecting the seed embryo) can be eliminated by thermotherapy (ling, ; paylan et al., ) . multiplex (rt)-pcr to detect simultaneously the seed-transmitted and seed-borne viruses for a crop can be a useful tool . incidence of plant viruses mechanically transmissible by contact, like those of the genus tobamovirus, can be reduced by hygienic measures such as using disposable gloves or washing hands with disinfectant, heat sterilization of tools and debris and limiting the access to crops (broadbent, ) . most plant viruses are transmitted by arthropod vectors, mainly aphids, whiteflies, and thrips. three strategies to prevent the spread of plant viruses by vectors have been used (antignus, ; fereres and raccah, ) : i) reducing vector populations by pesticides; biological control with natural enemies such as arthropod predators and parasitoids (teĺlez et al., ) or entomopathogenic fungi, nematodes, bacteria and viruses (kalha et al., ) ; and biotechnology-based approaches based on protease inhibitors, neurotoxins, or rna silencing of genes essential for insect development or metabolism (fereres and raccah, ; nandety et al., ; vogel et al., ) . ii) preventing the vector from reaching the crop with barriers (greenhouses and barrier plants), by interference of the insect vision with uv-absorbing plastics and reflective surfaces, by agronomical practices such as changing the planting or sowing dates to avoid high vector populations, or imposing a time gap between crops and/or space gap between plots to break the transmission cycle (antignus, ; fereres and raccah, ) . iii) interfering with the transmission process by spraying mineral oils, synthetic peptides or modified proteins to outcompete virus-encoded proteins needed for virus attachment to insect receptors (lecoq and desbiez, ; blanc et al., ) . the rate of insect transmission can be evaluated using different detection techniques, such as elisa, molecular hybridization and pcr, to detect the virus in the receptor plants and real-time qpcr to estimate the virus titer in the source plants, which affects transmissibility (olmos et al., ; rotenberg et al., ; ferriol et al., ; debreczeni et al., ) . the main challenge of agriculture in this century is to produce nutritious food for the growing world population in a sustainable manner while protecting the environment and human health (pretty, ; pretty et al., ) . damages caused by pests and diseases have a considerable negative economic impact in agriculture, being emergent viral diseases particularly important (anderson et al., ; mumford et al., ) . the correct identification of viruses is critical for disease management. however, the great ability of viruses to evolve and generate molecular and biological variation is a major difficulty for virus detection and disease management. presently, when a new virus-like disease appears, the first approach is to test for known viruses with well-established techniques ( figure a ). elisa is the most popular for routine analysis because of historical reasons, easiness to perform with little training and commercial availability of antibodies specific for the main plant viruses. however, antibody production is expensive, time-consuming, and unpredictable, and it cannot be designed to cope with viral variability. in contrast, the development of molecular techniques is fast and cheap, making them more appropriate to cope with the frequent cases of new virus emergence (boonham et al., ) . pcr techniques are the most widely used because of the easy design, versatility, and low cost of primers. real-time qpcr is becoming the molecular method of choice for routine virus analysis (especially for new viruses for which antibodies are not available or with low accumulation levels) and quantification. onsite detection techniques by lfa and isothermal amplification (rpa and lamp) allow an almost immediate response and are rapidly developing. when these techniques fail to detect the virus causing disease, the best approach is to use hts, which can identify all the viruses in one plant, albeit infectivity assays or field surveys are necessary to determine which of the viruses detected is likely the disease causal agent ( figure a ). in some cases, such as quarantine, using hts as first approach for virus detection can be more profitable than testing many viruses with elisa or molecular detection techniques. however, hts is still too expensive for most routine analyses and it is necessary to develop rapid and accurate detection techniques for each virus, being pcr the easiest to develop. the design of primers or probes figure | phylogenetic tree of citrus tristeza virus (ctv) isolates collected from the first outbreak of ctv in sicily, italy and a map showing the migration paths within sicily . rubio et al. virus variability: diagnosis and control for accurate detection requires to evaluate the genetic diversity of viral populations by analysis of nucleotide sequences. ( figure b ). presently, disease management relies on preventing introduction of new viruses by border control and certification of virus-free propagative material (e.g., seeds) and preventing virus dispersal in the field. quarantine and certification require sensitive and broad-spectrum detection methods to minimize escapes such as the use of conserved primers specific for virus genera or families and multiplex procedures ( figure c ). hts is the best procedure and it is becoming affordable in quarantine facilities given the devastating consequences of introducing new pathogens. prophylactic measures to prevent or minimize virus dispersal require information on the virus biology (host range, transmission way, etc.), virus incidence and epidemiology. obtaining this information needs using high-throughput screening techniques able to analyze a high number of samples such as elisa and molecular hybridization ( figure c ). the other keystone for plant disease control is the obtention of resistant cultivars, which is performed mainly by plant breeding and commercialized by seed companies. however, apart from the important effort involved in breeding programs, resistance is not available for most crops and viruses. genetic engineering, despite the great potential and some remarkable successes (fuchs and gonsalves, ) , faces heavy opposition in some countries due to the public concern on the potential ecological impact of transgenic plants (jones, ) . resistant plants should be evaluated with specific methods, as resistance depends on host and virus genotypes. partial resistance can be evaluated by real-time qpcr ( figure c ). genome editing by crispr and the application of dsrna-loaded clay particles to trigger rna silencing are promising research fields. in any case, to aim at more durable and effective protection, it is necessary to characterize the genetic variability and relationships of plant viruses, as well as the factors and mechanisms involved in genetic change. hts excels for its broad-spectrum and multiplex detection, sensitivity, and precise quantification. no previous knowledge is required, enabling unbiased detection and discovery of new viruses. the sequences obtained allow a precise taxonomic assignation and an estimation of genetic relationships with other viruses and viral isolates. it is reasonable to expect that hts, especially portable nanopore sequencing devices, will become the standard diagnostic procedure as costs will be dropping and analytical procedures improving. lr conceived the idea. lr, if, and lg wrote the manuscript. lr and if designed the figures. all authors contributed to the article and approved the submitted version. this work was partially funded by grants rta - -c - from spanish ministerio de ciencia e innovacioń co-financed by feder and from ivia. if acknowledges financial support from the spanish ministerio de economia y competitividad, through the "severo ochoa programme for centers of excellence in r&d" - (sev- - ). detection and quantitation of two cucurbit criniviruses in mixed infection by real-time rt-pcr mutations associated with resistancebreaking isolates of beet necrotic yellow vein virus and their allelic discrimination using taqman technology advances in plant virus evolution: translating evolutionary insights into better disease management codon usage bias amongst plant viruses virus adaptation by manipulation of host's gene expression ecogenomic survey of plant viruses infecting tobacco by next generation sequencing simultaneous detection and identification of pepino mosaic virus (pepmv) isolates by multiplex one-step rt-pcr genetic bottlenecks during systemic movement of cucumber mosaic virus vary in different host plants analysis of genetic bottlenecks during horizontal transmission of cucumber mosaic virus multiplex rt-pcr detection of three common viruses infecting orchids rapid generation of genetic heterogeneity in progenies from individual cdna clones of peach latent mosaic viroid in its natural host emerging infectious diseases of plants: pathogen pollution, climate change and agrotechnology drivers control methods of virus diseases in the mediterranean basin simultaneous detection of six rna plant viruses affecting tomato crops using a single digoxigenin-labelled polyprobe development of a molecular assay for the detection of cucumber mosaic virus and the discrimination of its subgroups i and ii detection of five seedborne legume viruses in one sensitive multiplex polymerase chain reaction test single-step multiplex rt-pcr for simultaneous and colourimetric detection of six rna viruses in olive trees real-time rt-pcr high-resolution melting curve analysis and multiplex rt-pcr to detect and differentiate grapevine leafroll-associated virus variant groups i, ii, iii and vi estimation of the effective number of founders that initiate an infection after aphid transmission of a multipartite plant virus localizing viruses in their insect vectors third generation sequencing: technology and its potential impact on evolutionary biodiversity research microarrays for rapid identification of plant viruses methods in virus diagnostics: from elisa to next generation sequencing real time portable genome sequencing for global food security epidemiology and control of tomato mosaic virus nanopore sequencing as a surveillance tool for plant pathogens in plant and insect tissues multiple virus resistance at a high frequency using a single transgene construct dna microarray: parallel detection of potato viruses design, synthesis, and functional analysis of highly specific artificial small rnas with antiviral activity in plants fast-forward generation of effective artificial small rnas for enhanced antiviral defense in plants multi-targeting of viral rnas with synthetic trans-acting small interfering rnas enhances plant antiviral resistance a real-time rt-pcr assay for quantifying the fitness of tobacco etch virus in competition experiments diagnosis of plant diseases using the nanopore sequencing platform development of broad virus resistance in non-transgenic cucumber using crispr/cas technology selection pressures in the capsid genes of plant rna viruses reflect mode of transmission a phylogenetic survey of recombination frequency in plant rna viruses antibody array in a multiwell plate format for the sensitive and multiplexed detection of important plant pathogens multiple virus resistance using artificial trans-acting sirnas next-generation diagnostics with crispr characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses the promiscuous evolutionary history of the family bromoviridae ngs of virus-derived small rnas as a diagnostic method used to determine viromes of hungarian vineyards lethal mutagenesis of an rna plant virus via lethal defection contribution of uneven distribution of genomic rna variants of citrus tristeza virus (ctv) within the plant to changes in the viral population following aphid transmission a multiplex reverse transcription pcr assay for simultaneous detection of five tobacco viruses in tobacco plants induction of silencing in plants by high-pressure spraying of in vitro-synthesized small rnas recombination profiles between tomato yellow leaf curl virus and tomato yellow leaf curl sardinia virus in laboratory and field conditions: evolutionary and taxonomic implications emergence and phylodynamics of citrus tristeza virus in sicily, italy molecular detection of papaya meleira virus in the latex of carica papaya by rt-pcr characterization of rna-mediated resistance to tomato spotted wilt virus in transgenic tobacco plants dominant resistance against plant viruses detection, discrimination and absolute quantitation of tomato spotted wilt virus isolates using real time rt-pcr with taqman ® mgb probes transmission of tomato spotted wilt virus isolates able and unable to overcome tomato or pepper resistance by its vector frankliniella occidentalis increase in zucchini yellow mosaic virus symptom severity in tolerant zucchini cultivars is related to a point mutation in p protein and is associated with a loss of relative fitness on susceptible plants differentiation of cucumber mosaic virus isolates by hybridization to rubio et al. virus variability: diagnosis and control oligonucleotides in a microarray format rapid identification of tomato sw- resistance-breaking isolates of tomato spotted wilt virus using high resolution melting and taqman snp genotyping assays as allelic discrimination techniques multiplexed point-of-care testing-xpoct basic concepts in rna virus evolution in-field molecular diagnosis of plant pathogens: recent trends and future perspectives mutation rates among rna viruses beast: bayesian evolutionary analysis by sampling trees highly sensitive field test lateral flow immunodiagnostics of pvx infection application of rna silencing to plant disease resistance evolution and emergence of plant viruses a diagnostic oligonucleotide microarray for simultaneous detection of grapevine viruses resistance to wheat streak mosaic virus generated by expression of an artificial polycistronic microrna in wheat artificial evolution extends the spectrum of viruses that are targeted by a disease-resistance gene from potato plant virus transmission by insects (chichester: els detection and identification of species of the genus fabavirus by rt-pcr with a single pair of primers new molecular methods for identification of broad bean wilt virus detection and absolute quantitation of broad bean wilt virus (bbwv- ) and bbwv- by real time rt-pcr transmissibility of broad bean wilt virus by aphids: influence of virus accumulation in plants, virus genotype and aphid species genetic variability and evolution of broad bean wilt virus : role of recombination, selection and gene flow rapid detection and discrimination of fabaviruses by flow-through hybridisation with genus-and species-specific riboprobes nanopore-based detection and characterization of yam viruses developing an understanding of cross-protection by citrus tristeza virus genetic structure and evolution of natural populations of viruses causing the tomato yellow leaf curl disease in spain cross-reacting and heterospecific monoclonal antibodies produced against arabis mosaic nepovirus recombination every day: abundant recombination in a virus during a single multi-cellular host infection safety of virus-resistant transgenic plants two decades after their introduction: lessons from realistic field risk assessment studies practices and precautions in the use of cross protection for plant virus disease control extremely high mutation rate of a hammerhead viroid cucumber vein yellowing virus isolate-specific expression of symptoms and viral rna accumulation in susceptible and resistant cucumber cultivars genetic variation and population structure of an isolate of citrus exocortis viroid (cevd) and of the progenies of two infectious sequence variants synergistic interaction between tomato chlorosis virus and tomato spotted wilt virus results in breakdown of resistance in tomato an analysis of the durability of resistance to plant viruses simultaneous detection and identification of four viruses infecting pepino by multiplex rt-pcr characterisation of isolates and strains of citrus tristeza closterovirus using restriction analysis of the coat protein gene amplified by the polymerase chain reaction resistance screening against cucumber vein yellowing virus using a real-time (taqman ® ) rt-pcr assay in cucumber (cucumis sativus) mixed infections of pepino mosaic virus strains modulate the evolutionary dynamics of this emergent virus phylodynamics of pepino mosaic virus in spain coming of age: ten years of next-generation sequencing technologies next-generation sequencing and genome editing in plant virology a broad-spectrum pcr assay combined with rflp analysis for detection and differentiation of plum pox virus isolates differentiation of closely related but biologically distinct cherry isolates of prunus necrotic ringspot virus by polymerase chain reaction virus variability: diagnosis and control cross-protection or enhanced symptom display in greenhouse tomato co-infected with different pepino mosaic virus isolates stepwise artificial evolution of a plant disease resistance gene recessive resistance to plant viruses: potential resistance genes beyond translation initiation factors deep sequencing reveals a novel closterovirus associated with wild rose leaf rosette disease the sequence of sequencers: the history of sequencing dna emergence of a resistance-breaking isolate of rice yellow mottle virus during serial inoculations is due to a single substitution in the genome-linked viral protein vpg simultaneous detection of six stone fruit viruses by non-isotopic molecular hybridization using a unique riboprobe or 'polyprobe detection and absolute quantitation of tomato torrado virus (totv) by real time rt-pcr clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice rapid evolution of rna genomes real time taqman rt-pcr assay for the detection of cucumber green mottle mosaic virus detection of four calla potyviruses by multiplex rt-pcr using nad mrna as an internal control detection and isolate differentiation of citrus tristeza virus in infected field trees based on reverse transcription-polymerase chain reaction mathews' plant virology nucleic acid hybridization in plant virus diagnosis and characterization single primer pair designs that facilitate simultaneous detection and differentiation of peach mosaic virus and cherry mottle leaf virus strategies for simultaneous detection of multiple plant viruses viral diagnostics in plants using next generation sequencing: computational analysis in practice control of plant virus diseases plant virus emergence and evolution: origins, new encounter scenarios, factors driving emergence, effects of changing world conditions, and prospects for control relative incidence, spatial distribution and genetic diversity of cucurbit viruses in eastern spain exogenously applied dsrna molecules deriving from the zucchini yellow mosaic virus (zymv) genome move systemically and protect cucurbits against zymv entomopathogenic viruses and bacteria for insect-pest control," in integrated pest management: current concepts and ecological perspective nonparametric estimation from incomplete observations plant virology and next generation sequencing: experiences with a potyvirus screening for plant viruses by next generation sequencing using a modified double strand rna extraction protocol with an internal amplification control polycistronic artificial mirna-mediated resistance to wheat dwarf virus in barley is highly efficient at low temperature current trends in diagnostics of viral infections of unknown etiology lateral flow assays population structure and genetic diversity within california citrus tristeza virus (ctv) isolates the phylogeny of rna-dependent rna polymerases of positive-strand rna viruses complete viral genome sequence and discovery of novel viruses by deep sequencing of small rnas: a generic method for diagnosis, discovery and sequencing of viruses mega x: molecular evolutionary genetics analysis across computing platforms multiple artificial micrornas targeting conserved motifs of the replicase gene confer robust transgenic resistance to negative-sense singlestranded rna plant virus tempo and mode of plant rna virus escape from rna interferencemediated resistance improving the effectiveness of artificial microrna (amir)-mediated resistance against turnip mosaic virus by combining two amirs or by targeting highly conserved viral genomic regions viruses of cucurbit crops in the mediterranean region: an ever-changing picture phylogenetic and recombination analysis of tomato spotted wilt virus simultaneous detection of three lilyinfecting viruses using a multiplex luminex bead array genetic diversity and biological variation among california isolates of cucumber mosaic virus molecular evolution of a viral non-coding sequence under the selective virus variability: diagnosis and control pressure of amirna-mediated silencing development of a one-step immunocapture real-time taqman rt-pcr assay for the broad spectrum detection of pepino mosaic virus effectiveness of chemo-and thermotherapeutic treatments on pepino mosaic virus in tomato seed microrna-mediated gene silencing in plant defense and viral counter-defense development of real-time and conventional rt-pcr assays for the detection of potato yellow vein virus (pyvv) evolutionary analysis of tomato sw- resistance-breaking isolates of tomato spotted wilt real-time multiplex rt-pcr for the simultaneous detection of the five main grapevine viruses rt-pcr and real-time rt-pcr methods for the detection of potato virus y in potato leaves and tubers simultaneous detection of three pome fruit tree viruses by one-step multiplex quantitative rt-pcr detection and differentiation of comoviridae species using a semi-nested rt-pcr and a phylogenetic analysis based on the polymerase protein the diagnosis of the tomato variant of pepino mosaic virus: an ic-rt-pcr approach application of hts for routine plant virus diagnostics: state of the art and challenges genetic variation of populations of citrus psorosis virus contribution of recombination and selection to molecular evolution of citrus tristeza virus rdp : detection and analysis of recombination patterns in virus genomes fate of artificial micrornamediated resistance to plant viruses in mixed infections a dna probe mix for the multiplex detection of ten artichoke viruses interfamilial recombination between viruses led to acquisition of a novel translation-enhancing rna element that allows resistance breaking clay nanosheets for topical delivery of rnai for sustained protection against plant viruses a single tube, quantitative real-time rt-pcr assay that detects four potato viruses simultaneously the population genetics and evolutionary epidemiology of rna viruses specific synthesis of dna in vitro via a polymerase-catalyzed chain reaction detection of potato mop top virus and tobacco rattle virus using a multiplex real-time fluorescent reverse-transcription polymerase chain reaction assay the role and challenges of new diagnostic technology in plant biosecurity metagenomic analysis of plant viruses associated with papaya ringspot disease in carica papaya l synergistic disease in pepper caused by the mixed infection of cucumber mosaic virus and pepper mottle virus properties of two monoclonal antibodies specific to the cherry strain of plum pox virus recent advances in virus elimination and tissue culture for quality potato seed production nanopore sequencing of a novel bipartite new world begomovirus infecting cowpea development of the large-scale oligonucleotide chip for the diagnosis of plant viruses and its practical use emerging strategies for rna interference (rnai) applications in insects a new procedure to differentiate citrus tristeza virus isolates by hybridisation with digoxigenin-labelled cdna probes an oligonucleotide-based microarray for detection of plant rna viruses specific differentiation of recombinant pvy (n: o) and pvy (ntn) isolates by multiplex rt-pcr synthetic biology approach for plant protection using dsrna expression of artificial micrornas in transgenic arabidopsis thaliana confers virus resistance local gene silencing in plants via synthetic dsrna and carrier peptide the evolutionary trajectory of turnip mosaic potyvirus populations adapting to a new host real-time assay for quantitative detection of non-persistently transmitted plum pox virus rna targets in single aphids molecular diagnostic methods for plant viruses pvx-potyvirus synergistic infections differentially alter microrna accumulation in nicotiana benthamiana possible emergence of new geminiviruses by frequent recombination recent advances on the multiplex molecular detection of plant viruses and viroids durability of plant major resistance genes to pathogens depends on the genetic background, experimental evidence and consequences for breeding strategies review. elimination of viruses in plants: twenty years of progress simultaneous detection of the seven main tomato-infecting rna viruses by two multiplex reverse transcription polymerase chain reactions detection and identification of fabavirus species by one-step rt-pcr and multiplex rt-pcr loop mediated isothermal amplification: principles and applications in plant virology oligonucleotide microarray-based detection and genotyping of plum pox virus effects of different treatments on the inactivation of various seedborne viruses in some vegetables experimental virus evolution reveals a role of plant microtubule dynamics and tortifolia /spiral in rna trafficking a new era for mild strain cross-protection next generation sequencing for detection and discovery of plant viruses and viroids: comparison of two approaches the movement protein (nsm) of tomato spotted wilt virus is the avirulence determinant in the tomato sw- gene-based resistance a monoclonal antibody that discriminates strains of citrus tristeza virus quantitative detection of cucumber vein yellowing virus in susceptible and partially resistant plants using real-time pcr an induced mutation in tomato eif e leads to immunity to two potyviruses insect transmission of plant viruses: a constraint on virus variability agricultural sustainability: concepts, principles and evidence engineering of crispr/cas -mediated potyvirus resistance in transgene-free arabidopsis plants tomato spotted wilt tospovirus adapts to the tswv n gene-derived resistance by genome reassortment artificial microrna-mediated virus resistance in plants a lateral flow assay for quantitative detection of amplified hiv- rna metagenomics of plant and fungal viruses reveals an abundance of persistent lifestyles deep sequencing for discovery and evolutionary analysis of plant viruses variation in tomato spotted wilt virus titer in frankliniella occidentalis and its association with frequency of transmission development and application of a multiplex reverse transcription polymerase chain reaction assay for screening a global collection of citrus tristeza virus isolates differentiation of citrus tristeza closterovirus (ctv) isolates by single-strand conformation polymorphism analysis of the coat protein gene geographic distribution and molecular variation of isolates of three whitefly-borne closteroviruses of cucurbits: lettuce infectious yellows virus, cucurbit yellow stunting disorder virus, and beet pseudo-yellows virus characterization of citrus tristeza virus isolates by single-strand conformation polymorphism analysis of dna complementary to their rna population genetic variation of citrus tristeza virus isolates from california and spain: evidence for mixed infections and recombination a new approach to evaluate relative resistance and tolerance of tomato cultivars to begomoviruses causing the tomato yellow leaf curl disease in spain rapid detection of cucumber vein yellowing virus by tissue-print hybridisation with digoxigenin-labelled cdna probes genetic variability and evolutionary dynamics of viruses of the family a real-time rt-pcr assay for detection and absolute quantitation of citrus tristeza virus in different plant tissues detection and quantitation of citrus leaf blotch virus by taqman real-time rt-pcr discrimination between mild and severe citrus tristeza virus isolates with a rapid and highly specific real-time reverse transcription-polymerase chain reaction method using taqman lna probes simultaneous detection of the three ilarviruses affecting stone fruit trees by nonisotopic molecular hybridization and multiplex reversetranscription polymerase chain reaction virus variability: diagnosis and control estimation of population bottlenecks during systemic movement of tobacco mosaic virus in tobacco plants multiarray on a test strip (mats): rapid multiplex immunodetection of priority potato pathogens comparison of viral rna populations of pathogenically distinct isolates of citrus tristeza virus: application to monitoring cross-protection sensitivity of small rna-based detection of plant viruses genetic diversity in rna virus quasispecies is controlled by host-virus interactions inner workings: portable dna sequencer helps farmers stymie devastating viruses development of sybr green i based realtime pcr assays for quantitative detection of rice tungro bacilliform virus and rice tungro spherical virus the concept of sensitivity and specificity in relation to two types of errors and its application in medical research sequence analysis of plum pox virus strain c isolates from russia revealed prevalence of the d e mutation in the universal epitope and interstrain recombination events molecular characterization of a new virus species identified in yam (dioscorea spp.) by high-throughput sequencing microrna-guided processing impairs plum pox virus replication, but the virus readily evolves to escape this silencing mechanism new virus discovery by deep sequencing of small rnas detection of viral infections by an oligonucleotide microarray a new capsicum baccatum accession shows tolerance to wild-type and resistance-breaking isolates of tomato spotted wilt virus evaluation of reverse transcription-polymerase chain reaction assays for detecting apple chlorotic leaf spot virus in certification and quarantine programs facilitative and antagonistic interactions between plant viruses in mixed infections rna-rna recombination in plant virus replication and evolution control of tomato leaf curl new delhi virus in zucchini using the predatory mite amblyseius swirskii full genome characterization of citrus tatter leaf virus isolates for the development of a detection assay double-stranded rna-mediated interference with plant virus infection optimization and improvement of oligonucleotide microarray-based detection of tomato viruses and pospiviroids oligonucleotide microarray-based detection and identification of major tomato viruses high-throughput sequencing reveals bell pepper endornavirus infection in pepper (capsicum annum) in slovakia and enables its further molecular characterization lava: an open-source approach to designing lamp (loop-mediated isothermal amplification) dna signatures recessive resistance to plant viruses a one-step reverse transcription-polymerase chain reaction system for the simultaneous detection and identification of multiple tospovirus infections synergistic interaction of sweet potato chlorotic stunt virus (crinivirus) with carla-, cucumo-, ipomo-, and potyviruses infecting sweet potato a bead-based suspension array for the multiplexed detection of begomoviruses and their whitefly vectors the third revolution in sequencing technology detection and differentiation of plum pox virus using real-time multiplex pcr with sybr green and melting curve analysis: a rapid method for strain typing sequencing viral sirnas to identify previously undescribed viruses and viroids in a panel of ornamental plant samples structured as a matrix of pools direct rna nanopore sequencing of full-length coronavirus genomes provides novel insights into structural variants and enables modification analysis low genetic variation between isolates of citrus leaf blotch virus from different host species and of different geographical origins evidence of multiple recombination events between two rna sequence variants within a citrus tristeza virus isolate identification of a new enamovirus associated with citrus vein enation disease by deep sequencing of small rnas rna interference in insects: protecting beneficials and controlling pests suppression of gene silencing: a general strategy used by diverse dna and rna viruses of plants virus variability: diagnosis and control frontiers in plant science | www characterization of synergy between cucumber mosaic virus and potyviruses in cucurbit hosts probing of rna structures in a positive sense rna virus reveals selection pressures for structural elements two amino acid substitutions in the tomato mosaic virus -kilodalton movement protein confer the ability to overcome the tm- resistance gene in the tomato development of a short oligonucleotide microarray for the detection and identification of multiple potyviruses persistent infection and promiscuous recombination of multiple genotypes of an rna virus within a single host generate extensive diversity development of a lamp assay with a portable device for real-time detection of begomoviruses under field conditions dna polymorphisms amplified by arbitrary primers are useful as genetic markers co-infection of beet mosaic virus with beet yellowing viruses leads to increased symptom expression on sugar beet identification of viruses and viroids by next-generation sequencing and homology-dependent and homology-independent algorithms analyses of virus/viroid communities in nectarine trees by next-generation sequencing and insight into viral synergisms implication in host disease symptoms oligonucleotide microarray with a minimal number of probes for the detection and identification of thirteen genera of plant viruses rapid diagnostic detection of plum pox virus in prunus plants by isothermal amplifyrp ® using reverse transcription-recombinase polymerase amplification a new mechanistic model for viral cross protection and superinfection exclusion multiplex detection of three banana viruses by reverse transcription loop-mediated isothermal amplification (rt-lamp) a novel pair of universal primers for the detection of potyviruses cross-protection: a century of mystery the authors would like to apologize for any reference or study not included in this review. we would like to thank the reviewers of this manuscript for their insightful comments and suggestions. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fpls. . / full#supplementary-material. key: cord- -narre e authors: aziz, muhammad abdul; khan, amir hasan; adnan, muhammad; ullah, habib title: traditional uses of medicinal plants used by indigenous communities for veterinary practices at bajaur agency, pakistan date: - - journal: j ethnobiol ethnomed doi: . /s - - - sha: doc_id: cord_uid: narre e background: the pastoral lifestyle of indigenous communities of bajaur agency is bringing them close to natural remedies for treating their domestic animals. several studies have been conducted across the globe describing the importance of traditional knowledge in veterinary care. therefore, this study was planned with the aim to record knowledge on ethnoveterinary practices from the remote areas and share sit with other communities through published literature. methods: data was gathered from community members through semi-structured interviews and analyzed through informant consensus factor (fic) to evaluate the consent of current ethnoveterinary practices among the local people. results: in total, medicinal plants were recorded under the ethnoveterinary practices. most widely used medicinal plants with maximum use reports (urs) were visnaga daucoides gaertn., foeniculum vulgare mill., solanum virginianum l., withania somnifera (l.) dunal, glycyrrhiza glabra l., and curcuma longa l. new medicinal values were found with confidential level of citations for species including heracleum candicans and glycerhiza glabra. family apiaceae was the utmost family with high number ( species) of medicinal plants. maximum number of medicinal plants ( ) was used for gastric problems. high fic was recorded for dermatological ( . ) followed by reproductive ( . ) and gastrointestinal disorders ( . ). the main route of remedies administration was oral. conclusions: current study revealed that the study area has sufficient knowledge on ethnoveterinary medicinal plants. this knowledge is in the custody of nomadic grazers, herders, and aged community members. plants with new medicinal uses need to be validated phytochemically and pharmacologically for the development of new alternative drugs for veterinary purposes. the historical utilization of plants as health remedies both for human and animal is centuries old. it has been recognized that plants have the capacity to combat several types of diseases ethnoveterinary medicines, a term generally used for folk skills, beliefs, knowledge, practices, methods related to animals' health, and cure of various ailments in the rural areas [ ] . ethnoveterinary practices have achieved immense significance for the last decade owing to the discovery of some effective ethnoveterinary products [ ] . the utilization of traditional remedies poses a cheaper, easier, and sustainable alternative to synthetic drugs and pharmaceuticals [ ] . it has been reported that due to lack of proper animal husbandry practices, about - % of the losses occur in the animals' breeding sectors especially in developing countries [ ] , where the rural people are heavily dependent on livestock farming for their livelihood activities [ ] . the indigenous communities living in rural and mountainous territories of developing world consider livestock a vital source for economy, social security, and food and is thought to be a symbol of prestige for a particular family [ ] . livestock being as a subsector contributes around % of value addition in the agriculture sector and approximately % towards the gross domestic product (gdp). about million people living in the rural areas of the country are involved with the livestock subsector [ ] . hence, livestock raring plays a significant role in poverty reduction strategies. according to the report of economic survey of pakistan [ ] , the national herd of pakistan includes . million goats, . million cattle, . million buffalos, . million sheep, and . million camels. people residing in the remote areas utilize medicinal plants for livestock's health. particularly, the conventional lifestyle of nomadic and pastoralists makes it difficult for them to reach veterinary extension services due to high costs and less availability of allopathic medicines [ ] . in south asia, several ethnoveterinary studies have been conducted [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] including pakistan [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, scarce studies on ethnoveterinary medicines have been reported from the federally administrated tribal areas (fata) of the country. the tribal areas mainly comprised of mountainous territories where people use medicinal plants to treat livestock's diseases. traditional ethnoveterinary knowledge is mainly transmitted orally from one generation to another generation in the form of folk remedies, drawing stories, poems, drawing stories, folk myths, songs, and proverbs. this transmission of indigenous knowledge through oral way faces critical threats of extinction. therefore, it is necessary to record, document, and encourage the ethnoveterinary medication and integrate them into the existing animal health care services [ ] . bajaur agency is among one of the federally administrated tribal areas (fata) of pakistan having diversity of medicinal plants being used for the livestock's healthcare services. due to remote nature and lack of quality education, the area has been little explored for the scientific documentations of traditional knowledge. there is a dire need to explore the folk knowledge about the utilization of herbal remedies for veterinary practices prior to being extinct. hence, the current study was planned to investigate and document the traditional ethnoveterinary knowledge and practices and release it from the custody of knowledge bearers for sharing it with other communities through publish literature. bajaur (khar: headquarter) is the smallest agency of the fata having a total area of km . it shares km border with afghanistan, which is of great importance to pakistan and the region. the study area lays at an altitude of m above the sea level and geographically exists between °- °and °- °latitudes and °- °and °- °longitudes. the agency is surrounded to the west by kunar valley of afghanistan being separated by the rugged hindukush hills and other mountain passes known as nawa pass, ghakhi pass, and letai sar being the notable ones. the agency borders on south with mohmand agency, on east with lower dir district and the panjkora river, and on north with the watershed between bajaur agency and district dir. moreover, the agency is situated at the extreme end of the himalayan range. the areas dominated by agricultural lands are receiving about mm of average rain fall per annum. the two main tribes of bajaur agency known as tarkani and utman khel are mainly populated into seven tehsils including barang, nawagai, khar, mamund, salarzai, utman khel, and chamarkand. by profession, mostly, the people are farmers, teacher, drivers, and doing small scale businesses and jobs inside/outside the country. almost every household has a herd of domestic animals for socioeconomic gains. there are only three degree-level colleges and five higher secondary schools. moreover, there are only two government hospitals in the study area, while most people are deprived of modern health facilities, which justify their reliance on local herbalists (hakims). the study area consists of one veterinary hospital and small dispensaries to treat the domestic cattle. however, the local people still rely on traditional recipes due to larger distances from the aforementioned health centers. the dominant vegetation in the area is comprised of ailanthus altissimo, eucalyptus camaldulensis, ficus carica, melia azedarach, morus indica, morus nigra, olea ferruginea, pinus roxburghii, quercus baloot, and rumex hastatus. in the month of april, respondents were targeted based on their strong reputation in the field of ethnomedicinal knowledge while field survey was conducted from may to august . field visits were carried out prior to medicinal data collection in order to acknowledge the cooperation of the indigenous communities. mr. amir hasan khan, the local occupant of the area, visited different sites with his volunteer team including a taxonomist and a pharmacist. he arranged several meetings with the local representatives known as maliks, to whom objectives of the study were presented. a semistructured questionnaire was developed to gather knowledge on ethnoveterinary plants by following the method adopted by martin [ ] . mostly, the folk knowledge was gathered from nomads, farmers, and aged community members. the interviews were conducted at various places and in the local language called "pashto." each informant was acknowledged by presenting the main theme of the study to them in order to gain their consent and trust, which allowed the informants to talk more freely and openly. the recorded information was once again redisplayed to the informants to avoid errors and falsification. data was collected from different sites known as pashat, tali, inayat kali, ghar shamozai, loe sum, barang, mandal, khar, mamund, and salarzai. accordingly, the sites were categorized into foot hill villages and mountainous villages (fig. ) . a total of key respondents were selected belonging to different age groups, i.e., males and females ( table ). the selection of respondent was based on their high reputation with respect to traditional knowledge on ethnoveterinary plants. continuous relationships were maintained with the indigenous communities throughout the course of survey for the strong validation of traditional knowledge. surveyed ethnoveterinary medicinal plants were collected and identified by taxonomist at the department of botany, shaheed benazir bhuto university sheringal, district dir (upper), khyber pakhtunkhwa, pakistan. species botanical names and their family names were corrected and verified through the website www.kew.org/mpns. after collection, plants were pressed and dried under the shade, were poisoned ( % hgcl solution), and were mounted properly on the herbarium sheets for future reference. each herbarium sheet was labeled with a voucher number and submitted to the aforementioned department [ , ] . for each of the specie, use reports (urs) (citations) were counted. ur may be defined as the utilization of part of a plant species for a particular disease mentioned by an informant. to determine the informant consensus factor (fic), the reported species were arranged in various groups according to the ailment treated [ ] . ten ailment categories were prepared from the data. to calculate the fic, we used the formula, i.e., fic = nur − nt/ nur − . here, nur indicates the number of citations in each use category and nt represents the number of species cited. prospects and challenges to traditional ethnoveterinary knowledge indigenous communities play significant role in reporting traditional uses of medicinal flora. indigenous knowledge can be used as a tool to conserve and maintain the green diversity, and could be further utilized for scientific validation [ ] . during the nd session of united nations educational, scientific and cultural organization (unesco), traditional knowledge on ethnoveterinary medicines was declared an important part of cultural heritage, which is required to be brought under study, sustenance, and protection [ ] . indigenous communities at bajaur agency are dependent on livestock for supporting their livelihood. medicinal plants have a pivot role in the treatment of livestock's ailments in the area. usually, this treatment process depends either on the traditional knowledge being orally transmitted to the current generation of local people from their ancestors or through personal experiences. previous scientific literature has focused on the correlation of traditional medical expertise to ethnobotanical knowledge for the treatment of human ailments [ , ] , although the same plants may be used to treat livestock [ , ] . in our study, we have observed that the herders, farmers, and older community members are more equipped with traditional knowledge and familiar with veterinary medications, diagnosis process, and treatment. indigenous people of the study area are rich in traditional knowledge on veterinary medicines, which may be due to their close observation on domestic animals being considered as an important part of traditional lifestyle. most commonly, the male community member grazes herds of animal, while females take part in households' management. figures and showed some of the images of the grazed domestic animals, which are treated with medicinal plant in the area. other studies have explained this in a different way that men due to close proximity tend to know more about the animal behavior than women [ ] . people of the study area use plants not only for medicinal purposes to treat their domestic animals but also as a fodder. local community also prevents their animals from such nutrition, which is not healthy in certain conditions and seasons. one may consider this prevention to be a part of ethnoveterinary practices. nutrition is playing an important role in ethnoveterinary practices in both prevention and cure of domestic animals [ ] . livestock usually ingests some extra and non-important food substances in the green fodder, which could be termed as food medicines or medicinal food [ ] . studies have highlighted the importance of "food as medicines" in the context of local traditional knowledge; however, possible health advantages of food in ethnoveterinary methods need further attention [ ] . testing the nutritional status of each traditional ethnoveterinary remedy is not necessary; however, it is essential to evaluate the biological efficacy from the phytochemical, pharmacological, toxicological, and clinical perspectives for wider application. a considerable proportion of the documented uses of plant taxa in our study are in accordance with the established pharmacological effects [ ] . the prevailing indigenous ethnoveterinary knowledge in the study area is facing certain constrains leading it towards extinction. as an example, the nature of traditional knowledge is making it more difficult to learn and then transfer it in an accurate way. furthermore, practicing traditional therapies are not being respected by the new generation. other challenges include low literacy rate in the study area, no proper documentation of indigenous knowledge, and introduction of modern allopathic medicines, rapid technological advancement, and environmental degradation. similar kinds of threats have also been reported in other communities across the world [ ] [ ] [ ] . informants with little education were found less familiar to the traditional knowledge while people having no formal education were more responsive in this regard. some studies have found that education can be correlated with expertise either positively [ , ] or negatively [ ] , while others found no relationship [ ] . moreover, it is also ambiguous to determine the effect of "modernity" on the loss of ethnomedicinal knowledge. modernity has an established association with greater medicinal competence in dominica [ ] but appeared unrelated to variation in expertise among tsimane horticulturalists in bolivia [ ] . furthermore, it is also unclear whether correlation of expertise exists between ethnomedicinal knowledge and ethnoveterinary approaches; however, livestock keepers hold extensive knowledge related to disease prevention, diagnosis, and both traditional and novel biomedical treatments [ ] . in summary, despite maintaining knowledge on ethnoveterinary practices by the locals, the tendency to utilize modern pharmaceuticals is increasing day by day. hence, the conservation of ethnomedicinal knowledge by the local communities is extremely important for the livestock's health in the remote areas. the use of plants for medical purpose to treat a wide array of maladies emanates traces since the recorded history and even before. in our study, plant species belonging to families were documented. table presents details on the documented medicinal plants including their botanical names, vernacular names, family names, specimen numbers, parts used, medicinal uses, and use reports. family apiaceae ( species) has the high number of individual species used in ethnoveterinary practices followed by fabaceae ( species). other studies have also reported apiaceae as the dominant plant family being used in traditional medications [ , ] . the rationale of high use of apiaceae species in the current study, though based on traditional evidence, may be referred to their chemical constituents such as phenolics, poly phenolics, lectins, alkaloids, terpenoids, and essential oils, which carry antimicrobial potential [ ] . due to the predominance of sheep, goats, cows, and donkeys in the study area, we have specifically recorded the ethnoveterinary practices used for the treatment of these four types of domestic animals. key informants declared extensive uses of visnaga daucoides gaertn. table ) . medicinal plants with high urs strengthen the concept that such species are more significant to the local population and useful in sharing the traditional knowledge with one another in the area. in our study, v. daucoides is used to treat diarrhea, abdominal pain, and retained placenta in domestic animals. a whole plant is subjected to powder and is combined with flour and black tea to treat digestive problems especially in cow and buffalo. amaryllidaceae bulb of the herb is crushed and added milk for orally given to animals ( - days) for curing digestive complaints. bulb is crushed and mixed with way to administered orally for several days in order to rate of fertility in domestic animals. narcissus tazetta l. "sbbu- " gul-e-nargas leaves along with gurr and flour, fresh leaves (¼ kg) are boiled and orally given to livestock for the retained placental removal. apiaceae tea is prepared from its fruit (¼ kg), and then, it is combined with flour and given to cattle for days in order to treat gastric problems. cuminum cyminum l. "sbbu- " half kilogram of fruit is boiled in black tea and orally given for - days on daily basis for the expulsion of intestinal worms and treated gastric problems. eryngium biehersteinianum (m. bieb.) nevski "sbbu- " stem and leaves and stem powder of its stem and leaves is orally given for the treatment of liver problems up to the duration of - days. foeniculum vulgare mill. "sbbu- " fruit, leaves decoction is made from it fresh leaves and fruit ( - g), and t hen, it is combined with gurr, given orally to livestock for appetite and as sedative for the duration of to days. wall. ex dc. "sbbu- " fresh root of the plant ( g) is combined with wheat flour and made to paste which is orally given to goat, cow, and sheep as sexual tonic and to enhance the rate of fertility up to days. trachyspermum ammi (l.) sprague "sbbu- " seeds (¼ kg) of the plant, allium cepa, wheat flour, and foeniculum vulgare are thoroughly mixed. the resultant blend is then orally given ( days) and is considered as good appetizer. tea is made from its fruit and given orally to sheep, goat, cow, and buffalo while treating diarrhea, abdominal pain, and retained placenta. the remedy is constantly utilized for the duration of days. calotropis procera (aiton) dryand. "sbbu- " plants' fresh leaves are taken and decoction is made, and after that, the decoction is combined with "ajuga integrifolia" and is used for dermal parasites for to days. nerium oleander l. "sbbu- " to relieve the external parasite, the decoction of its leaves is used for animal bathing especially goat and cow. the decoction obtained from its bark and is combined with butter which is administered orally to all type of domestic animals to treat skin problems and as blood purifier. cassia fistula l. "sbbu- " amaltas fruit fruit of the plant is subjected to boiling along with milk and administered orally up to days to all sort of domestic cattle to relieve fever and gastric complexities. glycyrrhiza glabra l. "sbbu- " khwaga waly roots root (¼ kg) is subjected to paste which is mixed with flour and oil and then is given to goat, sheep, cow, and buffalo to increase milk production and enhance the rate of fertility. the remedy is used for the duration of to days. lotus corniculatus l. "sbbu- " stem and leaves, stem and leaves are crushed in weight of ¼ kg and orally given to cattle along with bread or dough for to days as sexual tonic and for urinary tract infections (uti). trigonella foenum-graecum l. "sbbu- " malkhoozi seeds seeds ( g) are crushed and given in dough to animals ( - days) against gastric disorders. decoction is made from the leaves and then gurr is added. this remedy is given orally to cattle for blood purification and as vormifuge. the water is applied topically to treat skin ailments. mentha spicata l "sbbu- " powder is made and decoction is made and then mixed with gurr and taken by animals to cure digestive problems. ocimum basilicum l. "sbbu- " kashmaly leaves seed plant leaves and seeds are subjected to decoction and used topically for skin problems. salvia moorcroftiana wall. ex benth. "sbbu- " kharghwag leaves decoction of its leaves is given orally daily for the treatment of digestive problems. gossypium arboreum l. "sbbu- " pomba kal about ¼ kg of its powder is mixed with gurr and used for to days. this remedy is administered orally on daily basis as galactagogue. grewia optiva j.r.drumm. ex burret "sbbu- " whole plant dried plant powder is subjected to oil ( ml), administered orally and topically twice a day for to days for wound healing process. the usage mode of ethnoveterinary plant species by one ethnic community is different from other communities due to difference in traditional knowledge [ , ] . previous literature has shown that decoction of the fruit of v. daucoides is used during abdominal pain, which is used to enhance body temperature in the study area [ , ] . in the same way, the f. vulgare is considered as a strong appetite and sedative. in other cultures across the globe, f. vulgare is used for various livestock problems. for instance, this plant is effective in digestion and diarrhea, when mixed with camellia sinensis, trachyspermum ammi, ghee, and sugar [ , ] . pneumonia is also being treated by giving its seeds to the animals [ ] , while other uses include galactagogue and ruminative [ ] . various parts of s. virginianum are taken for the treatment of cough, fever, milk production, and pain. there is scarce literature on the use of s. virginianum as galactagogue, which shows the unique use of this plant species in the study area and familiarity of local population through longtime experiences. published literature has indicated that the plant is also used for wound healing process [ ] fever, cough, and intestinal infections [ ] . roots and leaves of w. somnifera are given to sheep, cow, and buffalo for milk production and used as antipyretic and sexual tonic. indigenous populations comprising of various cultures residing in lesser himalayas (pakistan) use w. somnifera for bovine mastitis [ ] , while in ethiopia, this plant is being used to protect animals from bad evils [ ] . the plant has carminative effects and is used to remove the flatulence [ ] . additionally, this plant is used as refrigerant and for abdominal pain, digestion, jaundice, skeletonmuscular ailments, and wound healing against sunstroke [ ] ; for treating diarrhea [ ] ; for trypanosomiasis [ ] ; and for anorexia [ ] . informant reported g. glabra as galactagogue and enhances the rate of fertility. mussarat et al. [ ] reported that this plant is culturally used for the treatment of cough by the indigenous communities residing near the indus river, pakistan. however, from the literature, no conclusive evidence was found on the reported uses of g. glabra in our study. such evidence-based observations could justify the idea of cultural diversity across the regional level in plant remedies. previous studies related to the human's uses of g. glabra have demonstrates its effectiveness in the treatment of sex hormone imbalances and menopausal symptoms in women [ ] . in the current investigation, rhizome of c. longa is used as antiparasitic and treating genital infection and problems. in other cultures, across the country, the dried rhizome of c. longa is mixed with eggs and given for mastitis [ ] , jaundice, and skeleton muscular ailments [ ] . decoction of its leaves is mixed sugar, which is used as wound healing agent [ , ] . a root of c. longa is used for hoof problems and sore joints [ ] . in our study, the mustard oil is mixed with whey and is taken orally to relieve abdominal pain. the cultural ethnoveterinary uses from the lesser himalayas (pakistan) include that the oil extracted from b. rapa seeds is utilized for stomach disorders, eye infection, and skin diseases [ ] . furthermore, brassica rapa l. seeds are used for the retention of fetal membrane, while its oil is effective in treating genital prolepses and sores [ ] . this plant is also used in placental retention and mastitis and as antiparasitic [ ] ; myiasis, mange, and helminthiasis [ ] ; and flatulence [ ] . all these researchbased findings showed that the same medicinal plants are being used in different parts of the country; however, their uses differ from area to area and from culture to culture [ ] . the ethnoveterinary plants use by one ethnic community is almost different from other communities due to several reasons. to make a comprehensive comparative cultural diversity analysis of plant utilization in ethnoveterinary practices, we have selected a study conducted by aziz et al. [ ] in the fata region of pakistan. in comparison, we have found that most widely used medicinal plant species in our study are v. daucoides, f. vulgare, s. virginianum, w. somnifera, g. glabra, and c. longa. while according to aziz et al. [ ] , the ethnic communities in south waziristan agency are widely utilizing plant species such as b. rapa, punica granatum, capparis decidua, mentha longifolia, withania coagulans, and c. longa, during comparative analysis, it was found that only medicinal plants were commonly used in both regions for ethnoveterinary practices, which include acacia modesta wall, allium cepa l., allium sativum l., b. rapa, calotropis procera (aiton) dryand., cannabis sativa l., chenopodium album l. c. longa, f. vulgare, juglans regia l., nicotiana tabacum l., peganum harmala l., quercus oblongata d. don, trachyspermum ammi (l.) sprague, and v. daucoides. certain variations in the utilization of these plants and their parts were observed in both areas. for instance, the bulb of a. cepa is used as galactagogue by waziristanian communities while in bajaur, it is used to treat digestive problems. a. sativum is utilized for genital prolapsed while the same plant is used as sexual tonic for animals in bajaur agency. the seeds of b. rapa are widely used as appetizer and tonic and for cough, seasonal allergies, stomach disorders, and skin infections in south waziristan agency, while in the other region, it is used only against gastro-intestinal disorders. the indigenous communities at south waziristan agency consider the leaves of c. procera useful in joint pain while on the other side, the residents of bajaur agency used the latex against skin problems. c. album is used for wound healing and flatulence at waziristan while as stomachic at bajaur agency. j. regia is given for the retention of placenta at waziristan while gastric problems in bajaur. p. harmala is extensively used for gastrointestinal problems, as antiparasitic, and for skin diseases by waziristanian communities, while it is used only for the riddance of external parasites in bajaur. the possible reason for low consensus of the two regions in ethnoveterinary medicinal plants may be due to unique vegetation and distinct socio-cultural values. according to a survey, out of plant-derived pure compounds, % ( plant species) were having the same potential as indicated in traditional medications [ ] . as an example, galegine is obtained from galega officinalis l. and is used in the production of metformin and other bisguanidine-type anti-diabetic drugs [ ] ; khellin, extracted from v. daucoides., led to the development of cromolyn in the form of sodium cromoglycate, which is used as a bronchodilator; and papaverine isolated from papaver somniferum forms the baseline for verapamil, which is generally utilized for hypertension [ ] . survey participants did not describe the standardized dosage and recovery time like other previous ethnoveterinary documentations. the main problem highlighted in other studies is the lack of accuracy in such ethnoveterinary practices, which also push the locals towards modern allopathic drugs for livestock health maintenance [ , ] . the main reason that veterinarian has always complained is the non-standardized dosage in traditional medicines. though this is an accusation, one ethnomedicine does not mean that they lack efficacy but require standardization, which could benefit the traditional system by minimizing risks and toxicities. according to kearns [ ] , ethnoveterinary medicines are facing a great intellectual challenge from social theory and postmodernism, and this challenge was focused while detecting variations in animal health practices, beliefs, and experiences of various social groups. generally, it is not possible for all ethnoveterinary practices to be effective and, at the same time, they have certain weakness in terms of their efficacy as compared to modern medications [ ] . though it is convincing that most of the traditional veterinary medications have clear and sound health effects, many modern allopathic drugs are based on these medicines [ ] . certain plants in our study were used in single form for more than one disease. for example, cedrus deodara (roxb. ex d. don) g. don is used in a condition, in which milk obtained from the cattle gives bad smell, then the oil is given orally to the cattle. it is also used as a cooling agent and in treating digestive problems. in large quantity, the oil have the potential to depress the sexual power of male animals [ ] . monteiro et al. [ ] also reported similar findings from pakistan and brazil, respectively, where they described multiple uses of a single medicinal plant. utilization of certain plant species for multiple diseases is a widespread practice in ethnoveterinary medications. in contrast, some ethnoveterinary remedies (polyherbal formulations) are being made by combing two or more plants and additives such as whey, ghee, and sugar. this addition is generally followed in remedies to counteract the astringent taste, dilute, and reduce the relative potency of the remedy [ ] . in the study area, a total of plants were reported for gastrointestinal problems with maximum use reports of (table ) , which is regarded as the most common disease category in domestic animals being represented by abdominal pain, diarrhea, and digestive problems. these health issues can be easily detected by the respondents and may explain the fact that why the gastric problem category is high in ours as well as in others studies. different ailments were categorized into groups such as dermatological, gastrointestinal, galactagogue, reproductive, respiratory disorders, tonic, wound healing fever, and miscellaneous. those medical conditions, which were not fully described by the interviewees, were placed into the miscellaneous category. these include eye problems, weakness, and abnormal conditions related to various organ systems of animal bodies. highest fic values were recorded for dermatological problems ( . ) followed by reproductive ailments ( . ) and gastric disorders ( . ) ( table ) . fic value is an indicator of showing the consent of the local people on a specific plant species and efficacy of a certain taxa [ ] . sharma et al. [ ] declared that when fic becomes , it means that the local population is exchanging their view, ideas, and information about traditional medications, while on the other side, if the fic value is , then it is vice versa. fic value in the current study was recorded in between . and . for various livestock ailments (table ). these findings indicate the highest consent among the local people on traditional herbal therapies. previous research studies conducted in other areas also agreed to high consent of local people on traditional animal therapies. for instance, the reported fic values for dermatological problems were . , . , and . [ , , ] ; for reproductive disorders, . and . [ , ] ; for gastric problems, . , . , . , . , and . [ , [ ] [ ] [ ] ; for galactagogue, . and . [ , ] ; and for wound healing, . and . [ , ] . heinrich et al. [ ] has submitted the idea that high fic values can be used as a tool to target the plants for the isolation of biologically active components. in our study, most livestock's ailments were mentioned to be seasonal and epidemic due to change in fodder. furthermore, the concept of hot and cold food is also famous in order to prevent animals from diseases. the local residents change the relative fodder in different seasons in order to minimize the chances of various health problems in cattle. as an example, the seeds of the nigella sativa l. and kernels of q. oblongata are given to the cattle to energize them during the cold season. similarly, the fruits of the streblus asper lour. produce cooling effects and considered to be a better remedy during hot summer season. in the same manner, local communities tend to give the infusion of cannabis sativa l to their livestock in the summer season. quinlan [ ] and raziq et al. [ ] has also mentioned the concept of hot and cold food in traditional veterinary medications. drugs derived from plants or their extracts have certain therapeutic properties. to replace antibiotics by suitable therapeutic agents, plants can play an important role in combating with bacterial pathogens. there are several essential oils, which can be used as alternate of antibiotics. these oils can be easily isolated, having low toxicity on mammalian cells, and can be easily degraded in soil and water [ ] . in this section, we will analyze the pharmacological evidences of the most utilized studied medicinal plant species in order to check their therapeutic efficacy. in f. vulgare, phenols, phenolic glycosides, and volatile aroma compounds such as transanethole, estragole, and fenchone are reportedly the key phytoconstituents and responsible for its antioxidant activity. f. vulgare is pharmacologically validated (in vitro and in vivo) in demonstrating activities such as antibacterial, antifungal, antioxidant, antithrombotic, and hepatoprotective [ ] . by investigation, it was found that the leaf extracts of s. virginianum is more active against candida albicans, salmonella typhi, staphylococcus aureus, and nematodes [ , ] . for various extracts obtained in alcohol and water, it was found that w. somnifera has antibacterial potential, antihypercholesterolemic activities as well as diuretic potential [ , ] . it has been reported that alcoholic and aqueous extracts of c. longa have shown antibacterial activity [ ] while its ethanol, petroleum, water, and chloroform extracts are effective against certain strains of viruses, bacteria, and fungi and also have shown anti-inflammatory effects [ ] . researchers have claimed that plant-derived medicines used in traditional systems across the globe can be used as an indicator to consider them more effective than modern drugs [ ] . livestock keepers are using several plant-derived remedies for various acute as well as chronic disorders of cattle. plant-derived medicines have been used by physicians for hundreds of years in traditional systems, and most of the world population rely on these products for health care systems [ ] . there are several thousand plants across the globe being utilized for various therapeutic purposes both animals and humans [ ] . out of these medicinal plants, very low proportion has been investigated and proved scientifically for their indigenous uses [ ] . the essential oils in medicinal plants are having strong antimicrobial potential. as an example, essential oils of cinnamon, thyme, and oregano are therapeutically effective [ ] . antibiotic resistance is an emerging global concern related to veterinary and human medications [ ] . hence, it is necessary to search for new compounds to combat antibiotic resistant bacteria. improper therapeutic utilization of antimicrobial medicines in fishery, poultry, agriculture, and animal farming facilitates the emergence and production of drug resistant strains. additionally, poor prevention and control of unhygienic practices contribute in resistance emergence. the world health organization, food and agriculture organization, and world organization for animal health are stressing to promote best practices to avoid the emergence and spread of antibacterial resistance. continuous attempts are in progress to promote the moderate use of antibiotics in human as well as in animals to tackle the problem of antimicrobial drug resistance [ ] . in general, plants should be used as an alternative to synthetic drugs and investigated for their therapeutic efficacy. certain plants in our study including boerhavia erecta l., celtis australis l., chamaecyparis obtusa (siebold & zucc.) endl., eryngium biehersteinianum (m. bieb.), gossypium arboreum l., h. candicans wall. ex dc., narcissus tazetta l., opuntia littoralis (engelm.) cockerell, and s. asper need comprehensive phytochemical, pharmacological, and toxicological investigations. current study, one health concept, and changing environment current study reports that there are several ailments being treated with medicinal plants by the indigenous populations. most prevalent disease categories were dermatological, reproductive, and gastric problems. the dominance of these diseases not only poses threats to the domestic animals but also increases the chances of zoonoses. local population uses various animal products; hence, there are maximum chances of the migration of infectious diseases from these animals to humans. linkage of the ethnoveterinary studies with the researches of other disciplines may form an interdisciplinary approach to combat several types of health issues in both animals and plants. this approach mainly led to the concept of one health, which contributes towards understanding the complexities in health problems of living beings [ ] . a recent surge in emerging infectious diseases and their putative associated costs to society have reignited interest in the drive of disease emergence. a number of pathogens have emerged in the last years, including the severe acute respiratory syndrome virus, hendra virus, and nipah virus. however, there is a growing concern about the h n influenza virus, which fuelled much of the recent debate around emerging infectious diseases (eids) [ ] . one of the benefits that accrued from the attention on eids has been an increased recognition across a range of disciplines that the health of animals (including humans) and the health of the broader ecosystem are inextricably linked, which certainly given momentum to one health movement. one health is not all about eids, but it also covers important issues of food security and food safety [ ] . there is a strong consensus that the climate is changing now and that human activities are the primary cause [ ] . however, it is clear that climate change will alter the distribution and incidence of a wide range of diseases either directly or indirectly (e.g., diseases with a development stage outside the host) [ , ] . the pathways by which climate change can affect host pathogen vector interactions have recently been well described by gallana et al et al. [ ] . one health initiative task force (ohitf) [ ] defines one health as "the promotion, improvement, and defense for the health and well being of all species by enhancing cooperation and collaboration between physicians, veterinarians, and other scientific health professionals and by promoting strengths in leadership and management to achieve these goals". the one health approach plays a significant role in the prevention and control of zoonoses. approximately % of new emerging human infectious diseases are defined as zoonotic [ , ] . of the infectious diseases, approximately % are caused by multi-host pathogens, characterized by their movement across various species [ ] . this gives significant credence to the importance of examining health effects across species, in order to fully understand the public health and economic impact of such diseases and to help implement treatment and preventive programs. the application of one health approach has been recognized as a critical need by international organizations as well as the preferred approach to address global health issues. it is also noted that knowledge in veterinary medicine and animal nutrition and husbandry could provide insights into human nutrition and growth. it is a widespread phenomenon that natural resources including plants are always prone to threats in their natural habitat due to rapid human intervention and destructions of natural resources. the collection process of medicinal plants for ethnic practices and other anthropogenic practices is not only destructing the indigenous flora but also posing a threat to the traditional knowledge. unesco has emphasized on the documentation and preservation of traditional knowledge in south asia generally and pakistan and india particularly. however, efforts are going on but they are not sufficient for the conservation of traditional knowledge persistent since several centuries, which can lead to valuable discoveries in modern healthcare system. the local perception of indigenous communities regarding the threats being faced to the ecological resources especially the medicinal plants was examined in the current study. the lack of awareness has been observed as a major threat to the conservation of plant resources. it was also observed that different factors including time of collection, processing, storage, and herbal preparations are important and necessary steps to be considered for both economic returns and conservation. mainly, the local healers are involved in the collection of medicinal plants. a study in the swat region of pakistan has shown that higher economic outcomes can be obtained from proper harvesting of wild medicinal plants as compared to the standard cash crop [ ] . other studies are supporting our results by showing an enormous potential in improving the harvesting, storage, use, preparation, and marketing of the herbal product as a source of income [ ] . in the remote areas of the study region, local inhabitants obtained significant economic advantages from forest products. similar advantages have been reported for other mountainous communities in the northern parts of pakistan [ ] . there are certain other threats to the medicinal plant resources of the study area, which include deforestation, heavy grazing pressure, uncontrolled collection of fodder, and other non-timber forest products by the local people and traders. several studies have reported a decrease in the number of medicinal plants due to over exploitation and environmental degradation [ , ] . it is therefore a dire need to manage and design the overall grazing system to encourage the sustainable regeneration and protection of medicinal plants. keeping the observation and findings of the current investigation, proper management steps should be taken with the active participation from the indigenous communities to conserve this precious flora. it is also important to aware the local people about the market value and sustainable harvesting of medicinal plants. rapid modernization and urbanization is not only a threat for plant species' degradation but also a threat for the associated folk knowledge. that is why that the disappearance of folk knowledge has been declared more in danger than the natural resources themselves [ ] . therefore, we present a strong recommendation that ethnobotany as a subject should be included into the curriculum to help students in recognizing the endangered and medicinally important species of their respective regions. in addition, incentives may be given to farmers for the cultivation of medicinal plants on marginal lands and home gardens. indigenous communities at bajaur agency are dependent on medicinal plants for ethnoveterinary practices. knowledge about the traditional medicinal system is restricted to the herders, farmers, and elder community members. the younger generation is unaware of this traditional treasure and takes no interest due to modernization. hence, this study is an attempt towards the preservation of traditional ethnoveterinary knowledge from being extinct. there are several medicinal plants, which are being used in traditional herbal system of veterinary disorders. some of the important are v. daucoides, f. vulgare, s. virginianum, w. somnifera, g. glabra, and c. longa. new ethnoveterinary uses used at the study area were found for h. candicans and g. glabra. apiaceae is utmost plant family being in use for various livestock ailments. thorough phytochemical and pharmacological investigations are required by isolating the active compounds and testing the in vitro or in vivo efficacy of the abovementioned plants against the targeted veterinary diseases. furthermore, critical toxicological investigations are also required to ensure the safe and secure use of documented ethnomedicines. in order to share and further maintain this knowledge, it is direly needed to aware the rural population about the significance of traditional ethnoveterinary knowledge and to motivate them on the conservation of natural flora. intersectoral health care delivery ethnoveterinary medicines used for ruminants in british columbia documentation of ethnoveterinary practices for mastitis in dairy animals in pakistan meas. contribution livest. househ. livelihoods: a livest. module multi-topic househ. surv botanical ethnoveterinary therapies in three districts of the lesser himalayas of pakistan dairy industry in pakistan: a scenario economic survey of pakistan. government of pakistan (gop), finance division, economic advisor wing ethnoveterinary treatments by dromedary camel herders in the suleiman mountainous region in pakistan. an observation and questionnaire study ethnoveterinary medicinal practices of the villagers of usilampatti taluk of madurai district, india ethnoveterinary medicines used by tribals of tadgarh-raoli wildlife sanctuary cattle wounds and ethnoveterinary medicine: a study in polasara block ethnoveterinary practices of aborigine tribes in odisha ethnoveterinary medicinal practices in tribal regions of andhra pradesh, india plants used as ethnoveterinary medicines in sikkim himalayas ethnoveterinary uses of medicinal plants among traditional herbal healers in alaknanda catchment of uttarakhand, india ethno-veterinary practices for ephemeral fever of yak. a participatory assessment by the monpa tribe of arunachal pradesh plants used in ethnoveterinary medicine by malayalitribals in salem district of tamil nadu ethnoveterinary study of medicinal plants in a tribal society of sulaiman range ethno veterinary practices for the treatment of parasitic diseases in livestock in cholistan desert pakistan ethnoveterinary study of medicinal plants in malakand valley ethnopharmacological assessment of medicinal plants used against livestock infections by the people living around indus river ethnoveterinary medicinal uses of plants of poonch valley azad kashmir documentation of ethnoveterinary practices used for treatment of different ailments in a selected hilly area of pakistan ethno veterinary uses of medicinal plants of district karak, pakistan ethnoveterinary medicinal plant knowledge and practice among the tribal communities of thakht-e-sulaiman hills, west pakistan a methods manual. london: chapman and hall flora of pakistan. nos. - flora of pakistan. nos. - ethnoveterinary health management practices using medicinal plants in south asia-a review do pharmaceuticals displace local knowledge and use of medicinal plants? estimates from a cross-sectional study in a rural indigenous community ethnomedicine of menstruation in rural dominica, west indies understanding interrelationships among predictors (age, gender, and origin) of local ecological knowledge a survey of plants and plant products traditionally used in livestock health management in buuri district xuan dung nn. food, feed or medicine: the multiple functions of edible wild plants in vietnam ethnoveterinary herbal remedies used by farmers in four north-eastern swiss cantons circum-mediterranean cultural heritage and medicinal plant uses in traditional animal healthcare: a field survey in eight selected areas within the rubia project medicinal plants used by tibetans in shangri-la discussion on the institutionalization of participatory livestock technological development comparison of health conditions treated with traditional and biomedical health care in a quechua community in rural bolivia market economy and the loss of folk knowledge of plant uses: estimates from the tsimane' of the bolivian amazon school and local environmental knowledge, what are the links? a case study among indigenous adolescents in oaxaca schooling and local environmental knowledge: do they complement or substitute each other secular changes of indigenous knowledge of useful plants: separating age and cohort effects modernization and medicinal plant knowledge in a caribbean horticultural village pharmaceutical ethnobotany in the montseny biosphere reserve (catalonia, iberian peninsula). general results and new or rarely reported medicinal plants curing animals with plants, traditional usage in tuscany cross-cultural analysis of medicinal plants commonly used in ethnoveterinary practices at south waziristan agency and bajaur agency, federally administrated tribal areas (fata) plant products as antimicrobial agents tobacco smoke is a source of toxic reaction glycation products preliminary evaluation of antinephritis and radical scavenging activities of glabridin from glycyrrhiza glabra ethnoveterinary medicines used for horses in trinidad and in british columbia an inventory of the ethno veterinary practices for reproductive disorders in cattle and buffaloes, sargodha district of pakistan medicinal plants in therapy the value of plants used in traditional medicine for drug discovery ethnoveterinary knowledge and practices at colares island, pará state, eastern amazon, brazil medical geography: making space for difference ethnoveterinary medicine an annotated bibliography iowa state university research foundation ethno veterinary medicine: harnessing its potential ethnoveterinary knowledge of the inhabitants of marajo' island, eastern amazonia an inventory of the ethnobotanicals used as anthelmintics in the southern punjab (pakistan) ethnobotanical study of medicinal plants used by people in zegie peninsula. northwestern ethiopia ethnoveterinary remedies of diseases among milk yielding animals in kathua ethnoveterinary plants for the treatment of camels in shiwalik regions of kathua district of jammu & kashmir, india traditional arabic palestinian ethnoveterinary practices in animal health care: a field survey in the west bank (palestine) quantitative traditional knowledge of medicinal plants used to treat livestock diseases from kudavasal taluk of thiruvarur district ethnoveterinary plants of ankober district medicinal plants in mexico, healer's consensus and cultural importance ethnomedicine and ethnobotany of fright, a caribbean culture bound psychiatric syndrome selected antimicrobial essential oils eradicate pseudomonas spp. and staphylococcus aureus biofilms foeniculum vulgare: a comprehensive review of its traditional use, phytochemistry, pharmacology and safety antimicrobial activity of certain indian medicinal plants used in folklore medicine antihelmintic properties of some indigenous plants screening of some indian medicinal plants for their antimicrobial properties new delihi: campus books international herbal medicine are medicinal plants a potential alternative for conventional antibiotics in animal husbandry antimicrobial activity spectra of pelargonium graveolens l. and cymbopogon winterianus jowitt oil constituents and acyl derivatives global action plan on antimicrobial resistance antimicrobial drug resistance in bacteria isolated from sick animals and their environment in year - at central disease diagnostic laboratory, indian veterinary research institute, izatnagar. noto-are med ( ) a review of the metrics for one health benefits infectious disease emergence and global change: thinking systemically in a shrinking world ecosystem sustainability and health: a practical approach the critical decade : climate change science, risks and response. common wealth of australia department of industry, innovation, climate change, science climate change effects on animal disease systems and implications for surveillance and control. in climate change: impact on the epidemiology and control of animal diseases climate change and infectious diseases of wildlife: altered interactions between pathogens, vectors and hosts a new professional imperative-one health initiative task force. american veterinary association the animal-human interface and infectious disease in industrial food animal production: rethinking biosecurity and biocontainment institute of medicine microbial threats to health, emergence detection, and response medicinal and aromatic plant cultivation in the swat valley, north-western pakistan, for economic development and biodiversity conservation ethnomedicinal survey of important plants practiced by indigenous community at ladha subdivision, south waziristan agency ethnobotanical profile of plants of shawar valley, district swat, pakistan an ethnobotanical study of medicinal plants in high mountainous region of chail valley (district swat-pakistan) conserving indigenous knowledge as the key to the current and future use of traditional vegetables the authors extend their high appreciation and acknowledgment towards the local communities by providing moral support to the authors. this research study did not receive any grant from any organization. data gathered during the course of the study has been included in the article.authors' contributions ahk and hu conducted the field work. maa wrote the draft manuscript. ahk and hu helped in the compilation of data. ma gave technical comments on the draft and indicated the language and grammatical mistakes. maa and ma supervised all the stages. all the authors read and approved the manuscript.ethics approval and consent to participate not applicable the authors declare that they have no competing interests.• we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- -gu mt zp authors: shanmugaraj, balamurugan; malla, ashwini; phoolcharoen, waranyoo title: emergence of novel coronavirus -ncov: need for rapid vaccine and biologics development date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: gu mt zp novel coronavirus ( -ncov) is an emerging pathogen that was first identified in wuhan, china in late december . this virus is responsible for the ongoing outbreak that causes severe respiratory illness and pneumonia-like infection in humans. due to the increasing number of cases in china and outside china, the who declared coronavirus as a global health emergency. nearly , cases were reported and at least other countries or territories have reported coronavirus cases as early on as february. inter-human transmission was reported in a few countries, including the united states. neither an effective anti-viral nor a vaccine is currently available to treat this infection. as the virus is a newly emerging pathogen, many questions remain unanswered regarding the virus’s reservoirs, pathogenesis, transmissibility, and much more is unknown. the collaborative efforts of researchers are needed to fill the knowledge gaps about this new virus, to develop the proper diagnostic tools, and effective treatment to combat this infection. recent advancements in plant biotechnology proved that plants have the ability to produce vaccines or biopharmaceuticals rapidly in a short time. in this review, the outbreak of -ncov in china, the need for rapid vaccine development, and the potential of a plant system for biopharmaceutical development are discussed. the global health system consists of a network of organizations, including many private and public health sectors operating at different regional or global levels that have developed a stringent system that can provide effective protection to humans against emerging and re-emerging diseases. though mortality associated with various infectious diseases have reduced in recent years and global life expectancy has increased in many parts of the world, infectious disease threats still remain one of the major global challenges and concerns even now [ ] . the global health system is often confronted by emerging pathogens responsible for expanding an array of infectious diseases such as zika, chikungunya, ebola, nipah, severe acute respiratory syndrome (sars), middle east respiratory syndrome (mers), and influenza. the emergence of the novel coronavirus ( -ncov) has recently added to the list of problematic emerging pathogens in the st century, which was suspected to originate from the persons exposed to a seafood or wet market in wuhan, hubei province, china, suggesting animal-to-human transmission [ , ] . this virus strain is previously unknown and was reported to infect humans for the first time. the virus continues to expand rapidly throughout the world. many confirmed and susceptible cases have been identified in wuhan, china, and exported cases have also been reported in neighboring countries including thailand, japan, korea, taiwan, and other countries including the united states, canada, and european countries, which proves that the virus has the potential for quick dissemination across borders. in response to the rapid spread of the virus, many countries have tightened their border security, investigating people showing symptoms, and have taken necessary emergency steps to control its spread. due to the increasing number of cases in china and other countries, the who has declared the -ncov outbreak a global health emergency of international concern on january [ ] . coronaviruses (covs) belongs to the family coronaviridae, subfamily coronavirinae, and the order nidovirales. they are classified into four genera such as alphacoronavirus and betacoronavirus, both of which infect mammals, whereas gammacoronavirus infect avian species, and deltacoronavirus infect both mammalian and avian species. it is a large enveloped virus with a positive sense, single-stranded rna genome of about to kb that is distributed broadly among birds, humans, and other mammals such as camels, bats, mice, dogs, and cats [ ] . the genome is surrounded by a helical capsid and an envelope; the spike protein forms large protrusions in the envelope in the shape of a crown, which gives the virus a coronal appearance. the word 'corona' in latin means crown [ , ] . human coronaviruses (hcovs) are a major group of coronaviruses that are known respiratory pathogens associated with respiratory and intestinal infections of varying severity, including the common cold, pneumonia, and bronchiolitis. human coronaviruses such as hcov- e, oc , nl , and hku , usually cause mild infection in humans. however, the onset of betacoronaviruses, severe acute respiratory syndrome coronavirus (sars-cov) outbreak in guangdong province, china in , and the middle east respiratory syndrome coronavirus (mers-cov) disease outbreak in in the middle east resulted in high pathogenicity in humans, which demonstrated the lethality of virus when they cross the species barrier from animals to humans. both viruses are believed to be originated from bats and subsequently transmitted to humans [ ] . hcovs has evolved rapidly in recent years due to mutation, high nucleotide substitution rates, its ability to establish infection in a new host, and cross-species transmission [ ] . in december , china detected many cases of viral pneumonia-like disease similar to sars that were confirmed to be caused by novel betacoronavirus, provisionally called novel coronavirus ( -ncov). since then, the novel coronavirus outbreak has raised attention throughout the world. although the potential cause of the disease is still unknown, initial reports predicted that the virus is possibly of zoonotic origin. -ncov is the causative agent for severe respiratory infection in humans termed as novel coronavirus-infected pneumonia (ncip) [ ] . ncov is the third known coronavirus that causes fatal respiratory diseases in humans after highly pathogenic viruses sars-cov and mers-cov. chinese researchers isolated the novel coronavirus from the infected patient in early . as the virus is closely related to other bat coronaviruses, it is suspected that the bats are the primary reservoir for the virus. however, it is still unclear that, if the virus transmitted to humans directly from the bats or whether through an intermediate host. detailed understanding of the enzootic patterns of the virus, its evolution, and surveillance are essential to control the disease and possibly to prevent the future epidemics of similar viruses. the explosive outbreak of -ncov in china has been spread rapidly in many countries, probably by human movement and travel. the geographical distribution of the virus has been increasing since the outbreak. in early february , thousands of cases were confirmed in china and more than cases were reported outside of china, and numbers of cases are escalating daily in all the provinces in china and other countries. the virus has transmitted rapidly and many cases were reported globally. as of february , , nearly , affected cases and fatalities were reported in china. most of the confirmed cases were from the wuhan city in hubei province. outside china, other cases were confirmed globally, and one death was reported in philippines. in singapore, cases were confirmed, in thailand, in japan, in korea, each in australia and malaysia, in united states, and some cases were reported in germany, france, vietnam, uae, canada, india, philippines, italy, uk, russia, nepal, sri lanka, cambodia, belgium, finland, sweden, and spain as on early february [ ] . chinese health authorities, who, and most of the countries have responded fast, and immediate actions were taken to contain the virus. all the countries have implemented stringent border control, screening travelers for possible infection, and travel restrictions in order to prevent its spread. the rapid spread of this infection is frightening and also it causes both mortality and financial loss, which present the global concern of this emerging disease. the transmission of -ncov is often spread from person to person through the respiratory droplets generated during coughs or sneezes from an infected person. human-to-human transmission is reported in countries such as germany, japan, vietnam, and the united states [ ] . the confirmed cases through inter-human transmission have increased the fear and panic accompanying the -ncov outbreak. it is still unknown whether the virus spreads only through human contact or if there is possible transmission through oral-fecal contact as well. the incubation time varies from - days after infection. the clinical presentation of this infection resembles sars-cov characterized with fever, dry cough, and shortness of breath in most of the cases, whereas non-respiratory symptoms such as headache, muscle ache, dyspnoea, rhinorrhoea, sneezing, sore throat, diarrhea, nausea, and vomiting are also reported in few patients. the affected persons also develop acute respiratory distress syndrome. cases with critical illness showed respiratory failure, septic shock, and organs failure, which require intensive care support [ , , ] . at this time, the knowledge about this virus is limited. new cases and mortalities are increasing daily. as a newly emerging viral infection, there is no vaccine or anti-viral therapeutics to treat human coronavirus infection till now. as of now, preventing infection is the current priority for disease control. the current protocol for infected patients is to quarantine and provide supportive management and palliative care. the best way to avoid the virus infection is to keep oneself away from infected people and the utmost personal hygienic care is essential. quarantine measures shall be taken to separate, restrict the movement of infected people, and also the normal population from the regions where there is an epidemic outbreak. the who recommended precautionary measures to the general public, such as frequently cleaning hands, wearing a face mask, avoiding close contact with the infected persons or farm animals, and avoiding consumption of raw or half-cooked meat/eggs and following good food safety practices [ ] . there is an urgent need to develop rapid diagnostic tools and vaccines or post-exposure prophylaxis to treat this infection. reliable, timely laboratory diagnosis and an effective vaccine are crucial for effective disease management and public health intervention. an effective vaccine should be affordable, and also the production platform should produce suitable vaccine candidates rapidly at low cost, especially during a disease outbreak. the advantages and disadvantages of the current expression systems for recombinant protein production are given in table . currently, plant expression system offers many advantages over other conventional systems that have the potential to tackle the production of vaccine candidates rapidly at affordable cost facilitating the global vaccination programs, especially in resource-poor nations where the vaccines are needed most [ ] . in recent years, plants are being focused more on the production of biopharmaceuticals and virus-like particles (vlp's). the technologies employed for the production of plant-made vaccines are stable nuclear expression, transient expression, and chloroplast expression. based on several vital factors like yield, quality, time, and cost, appropriate technology can be chosen for producing biopharmaceuticals. recent advancements in the plant expression strategies, especially the development of transient expression system or viral vectors, resulted in a huge increase in the protein yield that makes this plant host system, a promising system for the production of various biopharmaceutical proteins [ ] . several reports in the last two decades have enough evidence to prove that the plant produced biopharmaceuticals are as effective as the mammalian cell-based proteins and also elicit potent neutralizing antibodies, or shown therapeutic effects against the particular pathogen or infection [ ] [ ] [ ] . the use of plants for the production of recombinant proteins and biopharmaceuticals has been gaining importance since the plant produced biologic taliglucerase alfa has been commercialized in against gaucher's disease that proclaimed a new era for plant made biopharmaceutical and triggered the innovation in the field of biopharmaceuticals [ ] . furthermore, many plant-produced candidates are in the clinical pipeline close to commercialization. some of the examples of plant-produced recombinant antigens and monoclonal antibodies for infectious diseases are given in table . currently, countries, including thailand, india, japan, korea, and the european community, are majorly involved in developing plant biopharmaceuticals against several human diseases [ ] . many reports reviewed the importance of plant expression system for the rapid production of candidate vaccines and therapeutic antibodies against infectious diseases [ ] [ ] [ ] [ ] [ ] [ ] . west nile virus pe tobacco [ ] many biopharmaceutical companies have shifted their momentum towards the plant expression system by knowing its importance. various plants such as tobacco, duckweed, moss, alfalfa, and other plants have been used for the production of plant-made pharmaceuticals. in september , leaf bio, inc., the commercial partner of mapp biopharmaceutical, inc. (mapp), received fast track designation by the u.s fda for their plant-made biopharmaceutical called zmapp for the treatment of ebola virus disease. the authorization of plant-based biopharmaceutical zmapp for emergency use in the earlier ebv outbreak by fda is potentially a major breakthrough in the plant molecular farming field, as it serves as an endorsement of the technology to potential investors and grant funding agencies [ , , ] . many vaccine antigens expressed in plants are in clinical or advanced pre-clinical trials. major players in the global plant-based biologics market include plantform, ibio inc., mapp biopharmaceutical, inc., pfizer inc., ventria bioscience, medicago inc., greenovation biotech gmbh, kentucky bioprocessing, phycobiologics inc., synthon, fraunhofer ime, healthgen, planet biotechnology, and icon genetics gmbh. as plant-made biopharmaceuticals provide efficacious and cost-effective strategies to protect against emerging infectious diseases, plant expression systems can be employed for the development of vaccines against ncov. transient expression in plants can be adapted for biopharmaceutical protein production when it is necessary to produce 'rapid response vaccines' as it produces more protein in a short time. the plant-based biopharmaceutical production against -ncov will include the identification of potential epitopes and production of full-length viral surface proteins present in the envelope region or production of subunit vaccines expressing immunogenic region or chimeric proteins. the receptor-binding domain (rbd) in spike protein located on the outer membrane of coronavirus mediates receptor binding and plays a major role in virus entry into the host cell [ ] . this protein could be used as a potential vaccine candidate as it is the major target for neutralizing antibodies [ ] . hence, it could be considered to develop potential effective vaccines or therapeutics against coronavirus infection. as the virus uses angiotensin-converting enzyme (ace ), the host cell receptor for its cell entry similar like sars-cov [ ] [ ] [ ] , the monoclonal antibodies that are identified and tested to be effective against sars virus protein or specific to ace can be produced in plants and shall be tested for its efficacy against ncov. earlier reports showed several vaccines and monoclonal antibody candidates in response to sars-cov and mers-cov, which could be tested and used for passive immunotherapy for an immediate immune response [ ] [ ] [ ] . the possibility of producing a vlp based vaccine is also feasible by expressing all the structural viral proteins that can assemble into vlps in plants. the structural and functional studies of viral proteins in ncov might help in designing better vaccines and specific therapeutics. producing viral proteins in plants may further be helpful to evaluate their potential in developing diagnostic kits or the protection/therapeutic tools that can be manufactured fast in order to manage the highly infectious ncov. this will open an avenue to characterize recombinant immunogenic viral proteins and provide a proof of concept for using plants as a robust, rapid, and flexible platform for producing protein/research reagents, which are highly essential to face potential ncov outbreaks. the coronavirus outbreak has been declared a global health emergency and represents one of the greatest risks to global health, as the virus has a tendency to infect a large number of human populations, and the outbreak can cause severe medical complications with economic impact, particularly in middle-income countries where resources are limited for early diagnosis and preventive measures. human mobility, air travel, and international trade can likely increase the number of cases in other regions as well. continued surveillance along with the robust response of government agencies, medical practitioners, and researchers, is highly essential for the effective management of this emerging pathogen. public health officials need to identify the source and virus reservoir, transmission cycle, pathogenesis, inter-human transmission, and clinical manifestations, which might be helpful to develop animal models, diagnostic reagents, anti-viral therapies, and vaccines against this pathogen. as the virus emerged suddenly and became a serious global concern, there is a need for rapid vaccine development. although classical expression systems for biopharmaceutical proteins are still amenable, the development of transient expression in plants has deeply influenced the pharmaceutical sector to produce affordable vaccines and biologics rapidly at low cost. hence, the plant expression platform shall be employed for biopharmaceutical production to accelerate the fight against this deadly infectious disease. the collaborative efforts of researchers are highly desirable to use a plant expression platform for producing an efficient cost-effective vaccine to control this epidemic. the continuous effort of research in this direction might be helpful in producing high-value biologics and pharmaceuticals on a large scale in a short time, especially during epidemics. infectious disease threats in the twenty-first century: strengthening the global response world health organization. novel coronavirus-china clinical features of patients infected with novel coronavirus in wuhan statement on the second meeting of the international health regulations emergency committee regarding the outbreak of novel coronavirus ( -ncov) genomic characterization and epidemiology of novel coronavirus: implications for virus origins and receptor binding coronavirus pathogenesis structure, function, and evolution of coronavirus spike proteins origin and evolution of pathogenic coronaviruses development of one-step, real-time, quantitative reverse transcriptase pcr assays for absolute quantitation of human coronaviruses oc and e a novel coronavirus from patients with pneumonia in china world health organization. novel coronavirus ( -ncov) situation report. available online a familial cluster of pneumonia associated with the novel coronavirus indicating person-to-person transmission: a study of a family cluster a major outbreak of severe acute respiratory syndrome in hong kong epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study plant expression platform for the production of recombinant pharmaceutical proteins plant-made pharmaceuticals: leading products and production platforms plant-produced anti-enterovirus (ev ) monoclonal antibody efficiently protects mice against ev infection cold chain and virus-free oral polio booster vaccine made in lettuce chloroplasts confers protection against all three poliovirus serotypes production and immunogenicity of soluble plant-produced hiv- subtype c envelope gp immunogens. front taliglucerase alfa: an enzyme replacement therapy using plant cell expression technology molecular pharming for low and middle income countries plant-based vaccines against viruses plant-made oral vaccines against human infectious diseases-are we there yet? plant-produced candidate countermeasures against emerging and reemerging infections and bioterror agents a perspective on the development of plant-made vaccines in the fight against ebola virus expression and functional evaluation of biopharmaceuticals made in plant chloroplasts development of systems for the production of plant-derived biopharmaceuticals antibody responses in mice stimulated by various doses of the potato-derived major surface antigen of hepatitis b virus freeze-drying of plant tissue containing hbv surface antigen for the oral vaccine against hepatitis b supercritical fluid extraction provides an enhancement to the immune response for orally-delivered hepatitis b surface antigen immunogenicity of chloroplast-derived hiv- p and a p -nef fusion protein following subcutaneous and oral administration in mice a chloroplast-derived c v polypeptide from the human immunodeficiency virus (hiv) is orally immunogenic in mice a plant-derived multi-hiv antigen induces broad immune responses in orally immunized mice immunogenic properties of a lettuce-derived c (v ) multiepitopic hiv protein oral delivery of plant-derived hiv- p antigen in low doses shows a superior priming effect in mice compared to high doses transgenic tobacco expressed hpv -l and lt-b combined immunization induces strong mucosal and systemic immune responses in mice immunization with an hpv- l -based chimeric virus-like particle containing hpv- e and e epitopes elicits long-lasting prophylactic and therapeutic efficacy in an hpv- tumor mice model expression of h n nucleoprotein in maize seeds and immunogenicity in mice induction of a protective immune response to rabies virus in sheep after oral immunization with transgenic maize, expressing the rabies virus glycoprotein expression of rabies glycoprotein and ricin toxin b chain (rgp-rtb) fusion protein in tomato hairy roots: a step towards oral vaccination for rabies cold chain and virus-free chloroplast-made booster vaccine to confer immunity against different poliovirus serotypes plant-made polio type stabilized vlps-a candidate synthetic polio vaccine expression of an immunogenic ebola immune complex in nicotiana benthamiana plant-produced anti-dengue virus monoclonal antibodies exhibit reduced antibody-dependent enhancement of infection activity characterization of vrc , a potent and broadly neutralizing anti-hiv mab, produced in transiently and stably transformed tobacco regulatory approval and a first-in-human phase i clinical trial of a monoclonal antibody produced in transgenic tobacco plants engineering, expression in transgenic plants and characterisation of e , a rabies virus-neutralising monoclonal antibody production, characterization, and antigen specificity of recombinant - - , a candidate monoclonal antibody for rabies prophylaxis in humans enhanced transport of plant-produced rabies single-chain antibody-rvg peptide fusion protein across an in cellulo blood-brain barrier device a broad and potent h -specific human monoclonal antibody produced in plants prevents influenza virus infection and transmission in guinea pigs rapid transient production of a monoclonal antibody neutralizing the porcine epidemic diarrhea virus (pedv) in nicotiana benthamiana and lactuca sativa in vitro and in vivo efficacy of anti-chikungunya virus monoclonal antibodies produced in wild-type and glycoengineered nicotiana benthamiana plants structural and functional characterization of an anti-west nile virus monoclonal antibody and its single-chain variant produced in glycoengineered plants commercialization of new biotechnology: a systematic review of commercial case studies in a novel manufacturing sector plant-made pharmaceuticals: fromedible vaccines to ebola therapeutics from sars to mers, thrusting coronaviruses into the spotlight severe acute respiratory syndrome structure of sars coronavirus spike receptor-binding domain complexed with receptor angiotensin-converting enzyme is a functional receptor for the sars coronavirus receptor recognition by novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars neutralizing human monoclonal antibodies to severe acute respiratory syndrome coronavirus: target, mechanism of action, and therapeutic potential antibodies and vaccines against middle east respiratory syndrome coronavirus recent advances in the vaccine development against middle east respiratory syndrome-coronavirus this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -kmpbdvof authors: demurtas, olivia c.; massa, silvia; illiano, elena; de martinis, domenico; chan, paul k. s.; di bonito, paola; franconi, rosella title: antigen production in plant to tackle infectious diseases flare up: the case of sars date: - - journal: front plant sci doi: . /fpls. . sha: doc_id: cord_uid: kmpbdvof severe acute respiratory syndrome (sars) is a dangerous infection with pandemic potential. it emerged in and its aetiological agent, the sars coronavirus (sars-cov), crossed the species barrier to infect humans, showing high morbidity and mortality rates. no vaccines are currently licensed for sars-cov and important efforts have been performed during the first outbreak to develop diagnostic tools. here we demonstrate the transient expression in nicotiana benthamiana of two important antigenic determinants of the sars-cov, the nucleocapsid protein (n) and the membrane protein (m) using a virus-derived vector or agro-infiltration, respectively. for the m protein, this is the first description of production in plants, while for plant-derived n protein we demonstrate that it is recognized by sera of patients from the sars outbreak in hong kong in . the availability of recombinant n and m proteins from plants opens the way to further evaluation of their potential utility for the development of diagnostic and protection/therapy tools to be quickly manufactured, at low cost and with minimal risk, to face potential new highly infectious sars-cov outbreaks. severe acute respiratory syndrome (sars) is a dangerous infection with pandemic potential. it emerged in and its aetiological agent, the sars coronavirus (sars-cov), crossed the species barrier to infect humans, showing high morbidity and mortality rates. no vaccines are currently licensed for sars-cov and important efforts have been performed during the first outbreak to develop diagnostic tools. here we demonstrate the transient expression in nicotiana benthamiana of two important antigenic determinants of the sars-cov, the nucleocapsid protein (n) and the membrane protein (m) using a virus-derived vector or agro-infiltration, respectively. for the m protein, this is the first description of production in plants, while for plantderived n protein we demonstrate that it is recognized by sera of patients from the sars outbreak in hong kong in . the availability of recombinant n and m proteins from plants opens the way to further evaluation of their potential utility for the development of diagnostic and protection/therapy tools to be quickly manufactured, at low cost and with minimal risk, to face potential new highly infectious sars-cov outbreaks. recombinant proteins expressed in plants have emerged as a novel branch of the biopharmaceutical industry and hold great potential to produce different types of therapeutic proteins at low cost and with reduced risks of contamination with human and animal pathogens (moustafa et al., ; paul et al., ; sack et al., ) . transient expression of target proteins can be easily achieved by plant viruses or by agroinfiltration (gleba et al., ) , saving the time spent in the generation of transgenic plants, often allowing higher protein yield due to the absence of chromosomal integration and consequently of position effects (komarova et al., ) . transient expression can also be used as a means for preliminary evaluation of correct expression before starting the generation of transgenic plants, or related platforms, such as plant cell cultures or microalgae (franconi et al., ) . antigen preparation plays a crucial role in the development of a diagnostic test, and plants represent an ideal biofactory system. the approach could be extended to other cases when a pathogen cannot be grown in the lab or is highly virulent and needs a methodology for fast and affordable production. indeed, virus-specific and 'orphan' vaccine candidates and therapeutics represent one of the most interesting applications of the plant-based technology, especially when it is necessary to produce 'rapid response' vaccines such as those directed against bioterrorism agents and diseases with pandemic potential, like influenza. the capacity for such a response has already been demonstrated by the (four) companies involved in the us in the production of million doses of influenza vaccine a month (rybicki, ) . this opens the way for the use of this technology for other diseases such as, to name a few, ebola (henao-restrepo et al., ; heymann et al., ) , avian flu (su et al., ) , mers (al-tawfiq and memish, ; keener, ) and other viruses where the principles of the so-called "one health initiative" (strategies to control diseases across species) are important (http://www.onehealthinitiative.com/). the fact that the epidemiology of these diseases is associated to sudden and sometimes unforeseen contagious burst, results in an on/off attention about, in terms of research, prevention and pharma industry efforts (barber, ; lamattina, ) . for this reason, tools for a quick and relatively easy scale-up may provide a solution to such emergencies (streatfield et al., ) . among emerging and re-emerging diseases, severe acute respiratory syndrome (sars) appeared in china in november . the epidemic spread to countries over continents, leading to more than infected patients globally (world health organization [who] , ) with a fatality rate of . % (suresh et al., ) . the aetiological agent of the syndrome, the coronavirus sars-cov, was rapidly identified (drosten et al., ; marra et al., ; rota et al., ) . the end of the sars outbreak was declared by world health organization [who] ( ) in july, thanks to strong containment measures. however, several local outbreaks were subsequently reported in china, as a consequence of accidental laboratory contaminations or infections after contact with animals infected with sars-cov strains significantly different from those predominating in the - outbreak (peiris et al., ) . while no effective therapy is currently available, considerable efforts have been made to develop vaccines and drugs to prevent sars-cov infection, since a sars epidemic may recur at any time in the future (gimenez et al., ; chan and chan, ) . moreover, because of the highly contagious nature of the disease, and since sars-cov has been defined a potential biological weapon (centers for disease control and prevention [cdc] , ), it is still important to develop effective sars-cov sensitive diagnostic tools (suresh et al., ) . the large sars-cov genome, a polyadenylated rna of , nucleotides, encodes four major viral structural components, the spike (s), envelope (e), membrane (m), and nucleocapsid (n) proteins, and non-structural proteins (bartlam et al., ) . the structural n protein is the most abundant protein in the sars-cov virion. it is a highly basic protein of amino acids ( kda) of the helical nucleocapsid, playing an important role in viral pathogenesis, replication, rna binding, cell cytokinesis and proliferation (surjit and lal, ) . n protein has been recognized as the preferred target for detection of sars-cov infection by reverse transcription-polymerase chain reaction (rt-pcr; suresh et al., ) . in addition, the who guidelines for sars diagnosis, developed during the outbreak in , suggested the use of n-based elisa for specific igg detection as confirmatory test of sars-cov infection (world health organization [who] , sars: laboratory diagnostic tests) due to the ability of the host to mount an early antibody response against the n protein (che et al., ) . furthermore, since the n protein is able to induce a long-term cell-mediated immune response in animal models, it represents a potential vaccine candidate as well (roper and rehm, ) . to date, the production of recombinant n protein has been achieved in a variety of heterologous expression systems, including plants, (zheng et al., ) , providing proofs of concept for its use in vaccine formulations (roper and rehm, ). however, the immune response in animal models (both natural and nonnatural sars-cov hosts) might be not useful to predict the human immune response. the m protein is the most abundant protein in the sars-cov viral envelope. it is functionally involved in the assembly and budding of virions from the cell. m protein forms homo-oligomers and interacts with s, e, and n proteins (hogue and machamer, ) . it consists of amino acids ( kda), with a short glycosylated n-terminal domain, three membrane-spanning domains and a long immunogenic c-terminal cytoplasmic domain. it has been reported that rabbit antiserum raised against recombinant m protein produced in yeast has a potent neutralizing activity in vitro (pang et al., ) . antibodies to the m protein were detectable in convalescent sars patients and b-cell epitopes of the m protein have been identified (he et al., ) . it has also been shown that m acts as a dominant immunogen for ctl response in humans (liu et al., ) . moreover, it has been demonstrated that sars-cov m-specific memory cd + and cd + t cells were persistent in the peripheral blood of recovered sars patients more than year after infection . in a study, where different dna vaccines were used, m generated the strongest t-cell response in an animal model, and recovered sars patients had a long-lasting cd + and cd + memory for the m antigen (roper and rehm, ) . these data suggest that further research should be directed toward evaluating the potential efficacy of the m antigen for vaccine and diagnostic tools development. in this study, we demonstrate the feasibility of using plant transient expression systems (potato virus x [pvx]-mediated infection and agroinfiltration) to produce two sars-cov antigens, the n and m proteins, as useful tools to face sars-cov infection. in particular, we demonstrated that the sars-cov n protein produced in nicotiana benthamiana is recognized by the specific antibodies of convalescent sars patients. moreover, the expression of the sars-cov m protein was achieved for the first time in plant. the approach to rapidly get crucial antigens by transient expression in plants, potentially attains to other infectious, either emerging or re-emerging, diseases (e.g., mers, avian flu, ebola), that share with sars the features of rapid outbreak burst and need to rapidly produce diagnostic or therapeutic tools. escherichia coli xl blue strain was used as a host for cloning and protein expression. cells were grown in luria-bertani medium at • c with shaking at rpm. agrobacterium tumefaciens gv and c c strains were used to transiently express the m protein in n. benthamiana and were grown in yeb medium ( g/l beef extract, g/l yeast extract, g/l peptone, g/l sucrose, mm mgso ) at • c with shaking at rpm. when necessary, ampicillin ( µg/ml) or kanamycin ( µg/ml) was added to the culture medium. hek- cells were cultivated as a monolayer in dmem medium with % fetal bovin serum (fbs) and µg/ml gentamicin at • c with % of co and relative humidity of %. the nucleocapsid (n, genbank protein id. aap . ) and membrane (m, genbank protein id. aap . ) full-length genes of the human sars-cov frankfurt i isolate, acc. no. ay were cloned into pcr . -topo ta cloning vector (invitrogen; carattoli et al., ) . the inserted fragments were cut out by digestion with bamhi-noti and sub-cloned into the pqe- (qiagen) prokaryotic expression vector . the full-length n gene ( -bp long) was amplified by pcr on the template plasmid pqe -n with pfu polymerase with the forward primer -ggccatcgatgaattcggatccatcatgagaggatcgc atcacatcc- (clai restriction site: underlined, ecori site: italic, initiation translation codon: bold) and the reverse primer -gacttgtcgacgcggccgctta tgcctgagttgaatcagcag- (sali site: underlined, noti site: italic, stop codon: bold). for mammalian cells expression, the pcr product was cut out by digestion with ecori/noti and inserted into the pvax vector (invitrogen). for plant expression, the pcr product was cut out with clai/sali and cloned into the ppvx plant vector (baulcombe et al., ) . in this vector, the full-length viral cdna of the potato virus x (pvx) is inserted between the constitutive s promoter of the cauliflower mosaic virus (camv s) and the transcription terminator (nos-term) of the nopaline synthase gene of a. tumefaciens, necessary for the regulation of the viral genome expression upon infection with plasmid dna. characteristic components of the viral expression vector are the following: viral replicase gene (rdrp); triple gene block encoding protein for cell-to-cell movement (m - ); viral coat protein gene necessary for encapsidation of viral rna (cp); coat protein sub-genomic promoter (sgp; figure a) . the full-length m gene ( -bp long) from the pcr . -topo ta vector was amplified in two subsequent steps. first, the forward primer -ggccatcgatgaattcggatccatcatggcagacaacgg tactattac- (clai restriction site: underlined, ecori site: italic, initiation translation codon: bold) and the reverse primer -gtgatggtgatga tgctcgagtgcctgtactagcaaagcaatatt- (end of the his tag: italic, end of the m gene: bold) were used to add the his tag at the c-terminus. subsequently, the same forward primer and the reverse primer -gacttgtcgacgcggccgctcaatggtgatggtgatg atgctcg- (sali site: underlined, noti site: italic, stop translation codon: bold) were used in order to add restriction sites useful for cloning. for simplicity, we report the name of the recombinant genes n-his and m-his as n and m genes. the purified pcr products were cloned into the eco rv-linearized pbluescript sk(+; pbs) cloning vector (stratagene; pbs-n and pbs-m), sequenced for authenticity and sub-cloned by clai-sali in the ppvx plant vector (ppvx-n and ppvx-m) or by ecori/noti in the pvax vector (pvax-n and pvax-m). after xl blue cells transformation, ppvx-n and ppvx-m plasmid dnas were purified (maxiprep kit, qiagen) for plant infection, while pvax-n and pvax-m plasmids were purified with endotoxin-free purification kits (plasmid maxi kit lps-free, qiagen) for mammalian hek- cells transfection. the m gene was also sub-cloned by xbai/sali from the pbs-m construct into the pbi- plant binary vector (clontech laboratories, acc. no. af ), obtaining the pbi-m construct. in this construct, the m gene is inserted between the camv s promoter and the nos terminator. characteristic components of the vector are: right and left borders (rb, lb), gene for kanamycin resistance (npt ii) under the transcription promoter and terminator of the nopaline synthase gene of a. tumefaciens ( figure a) . the pbi-m construct, obtained by substitution of the gus gene with the m gene, was used to transform the gv and c c strains of a. tumefaciens. the expression and purification of the n and m proteins were done according to standard protocols (the qiaexpressionist, qiagen). since it was previously described that the m protein is toxic in bacteria (carattoli et al., ) , culture growth was performed at suboptimal temperature of • c. in these conditions, we isolated a clone able to express the m protein in e. coli. characterization revealed that it corresponded to a spontaneously mutated m gene, coding for a protein carrying the three substitutions k > r, f > l, and i > v (m rlv ). the m rlv and the n proteins were purified by ni-nta affinity chromatography in denaturing conditions. for the n protein, when purified in native conditions, imidazole concentration in the wash buffer was mm. quantification of the n protein purified in denaturing and native condition was performed by coomassie stained sds-page comparing the intensity of the band at kda to known amount of bsa using a chemidoc imagelab system with imagelab . software (bio-rad). hek- cell line was transfected in six wells plates with the pvax-n or pvax-m constructs according to standard protocols (effectene transfection reagent, qiagen). h post-transfection (pt) some wells were added with . µm of the proteasome inhibitor mg- . h pt both samples, treated or not with mg- , were harvested and prepared for protein expression analysis (immunoblotting and immunofluorescence). after three washes with cold phosphate-buffered saline (pbs: mm na hpo , . mm nah po , mm nacl, ph . ) cells were centrifuged at rpm for , recovered and re-suspended in sds-loading buffer ( % glycerol, mm tris-hcl ph . , . % bromophenol blue, % sds, % -mercaptoethanol) and boiled for min. for immunofluorescence analysis, hek cells were grown on multi-chamber glass slides and transfected at % of confluency. h post transfection cells were washed three times with pbs, fixed with % paraformaldehyde for min and permeabilized with . % triton x- in pbs. samples were blocked with % no-fat dry milk in pbs. for detection of the n and m proteins, cells were incubated with specific polyclonal antibodies (pab) validated in immuno-cytochemical assay on sars-cov infected and previously described (carattoli et al., ) . for n protein we used a mouse anti-n pab, (obtained after immunization of animals with the purified his -n protein produced in e. coli) at : dilution. for the m protein we used a mouse anti-m pab (obtained by immunizing mice with the recombinant cytoplasmic domain, amino acids - , of the m protein produced in e. coli) at : dilution. cells were then incubated with a : dilution of an anti-mouse biotinylated secondary antibody (ge healthcare) and a : dilution of streptavidin-fitc conjugated (sigma-aldrich, gmbh, steinheim, germany). nuclei were counterstained with dapi ( µg/ml) in pbs. slides were examined using a fluorescence "axiolab zeiss" microscope (oberkochen, germany) interfaced with a coolsnap ccd camera (roper scient ., princeton, nj, usa). two leaves of n. benthamiana plants ( week-old) were dusted with carborundum powder and inoculated with µg of the plasmids ppvx-n, ppvx-m, or ppvx (empty vector, negative control) diluted in µl bi-distilled water. plants were also treated with µl bi-distilled water (mock-infected) for monitoring viral infection symptoms. plants were grown under h daylight at • c into a containment greenhouse (bio-safety level ) and observed daily for pvx infection signs. inoculated and symptomatic leaves were harvested and stored at - • c until use. four week-old n. benthamiana plants were infiltrated with a. tumefaciens c c and gv cultures harboring the pbi-m construct that had been grown and induced as described (kapila et al., ) . plants were subsequently grown as described and the leaf disks collected , , , , , and days post-infiltration (dpi) were homogenized with an ultraturrax in three volumes (w/v) of sds-loading buffer and boiled for min. to analyze n and m protein accumulation in plant tissues, soluble proteins were extracted from plant material using different buffers, depending on their hydrophobic properties. crude plant extracts were prepared by grinding the tissue to a fine powder in liquid nitrogen. the powder was re-suspended and homogenized with an ultraturrax in three volumes (w/v) of pbs or, alternatively for the m protein, in one volume (w/v) of gb buffer ( mm tris-hcl ph . , % glycerol, mm sucrose, mm mgcl , mm kcl, mm -mercaptoethanol) containing protease inhibitors ("complete, edta-free, " roche diagnostics, gmbh, mannheim, germany). tissue homogenates were centrifuged at • c, , × g, for min. the supernatant was transferred to a fresh tube and kept on ice (or at • c) until use and total soluble protein (tsp) content was estimated by the bradford assay (bio-rad inc., segrate, italy). pellets were resuspended in appropriates volumes of sds loading buffer, and prepared as described in the paragraph below, constituting the insoluble fraction of leaf extracts. homogenized tissues of ppvx-n infected plants were also used to inoculate n. benthamiana plants to propagate the infectious recombinant pvx particles until the third round of infection. a small-scale purification of plant-produced n protein was performed. in brief, lyophilized leaf material was ground in liquid nitrogen, re-suspended and homogenized with an ultraturrax in m urea, mm nah po , mm tris, ph . , and incubated for h at rt with gentle shaking. leaf material was finally lysed on ice by sonication at hz output ( s each) for three times. cell debris collected by centrifugation was discarded. the recovered supernatant was filtered ( . µm) and incubated for h with the ni-nta resin. plant n protein purification was performed by decreasing the ph (the qiaexpressionist). protein elution was obtained at ph . . for immunoblotting analysis of the n protein, plant extracts containing µg tsp and purified protein were boiled for min in sds-loading buffer. immunoblotting detection of the m protein was performed on plant extracts incubated at • c for ' in sds-loading buffer. samples were separated by % sds-page and transferred onto pvdf membranes (immobilon-p, millipore). after membrane blocking with % non-fat dry milk in pbs (mpbs) over night (o/n) at • c, membranes were incubated for h at room temperature (rt) either with an anti-his monoclonal antibody (mab; h -clone his- , sigma) or with specific polyclonal antibodies previously described (carattoli et al., ) . for n protein detection, we used a rabbit anti-n pab, (obtained after immunization of animals with the purified his -n protein produced in e. coli) at : dilution. immune complexes were revealed by h incubation at rt with an antirabbit biotinylated secondary antibody (b , sigma), at : dilution, followed by horseradish peroxidase (hrp)-conjugated streptavidin (rpn , ge healthcare) at : dilution. for m protein detection, membranes were incubated with the mouse anti-m pab at : dilution, followed by incubation with an anti-mouse hrp-conjugated secondary antibody at : dilution (na , ge healthcare). for both proteins, the bound antibody was detected using the ecl plus system ("enhanced chemi-luminescence", ge healthcare). the amount of n protein in the plant extracts was estimated by a quantitative triple antibody sandwich (tas) elisa. symptomatic systemic leaves, deriving from plants infected with ppvx-n, were collected and pooled. three independent extractions were performed and analyzed. microtiter plates were coated with µl/well of rabbit anti-n pab (diluted : in pbs) for h at • c followed by coating with µl plant extract with a normalized tsp content ( µg) for h at • c. wells were then blocked with µl/well of % mpbs at • c for h. the captured n protein was detected by incubating at • c for h with µl/well of a : dilution in % mpbs of a mouse anti-n pab (obtained after immunization of animals with the purified his -n protein produced in e. coli, carattoli et al., ) followed by incubation with : dilution of hrp-conjugated goat anti-mouse igg antibody. after each step, wells were washed three times with pbs + . % tween and one time with pbs. enzymatic activity was measured by adding µl/well v/v h o /abts [ , -azino bis-( -etilbenzotiazolin) sulphuric acid] (kpl inc., gaithersburg, md, usa) at rt in darkness condition. the absorbance of the samples was read after at nm on an elisa microtiter plate reader. known amounts of e. coli-purified n protein ( . , , , , , , and ng) were used as a standard. in order to give a better estimation of n protein yields in plant, the standard was diluted in n. benthamiana extract. a 'multi-strip' western blot assay was chosen to evaluate the antigenicity of the plant produced n protein. it is an immunoblotting procedure modified from kiyatkin and aksamitiene ( ) , in which the sample, loaded in a single-well polyacrylamide gel (a standard gel where a single preparative well is cast), is blotted onto a membrane that is then cut into strips of the same width, each containing same amount of protein. each strip is then incubated with different antibodies or sera and developed by a colorimetric assay (see ciufolini et al., ; di bonito et al., ) . in our experiments, single-well % sds-page gels were loaded with one of the following preparations: (i) e. coli purified n protein ( . µg); (ii) plant-purified n protein ( . µg); (iii) plant extract from ppvx-n symptomatic leaves containing about . µg of n protein and mg tsp; (iv) plant extract from ppvx symptomatic leaves containing about mg tsp. samples (iii) and (iv) were prepared as following: the powder ground from ppvx-n or ppvx symptomatic leaves was resuspended and homogenized by an ultraturrax in one volume (w/v) of tn buffer ( mm tris-hcl ph . , mm nacl) containing protease inhibitors. tissue homogenates were centrifuged at • c, × g, for . tsp was calculated by bradford assay and precipitated by trichloroacetic acid (tca) at % final concentration. after ' incubation in ice, samples were centrifuged, pellets were washed with cold % acetone, dried, re-suspended in sds loading buffer. gels were blotted using a semi-dry system (biorad) onto pvdf membranes. after blocking with % mpbs, membranes were dried at rt and cut in strips, . cm width. each strip of samples (ii) and (iii) contained approximately ng of the n protein (as plantpurified protein or in plant extract, respectively). as controls, strips containing ng of e. coli-produced n protein [sample (i)], were prepared, as well as strips of plant extracts without the n protein [extract from ppvx symptomatic leaves, sample (iv)]. before incubation with human sera, one strip from either plant-derived or e. coli-expressed n protein was incubated with the mouse anti-n pab, as previously described (carattoli et al., ) , to confirm protein presence and to check that the amount of protein in the strips was sufficient for colorimetric detection. the strips were incubated with pools of sars sera collected during the sars outbreak in hong kong in (chan et al., ; sars patients pooled in seven groups) at : dilution in % mpbs, o/n at • c. blood samples were collected with informed written consent. the study was approved by the institutional human research ethics committee (the joint chinese university of hong kong-new territories east cluster clinical research ethics committee). as non-sars controls, strips were incubated with three pools of sera from patients affected by unrelated respiratory diseases (carattoli et al., ) . antigen-antibodies complexes were revealed by a rabbit anti-human igg (h + l) hrpconjugate (southern biotechnology associates, inc, cat.no. - ) diluted : in % mpbs. after h of incubation at rt colorimetric reaction on the strips was induced by adding , -diaminobenzidine tetrahydrochloride substrate, dab (sigma d- ) and hydrogen peroxide. to produce the recombinant sars-cov n protein, n. benthamiana plants ( weeks old) were infected with the ppvx-n dna plasmid harboring the n gene ( figure a) . as a control, plants were either mock infected or infected with the wild type ppvx vector. while mock infected plants showed no symptoms, typical symptoms (mainly chlorotic spots) generally appeared on the inoculated leaves of plants infected with ppvx-n or ppvx vectors - days post inoculation (dpi). the infection spread systemically to apical leaves, where symptoms appeared - dpi. to examine whether the n protein accumulated in infected plants, soluble protein extracts were analyzed by elisa and immunoblotting. interestingly, after infection with the ppvx-n plasmid, protein expression corresponded to symptoms in systemic leaves in all plants analyzed (about ). this result indicates that the construct is stable and that the recombinant virus pvx-n can spread systemically within the inoculated plant. immunoblotting of soluble protein extracts from plants infected with ppvx-n reveals a single band of about kda in systemic leaves (figure b) , suggesting the absence of proteolysis. on the contrary, when the n protein is expressed in e. coli, additional bands of lower molecular mass are present ( figure b) . these bands probably correspond to protein degradation, in accordance with previous work demonstrating the intrinsic instability and/or autolysis of this protein when expressed in bacteria (mark et al., ) . furthermore, ppvx-n-derived extracts, stored at - • c for months and then thawed (figure b) , as well as extracts obtained from freeze-dried ppvx-n-infected leaves showed an intact n protein (data not shown). immunoblotting of mammalian cells transfected with the pvax-n plasmid also revealed the presence of a single band of approximately kda corresponding to the n protein (figure a) . immunofluorescence revealed a cytoplasmic localization of the n protein ( figure b) . plant extracts deriving from pvx-n-infected leaves were also used for subsequent inoculations. n protein expression was confirmed at least until the third cycle of re-infection (supplementary figure s ) , further demonstrating the stability of the construct and of the recombinant virion. the amount of recombinant n protein expressed in leaves, as measured by tas-elisa, was approximately - µg/g fresh leaf weight, corresponding to . % tsp (figure ) . although the n protein accumulated mainly in the soluble fraction in all the expression systems used (plant, bacteria and mammalian cells), for purification the best recovery of the n protein from e. coli was obtained in denaturing conditions ( mg of protein/liter of culture under denaturing conditions versus . mg protein/liter of culture under native conditions, figure a ). therefore, we decided to perform n protein purification in denaturing conditions also from plant tissue. we performed a small-scale purification by loading plant extracts derived from freeze-dried leaves (obtained from a pool including primary-infected and re-infected systemic leaves) on a ni-nta affinity purification column. in this way, we obtained yields of about µg of purified n protein/g of fresh leaf weight ( figure b ). the elisa and immunoblot analysis gave a first indication of antigenic features of the plant-derived n protein. in fact, in such analysis the n protein was specifically recognized by rabbit and mouse anti-his -n hyper-immune sera that had previously been shown to recognize the n protein in sars-cov infected cells (carattoli et al., ) . to further characterize the n protein expressed in plant, we analyzed its reactivity with sera from sars patients, collected during the sars outbreak in hong kong in . these sars sera were previously screened by an elisa assay with an e. coliexpressed n protein (di bonito and chan, data not shown). here, to evaluate sars-positive sera reactivity with n plant expressed protein, we used a 'multi-strip' western blot assay. in a first experiment, we evaluated the reactivity of the purified n protein with a pool of sars sera. as shown in figure a , both preparations of purified n protein, from e. coli and from plant, are recognized by sars sera as well as by the mouse anti-n pab (positive control). then, we validated the results obtained for the plant-purified n protein analyzing its reactivity with other six groups of sars sera deriving from patients and with three groups of sera from patients affected by non-sars respiratory diseases ( figure b) . in this experiment, we observed that the n protein purified from plant is specifically recognized by all the groups of sars sera analyzed, while no reactivity was observed with sera from patients affected by other respiratory diseases. we also tested the reactivity of sars and non-sars sera with the unpurified n protein (soluble extract of ppvx-n symptomatic leaves) and with the extract from plant infected with the ppvx empty vector. a light reactivity with sars sera was observed only for the unpurified n protein, while no reactivity was observed for the same preparation when using non-sars sera (supplementary figure s ) . importantly, the ppvx leaf extract did not react with any human sera (supplementary figure s ) . to our knowledge, this is the first report of the antigenic properties of a plant-derived n protein as revealed by direct serology using sars patient sera, suggesting its possible use for the development of sars diagnostic assays. we investigated the ability of plant expression systems to cope with the synthesis of m protein. we started our studies on m protein expression in n. benthamiana plants ( -week old) by infection with the ppvx-m plasmid harboring the m gene as described for the n protein. also in this case typical symptoms generally appeared - dpi on inoculated leaves and - dpi on systemic leaves. however, m protein production was reported in just plants out of analyzed at detectable levels (data not shown). hence, we investigated the possibility to obtain the m protein by agroinfiltration. n. benthamiana leaves were infiltrated with a. tumefaciens suspensions (strains c and gv ) harboring the pbi binary vector containing the m gene (pbi-m vector, figure a ). with this simple technology, and without the use of any post-transcriptional gene silencing suppressors, we were able to detect sars-cov m protein production in plant. the time course experiment revealed that the m protein is expressed mainly at and dpi (data not shown). for immunoblotting analysis, the m protein was extracted with an appropriate buffer (gb buffer) and, since it was previously reported that boiling causes m protein aggregation and precipitation (lee et al., ) , samples boiling was avoided. interestingly, the m protein accumulated almost totally in the soluble fraction of the plant extract ( figure b ). as described in the previous paragraph for the n protein, to better characterize the plant-derived m protein we worked, at the same time, on m protein expression in bacteria and mammalian cells. due to toxic properties of m protein for e. coli (carattoli et al., ) , we performed colony selection and m protein expression under sub-optimal growth conditions ( • c). in this way, the m protein, with a mass of about kda was purified by ni-nta affinity chromatography in denaturing conditions, obtaining yields of about µg/l culture (data not shown). dna sequence analysis revealed the presence in the m gene of three spontaneous point mutations: r > k in the n-terminal domain, l > f in the first transmembrane domain and v > i in the cytoplasmic domain (m rlv ). as the plant-derived recombinant m protein, the m rlv was also specifically recognized by the mouse anti-m pab ( figure c ) that had previously validated by immunofluorescence antibody assay (ifa) in sars cov infected vero cells (carattoli et al., ) . although we were not able to express the m protein with its original aa sequence in bacteria, the m rlv protein was useful as standard for the plant-produced m protein characterization. immunoblotting revealed that, contrary to prokaryotic cells, the plant system allowed the expression of the full-length m protein, especially by the use of the agrobacterium c c strain ( figure b) . the m protein yield, calculated by immunoblotting using the quantified m rlv protein purified from e. coli as standard, was estimated to be . - . % tsp. the mobility of the plant-derived m protein in sds-page was reduced compared to the mutated bacterial form (figure c) , suggesting a higher molecular mass of plant-expressed compared to the e. coliexpressed m protein. no attempts to purify the m protein from plant were done due to the expression estimates. the m protein was also expressed in mammalian cells. immunofluorescence on m-transfected hek- cells using the mouse anti-m pab revealed that m protein is primarily localized in the plasma membrane (figure ) . similar results were previously reported by tseng and collaborators (tseng et al., ) , who described a perinuclear and plasma membrane localization of the m protein expressed in various cell lines. however, we did not observe m protein expression by immunoblotting, either in transfected cells or in the culture medium (not shown), probably because of its low expression level, as we reported in n. benthamiana plants by pvx-mediated infection. the importance to develop effective therapeutic and preventive strategies, to be readily applied to new emergent pathogens is established by the two novel coronaviruses that have emerged in humans in the twenty-first century: severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov). both viruses cause acute respiratory distress syndrome and are associated with high mortality rates. there are no clinically approved vaccines or antiviral drugs available for either of these infections, thus their development represents a research priority (graham et al., ) . severe acute respiratory syndrome coronavirus was the first massive infectious disease outbreak and it still has the potential to cause large-scale epidemics in the future. the key to preventing and controlling a future outbreak of sars is to develop rapid and specific diagnostic methods so that suspected patients can be correctly assessed. moreover, effective and safe treatment/vaccination will be extremely important in minimizing the damage of a new pandemic. enormous efforts have been undertaken to these purposes. the four major diagnostic methods available for sars include viral rna detection by rt-pcr, virus induced antibodies by immunofluorescence assay (ifa), or by enzyme linked immunosorbent assay (elisa) of nucleocapsid protein (n) and inoculation of patient specimens in cell culture (world health organization [who] , sars: laboratory diagnostic tests). the sars-cov n protein, expressed at early stage of infection and triggering a strong antibody response by the host, is considered to be the best diagnostic target (surjit and lal, ) . plasmon resonance-based biosensors (huang et al., ; park et al., ) and nanowire/carbon nanotube transistors (ishikawa et al., ) have been developed for the detection of sars-cov n protein in patient sera. such sensors offer real-time detection of nanomolar concentrations of the protein. nevertheless, sars tests should also have other useful features such as cost-effectiveness and ease of operation. moreover, combinations of antigens may be necessary to provide a definitive diagnosis of sars in humans and susceptible animals (roper and rehm, ) . the production of recombinant n protein has been achieved in a variety of heterologous expression systems. a synthetic gene with optimized codons has been expressed in e. coli at high yield (das and suresh, ) but it was demonstrated that bacterially expressed n protein produces false seropositivity owing to interference of bacterially derived antigens (leung et al., ; yip et al., ; surjit and lal, ) or cross-reacts with antisera of human coronaviruses (hcov-oc and hcov- e)-infected patients (woo et al., ). an interesting study correlated the phosphorylation state of the n protein with its antigenicity and specificity of antibodies recognition (shin et al., ) . these data underline the importance of producing the recombinant protein in eukaryotic platforms such as insect cells, yeast, or plants to set up more efficient and specific diagnostic tests. to date, it was demonstrated that the n protein produced in insect cells may be useful for the development of highly sensitive and specific assays to determine sars infection (shin et al., ) . later, the n protein was transiently expressed in plant by agroinfiltration, and its antigenicity was demonstrated in mice (zheng et al., ) giving a proof of concept of its use in vaccine formulations. previously, the s domain of sars-cov s protein had been stably expressed in tomato and low-nicotine tobacco plants obtaining about . % tsp (pogrebnyak et al., ) . the plant-derived antigen was able to induce systemic and mucosal immune responses in mice. while previous works have primarily focused on the s and n proteins, there is growing evidence of the potential of the m antigen both as an effective vaccine and diagnostic candidate. thus, the obtainment of the recombinant full-length m protein in an eukaryotic expression system also represents a good way to develop an effective and safe sars-cov vaccine. in addition to the knowledge that the m protein elicits a strong humoral responses, and that a specific humoral and cellular immune response can be obtained by co-expressing s, m and e (lu et al., ) , it has been demonstrated that the m protein also contains t cell epitopes (liu et al., ) . the availability of recombinant m protein, in combination with other viral proteins might overcome the concern about the sensitivity and the specificity of n nucleoprotein-based assay, as described when using the n and the s proteins together (woo et al., ; haynes et al., ; gimenez et al., ) . this would help the development of more efficient reagents to detect antibodies in the infected human host. here, we propose the use of plants as an alternative system to produce the n and m antigens that could be useful to formulate new vaccines and diagnostic assays against sars. since plant transformation and regeneration of stable transformants require considerable time, we used transient expression systems (pvx and agroinfiltration) to evaluate the ability of the plant expression system to cope with the synthesis of the sars-cov m and n proteins. the n and m full-length genes of the human sars-cov frankfurt i isolate were cloned, without codon optimization, into different expression vectors. for the sars-cov n protein, we assessed the successful ectopic expression in n. benthamiana plants by ppvx-mediated infection (figure ) . we were able to obtain the n protein in systemic leaves in most primaryinfected plants as well as in % re-infected plants. these results demonstrate the stability of the construct, a condition not easily obtained especially when large sequences are inserted in the ppvx-derived expression cassette (the n gene is about bp). in fact, several studies report that the use of 'first generation' plant viral vectors (like the ppvx series) for the expression of proteins in plants can lead to insert elimination by natural selection over replication cycles as early as the first infection passage with a positive correlation between insert length and elimination rate (avesani et al., ) . unlike the observed prokaryotic expression pattern, no proteolysis products were detected in immunoblotting by using polyclonal sera in ppvx-n-derived extracts, fresh or stored at - • c for months ( figure b) . these data demonstrate the stability of the recombinant n protein when transiently expressed in n. benthamiana. the same stability was observed when the n protein was expressed in mammalian cells, even in the absence of proteasome inhibitor (figure a) . the purified plant-produced n protein is specifically recognized by sera from chinese sars patients of the outbreak, and not from patients affected by unrelated respiratory diseases (figure ) . this result suggests that the plant-expressed sars-n protein is suitable in sars diagnosis. it is interesting to note that, when using crude plant extracts, sars human sera reveal a weak band, corresponding to the molecular weight of the n protein, without any cross-reaction with other components of the plant extract (supplementary figure s ). taken together, the results indicate that plant-derived n protein is specifically detected by antibodies of sars patients, using an assay where the antigen-antibody complex is revealed by a colorimetric method, less sensitive but more specific and suitable for hospital clinical laboratories. it should be noted that the only information available to date about plant-derived sars-cov n protein antigenicity were from zheng et al. ( ) who immunized mice and evaluated the impact on the regulation of cytokines and on the elicited igg subclasses. our study is the first demonstration by direct serology that plant-derived n protein is able to reveal human n-specific antibodies present in sera of sars patients, thus providing an adequate instrument to develop a rapid, low-cost, immune-based diagnostic assay to be used as an alternative or in association to molecular diagnosis. for the m protein, we obtained a spontaneously mutated m rlv protein in e. coli. this allowed to overcome toxicity of the wild type protein when expressed in bacteria (carattoli et al., ) and to purify the m rlv protein that was then used as positive control in our experiments. as the sars-cov m protein is difficult to express in recombinant form, the exact structure and function of the protein are not fully elucidated (neuman et al., ; tseng et al., ; siu et al., ) . unlike prokaryotic cells, the plant system allowed the expression of the full-length m protein by using a. tumefaciens (in particular the strain c c ), demonstrating for the first time the possibility to express the sars-cov m protein in plants, without the need of codon optimization or addition of any further modifications. on the contrary, by pvx-mediated infection we observed very low expression levels for the m protein, only in plants out of analyzed. we can speculate that membrane m protein, that already proved to be toxic in e. coli, may be difficult to express by using a 'living vector' like pvx due to interference with virus replication and/or expression/assembly of viral components, while it is tolerated by the plant when expressed by agro-infiltration. compared to the mutated m rlv protein produced in e. coli, the m protein produced in plant shows a reduced electrophoretic mobility, suggesting a higher molecular mass ( figure c) . the reason for this difference remains to be elucidated, but it could be due to modified residues in the m rlv protein or by the presence of glycosylation in the m protein produced in the eukaryotic system (the native protein is n-glycosylated at the fourth residue). the plant-produced m protein yields were not sufficient to perform the test with human sera. thus, for plant-derived m protein characterization, efforts should be made in order to enhance expression yields. this includes the use of secondgeneration viral vectors (gleba et al., ) or other implemented platforms that have been developed in the last years (moustafa et al., ; paul et al., ; sack et al., ) . our results add further insights to the characterization of the n and m proteins and provide a proof of principle for using plants as a robust, rapid and flexible production system for protein reagents suitable to face potential recurring sars-cov outbreaks. rf, pdb concept and design of the research, analysis and interpretation of data, writing and revising the article, final approval of the version to be published. ocd, sm design of the research, acquisition of data, analysis and interpretation of data, drafting and writing the article. ei acquisition of data, analysis and interpretation of data. ddm writing and revising the article critically for important intellectual content. pksc design and revising the article critically for important intellectual content and final approval of the version to be published. the work was partially supported by the italian 'ministero della salute, ' grant 'progetto speciale lotta alla sars.' an update on middle east respiratory syndrome: years later stability of potato virus x expression vectors is related to insert size: implications for replication models and risk assessment who pillories drug industry on failure to develop ebola vaccine structural proteomics of the sars coronavirus: a model response to emerging infectious diseases jellyfish green fluorescent protein as a reporter for virus infections recombinant protein-based elisa and immuno-cytochemical assay for the diagnosis of sars department of health, and human services (hhs): possession, use, and transfer of. select agents and toxins; biennial review. final rule tracing the sars-coronavirus evaluation of a recombinant nucleocapsid protein-based assay for anti-sars-cov igg detection nucleocapsid protein as early diagnostic marker for sars detection of toscana virus-specific immunoglobulins g and m by an enzymelinked immunosorbent assay based on recombinant viral nucleoprotein copious production of sars-cov nucleocapsid protein employing codon optimized synthetic gene human antibody response to toscana virus glycoproteins expressed by recombinant baculovirus identification of a novel coronavirus in patients with severe acute respiratory syndrome plant-derived vaccines and other therapeutics produced in contained systems development of an enzyme-linked immunosorbent assay-based test with a cocktail of nucleocapsid and spike proteins for detection of severe acute respiratory syndrome-associated coronavirus-specific antibody viral vectors for the expression of proteins in plants a decade after sars: strategies for controlling emerging coronaviruses recombinant protein-based assays for detection of antibodies to severe acute respiratory syndrome coronavirus spike and nucleocapsid proteins identification of immunodominant epitopes on the membrane protein of the severe acute respiratory syndrome-associated coronavirus efficacy and effectiveness of an rvsvvectored vaccine expressing ebola surface glycoprotein: interim results from the guinea ring vaccination cluster-randomised trial ebola vaccines: keep the clinical trial protocols on the shelf and ready to roll out coronavirus structural proteins and virus assembly detection of severe acute respiratory syndrome (sars) coronavirus nucleocapsid protein in human serum using a localized surface plasmon coupled fluorescence fiber-optic biosensor label-free, electrical detection of the sars virus n-protein with nanowire biosensors utilizing antibody mimics as capture probes an agrobacterium-mediated transient gene expression system for intact leaves mers research outpaced by outbreaks multistrip western blotting to increase quantitative data output transient expression systems for plant-derived biopharmaceuticals the inaccurate and unfair who attack on pharma ebola efforts thermal aggregation of sars-cov membrane protein extremely low exposure of a community to severe acute respiratory syndrome coronavirus: false seropositivity due to use of bacterially derived antigens the membrane protein of severe acute respiratory syndrome coronavirus acts as a dominant immunogen revealed by a clustering region of novel functionally and structurally defined cytotoxic t-lymphocyte epitopes immune responses against severe acute respiratory syndrome coronavirus induced by virus-like particles in mice sars coronavirus:unusual lability of the nucleocapsid protein the genome sequence of the sars-associated coronavirus molecular farming on rescue of pharma industry for next generations a structural analysis of m protein in coronavirus assembly and morphology protective humoral responses to severe acute respiratory syndrome-associated coronavirus: implications for the design of an effective protein-based vaccine a selfassembled fusion protein-based surface plasmon resonance biosensor for rapid diagnosis of severe acute respiratory syndrome commercialization of new biotechnology: a systematic review of commercial case studies in a novel manufacturing sector severe acute respiratory syndrome severe acute respiratory syndrome (sars) s protein production in plants: development of recombinant vaccine sars vaccines: where are we? characterization of a novel coronavirus associated with severe acute respiratory syndrome plant-based vaccines against viruses the increasing value of plant-made proteins antigenic characterization of severe acute respiratory syndromecoronavirus nucleocapsid protein expressed in insect cells: the effect of phosphorylation on immunoreactivity and specificity suppression of innate antiviral response by severe acute respiratory syndrome coronavirus m protein is mediated through the first transmembrane domain plant-produced candidate countermeasures against emerging and reemerging infections and bioterror agents the epidemiology, evolution and recent outbreaks of avian influenza viruses in china: a review molecular targets for diagnostics and therapeutics of severe acute respiratory syndrome the sars-cov nucleocapsid protein: a protein with multifarious activities identifying sars-cov membrane protein amino acid residues linked to viruslike particle assembly self-assembly of severe acute respiratory syndrome coronavirus membrane protein longitudinal profile of immunoglobulin g (igg), igm, and iga antibodies against the severe acute respiratory syndrome (sars) coronavirus nucleocapsid protein in patients with pneumonia due to the sars coronavirus severe acute respiratory syndrome (sars): laboratory diagnostic tests library cataloguing in publication data persistent memory cd + and cd + t-cell responses in recovered severe acute respiratory syndrome (sars) patients to sars coronavirus m antigen naturally occurring anti-escherichia coli protein antibodies in the sera of healthy humans cause analytical interference in a recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay for serodiagnosis of severe acute respiratory syndrome boosted expression of the sars-cov nucleocapsid protein in tobacco and its immunogenicity in mice we thank dr. alessandra carattoli from 'istituto superiore di sanità' for coordinating the sars project. we also thank orsola bitti for technical assistance. the supplementary material for this article can be found online at: http://journal.frontiersin.org/article/ . /fpls. . key: cord- -r jbm et authors: lagarda-diaz, irlanda; guzman-partida, ana maria; vazquez-moreno, luz title: legume lectins: proteins with diverse applications date: - - journal: int j mol sci doi: . /ijms sha: doc_id: cord_uid: r jbm et lectins are a diverse class of proteins distributed extensively in nature. among these proteins; legume lectins display a variety of interesting features including antimicrobial; insecticidal and antitumor activities. because lectins recognize and bind to specific glycoconjugates present on the surface of cells and intracellular structures; they can serve as potential target molecules for developing practical applications in the fields of food; agriculture; health and pharmaceutical research. this review presents the current knowledge of the main structural characteristics of legume lectins and the relationship of structure to the exhibited specificities; provides an overview of their particular antimicrobial; insecticidal and antitumor biological activities and describes possible applications based on the pattern of recognized glyco-targets. currently, lectins are defined as carbohydrate binding proteins of non-immune origin that can recognize and bind simple or complex carbohydrates in a reversible and highly specific manner. for a long time, these proteins were named hemagglutinins, due to their ability to agglutinate red blood cells. at present, this concept is used when the specificity is unknown. it is also known that monomeric lectins do not exhibit this agglutinating activity [ ] . lectins are ubiquitously present in fungi, animals, bacteria, viruses and plants. among plant lectins, those of legumes have been the most widely considered [ ] . these lectins are abundant in the seeds and belong to a group of highly homologous proteins. however, their carbohydrate specificities and quaternary structures vary extensively [ ] , which greatly influences the type of recognized targets. throughout the history of lectin research, legume seeds have been screened for lectin activity. the existence of binding sites for specific carbohydrates, the main characteristic of lectins, is undoubtedly an important factor for determining their activity and future applications based on their properties. this review centers on the lectins of legumes, highlighting their specificity, particularities, main reported activities and potential applications. legume lectins represent the largest family of carbohydrate binding proteins, and their physicochemical and biological properties have been broadly studied [ ] [ ] [ ] [ ] [ ] . research of the plant lectin family using newer biochemical and biophysical strategies has significantly moved the field forward, and provided a model framework for studying protein-carbohydrate interactions. although plant lectins share high sequence homology and exhibit comparable physicochemical and structural properties, the diversity of their carbohydrate recognition specificity is remarkable. generally, the three-dimensional structure of plant lectins is characterized by β-sheets that are connected by α turns, β turns and bends. in addition to short loops, the quaternary interfaces are formed between β-sheets. the β-sheets are connected by loops forming antiparallel chains usually devoid of α helices ( figure ) [ , , [ ] [ ] [ ] [ ] . analysis of legume lectin monomers reveals high similarity in sequence as well as structure, with only minor variations in the length of loops and strands. the monomer structures display a jellyroll motif best described as a sandwich of - kda containing a carbohydrate recognition domain (crd) and metal binding sites for divalent cations (ca + and mn + ). however, in spite of strong similarities in primary, secondary and tertiary structures, the quaternary structures show considerable variations that lead to changes in the monomer-monomer interactions and the presence/absence of protein modifications, such as glycosylation. the impact of these variations has functional implications as they dictate the specificity of multivalent glycan binding [ , , , [ ] [ ] [ ] [ ] [ ] [ ] . most legume lectins appear to assemble as homodimers or homo-tetramers (dimers of dimers), the stability of which is attributed to hydrophobic cooperation, hydrogen bonds and salt links [ , , ] . legume lectins are subdivided into two categories, those with indistinguishable or almost indistinguishable subunits, and those with a variety of subunits [ ] . phaseolus vulgaris lectins (pha) are well-studied lectins of the first group [ ] [ ] [ ] [ ] [ ] [ ] . there are more than forty structures of legume lectins reported in either the apo (ligand-free) or holo (sugar-bound) form in the protein data bank [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, data about lectins from wild legumes is rare. mexico has roughly species of wild legumes confined to various regions, and more than percent are within the sonoran desert [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the isolation and purification of lectins from seeds of native plants such as parkinsonia aculeata, olneya tesota, acacia constricta, prosopis juliflora, cercidium praecox, caesalpinia caladenia and phaseolus acutifolius has been described. lectins from these plants have monomers of either or kda that also can form tetramers or dimers. in addition, it has been shown that these lectins involve a group of different isolectins, compared to those found in lectins of the phaseolus genus [ ] [ ] [ ] [ ] [ ] . on the other hand, the primary sequences of plant lectins have been used to infer evolutionary relationships, to reflect processing routes, to suggest folding patterns as well as to provide clues on how carbohydrate ligands fit into their binding pockets [ ] . direct amino acid sequencing has revealed homology among the lectins from the wild desert legumes a. constricta [ ] and o. tesota [ , ] , and more domesticated species like p. vulgaris and p. coccineus [ , ] . furthermore, antibodies raised against pha show reactivity with lectins from a. constricta, c. praecox and c. caladenia [ ] . from these studies, it is clear that lectins have been conserved during the evolution of legumes and that the extensive homologies reflect taxonomical relationships among these plants [ ] . legume lectins represent a crucial point in the study of the molecular basis of protein-carbohydrate interactions. each has a crd with a basic architecture accessible to both monosaccharides and/or oligosaccharides that confers remarkable divergence in their specificities [ , ] . lectins can non-covalently interact with carbohydrates in a manner that is usually reversible and highly specific. recognition can occur in the terminal or intermediate position of the glycan structure [ ] . in all legume lectins, four invariant amino acid residues participate in the binding of the carbohydrate, yet each lectin shows different specificity [ , , ] . the crd pocket is formed by four loops adjacent to each other, but the loops are not close in sequence. these regions exhibit the highest residue variability and appear to be involved in specificity determination [ , ] . the crd of legume lectins lies in close proximity to the metal binding sites, which may aid in binding activity [ ] , but are not often directly involved in carbohydrate binding [ ] . most lectins bind to mono-and oligosaccharides; however, more recently discovered lectins show specificity for complex sugars and glycoproteins. generally, lectins show specificity for di-, tri-and tetra-saccharides with association constants significantly higher than those for the corresponding monosaccharides [ , , ] . some lectins have shown affinity towards carbohydrate structures not present in plants, such as the thomsen-nouveau antigen or complex n-glycan structures with terminal galactose and sialic acid residues [ ] . the specificity of legume lectins for some typical animal glycans, has led to the suggestion that legume lectins play a role in plant defense against insects and/or predator animals [ , ] . the legume lectins that have been purified and characterized as members of the complex type include, griffonia simplicifolia iv lectin (gs iv) [ , ] , maackia amurensis lectin (mah and mal- ) [ ] , cicer arietinum lectin [ ] , saraca indica lectin [ , ] and p. vulgaris e and l lectins (pha-e and pha-l) [ , , , [ ] [ ] [ ] [ ] [ ] . wild legume lectins from the sonoran desert also have shown varying degrees of specificity for some complex carbohydrates of glycoproteins. the hemagglutinating activity of a. constricta isolectins (vls) and the p. juliflora lectin is strongly inhibited in the presence of asialofetuin and to a lesser extent with fetuin and thyroglobulin [ , ] . c. praecox and c. caladenia lectins are inhibited by fetuin and mucine [ ] . o. tesota pf and pf lectins are inhibited by free monosaccharides and/or by complex carbohydrates of fetuin, thyroglobulin, immunoglobulin, mucine and ovalbumin, whereas pf is only inhibited by the intact glycoprotein [ ] . although glycan structures present in glycoproteins are known to have n and o linked oligosaccharides, desialylation of the glycan can result in an increase or decrease in lectin binding affinity [ ] . the galactose residues found in glycosylated proteins are often capped with sialic acid, a characteristic that may alter the interaction with lectins, as is seen for amaranthus caudatus and abrus pulchellus [ , ] . many studies on lectin-carbohydrate specificity have been published. unfortunately, often specificity analyses are used to "specifically" determine structural motifs in glycoconjugates without taking into account the rather complex data on this subject. furthermore, the choice of ligands used to assay lectin binding in some studies is limited either by natural or commercially available sources of glycans, or to simple mono-or disaccharides. therefore, a critical re-evaluation of lectin binding specificities is required. such studies using microarray techniques with immobilized glycans or immobilized lectins, dot blot assays to screen different lectins and antibodies with multiple oligosaccharides, combined with information gleaned using recombinant glycosyltransferases, are opening up new possibilities of generating a broader range of standard (neo)glycoconjugates with clearly defined structures [ ] . antimicrobials have been used to treat diseases caused by bacteria and fungi and such treatments have significantly reduced the mortality rate from these infections in humans and animals. however, the extensive use of antimicrobial substances in medical, agricultural, and veterinary practices is a topic of great concern to clinical microbiologists all over the world due to the growing emergence of opportunistic microorganism strains resistant to drugs that cause serious infections [ ] [ ] [ ] [ ] . microbial resistance is a genetic phenomenon resulting in a reduction in effectiveness of drug therapy; it may be caused by mutations during the reproductive process of the microorganisms or by imported genes acquired through transduction, conjugation, and transformation mechanisms [ ] . therefore, there is a growing interest in developing new strategies for inhibiting the growth or survival of microorganisms based on the screening of new sources of natural compounds as alternatives to commercially available drugs [ , ] . a large number of proteins with antibacterial, antifungal and/or antiviral activity have been isolated from the seeds, tubers, and rhizomes of different plants, where they accumulate to high levels and may function as a reserve source of amino acids and metal ions [ ] . these proteins may directly interfere with the growth, multiplication and spread of microbial agents by different mechanisms [ ] , as in the case of lectins, by agglutination and/or microorganism immobilization. plant lectins often are present at potential sites of microbial invasion and their binding to fungal structures led to the inhibition of fungal growth and germination. studies carried out with soy bean and common lentil agglutinins provide evidence for these roles [ , [ ] [ ] [ ] . a main characteristic of lectins is their ability to interact with glyco-components present on the cell membrane surface, in cytoplasmic and nuclear structures and in the extracellular matrix of cells and tissues from almost all kingdoms of life [ ] . therefore, plant lectins are thought to play important roles in the plant immune defense and emerge as potential antimicrobial candidates for drug therapies. the antimicrobial effect of legume lectins is shown in table . although many lectins show antimicrobial activity, clinical trials are needed to establish therapeutic efficacy, to optimize dosage, delivery and bioavailability, and to assess potential allergic reactions [ ] . the antibacterial activity of lectins results from their interaction with a wide variety of complex carbohydrates of the bacterial cell wall, such as teichoic and teichuronic acids, peptidoglycans and lipopolysaccharides [ , ] . lectins of triticum vulgare, dolichos lablab l., trigonella foenumgraecum, trifolium alexandrium l., bauhinia variegata l. and delonix regia have shown antimicrobial activity against mycobacterium rhodochrous, bacillus cereus, b. megaterium, b. sphaericus, escherichia coli, serratia marcescens, corynebacterium xerosis and staphylococcus aureus [ ] lectins from the vicieae tribe strongly react with the bacterial cell wall components, muramic acid, n-acetylmuramic acid and muramyl dipeptides [ ] . nevertheless, although legume seed lectins can recognize and bind to the constituents of the bacterial cell wall, such binding does not imply that these interactions occur in vivo, and certainly does not prove that these lectins are involved in the protection of seedlings against bacteria [ ] . it is thought that plant lectins do not alter the membrane structure and/or permeability nor disturb the normal intracellular processes of invading microbes, since they exert an indirect effect that is based on interactions with cell wall carbohydrates or extracellular glycans [ ] . however, electron microscopy studies revealed that treatment with araucaria angustifolia lectin promoted morphologic alterations, including the presence of pores in the membrane of gram-positive bacteria, and bubbling on the cell wall of gram-negative bacteria [ ] . others reported that the antibacterial activity of lectins occurred through the interactions of the lectins with n-acetylglucosamine, n-acetylmuramic acid and tetrapeptides linked to n-acetylmuramic acid present in the cell wall of gram-positive bacteria, or to lipopolysaccharides present in the cell wall of gram-negative bacteria [ ] . bind to the glycosylated envelope protein and block cellular entry (interfere with replication cycle) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] in addition to these findings, lectins from the legumes from canavalia ensiformis, t. foenumgraecum, arachis hypogaea, cajanus cajan, p. vulgaris and pisum sativum were shown to inhibit biofilm formation by streptococcus mutans, but the growth of planktonic cells was not affected [ ] . since bacterial biofilms make disinfection procedures more difficult by increasing bacterial resistance to detergents and antibiotics, the inhibition of biofilm formation and development by legume lectins could be a useful characteristic of these proteins. furthermore, the rapid evolution of bacteria with antibiotic resistance (in particular, multidrug resistant bacteria or superbugs) has reduced the efficacy of conventional treatments against their biofilms. hence, development of non-antibiotic alternatives that could efficiently treat or reduce biofilms should become a priority. furthermore, research focused on finding lectins that reduce superbugs could represent an interesting alternative drug therapy that deserves further attention. regardless of the considerable number of lectins that have been characterized, only a small number have shown antifungal effects. because plant lectins do not penetrate the cell wall or the membrane to reach the cytoplasm of fungi, direct inhibition of fungal growth by lectins is unlikely. in spite of this, indirect responses produced by the attachment of lectins to chitin and other glycans on the fungal surface could affect fungal survival or other activities [ , ] . lectin binding to hyphae could result in inhibition of fungi growth as a result of poor nutrient absorption as well as by interference with the spore germination process [ ] . lectins can cause different morphological changes that render fungi more vulnerable to differing stress conditions [ ] . for example, in one study, lectin interactions resulted in swollen hyphae, vacuolization of the cell content and improved lysis of hyphal cell wall, that, in turn, increased susceptibility of fungi to osmotic shock [ ] . in another case, it was suggested that small antifungal lectins could penetrate the fungal cell wall and reach the cell membrane where blocking of active sites of enzymes could alter cell wall morphogenesis [ , ] . some reports indicate that lectins from the legumes astragalus mongholicus, p. coccineus, archidendron jiringa nielsen, b. ungulata, glycine max, indigofera heterantha and a. hypogaea can exhibit antifungal activity against various species of phytopathogenic fungi, such as botrytis cinerea, fusarium oxysporum, f. moniliforme, f. solani, colletorichum sp., drechslera turcia, and exserohilum turcicum, as well as pathogenic fungi such as candida albicans, penicillium italicummm, and aspergillus sp. [ , [ ] [ ] [ ] , ] . additional studies are required to elucidate the molecular basis for the antifungal activity of individual lectins as the ability of lectins to inhibit growth differs among fungal species. plant lectins with antiviral activity are of substantial therapeutic interest. some of these carbohydrate binding proteins exhibit significant activity against human immunodeficiency virus (hiv) and other viruses [ ] . retroviruses, such as hiv, have a surface covered by highly glycosylated virally-encoded glycoproteins, e.g., gp (that contains high mannose and/or hybrid glycans) and gp . the carbohydrates on these proteins are particularly important because the glycans can be used as tools to render the virus easily recognized by the immune system, and thus susceptible to immunological neutralization [ ] . the interactions between host and proteins from the viral envelop also can be altered by compounds that specifically recognize and bind glycans. furthermore, lectins can crosslink surface viral glycans and thereby prevent interactions with other co-receptors [ ] . antiviral lectins used as therapeutic agents offer lower toxicity and can be included in topical applications. generally, lectins are odorless, and resistant to high temperatures and low ph, ideal properties for development of microbicide drugs [ ] . most current antiviral therapeutics prevent the viral life cycle, or inhibit the entrance of the virus into host cells [ ] . the antiviral action of plant lectins varies extensively depending upon their carbohydrate specificity. corona virus are highly susceptible to mannose specific lectins that interfere with the viral attachment in early phases of the replication cycle and suppress viral development by binding towards the end of the viral infection cycle [ ] . other antiviral non-legume lectins with encouraging properties include griffithsin (grft), cyanovirin (cv-n) and banana lectin (banlec), and banlec has been recommended as an antiviral microbicide. most often antiviral lectins are recommended for incorporation into vaginal and rectal gels, creams or suppositories that act as a barrier to prevent hiv transmission. in such processes, lectins bind the virus preventing its entry and fusion to target cells, consequently averting contamination [ ] . several legume lectins possess antiviral activity. lectins like c. ensiformis agglutinin (con a), lens culinaris agglutinin (lca), vicia faba agglutinin, p. sativum agglutinin (psa) and pha-e bind to the envelope glycoprotein gp and to inhibit fusion of hiv-infected cells with cd cells by a carbohydrate-specific interaction with the hiv-infected cells [ ] . in addition, g. max agglutinins inhibit hiv- reverse transcriptase activity [ ] . although glycan structures of viral proteins involve high mannose, further research in this area is needed to explore lectins that recognize structures with different sugars such as sialic acid, fucose and n-acetyl glucosamine with the aim of developing novel approaches and therapies in the field of virus biology. legume lectins are toxic to a broad spectrum of insects representing several orders including, coleoptera, diptera, lepidoptera, hymenoptera, isoptera, neuroptera and homoptera. in this regard, the family of leguminous lectins is the most studied due to its insecticidal potential. a great number of legume lectins have shown insecticidal activity including those from c. ensiformis, p. sativum, p. vulgaris, glechoma hederacea, g. simplicifolia, o. tesota (pf ) and b. monandra [ , , ] . these lectins showed harmful effects in the different developmental stages of insects (larvae and adults). in this regard, they may increase mortality, delay development and/or adult emergence and reduce fecundity. usually, the effect of lectins on insects is evaluated by feeding larvae with artificial seeds containing the lectin or with transgenic plants that overexpress the lectin of interest. the expression of a given lectin in transgenic plants is only possible if the lectin molecular sequence is well characterized and also requires an available transformation protocol for the desired plant species [ ] . in the last twenty years, research has focused on elucidating the mode of action of insecticidal lectins. in general, lectins can exert a toxic effect via binding to the peritrophic membrane (pm), peritrophic gel (pg) or the brush-border microvilli of epithelial cells (figure ) [ ] . the pm or pg is a film surrounding the food bolus in most insects and is composed of chitin and proteins. some non-legume lectins with insecticidal activity such as the rhizoctonia solani agglutinin and sambucus nigra agglutinin ii can pass through the pm of the red flour beetle, tribolium castaneum. this ability to reach the endoperitrophic space is governed by the dimensions of the molecule and the charge and size of the pm pores [ ] . in the case of insects lacking a pm, the insecticidal effect of lectins may be exerted by their direct interaction with glycoconjugates present on the cell epithelium [ ] . in fact, the insecticidal activity of lectins relies on their binding properties to sugars present on the surface of the insect gut epithelial cells. in insects, the surface of epithelial cells is rich in glycoproteins that, in addition to their determinant roles for the proper gut function, provide a number of available targets for lectin binding. the interaction of lectins with different glycoproteins or glycan structures in insects may interfere with important physiological processes ( table ) . although the key receptors for lectins could be localized on the surface of gut epithelial cells, if lectins are internalized, they may interact with a new set of targets located in the intracellular space, allowing the lectin to interfere with particular metabolic pathways. such internalization has been more studied for non-legume lectins. for example, the garlic leaf lectin, which affects survival and development of the moth helicoverpa armigera, was found to be internalized by binding to a glycosylated alkaline phosphatase anchored to the insect midgut membrane. in addition, galantus nivalis lectin was reported to cross the midgut epithelium of nilaparvata lugens and co-localize in the fat body and hemolymph. such co-localization was proposed to occur via a carrier-receptor, ferritin. the internalization of the colocasia esculenta tuber agglutinin into insect hemolymph also was demonstrated, suggesting it could interact with various n-glycosylated intracellular targets, which may, in part, explain the lectin toxicity [ ] . although the complete mechanism by which some lectins are able to cross midgut epithelial cells is not yet fully understood, available evidence has highlighted the implication of clathrin-mediated endocytosis as part of this process [ , ] . regardless of the mechanisms involved in lectin interactions with insect cells, in order to be toxic, a lectin must avoid degradation by the digestive enzymes from the insect gut. the insecticidal effect of some lectins is attributed to the resistance of these proteins to proteolysis and to their properties of stability in a wide ph range. for example, pf , an insecticidal lectin to zabrotes subfasciatus, is resistant to in vitro proteolysis by insect digestive enzymes. this is in contrast to pha-e lectin that is digested after h of incubation with these enzymes, and therefore is not toxic for this insect [ ] . other lectins with insecticidal activity that share the common feature of resistance to proteolysis by different digestive enzymes include, g. simplicifolia ii lectin (gs ii), ulex europeus agglutinin (uea) and b. monandra lectin [ , ] . the degree of resistance to digestive enzymes depends on the capacity of the lectin to bind to glycoconjugates of the insect gut. gs ii mutant lectins lacking the ability to bind carbohydrates displayed sensitivity to proteolysis by digestive enzymes with a consequent loss of toxicity [ ] . helicoverpa armigera [ ] alanyl aminopeptidase n sucrase acyrthosiphon pisum [ ] in summary, the insecticidal effect exhibited by several plant lectins is a complex phenomenon that relies on the capacity of the lectin to interfere with critical physiological processes during insect development. such interference often may be mediated by a lack of nutrient absorption that occurs when midgut epithelial cells are atrophied as a result of the interaction of lectins with glyco-targets located either on the surface or inside the cell. studies of the recognition of plant lectins by insect gut glyco-targets can allow one to develop hypotheses on which cellular pathways are affected by the insecticidal activity. these pathways vary depending on the type of insect and the lectin, and could be as diverse as the type of recognized glycan or receptors, and might include the effectors of energy metabolism, and redox and ionic homeostasis (summarized in table ). finally, the study of potential non-yet-identified insecticidal plant lectins may contribute to the development of distinctive tools for sustainable pest control. however, in this regard, care must be taken considering the effect lectins might exert on beneficial insects as well. recently, an increased interest in the potential of plant lectins as cancer therapeutic agents has become evident. lectins can be used to study the metastatic distribution patterns, the expression profiles of cancer cells glycoconjugates and their biological effects in various tumors. it is well known that carbohydrates present on the cell membrane are important for cell recognition, communication and adhesion. in cancer, these interactions are essential for tumor progression and metastasis. in tumor cells, glycosylation is generally altered in comparison with normal cells and these alterations can be detected by lectins [ , ] . litynska et al. compared the lectin-binding pattern in different human melanoma cell lines using the pha-l lectin and other non-legume lectins, including the galanthus nivalis lectin, s. nigra lectin, mal, datura stramonium agglutinin and wheat germ agglutinin (wga). studies demonstrated that acquisition of metastatic potential of tumor cells is correlated with the expression of branched and sialylated complex n-oligosaccharides [ ] . interestingly, the binding of mal-i to gastric cancer cells was proportional to their metastatic capacity, and a high expression of α ( , )-linked sialic acids was closely correlated with lymph node metastasis [ ] . korourian et al. used g. simplicifolia lectin-i (gs i) and vicia vilosa agglutinin (vva) to establish an expression analysis of carbohydrate antigens in human breast ductal carcinoma in situ (dcis). both lectins showed a significant association with nuclear grade (cell size and uniformity) of dcis [ ] . positive correlations also were found between the expression of vva-and gsi-reactive structures and tumor grade in dcis patients, suggesting that the glycoconjugates found in these antigens are associated with invasiveness in dcis [ ] . pha can be used to discriminate between hepatocellular carcinoma and benign liver disease [ ] . this suggests that lectins could potentially be used to develop prognostic tools for cancer diagnosis and the detection of metastasis, and to some extent to estimate the severity of the clinical case. in addition to these uses, plant lectins have shown in vivo and in vitro antitumor activity. lectins with the potential to induce inhibition of tumor cell growth, such as, con a, mistletoe type-i lectins and pha are currently in different phases of clinical trials [ , ] . plant lectins can modify the expression of interleukins and some protein kinases and in this way modulate the immune system. furthermore, in cancers, lectins could alter the signaling pathways involved in the expression of members of the bcl- , autophagy (atg) related, and caspase families, as well as p , erk, ras-raf, and bnip , and thereby induce both apoptosis and autophagy [ ] . soybean lectin produces reactive oxygen species in a dose-dependent manner in hela cells inducing apoptosis, autophagy and dna damage in the cells [ ] . the cell surface carbohydrates are key targets for lectins; in cancer, a general pathway involves recognition of carbohydrate receptors triggering the activation of enzymes such as mbl-associated serine proteases [ ] . however, the binding of lectins to glycans on the cell membrane may not be sufficient to induce apoptosis. kim et al. showed that cell death requires lectin endocytosis that triggers signaling for apoptosis [ ] . con a is cytotoxic to hepatoma cell lines. its toxicity is mediated primarily by its binding to the mannose moiety of cell membrane glycoproteins, with subsequent internalization and accumulation in the mitochondria. autophagy is then activated, leading to lysosomal degradation of the affected mitochondria and, ultimately, cell death [ , ] . overall, these findings of cancer research have revealed the mechanisms responsible for the antitumor action of different plant lectins. these mechanisms are varied and depend upon different factors such as the tumor cell origin and type as well as lectin concentration. the screening and characterization of novel lectins from legume resources has allowed the discovery of proteins with a great capacity to distinguish specific glycans from a diversity of glyco-targets found in a variety of organisms. legume lectins could be potential candidates for developing tools based on protein-carbohydrate interactions with variable specificities. lectin-mediated drugs focused on targeting specific cells could lead to promising anticancer and antimicrobial treatments, which would directly impact areas of economic importance, such as the pharmaceutical and food industries and agriculture. additionally, the use of lectins against insect pests could provide a form of sustainable pest control. more research is needed to explore and support the therapeutic effect of lectins. studies of action mechanisms, the relationship between the role of the lectins and their molecular features, and finally, their effect in modulating the expression of proteins and genes are required to move forward the use of lectins for clinical applications. domesticated legumes provide an accessible and abundant source of lectins, unlike wild legumes, which, in some regions, may even be considered protected species. in some cases, successful recombinant production of lectins would be a key factor in whether a specific lectin could find use in a feasible industrial application. the overexpression of recombinant lectins remains a great challenge, even more so for those lectins where the lack of adequate post-translational modifications could compromise their activity. the authors thank joy winzerling for her critical reading and editing of the manuscript. author contributions: all authors compiled the information, wrote the review, read and approved the final manuscript. the authors declare no conflict of interest. history of lectins: from hemagglutinins to biological recognition molecules proteins with diverse applications legume lectins a large family of homologous proteins purification of complex carbohydrate specific lectins from olneya tesota seeds using tandem affinity chromatography purification and characterization of complex carbohydrate specific isolectins from wild legume seeds: acacia constricta is (vinorama) highly homologous to phaseolus vulgaris lectins proteomic approaches to study structure, functions and toxicity of legume seeds lectins. perspectives for the assessment of food quality and safety insecticidal action of pf lectin from olneya tesota (palo fierro) against zabrotes subfasciatus larvae and midgut glycoconjugate binding analyses of carbohydrate recognition by legume lectins: size of the combining site loops and their primary specificity carbohydrate binding, quaternary structure and a novel hydrophobic binding site in two legume lectin oligomers from dolichos biflorus how proteins bind carbohydrates: lessons from legume lectins structural features of the legume lectins novel structures of plant lectins and their complexes with carbohydrates weak protein-protein interactions in lectins: the crystal structure of a vegetative lectin from the legume dolichos biflorus determinants of quaternary association in legume lectins l-type lectins lectins: tools for the molecular understanding of the glycocode signature of quaternary structure in the sequences of legume lectins plant lectins: occurrence, biochemistry, functions and applications purification of the phytohemagglutinin family of proteins from red kidney beans (phaseolus vulgaris) by affinity chromatography comparison of phaseolus vulgaris cultivars on the basis of isolectin differences characterization of the structural determinants required for the high affinity interaction of asparagine-linked oligosaccharides with immobilized phaseolus vulgaris leukoagglutinating and erythroagglutinating lectins a one-step procedure for isolation and resolution of the phaseolus vulgaris isolectins by affinity-chromatography the crystallographic structure of phytohemagglutinin-l phytohemagglutinin from phaseolus vulgaris (pha-e) displays a novel glycan recognition mode using a common legume lectin fold a portal for structural glycosciences. glycoinformatics crystallographic structure of metal-free concanavalin a at . angstrom resolution the monosaccharide binding-site of lentil lectin: an x-ray and molecular modeling study the structure of the pea lectin-d-mannopyranose complex at a . angstrom resolution the crystal structure of a plant lectin in complex with the tn antigen molecular modeling of native and mutated lima bean lectin: dissection of lectin/blood group a trisaccharide interactions agroecosystem diversity: a model from the sonoran desert mexican leguminosae: phytogeography, endemism, and origins estudio de algunas semillas de leguminosas del desierto de sonora. factores antinutricionales y calidad de sus proteínas y aceites aislamiento y caracterización de las lectinas de leguminosas silvestres del desierto de sonora: cercidium praecox (palo de brea) y caesalpinia caladenia (palo dorado) caracterización de los oligosacáridos de las lectinas de olneya tesota pf y su isoforma más abundante (if ) y establecer la relación con la función de reconocimiento isoformas de la lectina pf , características moleculares y reconocimiento de leucocitos de sangre periférica de adultos purificación y caracterización de la lectina de prosopis juliflora y evaluación del desarrollo larval de zabrotes subfasciatus en las semillas purification, biochemical characterization, and bioactive properties of a lectin purified from the seeds of white tepary bean (phaseolus acutifolius variety latifolius) identificación de la interacción de monocitos humanos con las lectinas de olneya tesota (if ) y phaseolus vulgaris (pha-l) por citometría de flujo phytohemagglutinin mitogenic proteins. structural evidence for a family of isomitogenic proteins biological and biochemical properties of phaseolus vulgaris isolectins subunit heterogeneity in the lima bean lectin lectin and lectin-related proteins in lima bean (phaseolus lunatus l.) seeds: biochemical and evolutionary studies lectin from phaseolus acutifolius var. escumite: chemical characterization, sugar specificity, and effect on human t-lymphocytes analysis of olneya tesota lectin peptides with liquid chromatography in two dimensions and tandem mass spectrometry (lc-ms/ms) characterization of phaseolus-vugaris phytohemagglutinin genes closely linked on the chromosome lectin-related resistance factors against bruchids evolved through a number of duplication events antimicrobial activity of lectins from antimicrobial activity of lectins from plants pattern recognition in legume lectins to extrapolate amino acid variability to sugar specificity las lectinas vegetales como modelo de estudio de las interacciones proteína-carbohidrato lectin-carbohydrate complexes of plants and animals: an atomic view molecular modeling of the dolichos-biflorus seed lectin and its specific interactions with carbohydrates: α-dn-acetyl-galactosamine, forssman disaccharide and blood-group-a trisaccharide analysis of sequence variation among legume lectins: a ring of hypervariable residues forms the perimeter of the carbohydrate-binding site a lectin from platypodium elegans with unusual specificity and affinity for asymmetric complex n-glycans comparison of the nature of interactions of two sialic acid specific lectins saraca indica and sambucus nigra with n-acetylneuraminic acid by spectroscopic techniques lectins, lectin genes, and their role in plant defense lectins as plant defense proteins structures of the lectin-iv of griffonia simplicifolia and its complex with the lewis b human blood-group determinant at . -angstrom resolution an unusual carbohydrate binding site revealed by the structures of two maackia amurensis lectins complexed with sialic acid-containing oligosaccharides properties of a lectin purified from the seeds of cicer-arietinum. hope-seyler's saracin a lectin from saraca-indica seed integument recognizes complex carbohydrates structure of a complex-type sugar chain of human glycophorin a mitogenic leukoagglutinin from phaseolus-vulgaris binds to a pentasaccharide unit in n-acetyllactosamine-type glycoprotein glycans structural determinants of phaseolus-vulgaris erythroagglutinating lectin for oligosaccharides purification and characterization of novel lectins from great northern bean, phaseolus vulgaris l. bba-gen the high specificities of phaseolus vulgaris erythro-and leukoagglutinating lectins for bisecting glcnac or beta - -linked branch structures, respectively, are attributable to loop b presence of beta-linked galnac residues on n-glycans of human thyroglobulin isolation and characterization of amaranthin, a lectin present in the seeds of amaranthus-caudatus, that recognizes the t-antigen (or cryptic-t)-antigen characterization of the sugar-binding specificity of the toxic lectins isolated from abrus pulchellus seeds specificity analysis of lectins and antibodies using remodeled glycoproteins antimicrobial lectin from schinus terebinthifolius leaf fluconazole-resistant candida albicans vulvovaginitis screening of aqueous extracts of medicinal herbs for antimicrobial activity against oral bacteria antimicrobial property of extracts of indian lichen against human pathogenic bacteria antimicrobial activity of cladonia verticillaris lichen preparations on bacteria and fungi of medical importance antimicrobial peptides from plants antimicrobial peptides in mammalian and insect host defence the structural basis for carbohydrate recognition by lectins antifungal and antibacterial activities of lectin from the seeds of archidendron jiringa nielsen medicinal applications of plant lectins purification of a lectin from eugenia uniflora l. seeds and its potential antibacterial activity antimicrobial activity of legume seed proteins interactions of plant-lectins with the components of the bacterial-cell wall peptidoglycan effects of a lectin-like protein isolated from acacia farnesiana seeds on phytopathogenic bacterial strains and root-knot nematode lectins: to combat infections interaction of a legume lectin with two components of the bacterial cell wall. a crystallographic study evaluation of antimicrobial activity of a lectin isolated and purified from indigofera heterantha a novel homodimeric lectin from astragalus mongholicus with antifungal activity a novel sialic acid-specific lectin from phaseolus coccineus seeds with potent antineoplastic and antifungal activities bul: a novel lectin from bauhinia ungulata l. seeds with fungistatic and antiproliferative activities studies on growth inhibition by lectins of penicillia and aspergilli isolation of a homodimeric lectin with antifungal and antiviral activities from red kidney bean (phaseolus vulgaris) seeds carbohydrate profiling of fungal cell wall surface glycoconjugates of aspergillus species in brain and lung tissues using lectin histochemistry lectins with anti-hiv activity: a review plant as a plenteous reserve of lectin bacterial detection using carbohydrate-functionalised cds quantum dots: a model study exploiting e. coli recognition of mannosides isolation and characterization of myrianthus holstii lectin, a potent hiv- inhibitory protein from the plant myrianthus holstii effects of succinylated concanavalin a on infectivity and syncytial formation of human-immunodeficiency-virus correlation between carbohydrate structures on the envelope glycoprotein gp of hiv- and hiv- and syncytium inhibition with lectins development and applications of lectins as biological tools in biomedical research proteins with antifungal properties and other medicinal applications from plants and mushrooms fungal cell wall chitinases and glucanases. microbiology lectins in higher plants fusarium sp growth inhibition by wheat germ agglutinin hevein: an antifungal protein from rubber-tree (hevea brasiliensis) latex insecticidal and antifungal activity of a protein from pouteria torta seeds with lectin-like properties three dimensional structure of the soybean agglutinin-gal/galnac complexes by homology modeling virus glycosylation: role in virulence and immune interactions structural studies of algal lectins with anti-hiv activity plant lectins are potent inhibitors of coronaviruses by interfering with two targets in the viral replication cycle griffithsin: an antiviral lectin with outstanding therapeutic potential bun ng, t. biochemical and functional properties of a lectin purified from korean large black soybeans a cultivar of glycine max insecticidal action of bauhinia monandra leaf lectin (bmoll) against anagasta kuehniella (lepidoptera: pyralidae), zabrotes subfasciatus and callosobruchus maculatus (coleoptera: bruchidae) pea lectin expressed transgenically in oilseed rape reduces growth rate of pollen beetle larvae plant-insect interactions: what can we learn from plant lectins? arch biodegradable microparticulate system of captopril penetration through the peritrophic matrix is a key to lectin toxicity against tribolium castaneum binding of insecticidal lectin colocasia esculenta tuber agglutinin (cea) to midgut receptors of bemisia tabaci and lipaphis erysimi provides clues to its insecticidal potential fungal lectin, xcl, is internalized via clathrin-dependent endocytosis and facilitates uptake of other molecules mechanism of entomotoxicity of the plant lectin from hippeastrum hybrid (amaryllis) in spodoptera littoralis larvae functional mechanics of the plant defensive griffonia simplicifolia lectin ii: resistance to proteolysis is independent of glycoconjugate binding in the insect gut the insecticidal activity of recombinant garlic lectins towards aphids ferritin acts as the most abundant binding protein for snowdrop lectin in the midgut of rice brown planthoppers (nilaparvata lugens) ferritin acts as a target site for the snowdrop lectin (gna) in the midgut of the cotton leafworm spodoptera littoralis effect of myracrodruon urundeuva leaf lectin on survival and digestive enzymes of aedes aegypti larvae entomotoxic action of jackbean lectin (con a) in bird cherry-oat aphid through the effect on insect enzymes insight to the mode of action of allium sativum leaf agglutinin (asal) expressing in t rice lines on brown planthopper identification of membrane proteins of the midgut of zabrotes subfasciatus larvae associated with the insecticidal mechanism of pf lectin binding of pf lectin from olneya tesota to gut proteins of zabrotes subfasciatus larvae associated with the insecticidal mechanism receptors of garlic (allium sativum) lectins and their role in insecticidal action enhanced expression of α , -linked sialic acids promotes gastric cancer cell metastasis and correlates with poor prognosis comparison of the lectin-binding pattern in different human melanoma cell lines expression analysis of carbohydrate antigens in ductal carcinoma in situ of the breast by lectin histochemistry alteration of protein glycosylation in liver diseases plant lectins: targeting programmed cell death pathways as antitumor agents mistletoe extracts standardised in terms of mistletoe lectins (ml i) in oncology: current state of clinical research plant lectins, from ancient sugar-binding proteins to emerging anti-cancer drugs in apoptosis and autophagy the biological functions of mbl-associated serine proteases (masps) antitumor effect of soybean lectin mediated through reactive oxygen species-dependent pathway lectin-induced apoptosis of tumour cells autophagy induction in t cell-independent acute hepatitis induced by concanavalin a in scid/nod mice this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -zq hpjs authors: gouda, sushanto; das, gitishree; sen, sandeep k.; shin, han-seung; patra, jayanta kumar title: endophytes: a treasure house of bioactive compounds of medicinal importance date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: zq hpjs endophytes are an endosymbiotic group of microorganisms that colonize in plants and microbes that can be readily isolated from any microbial or plant growth medium. they act as reservoirs of novel bioactive secondary metabolites, such as alkaloids, phenolic acids, quinones, steroids, saponins, tannins, and terpenoids that serve as a potential candidate for antimicrobial, anti-insect, anticancer and many more properties. while plant sources are being extensively explored for new chemical entities for therapeutic purposes, endophytic microbes also constitute an important source for drug discovery. this review aims to comprehend the contribution and uses of endophytes as an impending source of drugs against various forms of diseases and other possible medicinal use. plants have served as a source of medicinal bioactive compounds against numerous forms of ailments for centuries. ironically, in recent years, microorganisms associated with plants rather than plants themselves have proved to offer material and products with high therapeutic potential (subbulakshmi et al., ) . endophytes are an endosymbiotic group of microorganisms -often bacteria or fungi -that colonize the inter-and/or intracellular locations of plants (pimentel et al., ; singh and dubey, ) . for these organisms, all or part of their life cycle occurs within their hosts, without causing any apparent symptoms of disease. they are ubiquitous in nature and exhibit complex interactions with their hosts, which involve mutualism, antagonism and rarely parasitism (nair and padmavathy, ) . endophytes are known to enhance host growth and nutrient gain. they may improve the plant's ability to tolerate various types of abiotic and biotic stresses, and enhance the resistance of plants to insects and pests. they produce phytohormones and other bioactive compounds of biotechnological interest (enzymes and pharmaceutical drugs) (joseph and priya, ; parthasarathi et al., ) . researchers have indicated the presence of one or more types of endophytes in every single plant studied to date (strobel and daisy, ) . endophytes can colonize in the stem, roots, petioles, leaf segments, inflorescences of weeds, fruit, buds, seeds and also dead and hollow hyaline cells of plants (hata and sone, ; specian et al., ; stępniewska and kuzniar, ) . the population of endophytes in a plant species is highly variable and depends on various components, such as host species, host developmental stage, inoculum density and environmental condition (dudeja and giri, ) . however, very few studies have exploited these symbiotic groups of organisms and their bioactive metabolites. for the past few decades, it has become evident that the discovery rate of active novel chemical entities is declining. while plant sources are being extensively explored for the discovery of new chemical entities for various therapeutic purposes, endophytic microorganisms play an important role in this search for natural bioactive compounds, with potential use in the health sector and in drug discovery (lam, ) . this review highlights the various sources of endophytes, their secondary metabolites and role as source of drugs. such studies may improve the understanding of endophytes and address the need for new and useful compounds necessary to combat various pathogens associated with human health and other possible medicinal uses. numerous surveys, mostly on the pathogenicity interactions between plants and microorganisms associated with them, have been accomplished. however, after several explanations and studies on the role of microbial diversity associated with various plant species, it was assumed that only a small fraction of the microbes interacting with the plant are pathogenic in nature (andreote et al., ) . most of the microorganisms that inhabit plants play a major role in the plant's health and development, although, sometimes they are neutral (mendes et al., ; philippot et al., ) . it is considered that a single plant species could possess thousands of microbes, categorized as epiphytes (microbial inhabitants of the rhizosphere and phyllosphere; those near or on plant tissue) or endophytes (microbes residing within plant tissues in leaves, roots or stems), depending on their area of colonization in the plant species (oldroyd et al., ; turner et al., ; andreote et al., ) . apart from the disease-causing microorganisms, the presence of other (non-pathogenic) organisms inside the plants was first pronounced by de bary ( ), who detected the presence of microbial cells in the microscopically analyzed plant tissues. however, this observation continued to be unexplored until the definition of endophytes came into existence toward the end of last century. de bary ( ) provided the first definition of an endophyte, as "any organism that grows within plant tissues are termed as endophytes," however, the definition continues to change as per various researchers (wilson, ; hallmann et al., ; bacon and white, ) . petrini ( ) provided the most suitable definition for endophytes, which means any organism that at some part of its life cycle, colonizes the internal plant tissues without causing any type of harm to the host plant. furthermore, due to extensive studies of these groups of microorganisms, the endophytic communities have been divided into different subgroups, such as 'obligate' or 'facultative, ' which are associated with all types of plants (rosenblueth and martínez-romero, ) . endophytes that depend on the metabolism of plants for survival, being spread amongst plants by the activity of different types of vectors or by vertical transmission, are termed obligate endophytes (hardoim et al., ) . whereas, the facultative endophytes are those that live outside the host body during a certain stage of their life cycle and are mostly associated with plants from its neighboring soil environment and atmosphere (abreu-tarazi et al., ) . numerous attempts have been made during recent years to discover the origin of endophytic organisms in different species (hallmann et al., ; mitter et al., ) . initially, researchers choose the rhizosphere or the seed-born microbial communities as the major sources of endophytes. normally, the specific endophytes interaction with various plants and evidence of their strategy of existence and transmission is provided by their genome organization (andreote et al., ) . researchers have reviewed the genome sizes and origins of endophytes by correlating the genome size with the bacterial lifestyle (dini- andreote et al., ) . as per many researchers, the definition of endophytes could be suitable for the hypothesis that they live inside the plant species, where the environment is more stable compared to the soil, where the plant grows. however, there are also some endophytes that only appear in the plant during part of their lifecycle. thus, the endophytic community is made up of organisms from distinct origins, with those with larger genomes likely to live in variable environments, such as soils, while those with smaller genomes are likely to exist in the stable environment and are vertically transmitted (mitter et al., ) . endophytes are associated with plants in various forms, including bacteria (actinomycetes or mycoplasma) or fungi that have been colonized inside the plant tissues. more than genera from phyla of bacterial species have been reported to be associated with endophytes and among them, most of the species belong to the phyla actinobacteria, proteobacteria, and firmicutes (golinska et al., ) . the diversity of endophytic bacteria ranges from gram-positive to gram-negative bacteria, such as achromobacter, acinetobacter, agrobacterium, bacillus, brevibacterium, microbacterium, pseudomonas, xanthomonas etc. (sun et al., ) . bacterial endophytes are diverse in nature and are known to produce different bioactive metabolites that act as antimicrobial and anticancer compounds, for example, with % of them reported from the single genus, streptomyces (berdy, ) . actinomycetes are prokaryotic microorganisms that belong to the phylum actinobacteria and possess mycelium like fungus and forms spores (chaudhary et al., ; barka et al., ) . traditionally, these actinomycetes were considered transitional forms between the fungi and bacteria (barka et al., ) . however, the comparison of actinomycetes to fungi is only superficial because most of their properties are similar to those of bacteria but unlike bacterial cells, the cells of actinomycetes are thin with a chromosome that is organized in a prokaryotic nucleoid and a peptidoglycan cell wall. endophytic actinomycetes are known to produce various chemical entities with unique structures of considerable medicinal importance (gayathri and muralikrishnan, ; singh and dubey, ) . many antimicrobial compounds have been reported from various types of endophytic actinomycetes. streptomyces is one of the dominant genera, which is most commonly isolated as endophytic actinomycetes (zhao k. et al., ; golinska et al., ) . compounds of biological interest isolated from streptomyces sp. include munumbicins (a and b), naphthomycin (a and k), clethramycin, coronamycin, cedarmycin (a and b), saadamycin, and kakadumycins. other active compounds isolated from actinomycetes are paclitaxel extracted from kitasatospora sp. associated with taxus baccata, and tyrosol from emblica officinalis, which is suggested to inhibit food-borne microbes (zhao k. et al., ; gangwar et al., ; golinska et al., ) . mycoplasma species are also reported as plant endophytes. endophytic mycoplasma species were conveyed to be in a symbiotic relationship with some red algae, such as bryopsis pennata, b. hypnoides and also in arcobacte (hollants et al., ) . however, there is no confirmed evidence of its uses, the source of extraction or use against foodborne diseases or other pathogens. fungi are a heterotrophic group of organisms with various life cycles that include symbiotic relationships with a wide variety of autotrophic organisms (dayle et al., ) . endophytic fungi have been classified into two broad groups based on their phylogeny and life history traits. these include the clavicipitaceous, which infect some grasses confined to cool regions and the non-clavicipitaceous endophytes, which are from asymptomatic tissues of non-vascular plants, ferns and allies, conifers and angiosperms and are limited to the ascomycota or basidiomycota group (jalgaonwala et al., ; bhardwaj and agrawal, ) . endophytic fungi produce some of the most broadly used antibiotic and anticancer drugs. penicillenols, extracted from penicillium sp., is cytotoxic to numerous cell lines. taxol, isolated from taxomyces andreanae, is the most effective and successful anticancer drug extracted from endophytic fungi to date. clavatol (torreya mairei), sordaricin (fusarium sp.), jesterone (pestalotiopsis jesteri), and javanicin (chloridium sp.) are all known to possess strong antibacterial and antifungal properties against numerous foodborne infectious agents (jalgaonwala et al., ) . pestacin, isolated from p. microspora, has excellent antioxidant properties. though the endophytes were overlooked for a long time and are considered as pathogen-causing contaminations, many that inhabit inside the plants are often recognized as symbionts, with a distinctive and cherished interaction with the plants with whom they grow (mitter et al., ; berg et al., ) . recent studies have confirmed the occurrence of endophytes by various cultivation-independent assays and by fluorescence in situ hybridization-confocal laser scanning microscopy studies (mitter et al., ; berg et al., ) . cultivation-based techniques, use the recovery and testing of isolates, whereas cultivation-independent techniques screen for variations in the total endophytic communities (menpara and chanda, ) . endophytes can be easily isolated on any microbial or plant growth, such as agar, potato dextrose agar and any nitrogenor carbon-containing media. the most frequent method used to detect and enumerate endophytes involves isolation from surface-sterilized host plant tissue. the main factors that may regulate entophyte colonization within a plant or microbial species, include the genotype of the plant, the growth stage of the plant, the physiological status of the plant, the type of plant tissues, the environmental condition of the soil in which it is grown, the sampling season, the surface sterility, selective media and culture conditions as well as different agricultural practices (gaiero et al., ; golinska et al., ) ecological awareness on the role of endophytes in nature, can also provide the best clues for targeting a particular type of endophytic bioactivity with the greatest potential for bioprospecting (strobel and daisy, ) . endophytes are reported to produce a number of bioactive metabolites in a single plant or microbe which served as an excellent source of drugs for treatment against various diseases and with potential applications in agriculture, medicine, food and cosmetics industries (strobel and daisy, ; jalgaonwala et al., ; godstime et al., ; shukla et al., ) . these secondary metabolites were categorized into various functional groups, alkaloids, benzopyranones, chinones, flavonoids, phenolic acids, quinones, steroids, saponins, tannins, terpenoids, tetralones, xanthones, and many others (figures a,b) (schulz et al., ; strobel and daisy, ; jalgaonwala et al., ; joseph and priya, ; pimentel et al., ; godstime et al., ) . extraction of metabolites from endophytes is affected by various factors, such as the season of sample collection, climatic condition and geographical location . however, with a revolutionary synthetic process that has been developed during the past few years, extraction from plants and other natural sources has now become more feasible, efficient and convenient (hussain et al., ) . the production of bioactive substances by endophytes, has been directly associated with the evolution of the host microorganisms, which may have incorporated genetic information from higher plants, allowing them to better adapt to the host plant and perform some functions, such as protection from various types of pathogens, insects, and grazing animals (strobel, ) . some of the commonly found secondary bioactive compounds from endophytes are described below. taxol (paclitaxol), a complex diterpene alkaloid produced by the endophyte metarhizium anisopliae found in the bark of taxus tree, is one of the most promising anticancer yersinia enterocolitica swine meat and meat products, milk and dairy products joseph and priya, meca et al., frontiers in microbiology | www.frontiersin.org agents developed or synthesized to date (zhang et al., ; visalakchi and muthumary, ; jalgaonwala et al., ) . camptothecin, from nothapodytes foetida is known to have cytotoxic and antifungal properties (joseph and priya, ; han and rahman, ) . huperzine a (hupa), from huperzia serrata, can act as a cholinesterase inhibitor (nair and padmavathy, ) . lignans, such as cathartics, emetics and cholagogue, isolated from endophytic podophyllum hexandrum, are reported to act as anticancer agents (konuklugil, ) . resins, such as etoposide and teniposide extracted from p. emodi, possess strong anticancer activity (konuklugil, ) . compounds such as oxacillin, ampicillin, catechin, gallic acid, and cefalexin are known to possess bactericidal activities (akiyama et al., ) . terpenoids possess antineoplastic, antibacterial, and antiviral effects as well as gastrointestinal stimulation (jalgaonwala et al., ; godstime et al., ) . the endophytic fungus, cytonaema sp., produces triterpenoid helvolic acid with strong antibacterial activity (kumar et al., ) . infectious and parasitic diseases account for approximately half of the deaths worldwide (menpara and chanda, ) . although it is the generation of nano to pico drugs, natural sources have been proven as the best source for drug discovery. medicinal plants and their endophytes are an important source of precious bioactive compounds and secondary metabolites that contribute to more than % of the natural drugs available in the market (singh and dubey, ) . endophytic microorganisms are the storehouse of novel secondary metabolites that can serve as an excellent source of drugs for antiarthritic, antimicrobial, anticancer, antidiabetic, anti-insect, and immunosuppressant activities (jalgaonwala et al., ; godstime et al., ) . to date, only a few plants have been investigated for their endophytic diversity and potential to produce bioactive secondary metabolites. the discovery of novel antimicrobial secondary metabolites and bioactive compounds from different types of endophytic microorganisms is an important alternative to overcome the increasing levels of drugs resistance to various pathogenic microorganisms (godstime et al., ) . there are a number of bioactive compounds, such as camptothecin, diosgenin, hypericin, paclitaxel, podophyllotoxin, and vinblastine, which have been commercially produced by different endophytic fungi present in respective plants and they are of both agricultural as well pharmaceutical importance (joseph and priya, ; zhao et al., a) . these compounds are analogs of various types of phytohormones, essential oils etc. isolated from various endophytes (zhao et al., b; molina et al., ; nicoletti and fiorentino, ) . a detailed list of the endophytes isolated from various sources, their bioactive metabolites and the uses of endophytes as a source of medicine against various diseases is presented in table . plant roots and the gastrointestinal system of animals play a major role in the absorption of various nutrients and thus harbor a large, complex and dynamic group of microorganisms that help to degrade nutrients to more easily absorbed forms (ramirez-puebla et al., ) . the animal gastrointestinal system is inhibited by a number of microorganisms, starting from the archaea to the eukaryotes along with a number of plant-associated bacteria, particularly the endophytes (rosenblueth and martínez-romero, ; parniske, ; ramirez-puebla et al., ) . similarly, various soil types, moisture, plant genotypes, root lysates etc. are the determinants of the root microbiota (doornbos et al., ) . the diversity of microorganisms in healthy humans vary with diet, maintenance of hygiene, hormonal cycles, infections, uptake of medicine, sexual activity, etc. (muzny et al., ; gerber, ) . in this context, various studies associated with the microbiome of humans have been undertaken during recent times. a study from the human microbiome project has also stated the role of the gut microbiome in regulating the host circadian clock, which in mammals is located in the brain (leone et al., ) . these studies have provided evidence that a high-fat diet could alter the microbiome circadian rhythm, thereby suggesting a link between the diet, gut microbiota and obesity (leone et al., ) . furthermore, there are a number of microbiome studies undertaken to find out the associations and diversity of microbiota in both plant and animal systems. longitudinal microbiome studies are undertaken to gain insights into the dynamic behaviors of the microbiota, such as the microbial succession events during maturation of the infant's gut, normal temporal inconsistency in healthy adults, responses to various dietary changes and the use of different antibiotics and in instances of dysbiotic alterations that signal symptomatic diseases. furthermore, the multi-omic analyses that combine information from multiple data sources, such as metabolomes, proteomes and transcriptomes, provide a deep vision into the purposeful changes of the internal microbiome with respect to time. similarly, the computational tools to analyze microbiome time-series data is another area that shows tremendous growth. these techniques could model the inter-individual variability, while automatically capturing commonalities at appropriate levels in the ecosystems (gerber, ) . endophytes are a poorly investigated group of microorganisms capable of synthesizing bioactive compounds that can be used to combat numerous pathogens. these have been dependable sources of bioactive and chemically novel compounds and have proven to be useful for novel drug discovery. biotransformation methods have a wide range of uses, particularly in the production of numerous bioactive compounds, as antimicrobial (vanillin, essential oils), antifungal and antiviral (alkaloids), antioxidant(eugenol), antiinflammatory (cineole) etc. it is imperative to review and highlight the previous successes, ongoing research and latest developments in research associated with endophytic microorganisms to draw the attention of the research community toward this emerging field and possible exploitation of the available sources for their therapeutic uses in various fields, such as the medical, pharmaceutical, food and cosmetics. jkp, sg, gd, and ss wrote the manuscript. h-ss and jkp designed the concept, edited the manuscript. all the authors read and approved the manuscript. this study was supported by the agricultural research center, ministry of food, forestry, and fisheries, republic of korea. endophytic bacteria in long-term in vitro cultivated axenic pineapple microplants revealed by pcr dgge antibacterial action of several tannins against staphylococcus aureus exploring the potential of endophytes from medicinal plants as sources of antimycobacterial compounds exploring interactions of plant microbiomes microbial endophytes taxonomy, physiology, and natural products of thoughts and facts about antibiotics: where we are now and where we are heading unraveling the plant microbiome: looking back and future perspectives a review fungal endophytes: as a store house of bioactive compound diversity and versatility of actinomycetes and its role in antibiotic production angiosperm dna contamination by endophytic fungi: detection and methods of avoidance leipzig: hofmeister's handbook of physiological botany bacterial genomes: habitat specificity and uncharted organisms impact of root exudates and plant defense signaling on bacterial communities in the rhizosphere beneficial properties, colonization, establishment and molecular diversity of endophytic bacteria in legume and non-legume endophytes: exploitation as a tool in plant protection production and genetic improvement of a novel antimycotic agent, saadamycin, against dermatophytes and other clinical fungi from endophytic streptomyces sp. hedaya coronamycins, peptide antibiotics produced by a verticillate streptomyces sp. (msu- ) endophytic on monstera sp identification of the biosynthetic gene cluster and an additional gene for resistance to the anti-tuberculosis drug capreomycin inside the root microbiome: bacterial root endophytes and plant growth promotion diversity and biopotential of endophytic actinomycetes from three medicinal plants in india isolation and characterization of endophytic actinomycetes from mangrove plants for antimicrobial activity the dynamic microbiome mechanisms of antimicrobial actions of phytochemicals against enteric pathogens -a review endophytic actinobacteria of medicinal plants: diversity and bioactivity bacterial endophytes in agricultural crops alkaloids produced by endophytic fungi: a review properties of bacterial endophytes and their proposed role in plant growth isolation of endophytes from leaves of neolitsea sericea in broadleaf and conifer stands purification, identification and activity of phomodione, a furandione from an endophytic phoma species who is in there? exploration of endophytic bacteria within the siphonous green seaweed bryopsis (bryopsidales, chlorophyta) current approaches toward production of secondary plant metabolites screening for potential antimicrobial compounds from ganoderma boninense against selected food borne and skin disease pathogens natural products from plant associated endophytic fungi bioactive compounds from endophytes and their potential in pharmaceutical effect: a review endophytic fungi from medicinal plants: a treasure hunt for bioactive metabolites the importance of aryltetralin (podophyllum) lignans and their distribution in the plant kingdom endophytic fungi: as a source of antimicrobials bioactive compounds new aspects of natural products in drug discovery effects of diurnal variation of gut microbes and high-fat feeding on host circadian clock function and metabolism antibacterial effect of the bioactive compound beauvericin produced by fusarium proliferatum on solid medium of wheat the rhizosphere microbiome: significance of plant beneficial, plant pathogenic, and human pathogenic microorganisms endophytic bacteria -unexplored reservoir of antimicrobials for combating microbial pathogens comparative genome analysis of burkholderia phytofirmans psjn reveals a wide spectrum of endophytic lifestyles based on interaction strategies with host plants application of fungal endophytes in biotechnological processes characterization of the vaginal microbiota among sexual risk behavior groups of women with bacterial vaginosis impact of endophytic microorganisms on plants, environment and humans plant bioactive metabolites and drugs produced by endophytic fungi of spermatophyta bioactive metabolite produced by phomopsis sp., an endophytic fungus in allamanda cathartica linn the rules of engagement in the legume-rhizobial symbiosis arbuscular mycorrhiza: the mother of plant root endosymbioses isolation and characterization of antimicrobial compound from marine streptomyces hygroscopicus bdus fungal endophytes of tree leaves going back to the roots: the microbial ecology of the rhizosphere use of endophytes to obtain bioactive compounds and their application in biotransformation process an anti-herpes simplex virus type- agent from xylaria mellisii (bcc ) gut and root microbiota communalities bacterial endophytes and their interactions with hosts endophytic fungi: a source of novel biologically active secondary metabolites isolation and screening of endophytic fungi from medicinal plants of virudhunagar district for antimicrobial activity endophytic microbes: a novel source for biologically/pharmacologically active secondary metabolites endophytic actinomycetes as emerging source for therapeutic compounds chemical characterization of bioactive compounds from the endophytic fungus diaporthe helianthi isolated from luehea divaricata endophytic microorganisms-promising applications in bioremediation of greenhouse gases endophytes as sources of bioactive products bioprospecting for microbial endophytes and their natural products. microbiol bioactive endophytic fungal isolates of biota orientalis (l) endl., pinus excelsa wall. and thuja occidentalis l isolation, characterization, and antimicrobial activity of endophytic bacteria from polygonum cuspidatum the plant microbiome taxol (anticancer drug) producing endophytic fungi: an overview endophyte: the evolution of a term, and clarification of its use and definition an endophytic taxol-producing fungus from taxus media, cladosporium cladosporioides md plant-derived bioactive compounds produced by endophytic fungi endophytic fungi for producing bioactive compounds originally from their host plants the diversity and anti-microbial activity of endophytic actinomycetes isolated from medicinal plants in panxi plateau china key: cord- -xlwsj v authors: shanmugaraj, balamurugan; i. bulaon, christine joy; phoolcharoen, waranyoo title: plant molecular farming: a viable platform for recombinant biopharmaceutical production date: - - journal: plants (basel) doi: . /plants sha: doc_id: cord_uid: xlwsj v the demand for recombinant proteins in terms of quality, quantity, and diversity is increasing steadily, which is attracting global attention for the development of new recombinant protein production technologies and the engineering of conventional established expression systems based on bacteria or mammalian cell cultures. since the advancements of plant genetic engineering in the s, plants have been used for the production of economically valuable, biologically active non-native proteins or biopharmaceuticals, the concept termed as plant molecular farming (pmf). pmf is considered as a cost-effective technology that has grown and advanced tremendously over the past two decades. the development and improvement of the transient expression system has significantly reduced the protein production timeline and greatly improved the protein yield in plants. the major factors that drive the plant-based platform towards potential competitors for the conventional expression system are cost-effectiveness, scalability, flexibility, versatility, and robustness of the system. many biopharmaceuticals including recombinant vaccine antigens, monoclonal antibodies, and other commercially viable proteins are produced in plants, some of which are in the pre-clinical and clinical pipeline. in this review, we consider the importance of a plant- based production system for recombinant protein production, and its potential to produce biopharmaceuticals is discussed. recombinant proteins are complex exogenous ("foreign") proteins that are produced in expression hosts, and mainly used as medical diagnostic reagents and in human healthcare as vaccines, drugs, or monoclonal antibodies [ ] . the prominent role and increasing market demand for high-value recombinant proteins in novel drug discovery creates an opportunity for the development of various protein expression hosts to manufacture proteins by following the existing rigid standards laid down for veterinary and human applications. the industry is focusing mainly on already established production platforms using prokaryotic and eukaryotic expression host systems such as escherichia coli, a selection of yeast, insect, and mammalian cell cultures, due to their well-defined processes in-line with current good manufacturing practice (cgmp) [ ] . moreover, industrially established mammalian and other cell cultures have stringent regulatory approval in place, which hinders the industrial acceptance of the new technology or production system. bacterial expression systems offer rapid production with high product yield, whereas saccharomyces cerevisiae and pichia pastoris (yeast) offer post-translational modifications (ptms) which are essential for functional activity of the recombinant proteins [ ] . the majority of the table . available expression platforms for recombinant protein production with potential advantages and disadvantages (adapted from shanmugaraj et al., ) [ ] . the concept of using plants for the production of foreign proteins including pharmaceutical and non-pharmaceutical proteins has been well explored and documented. many reports proved the ability of in vivo and in vitro plant systems to produce vaccine candidates both for veterinary and human applications and showed that plant-produced antigens elicit potential immune responses in animal models and even confer protection in animal challenge experiments. examples of the variety of pharmaceutical and non-pharmaceutical proteins expressed in plant systems are illustrated in tables and . tobacco has been engineered to express a variety of antigens in the nucleus and chloroplast including, but not limited to, chikungunya, dengue, ebola, influenza, and zika. the transformation protocols for recombinant protein production are also established for fruits and vegetables such as tomatoes and potatoes. transgenic potatoes expressing the s glycoprotein of the infectious bronchitis virus confers protection to chickens upon virus challenge [ ] . leafy crops such as lettuce, alfalfa, and clover have been investigated for molecular farming to obtain the oral delivery of vaccine antigens eliminating purification and injections. the lettuce chloroplast-derived booster vaccine using lyophilized plant cells expressing the poliovirus capsid protein induced neutralization antibodies in mice primed with inactivated poliovirus vaccine (ipv) and conferred protection against all polio serotypes [ ] . plant systems have also been evaluated for the expression of virus-like particles (vlp) of many viruses including norovirus, poliovirus, foot-and-mouth disease virus, influenza, [ ] [ ] [ ] [ ] , and the potential for plant-derived vlps to be used as candidate vaccines and reagents has been reviewed in detail elsewhere [ , , ] . apart from expressing antigens for human diseases, several antigens for veterinary applications and non-pharmaceutical proteins have also been well tested for expression in plants, and are particularly gaining attention due to the fact that these products can quickly reach the market due to lower regulatory burden [ ] . this was clearly evidenced by the commercialization of avidin [ ] , β-glucuronidase [ ] , and trypsin [ ] by the us-based biotechnology company prodigene, inc. the vaccine against newcastle disease virus (ndv) was the first plant-based poultry vaccine (dow agrosciences) that obtained regulatory approval from the united states department of agriculture in , opening a new avenue for the commercialization of plant-derived vaccines. currently, many plant-derived non-pharmaceutical and pharmaceutical proteins are in clinical development. although proof-of-concept and efficacy of many vaccine candidates proved the feasibility and scalability of the robust plant system, it is high time to compete with the established expression systems. now the plant-based good manufacturing practices (gmp) complaint production facilities such as fraunhofer (germany), kentucky bioprocessing (usa), medicago (canada), and protalix biotherapeutics (israel) are available to manufacture gmp materials for human clinical trials. fraunhofer ime received a gmp license for the production of neutralizing anti-hiv antibody g in tobacco for phase i clinical testing [ ] . the plant molecular farming research community continuously thrives to set up a regulatory framework for plant-derived products. the expression methods used for the recombinant protein production in plants can be either stable or transient expression. pmf relies on following approaches for the expression of vaccine candidates, i.e., stable nuclear transformation, stable chloroplast transformation, or transient expression, by using plant viral vectors and stable transformation of hydroponically grown plants in which recombinant proteins are recovered from the medium [ ] (figure ). stable nuclear transformation is the traditional strategy of genetic manipulation in plants for recombinant protein production. the transgene in the plant expression vector can be introduced into the in vitro grown plantlets either with agrobacterium tumefaciens-mediated transformation or particle bombardment, and stable transgenic lines can be developed. the best transgenic line for protein production will be subsequently screened from the pool of transgenic lines. by this method, recombinant proteins can be produced in successive generations, as the transgene has been stably integrated into the plant genome. the model plants such as arabidopsis thaliana and tobacco were more commonly used during the early stages of genetic transformation to develop stable transformants [ ] . stable transformation in plants requires substantial time and is a labor-intensive process, and the protein expression is insufficient to meet the industrial-level protein production. however, the antigen expression in stable transgenic line could be used for developing oral vaccines that could reduce the cost associated with protein purification [ , ] . alternatively, transient expression based on agroinfiltration or virus-based vectors have been developed to complement transgenic plants that offer rapid and high-level protein expression within a few days. the drawbacks and challenges associated with stable expression, such as stable nuclear transformation is the traditional strategy of genetic manipulation in plants for recombinant protein production. the transgene in the plant expression vector can be introduced into the in vitro grown plantlets either with agrobacterium tumefaciens-mediated transformation or particle bombardment, and stable transgenic lines can be developed. the best transgenic line for protein production will be subsequently screened from the pool of transgenic lines. by this method, recombinant proteins can be produced in successive generations, as the transgene has been stably integrated into the plant genome. the model plants such as arabidopsis thaliana and tobacco were more commonly used during the early stages of genetic transformation to develop stable transformants [ ] . stable transformation in plants requires substantial time and is a labor-intensive process, and the protein expression is insufficient to meet the industrial-level protein production. however, the antigen expression in stable transgenic line could be used for developing oral vaccines that could reduce the cost associated with protein purification [ , ] . alternatively, transient expression based on agroinfiltration or virus-based vectors have been developed to complement transgenic plants that offer rapid and high-level protein expression within a few days. the drawbacks and challenges associated with stable expression, such as insufficient protein expression, time, and consistency, have been overcome by the development of novel strategies involving deconstructed viral vector systems such as magnicon ® technology, geminiviral, and peaq, which allows rapid accumulation of recombinant proteins in a short time [ ] . hence, it is considered as a suitable convenient platform, especially for the production of vaccine antigens or monoclonal antibodies against infectious diseases (figure ). gleba et al. ( ) summarized the application of plant viral vectors for the transient expression of heterologous proteins in plants [ ] . plant transient expression holds tremendous potential to produce rapid-response proteins, emergency vaccines, or biologics, which was impressively shown during the ebola outbreak in . mapp biopharmaceutical inc., usa produced an experimental drug zmapp, an anti-ebola antibody cocktail of three chimeric monoclonal antibodies manufactured in tobacco plants (nicotiana benthamiana) to treat humans during the recent ebola outbreak [ ] . during a pandemic situation, in order to cope with a rapidly spreading infectious disease, production methods should meet the demand for production targets of strategic vaccines to control the disease. one of the recent examples is the pandemic, corona virus disease (covid- ) . the virus has spread rapidly, and millions of people have been affected across continents in few months, posing a constant threat to global health. this infection has created a massive demand for diagnostic reagents, vaccines, and therapeutic development. given the speed advantages, and proven viability of the plant production platform, the transient expression system in particular could be employed to produce recombinant proteins at high levels to meet the sudden demand for production of viral antigens or antiviral proteins that could be used as research reagents, emergency vaccines (sars-cov- subunit and virus-like particle vaccines), or other biopharmaceuticals to fight against covid- [ , ] . the neutralizing monoclonal antibodies against sars-cov- could also be produced in plants with minimal investment, which could be used for passive immunotherapy [ ] . recently, medicago (quebec, canada), kentucky bioprocessing (owensboro, kt, usa), and ibio (bryan, tx, usa) joined the global race for developing potential plant-based vaccines for covid- [ ] . by using the transient expression platform, recombinant protein production in plants could be scaled up rapidly, and milligram quantities of proteins could be produced in a timeframe of less than weeks after receiving the corresponding gene construct [ , , ] . plants , , x for peer review of has spread rapidly, and millions of people have been affected across continents in few months, posing a constant threat to global health. this infection has created a massive demand for diagnostic reagents, vaccines, and therapeutic development. given the speed advantages, and proven viability of the plant production platform, the transient expression system in particular could be employed to produce recombinant proteins at high levels to meet the sudden demand for production of viral antigens or antiviral proteins that could be used as research reagents, emergency vaccines (sars-cov- subunit and virus-like particle vaccines), or other biopharmaceuticals to fight against covid- [ , ] . the neutralizing monoclonal antibodies against sars-cov- could also be produced in plants with minimal investment, which could be used for passive immunotherapy [ ] . recently, medicago (quebec, canada), kentucky bioprocessing (owensboro, kt, usa), and ibio (bryan, tx, usa) joined the global race for developing potential plant-based vaccines for covid- [ ] . by using the transient expression platform, recombinant protein production in plants could be scaled up rapidly, and milligram quantities of proteins could be produced in a timeframe of less than weeks after receiving the corresponding gene construct [ , , ] . alternately, chloroplast expression focuses on expressing the transgenes in chloroplast by the precise insertion of foreign dna by homologous recombination into the chloroplast genome. much progress has been made in chloroplast engineering in recent years. the transformation of the chloroplast genome has many advantages over nuclear transformation which includes higher protein production, lack of gene silencing and position effect, polycistronic mrna expression, and prevention of transmission of foreign dna through pollen by uniparental plastid gene inheritance (maternal inheritance) in crop plants [ ] [ ] [ ] [ ] . similar to bacterial and mammalian cells, heterologous protein production can be achieved by using individual suspension of plant cells rather than whole plants. the cell suspension derived from undifferentiated callus grown in liquid medium can be scaled up in bioreactors for large-scale protein production under an aseptic environment. the first usda-approved poultry vaccine and the first fda-approved recombinant plant-produced pharmaceutical protein "elelyso" were produced in tobacco and carrot cell suspension cultures, respectively, which proved the importance and competitiveness of plant suspension culture in high-value protein production in the biopharmaceutical industry [ , [ ] [ ] [ ] . hairy root cultures are also being explored as an alternative recombinant protein production system due to their ease in protein recovery and low costs. the recombinant proteins are secreted from the transgenic plant roots into the culture medium viz., rhizosecretion; hence, this allows continuous protein production and recovery from the culture medium without the requirement of cell lysis during extraction. moreover, recombinant proteins produced from root cultures attribute to the improved protein quality and quantity without complex downstream processing that could eventually reduce production costs as well [ ] . a recent review on the applications of hairy root cultures for protein production has been extensively discussed by gutierrez-valdes et al. ( ) [ ] . although plants are attractive with several unique advantages, they are unable to compete with the existing microbial and mammalian systems, as both are well established and characterized, especially in terms of gmp manufacturing and regulatory approval in an industrial setting. even after many years of research, which has shown the proof-of-concept of expressing many therapeutic proteins in plants, the process of producing therapeutic proteins from the lab bench to commercialization is slow. hence, in order to move forward, the commercial potential and economic sustainability of technology needs to be exploited by developing veterinary vaccines, non-pharmaceutical diagnostic, cosmetic products, and industrial enzymes in plants, as they have a low regulatory burden compared to therapeutic proteins [ , ] . this technology can also be employed to reproduce rapid response vaccines or diagnostic reagents against emerging infections. for the past few years, extensive research has been carried out to combat the several emerging diseases including zika, chikungunya, nipah, sars-cov, mers-cov, and more recently sars-cov- . even though several efforts have been made for many years to develop effective vaccine candidates for many of those emerging and zoonotic diseases, still, there are no vaccine candidates or therapeutic measures available commercially. even if a successful vaccine or drug developed against such diseases, it is unlikely that it would have a significant impact on developing and under-developed countries, due to the high cost associated with it, and scalability concern. in such a scenario, a plant-derived vaccine or diagnostic reagent would be a feasible approach to rapidly respond to the demand and need for recombinant proteins. however, harnessing the full potential of this plant molecular farming technology for cost-effective vaccines or drug development will be evident in the upcoming years. plants have both economic and technical advantages over conventional expression systems for the production of pharmaceutical and non-pharmaceutical products. the different pmf technologies such as nuclear, chloroplast expression, viral transfection, and transient expression systems have their unique features, enabling them to address a production of diversified product "targets" with less production constraints in a short time. many scientific and technical challenges associated with the plant platform were met in recent years. however, the regulatory burden associated with therapeutic protein production is a major barrier that hinders the widespread acceptance of the plant system. considering the low costs and greater scalability of plant production systems, the commercialization of non-pharmaceutical proteins is straightforward and faster due to lower regulatory challenges. hence, the universal acceptance of the technology will be strongly influenced by the regulatory framework and restrictions applied to plant-derived products worldwide. the demand for industrially or pharmaceutically useful recombinant proteins, together with demonstrated production capability and economic feasibility of the plant system, suggests a bright future for the plant-made biologics. the authors declare no conflict of interest. therapeutic recombinant protein production in plants: challenges and opportunities. plants people planet critical analysis of the commercial potential of plants for the production of recombinant proteins comparison of yeasts as hosts for recombinant protein production therapeutic glycoprotein production in mammalian cells transient expressions of synthetic biology in plants plant expression platform for the production of recombinant pharmaceutical proteins production of antibodies in transgenic plants first plant-made biologic approved plant molecular farming: much more than medicines plant-made vaccine antigens and biopharmaceuticals plant-produced biopharmaceuticals: a case of technical developments driving clinical deployment potential of plants to produce recombinant protein products the case for plant-made veterinary immunotherapeutics recent development and future prospects of plant-based vaccines plant-made vaccines in the fight against cancer the algal chloroplast as a platform for making biopharmaceuticals current state-of-the-art in plant-based antibody production systems plant molecular farming of virus-like nanoparticles as vaccines and reagents the potential of plants as a system for the development and production of human biologics production of active eukaryotic proteins through bacterial expression systems: a review of the existing biotechnology strategies generation of glyco-engineered nicotiana benthamiana for the production of monoclonal antibodies with a homogeneous human-like n-glycan structure advanced plant-based glycan engineering plant-made pharmaceuticals: leading products and production platforms plants as factories for human pharmaceuticals: applications and challenges emergence of novel coronavirus -ncov: need for rapid vaccine and biologics development generation of the transgenic potato expressing full-length spike protein of infectious bronchitis virus cold chain and virus-free oral polio booster vaccine made in lettuce chloroplasts confers protection against all three poliovirus serotypes plant-made polio type stabilized vlps-a candidate synthetic polio vaccine high-level expression and enrichment of norovirus virus-like particles in plants using modified geminiviral vectors novel expression of immunogenic foot-and-mouth disease virus-like particles in nicotiana benthamiana efficacy of a plant-produced virus-like particle vaccine in chickens challenged with influenza a h n virus virus-like particles produced in plants as potential vaccines plant-based vaccines against viruses commercial production of avidin from transgenic maize: characterization of transformant, production, processing, extraction and purification commercial production of β-glucuronidase (gus): a model system for the production of proteins in plants maize (zea mays)-derived bovine trypsin: characterization of the first large-scale, commercial protein product from transgenic plants regulatory approval and a first-in-human phase i clinical trial of a monoclonal antibody produced in transgenic tobacco plants expression of hepatitis b surface antigen in transgenic plants immunization with potato plants expressing vp protein protects against rabbit hemorrhagic disease virus immunogenicity of porcine transmissible gastroenteritis virus spike protein expressed in plants oral immunization of cattle with hemagglutinin protein of rinderpest virus expressed in transgenic peanut induces specific immune responses bovine herpes virus gd protein produced in plants using a recombinant tobacco mosaic virus (tmv) vector possesses authentic antigenicity expression of human papillomavirus type major capsid protein in transgenic a corn-based delivery system for animal vaccines: an oral transmissible gastroenteritis virus vaccine boosts lactogenic immunity in swine plant-based vaccine: mice immunized with chloroplast-derived anthrax protective antigen survive anthrax lethal toxin challenge immunogenicity in humans of an edible vaccine for hepatitis b expression of the newcastle disease virus fusion protein in transgenic maize and immunological studies f (k ) fimbrial adhesin faeg expressed in alfalfa reduces f + enterotoxigenic escherichia coli excretion in weaned piglets plant-produced cottontail rabbit papillomavirus l protein protects against tumor challenge: a proof-of-concept study oral immunization with transgenic rice seeds expressing vp protein of infectious bursal disease virus induces protective immune responses in chickens high-yield rapid production of hepatitis b surface antigen in plant leaf by a viral expression system influenza virus-like particles produced by transient expression in nicotiana benthamiana induce a protective immune response against a lethal viral challenge in mice ingestion of transgenic carrots expressing the escherichia coli heat-labile enterotoxin b subunit protects mice against cholera toxin challenge an efficient plant viral expression system generating orally immunogenic norwalk virus-like particles induction of a protective antibody response to fmdv in mice following oral immunization with transgenic stylosanthes spp. as a feedstuff additive high-level expression of the hiv- pr gag polyprotein in transgenic tobacco chloroplasts generation and immunogenicity of japanese encephalitis virus envelope protein expressed in transgenic rice immunization with plant-expressed hemagglutinin protects chickens from lethal highly pathogenic avian influenza virus h n challenge infection a plant-based system for rapid production of influenza vaccine antigens. influenza other respir transient expression of hemagglutinin antigen from low pathogenic avian influenza a (h n ) in nicotiana benthamiana transient expression of vp in nicotiana benthamiana and its use as a plant-based vaccine against infectious bursal disease virus efficacy of a bvdv subunit vaccine produced in alfalfa transgenic plants a method for rapid production of heteromultimeric protein complexes in plants: assembly of protective bluetongue virus-like particles norovirus narita virus-like particles expressed in nicotiana benthamiana induce serum and mucosal immune responses boosting in planta production of antigens derived from the porcine reproductive and respiratory syndrome virus (prrsv) and subsequent evaluation of their immunogenicity high-yield expression of m e peptide of avian influenza virus h n in transgenic duckweed plants production of h n influenza virus matrix protein ectodomain protein bodies in tobacco plants and in insect cells as a candidate universal influenza vaccine high expression of consensus dengue virus envelope glycoprotein domain iii using a viral expression system in tobacco production of dengue virus envelope protein domain iii-based antigens in tobacco chloroplasts using inducible and constitutive expression systems subunit vaccine based on plant expressed recombinant eimeria gametocyte antigen gam elicit protective immune response against chicken coccidiosis potential of plant biologics to tackle the epidemic like situations-case studies involving viral and bacterial candidates plant-produced zika virus envelope protein elicits neutralizing immune responses that correlate with protective immunity against zika virus in mice immunogenicity of plant-produced porcine circovirus-like particles in mice production and immunogenicity of soluble plant-produced hiv- subtype c envelope gp immunogens. front transient expression of a tumor-specific single-chain fragment and a chimeric antibody in tobacco leaves rice cell culture as an alternative production system for functional diagnostic and therapeutic antibodies cereal crops as viable production and storage systems for pharmaceutical scfv antibodies plant-derived anti-lewis y mab exhibits biological activities for efficient immunotherapy against human cancer cells biochemical and functional characterization of anti-hiv antibody-elp fusion proteins from transgenic plants extremely high-level and rapid transient protein production in plants without the use of viral replication optimal nitrogen supply as a key to increased and sustained production of a monoclonal full-size antibody in by- suspension culture high-level rapid production of full-size monoclonal antibodies in plants by a single-vector dna replicon system robust production of virus-like particles and monoclonal antibodies with geminiviral replicon vectors in lettuce glycomodification and characterization of anti-colorectal cancer immunotherapeutic monoclonal antibodies in transgenic tobacco prophylactic and therapeutic testing of nicotiana-derived rsv-neutralizing human monoclonal antibodies in the cotton rat model engineering, expression in transgenic plants and characterisation of e , a rabies virus-neutralising monoclonal antibody structural and functional characterization of an anti-west nile virus monoclonal antibody and its single-chain variant produced in glycoengineered plants plant-produced anti-dengue virus monoclonal antibodies exhibit reduced antibody-dependent enhancement of infection activity rice endosperm produces an underglycosylated and potent form of the hiv-neutralizing monoclonal antibody g transient expression of biologically active anti-rabies virus monoclonal antibody in tobacco leaves plant-produced anti-enterovirus (ev ) monoclonal antibody efficiently protects mice against ev infection structural and in vitro functional analyses of novel plant-produced anti-human pd antibody high level production of monoclonal antibodies using an optimized plant expression system in vitro and in vivo efficacy of anti-chikungunya virus monoclonal antibodies produced in wild-type and glycoengineered nicotiana benthamiana plants production of correctly processed human serum albumin in transgenic plants characterization of a human glycoprotein (erythropoietin) produced in cultured tobacco cells production of functional human α -antitrypsin by plant cell culture commercial production of aprotinin in transgenic maize seeds raskin, i. production of recombinant proteins in tobacco guttation fluid triple helix assembly and processing of human collagen produced in transgenic tobacco plants high-yield production of a human therapeutic protein in tobacco chloroplasts overexpression of the bt cry aa operon in chloroplasts leads to formation of insecticidal crystals a chloroplast transgenic approach to hyper-express and purify human serum albumin, a protein highly susceptible to proteolytic degradation expression of active human epidermal growth factor (hegf) in tobacco plants by integrative and non-integrative systems high-level expression of basic fibroblast growth factor in transgenic soybean seeds and characterization of its biological activity field production and functional evaluation of chloroplast-derived interferon-alpha b expression of human growth hormone in transgenic rice cell suspension culture exhaustion of the chloroplast protein synthesis capacity by massive expression of a highly stable protein antibiotic human growth hormone expressed in tobacco cells as an arabinogalactan-protein fusion glycoprotein has a prolonged serum life expression of a functional recombinant human basic fibroblast growth factor from transgenic rice seeds expression of biologically active anti-thrombosis protein lumbrokinase in edible sunflower seed kernel ectopic expression of human acidic fibroblast growth factor in the medicinal plant, salvia miltiorrhiza, accelerates the healing of burn wounds the combination of plant translational enhancers and terminator increase the expression of human glucocerebrosidase in nicotiana benthamiana plants oral delivery of acid alpha glucosidase epitopes expressed in plant chloroplasts suppresses antibody formation in treatment of pompe mice stable expression of basic fibroblast growth factor in chloroplasts of tobacco high-level expression of thermostable cellulolytic enzymes in tobacco transplastomic plants and their use in hydrolysis of an industrially pretreated arundo donax l. biomass recombinant human osteopontin expressed in nicotiana benthamiana stimulates osteogenesis related genes in human periodontal ligament cells recombinant human dentin matrix protein (hdmp ) expressed in nicotiana benthamiana potentially induces osteogenic differentiation shotguns vs lasers: identifying barriers and facilitators to scaling-up plant molecular farming for high-value health products molecular farming in plants: host systems and expression technology recent progress in the development of plant derived vaccines viral vectors for the expression of proteins in plants reversion of advanced ebola virus disease in nonhuman primates with zmapp will plant-made biopharmaceuticals play a role in the fight against covid- ? expert opin perspectives on monoclonal antibody therapy as potential therapeutic intervention for coronavirus disease- (covid- ). asian pac potential applications of plant biotechnology against sars-cov- plant molecular farming-integration and exploitation of side streams to achieve sustainable biomanufacturing. front engineering the plastid genome of higher plants plastid biotechnology: prospects for herbicide and insect resistance, metabolic engineering and molecular farming the engineered chloroplast genome just got smarter expression and functional evaluation of biopharmaceuticals made in plant chloroplasts on the way to commercializing plant cell culture platform for biopharmaceuticals: present status and prospect large-scale production of pharmaceutical proteins in plant cell culture-the protalix experience putting the spotlight back on plant suspension cultures. front biologically active recombinant human erythropoietin expressed in hairy root cultures and regenerated plantlets of nicotiana tabacum l hairy root cultures-a versatile tool with multiple applications. front this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -eoabmyg authors: nicoletti, marcello title: new solutions using natural products date: - - journal: insect-borne diseases in the st century doi: . /b - - - - . - sha: doc_id: cord_uid: eoabmyg most antibiotics are derived from natural products, like penicillin, as well as recent insecticides, like pyrethroids. secondary metabolites are produced by plants as ecological chemical mediators, and can therefore possess intrinsic physiological properties against other organisms. these benefits are far from being fully explored. in particular, attention is here focused on the multipurpose neem tree (azadirachta indica), reporting several experiments of applications in the field of seed oil and neem cake. the latter product seems to be promising because of the low cost, the possible production on a large scale, and the selection of effects in favor of beneficial organisms. neem cake is able to act on different sites, as required by integrated pest management. several utilizations of neem products are reported and their potentiality evidenced. some considerations in this chapter may appear distant from the title of the book, but only by applying the general natural rules can the reason of the single phenomenon be understood. other studies on resistance mechanisms of plasmodium are enabling new possible methods of control always based on natural products activity. and biotic targets, which are important to the individual homeostasis and survival. one form of evidence in accordance with this interpretation is the strong correlation between natural products' producers and biodiversity, therefore indicating a matter of adaptation. however, it is possible to argue that if differences can characterize each organism from another, the some is not true in case of a molecule. once determined the structure, a molecule is a molecule, even if generated by the metabolism or by a synthetic route, and therefore the living rules cannot be applied to the molecular world. this is true only in part. we must consider, in particular for natural products, the conformational forms, which are different ways of the same molecule to react. an organic molecule can change easily its d structure adapting to the environmental needs, like we open and close our hands. furthermore, once inside the metabolism, the molecule, even if inorganic, is integrated in the organic network and it is forced to collaborate to an integrated living system. the cell environment is far different from the test tube of the chemist. in practice, the molecule is simply part of an advanced complex integrated dynamic system, limiting its freedom. beside the consideration that physical forces work in the same way in the assembling matter, as we can know and distinguish a giraffe from a wizard, we can study natural compounds and distinguish them on basis of the structures and assign their place and role. about the role, we must remember that natural products are chemical mediators inside the environment, meaning that they must act on the receptor of the target organisms. therefore, the structures of natural products are derived from their activity, the same argument we use in the consideration of a pharmacological drug. here, the importance of natural products inspiring all the molecules tailored to affect living organisms. in particular, among the detected activities in plant species, the defensive against herbivorous is highly reported. plants cannot counteract by movements like animals and therefore an arsenal of chemical weapons is essential in their fight for survival. therefore, it is highly possible to find natural products toxic or repellent to phytophagous, like insects, as well as active constituents against pathogens. natural products with such properties should be extracted and found in the complex and confused reservoir of secondary metabolites, but the presence into the plant is not sufficient. it is necessary to realize the mechanism of action and the potentiality to be used as marketed products. therefore, the pathway should comprehend the discovery, the isolation, the sources, the bioactivities, and the possibility to be obtained in high quantity, the method of utilization, the environmental impact and the cost of production. the natural products have been already reported for antibiotic activity. in particular, essential oils and phenols are stated in many papers as being responsible of antibacterial and insecticide properties (ghosh et al., ; gibbons, ) . however, the activity seems to be too general. most essential oils are composed of the same main constituents, the difference being mainly quantitative for each compound or hidden deeply inside the plethora of secondary constituents. furthermore, excluding the phenols typical of essential oils, the quantity of known phenols is very large and complicated, with thousands of structures reported and different activities connected. therefore, special research must be performed, including the possibility to test new substances never reported. among secondary natural products with insecticide activity, a special place must be assigned to essential oils. in this regard, several papers report the utilization of essential oils as the active ingredient against pests. the antimicrobial activity of essential oils is also well-reported and evident in nature. several plants accumulate essential oils in the inner parts of roots and rhizomes, in order to avoid the devastating attack of micropathogens, wherein the plant often accumulates precious reserve substances. in other cases, the aerial parts are focused on defensive or cooperative actions. some birds defend their clutch by surrounding the nest with aromatic plants. the human utilization of essential oils of different kinds against insects has a long story. herodotus reported the use in ancient egypt of mosquito nets and towers impregnated with fish odor to avoid mosquito bites. an essential oil is a complex chemical mixture of substances volatile at ordinary temperature (figs. . and . ), and therefore the constituents must have low molecular weight. in other words, they are micromolecules, with average molecular weight of - uma and hydrocarbon prevalence. essential oils can be extracted from the raw materials by utilizing their volatile properties, such as in the steam distillation method. the antimicrobial activity of essential oils is usually a consequence of the content of phenols, but other properties must be considered. on the basis of the structures of the active constituents, there are two types of essential oils. the first one, mainly present in less advanced angiosperm dicotyledons, like magnolidae, contains mainly root and fruit drugs rich in simple phenolic phenylpropanoids, which are mainly utilized by the plant in protection of pathogens. in rosidae and sympetalae, the terpenoids progressively become predominant. the new volatile constituents, in addition to the protective and toxic effects, afford a positive attraction on pollinatory agents, evidencing the plant position and allowing a memory of the selected species. in this way, the scenario of the interaction between animals and plants changes from defensive to collaborative. the new plants to be selected and appreciated can enrich the offer to the collaborative animals fig. . a typical current apparatus for the production of essential oils by steam distillation. with fruits of inebriant flavors, colored flowers and other nice experiences. therefore, utilized essential oils are mainly complex mixtures of volatile plant secondary metabolism and consist mainly of monoterpenes and sesquiterpenes, which means lipid secondary metabolites, and, to a lesser extent, of aromatic compounds. the choice of essential oil depends firstly on the taxonomy of the selected plant and the effects depend on the nature of the constituents of the essential oil. the conclusion, based also on direct experiments, is the presence of a general antibiotic and insecticide activity; however, another real need is a selective toxicity in favor of the useful organisms. therefore, some kind of activity is expected for an essential oil, but there is a necessity to maximize its effectiveness. they are exploited in several fields, such as perfumery, food, pharmaceutics, and cosmetics, but essential oils have also long-standing uses in the treatment of infectious diseases and parasitosis in humans and animals. essential oils, currently more than of which are known, are highly variable in their complex composition. usually, at least a mixture of more than main different constituents of low molecular weight is present. among the single species, the qualitative composition of the essential oil is respected, although a quantitative variability is common between population according to the environmental pressures. however, some terpenes can be easily found, like the hydrocarbons (myrcene, pinene, terpinene, limonene, cymene, αand β-phellandrene) and the oxygenated ones, like the alcohols (geraniol, linalool, menthol, terpineol, borneol) , the aldehydes (citral, citronellal), ketones (menthone, pulegone, carvone), bicyclic monoterpene ketones (thujone, verbenone, fenchone), acids (citronellic acid, cinnamic acid), oxides ( , -cineole) and esters (linalyl acetate), but the aromatic phenols (carvacrol, thymol, safrole, eugenol) are also common. a few essential oils may also contain sulfur-containing constituents, methyl anthranilate, coumarins, and special sesquiterpenes such as zingiberene, curcumin, farnesol, sesquiphellandrene, turmerone, nerolidol, etc. often these components are at low concentrations (less than % each), but the opposite is also possible, with major compounds that can represent up to % of the total volume of oil, as much as % like eucalyptol in eucalyptus or limonene in citrus or pinenes in turpentine of pinus. therefore, the antiparasitic activity of an essential oil can vary according to differences in its chemical composition, but it is usually present. nowadays, there is an increasing interest in the utilization of essential oils against endoparasites and ectoparasites of animals and humans, in particular when they are resistant to conventional drugs. however, the use of essential oils is in general restricted for the high cost and considering that usually they are not adequately specified for the considered target (bagavan and rahuman, ; shaalan et al., ; tikar et al., ) . again, also in the case of essential oils, the insurgence of the insecticide resistance must be considered (brown, ) , although usually less common in these cases. therefore, in consideration of their general but also weaker effectiveness of essential oils in comparison with synthetic insecticides, their utilization requires the insecticidal properties of essential oils to be investigated in different approaches of selection of the studied plants and their uses. some examples of researches, in which i had occasion to participate, involving essential oils in insect-borne diseases are reported here. the leading idea was to utilize the essential oil properties in an innovative way, such as in mixture or selected types. in (benelli et al., a,b) , the activities of five essential oils were investigated. the essential oils were obtained from different plants: pinus nigra var. italica (pinaceae), hyssopus officinalis subsp. aristatus (lamiaceae), satureja montana subsp. montana (lamiaceae), aloysia citriodora (verbenaceae), and pelargonium graveolens (geraniaceae) against culex quinquefasciatus (diptera: culicidae), which is a vector of lymphatic filariasis and of dangerous arboviral diseases, such as west nile and st. louis encephalitis. the research was original in its focus on the potential synergistic and antagonistic effects, testing them in binary mixtures on c. quinquefasciatus larvae. mixtures of essential oils are very easy to obtain, since the constituents are perfectly soluble in the final solution and the selected oils were cheap and easy to find on the market. in such a way, knowing the composition, it is possible to combine constituents, enhancing the range and the quality of activity. the pool of the investigated species was highly varied, but this was considered a positive factor. first, the chemical composition of each essential oil was investigated by gc-ms analysis, which is the best analytic method in such mixtures of volatile compounds. therefore, it was also necessary to test the activity of each essential oil and later to try the best combination on the basis of its effectiveness. the highest effectiveness was obtained by s. montana subsp. montana essential oil (lc ¼ . μl l À ), followed by p. nigra var. italica (lc ¼ . μl l À ), and a. citriodora (lc ¼ . μl l À ). it was possible to obtain an enhancement of the larvicidal activity by preparing simple binary mixtures of essential oils (ratio : ), such as s. montana+a. citriodora, which showed higher larvicidal toxicity (lc ¼ . μl l À ). on the other hand, testing s. montana + p. nigra ( : ), an antagonistic effect was detected, leading to an lc ( . μl l À ) higher than the lc values calculated for the two oils tested separately. therefore, these results indicate the extreme need for innovation and imagination in natural products research, against many papers repeating the same procedure that change only the plant used. another work (pavela et al., a,b) was based on geographic distribution and the traditional use. six medicinal and aromatic plants-azadirachta indica (see later in this chapter), aframomum melegueta, aframomum daniellii, clausena anisata, dichrostachys cinerea, and echinops giganteus-have been traditionally used in cameroon to treat several disorders, including infections and parasitic diseases. the aim was to evaluate the activity of the essential oils of these plants against trypanosma brucei tc and determine their selectivity with balb/ t (mouse embryonic fibroblast cell line) cells as a reference. essential oils from a. indica, a. daniellii, and e. giganteus proved to be the most active ones, with half maximal inhibitory concentration (ic ) values of . , . , and . μg/ml, respectively. these essential oils were characterized by different chemical compounds, including monoterpenes and sesquiterpene hydrocarbons and oxygenated sesquiterpenes. some of their main components were assayed as well on t. brucei tc , and their effects were linked to those of essential oils. in this way, the research partially confirmed the ethnopharmacological indications, validating their traditional use and confirming the utility of popular information in the search for useful plants. the synergic action of binary mixtures of similar constituents of essential oils against larvae of the filariasis vector culex quinquefasciatus was also the inspiration behind research (benelli et al., a,b) on four apiaceae species: trachyspermum ammi, smyrnium olusatrum, pimpinella anisum, and helosciadium nodiflorum. initially, all the essential oils proved to be highly toxic to the larvae, but short-term exposure to both binary mixtures strongly reduced emergence rates, fertility, and natality of the c. quinquefasciatus that survived after the treatment at the larval stage. in addition, larvicidal acute toxicity of essential oils main constituents, i.e., germacrone, isofuranodiene, and (e)-anethole, were carried out, with lc being . mg l À , . mg l À , and . μl l À . the results demonstrated the promise of these essential oils and their constituents to develop cheap and effective mosquito larvicides. in another paper (pavela et al., b) published in the same year, the vector target was the same but the selection of the plant totally different, as endemic to madagascar. the reason is that in some parts of the world, there are interesting examples of endemic flora whose species could contain different essential oils and therefore different activity. for this reason, pharmaceutical companies often explore remote parts of amazonia or isolated zones in search of new active compounds. there were examples of exploitation of rare african rauwolfia species to obtain their indole alkaloids. working with endemic species is important considering that in many cases, populations are in limited numbers and at risk of extinction, and we need to identify their molecular treasure before they disappear. this was also the motivation for my trips to several parts of the world, focusing in particular on deserts and islands, in search of special plants. madagascar's fauna and flora are diverse and unique. when the unique gondwana continent braked up in several pieces, india started to move to asia living africa. however, a consistent block remained near to africa, becoming a great island, now known as madagascar. this happened more than million years ago. the isolation of madagascar gave rise to a particular case of biodiversity. this is the story of the beginning of madagascar, as far as we know. here, it is important to report that the island is characterized by at least seven very different habitats, each with different endemisms. the potentiality of cinnamosma madagascariensis, an endemic species widely present in the forests of madagascar, was reported to us thanks to the exceptional collaboration with professor philippe rasoanaivo, who had a deep knowledge of the flora of the island and their economic importance. this plant has important traditional uses ranging from management of dementia, epilepsy, and headache to malaria (rakotosaona et al., ) . few data have been reported about the chemical composition of its essential oils, and no studies have been published on its bioactivity against mosquitoes. once again, we first investigated the chemical composition of essential oils extracted from stem bark and leaves of the plant, and later their larvicidal potential against the filariasis vector culex quinquefasciatus. the reason was that when you have little information, you must consider that different parts of a plant can contain very different essential oils. in fact, gc-ms analysis revealed differences between the volatile profiles of leaves and bark oils. in the former, linalool ( . %), limonene ( . %), myrcene ( . %), and α-pinene ( . %) were the major constituents, while in the latter one, β-pinene ( . %), α-pinene ( . %), and limonene ( . %) were the most representative compounds. acute toxicity experiments conducted on larvae of the filariasis vector c. quinquefasciatus led to an lc of . and . μl l À for the bark and leaf essential oils, respectively. overall, cinnamosma madagascariensis bark and leaf essential oils against filariasis vectors proved to be promising, since they are effective at moderate doses. the insecticidal activity of the essential oil of another malagasy plant was also studied (benelli et al., ) . hazomalania voyronii is popularly known as hazomalana and its use to repel mosquitoes and resist against insect attacks has been handed down from generation to generation in madagascar. the property of the essential oils obtained from the stem wood, fresh and dry bark of h. voyronii were able to repel important mosquito vectors (aedes aegypti and culex quinquefasciatus). furthermore, the toxicity of the aforementioned essential oils was investigated by who on three insect species of agricultural and public health importance (cx. quinquefasciatus, musca domestica, and spodoptera littoralis), respectively, as well as the adequate topical application methods and compared with the commercial repellent n,n-diethyl-m-toluamide (deet). repellence assay revealed almost complete protection (> %) from both mosquito species for min when pure fresh bark essential oil was applied on the volunteers' arms, while deet % repelled more than % of the mosquitoes up to min from application. the research validated the traditional use of the bark essential oil to repel insects, although an extended-release formulation based on h. voyronii essential oils is needed to increase the repellent effect over time. furthermore, it evidenced the wide spectrum of insecticidal plants potentially useful in the fabrication of green repellents and insecticides useful to control mosquito vectors and agricultural pests, avoiding the utilization of synthetic products. another interesting study (benelli et al., b) was dedicated to helichrysum faradifani (asteraceae), which is a perennial endemic shrub growing in rocky and sandy places of madagascar. the ethnopharmacological about malagasy traditional medicine reports that this plant is used as a wound-healing agent, disinfectant, and for the treatment of syphilis, diarrhea, cough, and headache. the chemical composition of the essential oil distilled from the aerial parts of h. faradifani, and analyzed by gc-ms, evidenced that monoterpene hydrocarbons ( . %) were the major fraction of the essential oil, with bicyclic α-fenchene ( . %) being the predominant component. sesquiterpene hydrocarbons ( . %) were the second major group characterizing the oil, with γ-curcumene ( . %) being the most abundant component. its insecticidal activity was evaluated against second, third, and fourth instar larvae of the lymphatic filariasis vector culex quinquefasciatus by acute toxicity assays. the most sensitive were second instar (lc ¼ . μl l À ) larvae. for the third and fourth instar larvae, the estimated lc were . and . μl l À , respectively. finally, a different approach to volatile substances was performed, considering that smoke is often traditionally used against mosquitos (ansari and razdan, ) . therefore, the larvicidal, pupicidal, and smoke toxicity of senna occidentalis and ocimum basilicum leaf extracts against the malaria vector anopheles stephensi were evaluated (murugan et al., ) . in larvicidal and pupicidal experiments, s. occidentalis lc ranged from . (i instar larvae) to . ppm (pupae), and o. basilicum lc ranged from . (i instar larvae) to ppm (pupae). smoke toxicity experiments conducted against adults showed that s. occidentalis and o. basilicum coils evoked mortality rates comparable to the pyrethrin-based positive control ( %, %, and %, respectively). furthermore, the antiplasmodial activity of these plant extracts in antiplasmodial assays was evaluated against chloroquine (cq)-resistant (cq-r) and cq-sensitive (cq-s) strains of plasmodium falciparum. the s. occidentalis % inhibitory concentrations (ic ) were . μg ml À (cq-s) and . μg ml À (cq-r), while those for o. basilicum ic were . μg ml À (cq-s) and . μg ml À (cq-r). the high potentiality of the reported data must be considered. these smokes, as the essential oils, can be quite easily obtained in good quantity and low cost and therefore locally produced and directly utilized. this is important for countries with limited economic resources. the distribution of individuals in accordance with the boltzmann curve is the result of the current chemical-physical environmental pressure, concentrating the organisms of the species in the most adapted form. however, sooner or later situations are destined to change, and some of the individuals confined in the wings of the curve are ready to profit of the change and enter in the center, as soon as the conditions will be favorable to them. another consequence of this typical statistical distribution is the careful preservation of individual types inside the population. in practice, on the genetic point of view, the best species or the favored community do not exist in absolute, and any declaration or pseudo-scientific argumentation about the primacy of a race, also human, must be considered as a guilty stretch. as confirmation, this is also in accordance with the distribution of the constituents of matter at a subatomic level. the final consideration is that the chemical composition of a plant is limited, being the expression of the genome of the species, but it may change at any time in response to internal and external stimuli. let us use these concepts to evaluate insecticides used in insect-borne diseases. the interest in the use of biocidal products of natural origin began in the s and grew until the s, when it was obscured by the arrival of synthetic insecticides on the scene. for a long time, the pesticides scenario was dominated by synthetic products, until several factors caused a decline in their utilization. however, in the last years, interest in natural products has reappeared intensely, especially for the control of noxious insects at larval stage. this situation has matured, as is well known, following the indiscriminate (and not always necessary) use of excessive amounts of pesticides which, once released into the environment, are difficult to eliminate, as evidenced by the paradigmatic case of ddt (see chapter ). at the same time, incidence of insect resistance has increased, resulting in partial product inactivity and/or increasingly massive dosage requirements. all this led to a need for the formulation of a new generation of pesticides, and to focus research and production efforts on natural products. in , the world health assembly reported in resolution . , section . , the need to develop bio-insecticides. slowly but inexorably, the pesticides market registered the rise of biopesticides from natural products. the change in favor of natural products is the result of two concomitant facts: the evidence of the environmental damage due to massive utilization of synthetic products, and a new and growing sensibility in favor of respect for habitats, asking for more compatible solutions. in the current search for the production of a new generation of pesticides, useful for mankind's battle against superbugs and other threats, to face challenges to food supply and health, plant sources play a relevant role. the current prevalence of natural products evidences that a consistent number of biocides and antibiotics have been obtained from substances produced by living organisms, which are part of the great book of mother nature, whose lessons are still useful. that probably means that attention in chemistry is finally moving from the free synthetic approach to natural products, already selected during the long story of molecular evolution. in an article that appeared in acs' journal of natural products, charles l. cantrell and colleagues pointed out the impact of natural productssubstances produced by living plants, animals, and other organisms-on the production of pesticides. the article reports the percentages for registered insecticides obtained from new active ingredients in the period - (cantrel et al., ) . the paper's aim was focused on the impact of natural product and natural product-based pesticides on the u.s. market, obtained on the basis of nai registrations of new active ingredient registrations with the u.s. environmental protection agency (epa). the ingredients are categorized into four categories: biological (b), natural product (np), synthetic (s), and synthetic natural derived (snd). in particular, nps are considered substances produced by living plants, animals, and other organisms. the report evidences that nps, snds, and bs all have origins in natural product research. nps accounted for . %, ss for . %, bs for . %, and snds for . %, arising from the combination of conventional pesticides and biopesticides. in the registered conventional pesticides, the category of biopesticides alone registered an evident majority of nps (with . %), followed by bs ( . %), snds ( . %), and ss ( %). in contrast, on the conventional pesticides alone, the category s clearly dominated with %, followed by snd with . %, np . %, and b . %. the review indicates that in the same period, more natural products were registered as nais for conventional pesticides and biopesticides than any other type of ingredient. the authors report that when biological ingredients and natural products recreated in laboratories are included, more than % of all nais registered in that time frame have natural origins. more than two out of every three new insecticides approved in the last years are directly derived from natural substances produced in plants or animals or have significant roots in them. it is noteworthy that these numbers are very similar to those obtained if we compare with a similar projection concerning registered medical drugs in a similar period, and published in the same scientific journal. it is also noteworthy that a similar trend can be observed in the case of the registration of medical drugs in the period - , as previously reported in the same scientific journal by david j. newman and gordon m. cragg (newman and cragg, ): % of registered drugs directly or indirectly derived from np of secondary metabolism, % from b, % from snd, and only % from s. again, the details reveal differences in the sectors, with np and b dominating in anticancer and antibiotics, whereas the opposite concerns antiinflammatories, with s clearly dominating. these data, obtained on a total number of new approved drugs, are the results of several reviews that confirmed these percentages. in particular, the authors stress the role of microbes in the production of new drugs derived from natural products: "we wish to draw the attention of readers to the rapidly evolving recognition that a significant number of natural product drugs/leads are actually produced by microbes and/or microbial interactions with the "host from whence it was isolated," and therefore "it is considered that this area of natural product research should be expanded significantly." in other words, the future of pharmaceutical drugs could be related to natural products obtained by natural synthesis. once conceivable that the shift from synthetic pesticides to biopesticides seems to be incontrovertible, as fueled by the resistance phenomenon and the general tendency for "natural," the key argument is the choice of the raw material for the best bioinsecticide. as evident from the above reviews, bioinsecticides could be extracted from a living organism, like a plant, or produced by a living organism, like a bacterial strain by hemisynthesis, or obtained by synthesis in accordance with the structure of the active natural product. here, the debate is open between those affirming that "a molecule is a molecule" and those in favor of "original" natural products. anyway, in the case of an extract, the complexity of ingredients cannot be reproduced or performed by synthesis. the main characters of a natural "ideal" insecticide should be: biodegradability, environmental care, sustainability (obtained from renewable materials) and selectively (harmful to beneficial insects). it should also satisfy some conditions to be economic appealing and relevant, like be easy to produce, low cost, derived from raw materials that available and abundant in the country where the insecticide should be utilized. the last conditions are important to avoid accumulation by multinational agencies, as is happening for coffee and cacao. finally, but not in order of importance, the ideal natural insecticide must be able to compete in the market with the insecticides currently in use. the research for the ideal bioinsecticide is open, starting from the plant to be used. evident that this role should be assigned to neem. at that time, the tree was practically unknown in the occident, since its distribution and utilization were confined to the indian subcontinent. therefore, i wrote an article about neem to explain the importance of this plant, and some years later, i was contacted by an italian industry using neem oil because of the curiosity and interest raised by that article. the references about this plant and its use appear since time immemorial, as reported in the ayurveda and unani systems of traditional medicines, and even in the earliest sanskrit writings referring to the medical uses of fruits, seeds, oil, leaves, roots, and bark (gupta, ) . neem can be found all over the indian subcontinent since the time of the sanskrit-speaking aryans. so important was the species for the aryans, they even mentioned and included it in their sacred ayurveda, which is the sacred book of indian medicine. the species was later dispersed throughout the old tropics, including indonesia, either naturally or brought back by the ancient austronesian sailors after visiting and trading in india at least around years ago. through the centuries (kumar and navaratnam, ) , the medical importance of neem never waned in the indian subcontinent and it is now considered the "village pharmacy" for its importance in the ordinary life of indians, who use this plant to treat several illnesses (nix, ; girish and bhat, ) . many other news and references increased my interest. the marvelous tree, the problem-solving tree, the divine tree, india's tree of life, nature's drugstore, the pharmacy tree, the panacea for all diseasesthese are just some of the terms used to evidence the respect for this plant and its importance (ruskin, ; brahmachari, ; puri, ; national research council, ) . neem's relevance and its beneficial properties has been reported by the who/unep ( ), which considered neem as an effective source of environmentally powerful natural pesticide and one of the most promising trees of the st century for its great potential in pest management, environmental protection, and medicine koul and wahab, ) . furthermore, the u.s. national academy of sciences dedicated a report to neem, significantly titled "neem-a tree for solving global problems" (nas, ) . the importance of neem has increased exponentially in recent years. considering the enormous quantity of results and scientific data concerning the validation of medicinal and biological properties, the international scientific community included neem on the list of the top plants to investigate and use for the sustainable development of the planet and the health of mankind (tewari, ; foster and moser, ) . however, in the occident, insecticidal activity is the most common application for neem oil and its derived products. the plant, besides neem, is also known as nimba, nimtree, margosa, and indian lilac. in botany, neem is azadirachta indica a. juss and belongs to the meliaceae family. meliaceae are angiosperm rosidae, closely related to simaroubaceae and rutaceae (schumutterer, ) . the family includes genera and c. species. these are woody plants, like trees, shrubs, and shrublets, pantropically present, with a few temperate representatives in china and south africa. it is estimated that c. million years ago, two subfamilies diverged, cedreloideae with genera and melioideae with genera, including aglaia, dominating with c. species, and azadirachta with only two species. meliaceae are commonly known as the mahogany family, being known mainly for some important timber species, such as the true mahogany (swietenia mahagony). however, losses due to overexploitation and genetic erosion, as well as toxic effects on workers, limited the use of this true mahogany, nowadays widely replaced by spanish mahogany (s. macrophylla). azadirachta indica is an evergreen tree that grows up to a height of - m, but in favorable conditions it can to a height of about - m, with a trunk diameter up to . m (fig. . ) . the leaves of neem, composed of - leaflets, meaning - leaflets with a single terminal, are abundant, suspended by a strong and long petiole which lacks stipules, crowded near the ends of the branches. the leaflets are toothed, deeply serrated, their margins irregularly serrated, sharply pointed, and curved like a scythe. young leaves are pale, tender green, and tinted with rust, but during the favorable season the tree profits from the fresh, green color and shining surface of the leaves, giving a delicate and charming appearance. white small flowers are abundant, very fragrant, bisexual, or staminate in male exemplars. they are arranged in clusters at the axils of the leaves. they present five separated petals arranged in the form of a star. they appear in spring, and open in the afternoon giving out a delicate smell, which increases during the night. the fruit is a smooth, yellow-green, small, round drupe with a sweetflavored pulp. during the monsoon, when the flowers have fallen and the tree is in full foliage, the curved, toothed leaves, massed round the branches, have a distinctive appearance, which is easy to recognize. from march to may, the flowers, with five whitish petals, appear in great numbers on long, drooping stems. flowers are used to produce a bitter honey. the fleshy fruits are purplish-black, single-seeded drupes, which turn yellowish when ripe. elliptical in shape, they have a sweet-tasting juice loved by birds and bees. however, after the rains, the fruits change, giving off a strong unpleasant smell. in autumn, fruits fall down in great quantities if not harvested. the tree is believed to be native at least of north-east india and burma, or indonesia, but now widely distributed in the indian subcontinent, and it grows naturally throughout the dry regions of the country. it is usually planted along roads and avenues in towns and villages, because it grows fast and easily, and has an irregularly rounded crown with a canopy of leaves, making it a useful shade tree ( fig. . ) . the central regions of india are considered the patria of neem. if you go to coimbatore, in tamil nadu, you can find neem trees everywhere, in towns and in the countryside. it is mainly used for shade, lining streets or in most people's back yards. in india, it grows throughout the states of uttar pradesh, bihar, west bengal, orissa, delhi, maharashtra, gujarat, and andhra pradesh, but the original natural distribution is obscured by widespread cultivation. cultivation is easy, since neem usually grown from seed but can be propagated also from cuttings or root suckers, and it is a fast-growing species. potential utilizations of neem concern human, animal, and environmental health. the last one is a recent but very important acquisition. neem is cultivated for two main reasons: environmental care and the production of seed neem oil. the tree's tolerance and adaptation to hot and dry climates has made it one of the most commonly planted species in arid and semi-arid areas (tiwari et al., ) . the survival capacity of neem is mainly due to its highly expanded root system. neem trees are extremely useful to counteract desertification and furnish the only source of wood in arid and nutrient-deficient zones. the plant does not need particular care and grows rapidly up to m tall. neem trees attain maturity in - years in areas where the sunlight is intense, weather is warm, and good well-drained soil is found. the tree is stable in windy zones and can live for years or more. to survive in arid climates, neem depends on a wide strong root system with a deep tap root and extensive lateral roots, which are ideal for soil conservation. furthermore, its planetary presence can contribute to positive carbon sequestration to minimize climate changes, considering that adult neem trees can retain ae . g of co per m and per hour, which means - tons of co per hectare. therefore, neem trees can be considered to be air purifiers as well as air fresheners. for all these reasons, neem is widely cultivated in warm countries, and its areal distribution is expanding rapidly by massive cultivations in sub-tropical regions of america (caribbean cuba, central and southern america), asia (nepal, pakistan, bangladesh, sri lanka, myanmar, thailand, malaysia, indonesia, iran, china, turkey, indonesia), africa (kenya, cameroon), and australia. the cultivation of neem trees is in particular increasing in the drought-prone areas, like in south arabia (arafat valley) and in the uae. in , northern nigeria, thanks to the governmental project arid zone afforestation (azap), saw , neem trees being planted. in europe, some cultivations are reported only in southern spain and portugal (sara and folorunso, ) . the presumed current global neem trees presence and production are reported in table . . the data were obtained by cross-referencing several sources and are only indicative, considering that a real census was never completed in many countries (in particular in china) and many plantations are in progress. india is still by far the homeland of neem, but the scenario has changed rapidly in the last years and will continue to do so in the future, as can be deduced from the data in table . . visiting oman, i noted the presence of planted neem trees in areas where acacia was the only other woody plant (usually the only plant) able to survive. the neem tree is resistant to drought and it grows in many different types of soil, but it thrives best in well-drained deep and sandy soils. normally, it flourishes in areas wherein the neem is a life-giving tree, especially for dry coastal, southern districts. its capacity to survive in arid zones improved cultivation in sub-arid to sub-humid conditions, with annual rainfalls between and mm. it can tolerate high to very high temperatures, but it does not tolerate temperatures below °c, making cultivation in temperate climate very difficult. however, the future worldwide distribution of neem is not predictable. indians are convinced that it will be difficult to obtain similar ideal conditions to their country and others in the orient; however, the story of the cultivation of cinchona by dutch botanists in indonesia tells us that the results of the cultivation can be successful and the results even superior. in the complete linnaean binomial name of neem we found a. juss, which is the mark of the author of this species, designating the scientist who first published the name and a complete and scientifically reliable description of the species. a. juss refers to antoine laurent de jussieu ( - ), a great french botanist, who was the first to publish a complete and valid natural classification of flowering plants, named genera plantarum, published in , the same year as the french revolution, surpassing the sexual system presented by linneus. antoine laurent jussieu was the member of a family of a plant enthusiasts; his uncle was the botanist bernard de jussieu, whose transferred knowledge and unpublished work were the starting point of the book of antoine laurent, and his son andrien-henri also became a botanist. the merit of his work was the use of multiple characters to define taxa. in this approach, he achieved a significant improvement over the "artificial" system of linneus, mainly based on the number of the reproductive characters, i.e., stamens and pistils. many people know the work and name of linnaeus, but the impact of jussieu's work was fundamental in taxonomy, founding the principles that served as the foundation of plant classification in a natural system. many presentday plant families are still attributed to him, as the species is to linnaeus. that's what who can find in ordinary sources of information. the consequent idea is that jussieu, though living in france at a historical revolutionary time, was totally dedicated to botany, but his work was also revolutionary, through a strange pathway. deeper information about his life is a source of important lessons. his uncle, bernard de jussieu, invited the young antoine to paris, where he was trained in medicine for years. however, his uncle, via his position as a demonstrator at the jardin du roi (royal garden), had other plans for antoine. he guided his nephew's studies and prepared him for a lecturer's position at the garden, which was soon to become vacant. at just -years old, antoine was transferred to that position and his botanical training was limited because the subject then was viewed only as an accessory to his medical course. the inexperienced jussieu had to study botanical topics by night, since he had to teach by day. using the plants in the garden to teach plant morphology, which were arranged according to the current artificial system of joseph pitton de tournefort, jussieu started to realize the inadequacy of that system. this progressively changed his interest into a true passion for botany, and classification began until, as part of an application for a place at the academy of sciences, he produced a treatment of the ranunculaceae, starting a complete revision of the plant taxonomy system. continuing in his study, it became apparent to jussieu that the artificial system of tournefort was inadequate, and from he began arranging the plants in the royal garden in his own way and finally transferring the knowledge in the construction of his own system of plant classification. despite the initial success obtained by linnaeus' sexual system, it was clear to jussieu that the swedish naturalist had used a counting method, whereas it was necessary to grade the characters, considering some of them more important than others, depending on how variable they are within a species. this was a necessary lesson to consider the variability of living organisms correctly. however, he continued practicing medicine, chiefly devoting himself to the health of very poor people. in , he was put in charge of the hospitals and charities of paris. in the final years of his life, jussieu, by then almost blind as well as deaf, dedicated the last part of his extraordinary life to meditation and prayer. the neem tree has few diseases and enemies (boa, ; schmutterer, ) . in general, it is considered a very resistant and healthy plant . however, it is possible that after its spread around the world, something is changing. we must move our focus from india to the new settlements, in africa. in northern nigeria, neem now is planted in towns and villages, as a highly evaluated source of shade and firewood, as well in the establishment of shelterbelts. in nigeria, like in other parts of africa, the small twigs of neem are used to clean and whiten teeth, in consideration of its antibacterial properties. in particular, reports of pests and damages comes from east africa, like gall mites (phyllocoptes sp.) on older plants, but the most potentially dangerous pest is aonidiella orientalis, known as oriental scale (ofek et al., ; lale, ; elder et al., ) . this insect was widespread in western kenya but is not currently harmful. likely to be native to asia, it has been introduced to many regions via shipments of plants and then began its slow spread. some ports check for this and other scales in plant shipments. in africa, neem has been widely planted in the sahel region. oriental scale first appeared in the sahel during the mid- s and caused widespread damage to neem trees planted there. infestations were first detected in nigeria in along its border with cameroon and by the mid- s, widespread damage had been reported to neem trees throughout northeastern nigeria. the oriental scale is a flattened, circular or oblong insect, about . mm in diameter. it varies in color from yellow to light reddish brown. it frequently forms large colonies and sucks the sap of small stems and branches, which is phloematic sap, rich in sugars and other organic substances. infestations often spread to the foliage, fruits, and even seeds. feeding damage causes the foliage to die, giving infested trees a burnt appearance. this is followed by progressive dieback of branches and eventual tree mortality. the heaviest infestations appear to be on large trees located either in marketplaces or around human settlements. the female attaches to the surface of a plant and causes the disease by the larvae, which roam the plant, feeding on sap by inserting their stylets, sucking sap, and weakening the plant progressively. the physical damage includes discoloration and deformation of leaves. flowers and fruits fail to develop. it is noteworthy that the effects of the pest attacks are very similar to those already reported for olive trees, including exsiccation of leaves and wood. the lesson is that, in this outbreak, we have an explosive negative mixture of alien species and man's activity on the habitat. the enormous spread of neem trees in recent years is an unusual phenomenon, whose consequences should be better monitored. before going into detail about the constituents, we must consider the typologies of the forms of the marketed products containing natural products. we are referring to insecticides, but the same considerations are simply applicable to nutraceuticals, phytomedicines, cosmetics, and even food. it is possible to find the plant drug, meaning a part of the plant utilized. the plant drug is usually utilized exsiccated, or as a derived product, like an extract, resin or oil, which can be obtained as such, or be enriched in one or more constituents, which are considered responsible for the activity. in this sequence, the original starting point, which makes the plant useful, has been betrayed in favor of increased efficacy. however, following this treatment, there are products registered as food supplements containing % of a pure substance and usually the origin is not at all natural (although this is not declared on the label). in such cases, the product is more similar to a medicinal drug than an extract and it should be considered and used in medicinal form. the distinction between such marketed products is not evident and not reported to the unaware consumer, who may well prefer the product due to its apparently "natural" origin. knowledge of the chemistry is therefore fundamental and the basis of any decision about the appropriate use of a plant. however, we cannot know exactly what is inside a plant. all our methods of investigation are limited and may be misleading, although papers and books are full of information about the compounds contained in plants or their derived products. this is the consequence of plants' extreme chemical complexity. in a single leaf of hemp, more than constituents have been detected, considering the secondary metabolites alone, and hemp can contain high levels of thc or cannabinoids can be practically absent. in such cases, the morphology does not give any help and only a reliable chemical analysis can indicate what kind of hemp we are handling. since its beginning, phytochemistry has sought knowledge of all natural products. in about one hundred of years of activity, innumerable analyses were made and an enormous quantity of data collected in a very useful data base, but recent advancement in analytical devices and novel interpretation ask for a revision of the result of this job (kaushik et al., ; forin et al., ) . we must remember that the molecular world is not detectable by our senses and therefore we have secondhand and probably only partial information. sometimes, this information is considered sufficient to assign the compound/activity relationship, but only until other analyses confirm the presence of other molecular candidates. the seeds of neem contain at least identified biologically active compounds (govindachari, ) . among them, major constituents are nortriterpenes, named limonoids, i.e., azadirachtin, nimbin, nimbidin, nimbolides, and many others (ragasa et al., ) . however, each year other new limonoids are discovered. more constituents mean more possible activities. preparations from the leaves or oils of the seeds are used as general antiseptics (mossini et al., ) . due to neem's antibacterial properties, it is effective in fighting most epidermal dysfunction, such as acne, psoriasis, and eczema. ancient ayurvedic practitioners believed high sugar levels in the body caused skin disease. neem's bitter quality was said to counteract this sweetness. during the last desert locust plague in africa, it was noticed that these insect, schistocerca gregaria (forskal), ate almost any vegetal around, leaving a bare landscape when they fly away, except for neem trees, probably because of the antifeedant effect of the very bitter leaves, due to the presence of limonoids. traditionally, indians bathed in neem leaves steeped in hot water. since there have been no reports of topical application of neem causing adverse side effects, this is a common procedure to cure skin ailments or allergic reactions. neem also may provide antiviral treatment for smallpox, chicken pox, and warts, especially when applied directly to the skin. its twigs are commonly used to clean and disinfect teeth. the brushing of teeth with neem to prevent gum diseases and for teeth whitening is very common, and not only in india. there are also various kinds of natural toothpaste on the market that contain neem extracts. it is also possible to prepare a homemade toothpaste to achieve shiny, cleaner teeth. neem powder is made by grinding dried neem leaves, which is traditionally used by mixing one teaspoon of neem powder with one teaspoon of baking soda and enough water to make a useful paste. these preparations can help to avoid the plaque and tartar that build up in gums, which are the root causes of bad breath. in addition to cosmetic uses, neem's antimicrobial activity to maintain dental health is also worth noting (chava et al., ) . a preparation can be obtained by boiling neem leaves in water until they reduce to a quarter of the original volume. finally, gargling with this concoction contributes to good breath and whiter teeth, as it kills the bacteria inside the mouth. the teas of neem leaves are utilized in indonesia and oman for their digestive properties (sujarwo et al., ) . neem's effectiveness is due in part to its ability to inhibit pathogens from multiplying and spreading (benelli et al., ) . neem produces painrelieving, antiinflammatory, and fever-reducing compounds that can aid in the healing of cuts, burns, sprains, earaches, and headaches, as well as fevers (chopra et al., ) . several studies of neem extracts in suppressing malaria have been conducted, all supporting its use in treatment. neem has broad applications to human and animal health, as well as organic farming (bhowmik et al., ) . it is reported as a powerful antiviral and antibacterial, with peculiarities that set it apart from other herbs in that class of broad antimicrobials (sandanasamy et al., ) . neem oil is also commonly added to a variety of creams and salves. it is effective against a broad spectrum of skin diseases including eczema, psoriasis, dry skin, wrinkles, rashes, and dandruff. these are just a few examples of the possible utilization of neem, and the potentiality of neem is considered by everybody, including the onu and other institutions, to be very high, but so far little has been done to develop appropriate products from it to help mankind. in particular, considering insect-borne diseases, in vivo activity of neem seed oil (nso) against malaria plasmodium has also been reported (dahiya et al., ; trapanelli et al., ) . today's exploding growth in human population is seriously depleting the world's natural reserves and economic resources. unless the runaway human population growth rate is slowed down, there will be little hope for raising everyone out of poverty in the developing world. besides educational constraints, the nonavailability of inexpensive methods of contraception, which do not cause trauma or impose on the esthetic, cultural, and religious sensitivities of people, limit the success of birth regulation programs. however, recent findings indicate that some neem derivatives may serve as affordable and widely available contraceptives. a recent controlled study in the indian army proved the efficacy of neem as a contraceptive. in , the report of the washington-based international food policy research institute predicted a world even more unequal than the present, with food surpluses in the industrialized world and with chronic instability and food shortages in the global south, particularly in african countries. the main product of neem seed oil (nso) is the fixed oil obtained by expressing the seeds, still enclosed in the kernels. therefore, the fleshy pulp is removed or dried, to obtain the inner part. nso can be obtained by different extraction methods. most nso is produced in india by small-scale producers at ordinary temperatures using very simple machinery, which is utilized in other periods of the years for other oily extraction, like arachnids or soya seeds (figs. . - . ). however, modern apparatus is also used in india and many other countries are now producing and refining nsos. therefore, considering also the possible different geographical origin of the raw material, combined pre-and postharvesting factors can result in great differences in constituents present in marketed nsos, as already reported. therefore, despite the common definition for all the oils obtained from kernels of neem, it is necessary to consider that there is no single nso, but many nsos differing in shape, color, viscosity, chemical constitution, and activity. medicinal and cosmetic utilizations are relevant and continuously increasing. cold pressed neem oil is commercially known as margose oil and considered as pressed directly from seeds. there are hundreds of marketed products worldwide based on margose oil. neem, or margose, oil has a brown color, a bitter taste, and a garlic/sulfuric smell. the oil is usually obtained by simple pressing, but extraction by organic solvents, like hexane, is also used, though in such cases traces of the residue of solvent are always present in the final product. the type of insecticide is commonly registered as biocide, insect repellent, and antifeedant, intended for use on outdoor and greenhouse agricultural food and ornamental crops as a repellent and insect growth regulator. the products are considered to have no risk to human health because of their low toxicity via all routes of exposure. there is no reason to believe that any nontarget organisms, including honeybees and other beneficial insects, would be adversely affected, as tested by the environmental protection agency (epa). the environmental protection agency (epa) is probably the most important agency in charge of environmental care and health. it is an agency of the united states federal government whose mission is to protect human and environmental health. the epa is also in charge of the regulations of carbon emissions from power plants, automobiles, and other contributors to climate change. the epa became popular in europe because of a civil enforcement case against volkswagen and other car manufacturers, subject to reservations set forth in each of the partial settlements. in , the u.s. department of justice resolved a criminal case against volkswagen ag with a plea agreement for the offenses of conspiracy, obstruction of justice, and entry of goods by false statement, and the u.s. customs and border protection resolved civil fraud claims with volkswagen arising from the illegal importation of affected vehicles. the epa was established in december by an executive order of president richard nixon, with headquarters in washington, d.c., in response to widespread public environmental concerns that gained momentum in the s and s. epa is a giant public agency with a budget of billion us $, and it is born as reaction to the public movement in favor of the environment due to the carson's book (see chapter ). it is considered reliable because independent. the epa is responsible for creating standards and laws to protect and preserve the natural environment and improve the health of humans by researching the effects of and mandating limits on the use of pollutants. the epa's aims include the regulation of the manufacturing, processing, distribution, and use of chemicals and other pollutants. in addition, the epa is charged with determining safe tolerance levels for chemicals and other pollutants in food, animal feed, and water. the best presentation of neem insecticide properties and the rationale of its utilization is the report of the epa about the registration of cold pressed neem oil, concerning a product named plasma neem. the report has a significant subtitle: "reasons why neem oil is an effective way to control insect hoppers" (epa (us environmental protection agency), ). the target of the product cold pressed neem oil is insect hoppers, because of their special liking for cash crops. the consequence is a crisis because of insect hoppers infesting these cash crops by chewing and sucking the leaves, as reported by many farmers. the reasons to use neem are focused on the effects of chemical fertilizers, which "provide a remedy but tend to kill beneficial insects as well." the report identifies three reasons why neem oil should be considered an effective way to control insect hoppers which feed on cash crops. reason : selectivity. "neem oil is usually sprayed on the leaves and stalks of a cash crop. so it is aimed at only the insect hoppers who spoil the cash crop by chewing the leaves or biting off bits of the stalk. the insect hopper which attacks the plant by consuming it will end up consuming the neem oil as well and hence die as an aftermath. beneficial insects, who replenish the soil in a natural fashion, do not consume the plant and hence do not get affected by neem oil." reason : eco-friendly efficacy. the content of azadirachtin in neem oil is considered very good at controlling and eliminating insect hoppers, avoiding the negative effects on the productivity of cash crops. furthermore, the neem oil exerts no damage to the soil or disruption of the chemical composition of a fertile soil. the product affects biological functioning of the insect hopper, and therefore the insect hopper forgets to feed or breed after devouring a cash crop with neem oil sprayed on it. the conclusion about this point is clear: "this leads to the complete elimination of the insect hopper and the infestation cycle without any other adverse chemical side effects." the action is mainly larvicide, but also deterrent and adult insecticide in most species. reason : cost-effectiveness. the utilization of neem oil for preserving cash crops from insect hoppers is considered very cost-effective. in comparison to chemical fertilizers, neem oil is expensive, but other positive effects must be evaluated. there is another consideration about the cost/benefit effect: eliminating negative insects and worms by neem oil, farmers need not spend money on buying supplements for the enrichment of soil. the heavy investment to preserve soil quality is totally avoided when a farmer uses neem oil. in conclusion: "overall, it can be said that neem oil as a source to control insect hoppers in cash crops is extremely beneficial for farmers." the final consideration of the epa is the advice not to use the content of the report as an endorsement of nso. in fact, it is important to remember that the report is based on scientific and experimental data. the epa is not working in favor of the industry or the market, but its judgments must be considered objective although its aim is environmental improvement. nso, obtained by the cold expression method, is the only natural productderived insecticide, whose registration was approved by epa. the epa's authority is so far internationally recognized. therefore, in the report there are three main key items of information: the evaluation of the selective insecticide activity; the preference for synthetic products in consideration of the eco-friendly properties; and the indication of azadirachtin as necessary for the activity. let us consider the report as a guide and evaluate each of these points. later, we will add other considerations. it is noteworthy that all these indications can be found already reported and present in the information about the ethnobotany and traditional medicine of neem. in the cultivation of rice, when the plantlets are underwater, the addition of extracts of neem to the usual fertilizer, not only decreases the mosquitoes number, but also has beneficial effects on the production. the utilization of neem to increase soil fertility and treat medicinally plants and livestock is recommended in the upavanavinos, an ancient sanskrit book on agriculture. the dried leaves and the oil are used as preservatives against insects and microorganisms for the postharvesting conservation of foods. conservation is guaranteed for more than a year. panels derived by the extraction of the oil are used as food additives for livestock, and the animals are washed with this diluted oil to prevent the attacks of parasites or harmful insects. in the last decade, the focus was on the potentiality of a new analytical technique, hptlc (high performance thin layer chromatography) ( fig. . ) . hptlc is the last evolution of planar chromatography and allows the evidence of most of the constituents of an extract in an identifying track, named fingerprint, wherein identification of constituents can be obtained by direct comparison in the same plate with the correct standards, utilizing the rf value and the reaction with adequate derivatization (nicoletti et al., a,b) . improvements in separation and visualization of the spots are obtained by reduction of the size of the particles of the silica gel, constituting the fixed phase. the mobile phase can be selected on the ample repertory of solvents and their mixtures, as well as the several methods of derivatization and detection. the final effect, in comparison with ordinary planar chromatography (tlc), is like a myopic person wearing glasses. the advantages in comparison to the tlc are in the total control of the environmental conditions and the automatization of the procedures. each step of the analysis is performed entirely by a series of devices, and the operator is only asked to produce the program of actions by the software. plates can be visualized and derived in several ways, obtaining multiple information (figs. . and . ). they can be easily preserved and stored as digitalized images inside the computer, and immediately sent everywhere or compared with a data bank. however, the most important feature of hptlc is that the results of the analyses are very clear, thanks to careful preparation work that enabled optimal chromatographic conditions, as demonstrated by the quality of the images. in other words, our idea was to "see the molecules" (nicoletti, ) and obtain simple and clear evidence of the metabolic production. molecules are too small to feel their presence with our senses or directly by any sort of device, but it is possible to evidence their chemical properties in the plate, recording the rf value, the fluorescence, and the color after derivatization. an hptlc or nmr graphic needs an expert for correct interpretation, such as any specialized analysis, like a cardiogram . in hptlc, without any knowledge of chemistry, the presence or absence of a determined spot is evident (figs. . and . ). hptlc was selected to obtain a metabolomic approach, meaning the study of many constituents as possible, focusing on secondary metabolites (toniolo et al., ) . metabolomic is one of the -omic sciences generated by the dissection of the dogma of biology, based on the sequence dna➔rna➔proteins. it is necessary to propose some comments about the dogma of biology and why its crisis generated a series of other points of view. first of all, the use of the word "dogma" should be avoided in biology, since the matter is more complicated than a simple sequence, as actually happened. the central dogma of biology was first proposed in by francis crick, as a consequence of his discovery of the structure of dna. the dogma describes process by which the instructions contained in dna are converted into a functional product. another definition is: "the coded genetic information hard-wired into dna is transcribed into individual transportable cassettes, composed of messenger rna (mrna); each mrna cassette contains the program for synthesis of a particular protein (or small number of proteins)" (sources: definition from chapter : the dynamic cell, of molecular cell biology). the flow of genetic information within a cell follows the sequence: dna codes for rna via the process of transcription (occurring within the nucleus), rna codes for protein via the process of translation (occurring at the ribosomes), and proteins are responsible for the synthesis of the other metabolites (proteins are spread everywhere). cell data are organized within the database of dna and reversed in the metabolic flux, through rna. although clearly deficient, the central dogma of biology dominated genetics for decades, but through ongoing research, many exceptions were discovered. for example, most dna is silent, since it does not encode proteins. retroviruses, which are relevant for our arguments, present the possibility that rna transcribes into dna through the use of a special enzyme called reverse transcriptase, and other cases of deviance can be reported. however, the biggest revolution consists in the direction of the arrows. it is necessary that information could follow also the reverse pathway, allowing an appropriate response by the genome potentiality. therefore, at least the dogma must be rewritten with two-way arrows. in principle, a metabolomics study should be the determination of the pathway of cell production from the genome through transcription, but the term "metabolome" is now used to evidence the whole pool of metabolites, in particular for natural products, whereas transcriptomics is related to proteins. transcriptomics involves serious difficulty to obtain reliable results. a protein seems perfectly comfortable inside the cytoplasm, but outside, irreversible denaturation causes definitive degradation and consequent difficulty in understanding the protein's functionality. in contrast, small molecules are more stable in any environment and their molecular structures at least can be determined by phytochemical analysis (nicoletti and toniolo, ; toniolo et al., ) . however, in the metabolome we have hundreds of thousands of different constituents to be studied, and the classic approach to study the molecules one by one is impracticable, and other methods must be utilized. the lesson is that the role of any metabolite cannot be discarded a priori, and also a secondary influence in the evaluation of the property of an extract can be important to definite and obtain the final reaction. once again, the "magic bullet" paradigm is under discussion, but the total utilization of plant extracts must also be considered an unsatisfactory solution. the aim of our approach was to adapt the method to other subjects outside the pharmaceutical applications. therefore, our first studies focused on the determination of adulterants in nutraceuticals and other pharmaceutical products, like the "green viagras." later, we adapted the method and the devices to use the metabolome as a source of information about what is going on in a complex system in which living organisms are acting. therefore, we are able to study the effect of environmental factors, like ozone, on the quality of wine (valletta et al., ) . however, probably the most impressive application was the study of the environmental effects of the costa concordia disaster (toniolo et al., ) . on the night of january , , the costa concordia, a giant yacht with approximately cabins, passengers, and crew, was wrecked off the rocks of the italian coast a few hundred meters from the port of giglio, a little island on the tyrrhenian coast in tuscany. like an injured helpless mastodon, the cruise ship inclined dangerously, until the inclination stopped with most of its starboard side under water. because of the inclination and the amount of people, the overnight evacuation of the costa concordia was a challenging process, and people died. the cost of removing the ship was us$ million. for scientists like us, interested in environmental damage, it was a unique occasion. for year, months, and days, the enormous hull of the boat altered the underlying marine habitat, interrupting the normal flow of sunlight over a surface of more than , m . the seagrass posidonia oceanica was chosen as the target organism of the impact evaluation, since, like in other parts of the mediterranean sea, it forms large underwater meadows. using hptlc analysis, it was possible to determine the health of each collected plant and make a map of the metabolic damage, which accorded with the shadowed area. however, albeit the negative conditions, the rhizomes turned out to be mostly still alive and able to reproduce the meadow again. therefore, the final task of our research was simply to wait until nature carried out its work. however, there is a further chapter of this story, written after our study. to remove the ship, a platform was transported from north europe. the problem was that the bottom of the platform was full of mytilus. when the platform was exposed to the hot mediterranean sea temperature, the mussels died, releasing their bodies, covering down the sea background and causing a further source of damage. a clear example of human stupidity and superficiality. anyway, devoted to our task, we are now repeating our analyses to understand what happened and what is still going on, relying on the quality and reliability of our indisputable results. the study of neem oil was based on the experiences obtained by improving the hptlc devices via the metabolomics approach. the central idea was to collect as much information as possible about the constituents of the neem products, without any preference for any kind of metabolite, considering any product and any extract like a unique molecular system. in the hptlc analyses on nsos, the objective was to achieve the total chemical characterization of the used oil, and then the derived products, by means of the production of a chromatographic reference profile of the metabolites' production. this objective is not easy to achieve due to the complexity and variability of neem oil. neem products are subjected to great variation in composition, due to preharvesting factors, like environmental situation, genomic differences, influences of the habitat, and others, including postharvesting situations, like harvesting and stocking, treatment of the raw material, separation of different parts, extraction methods, production of the final product, and others. in fact, analyzing different marketed neem oil from several productions and countries, we decided that it was not possible to refer to a single neem oil, but to neem oils in the plural, due the great differences in composition. therefore, we decided to obtain and adopt a reliable reference metabolomic hptlc profile for the neem extracts or products to be utilized in our biological experiments in vitro and in the field. in fact, one of the typical problems in activity tests is the differences in raw material giving rise necessarily to different results in activity and utilization. another important aspect of our metabolomic study was that the complexity of the neem profile was even greater than expected. this result is the consequence of the generalist approach. in other molecular chromatographic or spectroscopic analyses, like hplc, the result is sub judice on the detector's settlement. therefore, if the molecule does not possess the adapt chromophore, the molecule, even if it is the main component, is invisible to the detector. in hptlc, there are universal derivatization methods, like h so , to reveal the organic substances, but it is possible when necessary to adopt a particular agent. in this way, it was possible to exclude the presence of a relevant percentage of azadirachtins and the occurrence of other constituents relevant for the activity. this is another recurring lesson for those studying natural products. although a plant has been the object of several phytochemical studies, new constituents can be obtained. an example is the discovery of gossypol in cotton oil. insecticidal activity is reported in a hundred or so published papers, concerning a wide range of species of arthropods, as confirmations of many traditional uses (schumutterer, ; amirthalingam, ; jones et al., ; van der nat et al., ; biswas et al., ) . leaves are used in houses to repel and keep away insects. when half of a sample of soya leaves are sprayed with nso and offered as food to the japanese coleopteran (popillia japonica), the insects feed only on the nontreated parts of the leaves. in nicaragua, farmers spray their cultures with an aqueous extract obtained by leaving the seeds for h in water. in general, nso-based products have proven to be very effective against a huge range of pests of medical and veterinary importance, mainly including mosquitoes. the insecticidal properties of neem and its many formulations are based on experimented antifeedant, fecundity suppression, ovicidal and larvicidal activities, including growth regulation and repellence against a great number (around ) of different insects, also at very low dosages, whereas useful insects were shown to be unaffected isman, ; sharma et al., ; schumetter and singh, ; forin et al., ) . the deterrent activity was also important, which can be easily determined as reported in fig. . . other studies, like the molting and the growing of the selection under investigation, need special devices, as those reported in fig. . . in particular, a concentrated extract of neem seeds, named mitestop, developed by the university spin-off company alpha-biocare (d€ usseldorf, germany), proved to be very effective against a huge range of pests of medical and veterinary importance, including ixodes and rhipicephalus ticks, house dust mites, cockroaches (blatta, blattella, and gomphadorhina), raptor bugs (triatoma), cat fleas, bed bugs, biting and bloodsucking lice, poultry mites, and beetle larvae parasitizing the plumage of poultry. neem leaves can also be used to protect stored woolen and silk clothes from insects. concerning mosquitoes, emulsified formulations of a. indica oil showed excellent larvicidal potential against different mosquito genera, including aedes, anopheles, and culex, also under field conditions. insect growth regulatory activity of neem-borne molecules alter or block the metamorphoses of larvae (toniolo et al., ) . neem weakens the cuticle defense system of the young instars, causing easy penetration of pathogenic organisms, or interferes with the molting mechanism. concerning biological control, an increase of the control of aedes populations was observed after the combined application of predatory copepods and neem-based larvicidal products, since repeated application of nso does not affect populations of predatory copepods. however, relevant limitations are related to the relatively high cost of refined products and the low persistence on treated surfaces exposed to sunlight. in the soil, the half-life of azadiracthins, meaning the time necessary to degradate the compounds, is from min to days, depending on the environmental conditions, like moisture, high temperature, and sun. the breakdown is faster on plant leaves, due to the exposure and the surface. in the attempt to assign the active constituents of nso, we must consider that the chemistry of neem is very complicated in terms of numbers and types of constituents. despite the great quantity of dedicated research, chemical research is far from complete. hundreds of compounds have been isolated and identified from various parts of neem, with seeds being the most investigated for their commercial value. the seeds may contain approximately % of a brownish yellow oil, mainly constituted by several fatty acids, i.e., oleic acid cis- -ottadecenoic ( - %), palmitic esadecanoic acid ( %- %), stearic acid ottadecanoic ( %- %), linoleic acid cis, cis- , -ottadecadienoic ( %- %), and arachidic acid ( %- %), although several other compositions have been reported. after a certain time, fatty constituents tend to separate and appear as white amorphous material. the main characteristics of the oil are its unpleasant strong alliaceous odor and acrid taste, attributed to sulfurous constituents. the shape and consistence can be very different according to the extraction method and the source. in fact, the composition of neem oil is highly variable, depending on preharvesting factors, like the cultivar, the geographic and environmental origin of the raw material, collection seasons, and postharvesting, like the extraction method, preservation, and conservation. extraction can be executed with different apparatus, temperatures, pressures, and methods, affecting the yield as well as the content. as later reported, these aspects have been deeply considered and hptlc analyses can be utilized to ensure the chemical composition of the neem oil utilized in the biological experiments. among the c. compounds characterized from the neem seeds, more than one-third of them are nortriterpenoids, which are triterpenoids lacking some carbon atoms (kaushik et al., ) . nortriterpenoids are chemotaxomically well located in a few related families of rosidae angiosperm dicotyledons, i.e., rutaceae, simarubaceae, cucurbitaceae, and meliaceae, within the rutales order. generally, in the plants of the rutales order, the partial loss of the lateral chain is followed by a complicated rearranging of the remaining part, giving rise to different polycyclic molecular skeletons, full of oxygenated functional groups, partially acylated. syntheses of complex natural compounds are costly and therefore they are usually used only for special activities. it is necessary to consider this point, which is in favor of the use of the plants as source of these compounds, since the synthesis can reproduce the chirality of nortriterpenoids only with extreme difficulty and cost. nortriterpenes are a very interesting part of the plant's chemical ability, that we call biosynthesis, to produce active complex molecules. nortriterpenes present very complicated structures and high numbers of active parts. we must remember that in natural products, activity is based on the presence of functional groups, made by heteroatoms, which means mainly o and/or n. if nitrogen is present, you have alkaloids, otherwise the range is higher, comprising phenols, alcohols, ketones, and others. however, the introduction of an oxygen inside a derivative usually is obtained to increase the activity, but also introduces instability in the molecule. we must remember that a natural product started from co and is likely to become co again at the end of its life. this is the necessary turnover of atoms and energy in organic matter. the first process accumulates energy and it is based on reductive reactions (endothermic reactions), whereas the second one is based on oxidation and produces energy (exothermic reactions). in other words, life is based on subtraction of negative entropy from the habitat, and at the end of its life, the organism releases this energy to the system. during its life, the molecule is expected to carry out its role inside the biosystem, which is the reason for its synthesis inside the plant. to understand the role, the nature of the target is essential. in insect-borne diseases, the natural product should interfere in the life of herbivorous insects or dangerous pathogens. in the case of neem, the activity is mainly larvicidal, blocking the metamorphosis to the next pupal stage. the larvae are unable to develop and change their state. to obtain this result, a lot of chemical and finalized activity are necessary, in this case consisting of hormonal interference in the insect metamorphosis process. in other words, the molecule must be able to mimic the internal complex chemical apparatus that allows drastic changes in the forms of the insect, until it stops the process. let us consider in particular the class of nortriterpenes. we have already had occasion to meet monoterpenes in the essential oil constituents. owing to their biosynthetic origin derived from progressive accumulation of isoprene units, each made by five carbon atoms, terpenes are classified according to the increasing molecular weight in monoterpenes (c ), diterpenes (c ), triterpenes (c ), and tetraterpenes (c ). squalene, the unique precursor of all triterpenes, is a linear unsaturated hydrocarbon, but its derivatives, for stability reason, are all cyclized with hexagonal and pentagonal cycles in the final structure. among triterpenes, the most famous class is certainly the steroidal one. steroids are present in any organisms, where they carry out several fundamental roles. without steroids, starting from the cholesterol stabilization of cell membranes to the influence on metabolism, no cell, and therefore no organism, could survive. however, each organism synthetizes its own steroids. in fact, animals possess simpler steroids usually with few oxygenated functional groups, bacteria produces steroidal triterpenes mainly dedicated to the stability of the cell envelope, and plants biosynthetize quite complex structures, named phytosterols. generally, phytosterols possess more cycles respect to the ordinary structure of the steroid model and an increase of the number of functional groups (roy and saraf, ) . animal steroids are based on a simple, easy-to-remember sequence of four fused cycles: three hexagonal and the last pentagonal. the basic structure of a steroid is quite easy to write and remember by students, including the stereochemistry, whereas the structures of limonoids are very complex and not so easy to remember. the problem is that in the basic structure of a steroid the sequence of the cycles is linear, whereas in limonoids there are complicated re-arrangements, causing a circular total structure. the insect's molt and metamorphosis are triggered and directed by hormones, usually consisting of steroids, such as prohormones (pheromones or juvenile hormones) and ecdysones. the term "ecdysone" was introduced by the german biochemist peter karlson ( karlson ( - in in "chemische untersuchungen € uber die metamorphosehormone der insekten" (karlson, ) . the etymology of this word is interesting, from the greek ekdusis "shedding," or more precisely ἐκ(ς) ("external" or "from inner to out") + δύω ("dress oneself") + -si(s) ("action") + -ona ("hormone"). however, ecdysones have typical steroidal structures, whereas limonoids are the result of a complex chemical rearrangement. thus, to interfere in insect life, special triterpenes are necessary. several plants, like those in rutales and sapindales and related families, are specialized in the synthesis of such molecules, probably made to defend the plant from phytophagous insects. to obtain this result, great chemical ability is necessary. first, part of the typical hydrocarburic lateral chain, typical of most steroids, is lost ("nor" in chemistry means exactly this passage obtained by cutting c-c bonds), and the remaining part is both oxygenated and compressed in a complicated polycyclic structure, which is quite stable in the cell environment, but easily degradable in contact with atmospheric oxygen and sunlight. in this way, the nortriterpenes can be produced in the plant and transferred to the insect with mortal effects through a subtle toxic effect. the idea is to interfere with the growth regulators, interrupting the balance of hormones, named juvenile, in particular interrupting the transition process from the larval instar stages to pupae and adults by juvenile hormone analogues. therefore, on learning this lesson from nature, we can find solutions and inspirations. on the basis of these considerations and the structural diversity ( fig. . ), nortriterpenes can be divided into two main groups: limonoids (c ), with partial loss of the lateral chain (manners, ) , and quassinoids (c and c ), with total loss of the lateral chain (vieira and braz-filho, ) . in ancient times, plants containing these kinds of compounds were mainly famous for the bitterness of their drugs, utilized in the production of tonics, digestifs, and medicines. limonoids are part of our ordinary experience with some fruits and are crucial for the dissemination process. when we eat a citrus fruit, such as a lemon or an orange, we taste the agreeable flavor of the juice, but the seeds are discarded because they contain the nortriterpene limonin, which is very bitter and unpleasant. by throwing away the seed, we contribute to the reproduction of the plant. several other properties of limonoids have also been reported, including antioxidant, antimicrobial, and antitumoral activities, the insecticidal of neem being so far the most important. limonoids are considered the most active ingredient of insecticide neem products. they are classified into nine basic structures, with three main skeleton types: (a) azadiracthins, highly polioxygenated and acylated, with a saturated first ring, a tetrahydrofurane ring between the two first rings, and a final dihydrofurane ring chained with the other part of the molecule; (b) nimbins, less oxygenated and acylated with a skeleton evidently similar to that of the steroids, the furane ring with only a link with the remaining part of the molecule; and (c) a third type similar to the azadirachtins one, but the polycyclic part containing the dihydrofurane ring is less complicated, giving rise to a more linear general skeleton. such variability is necessary to sustain the large range of targets. in fact, these differences are just as important for the biological activity as for the decomposition. however, in terms of market considerations, azadirachtins, in particular azadirachtin a, are considered the reference constituents to evaluate the quality, and therefore the activity, of neem oil. azadirachtin a is a highly oxygenated c-secolimonoid, whose content in the seeds is highly variable ( . %- %), mainly depending on the producing zone and the seasonal trend. this compound acts as a biocidal on insects after ingestion or contact, with several effects: (a) interference on the growing processes, inhibiting the molting or blocking the hormone ecdysone synthesis; (b) antifeedant, with reduction of feeding; (c) negative effects on adult fecundity and egg fertility; and (d) diminution of the defense capacity of the cuticle, easing the penetration of pathogens. in particular, the larvicidal effect consists in the formation of the "permanent larvae," i.e., larvae are able to complete the molt as a consequence of destruction of the cuticle or of hormonal perturbation of the metamorphoses. this study consists in the careful observation of the larvae transformations and in the daily count of the consequence of azadirachtins and related compounds on the molting of phytophagous with buccal apparatus, either biting-sucking and chewing, comprised in all systematic categories: orthoptera including grasshoppers, locusts and crickets, etheroptera, homoptera, aphides, cicadellidae, hymenopterous, thysanoptera, aleurodidae, dipera, beetles, and others, including acarus and nematodes. neem's oil formulations usually show a range of different azadirachtins amounts, ranging from to mg/kg, meaning that products can be obtained either by using directly poor neem oil or a dilution process of neem extracts containing different quantity of azadirachtins, up to %. in addition to neem oil, azadirachtins are also marketed, in particular azadirachtin a. the amount of production of this substance amounts to about tons, with % coming from india, and china as the second producer. other data about the activities of nso and its products can be found in the references at the end of this chapter. our first experiments clearly demonstrated strong larvicidal activity of nso and neem cake on asian tiger mosquito (nicoletti et al., a,b) . however, our hplc and hptlc analyses showed a low content in azadirachtins in the nso and in the methanol extract . the result was interesting, since it is well-known that insecticidal activity is strictly related to the chemical composition, but in contrast to most reports evidenced a relation between the activity and the presence of these limonoids. this consideration prompted research to identify a relationship between composition and activity in the case of nsos marketed by different producers. first, the hptlc analysis indicated great differences in the fingerprints of the analyzed oils, with special reference to limonoids (nicoletti, ; toniolo et al., ) . a second analytical step consisted of a fractionation of three selected neem oils in three fractions of increasing polarities (i.e., ethyl acetate fraction (ea), butanol fraction (bu), and water (we)). the initial neem oil and the obtained fractions were evaluated for larvicidal toxicity and field oviposition deterrence against the asian tiger mosquito, aedes albopictus. the experiments showed good toxicity of the entire neem oil and ea fractions against a. albopictus fourth instar larvae (with lc values ranging from . to . ppm), while little toxicity was exerted by bu and we fractions. the differences of activity were in accordance with the results of hptlc analyses, since the nsos more concentrated proved to be more active. these results were confirmed by deterrence of a. albopictus oviposition in the field (effective repellence values ranging from . % to . %), while no effectiveness of bu fractions was found. concerning ovideterrent activity, no difference due to the production site was found. these experimental data evidenced the possible use of neem constituents against culicidae in the field. the constituents must be found in the apolar fraction, but the hptlc analysis showed a complex composition, wherein limonoids were not prevalent. therefore, neem oil and ea fraction seem promising, since they are effective at lower doses, if compared to synthetic products currently marketed, and could be advantageous alternatives to develop newer and safer mosquito control tools, but other studies are necessary to obtain a better definition of the active constituents and tailor the neem products in accordance with the required utilization (benelli et al., c; . therefore, when we started our work on neem products, we found several incongruities between the reported studies in the literature and our results . in case of incongruence of the experimental data, two main interpretations are possible. the anomalous data could be the consequence of some error in the experimental procedure or the previous reported data must be reconsidered on the light of the new ones. in fact, many scientific important discoveries have been as consequences of unexpected results. there is a strong tendency in pharmacology to assign the activity of a plant drug to one constituent, or eventually a few of the same chemical class. this is mainly a consequence of the pharmacological tests, which are tailored on the magic bullet axiom and the difficulties in determining precisely the composition of an extract. however, in an extract, and consequently in the plant, there are hundreds of compounds, with effects on bioavailability, solubility, and synergic and antagonist activity. in opposition to the magic bullet, there is the approach of the phytocomplex, invoked by many researchers in phytochemistry and pharmacognosy. an important part of research on neem was dedicated to increase its availability and properties, focusing in particular on stability and cost, toward the production of the ideal insecticide. the first aspect was assigned to the production of nanobioparticles containing neem extracts, which demonstrated clear larvicidal and deterrent activity on vectors, like ae. aldopictus, also in field conditions (chandramohan et al., ; murugan et al., ) . several factors must be considered in the case of a product based on natural substances. in theory, the plant could be available for everyone and therefore it cannot be patented. therefore, so far natural products are available for everyone and thus have been explored very little. natural products are the chemical part of the environmental interactions between living organisms and therefore they are natural candidates for the production of active drugs. the chemical production of a plant is strictly subjected to the environmental conditions that can highly influence this production. first, the exactly determined species must be used and determined in composition. once the raw material is obtained, the process of transformation can significantly influence the composition of the product. the technological transformation is essential to the quality and efficacy of the product. therefore, this second step is vital for the success of the product. the third step consists of the target being assigned to the product and the consequent marketing. in future, natural products will be even more important in the production of new drugs and foods and feeds, able to face the challenges of a continuously changing market. technology is key to this. the natural products market is expanding rapidly in previously unexplored areas, in particular as an alternative to products based on synthetic compounds. the prospects and possibilities in this situation are immense, but knowledge of nature and activity of natural products must be revised utilizing recent devices and research approaches. importance and role of natural products will increase if the multidrug resistance continues, asking for new bacterial and insect possibilities of control. the common composition of a botanical product is based on a single herb or on the combination of more species based on recipes and formulae mainly derived from the historical literature and empirical experiences. the long and accurate work of phytochemistry based on the sequence extraction-separation-identification, derived from the correspondence of one drug to one illness, generated a huge catalogue of identified natural substances that can be employed as useful standards to determine the composition of the botanical drug. the knowledge about composition must be as complete as possible; not a single constituent should be unused, and utility depends strictly on the utilization. natural products are derived mainly from plants as the result of coevolution between organisms and environment (tehri and singh, ) . for this reason, they have been used for centuries in popular and traditional medicines, as well as often being employed as spices and insecticides. unlike modern pharmacology and drug development, which are based on a single chemical entity, natural product preparations are multiingredient. a single herbal drug contains at least compounds making a complex matrix, named a phytocomplex, in which the single active constituent is not considered solely responsible for the overall efficacy. the utilization of the phytocomplex is based on experimental basis, since many data afford the validity of this approach, although further confirmations could be obtained using modern pharmacological devices. in other words, the same botanical raw material can be used directly, or extracted in different ways or used as a source of selected substances, or modified according to the product and target. in , a mixed team of experts from mit (massachusetts institute of technology) and the broad institute of harvard university, both in the usa, reported an interesting and innovative study for a scientific evaluation of the effectiveness of natural products. the argument is strictly inherent to the endless debate about the role of natural products and their efficacy, causing a fighting contrast, but useless and boring, between supporters of "natural" versus defenders of synthetic drugs. the key aim of the study was to understand what is going on between the two main levels of the metabolism (primary and secondary), on the basis of the consideration of the functional connection between genes and gene products, as well as between genes and targets. an innovative feature of the study is that the researchers decided to commit the argument to neutral judgment, submitting the elaboration of collected data to the computational work of artificial intelligence. the work was based on the comparison of cumulative connectivity distribution of small molecules, natural or synthetic, grouped according to connectivity associated with the target. assuming that proteins form biological networks and that metabolism and health depend on these networks, we should be able to assign a role to the molecules considered as possible medicines. the result showed that natural products target the proteins with a high number of protein-protein functional interactions (higher network connectivity), whereas the synthetic ones act on a limited protein network. the conclusions of the study, based on a computational approach, were evident: "we observe that approved drug targets that are not also natural product targets exhibit a connection distribution much closer to the case for human disease genes that natural product targets, which remain the most highly connected targets." this sentence indicates a positive and useful consideration about the role and activity of natural products. natural products tend to target more essential and general protein networks to an organism than other groups of small-molecule targets, like those more related to specific disease genes. therefore, the dichotomy between natural and synthetic active constituents must be considered mainly as a consequence of a cultural heritage, unable to assign a complementary or differently appropriate role to the two classes of molecules. the results of the study are coherent with the nature of natural products, whose production is the consequence of environmental interactions, including defense against predators or pathogens. this kind of defense cannot be specific, and therefore natural products act on more highly connected network of proteins, interrupting or limiting the activity of the essential proteins in environmental competitors or invaders. they may be tailored for a positive or negative influence in physiologic activities and basic metabolism of an ample range of organic targets. these arguments are in favor of the potential use of natural products as insecticides. in any case, there are several difficulties in assigning the activity to single constituents, causing several cases of wrong or misleading assignments of all the activity to single substances in the case of a plant extract, like in valerian (valeriana officinalis), whose extracts are largely marketed and utilized for their mild sedative effects. with the discovery of valepotriates, the effects were assigned to these constituents, but after the evidence that extracts with low content in instable valepotriates also exerted similar action, the essential oil was considered additionally responsible for the effect. another case consists of a current debate about hemp. besides cannabinoids, its essential oil and other constituents are now considered important for the multiple activities of hemp. in other words, there are hundreds of marketed products of hemp and many related claimed activities, and this can be related to the complex cannabidioma and/or the different compositions of the products, although they are all derived from the same raw material. it is very important to stress that important new features can appear, also in the case of species highly studied in their chemical composition, as shown in the scientific literature. recently, a new cannabinoid was isolated from cannabis sativa (citti, ) . as is wellknown, (À)-trans-Δ -tetrahydrocannabinol (Δ -thc) is the main compound of hemp and it is considered the main one responsible for intoxicant activity. however, the chemical constitution of this species is subject to high differences in accordance with its varieties and cultivars. cannabinoids possess a unique structure, derived by junction of a monoterpene and a polyketide unit. most of them have a side alkyl chain, whose length influences the biological activity of this cannabinoid. in fact, analogues of Δ -thc with a longer side chain were synthetized and they have shown cannabimimetic properties far higher than Δ -thc itself (seven c against five). in this study, a new phytocannabinoid with the same structure of Δ -thc, but with a seven-term alkyl side chain, was isolated and identified, and its stereochemical configuration confirmed by a stereoselective synthesis. this new phytocannabinoid has been called (-)-trans-Δ -tetrahydrocannabiphorol (Δ -thcp). the binding activity of Δ -thcp against human cb receptor in vitro (k i ¼ . nm) proved to be similar to that of cp (k i ¼ . nm), a potent full cb agonist. in the cannabinoid tetrad pharmacological test, Δ -thcp induced hypomotility, analgesia, catalepsy, and decreased rectal temperature, indicating a thc-like cannabimimetic activity. as confirmation, the corresponding cannabidiol (cbd) homolog with a seven-term side alkyl chain (cbdp) was also isolated and unambiguously identified by matching with its synthetic counterpart. the presence of this new phytocannabinoid could account for the pharmacological properties of some cannabis varieties that are difficult to explain by the presence of the sole Δ -thc and indicate the importance of the interaction between constituents of the so-called cannabidiome. therefore, we were not totally surprised when we found good larvicidal activity against aedes albopictus also in nsos with low content in azadirachtins . this was quite a novelty on the basis of the literature, but it is necessary to consider the importance of the metabolomics approach and the possibility with hptlc to obtain several views of the same plates. each view means a revelation of different compounds on the basis of their chemical structure and present functional groups. using an appropriate revelation agent, it is possible to see compounds that are not visible with another derivatization. this approach is contrary to the tendency of current analytical chemistry to focus on a single class of compounds or even unique constituents, which obtain perfect and reliable but limited results. another incongruence consisted of the presence of insecticide activity also in neem products after years of production, when limonoids should be highly degradated. the first experimental evidence we obtained on the activity of neem oil was the inability of the larvae of ae. albopictus to complete the molt from larva to pupa. the larvae proved to be initially immature, their bodies imperfect, and finally before the third instar, most insects died and none was able to fly. the delicate mechanism of the development stage was jammed and the cruel destiny of the unfortunate insects assigned. each organism has its weakness. mosquitos, like any arthropod, possess a rigid exoskeleton, which offers efficient strong and secure protection, also against pesticides, which is one of the reasons for the success of these creatures. the exoskeleton of insects is primarily made of proteins (sclerotin) and chitin (a polysaccharide), which are interwoven and linked together to form strong but flexible bundles. interestingly, chitin is also the main constituent of the fungal cell wall. the ratio of the components of the exoskeleton varies from one body part to another on an insect. however, the exoskeleton is too rigid, and acts like a cover that encases the entire insect, and being a nonliving formation, the exoskeleton does not change size and grow with the insect. the exoskeleton is too ridged to be recycled or modified, and it must be substituted, but it must also protect the insect until the new exoskeleton is ready. during the growth period, insects must shed the exoskeleton in order to assume a new form. as a result, it is necessary for the insect to shed its old exoskeleton to make way for a new, larger one through a process called molting. this is a hormone-controlled phenomenon. during the molting stages, the hormones are released to start and finalize each step of the metamorphosis, until the mature insect finally emerges. however, the chemical constitution of the exoskeleton is variable in each insect species and this is the reason for the selective toxic effects, such as those reported in the case of neem. regarding the structures of insecticides acting as growth regulators, albeit in the case of ecdysones the relation with insect hormones is evident, in other cases the similarity is not so clear, as well as the real mechanism of action. the stages between the subsequent molts are generally called instars. these correspond to altered body proportions, colors, patterns, and changes in the number of body segments or head width. for most insect species, an instar is the developmental stage of the larval forms, but an instar can be any developmental stage including pupa or imago. the larval stage is in particular a delicate stage of the insect metamorphosis. however, we were totally aware that confirmation of the neem insecticide activity, albeit with a demonstrated chemical constitution, in a laboratory experiment was a weak starting point. the open questions were numerous: (a) how to obtain the same result in the field; (b) whether the larvicidal activity could be connected to other properties, in order to improve the use; (c) what the cost of neem oil would be, considering the large-scale spread of the insects; (d) how limited the stability of the active ingredients of neem would be; (e) what determination of chemical content of neem oil would be required, to be connected to the determination of the activity; and (f) what the ambit of utilization would be and the possible damage to the habitat. other advantages arising from the use of neem-based products are the rare induction of resistance, due to their multiple mode of action against pests, the low toxicity rates that have been detected against vertebrates, and finally the necessary environmental care. there is a little confusion about the plant species named azedarach, and very similar denominations. the name azedarach was given by the famous persian physician avicenna ( - ) to indicate some poisonous trees; however, azadirakhti literally means "free book of india." in , linnaeus reported about melia azadarachta in his species plantarum ( : with habitat: india). in the same book ( : ), we can find melia azedarach (habitat: syria) and melia azedarach var. sempervirens (habitat: zeylona). actually there are two distinct species, azadirachta indica a. juss, attributed to neem (or nimba, meaning "who gives good health," as reported in the sanskrit books) and melia azedaracht linneus, attributed to melia, a very similar tree. this is the typical taxonomic situation in botany and zoology. the differences between taxa are often very narrow and only specialists are able to find them. in any case, the problem of the significance of these differences is always a matter of debate. god bless taxonomists, because they are necessary to obtain order out chaos, but please do not spend your precious intellect on endless discussions with no final consistent result! in fact, the matter is complicated by synonymous, parental disputes, errors of any type, including wrong transliteration (i.e., gingko and ginko), disputable rules of the international codices, and more. neem and melia are very similar, but there are several tricks to distinguish between the two species. the first is commonly known also as indian lilac and the second one as persian lilac or simply melia. neem has usually white flowers whereas melia presents an explosion of blue flowers; the fruits of the former have an elongated shape, whereas the latter's are totally rounded. if the trees do not have flowers or fruits, and you are not a botanist, you may be in trouble, but you can remember that neem cannot live in temperate climate regions, whereas melia can be easily cultivated in such places. therefore, if you are in europe or the usa, you can be % sure on the matter. melia azedarach is known by several common names, such as melia, chinaberry tree, pride of india, bead-tree, cape lilac, syringa berrytree, persian lilac, and others. it is usually a large tree growing up to m tall, with leaves -pinnate, rarely -, with primary pinnae in two to six pairs, usually three to seven leaflets per pinna, narrowly ovate or subovate, serrate, acuminate, irregularly toothed, or crenate. flowers are abundant and small, sweet-scented, in large axillary panicles. all parts of this tree are reported to have medicinal uses, but in particular, in terms of insecticide properties, seedlings are reported to present aphid attacks. a leaf used as a bookmark will deter insect pests. in italy, the tree is known as the tree of rosary, since in the past, before the advent of plastic, its hard and round kernels were used to make the grains of a rosary. our research on melia azedarach, as well as the references on this plant, evidences a significant difference in the chemical composition. limonoids are present, but different from azadirachtins and other constituents make a marked relevant dissimilarity in composition. the initial conclusion was that melia probably cannot compete with neem as an insecticide, but other utilizations can be explored. however, once again a limit in the references is an irresistible task for a researcher in search of innovations. in addition to the insecticidal properties, we were initially particularly interested in the antimicrobial activity. people often associate antimicrobial activity with infection and effects on their health, but microbes are everywhere and most damage affects cultivation of plants. agricultural methods of reproduction of plants with economic value were totally transformed by the introduction of micropropagation and stem cell culture. micropropagation allows the rapid cultivation of selected cultivars, saving time and resources. however, although the first steps of micropropagation were performed in aseptic conditions, the possibility of infection of calla, shoots, and seedlings is high. avoiding the infection must be done via an appropriate and sensitive approach, avoiding damage to the delicate meristems-a typical job for natural products. the antibacterial study (marino et al., ) aimed to investigate the antibacterial activity of unripe fruits of melia azedarach collected in different periods. the activity was tested on the shoots of a hybrid of prunus cerasifera x prunus spinosa and calla lily of zantedeschia aethiopica against several bacterial species. the data reported evidenced a positive antibacterial activity and the absence of any negative effect on the growth of shoots surviving at the second subculture on a standard medium. hptlc analysis showed the prevalence of polyphenols, such as chlorogenic and caffeic acids, which, on the basis of the literature, are consistent with the antimicrobial activity. this activity is important considering that many plant species of economic relevance are now obtained by micropropagation, and this cultivation in vitro is necessary to avoid any sort of contamination. further research is essentially the rational collection of most of the arguments previously considered, as evident in the title: "green-synthesised nanoparticles from melia azedarach seeds and the cyclopoid crustacean cyclops vernalis: an eco-friendly route to control the malaria vector anopheles stephensi?" (anbu et al., ) . in this research, once a single-step greensynthesis of silver nanoparticles (agnp) using the seed extract of m. azedarach was obtained, we tested its mosquitocidal activity. in laboratory assays on anopheles stephensi, ag np showed lc ranging from . (i instar larvae) to . ppm (pupae). in the field, the application of ag np ( Âlc ) led to complete elimination of larval populations after h. finally, we decided to test the nanoparticles on nontarget aquatic predators. the application of ag np in the aquatic environment did not show negative adverse effects on predatory efficiency of the mosquito natural enemy cyclops vernalis. the reason for this additional research lies in the fact that numerous aquatic arthropods attack and devour preparasites. as we already know, the utilization of the insecticides, though with plant-derived active constituents, could be dangerous for the environmental equilibria. in particular, it could affect the natural biological control, based on the presence predators of the vector in the common habitat, remembering that all the insect stages, except the adult insect, need water. in such sites, there is fresh water everywhere, such as lakes, pools, and similar places, enabling life along the plant-covered banks of stagnant and slow-flowing bodies of water. in such places, mosquitos can proliferate as can any other predator, which in an aquatic environment is fundamental to limit the proliferation of the vector. in fact, after coupling, and the consequent blood feeding necessary to assume the proteins necessary for the eggs maturation, the female is looking for an appropriate place for the deposition of - eggs. a single anopheles, like other insect, is able to produce a quantity of eggs and larvae enough to invade all the neighboring habitats, as in the classic case of a locust invasion. this is not possible only thanks to the natural enemies. the microaquatic environments are the scenario of a continuous fight for survival, where often two or more species of arthropods are involved, as predator or as prey. in our study, we selected the genus cyclops, which is one of the most common of freshwater copepods, comprising more than species. copepods are very little crustaceans, commonly called water fleas. they have a single large eye, which may be either red or black, and therefore they are named for the cyclops of greek mythology. cyclops prefers fresh water, and is less frequent in brackish water, where it feeds on small fragments of plant material, animals, or carrion. it swims with characteristic jerky movements and has the capacity to survive unsuitable conditions by forming a cloak of slime, with an average lifespan of about months. several microscopic crustaceous, including copepod species, feed small and very small preys. in high-density, unstructured environments such as eutrophic lakes, predatory copepods commonly coexist with certain smallbodied prey, where encounters are frequent with ineptness on the part of the predator and counter-tactics by the prey. in particular, laboratory studies showed that copepods are effective predators on early-instar culex larvae, involving an important role in suppressing mosquito populations, because of their feeding behavior and abundance. they are very efficient in this role, since the presence of alternative abundant food, like bacteria and protozoa, does not deter their attacks on their preferred prey. copepods are capable of killing and eating at least four preparasites within min and a predator density of copepods/liter is expected to reduce the mosquito larvae by %, with the rate of predation inversely proportional to the water volume. neem cake: from by-product of an industrial process to multipurpose resource for a sustainable agriculture chain during our research activity, we were highly interested in industrial plantborne by-products, since they can offer new products to the market with lower cost and high usefulness. our attention was immediately attracted by neem cake, a cheap by-product of nso extraction, obtained as a residue after mechanical pressing of the neem seeds, considered of low economic value and utilized to enrich the soil of some mineral components, such as nitrogen. the laboratory test indicated neem cake activity against aedes albopictus and a number of culicidae species (nicoletti et al., ) . in the case of biocidal treatments, it is important to demonstrate that insecticide activity is associated with antimicrobial activities in consideration of the high possibility of infection and the severe consequences for health, in particular in the case of the animals, both pets and livestock. the importance of insecticidal and antimicrobial activities for animal treatment has been evidenced in the experiments with nso and neem cake further reported, including larvicide, deterrent, and repellent activities (benelli et al., a,b; nicoletti et al., a,b) . the complex range of different compounds in the neem seeds open the possibility to utilize the derived products to solve many current problems. the challenge now is to obtain marketed products tailored for different utilizations. the reported experiments evidenced these potentialities, which are only waiting a realization and a wider utilization. despite diseases, wars, and environmental disasters, the human population is growing. first of all, more people will need more food. this forecast shows in particular a massive increase in animal protein demand, needed to satisfy the growth in the human population, wherein billions of people require an increase of caloric input and better food. therefore, attention is focused on the sources of feed protein and their suitability, quality, and safety for future supply. in addition, the quantitative production aspect is causing a series of problems. there will need to be a considerable increase in feed manufacture, requiring a thriving, successful, and modern feed industry, including a key aspect concerning the protection and preservation of the food produced and marketed. this aspect is strictly related to safety issues, which will remain paramount in the minds of consumers following recent food crises. it is time to consider that the need of more food to feed due to increasing planet population perhaps cannot simply be solved by massive production, but reduction of food waste and conservation can increase food availability by %- %. "feed the planet and energy for life" was the theme of world expo in milan, italy. among the activities occurring at the expo, research and proposals concerning the utilization of neem products were presented in a call for projects in favor of sustainable progress and production of future foods. the neem project was selected as the best one due to its possible applications in the production of food and feed. the expo event projected feeding as the main challenge for humankind and showed the extreme urgency of elements of innovation in technology and science connected to the production and conservation of food. it was demonstrated how serious feed problems still plague several areas of the world today, and the possibility of new solutions was mentioned. the neem project was focused on the agricultural utilizations of neem cake concerning its advantages as soil fertilizer and as a natural ectoparasiticide for the treatment of sheep and goats. neem products were proposed as being able to affect the biotic composition of the soil. neem cake must be preferred to neem oil for its cost and its form as a powder immediately available. several experiments evidenced the improvements of the utilization of neem cake in agrarian ecosystems: ( ) availability of nutrients, in particular nitrogen and phosphorus, more consistent with the requirements of the crop; ( ) development of the microbial beneficial biomass of the soil, which increases in quantity and activity, but with selective influence against nematodes and other negative components. in agricultural practices, plants in addition to nutrients should count on a greater variety of useful microorganisms and on acquisition of nutrients themselves, through the activation of complex symbiotic systems. if you want to understand the state of health of a tree, you must look down, not up; and ( ) development of pest control system of insects and other arthropods of agricultural and livestock interest. neem cake, as an industrial by-product, is a heterogeneous material that maintains a high added value due in large part to its chemical composition, which confers its biological activity. neem cake is widely available on the global market, considering the increasing presence of neem trees in the world and production of nso. the exploitation of its characteristics in the food chain to improve consumer health, increase the productivity of agricultural products, and feed the planet is the logical consequence of the urgent need to develop new sustainable agricultural systems in a world where many highly polluting pesticides are no longer allowed to be used. however, more research, in particular in field conditions, is necessary to understand the real value of its microbiological, insecticide, fertilizer, and nematocide activity, involving collaborations between different experts in individual sectors-import companies, organic farms, and research institutions-in order to determine the manner and timing of land application of this valuable product of "waste," still underestimated. neem cake could lead to a revolutionary improvement in the fertilization of agricultural plants, adding to the characteristics of chemical fertilizers those of a soil improver. in agriculture, we could define neem cake as a prompt nutrient-release fertilizer, effective in allowing rapid absorption of nutrients and promoting development of the plant, with the capacity to increase the activity of the microbial biomass and organic matter, favoring the sequestration of carbon. the idea was to join the fertilizer, insecticide, and antimicrobial utilities of neem cake. exploitation of the use of neem cake as an insecticide came from this first test: some pots of impatiens (impatiens balsamina) were fertilized with % by volume of neem cake, and mosquito larvae were reared starting from eggs. the eggs were hatched in control and treated in pot saucers, but none of the newborn larvae survived in the water saucers of pots treated with neem cake, while in the water saucers of pots unfertilized with cake, the control larvae completed their development in less than a week, becoming adult mosquitoes. other major beneficiaries of the use of neem cake as insecticide are undoubtedly sheep farmers, who can use an organic product of natural origin and low cost that is simultaneously effective against the larvae of culicoides and other pests, while respecting the natural biotic communities. direct beneficiaries of neem cake, as a fertilizer, are farmers seeking pest and nematode control, in particular for nematodes. currently, some highly toxic products are still on the market by virtue of the absence of suitable alternatives. particular attention must be focused on the changes on soil micro-composition, as evidenced in several field experiments. in conclusion, we can report the following important advances in the use of neem cake as a functional fertilizer: (a) energy saving flows from the use of a waste of an industrial chain; (b) environmental sustainability, as documented by the analyses attesting the absence of heavy metals, aflatoxins and residues of pesticides; (c) neem cake is an excellent alternative to methyl bromide (bm) (banned as being responsible for the "thinning" of the ozone layer of the atmosphere); (d) neem cake is an excellent alternative to temephos and other organophosphates used to treat water infested with disease-carrying insects including mosquitoes, midges, and blackfly larvae; (e) neem cake is a great alternative to nematicides, like , -dichloropropene; and (f) neem cake in the field trials carried out in sardinia had efficacy similar to azadirachtin biological products already established in organic farming, but were very expensive and not really effective. in addition, neem cake showed very low effect on "nontarget" insects that live in the same environments as culicoides larvae. ectoparasites are organisms that inhabit the skin of another organism, causing significant infestations and pathologies. many micropathogens can profit from the work of ectoparasites, either to colonize the skin injury and lesion, or be inserted in the host during the feeding. the vast majority of ectoparasites are arthropods, e.g., insects and arachnids. again the triangle host-vector-etiological agent is reproduced. many ectoparasites are vectors of pathogens, which are typically transmitted while feeding on or from other hosts. several ectoparasites (e.g., most lice) are host-specific, including livestock, pets, poultry, fish, and bees, but others parasitize a wide range of hosts, including humans. typical effects of infection on the host are irritability, dermatitis, secondary infection (other parasites profit of the skin necrosis), fecal hemorrhages, blockage of orifices, inoculation of toxins, and exsanguination. as a consequence, the host's general health can be seriously affected with low weight gains, particularly important in livestock. subdermally located parasitic larval stages of certain flies can be favored by the ectoparasited infection, causing a condition termed "myiasis." when insects (order hemiptera) are involved, the infection mechanism is similar to that previously described for any insect-borne disease. the vector contains several hematophagous ectoparasites, including approximately species of kissing ("cone-nose") bugs (reduviidae, triatominae) and bed bugs and bat bugs (cimicidae). these parasites make physical contact with the host principally when ingesting a blood meal. these kissing bugs usually prefer domestic animals, from which relatively large blood volumes may be imbibed; in such a way they can cause a great deal of damage and transmit important diseases. ectoparasites play a very detrimental role in terms of decreasing the productivity of livestock, such as sheep and goats. nso was utilized in the field as an antibacterial in the case of ectoparasites' stings and bites resulting from goat wounds. common external sheep and goat parasites include ticks, lice, and mites. they cause restlessness and irritation. weight loss and reduction in milk production may occur as a result of nervousness and improper nutrition, because animals spend less time eating. bites can damage sensitive areas of skin (teats, vagina, eyes, etc.) . some parasites feed on blood, causing anemia, especially in young animals. the bite and the sting of ectoparasites allow bacteria to proliferate in wounds from abrasions or lesions from scratching, and cause levels of tissue reaction of different entities, super-infection, and cervical lymphadenopathy. ectoparasites cause many problems in livestock production. they seriously damage sheep and goat skins, resulting in the rejection or downgrading of the skins. this causes huge economic loss, as this skin damage renders it unsuitable for the leather industry due to the decrease in quality. lower production of meat is also a typical consequence. pseudomonas aeruginosa wound infections are characterized by a change in the color of the skin around the wound area and the formation of lesions. the bacterium products and pigment cause yellow discoloration of wool and consequently reduced quality and market value. nso treatment in the field on as a natural ectoparasiticide for sheep and goats proved to be successful in preventing and curing the attacks of endoparasites ( fig. . ) . the experiments were performed on selected livestock (fig. . ) by a specialized team of crea researchers (de matteis et al., ) . the effects on the parasites were evident (figs. . and . ) and even after the first treatment with nso, protection against ectoparasites was obtained. more important, the health of the treated livestock improved, as testified by the hematological profile of goats. in vivo and in vitro tests on blood cells from siriana, sanen, cashmere, and maltese goat (capra hircus) breeds showed no significant difference (p <. ) between nso treated and untreated goat hematological parameters at each sampling time considered. in addition, the nso effect on goat pbmc cultured in rpmi medium was evaluated at : Â to : Â dilutions at , , and h of exposure. the in vitro test revealed that the response of goat pbmc viability is dependent on concentration, incubation time, and nso dose. in conclusion, the nso should be considered useful, safe, and innovative for development of topical solutions for the care of wounds. among the most relevant typology of neem products, we focused on selectivity. the antibacterial activity of nso was assayed against isolates of escherichia coli, considering that this bacterium can produce beneficial and pathologic populations. the molecular biology characterization showed that isolates resulted in diarrheagenic e. coli. nso showed biological activity against all isolates. however, there were significant differences between the antibacterial activities against pathogenic and nonpathogenic e. coli, as well as between nso and ciprofloxacin activities. on the basis of the results obtained, nso is able to counteract e. coli and also influence the virulence of e. coli-viable cells after treatment with nso. the preservation of marketed food is an important aspect of the smart utilization of the produced food (maruchecka et al., ) . furthermore, the consequent waste of unutilized food is a relevant problem in overcrowded towns (hlpe, ) . a large quantity of food is lost or wasted throughout the supply chain, from initial agricultural production down to final household consumption (hlpe, ; kader, ) . the loss or waste for high perishable food, such as fresh fruit and vegetables, fish and livestock products, has been estimated at as much as half of all food grown before and after it reaches the consumer. approximately one-third of all ffvs produced worldwide are lost during food supply chain production. shelf life plays a central role in food spoilage. the impact of the enormous quantity of packaging is evident in any planet environments. increase of the shelf life means reduction of cost and waste. everything pivots around the material utilized for packaging, and new solutions are emerging (otoni et al., ; singh and singh, ; cooksey, ; appendini and hotchkiss, ) , including passive packaging (brockgreitens and abbas, ; ozdemir and floros, ) , active packaging (coma, ) , intelligent packaging (lee et al., ; de kruijf et al., ) , and smart packaging (dobrucka and cierpiszewski, ) . although the results are not evident in our ordinary life, the galaxy of packaging is rapidly moving and increasing in research and proposals, based on new technologies and advanced techniques recently available, like nanotechnology and molecular biology. efforts are focused on solving the food preservative problems, to extend the shelf life of perishable foods, by reducing the need for additives and preservatives. "smart packaging" is based on the production of functional methods to obtain the following goals: be tailored depending on the product being packaged, including several types of food, beverages, pharmaceuticals, household products, etc.; reduce food waste, increasing the shelf life; and maintain, and eventually enhance, product attributes (e.g., look, taste, flavor, aroma). the key words are protect, preserve, and present. several methods and approaches, such as oxygen scavenging and antimicrobial technologies associated to the production of modified films, have been considered . they are different solutions to serve the basic and fundamental properties of packaging. so far, the dominant packaging is the basic one, using low-cost material and involving no interaction with the food inside. this is passive packaging, wherein the traditional packaging systems are included, as the use of covering material characterized by some inherent insulating, protective, or ease-ofhandling qualities. usually, the ordinary packaging of food is mainly a used to method to attract and select the consumer, beside a preservation. the consequence is the enormous amount of waste, and the consequent damage to the environment. this situation is increasing due to the increasing numbers of consumers in emerging countries, where these consequences are not adequately considered. packaging is considered active when it can interact in the same way and/or react to various stimuli, in order to keep the internal environment favorable for the maintenance of product quality. several environmental, biotic, and abiotic factors must be considered, in order to respond to the degradation process successfully. the activity involved could be the presence of an oxygen scavenger (this can absorb high-energy oxygen inside a package and therefore increase the shelf life of a product) or an anti-ros (a scavenger of radicals by oxygen or other origins), such as in the typical case of phenolic natural products. smart packaging relies on the use of chemicals, electrical, electronic, or mechanical technology, or any combination of these. technology is used to modify the packaging by adding constituents to change its features and properties (kerry et al., ; malhotra et al., ) . active and intelligent packaging is particularly dedicated to the preservation of fresh products, like vegetables, in accordance with increasing requirements for this kind of food (nicoletti and del serrone, ; nicoletti, a,b) . intelligent packaging systems monitor the condition of packaged foods to give information regarding the quality of the packaged food during transport and storage (aguilera et al., ) . probably the most innovative aspect of intelligent packaging is that it can be supported by the utilization of systems of detection in meat and meat products, obtained through the use of sensor technologies indicators (thakur and ragavan, ) , including integrity, freshness, and time-temperature indicators (ttis) and radio frequency identification (rfid). therefore, active and smart packaging performs additional functions to the basic one by the introduction of innovations in the design of packaging, with the aim of increase the shelf life, but also to add conveniences for the user and usefulness for the consumer, to be introduced also in the supply chain. in this way, the product can respond not only to the need for a longer life, but also make the product more available, more useful, and more safe. since our invisible enemies are asked to play their role again, antibiotic activity is required. packaging is mainly used to separate food from environmental conditions, utilizing simple material made of paper or plastic. however, it cannot prevent internal attacks by microorganisms, but can only limit or delay the effects. therefore, additional treatments are required to limit their action, like the utilization of low temperatures, which involves additional costs and energy consumption and pollution. a new idea is to associate to the packaging some antimicrobial agent. before and during packaging, storage, and shelf life, food is subjected to a continuous attack by microorganisms. these microorganisms are working to benefit themselves by demolishing progressively the molecular structure of the food, as soon and as completely as possible. therefore, by preserving the food, we are working in a thermodynamically unfavorable situation. in term of shelf life, the food is in competition with its natural recycling, and, working to maintain as possible this limbo, we can utilize the food efficiently as it is possible. the resistance phenomenon interests also zoonotic food-and waterborne pathogens becoming more resistant to antibiotics (del serrone et al., ) . resistant strains of pathogens have been isolated from food, causing an increasing incidence of food-borne diseases. through the food, these microorganisms could be entering the human gastrointestinal tract on an almost daily basis. the antimicrobial activity of nso and related products have already been reported (palanappian and holley, ; baswa et al., ; sairam et al., ) . a possible utilization of antibacterial activity of neem cake against meat spoilage bacteria was tested using a broth model meat system . the tests were positive, since the growth inhibition zone (mm) varied significantly (p!. ). with respect to ciprofloxacin activity, the antibiotic value ranged as follows: . ae . to . ae . mm and . ae . to . ae . mm, respectively. the percentage of bacterial growth reduction (gr%) also varied significantly (p !. ) in function of considered nce concentrations ( : - : , ), with the highest gr% for μg nce ( . ae . to . ae . ). the numbers of viable bacterial cells never significantly (p . ) exceeded inocula concentrations used to contaminate the meat. all the results of the experiments showed that neem cake is able to counteract the main microorganisms responsible of meat spoilage, like strains of gram-positive and gram-negative, as well as facultative anaerobic bacteria. the antimicrobial activity of neem products was confirmed also for nso against spoilage bacteria, such as carnobacterium maltaromaticum, brochothrix thermosphacta, escherichia coli, pseudomonas fluorescens, lactobacillus curvatus, and l. sakei. after the second day after nso, only c. maltaromaticum-viable bacterial cells were detected. these data could be used to create new intelligent packaging. utilizing a nanotechnology already employed for other materials, neem cake may be incorporated into the cavities of nanoparticles, maintaining its antiparasite activity. once incorporated into the packaging material, the neem cake, also in minimum quantity, should be able to effect its preservative food action, acting against the demolishing microorganisms. the increase of the shelf life of meat should compensate for the additional cost of the packaging material, not considering the decrease of waste. it is possible that the first activity of luca was to find the energetic source for survival, and the second was to compete with the other lucas. the results are an endless transformation of forms and production of new molecules. the living organisms had a long time to organize their molecular weapons and the secondary metabolites are there, produced and organized to be considered and utilized in the right way. the neem tree is an example of nature's treasure. the advent of homo sapiens (lucy) changed in part the rules of the natural game, but natural products still remain a necessity for our life. recently, parenteral artesunate has replaced quinine and many other antimalarial products for the treatment of severe malaria. however, several reports have demonstrated the emergence of resistance to the efficacy of artemisinin-based combination therapy monotherapy, such as in western cambodia and other regions in south-east asia. to face the phenomenon, artemisinin-based combination therapies are now recommended by the who. the aim is to reduce the morbidity and mortality associated with malaria with artemisinin-based combination therapies, including chloroquine plus other drugs, like sulfadoxine-pyrimethamine. meanwhile, with increasing resistance to chloroquine, quinine is reconsidered, being so far the only substance for which plasmodium did not develop resistance. the consequences are that in uganda quinine was prescribed for up to % of children younger than years with uncomplicated malaria, and from , african countries recommended quinine as a second-line treatment for uncomplicated malaria, as a first-line treatment for severe malaria, and for treatment of malaria in the first trimester of pregnancy. recent surveillance data from other sites are in accordance. however, quinine was substituted due to its limits and therefore in , who ( ) guidelines recommended reinforcing quinine's activity by combining it with other antimalarial agents, like doxycycline, tetracycline, or clindamycin as a second-line treatment for uncomplicated malaria (to be used when the first-line drug fails or is not available) or quinine plus clindamycin for treatment of malaria in uncomplicated cases and in the first trimester of pregnancy. the development of effective cocktails is a current trend of medical treatment of several diseases, including forms of cancer. in addition, the combination of natural products and synthetic drugs is recommended. natural products can be utilized as resistance-modifiers or chemosensitizers, and may be able to restore chloroquine sensitivity in resistant strains of plasmodium. the idea of years of research from different research groups was that the antimalarial treatment combined with natural products could be based on lower doses of chloroquine, in order to minimize the resistance insurgence and to avoid the collateral effects in the case of prolonged use, necessary in areas where the disease is endemic. this approach came from an ethnopharmacological investigation by professor rasoanaivo (rasoanaivo et al., ) . most people consider ethnopharmacology to be a collection of ancient utilizations of natural sources, and as knowledge that is going to disappear. on the contrary, in addition to traditional uses there are new ones emerging, even as consequences of the utilization of modern drugs. considering that oms reports that % of the planet population relies on traditional medicine, the utilization of medicinal plants is not limited to ancient times and past populations, but it changes according to needs and evolution of treatments. ethnobotanical knowledge is still passed from one generation to another in the majority of populations living in rural areas, and in urban areas, where malaria has been revealed to be resistant or incurable by modern scientific medicines, people have turned to traditional treatments. it is therefore of paramount importance to preserve and transmit this ethnobotanical heritage. therefore, this discipline must be regarded as a multidisciplinary science in movement, where botany, chemistry, and pharmacology play central roles for scientific evaluation and validation of popular uses. however, economic and social aspects must also be considered, in order to develop new drugs and treatments of both old and new diseases. most antimalarial drugs currently in use belong to the classes of aminoquinolines (chloroquine, amodiaquine, primaquine), quimolinomethanol derivatives (quinine, mefloquine, halofantrine), diaminopyrimidines (pyrimethamine), sulfonamides (sulfadoxine, sulfadiazine), biguanides (proguanil and derivatives), antibiotics (tetracyclines, doxycyclin, clindamycin), sesquiterpenes (artemisinin, dihydroartemisinin, arteether, artemether, artesunate) , and naphtoquinones (atovaquone). among them, only quinine and artemisinins are natural products, but also a relevant part of the current antimalarial arsenal. the potentiality of natural products is very high. a review by willcox and bodeker ( ) on traditional herbal medicines for malaria in three continents reported plant species from families. however, the clinical trials are largely lacking, since only eight clinically controlled trials have been reported, involving p. falciparum and p. vivax. in the case of malaria, alkaloids are the first candidates responsible for the activity. there is a long tradition in popular medicine of plants containing these compounds to control fever. these plants also have a bitter taste, which is usually connected to the alkaloid presence, as already reported for the aforementioned quinine bark case. two important considerations attracted our attention, in view of the possibility to explore new strategies: the special endemic flora of madagascar and the occurrence of information about a popular treatment of malaria as yet unreported. madagascar is a land of endemism, consisting about , species of vascular plants, of which % are endemic, and eight families totally endemic. malaria is practically endemic in all madagascar and therefore the population harbors a very rich and unique knowledge on antimalarial plants. after a resurgence of malaria in the early s, as a consequence of plasmodium falciparum resistance and due to the high costs of conventional drugs, local populations returned to the uses of herbal remedies. two hundred and thirty-nine plant species, of which about % are endemic to madagascar, have been reported as having antimalarial uses in malagasy traditional medicine. prof. rasoanaivo discovered the use by some populations in madagascar of decoctions of some local plants in association with low doses of chloroquine to complement chloroquine action against chronic malaria . the lower use, one or two tablets of chloroquine ( - mg), is probably adopted to avoid collateral effects due to prolonged use of chloroquine, but such a dose could be considered inadequate to favor chloroquine resistance. therefore, we have a mixture of recent learning and ancient knowledge, evidencing the reality of ethnopharmacology. however, popular uses of medicinal plants need scientific validation with advanced tools. therefore, research started from the knowledge that some populations in madagascar use decoctions of some local plants in association with low doses of chloroquine to complement chloroquine action against chronic malaria. in such a way, resistance insurgence and collateral effects are both lowered. on the basis of the ethnobotanical work conducted by rasoanaivo and his collaborators, plants were selected and investigated for in vitro and in vivo antimalarial activity and a chloroquine-potentiating effect. in the case of validation of the activity, the determination of the active constituents followed. the results of these selections were that the alkaloids of loganiaceae, menispermiaceae, and rutaceae were the most promising compounds showing significant effects, some of them potentiating the action of chloroquine. from a phytochemical point of view, alkaloids are in pole position among natural products utilized in traditional medicine against malaria. mono-and bis-indole alkaloids have been isolated from several plants that are traditionally used to treat malaria on different continents. the most active compounds are those that originate from plants belonging to the genera strychnos (loganiaceae) and alstonia (apocynaceae). a review covering the indole alkaloids that have high antiplasmodial activities in vitro and in vivo, and favorable selectivity indices (si¼cc /ic ), was published by frederich et al. ( ) . in the case of malaria, alkaloids are the first candidates as being potentially responsible for the activity. there is a long tradition in popular medicine of plants containing these compounds to control fever. these plants also have a bitter taste, which it is usually connected to the alkaloid presence, as already reported for the aforementioned quinine bark case. two important considerations attracted our attention, in view of a possibility to explore new strategies: the special endemic flora of madagascar and the occurrence of information about a popular treatment of malaria so far never reported. madagascar is land of endemism, consisting about , species of vascular plants, whose % are endemic, and even families totally endemic. malaria is practically endemic in all madagascar and therefore the population harbours a very rich and unique knowledge on antimalarial plants. after resurgence of malaria in the early 's, as a consequence of plasmodium falciparum resistance and due to high costs of conventional drugs, local populations back to the use of herbal remedies (blanchard, ; maggi et al., ) . two hundreds and thirty-nine plant species, of which about % are endemic to madagascar, have been reported as having antimalarial uses in the malagasy traditional medicine. prof. rasoanaivo discovered the use by some populations in madagascar of decoctions of some local plants in association with low doses of chloroquine to complement cq action against chronic malaria . the lower use, one or two tablets of chloroquine ( - mg) , is probably adopted to avoid the collateral effects due to a prolonged utilization of chloroquine, but such dose could be presumed inadequate to favour chloroquine resistance. therefore, we have a mixture of recent acquirement and ancient knowledge, evidencing the actuality of ethnopharmacology. however, popular uses medicinal plants need a scientific validation with advanced tools. therefore, the researches started from the knowledge that some populations in madagascar use decoctions of some local plants in association with low doses of chloroquine to complement chloroquine action against chronic malaria. in such way, resistance insurgence is lowered, as well as collateral effects. on the basis of the ethnobotanical work conducted by rasoanaivo and his collaborators plants were selected and therefore investigated for in vitro and in vivo antimalarial activity and a chloroquinepotentiating effect (maggi et al., ) . in case of validation of the activity, the determination of the active constituents followed. the results of this selection were that the alkaloids of loganiaceae, menispermiaceae and rutaceae were the most promising compounds showing significant effects, some of them potentiating the action of chloroquine. mono-and bis-indole alkaloids are traditionally used to treat malaria in different continents (ramanitrahasimbola et al., (ramanitrahasimbola et al., , . the most active compounds were mainly related to the genera strychnos (loganiaceae) and alstonia (apocynaceae). a review covering the indole alkaloids that have high antiplasmodial activities in vitro and in vivo, and favourable selectivity indices (si¼cc /ic ) was published by frederich et al. ( ) . strychnos is a pantropic genus, with about species, present in three continents: in africa, in america, and in asia and oceania (only s. potatorum is present in both asia and africa). asiatic species are mainly small trees, whereas in the new world lianes are generally dominant. the most famous strychnos species is the asiatic s. nux-vomica, because of strychnine contained in the seeds with other related alkaloids. strychnine is also known and used for its bitter taste. south american species are characterized by different mono and bisindole alkaloids, important as constituents of some curare preparations of indios amazonia tribes (see introduction) . during the preparation of curare, the tribe curandero selects local plants and extracts the mixture by hot water. finally, the extract is filtered on leaves and concentrated to obtain a paste, which is preserved into a container, like a calebassa or a tube, maiden by a cane, or a pottery. active constituents in curare are bis-indole alkaloids from bark of strychnos ssp. and bis-tetrahydroisoquinoline alkaloids from menispermaceae. the genus strychnos is represented in madagascar by species, of which five are endemic to the island. among them, s. diplotrocha leeuwenberg and s. myrtoides gilg & bussse are used as antimalarial in the northeastern part of the country (rasoanaivo et al., ) . the phytochemical analysis allowed the separation and the structural determination of several indole alkaloids, some already known and others never reported, including mixtures of epimers, which is very unusual in the same plant (rasoanaivo et al., (rasoanaivo et al., , (rasoanaivo et al., , . the in vitro and in vivo chloroquine-potentiating effect of the crude extract of dried and powdered stem barks of s. myrtoides exerted chloroquine-potentiating effects on p. falciparum fcm , but it was devoid of intrinsic antimalarial activity. the extract was also devoid of cytotoxic effects on hela and l fibroblast cells. the two compounds exhibit a closely related structure but different basicity. therefore, the latter parameter can be excluded from the factors affecting the chloroquine-potentiating effect. these results were confirmed by other experiments, demonstrating that the crude extract of s. myrtoides showed higher chloroquine-enhancing activity than its major bioactive constituents. these data support the use of the plant as a phytomedicine to treat malaria, but minor components of the extract may act synergistically. among the main isolated alkaloids, malagashanine was very interesting. malagashanine is an unusual indole alkaloid of the strychnos type. its pentacyclic structure contains seven consecutive stereogenic centers and, most important, a transfusion between the c and d rings, against all the other similar natural alkaloids. therefore, malagashanine is the parent compound of a new type of indole alkaloids (fig. . ) (kong et al., ) , named n b c( )-secocuran, isolated so far from the malagasy strychnos species, which are traditionally used as chloroquine adjuvants in the treatment of chronic malaria (rasoanaivo et al., a (rasoanaivo et al., , . malagashanine showed only weak in vitro intrinsic antiplasmodial activity (ic ¼ . ae . μm), but did display marked in vitro chloroquine-potentiating action against the fcm chloroquine-resistant strain of plasmodium falciparum. another study allowed clarification of the mechanism of action of the major constituent, malagashanine, being able to prevent chloroquine efflux from the cell, and stimulates chloroquine uptake into drug-resistant p. falciparum strains. malagashanine appears able to act more on plasma membrane than inside the parasite, allowing the toxicity of chloroquine against plasmodium, even at sublethal doses. in the attempt to confirm the reversal of chloroquine resistance by the bark of s. myrtoides, a double-blind randomized controlled clinical trial of a standardized alkaloid extract titrated at % malagashanine took place in a government-run outpatient clinic in the town of ankazobe (northwest central highlands of madagascar), but the results of the treatment showed no significant efficacy, indicating a need for other confirmations. however, in conclusion, the approach, in accordance with recent tendencies on multidrug resistance control, based on mixtures of natural products and classic antimalarial drugs, with a relevant coincidence between the ethnobotanical reports and the scientific evidence, may offer interesting possible solutions for the treatment of malaria. many aspects about the mechanism of action of malagashanine as chloroquine adjuvant to reverse the resistance need further study. malagashanine could increase drug accumulation by interacting with a dysregulated ion exchanger, avoiding the decrease inside the food vacuole, or acts by a mechanism related to drug binding to hematin (perisco et al., ; rafatro et al., ) . in particular, in relation to the ph role in the blood red cell, it would be necessary to determine if malagashanine acts inside or outside the food vacuole, including the membrane periphery. the capacity of malagashanine to reverse cq resistance may be related to the well-known properties of verapamil ( fig. . ) and related substances (martin et al., ; martiney fig. . verapamil and other compounds studied for cq-resistant reversal by membrane calcium channels blocking. adovelande et al., ) . verapamil was the first calcium channel antagonist to be introduced into therapy in the early s. it is a phenylalkylamine calcium channel blocker used in the treatment of high blood pressure, heart arrhythmias, and angina. in short-term incubations, verapamil was found to increase chloroquine accumulation in the lysosome of erythrocytes infected with both chloroquine-sensitive and -resistant organisms, but only to affect the chloroquine susceptibility of the latter. verapamil works independently of the overall ph gradient concentrating cq into a trophozoite's digestive vacuole. the activity is therefore related to the inhibition of membrane ion channels, interfering in the chloroquine transit within the parasite's cytoplasm. other substances like chlorpheniramine and others are reported as candidates for cq-resistant reversers. in any case, again the key role of natural products and ethnoparmacology information, such as for quinine (cinchona sp.) and artemisinin (artemisia annua), is fully confirmed. another attempt to explain the activity of malagasy plants alkaloids explored the role of glutathione. l-glutathione reduced (gsh) (fig. . ) is a simple tripeptide, consisting of glutamic acid, cysteine, and glycine. it is considered one of the most powerful endogenous antioxidants, capable of preventing damage to cellular components caused by reactive forms of oxygen, radicals, and heavy metals, although its role in stress management and efficient defense against pathogens are still under study (mangoyi et al., ) . besides its antioxidant defense and free radical scavenging, glutathione regenerates important antioxidants such as vitamins c and e. gsh exists in every cell of the human body, but it is also present in many other organisms, including fungi and bacteria. there is a linkage between gsh and malaria. some parasites are superprotected by gsh. they are endowed with powerful and host-independent mechanisms, which de novo synthesize or regenerate gsh and protect the parasites from oxidative damage and other outside attacks. gsh in particular protects the gametocytes against oxidative stress and inhibits the action of arginine, which produces no and expels it from the food vacuole. at the trophozoite stage of p. falciparum in human erythrocytes, gsh takes part in detoxifying processes of heme, produced by hemoglobin digestion, by polymerizing some % of heme to insoluble hemozoin. some authors suggest that the nonpolymerized heme, existing in the food vacuole, is subsequently degraded by gsh, increasing the role of this metabolite. chloroquine could interact with gsh, competitively inhibiting the degradation of heme by gsh or allowing toxic heme to accumulate in membranes and damaging parasites. this argument merits some explanation. the prooxidant damage and inflammation process created by excessive heme, hemozoin, and fragments from rupture of the digestive vacuoles in blood vessels and plasma can be mitigated by glutathione. in other words, the inside of the infected erythrocyte glutathione is beneficial to the parasite; outside of the erythrocyte it reduces the negative effects of the malarial infection. high oxidative stress could actually be detrimental for the survival of young parasites (gallo, ; patzewitz and m€ uller, ) . glutathione transferases (gsts) are versatile enzymes involved in the intracellular detoxification of numerous substances. gsts have been investigated in parasite protozoans, like those involved in malaria, with respect to their biochemistry and as targets in synthesis of new antiparasitic agents. p. falciparum possesses high quantity of these enzymes (pfgst) and their activity was found to be increased in chloroquine-resistant cells, and it has been shown to act as a ligand for parasitotoxic hemin. pfgst represents a promising target for antimalarial drug development. a pfgst isolated from p. falciparum has been associated with chloroquine resistance. plant extracts have been found to act at different vulnerable metabolic sites of pfgst, disturbing gsh-dependent detoxification processes, increasing cytotoxic peroxides levels and possibly increasing the concentrations of toxic hemin in the parasites. in the case of s. myrtoides alkaloids, malagashanine was found to prevent chloroquine efflux from and stimulated chloroquine influx into drug resistant p. falciparum, suggesting that its effects are more on the plasma membrane than inside the parasite. malagashanine ( μm) reduced the activity of pfgst to %, but showed a time-dependent inactivation of pfgst, suggesting a role of malagashanine as a chemomodulator in cases of pfgst overexpression in chloroquine-resistant strains. the malaria cycle of a parasite is based on two cycles, one involving the host and the other affecting the vector. during the mosquito cycle, again there are metamorphoses and reproduction by the parasite. in consideration of the resistance phenomenon, new transmissionblocking agents, able to interrupt malaria transmission, are required. these blocking drug components can be effective in reducing gametocyte density in the human host (gametocytocidal activity) or disrupting parasite development in the vector (sporontocidal activity), resulting in a reduced number of infective vectors and, as a consequence, decreased incidence of malaria cases. in other words, control malaria's parasites through the cure of the vectors infested by the disease. in the sexual stages of plasmodium parasites, gametocytes are critical for the transmission of the parasite to its vectors. p. falciparum gametocytes are also important in the disease diffusion, since being exceptionally long-lived, they cause clinically cured patients to be reservoirs of infection. the cycle of propagation of the malaria parasites starts when the female anopheles feeds on blood from an infected vertebrate. immediately, the first metamorphosis starts. by the ingestion, the mature male and female gametocytes, namely micro-and macrogametocytes, enter the mosquito host. immediately after reaching the mosquito's midgut, the two types of gametocytes undergo dramatic metamorphoses. we must remember that such transformations are a response to environmental stimulation, like a decrease in temperature, an increase in ph, and an influence of xanthurenic acid. within - min, the rounded macrogametes leave the erythrocytes and diffuse inside the blood, together with the flagellates microgametes. now comes the last change. within the next h, the motile male gametes can fecundate the macrogametes, and round zygotes develop that mature to elongated motile ookinets and move to the outer midgut surface, completing early sporogonic development. these changes can be obtained by severe transformation inside the intrinsic cell organization, involving the cytoskeleton directly. an equatorial position of chromosomes in the metaphase plate in the middle of the spindle is necessary for mitosis and symmetric cell divisions. a symmetric metaphase plate position is essential for symmetric cell divisions, explaining why it is conserved in all metazoans, plants, and many fungi. control of this parameter is essential, since differences in cell size have been linked to cell fate and generate a class of anticancer drugs. movements of chromosomes are in charge of microtubules, which are elements of the cytoskeleton. the cytoskeleton is a network of protein fibers forming the "infrastructure" of eukaryotic and prokaryotic cells. in eukaryotic cells, protein filaments and motor proteins form a complex mesh of protein filaments and motor proteins. the cytoskeleton aids the inside cell movement and transportation of subunits, like organelles and molecule groups, stabilizes and maintains cell shape, and gives support and order. the cytoskeleton is not a static structure but it is able to disassemble and reassemble its parts in order to enable internal and overall cell mobility. intracellular movements include in particular manipulation of chromosomes during mitosis and meiosis from the equatorial plaque to the polar positions, in the formation of daughter cells, and also it is implicated in the immune cell response to pathogens. the cytoskeleton is composed of at least three different types of fibers: microtubules, microfilaments, and intermediate filaments. these fibers are distinguished by their size, with microtubules being the thickest and microfilaments being the thinnest. the assemblement of the proteins, tubulines a and b, makes microtubules, in form of long cave filaments. these hollow rods function primarily to help support and shape the cell and as "routes" along which organelles can move. therefore, without the action of microtubules, the cell is unable to reproduce. the cell is blocked in a limb, with part of the mitosis already done and the final act in progress. the result is a polyploid cell, meaning a cell with double or more than the normal number of chromosomes. because chromosomes cannot move alone, they must be dragged by the cytoskeleton. the mechanisms of action of several important antitumoral drugs derived from natural products are characterized by promotion of the assembly or disassembly of microtubules, meaning stabilization or destabilization of the tubules against depolymerization, resulting in mitotic arrest. treated cells have defects in mitotic spindle assembly, chromosome segregation and movements, and consequently in cell division. the main problem of the utilization of these compounds in combination chemotherapy for sensitive tumor types concerns their selectivity against malignant cells. cancer is basically a disease of uncontrolled cell division, including too-active mitosis, multiplying the cancerous mass. in most cases, these changes in activity are due to mutations in the genes that encode cell cycle regulator proteins. however, although cancer cells are a selected target, in consideration of their high level of mitosis, other tissues can be involved in the action of positive regulators of cell division. molecular agents of plant origin are of primary importance in cancer treatment. those acting on the cytoskeleton can be classified into two main groups: antimicrotubule agents like colchicine and the vinca alkaloids, which induce depolymerization of microtubules, and taxol and taxotere, which induce tubulin polymerization and form extremely stable and nonfunctional microtubules (rowinsky et al., ) . neem products have been seriously explored in recent years, in several sectors, mainly in the fight against insect-borne diseases. however, it seems that so far the potentiality of neem has been only lightly touched on. neemazal is a marketed neem product consisting of a quantified alcoholic extract obtained from azadirachta indica seeds, with a reported limonoid concentration of . %, consisting of azadirachtin a %, azadirachtins b-k . %, salanins %, and nimbins % (dembo et al., ; habluetzel et al., ) . neemazal completely blocks transmission of the rodent malaria parasite p. berghei to anopheles stephensii in vivo, when administered to gametocytemic mice at a corresponding azadirachtin a dose of mg/kg. other in vivo transmission blocking studies suggested that na may have stronger transmission blocking activity than azadirachtin a alone, evaluating the activity of nonazadirachtin a constituents of neemazal. in an ex vivo assay, which exploits a major target process of azadirachtin a against p. berghei, microgamete formation inhibition of plasmodium was used to estimate the pharmacodynamics of two varying doses of neemazal and azadirachtin a. a team led by prof. g. chianese (university of salerno, italy) explored the possibility of influencing plasmodium gametocytes by neem products, demonstrating the potential of blocking the reproduction stages of the parasite. neemazal is a marketed neem product consisting in a quantified alcoholic extract obtained from azadirachta indica seeds, with a reported limonoid concentration of . %, consisting in azadirachtin a %, azadirachtins b-k . %, salanins %, and nimbins %. neemazal completely blocks transmission of the rodent malaria parasite p. berghei to anopheles stephensii in vivo, when administered to gametocytemic mice at a corresponding azadirachtin a dose of mg/kg. other in vivo transmission blocking studies suggested that na may have stronger transmission blocking activity than azadirachtin a alone, evaluating the activity of nonazadirachtin a constituents of neemazal. azadirachtins exert relevant effects on microtubules assembly and organization, interfering with the expression and/or function of adhesive proteins during the genesis of microgametocytes, through disruption of the organization of mitotic spindles and cytoskeleton formation and activity. these molecules can interfere with cytoplasmic microtubule organization and distribution, causing severe depletion of actin levels. in this action, neemazal proved to be more effective than azadirachtin a. in confirmation, another study showed that the product completely inhibits the growth of p. falciparum field isolates in an. coluzzii mosquitoes at a dose of ppm in direct membrane feeding assays. microorganisms have not finished producing surprises and breaking the boundaries reported in books. meanwhile researchers are investigating malaria parasites more and more deeply in search of their weak points, but their study is complicated by the parasite's metamorphosis, which involves not only the shape but also fundamental aspects of the metabolism (becker and kirk, ) . asexual stages of the parasite contain a single mitochondrion, whereas gametocytes can have several mitochondria. the energy production is very important. plasmodium falciparum, as well as other similar apocomplexa protozoans, possesses an intriguing nonphotosynthetic plastic, discovered in the s. the surprise was that apicoplastides possess their own nucleic acid. regarding their role, they were considered by kilejian ( ) as "a source of some substrate essential for energy production of mitochondrion." in view of their other characteristics, they could be considered a possible bridge between organisms or the ancestral point of divergence from green algae and protozoans. in conclusion, apicoplastides could be part of the endosymbiosis pathway, wherein degenerated chloroplasts were useful to increase a mitochondrial efficiency still in evolution. thus, endosymbiosis started with the inclusion of the two main bacterial forms, the hetero-and the autotrophic one, but later the ancestral (green or red) primordial alga degenerated the chloroplast in favor of a clear evolution toward the heterotrophic metabolism ( fig. . ) . usually the shift to the eukaryotic cell is considered a consequence of environmental factors, such as the increase of oxygen in the oxidative atmosphere; however, it is possible that in some cases interactions between organisms could also have played an important role. the study on apicoplastides allowed researchers to evidence similarities (keeling, ; kilejian, ; k€ ohler et al., ) between different arthropod-borne diseases, such as avian malaria, eimoriosis, and toxoplasmosis, confirming once more the occurrence of common survival strategies in different organisms. other differences concern the enzymes network and the membrane transport mechanisms. the new knowledge about parasitespecific organelles could be of fundamental importance to the development of future antimalarial drugs, increasing efficiency and decreasing side effects, like resistance. another important research front full of possibilities is focused on the membrane mechanism of cq's extrusion by permeability pathways induced by the parasite in the host red blood cells (saliba et al., ) . this is related to the cq of interfering with the detoxification of toxic heme monomers. the studies showed that - h after the invasion by the parasite, the socalled new permeability pathways act on the interchanges, i.e., the entry of nutrients, as well as mediating the efflux of metabolic wastes. several groups advanced the hypothesis of a number of channel types, activated by particular stress or stimuli (ginsburg and stein, ; kirk et al., ; duranton et al., ; staines et al., ; thomas and lew, ) . all these references testify to and confirm the presence of a wide range of studies in search of an answer to the challenge of resistance. the front is still too large and undetermined, but every year the knowledge of host cell reaction is increasing and there is a high probability that the problem will be solved in the coming years. during the development of the arguments contained in this book, it was necessary link the insect-borne diseases argument to several collateral items. the idea in particular was concentrated on a possible utilization of this particular topic as an epiphany, meaning an enlightening subject, which allows a revision of the problem from a new perspective. the interpretation of a new and key piece of information can allow the process of significant thought about a problem, until, in accordance with the original significance of the term in ancient greek, the ἐπιφάνεια (epiphanea) appears like a manifestation, with a striking appearance. this book started with considerations about gaps and books. let us now return to these two points. the lesson from carson's book about the fundamental role of beneficial insects in the survival of mankind, has arguably not been understood. throughout all warm terrestrial ecosystems, insects are a dominant component and they are part of the lives of any organism. the insect-plant relationship is a fundamental biotic interaction, and plants account for a large part of the planet's biomass, many times the biomass of all animals together (new, ; jankielson, ; dunn, ) . the animal biodiversity is dominated by that of insects. they are a beautiful example of variability, in terms of both number of species (more than million) and abundance (more than half of all living organisms), although at most only about %- % of insects are scientifically described. this diversity, consisting of large numbers of individuals and great intra-and interspecific variety, is a consequence of the enormous functional significance of insects in habitats. primitive insects appeared very early in the silurian period, when plants and animals finally emerged from the sea and colonized dry land, and over the last million years the number of insect families has been rising. they were able to colonize any part of the territory, including the sky. today, the number of reported insect families is about and they have survived various negative major impacts, including the mass extinction event at the end of the cretaceous period. a review analysis, published in the journal biological conservation by francisco sánchez-bayo, at the university of sydney, australia, and kris wyckhuys, at the china academy of agricultural sciences, beijing china, attests a current insect collapse. the decline's hypothesis is based on a study of recent selected studies. the causes and significant factors include intensive agriculture, the heavy use of pesticides, urbanization, and climate change. the loss of insect population is calculated in an annual . % rate over the last - years, and the future tendency is evaluated to % in the next years and increasing continuously until only half left in years. this scenario is already underway. in puerto rico, a recent study revealed a % fall in ground insects over years. the catastrophic cascade effects on the planet's ecosystems include ants, aphids, shield bugs, and crickets, which are the food for many birds, reptiles, amphibians, and fish that eat insects. there are many indicators supporting the scenario (sánchez-bayo and wyckhuys, ; diamond, ) . in england, between and , the number of widespread butterfly species fell by % on farmed land, suffering the biggest recorded insect falls overall-though that is probably a result of this area being more intensely studied than most places. a particular alert concerning bees being seriously affected has also been raised in europe and the usa; for example, only half of the bumblebee species found in oklahoma in the usa in were present in (alburaki et al., (alburaki et al., , aizen, ). the number of honeybee colonies in the usa was million in , but . million have since been lost. in , according to eu data, there were around , beekeepers and million hives in the eu, producing , tons of honey per year, but the same source tells us that many insect pollinator populations are now in clear decline. there is similar news from brazil, with half a million bees dead. on one side, this is considered the effect of the use of some pesticides, toxic to bees. on the other side, it is a classic example of rapid and intense environmental change to improve agricultural intensification and pasture, with the systematic elimination of all trees and shrubs that normally surround the fields, so there are plain, bare fields that are treated with synthetic fertilizers and pesticides. dr. sanchez-bayo said: "we are not alarmists, we are realists. we are experiencing the sixth mass extinction on earth. if we destroy the basis of the ecosystem, which are the insects, then we destroy all the other animals that rely on them for a food source." he added, "it will collapse altogether and that's why we think it's not dramatic, it's a reality." the situation comprehends micro-and macroepisodes, like the continuous devastation of equatorial tropical forests, in particular the amazonia territory. the sequence is clear and well-known, and it always works: first, the fire destroys the vegetation, in particular the woody plants; second, the soil is cleaned, otherwise the plants could replace the habitat rapidly; and third, the territory is declared totally compromised and ready for further utilizations. however, as observed by samways in biodiversity and conservation ( , and later confirmed by this author in a series of further papers) in a paper titled "insects in biodiversity conservation: some perspectives and directives," the main concerns are the "lack of human appreciation of importance, coupled with the general disregard and dislike of insects, is an enormous perception impediment to their conservation. this impediment coupled with the taxonomic impediment must be overcome for realistic biodiversity conservation management. as it is not possible to know all the species relative to the rate at which they are becoming extinct, it is essential to conserve as many biotopes and landscapes as possible." there is a sentiment of urgency for measures "essential to preserve species dynamo areas as an insurance for future biodiversity," such that "preserved areas must also be linked by movement and gene-flow corridors as much as possible." the last point of view is crucial. preservation must be considered not only as an opportunity to maintain the presence of species in selected habitats against their disappearance, but it must be considered changes as opportunities to perform a positive future. in this regard, entomologists are asked to contribute in control of vectors affecting humans, crops, and livestock, but also to take an active part in the consideration due to the beneficial species. the central task is the possibility to predict accurately the environmental effects of any intervention. once the inherent risks connected with traditional control methods have been considered, the consequences of new introductions must be carefully predicted, including any synergist effect. the rate of insect species extinction is estimated as being eight times faster than those of mammals, birds, and reptiles (barnosky et al., ; dirzo et al., ) . another important current gap concerns scientific information. most ordinary people do not have access to data obtained by the scientific community, as well as opinions and models produced by experts and scientists. information, when available, is usually distorted and adapted to the dominant axioms by a plethora of generalist supposed experts. the proposed idea is that these kinds of people are able to know and comment on everything. the distortion, sometimes voluntarily pursued and often a consequence of general confusion, generates progressive modification of the starting points and even the concealment of important facts. the recent phenomenon of fake news is clearly generated from the same situation. although most research information is now easily accessible and can be obtained directly from the internet, its utilization remains restricted to dedicated people. in contrast, some scientific information is amplified far away from its real impact. how many times did you read about the discovery of a definitive cure to cancer? or about the already obtained solution to any physiological problem using staminal cells? in our era of globalized knowledge, news are obtained and fluxed indirectly, without few possibility of checking the origin and the reliability. it is necessary to consider that more than % of the human population, consisting of . billion people, are connected via the internet, and . billion utilize social networks regularly. these numbers are likely to increase % every year. all these people have access to information only through selected channels and although they are in a condition to verify it, science and general information are on different and distant levels. the main problem is that the information is reduced to a few soundbites, and there is no place for elaboration or proposals of other possible interpretations or points of view. this is not a recent case, produced by digitalization of communication. beside the sources, the problem of the quality of scientific information was fully evidenced more than years ago, in the "public understanding of science." this is the title of a report requested in by the royal society and prepared by a group of experts, whose leader was the geneticist sir walter fred bodmer. the report evidenced the general lack of knowledge about scientific themes. on one side, most of the population, accounting for two-thirds of europeans, was confident about science and technologies, considering that scientists were able to solve human problems and make human life "easier, healthier and comfortable." on the other side, the sequence "more communication ¼more knowledge¼more social adherence to scientific arguments" appears largely inadequate. the dominant problem about scientific communication is that ordinary people need an alphabetization to understand and meet the complexity of the scientific items. the conversion of the original scientific information is usually distorted and changed, at best "adapted," but more often polluted by political, social, and cultural interests. the result is a reductive metamorphosis, in the best case, or complete revision to be adapted and useful to already-made opinions. among the various examples of this operation we find neverending debates, such as those concerning ogm, vaccines, or the consequences of climate changes, without considering abnormal and artificially created themes, such as the contraposition between vegans and meat-eaters. the manipulation is based on a presumed "democratic" interpretation of scientific data. no vote is necessary to assure the consistency of a scientific law based on adequate experimentation, but the aim is that reliability must be obtained by public consensus and even agreement. independence has always been a necessary character of science, but manipulation was never pursued. history tells us that any political or social manipulation of science led to disaster. in contrast, priorities, when based on correct scientific information, as well as consequent implications and decisions, must be subject to the most ample democracy. at the end of this little journey through macro-, micro-, and nanoworlds, it is undeniable how long the road still is to understand and discover the mysteries of insect-borne diseases. in the meantime, we await the next surprises. the covid- pandemy dramatically evidenced all the current limits of science and technology to face this kind of challenges. the virus was faster and clever. predictively and prevention were insufficient. despite the potentiality, the debacle and medicine was evident and the consequent economic and social damages were enormous. microorganisms will continue to play their role inside the habitats and next time their target could be the industrialized sources of our food. however, it is clear that is society will continue to ignore the alerts of researchers and scientists, the next pandemy will be the worst one. synergy between two calcium channel blockers, verapamil and fantofarone (sr ), in reversing chloroquine resistance in plasmodium falciparum active and intelligent packaging: an introduction how much does agriculture depend on pollinators? lessons from long-term trends in crop production neonicotinoid-coated zea mays seeds indirectly affect honeybee performance and pathogen susceptibility in field trials honey bee survival and pathogen prevalence: from the perspective of landscape and exposure to pesticides green-synthesised nanoparticles from melia azedarach seeds and the cyclopoid crustacean cyclops vernalis: an eco-friendly route to control the malaria vector anopheles stephensi? operational feasibility of malaria control by burning neem oil in kerosene lamp in beel akbarpur village, district ghaziabad, india review of antimicrobial food packaging evaluation of larvicidal activity of medicinal plant extracts against three mosquito vectors has the earth's sixth mass extinction already arrived? antibacterial activity of karanj (pongamia pinnata) and neem (azadirachta indica) seed oil: a preliminary report of malaria, metabolism and membrane transport old ingredients for a new recipe? neem cake, a low-cost botanical by-product in the fight against mosquito-borne diseases larvicidal and ovideterrent properties of neem oil and fractions against the filariasis vector aedes albopictus (diptera: culicidae): a bioactivity survey across production sites shedding light on bioactivity of botanical by-products: neem cake compounds deter oviposition of the arbovirus vector aedes albopictus (diptera: culicidae) in the field neem (azadirachta indica): towards the ideal insecticide? synergized mixtures of apiaceae essential oils and related plantborne compounds: larvicidal effectiveness on the filariasis vector culex quinquefasciatus say chemical composition and insecticidal activity of the essential oil from helichrysum faradifani endemic to madagascar ethnopharmacology in the fight against plasmodium parasites and brain disorders: in memoriam of philippe rasoanaivo insecticidal and mosquito repellent efficacy of the essential oils from stem bark and wood of hazomalania voyronii herbal remedies of azadirachta indica and its medicinal application biological activities and medicinal properties of neem (azadirachta indica) le paludisme à madagascar a guide to the identification of diseases and pests of neem (azadirachta indica). rap publ neem-an omnipotent plant: a retrospection responsive food packaging: recent progress and technological prospects insecticide resistance in mosquitoes: a pragmatic review natural products as sources for new pesticides emergency and mosquitocidal potential of neem cake-synthesized silver nanoparticles: genotoxicity and impact on predation efficiency of mosquito natural enemies the efficacy of neem extract on four microorganisms responsible for causing dental caries viz streptococcus mutans, streptococcus salivarius, streptococcus mitis and streptococcus sanguis: an in vitro study biological activities and medicinal properties of neem (azadirachta indica) a novel phytocannabinoid isolated from cannabis sativa l. with an in vivo cannabimimetic activity higher than Δ -tetrahydrocannabinol: Δ bioactive packaging technologies for extended shelf life of meat-based products effectiveness of antimicrobial food packaging materials in vitro and ex vivo activity of an azadirachta indica a. juss seed kernel extract on early sporogonic development of plasmidium in comparison with azadirachtin a, its most abundant constituent active and intelligent packaging: applications and regulatory aspects assessment of neem oil effect on haematological profile and towards peripheral blood mononuclear cells of goat antimicrobial activity of a neem cake extract in a broth model meat system evaluation of a mono-component and a multicomponent herbal extracts as candidates for antimicrobial packaging of fresh retail meat assessment of microbiological quality of retail fresh pork meat in central italy neem (azadirachta indica a. juss) oil to tackle enteropathogenic escherichia coli impact of repeated neemazal ® treated blood meals on the fitness of anopheles stephensi mosquitoes the present, past and future of human-caused extinctions defaunation in the anthropocene active and intelligent packaging food-research and development-a review modern insect extinctions, the neglected majority organic osmolyte permeabilities of the malaria-induced anion conductances in human erythrocytes successful parasitoid control of aonidiella orientalis (newstead) (hemiptera: diaspididae) on carica papaya l biopesticide registration action document. office of pesticide programs. cold pressed neem oil. pc code . margosa extract pt- secondary metabolism as a measurement of the efficacy of botanical extracts: the use of azadirachta indica (neem) as a model insecticide status report on global neem usage indole alkaloids from strychnos species and their antiplasmodial and cytotoxic activities inherited glutathione reductase deficiency and plasmodium falciparum malaria-a case study plant extracts as potential mosquito larvicides phytochemicals for bacterial resistance-strengths, weaknesses and opportunities the new permeability pathways induced by the malaria parasite in the membrane of the infected erythrocyte: comparison of results using different experimental techniques neem-a green treasure chemical and biological investigations on azadirachta indica (the neem tree) plants myths and traditions in india impact of the botanical insecticide neem azal ® on survival and reproduction of the biting louse damalinia limbata on angora goats food losses and waste in the context of sustainable food systems neem pesticides the importance of insect in agricultural ecosystems focus on phytochemical pesticides. the neem tree increasing food availability by reducing postharvest losses of fresh produce chemische untersuchungen € uber die metamorphosehormone der insekten neemdb: convenient database for neem secondary metabolites bridge over troublesome plastids past, current and potential utilization of active and intelligent packaging systems for meat and muscle-based products: a review circular mitochondrial dna from the avian malarial parasite plasmodium lophurae spherical bodies transport of diverse substrates into malariainfected erythrocytes via a pathway showing functional characteristics of a chloride channel a plastid of probable green algal origin in apicomplexan parasites malagashanine: a chloroquine potentiating indole alkaloid with unusual stereochemistry neem: today and in the new millennium neem (azadirachta indica): prehistory to contemporary medicinal uses to humankind neem in the conventional lake chad basin area and the threat of oriental yellow scale insect (aonidiella orientalis newstead) (homoptera: diaspididae) current topics in active and intelligent food packaging for preservation of fresh foods not ordinary antimalarial drugs: madagascar plant decoctions potentiating the chloroquine action against plasmodium parasites glutathione transferase from plasmodium falciparum-interaction with malagashanine and selected plant natural products antimicrobial food packaging: potential and pitfalls citrus limonoids: analysis, bioactivity, and biomedical prospects antilarval activity of neem cake extracts against aedes albopictus composizione biologica con proprietà fortemente biocide a basso contenuto di azadiractina e procedimento per la sua realizzazione antimicrobial activity of melia azadirachta fruit extracts for control of bacteria in inoculated in vitro shoots of 'mrs- / ' plum hybrid and calla lily and extract influence on the shoot cultures reversal of chloroquine resistance in plasmodium falciparum by verapamil verapamil reversal of chloroquine resistance in the malaria parasite plasmodium falciparum is specific for resistant parasites and independent of the weak base effect product safety and security in the global supply chain: issues, challenges and research opportunities effect of neem leaf extract and on penicillum growth, sporulation, morphology and ochratoxin a production mosquitocidal and antiplasmodial activity of senna occidentalis (cassiae) and ocimum basilicum (lamiaceae) from maruthamalai hills against anopheles stephensi and plasmodium falciparum in vivo and in vitro effectiveness of azadirachta indica-synthesized silver nanocrystals against plasmodium berghei and plasmodium falciparum, and their potential against malaria mosquitoes report of an ad hoc panel of the board on science and technology for international development natural products as sources of new drugs from to hptlc fingerprint: a modern approach for the analytical determination of botanicals traceability in multi-ingredient botanicals by hptlc fingerprint approach advances in production of functional foods and nutraceuticals advanced in production of functional foods and nutraceuticals intelligent and smart packaging hptlc fingerprint analysis of plant staminal products analysis of multi-ingredient food supplements by fingerprint hptlc approach toxic effects of neem cake extracts on aedes albopictus (skuse) larvae neem cake: chemical composition and larvicidal activity on asian tiger mosquito the modern analytical determination of botanicals and similar novel natural products by the hptlc fingerprint approach current mosquito-borne disease emergencies in italy and climate changes. the neem opportunity neem-borne molecules as eco-friendly control tools against mosquito vectors of economic importance professor philippe rasoanaivo neem tree-"the village pharmacy the control of the oriental red scale, aonidiella orientalis newstead and the california red scale, a. aurantii (maskell) (homoptera: diaspididae) in mango orchards in hevel habsor (israel) trends in antimicrobial food packaging systems: emitting sachets and absorbent pads active food packaging technologies use of natural antimicrobials to increase antibiotic susceptibility of drug resistant bacteria glutathione biosynthesis and metabolism in plasmodium falciparum traditional herbal remedies and dietary spices from cameroon as novel sources of larvicides against filariasis mosquitoes? chemical composition of cinnamosma madagascariensis (cannelaceae) essential oil and its larvicidal potential against the filariasis vector culex quinquefasciatus say the interaction of heme with plakotin and a synthetic endoperoxide analogue: new insights into the heme-activated antimalarial mechamism neem: the divine tree azadirachta indica reversal activity of the naturally occurring chemosensitizer malagashanine in plasmodium malaria tetranortriterpenoids from azadirachta indica effect of the leaf essential oil from cinnamosma madagascariensis danguy on pentylenetetrazol-induced seizure in rats biological activities of the plant-derived bisindole voacamine with reference to malaria malagashanine potentiates chloroquine antimalarial activity in drug resistant plasmodium malaria by modifying both its efflux and influx malagashanine and malagashine, the alkaloids of strychnos mostuoides medicinal plants used to treat malaria in madagascar revised structure of malagashanine: a new nb,c( )-secocuran alkaloid reversing agents in treatment of drug-resistance malaria the co-occurrence of c( ) epimer nb,c( )-secocuran alkaloids in strychnos diplotricha and strychnos myrtoides screening extracts of madagascan plants in search of antiplasmodial compounds taxol: a novel investigational antimicrotubule agent limonoids: overview of significant bioactive triterpenes distributed in plants kingdom neem, a tree for solving global problems anti-microbial activity of a new vaginal contraceptive nim- from neem oil (azadirachta indica) transport and metabolism of the essential vitamin pantothenic acid in human erythrocytes infected with the malaria parasite plasmodium falciparum insects in biodiversity conservation: some perspectives and directives worldwide decline of the entomofauna: a review of its drivers fatty acid composition and antibacterial activity of neem (azadirachta indica) seed oil sustainable industrial utilization of neem tree (azadirachta indica) in nigeria some arthropod pests and a semi-parasitic plant attacking neem (azadirachta indica) in kenya the neem tree: azadirachta indica a. juss. and other meliaceous plants, sources of unique natural products for integrated pest management, medicine, industry and other purposes the neem tree: source of unique natural products for integrated pest management, medicine, industry and other purposes the neem tree (azadirachta indica a. juss) and other meliaceous plants: sources of unique natural products for integrated pest management a review of botanical phytochemicals with mosquitocidal potential mosquito repellent action of neem (azadirachta indica) oil quality of packaged foods plasmodium falciparum-induced channels ethnobotanical uses of neem (azadirachta indica a. juss.; meliaceae) leaves in bali (indonesia) and the indian subcontinent in relation with historical background and phytochemical properties the role of botanicals as green pesticides in integrate mosquito management-review monograph on neem (azadirachta indica a. juss.). international book distributors biosensors in food processing plasmodium falciparum and the permeation pathway of the host red blood cell susceptibility of immature stages of aedes aegypti, the vector of dengue and chikungunya to insecticides from india neem (azadirachta indica) and its potential for safeguarding animals and humans the hptlc approach to metabolomic determination of neem products composition determination by hptlc of chemical composition variability in raw material used in botanicals costa concordia disaster: environmental impact from phytochemical point of view trasmission blocking effects of neem (azadiracha indica) seed kernel limonoids on plasmodium berghei sporogonic development ecophysiological and phytochemical response to ozone of wine grape cultivars of vitis vinifera ethnopharmacognostical survey of azadirachta indica a juss quassinoids: structural diversity, biological activity and synthetic studies public health impact of pesticides used in agriculture: reportage of a world health organization and u.n. environmental programme malaria treatment guidelines. world health organization key: cord- - vnz zzg authors: garcia, sònia title: pandemics and traditional plant-based remedies. a historical-botanical review in the era of covid date: - - journal: front plant sci doi: . /fpls. . sha: doc_id: cord_uid: vnz zzg pandemics are as old as humanity and since ancient times we have turned to plants to find solutions to health-related problems. traditional medicines based mostly on plants are still the only therapeutic possibility in many developing countries, but even in the richest ones, herbal formulation currently receives increased attention. plants are natural laboratories whose complex secondary metabolism produces a wealth of chemical compounds, leading to drug discovery – % of widespread use drugs are indeed of plant origin. their therapeutic potential is even bigger: although many plant-based compounds show inhibitory effects against a myriad of pathogens, few reach the stage of clinical trials. their mechanism of action is often unknown, yet traditional plant-based remedies have the advantage of a long-term experience in their use, usually of hundreds to thousands of years, and thus a precious experience on their safety and effects. here i am providing a non-systematic historical-botanical review of some of the most devastating pandemics that humanity has faced, with a focus on plant therapeutic uses. i will revisit the middle ages black death, in which a plant-based lotion (the four thieves vinegar) showed some effectiveness; the smallpox, a viral disease that lead to the discovery of vaccination but for which the native americans had a plant ally, an interesting carnivorous plant species; tuberculosis and the use of garlic; the spanish flu and the widespread recommendation of eating onions, among other plant-based treatments; and malaria, whose first effective treatment, quinine, came from the bark of a peruvian tree, properties already known by the quechua people. synthetic analogues of quinine such as chloroquine or hydroxychloroquine are now being revisited for the treatment of covid symptoms, as they are artemisinin and derivatives, other plant-based compounds effective against malaria. finally, i will give some hints on another facet of plants to aid us in the prevention of infectious diseases: the production of biotechnological plant-based vaccines. altogether, my aim is to stress the significant role of plants in global health (past, present and future) and the need of enhancing and protecting the botanical knowledge, from systematics to conservation, from ecology to ethnobotany. pandemics have shaped the history of mankind, and plants were usually the first available therapeutic choice. there is evidence of herbal preparations by egyptians around bc, later improved by greeks and romans, and widely documented in official drug books known as pharmacopoeias. still in our days, traditional medicines based mostly on plants are the only therapeutic possibility for many people in developing countries (akerele, ) . but, also in the first world, with wide access to the most modern drugs, the use of plant-based traditional medicine is experiencing a revival, as it is seen as safer and healthier than synthetic drugs. indeed, one advantage of traditional remedies over modern drugs is that their effects and margin of safety have been known for long. there is also a renewed scientific interest on plant-derived drug discovery, according to the current increasing publication trend on the topic (atanasov et al., ) . the rich secondary metabolism that characterises plants make them a source of compounds that may have a yet unknown therapeutic potential, only limited by the availability of resources to perform clinical trials. it is claimed that natural products (mostly from plant origin) will be the most important source of new drugs in the future (atanasov et al., ) . a recent editorial (nature plants, ) highlighted the need of funding and understanding botanical knowledge in the context of the current, and possibly future, pandemics. it is urgent to develop therapeutic tools to protect from high risk of infection (mitjà and clotet, ) and plant-based remedies with proven safety profiles could be one of the faster solutions. here i present a non-systematic review with a historical-botanical perspective on some of the most important pandemics that humanity has faced, and in some cases is still facing, and how certain plants or plantbased remedies have been used, and may continue being used, to treat these diseases, possibly including covid . the black death or black plague took place in the middle ages ( ) ( ) ( ) ( ) ( ) in eurasia, and still is the deadliest pandemic ever, with an estimated loss of million of human lives wiping out to % of european population in roughly four years (deleo and hinnebusch, ) . although this is the most know outbreak of the bacterium yersinia pestis, the much earlier -and longer-plague of justinian (from to ad) was also caused by the same pathogen (harbeck et al., ) , killing about - million people during two centuries. there have been other less spectacular, but still important plague outbreaks, arriving to the most recent ones in madagascar during the present decade. originated in china, the plague was usually spread by trade boats, whose rats carried fleas with the bacterium, which was transmitted to humans directly by the bite of the flea, and then between humans by contact or aerosol inhalation. there are several forms of the disease, the most common being the bubonic plague, which provokes the inflammation of the lymph nodes (buboes) as its most recognizable sign; a second form is the pneumonic plague which affects the respiratory system and is more deadly; the third form, the septicaemic plague, is the least common but has a mortality ca. % (byrne, ) . the antibiotic treatment, starting in early xxth century, reduced the death rate to about %- % which previously was between %- %; however, little is known on the remedies used before antibiotics were a reality and the major plague outbreaks occurred much earlier. in the middle ages, some preventive measures included, among others, carrying sweet smelling herbs to clear "the evil air" (which was believed to carry the pathogen) around the person (jones, ) , garlic for cleaning kidneys and liver, and lavender or chamomile teas to calm the stomach bile (khaytin, ) . a remedy named "the four thieves vinegar" was very popular: it consisted in several herbs, such as angelica (angelica archangelica), camphor (cinnamomum camphora), cloves (syzygium aromaticum), garlic (allium sativum), marjoram (origanum majorana), meadowsweet (filipendula ulmaria), wormwood (artemisia absinthium), and sage (salvia officinalis), brewed in vinegar (gattefosse, ) . before going out, people should apply it on hands and face for avoiding to contract the plague. some of these plants are well known flea repellents, so this may be one of the reasons for its efficacy. other herbs such as meadowsweet might have been included to release pain (as it contains salicylic acid, a precursor of aspirin) and to mask odours, a very helpful property considering that decomposing bodies were usually encounteredthe legend states that the name of the remedy might refer to thieves using it to rob the plague dead or sick (lucas, ) . another treatment coming from ancient greeks also gained popularity: the king mithridates antidote (totelin, ) , an extract of about fifty plants in a mixture with opium (papaver somniferum) paste, which if any, at least eased the pain or promoted a peaceful death. other prescriptions included lavender or rosewater baths, probably due to their antimicrobial and buboes healing properties. willow bark (another source of salicylates) was also given as a painkiller (khaytin, ) . a curious "prophylactic" plant-based remedy was recommended by the napolitan doctor angelerio during a plague outbreak in alguero in the xvi century ( - ) ( figure ): "any person going out from home must carry a cane (note: probably from the species arundo donax) six spans long, and as long as the cane is, one must not approach other people" (bianucci et al., ) . the origin of smallpox, a viral infectious disease caused by variola virus (two variants: v. major and v. minor) is unknown, but it dates back at least to the ancient egypt (third century bc), since some mummies showed smallpox-like eruption, the characteristic macules of the disease. as with the plague and other pandemics, the disease has occurred in several outbreaks around the world, the most recent in the late s. this virus has killed between and million people during the th century (koplow, ) until the global eradication campaign by the world health organisation (who) in . smallpox was the first infectious disease to have been eradicated ( ), the (only) second one being rinderpest, a viral illness of cattle. the smallpox vaccine (the first ever) was based on edward jenner's demonstration, by the end of the xviii th century, that inoculation with cowpox (a variant of the smallpox virus infecting cows) protected against the disease. actually, jenner's contribution popularized the practice of vaccination, a word coined by himself coming from the latin word vaccinus (i.e., or/from the cow) for the prevention of several other infectious diseases. however, before vaccination was discovered, how did people deal with the illness? particularly interesting was the approach of the native americans, which were deeply affected by the disease. by the end of xix th century several surgeons and practitioners related to the us army, as well as the prestigious botanist charles f. millspaugh ( ) , described the use of poultices and infusions from the indigenous medical flora based on the plant sarracenia purpurea (family sarraceniaceae) to be effective for treating smallpox, in a likely case of medical appropriation of the indigenous therapeutic knowledge (lawrence-mackey, ). known by native americans (mi'kmaq people) as mqo'oqewi'k, also named purple pitcher plant, it belongs to a genus of carnivorous species that use modified pitcher-shaped leaves to trap insects. possibly, the spotted appearance of the plant (figure ), resembling one of the main clinical signs of the disease (clarke, ) , inspired its use to the indigenous people. this may be another example of the doctrine of signatures, an ancient concept by which god somehow indicated to men what plants would be useful for, by certain signs (coles, ) , a pseudoscience which has caused more harm than good in general, although exceptions appear. compelling descriptions of their effectiveness were recorded, such as ''the greatest remedy known for the dreadful scourge'' or "'it seemed to arrest the development of the pustules, killing, as it were, the virus from within" (clarke, ) . the advent of vaccination put forward the botanical remedy, but the antiviral properties of sarracenia purpurea have been later demonstrated in vitro (arndt et al., ) . the authors showed that the plant extract was not only active against smallpox, but also against other poxviruses, papovirus sv- and various herpes viruses, including papillomavirus and epstein-barr virus-associated carcinomas, usually by inhibiting the virus replication at the level of early transcription (moore and langland, ) . another global and persistent pandemic is tuberculosis, caused by mycobacterium tuberculosis. together with smallpox, it is one of the oldest known diseases, since molecular data and archaeological evidence support that it coexisted with humans from the neolithic (gutierrez et al., ; nicklisch et al., ) . relevant figures in the history of medicine such as hippocrates or avicenna identified the disease, which involved coughing blood and fever and which was often lethal. actually, avicenna detected the infectious nature of the illness and based on tuberculosis, was probably the first to come up with the idea of quarantine to stop the spread of infectious diseases (shephard, ) . tuberculosis infected million people only in , of which . million died (unaids, ). it is found in every country, being the first infectious cause of death worldwide. from the hiv/aids outbreak, the combination of both is usually fatal where tuberculosis is endemic (mainly developing countries), as the immune weakening caused by hiv facilitates the onset of tuberculosis. this disease is indeed the final cause of death of many hiv infected people; paradoxically, while many could live now with aids they are dying from tuberculosis. the main reason is that the bacterium has developed resistance to most antibiotics, which usually have to be taken during long and tedious treatments. this is why researchers have turned to the search of effective alternatives also among medicinal plants, as some of them have already demonstrated anti-tuberculosis activity. besides, an effective plant-based treatment would be more affordable in poor countries which are those more affected by the disease. among the plants that are being investigated, garlic (allium sativum, family alliaceae), a former remedy already used to treat the plague (see above) stands out for its renowned properties, although it still far from being an alternative. the benefits of garlic (of which there are about varieties) are well-known already from the ayurvedic and unani medicine systems (raghunandana et al., ) as well as from the chinese traditional medicine, but also ancient greeks, romans and even egyptians used it to treat illnesses. it has been used as a food and folk medicine for centuries by many cultures. garlic has a variety of pharmacological virtues, including antimicrobial, anticancer, antioxidant (dini et al., ) , fungicidal and as a cure for heart diseases, among others (majewski, ) . the antitubercular and other antimicrobial activities of garlic, however, have been demonstrated in vitro but still, seldom in vivo. although garlic has more than biologically active compounds, allicin is the most relevant, albeit highly unstable; therefore, depending on the preparation of the garlic-based remedy the efficacy may not be as high as expected (majewski, ) . the wide antimicrobial and even antifungal spectrum of allicin is explained by its inhibitory effects on sulfhydryl metabolic enzymes. by interacting with these enzymes, allicin induces thiol stress in bacteria, which, among others, inhibits the growth of the microorganisms (müller et al., ) . malaria, caused by protozoan species of the plasmodium group, is an infectious disease coming from the bite of a mosquito, usually an infected anopheles sp. female. the current name of the illness was given by the italians around xix th century, as a contraction of the words "mal aria" (i.e., bad air) from the belief that the disease was transmitted by the "miasma" coming from marshes (macip, ) . the most typical symptom is fever, together with nausea and vomiting, tiredness, headache, occasionally yellow skin and in severe cases it can lead to seizures, coma and death. it is another ancient disease, spanning from the neolithic to our days, and currently found in all intertropical continents. recent studies detected the parasite in african monkeys, probably being the source of the disease, although it is still debated how it spread worldwide (molina-cruz and barillas-mury, ) . it is known that malaria arrived to europe by the first century ad, probably coming from the african rainforests and travelling by the nile to the mediterranean, where it spread to the middle east and from there to greece, italyhistorians hypothesize on the triggering role of malaria in the fall of the roman empire -and the rest of europe, even as far as england and denmark (karlen, ) . between the xvi th -xix th centuries the disease crossed the atlantic ocean probably on slave ships to reach the american continent (yalcindag et al., ) . it was suffered by presidents of north america such as g. washington or a. lincoln and it raged specially with native americans, taking thousands of their lives. the centers for disease control and prevention, the leading national public health institute of the united states, was founded because of malaria in . in the last century, probably - million people have died from the disease, accounting for %- % of deaths (carter and mendis, ) . at present malaria is mostworrying in sub-saharan africa, accounting for ca. % of current cases, although there is also a resurge in southern asia (arrow, ) . the most well-known and one of the most effective historical treatments against malaria is quinine, an alkaloid extracted from the bark of the cinchona tree (cinchona officinalis) belonging to family rubiaceae, the same of coffee. it is original from peru (where it is the national tree, although currently it is considered an endangered species) and the quechua traditionally used the ground bark of these trees to stop shivering because of cold, not for malaria treatment per se. most likely, spanish jesuits missionaries brought cinchona to europe for the first time, having observed how the quechuas used it to threat shivering, by the end of xvi th century -a second case of medical appropriation in this story. the tree was named (by c. linnaeus) after the spanish countess of chinchon, who was treated with its bark in peru back in the early xvi th -linneaus misspelled the name of the countess, omitting the first "h" in the name (meshnick, ) . it is also said that the countess may have introduced the curative bark to europe when she returned to spain, but it is currently considered that this a legend rather than what actually happened (haggis, ) . quinine is effective on the "cessation of febrile paroxysms" (stephens et al., ) , one of the main symptoms of malaria, and which has given its popular name to the species (fever tree). malaria outbreaks, however, continued to appear during centuries with no alternative to quinine. during world war i the german army was strongly affected by the parasite in the troops of east africa, as the allies controlled java, the main worldwide quinine producer. in an interesting historical moment that impelled science, the german government commissioned a search for a substitute to quinine, determined not to suffer again from its shortage. in chloroquine, a synthetic compound similar to quinine was synthesized at the bayer laboratories. much later ( ) another very similar derivative, hydroxychloroquine, was produced in the us. both chloroquine and hydroxychloroquine are used to prevent and treat malaria, being some of the antimalarial drugs of choice in areas where the disease is not resistant to them (a recurrent problem in this disease); they were preferred over quinine because of much less severe adverse effects, although at present there are many other even safer alternative drugs for the treatment of malaria. in recent years, these antimalarials have shown several immunomodulatory effects and they currently treat, mostly, diseases such as lupus erythematosus or rheumatoid arthritis. as with tuberculosis, the parasite tended to develop resistance to these treatments sooner or later, and researchers were urged to look for alternatives. a very popular one was found in the plant artemisia annua (sweet wormwood, from family asteraceae) (figure ) a remedy known in chinese traditional medicine as qing-hao for more than years (klayman, ) . chinese herbalists had been using it for treating haemorrhoids, chills, and fevers (trigg and kondrachine, ) . the species, as other members of genus artemisia such as absinthe or tarragon, is aromatic and bitter. one of the compounds responsible for its bitterness, a sesquiterpene lactone extracted from the glandular trichomes named artemisinin, is the active compound against malaria. the discovery of artemisinin is also very remarkable from the historical point of view. in , during the chinese cultural revolution under mao zedong's mandate, the secret "project " was a plant screening research program to find an alternative treatment for malaria, which was ravaging vietnamese army during vietnam war. in , dr. youyou tu a researcher of that program, isolated artemisinin, "rediscovering" the ancient remedy qing-hao. the drug started to be used in , a relatively short-period to establish a new medicine in the market, but it was also based on thousands of years of experience by the chinese traditional practitioners. nowadays artemisinin and its synthetic derivatives are one of the main defences against drug-resistant malaria in the asiatic southeast. however, who recommends it in combined therapy with other drugs, in part to avoid the development of resistance and in part to counteract the short half-life of artemisinin in plasma, leading to the artemisinin combination therapies (acts) which include companion drugs such as some cloroquine derivatives (e.g., mefloquine). in tu was awarded the nobel prize for the discovery of artemisinin, which represents an important contribution of china to the global health, as well as the first and awaited nobel prize in the sciences for china. it is considered the most significant milestone of tropical medicine of the last century, contributing to a better health and saving tens of thousands of lives every year in tropical developing countries of south asia, south america and africa. in the spring of started one of the deadliest pandemics in recent history, caused by one aggressive strain of the h n influenza virus (the same virus that caused the swine flu pandemic). it was popularly known as the spanish flu because in the context of world war i censorship minimized the effects of the pandemic to keep people's morale in the countries involved in the conflict, while in the neutral spain newspapers were free to report its effects, giving the impression that this country had been particularly devastated by the disease. during months, until summer , there were three waves of the pandemic, being the second the worst. a fourth, much fainter wave, took place in the spring of and after this one, the virus disappeared as it had arrived. it infected about half a billion people (ca. / of the world's population) and killed about million (with some estimates as high as million); for a reference world war i estimates range from to million deaths. the origin of the virus is unclear but it is thought that it started as a zoonosis from birds to humans which later was transmitted from humans to swine. the symptoms were an amplified version of those of normal flu, but typically deaths were caused by complications derived from a secondary pneumonia. contrary to other h n flu strains, this one was unusually lethal among young people, and almost one century later its high-virulence is only partly understood (tumpey et al., ) . since at that time there was neither vaccine, nor antibiotics to treat secondary pneumonia, the main prophylactic options were, as with the covid pandemic, to avoid contact through lockdown and quarantines, to increase personal hygiene and to use disinfectants widely. also, people turned to folk remedies and some recommendations got popular, such as the widespread advice "eat more onions!" (figure ) (arnold, ) . as with garlic, onion (allium cepa) has certain compounds (particularly a polyphenol named quercetin) which have demonstrated antiviral properties (lee et al., ; sharma, ) but still more research and clinical trials are needed. besides, in the usa a group of doctors known as "the eclectics" got positive results by treating the flu symptoms with plant remedies, together with other measures that included exercise. they reported a fatality rate ca. . % for their patients while the average in that pandemic was ca. % (abascal, ) . by selecting the herbs to match the symptoms, they used a wide variety of species. the most remarkable among them, and that have later proved therapeutic, were: gelsemium sempervirens (known as yellow yasmine, with antipyretic properties), eupatorium perfoliatum (boneset, already known by native americans to treat cold-related symptoms), actea racemosa (black cohosh, also used by native americans as a painkiller, probably due to the content in salicylic acids of its roots) and asclepias tuberosa (pleurisy root, used to treat respiratory problems and with expectorating properties) (abascal, ) . however, despite their long history of use, again there is little applied research on these plants. currently, flu is partly under control by the release of annual vaccination campaigns with newly synthesized vaccines that collect most of the virus' seasonal variability. in the latter most important flu pandemic ( ) besides the vaccine, oseltamivir (tamiflu ® ) a drug derived from the species ilicium verum (star anise, from family schisandraceae) was also crucial to treat most severe cases, although the production of this compound is limited by the low productivity of the tree, and synthetic derivatives are being developed (macip, ) . finding adequate treatments for flu is still and urgent task as the fear of a pandemic similar to the one in is a still a sword of damocles in the concerns of most epidemiologists. from the s, the science of "molecular farming" gives another potential role to plants on the prevention of infectious diseases, involving plants or plant cell cultures to produce recombinant proteins (rybicki, ) . the first steps of this approach were the "manufacturing" of the human growth hormone, monoclonal antibodies or human serum albumin in transgenic tobacco or sunflower plants ( barta et al., ; hiatt et al., ; sijmons et al., ) . other recombinant proteins more recently produced in plants -shifted from bacterial, mammalian or fungal cell to plants and plant cell cultures-and commercialized, include human type collagen i manufactured in tobacco, bovine trypsin in maize or human lysozyme and lactoferrin in rice (yang et al., ; hennegan et al., ; shoseyov et al., ; takeyama et al., ) . as with the mentioned proteins, the vaccine production would follow similar steps: isolation of a specific antigen protein, the one that triggers an immune response from the virus; the gene(s) encoding that protein is transferred to bacteria and these bacteria are used to infect plants, so the plant will in turn produce the antigen proteinthe vaccine. plants would provide a flexible, cheap and easily scalable method to manufacture vaccines. they would also be safer than traditional vaccines, because of the absence of pathogens of animal origin. plant-based vaccines for humans are not yet in the market, although some candidates have entered clinical trials (rosales-mendoza et al., ) . it is likely, however, that they will start being approved in the mid-term, at least in the cases of ebola or rabies, or in a longer term for seasonal influenza (rybicki, ) . the outbreak of covid caused by the coronavirus sars-cov- , originated in china (province of wuhan) in december and has caused , , infections and , deaths worldwide (updated th june ). even earlier than the pandemic status was declared by who ( th march ) researchers across the world engaged in hundreds of clinical trials, in an unprecedented quest for a cure to the disease, vaccine, drug or both. given the long time-frames that usually imply finding a good candidate, many research groups have turned to repurpose other drugs. the reasons are that the effects (including adverse or side effects) of these drugs are well known and have been used in broad population groups with different ages and idiosyncrasies, so the security margin is increased, allowing to save precious time in long trials. in this regard, antimalarials are potential candidates (schlagenhauf et al., ) and both chloroquine but particularly hydroxychloroquine (as explained above, synthetic derivatives of quinine, the antimalarial alkaloid coming from the bark of the fever tree) are being studied to fight covid , although it is perhaps too soon to draw conclusions on their efficacy. several studies have reported their utility for some patients and some national guidelines have recommended both drugs for treatment of covid (see colson et al., and singh et al., ) despite there has been certain controversy. the who halted studies on these drugs by the end of may prompted by an observational study reporting that hydroxychloroquine produced a higher mortality rate in hospitalised patients, but the study was soon retracted on the basis of questionable veracity of data and analysis (mehra et al., ) and trials on the drug have been resumed shortly after. the effectiveness of hydroxycloroquine taken at initial stages of the disease was recently tested in a multicentre randomised controlled trial based on previous experiences of post exposure prophylaxis (pep) drugs to prevent infections (mitjà and clotet, ) , but no benefit was observed beyond the usual care . other plant-based antimalarials, artemisinin and derivatives, are also being tested against sars-cov- , again not without controversy. in many african countries, an elixir based on artemisia annua extract, "covid-organics" is being distributed as a cure against covid . however, there is little scientific evidence of the effectiveness of such elixir and its extended consumption can have associated problems, the most important the development of resistance to the drug by the malaria parasites in a continent particularly sensitive to the disease. nevertheless, there is evidence that the extract of artemisia annua has antiviral properties, being active against sars-cov- (li et al., ) , herpes simplex (karamoddini et al., ) , hepatitis a (seo et al., ) , hepatitis b, bovine viral diarrhoea, and epstein-barr (haq et al., ) . this has stimulated the research of the potential use of artemisinin and derivatives (such as artesunate) to treat covid , which is now being conducted by several biotech companies (e.g., mateon therapeutics, artemilife) as well as by public research institutions (e.g., the liverpool school of tropical medicine, the max planck institute of colloids and interfaces). traditional chinese medicine has also had a say in the cure of covid : the national administration of traditional chinese medicine (natcm) organized a study in late january to identify potential treatments, and the lung cleansing and detoxifying decoction (lcdd) was widely used and studied through clinical trials; its apparently high effectiveness made that the natcm officially recommended lcdd as a treatment for covid (weng, ) . among the lcdd ingredients, there were species such as ephedra sinica (well known as decongestant and bronchodilator through the active compound ephedrine), atractylodes macrocephala (showing antiviral activity against influenza viruses in experimental assays) or scutellaria baicalensis (containing anti-inflammatory flavonoids), and the combination of these effects and others from the remaining ingredients likely counteracts covid by their synergistic activities. however, as weng ( ) points out, it is difficult to transfer the success of the lcdd treatment to other countries, both because the cultural acceptance of tcm is not present outsides china, and due to the lack of knowledge on the precise chemical composition and mechanism of action, which are required in modern therapy. once the first vaccine for covid is finally developed (with estimates ranging from september to several years) another plant may also play an important role in order to produce it in large amounts: nicotiana benthamiana, a close relative to tobacco. this species is the focus of the eu project newcotiana, coordinated by researcher dr. diego orzaéz at the csic, the leading public research body in spain. in this project, genome editing practises (e.g., crispr) will be used to transform the plant in a biofactory for the large-scale production of the vaccine once it is available. moreover, one company may be currently developing a covid vaccine based on the expression of a sars-cov- protein in tobacco -kentuchy bioprocessing, a biotechnological branch of british american tobacco (capell et al., ) . as rosales-mendoza et al. ( ) stated in a recent review, the production of a plant-based vaccine in the context of the current pandemic would have the advantages of low cost, fast and escalable production, easy administration, and safety. beyond plant-based vaccines, molecular farming through plants, usually by transient expression of target proteins, can also be deployed to produce diagnostic reagents, as well as antibodies and antiviral proteins for therapeutic use. the italian biotechnology company diamante is generating antigens, to use in elisa tests (serological), based on a sars-cov- protein also in tobacco plants (capell et al., ) . another eu consortium, pharma-factory is also developing plant-based platforms to produce medical, veterinary and diagnostic products, for dealing with covid and also other diseases. i hope that the reader finds this review useful to call for the important role that plants have played and still play in human health. as schlagenhauf et al. ( ) commented, plant-based remedies are, more than an "alternative medicine", the organisms to which we owe some of the most useful therapeutic tools. still in the era of wide implementation of (synthetic) drug treatments, we turn to plants in many cases when resistances appear, as shown. paradoxically, there is a human tendency to ignore plants, a form of cognitive bias known as "plant blindness" (allen, ) that should be opposed, perhaps by enhancing and implementing more widely the botanical education. in this context, it is also essential not only to maintain but to increase societal funding into basic sciences such as botany, as well as to foster collaboration between scientists from different disciplines, whose interaction may open new therapeutic possibilities. finally, i would like that this review serves as a little recognition to the usually ignored ethnobotanical traditional knowledge of many indigenous peoples across the world of which the so-called western culture has in most occasions, illegitimately appropriated. the author confirms being the sole contributor of this work and has approved it for publication. i am thankful to roi rodriguez (institut botànic de barcelona) who kindly revised this paper and contributed interesting observations, as well as to the reviewer. herbs and influenza: how herbs used in the flu pandemic can be effective in the next nature's medicinal bounty: don't throw it away plant blindness in vitro characterization of a nineteenth-century therapy for smallpox pandemic : the story of the deadliest influenza in history the locus of decisions about aids/hiv, malaria treatment: what does welfare economics say? a question discovery and resupply of pharmacologically active plantderived natural products: a review the expression of a nopaline synthase -human growth hormone chimaeric gene in transformed tobacco and sunflower callus tissue quinto tiberio angelerio and new measures for controlling plague in th-century alghero the black death potential applications of plant biotechnology against sars-cov- evolutionary and historical aspects of the burden of malaria dictionary of practical materia medica in three volumes adam in eden, or nature's paradise chloroquine and hydroxychloroquine as available weapons to fight covid- a plague upon the phagocytes the potential role of garlic (allium sativum) against the multi-drug resistant tuberculosis pandemic: a review aromatheŕapie -les huiles essentielles hormones veǵetales ancient origin and gene mosaicism of the progenitor of mycobacterium tuberculosis fundamental errors in the early history of cinchona artemisia annua: trials are needed for covid- yersinia pestis dna from skeletal remains from the th century ad reveals insights into justinianic plague improvement of human lysozyme expression in transgenic rice grain by combining wheat (triticum aestivum) puroindoline b and rice (oryza sativa) gt promoters and signal peptides production of antibodies in transgenic plants the new cambridge medieval history: c. -c. antiviral activities of aerial subsets of artemisia species against herpes simplex virus type (hsv ) in vitro man and microbes: disease and plagues in history and modern times arcgis storymaps. herbal medicine in the black plague qinghaosu (artemisinin): an antimalarial drug from china smallpox: the fight to eradicate a global scourge medical appropriation in the 'red'atlantic: translating a mi'kmaq smallpox cure in the mid-nineteenth century anti-influenza a virus effects of fructan from welsh onion (allium fistulosum l.) identification of natural compounds with antiviral activities against sarsassociated coronavirus nature's medicines; the folklore, romance, and value of herbal remedies les grans epidèmies modernes (edicióactualitzada): la lluita de l'home contra els enemics invisibles allium sativum: facts and myths regarding human health retraction-hydroxychloroquine or chloroquine with or without a macrolide for treatment of covid- : a multinational registry analysis from quinine to qinghaosu: historical perspectives medicinal plants: an illustrated and descriptive guide to plants indigenous to and naturalized in the united states which are used in medicine, their description, origin, history, preparation, chemistry and physiological effects fully described use of antiviral drugs to reduce covid- transmission hydroxychloroquine for early treatment of adults with mild covid- : a randomized-controlled trial the remarkable journey of adaptation of the plasmodium falciparum malaria parasite to new world anopheline mosquitoes sarracenia purpurea: a botanical extract with anti-papilloma virus and oncolytic activity allicin induces thiol stress in bacteria through s-allylmercapto modification of protein cysteines rib lesions in skeletons from early neolithic sites in central germany: on the trail of tuberculosis at the onset of agriculture redeploying plant defences investigations on plant antibiotics; studies on allicin, the antibacterial principle of allium sativum (garlic) what does plant-based vaccine technology offer to the fight against covid- ? vaccines plant-based vaccines against viruses repurposing antimalarials and other drugs for covid- antiviral activity of herbal extracts against the hepatitis a virus efficacy of garlic and onion against virus the middle-ages: monasteries, medical schools and the dawn of state health care," in an illustrated history of health and fitness, from pre-history to our post-modern world human collagen produced in plants: more than just another molecule production of correctly processed human serum albumin in transgenic plants chloroquine or hydroxychloroquine for prevention and treatment of covid- syst. rev. , cd studies in the treatment of malaria: v. intramuscular injections of quinine alkaloid in simple tertian malaria plant-based vaccines for animals and humans: recent advances in technology and clinical trials mithradates' antidote-a pharmacological ghost commentary: malaria control in the s characterization of the reconstructed spanish influenza pandemic virus tuberculosis-good progress, but not enough plant solutions for the covid- pandemic and beyond: historical reflections and future perspectives multiple independent introductions of plasmodium falciparum in south america expression and localization of human lysozyme in the endosperm of transgenic rice key: cord- -ku tmjd authors: sabotič, jerica; kos, janko title: microbial and fungal protease inhibitors—current and potential applications date: - - journal: appl microbiol biotechnol doi: . /s - - -x sha: doc_id: cord_uid: ku tmjd proteolytic enzymes play essential metabolic and regulatory functions in many biological processes and also offer a wide range of biotechnological applications. because of their essential roles, their proteolytic activity needs to be tightly regulated. therefore, small molecules and proteins that inhibit proteases can be versatile tools in the fields of medicine, agriculture and biotechnology. in medicine, protease inhibitors can be used as diagnostic or therapeutic agents for viral, bacterial, fungal and parasitic diseases as well as for treating cancer and immunological, neurodegenerative and cardiovascular diseases. they can be involved in crop protection against plant pathogens and herbivorous pests as well as against abiotic stress such as drought. furthermore, protease inhibitors are indispensable in protein purification procedures to prevent undesired proteolysis during heterologous expression or protein extraction. they are also valuable tools for simple and effective purification of proteases, using affinity chromatography. because there are such a large number and diversity of proteases in prokaryotes, yeasts, filamentous fungi and mushrooms, we can expect them to be a rich source of protease inhibitors as well. applications of protease inhibitors are intimately connected to the proteases they inhibit. so, as a preface to protease inhibitors, an overview of proteases with the modes of regulation of their proteolytic activity is provided. then, known microbial and fungal protease inhibitors are reviewed, with the emphasis on protein (tables and ) rather than small-molecule protease inhibitors (table ) . finally, their potential applications in the fields of medicine, crop protection and biotechnology are described, based on their target proteases. microorganisms (prokaryotes, yeasts and filamentous fungi) and higher fungi or mushrooms have been selected for review since protease inhibitors of microbial origin have already proven useful in many different applications. higher fungi have emerged as a valuable source of new protease inhibitors with unique characteristics only in the last decade and therefore offer great potential for future applications. proteases, also called peptidases or proteolytic enzymes, constitute a large group of enzymes that catalyse the hydrolysis of peptide bonds. cleavage of peptide bonds can be general, leading to complete degradation of protein substrates into their constituent amino acids, or it can be specific, leading to selective protein cleavage for post-translational modification and processing. peptidases that cleave peptide bonds at the termini of polypeptide chains are called exopeptidases, while endopeptidases cleave peptide bonds within the polypeptide chain. peptidases are classified according to their catalytic type into aspartic, cysteine, glutamic, serine and threonine peptidases, according to the main, functional amino acid residue at the active site. metallopeptidases, on the other hand, are those whose catalytic activity depends on the presence of a divalent metal ion bound within the active site. in the mer-ops database (http://merops.sanger.ac.uk/), peptidases are classified further into families, according to their sequence similarity, and into clans, according to their structural similarity. there are peptidase families assigned in the merops database (release . , july ) and clans, based on structural data (barrett ; rawlings et al. ) . peptidases are present in all living organisms, including viruses, bacteria, archaea, protists, fungi, plants and animals. serine peptidases form the most abundant class, followed by metallo-, cysteine, aspartic and threonine peptidases. there has been an explosive growth of the number of peptidase families observed in eukaryotic organisms, there being peptidases in bacterial genomes and half as many in archaeal genomes and from to peptidase genes in plant and mammal genomes. furthermore, there is a striking difference between the compositions of eubacterial and eukaryotic degradomes, (i.e. the complete set of proteases present in an organism). sixteen peptidase families constitute the core of the nearly ubiquitous peptidase families present in all living forms. additional peptidase families are widely distributed in eukaryotic organisms, while another ten are unique to higher metazoan organisms, performing mainly limited proteolysis in extracellular environments (page and di cera ; rawlings et al. ) . in addition to the merops database, information on proteases can be found in several other online databases, including the degradome database (http:// degradome.uniovi.es/) (quesada et al. ) and the proteolysis map (pmap) (http://www.proteolysis.org/) that comprises five α m a a, a a, c a, c a, c , m , m a, m b, m a, m b, s a, s b, s a a underlined families include protease inhibitors exclusively of microbial and/or fungal origin b × denotes the number of sequence homologues found in each group of organisms: × less than , ×× - , ××× - and ×××× more than (armstrong and quigley ; sottrup-jensen ) serine protease inhibitors ovomucoid (kazal-type) chymotrypsin (s ) and subtilisin (s ) families tight-binding, laskowski described in stramenopiles oomycetes (fungus-like microorganisms distantly related to fungi); e.g. involved in pathogenicity of phytophtora infestans (tian et al. ; rawlings and barrett ) aprotinin i chymotrypsin family (s ) broad inhibitory specificity (ascenzi et al. ; rawlings and barrett ) peptidase b inhibitor i subtilisin family (s ) bacterial inhibitors are propeptides of subtilisin-like proteases. fungal inhibitors are separate polypeptides, e.g. pleurotus ostreatus proteinase a inhibitor poia and yeast proteinase inhibitor yib they are potent but unstable inhibitors, gradually degraded by subtilisin (ascenzi et al. ; dohmae et al. ; kojima et al. ; kojima et al. ; kojima et al. ; rawlings and barrett ) marinostatin i proteases of family s (subtilisin) and certain proteases of family s (chymotrypsin) tight-binding, laskowski. structure stabilized by two internal ester bonds that are essential for their inhibitory activity exclusive to marine bacteria (kanaori et al. ; rawlings and barrett ) ecotin i chymotrypsin family (s ) tight-binding, laskowski for primary binding site. active as dimers, each monomer binds the protease at two binding sites ecotins from enterobacteria and parasites perform a protective role against host digestive proteases and target host proteases to facilitate colonization. structure enables inhibition of multiple proteases with the chymotrypsin fold (eggers et al. ; eschenlauer et al. ; rawlings and barrett ) streptomyces subtilisin inhibitor (ssi) family s (subtilisin, kexin), family s (trypsin, plasmin) and the metalloprotease griselysin (family m ) tight-binding, laskowski exclusive to bacterial actinomycetales order. they probably control endogenous proteases involved in proteolytic activation of transglutaminase (kantyka et al. ; taguchi et al. ; tsuyuki et al. ; rawlings and barrett ) carboxypeptidase y inhibitor i serine carboxypeptidase y (family s ) a defensive role against predatory insects has been shown, but a regulatory endogenous role is also possible (avanzo et al. ; odani et al. ; sabotič et al. ; rawlings and barrett ) human elastase- and endogenous aspergillus elastases (family s ) unknown homologues have been found in a few other ascomycete species and in proteobacteria, but none was characterized biochemically (okumura et al. ; okumura et al. ; rawlings and barrett ) serpin i chymotrypsin (s ) and subtilisin (s ) families. some members inhibit also cysteine proteases of papain (c ) and caspase (c ) families trapping. suicide inhibitors in which a rapid conformational change traps the cognate protease in a covalent complex the physiological role of microbial serpins has been proposed to be to protect the cellulosedegrading apparatus (cellulosome) against proteolytic degradation. the only fungal serpin (celpin), was characterized from the anaerobic fungus piromyces sp. e (kantyka et al. ; law et al. ; roberts et al. ; steenbakkers et al. ) cysteine protease inhibitors thyropin i papain-like proteases (family c ), and equistatin inhibitor unit inhibits an aspartic protease cathepsin d (family a ) tight-binding present in animals and in one bacterial pathogen (coxiella burnetii) that has presumably acquired the gene by lateral transfer (kantyka et al. ; rawlings and barrett ) survivin i caspases-aspartate-specific cysteine proteases (family c ) tight-binding, several mechanisms survivin plays a dual role as a mitotic regulator of cell division and as an inhibitor of caspase activation in the process of apoptosis. fungal homologues have been identified in ascomycete and a few basidiomycete genomes. the fission yeast homologue is a conserved chromosomal passenger protein (huang et al. ; luthringer et al. ; rawlings ) chagasin i protozoan and mammalian papain-like cysteine proteases (family c ) tight-binding parasitic chagasins are involved in regulating endogenous cysteine proteases essential for their life cycle. in bacteria and archaea, chagasins serve as endogenous regulators, and in some pathogenic species, they also serve a protective role against host proteases (kantyka et al. ; santos et al. ) clitocypin (i ) and macrocypin (i ); together named mycocypins i , i papain-like cysteine proteases (family c ) and legumain (family c ) and serine protease trypsin (family s ) tight-binding. mycocypins are small and exceptionally stable proteins. they have a β-trefoil fold formed by the core six-stranded β-barrel that supports loops which provide a versatile surface for the inhibition of several types of proteases unique to basidiomycetes. they probably have an endogenous regulatory role or a role in defence against pathogen infection and/or predation by pests. a defensive role for mycocypins is further supported by their high genetic variability and conformational stability as well as a broad inhibitory profile (brzin et al. ; kidrič et al. ; renko et al. ; sabotič et al. a; sabotič et al. ; sabotič et al. b the high specificity is the result of the structural stabilization of the ia inhibitor in complex with saccharopepsin since the unstructured inhibitor in solution forms an alpha helix upon interaction with the enzyme active site (green et al. ; phylip et al. ) different databases (cutdb, pathwaydb, proteasedb, substratedb and profiledb) (igarashi et al. ). the occurrence of proteases in all living organisms indicates their critical role in essential metabolic and regulatory functions in many biological processes. proteases are important in the production of nutrients for growth and proliferation. extracellular proteases catalyse the hydrolysis of proteins into smaller peptides and amino acids for subsequent absorption into cells, constituting a very important step in nitrogen metabolism. proteases perform critical regulatory functions in numerous physiological processes since they regulate the fate, localization and activity of many proteins, modulate protein-protein interactions and contribute to the generation, transduction and amplification of molecular signals. proteases are involved in a wide span of cellular and metabolic processes, including regulation of gene expression, dna replication, transport of proteins, cell growth and differentiation, cell cycle, heat shock response, sos response to dna damage, misfolded protein response, oxidative stress response and programmed cell death (lopez-otin and bond ; rao et al. ). furthermore, in plants, proteases are important in the build-up and breakdown of seed storage proteins during seed germination, protein remobilization upon organ senescence and in many developmental processes such as embryogenesis, chloroplast biogenesis, photomorphogenesis, hormone signalling, flower development, pollen-pistil interaction and local and systemic defence responses against pathogens and herbivores (simoes and faro ; salas et al. ; schaller ; van der hoorn ) . moreover, in animals, proteases are involved in tissue morphogenesis and remodelling, angiogenesis, neurogenesis, ovulation, fertilization, wound repair, stem cell mobilization, haemostasis, blood coagulation, inflammation, immunity, autophagy and senescence (lopez-otin and bond ). proteases of microbial and fungal origin offer a wide range of biotechnological applications. alkaline proteases have been used in the detergent industry for over years. proteases with elastolytic and keratinolytic activities have been used in the leather industry for de-hairing and baiting of skins and hides. the food industry uses proteases in cheese making, baking, preparation of various protein hydrolysates, meat tenderization and manufacturing protein-rich diets. in the pharmaceutical industry, proteases have found uses as therapeutic agents as well as additives in preparations of slow-release dosage forms. bioprocessing of used x-ray films for silver recovery involves the use of alkaline proteases. proteases allow potential applications for the management of wastes from various food processing industries and from household activities. in addition to industrial and medical applications, proteases are used in basic research; for example, proteases with very selective peptide bond cleavage are used in protein sequencing, unselective proteinase k is used in nucleic acid isolation, and trypsin is widely used in maintaining animal cell cultures (kumar and takagi ; rao et al. ) . there is also the downside to proteases as some are important virulence factors of many pathogenic bacteria, parasites and viruses. these proteases are involved in acquiring nutrients for growth and proliferation through host tissue degradation and evasion of host immune defences. in addition to colonizing and facilitating dissemination functions, they are also involved in evading the host immune system by interrupting the cascade pathways, disrupting the cytokine network, excising cell surface receptors and inactivating host protease inhibitors (maeda ; travis and potempa ; supuran et al. ) . because proteases play essential roles in life and death processes in all living organisms and because peptide bond hydrolysis is irreversible, anomalies in proteolytic activities lead to numerous pathological conditions, including cancer, neurodegenerative disorders and inflammatory and cardiovascular diseases, as well as bacterial, viral and parasitic diseases (lopez-otin and bond ; turk ) . activity of proteases is regulated on several levels, including regulation of gene expression at transcriptional and post-transcriptional levels, synthesis as inactive zymogens, blockade by endogenous inhibitory proteins, spatial and temporal compartmentalization, post-translational modification (glycosylation, phosphorylation, co-factor binding), proteolysis and degradation (lopez-otin and bond ). protein protease inhibitors constitute a very important mechanism for regulating proteolytic activity. they can be classified approximately according to the class of proteases they inhibit (for example, serine or cysteine protease inhibitors). however, those composed of multiple inhibitor units and pan-inhibitors (such as α -macroglobulin of family i ) that target proteases of different catalytic classes prevent unambiguous classification. a more detailed classification is included in the merops database (http://merops.sanger. ac.uk/), which follows a hierarchy similar to that for proteases. protease inhibitors are grouped into families based on sequence homology and into clans based on protein tertiary structure. in release . ( july ) of the mer-ops database, there are families of protease inhibitors, and those with available three-dimensional structural data have been assigned to different clans (rawlings ) . of the families, include members of microbial and fungal origin (tables and ). of these, seven families include members of exclusively bacterial origin (i , i , i , i , i , i , i ), and five families include members of exclusively fungal origin (i , i , i , i , i ). in addition to protein protease inhibitors, the merops database includes a list of small-molecule inhibitors that are well known and widely used. many of them have been synthesized in the laboratory; however, those that occur naturally (table ) have been isolated from bacteria and fungi (rawlings ) . there are two general mechanisms of protease inhibition, namely, irreversible "trapping" reactions and reversible tight-binding reactions. trapping reactions work only on endopeptidases and are the result of a conformational change of the inhibitor triggered by cleavage of an internal peptide bond by the host protease ( fig. a ). only three families utilize a trapping mechanism: i (serpins), i (α -macroglobulin) and i (viral caspase inhibitors). reversible tight-binding inhibition is widespread, the best known being the "standard canonical" or "laskowski mechanism", in which the inhibitor has a peptide bond that is cleaved by the peptidase active site in a substrate-like manner. the inhibitor is only slowly released due to the conformational stability of the stabilized loop that can mimic a substrate. this mechanism has been conclusively demonstrated only for inhibitors of serine proteases. other reversible tight-binding protease inhibitors physically block the protease active site by high-affinity binding to sites on either side of the active site (fig. b, c) . binding of an inhibitor to the active site can also be irreversible, when an electrophilic reactive group of the inhibitor forms a covalent bond with an amino acid residue in the enzyme active site. there are also some inhibitors that block the exosites, to which substrate binding occurs in addition to the active site in some proteases (christeller ; rawlings ; ). the families of protein protease inhibitors that include members of microbial and fungal origin are described in table , and information of their distribution in taxonomic groups is given in table . protease inhibitors are described in groups according to the catalytic class of protease they inhibit and following the merops inhibitor classification (rawlings ; rawlings and barrett ) . since prokaryote-derived protease inhibitors have been reviewed recently (kantyka et al. ), more information on protease inhibitors of fungal origin, including yeasts, filamentous fungi and mushrooms, is provided here. among the small-molecule protease inhibitors isolated from bacteria and fungi (table ) , there are several that show broad inhibitory specificity and inhibit proteases of different catalytic classes. several inhibit both serine and cysteine proteases (antipain, chymostatin, leupeptin), serine and metalloproteases (bestatin, puromycin), metallo-and cysteine proteases (amastatin) or metallo-, cysteine and serine proteases (bacitracin a). of the small-molecule cysteine protease inhibitors, the best known is e- ( -[l-n-(trans-epoxysuccinyl) leucyl] amino- -guanidinobutane), an irreversible inhibitor originally isolated from aspergillus japonicus (hanada et al. ) and routinely used as a class-specific cysteine protease inhibitor. a number of e- analogues have been synthesized in order to improve selectivity for a particular cysteine protease. several inhibitors are specific for metalloproteases and inhibit more than one protease family (e.g. phosphoramidon). the only natural small-molecule inhibitor of aspartic proteases is pepstatin, originally isolated from various species of actinomycetes (umezawa et al. ) , which inhibits several families of aspartic proteases. it is a hexa-peptide containing the unusual amino acid statine (rawlings ) . proteases play an important part in almost every biological process; therefore, unregulated activity often leads to disease. in this review, only excessive proteolysis will be addressed as it is the one that can be reversed by protease inhibitors. excessive proteolysis plays an important role in cancer and in cardiovascular, inflammatory, neurodegenerative, bacterial, viral and parasitic diseases. due to the obvious relevance of protease inhibitors, they have been studied extensively with the intent to develop therapeutic drugs (drag and salvesen ; turk ; haq et al. ) . proteases that have a potential as therapeutic targets are reviewed, according to their catalytic type, for each group of disease-causing organisms and for other human diseases. information on the availability of protease inhibitors for each protease described is provided, with the emphasis on those for which specific inhibitors have not yet been identified. proteolytic processing of virus polyprotein into structural and non-structural proteins is an essential part of the viral replication cycle, making the proteases an important antiviral drug target. several viral proteases have been studied as therapeutic targets. although proteases of any virus could be potential antiviral targets, viruses that cause chronic diseases (e.g. hiv, herpes virus) and those that could cause large-scale epidemics (e.g. sars coronavirus, dengue virus) have received most attention. several protease inhibitors acting against the human immunodeficiency virus (hiv- ) protease, a homodimeric aspartic protease, have been used in treating hiv- infection. fig. examples of protease inhibitors utilizing irreversible "trapping" reaction (a) and reversible tight-binding reactions (b and c). proteases are shown in light grey, their active site residues in black and inhibitors in dark grey. a serine protease trypsin in complex with serpin (family i ) (pdb id k o). the protease cleaves the reactive centre loop of serpin, which triggers a conformational change in the inhibitor and trapping of the protease in an inactive covalent complex. b cysteine protease cathepsin v in complex with clitocypin (family i ) (pdb id h s). the inhibitor binds to the protease active site cleft and obstructs access of substrate. c aspartic protease plasmepsin iv in complex with the small-molecule inhibitor pepstatin a (pdb id ls ). the inhibitor binds in the active site of the protease these are all low molecular weight peptidomimetic inhibitors whose design has been based on the structures of the compounds binding to the protease active site. in therapy, they are usually used in combination with inhibitors of reverse transcriptase (abbenante and fairlie ; anderson et al. ). in addition to a number of designed synthetic inhibitors, a potent peptidic inhibitor of hiv- protease of bacterial origin (atbi) has been found in an extremophilic bacillus sp. (dash and rao ; vathipadiekal et al. ) . other targeted viral proteases belong to the serine catalytic type. human cytomegalovirus (hcmv) is one of eight human herpes viruses and is widespread in populations worldwide, with infection rates of - %. it causes asymptomatic infections in healthy individuals but high morbidity and mortality in immunocompromised individuals. a few inhibitors of the cytomegalovirus protease have been described from bacterial (streptomyces) and fungal (cytonaema) origins (stoeva and efferth ; anderson et al. ). an additional target in antiviral therapy against cytomegalovirus is the proteasome, where the aim is to hinder the recruitment of host proteins by the virus for its replication. several synthetic and a few natural proteasome inhibitors (e.g. lactacystin from streptomyces lactacystinaeus) are known and have been reported to obstruct replication of several viruses, including influenza virus, herpes simplex virus type , paramyxovirus and rhabdoviruses, as well as cytomegalovirus (kaspari and bogner ). the serine proteases ns and ns of flaviviruses are targets for antiviral drug development against hepatitis c virus and dengue virus, with the former blood-transmitted virus causing various liver diseases (including cirrhosis and liver cancer) and the latter being a mosquitotransmitted disease causing dengue hemorrhagic fever. several structure-based designed low molecular weight inhibitors are in different phases of clinical evaluation (anderson et al. ; lange et al. ; tomlinson et al. ). the coronavirus associated with severe acute respiratory syndrome, sars, encodes a chymotrypsin-like cysteine protease m pro that is similar to picornavirus c protease. since the sars global outbreak, several strategies of structure-based design of low molecular weight protease inhibitors have been applied in the search for antiviral drugs against sars (anderson et al. ; sirois et al. ). the first to be considered were the protease inhibitors targeting the picornavirus c protease. the picornaviruses, which encode a c protease, are important human and animal pathogens such as poliovirus, hepatitis a virus, coxsackievirus, human rhinovirus and foot-and-mouth disease virus. inhibitors targeting the c protease of human rhinoviruses that cause common cold, as well as c proteases of other picornaviruses and coronaviruses, have been developed based on structural data, but none has yet successfully passed all the phases of clinical evaluation (neubauer et al. ; wang and liang ) . due to the rapid development of resistance in viruses, the search for novel strategies for developing inhibitors targeting different sites on proteases is encouraged, including the search for novel lead compounds from natural sources and structure-based drug development. bacterial pathogens employ an array of virulence factors that enable their colonization, evasion of host defences and dissemination. proteases are important virulence factors of many pathogenic bacteria, which play roles in acquiring nutrients by direct degradation of host tissue components. the even more important aim of disrupting host immune response and signalling cascades has been reviewed by potempa and pike ( ) . most currently available antibiotics target bacterial cell wall synthesis or protein synthesis. in the light of rapidly spreading antibiotic resistance, bacterial proteases are promising targets for the design of novel antibiotics. metalloproteases are most abundantly represented in primary and opportunistic pathogens, although all catalytic classes are found. these proteases are often associated with mobile genetic elements (plasmids, pathogenicity islands, integrated phages), and their expression is not constitutive but regulated through environmental or cellular signals (travis and potempa ; wladyka and pustelny ) . omptins (outer membrane proteins t) are aspartic proteases (family a ) found in several gram-negative bacteria, including the pathogenic species escherichia coli (ompt), yersinia pestis (pla), shigella flexneri, shigella dysenteriae (sopa), salmonella enterica (pgte), legionella pneumophila (lpa) and plant pathogens agrobacterium tumefaciens and erwinia pyrifoliae (plaa). omptins are bacterial virulence factors and, in addition to their proteolytic activity, possess adhesive and invasive activities. they modulate the coagulation system since they act as plasminogen activators (ompt, pla, pgte, lpa), inactivate tissue factor pathway inhibitor (ompt, pla, pgte), degrade thrombin-activatable fibrinolysis inhibitor (pla, pgte), degrade the complement system proteins (pla, pgte) and antimicrobial peptides (ompt, pla, pgte), and process autotransporters (ompt, sopa). lipopolysaccharide (lps) is required for their enzymatic activity. omptins have a unique catalytic mechanism that combines the elements of both serine and aspartic proteases, and partial inhibition by serine protease inhibitors has been reported (hritonenko and stathopoulos ; valls seron et al. ; yun et al. ). other than a weak substrate-based peptide inhibitor with a d-arg, shown to inhibit ompt (dekker et al. ) , and a colicin immunity protein shown to protect colicin e from degradation by ompt in escherichia coli (duche et al. ) , no specific inhibitors have been reported. no specific inhibitor has so far been found for the exfoliative toxin a, a glutamate-specific serine protease (family s ) produced by staphylococcus aureus, which is the causative agent in staphylococcal scalded skin syndrome. the target of the toxin is a transmembrane glycoprotein desmoglein- of the cadherin superfamily, which plays an important role in keratinocyte cell-cell adhesion (ladhani ) . immunoglobulin a proteases (iga proteases) are serine proteases (family s ) produced by several pathogenic bacteria, including species causing bacterial meningitis, haemophilus influenzae, neisseria meningitidis and streptococcus pneumoniae. the iga proteases enable colonization of human mucosal surfaces by cleaving the secretory iga antibodies, thus disrupting the specific immunity response. except for an early report on substrate analogue inhibitors (burton et al. ) and small synthetic peptide inhibitors (bachovchin et al. ) of neisseria gonorrhoaeae iga protease, there has been no further development of iga protease inhibitors (mistry and stockley ) . other immunomodulating serine proteases are c a peptidases from streptococci (family s )-those of group a (streptococcus pyogenes scpa) and group b (streptococcus agalaciae scpb) streptococci have been described in more detail. streptococcus pyogenes is the causative agent of pharyngitis and also causes rheumatic fever and skin infections, which can develop severe complications, including toxic shock syndrome. c a peptidases are important for streptococcal pathogenesis as they specifically cleave complement c a and therefore prevent the recruitment of phagocytic cells to the infection site (cheng et al. ; collin and olsen ) . antibodies raised against c a peptidase were used to inhibit c a peptidase in vivo (park and cleary ) , but no peptidase inhibitors have been described. serine proteases are important pathogenesis factors in bacteria involved in dental diseases. treponema denticola is a spirochete implicated in the progression of periodontal diseases. a serine protease, trepolisin (also called dentilisin), of family s is an important pathogenesis factor, a mediator of cytopathic effects by degrading host proteins, including extracellular matrix components and host protease inhibitors (sela ) . the broad-range inhibitor of serine proteases (families s and s ), chymostatin, inhibits trepolisin; however, no specific inhibitors have been described. another important oral cavity pathogen involved in periodontal disease, porphyromonas gingivalis, in addition to a few cysteine proteases (discussed further in the following), produces a serine protease, a prolyl tripeptidyl peptidase ptpa (family s ), which is involved in degrading host connective tissue, providing nutrients for bacterial growth (banbula et al. ) . a substrate-based specific inhibitor of ptpa has been developed (xu et al. ). bacterial type i signal peptidases spase (family s ) are serine proteases widespread among gram-negative and gram-positive bacteria and are membrane proteases required for processing newly synthesized secreted proteins. in addition to their essential role in bacterial viability, they are important antimicrobial drug targets as they are involved in the secretion of many virulence factors (paetzel et al. ) . synthetic penem inhibitors have been developed for inhibiting both gram-negative (escherichia coli) and grampositive (staphylococcus aureus, staphylococcus epidermidis) spases. the various penem derivatives display different degrees of activity against these pathogenic bacteria spases (allsop et al. ; harris et al. ). recently, substratebased peptide aldehydes have been shown to be promising inhibitors of escherichia coli and staphylococcus aureus spases (buzder-lantos et al. ). however, the in vitro inhibitory activity and in vivo antimicrobial activities of these inhibitors did not correlate well, so further optimization of and search for novel spase i inhibitors are expected. no specific inhibitors have been described for the streptococcal pyrogenic exotoxin b (speb, streptopain), a cysteine protease (family c ) produced by all strains of streptococcus pyogenes. it is a multifunctional protease and an important pathogenesis factor with several immunomodulating activities, including release of proinflammatory molecules, degradation of extracellular matrix, cleavage of igg in the hinge region and degradation of other immunoglobulins. in addition to class-specific cysteine protease inhibitors, a peptide derivative was shown to inhibit speb, as well as α -macroglobulin and an s-nitrosylated form of α -protease inhibitor (collin and olsen ) . ides (family c ) is another cysteine protease from streptococcus pyogenes that specifically cleaves igg, its only known substrate (vincents et al. ). in addition to specific, inhibitory igg antibodies (akesson et al. ) , synthetic reversible inhibitors were designed, with aldehyde compounds being the most promising; however, no specificity data are yet available for these inhibitors (berggren et al. ). staphylococcus aureus causes a range of diseases, from mild skin infections to life-threatening disorders, including septicaemia, endocarditis, toxic shock syndrome and pneumonia (lowy ) . it expresses several extracellular proteases with proposed roles in pathogenicity, including a serine protease v (sspa), cysteine proteases staphopains a and b (scpa, sspb) and a metalloprotease aureolysin (aur) (shaw et al. ) . staphopains a and b (family c ) are papain-like cysteine proteases that are co-expressed with their respective specific inhibitors staphostatins a and b (dubin ) . their role in pathogenicity has not been determined. staphopain b (sspb) is activated by the glutamyl peptidase sspa (v protease, family s ), which is expressed from the same operon (shaw et al. ). v protease modulates the surface protein profile and so influences the binding of fibronectin by staphylococcus aureus (mcgavin et al. ) . sortases are cysteine proteases (family c ) of grampositive bacteria that catalyse the covalent attachment of proteins to the cell wall peptidoglycan. they have been shown to contribute to the virulence of several important pathogens, including staphylococcus aureus, bacillus anthracis, listeria monocytogenes and streptococcus pneumoniae. therefore, they have been considered to be important targets for the development of novel antiinfective agents (suree et al. ; clancy et al. ) . staphylococcus aureus sortase (srta) has been at the focus of sortase inhibitor development due to the emergence of antibiotic-resistant strains and the need for novel antimicrobial strategies. several types of srta inhibitors have been investigated, including nonspecific sulfhydryl modifiers, peptide analogues of the sorting signal, compounds from plants and marine organisms, and synthetic small-molecule inhibitors. several inhibitors of srta have been described with varying strength and specificity; however, good in vitro inhibitory activity has not yet led to an effective in vivo sortase inhibitor (maresso and schneewind ; suree et al. ; clancy et al. ) . the cysteine protease clostripain (family c ) is a secreted protease of the anaerobic bacterium clostridium histolyticum, a causative agent of gas gangrene (clostridial myonecrosis). clostripain selectively hydrolyses arginyl bonds and constitutes an important clostridial virulence factor (jozwiak et al. ; manabe et al. ). in addition to oxidizing agents, thiol-blocking agents and heavy-metal ions that all inhibit clostripain, good reversible inhibitors have been described, namely, aziridine peptide esters, which are thus promising lead compounds for the development of specific clostripain inhibitors (schirmeister and peric ; barrett et al. ) . gingipains are extracellular cysteine proteases (family c ) produced by the oral pathogenic bacterium porphyromonas gingivalis, a major etiological bacterium of chronic periodontal disease. gingipains comprise two argininespecific cysteine proteases (rgpa and rgpb) and a lysinespecific cysteine protease (kgp). they constitute the major virulence factor of this periodontopathogenic bacterium as they are involved in multiple facets of its virulence and survival, including the destruction of periodontal tissues, disruption of the host immune system by inactivation of host proteinase inhibitors and deregulation of several proteinase cascades, as well as acquisition of nutrients required for bacterial growth and survival in the periodontal pocket (fitzpatrick et al. ; travis and potempa ) . due to their importance in pathogenesis, considerable efforts have been put into discovering or designing specific inhibitors of gingipains. tetracyclines, inhibitors of prokaryotic protein synthesis, have been shown to have, in addition to their antibiotic activity, inhibitory activity against cysteine proteases through binding to the proteinase outside the substrate binding site (imamura et al. ). peptidyl chloromethanes have been used as specific rgp and kgp inhibitors for their characterization (potempa et al. ) , and compound a was shown to attenuate porphyromonas gingivalis virulence through specific kgp inhibition (curtis et al. ) . based on histatin cleavage specificity, small peptide analogues were designed, which specifically inhibit rgp and kgp (kyt- and kyt- , respectively), which display attenuation of several virulence traits of porphyromonas gingivalis (kadowaki et al. ) . chlorhexidine, which has been used in oral healthcare preparations on account of its antimicrobial effects, also inhibits proteolytic activities, including those of gingipains. moreover, chlorhexidine inhibitory activity against r-gingipains was enhanced by the addition of zn(ii), which has also been used in human oral health care (cronan et al. ) . metalloproteases are important virulence factors of many primary and opportunistic pathogenic bacteria and cause major infectious diseases such as cholera, salmonellosis, legionnaires' disease, cystic fibrosis, botulism, tetanus and anthrax (miyoshi and shinoda ) . they have either direct roles in host interaction or indirect roles in processing other important virulence factors. therefore, much has been invested in the search for an effective protease inhibitor for use in treatment (jacobsen et al. ), but none has yet been developed, which would be used in clinic. metal chelators, including edta (ethylenediaminetetraacetic acid), egta (ethylene glycol-bis( -aminoethylether)-n, n,n′,n′-tetraacetic acid) and , -phenanthroline, inhibit metalloproteases in general. the ubiquitous presence of metalloproteases prevents the use of broad-spectrum inhibitors, and the search for potent and specific inhibitors of individual metalloproteases that could find clinical applications is important. the most studied bacterial metalloproteases are those of the thermolysin family (m ), including mpi protease of listeria monocytogenes, coccolysin of enterococcus faecalis, hemagglutinin/proteinase of vibrio cholerae and helicobycter pylori, pseudolysin of pseudomonas aeruginosa, aureolysin of staphylococcus aureus, legionella pneumophila protease and λ-toxin of clostridium perfringens. inhibitors of thermolysin family proteases are of bacterial origin, including those isolated from streptomyces, the small-molecule inhibitor phosphoramidon and protein inhibitor smpi (streptomyces metalloproteinase inhibitor) of family i . another family of inhibitors targeting bacterial thermolysins is family i of animal origin (adekoya and sylte ; supuran et al. ; rawlings and barrett ) . of the bacterial metalloproteases, the light chain domains that are zinc metalloproteases (family m ) of tetanus and botulinum neurotoxins (tent and bonts) from clostridium tetani and clostridium botulinum, respectively, have also been studied extensively. various β-aminothiols have been considered as selective bont and tent inhibitors and have been further developed into strong and selective pseudotripeptide inhibitors of bont/b (blommaert et al. ; supuran et al. ) . clostridium histolyticum collagenases and their homologues from vibrio (family m ) are very effective in connective tissue degradation and hydrolyse triple helical regions of collagen under physiological conditions. they are targeted for both therapy and diagnosis of clostridial infections, and several types of compounds have been found to inhibit them. however, in addition to bacterial collagenases, they also inhibit vertebrate collagenases (supuran et al. ; barla et al. ) . no potent and selective inhibitor has yet been found for metalloproteases of family m from pathogenic bacteria, including serralysin from serratia, pseudomonas and erwinia, aeruginolysin from pseudomonas aeruginosa, mirabilysin from proteus mirabilis and fragilysin from bacteroides fragilis. they are, however, inhibited by protein inhibitors of bacterial origin belonging to family i and hydroxamate inhibitors, including batimastat (supuran et al. ; rawlings and barrett ) . another group of medicinally important bacterial proteases for which specific inhibitors have not yet been described comprises the metalloexopeptidases, which belong to several merops families (m , m , m , m , m , m , m , m , m , m , m , m , m , m , m and m ). of the metallocarboxypeptidases (belonging to families m , m , m , m ) the zinc-containing d-ala-d-ala dipeptidase vanx (family m ) has been studied in view of its ability to mediate antibiotic resistance against vancomycin (crowder ) . similarly, the family m of membrane dipeptidases includes members that degrade β-lactam antibiotics. other carboxypeptidases, such as glutamate carboxypeptidases (family m ), have been studied with a view to clinical use in treating different types of cancer (holz et al. ) . bacterial metalloaminopeptidases, which perform essential cellular functions in protein synthesis and maintenance, have been studied as targets for novel antibiotics. a few reviews on inhibitors designed to inhibit bacterial aminopeptidases of families m (e.g. leucyl aminopeptidases or laps), m (alanyl aminopeptidase) and m (methionine aminopeptidases) cover the natural and designed compounds that could serve as lead compounds for inhibitors aimed at this group of proteases (mucha et al. ; holz et al. ; supuran et al. ; rawlings and barrett ) . bacterial metalloproteases that cleave immunoglobulin a (iga proteinases, families m and m ) constitute important colonization factors for several pathogenic bacteria (e.g. streptococcus, neisseria, haemophilus, clostridium, prevotella, capnocytophaga, bacteroides). no strong and specific inhibitor is available for these enzymes. the same is true also for another family of pathogenesis-related metalloproteases (family m ), including staphylolysin from pseudomonas aeruginosa and lysostaphin from staphylococcus simulans. in addition to their function in increasing virulence, staphylolysin and lysostaphin show bactericidal activity against staphylococcus aureus and have been studied with a view to their use in countering drug-resistant staphylococci (e.g. methicillin-and vancomycin-resistant staphylococcus aureus) (barequet et al. ; desbois and coote ) . the metalloprotease anthrax lethal factor lf (family m ) is a component of the anthrax toxin responsible for the major symptoms and death associated with bacillus anthracis infection (kim and yoon ). an increased interest in anthrax vaccination and treatment methods has been provoked by the use of bacillus anthracis spores as a bioweapon. lethal factor (lf) inhibitors would provide two-fold protection, namely, in preventing early spore protection in macrophages and, later, inhibiting lf disruption of signalling pathways through inactivation of mitogen-activated protein (map) kinase kinases. the search for an lf inhibitor to use in combination with antibiotic treatment is aimed at finding a selective and potent lf inhibitor (shoop et al. ; turk ), which would be non-(cyto) toxic and have good biological stability and bioavailability (johnson et al. ; kim et al. ; li et al. ). the predominant fungal diseases afflict immunocompromised patients and are caused by opportunistic pathogens candida sp. (e.g. candida albicans, candida glabrata, candida parapsilosis) and aspergillus sp. (e.g. aspergillus fumigatus, aspergillus niger, aspergillus nidulans, aspergillus calidoustus), followed by other ascomycetes of genus fusarium, basidiomycete genera malassezia, cryptococcus and trichosporon, and zygomycete genera rhizopus and mucor (boekhout et al. ). the importance of proteases in the pathogenicity of these opportunists is controversial; however aspartic, serine and metalloendopeptidases, as well as aminopeptidases, carboxypeptidases and dipeptidylpeptidases that are secreted by these species, have been proposed as the virulence factors that facilitate colonization and invasion by hydrolysis of host proteins or damage cells and molecules of the host defence system (segal ; yike ). the most studied are the secreted aspartic proteinases of candida albicans (saps) (naglik et al. ) . interestingly, the protease inhibitors targeted against the viral aspartic protease used in treating hiv infection also inhibited candida saps and reduced occurrence of candidiasis in these patients. secreted aspartic proteases are thus an important target for the development of new protease inhibitor based compounds for treating candidiasis (braga-silva and santos ; naglik et al. ; dash et al. ) . fungal proteases are also important fungal allergens, and most belong to the serine catalytic type. addition of protease inhibitor during aspergillus fumigatus and aspergillus niger protease and antigen sensitization attenuated allergic inflammation and hyper-responsiveness in an animal model (yike ) . secreted alkaline serine protease of aspergillus fumigatus was shown to help evade the host immune response by degrading human complement proteins, and it is therefore a good target for drug development (behnsen et al. ) . a network of proteolytic enzymes is very important for the survival of dermatophytes such as microsporum canis and trichophyton rubrum, the specialized pathogenic fungi that infect stratum corneum, nails or hair of healthy individuals. it includes the metalloendopeptidases, fungalysins (family m ), serine endopeptidases, subtilisins (family s a) and several exopeptidases (dipeptidyl peptidases (family s ), aminopeptidases (family m ) and carboxypeptidases (family s )) (monod ) . although proteases are only one of the several groups of virulence factors of pathogenic fungi, they aid in the invasion of tissues and evasion of immune responses. therefore, specific protease inhibitors aimed at them would constitute a valuable addition to the presently used antifungal drugs that target fungal cell wall and cell membrane integrity or dna replication-and to which many pathogenic strains have acquired resistance (marie and white ) . the classspecific (broad-spectrum) aspartic protease inhibitor pepstatin has been shown to inhibit adhesion of candida albicans and prevent invasion or mucosal tissue damage by inhibiting the saps (naglik et al. ) . recently, it has been shown that ergosterol production transcriptional regulator (sre ), which is activated by a metalloprotease stp , is essential for the survival of cryptococcus neoformans in the presence of antifungal drugs that inhibit sterol biosynthesis. therefore, regulators of the ergosterol pathway, including the metalloprotease stp , constitute promising targets for novel antifungal therapeutics to be used in combination with a sterol synthesis inhibitor for treating cryptococcosis in immunocompromised individuals (bien et al. ). protozoan parasitic diseases, including malaria, leishmaniasis and african and american trypanosomiasis, are some of the most important infectious diseases in the world, with high mortality and morbidity rates in developing countries. reasons for their persistence, despite prolonged use of antiparasitic drugs, include their toxic side effects and the increasing emergence of drug resistance. therefore, research over the past years has been focused on identifying new targets for antiparasite treatment and on developing substances suitable for human therapy. parasitic proteases constitute one of the very important druggable targets since they are key virulence factors due to their essential roles in cell metabolism and interaction with the host (zucca and savoia ; renslo and mckerrow ; mckerrow et al. ) . aspartic proteases plasmepsins (family a ), which are important for haemoglobin catabolism in parasites causing malaria (plasmodium falciparum, plasmodium vivax, plasmodium ovale, plasmodium malariae), have been targeted for the design of novel antimalarial drugs, i.e. selective protease inhibitors. although plasmepsin inhibitors (including the broad-spectrum pepstatin) have been shown to have antimalarial effects, the main challenge still remains to design an inhibitor that would be active against different plasmepsins i, ii and iv, and the histoaspartic protease hap involved in haemoglobin degradation, but also inactive against the homologous human aspartic proteases (cathepsins d and e) (zucca and savoia ; rosenthal ; ersmark et al. ; gil et al. ). in addition to plasmepsins, the cysteine proteases falcipains (family c ), metalloprotease falcilysin (family m ) and aminopeptidases have been targeted for development of new antimalarial protease inhibitors. a synergistic effect has been observed for their combined use (e.g. a cocktail of aspartic and cysteine protease inhibitor) (mckerrow et al. ; rosenthal ; zucca and savoia ; trenholme et al. ) . cysteine proteases similar to falcipain (family c ) have been associated with the pathogenesis of trypanosomiasescruzipain of trypanosoma cruzi (chagas disease) and brucipain or rhodesain of trypanosoma brucei (sleeping sickness). cruzipain (or cruzain) has been extensively studied and is considered to be a promising target for chemotherapy since it is critical for parasite viability in all stages of infection, especially for nutrition acquisition, tissue invasion and host immune response evasion (duschak and couto ). an irreversible inhibitor, k , was effective in pre-clinical models of chagas disease; however, a safer treatment would be achieved by utilizing a reversible and highly specific cruzipain inhibitor (beaulieu et al. ; mckerrow et al. ; brak et al. ). cysteine proteases have been implicated in the pathogenesis of leishmania species and are major virulence factors as they substantially modify the immune response. a surface metalloprotease gp or leishmanolysin (family m ) has been shown to be essential for establishing and maintaining the infection and would make a promising target for development of a selective protease inhibitor for antiparasitic chemotherapy (olivier and hassani ; yao ) . in addition to the computational design, development and optimization of a suitable protease inhibitor, based on the d structure of the target protease, the search for novel types of inhibitors from natural sources (pereira et al. ) , such as fungi and microbes, is important for identifying new lead compounds. serine proteases are also a neglected group of potentially targetable protozoan proteases involved in their pathogenicity, although several subtilisins (family s ) have been described from plasmodium falciparum and toxoplasma gondii, and two oligopeptidases (family s ) from trypanosoma cruzi (mckerrow et al. ) . in addition to protozoan parasites, helminths, which include parasitic roundworms (nematodes) and flatworms (trematodes and cestodes), also cause important parasitic infections. cysteine cathepsins (family c ), which are important for many aspects of the helminth-host relationship, are a potential target for developing antihelminthic drugs. cysteine protease inhibitors have been shown to impair fecundity of the liver fluke (fasciola hepatica) and blood fluke (schistosoma mansoni) in animal models. however, the similarity of host cathepsins, together with their importance, calls for the design of a very specific and selective protease inhibitor. alternatively, drug design could exploit bioavailability and pharmaco-kinetic and -dynamic properties to target parasitic proteases preferentially (robinson et al. ). in addition to proteases, parasite-derived protease inhibitors play important roles in manipulating the host immune system and establishing a niche for the successful feeding and reproduction of helminth parasites. therefore, protease inhibitors are also important targets for immunological control of helminth parasitic infections and make proteases that are insensitive to parasite-derived protease inhibitors valuable candidates for new types of antihelminthic therapy (knox ; stepek et al. ) . the ability of tumour cells to invade extracellular barriers and to metastasize to distant sites is associated with the activity of proteases (kos and lah ) . the major molecular mechanism, which involves the active role of various intra-and extracellular proteases, is the dissolution and remodelling of connective tissue and the basement membrane. it includes matrix metalloproteases (mmp), serine proteinases such as urokinase, tissue types of plasminogen activator (upa, tpa) and plasmin, aspartic proteinase cathepsin d and cysteine proteinases cathepsins b, h, s and l (schmitt et al. ). in addition to extracellular matrix remodelling, proteases regulate several other processes, leading to the progression of malignant disease, such as cell adhesion, migration, differentiation, proliferation and signalling of tumour cells. it is well accepted that tumourassociated proteolytic activity escapes the control of endogenous protease inhibitors and that treatment of patients with exogenous protease inhibitors may suppress the progression of the disease and improve the outcome of cancer patients. however, the treatment should specifically target the tumour-associated proteases that cause harmful actions and not those involved in the numerous physiological processes in cells and tissues. several protease inhibitors failed in clinical trials due to lack of specificity, resulting in severe side effects and/or lack of clinical benefit in treated patients (turk ; coussens et al. ) . new approaches to developing protease inhibitors applicable in therapeutic interventions include structure-based medicinal chemistry and development of molecular systems to deliver inhibitors to the site of action. among the small-molecule inhibitors of bacterial and fungal origin, peptidyl aldehydes such as leupeptin and antipain, hexapeptide pepstatin and epoxysuccinyl peptide e- and their analogues have been studied as anticancer agents. the thiol-protease specific inhibitor, e- , originally isolated from aspergillus japonicus (hanada et al. ) , has been studied extensively as a potential antitumour agent in cell culture and animal models. derivatives of e- , displaying selectivity between different cysteine proteases (frlan and gobec ) , represent the next step towards their application in treating cancer and other diseases. they were designed on the basis of the x-ray crystal structures of individual cathepsins, and the most studied were cathepsin b specific inhibitors ca- and ca- , cathepsin l specific inhibitors clik- and clik- , and cathepsin x specific inhibitor ams- . cathepsin s specific inhibitor clik- was designed on the basis of the structure of leupeptin and antipain (katunuma ) . antitumour activity was exhibited particularly by ca- , a specific inhibitor of the cysteine protease cathepsin b (johansson et al. ) , which appears to be crucial for tumour cell invasion (lah et al. ) . in animal models, it was shown that ca- reduces tumour growth, invasion and angiogenesis of many cancer types, including pancreatic cancer, melanoma and breast tumours, all tested on animal models. the cell permeability of epoxysuccinyl inhibitors was improved by esterification. the esters are less active than free acids; however, in cells, they are rapidly hydrolysed to their active form. better cell permeability was demonstrated for ethyl ester e- d and the methyl ester of ca- , which are also highly soluble and effective for prolonged periods (frlan and gobec ) . metalloproteases are an important group of proteases that have been considered extensively as targets for cancer therapy due to the many roles they play in carcinogenesis and tumour invasion, growth and dispersion. however, the diversity of endogenous metalloproteases and their numerous and versatile physiological roles have prevented the use of broad-spectrum metalloprotease inhibitors. much effort is invested in determining which metalloproteases to target and in designing highly selective and potent protease inhibitors based on the structural characteristics of individual target metalloproteases (coussens et al. ; bialas and kafarski ; dorman et al. ; overall and kleifeld ) . several proteases of the serine catalytic type have also been targeted for the design of specific protease inhibitors for use in cancer treatment, including the urokinase plasminogen activator and matriptase (abbenante and fairlie ; bialas and kafarski ; ulisse et al. ). the broad-range microbial inhibitors of serine, cysteine and threonine proteases, leupeptin and antipain, were also shown to inhibit malignant transformation (vaccari et al. ) or tumourigenesis (hozumi et al. ) . threonine catalytic type proteolysis is present in proteasomes, and several chemical classes of natural and synthetic proteasome inhibitors have been considered as anticancer agents because of their preferential antiproliferative and proapoptotic activity on cancer cells (kisselev and goldberg ; abbenante and fairlie ; bialas and kafarski ; cecarini et al. ; chen et al. ) . the aspartic protease inhibitor pepstatin has also been frequently tested as an antitumour agent since cathepsin d was identified as an important tumour promoting factor (greenbaum and sutherland ; benes et al. ). irregular function of proteases is associated with a variety of other diseases, representing targets for therapeutic application of all catalytic types of protease inhibitors. smallmolecule inhibitors of bacterial and fungal origin, described already in the previous section, particularly epoxysuccinyl inhibitors, have been reported as potential protective agents in autoimmune, neurodegenerative, antiinflammatory and cardiovascular diseases; osteoporosis; muscular dystrophy; diabetes and others. ca- and clik- were demonstrated to cause a switch between th and th t cell response due to the different roles of cysteine cathepsins b and l in antigen processing and presentation (katunuma ) . cathepsin s inhibitor clik- has been shown to suppress sj gren syndrome, an autoimmune disease associated with the processing of α-foldin by cathepsin s (saegusa et al. ) . the cathepsin x inhibitor ams- reduces the activation of integrin receptor lfa- (lymphocyte functionassociated antigen ), a molecule involved in t cell adhesion, proliferation and migration (jevnikar et al. ) . lfa- overexpression and activation by cathepsin x is typical of autoimmune diseases, particularly psoriasis. the same inhibitor significantly enhanced the proliferation of neuronal cells and neuritogenesis, preventing the processing of neurotrophic factor γ-enolase by cathepsin x (obermajer et al. ). epoxysuccinyl inhibitors have been tested in several animal models of neurodegenerative diseases. e- was shown to restore normal synaptic function in the app/ps mouse model of alzheimer's disease with overexpressed amyloid precursor protein (app) and presenilin (ps ) (trinchese et al. ). e- d and ca- me also reduced the accumulation of neurotoxic beta-amyloid peptides in brains, presumably inhibiting cathepsin b involved in processing the amyloid precursor protein (hook et al. ). aspartic proteases β-secretase (also known as memapsin- or bace and belonging to merops family a ) and γ-secretase (composed of two presenilins belonging to merops family a ) are important therapeutic targets for treating alzheimer's disease, and several compounds designed to reduce their activity are in clinical trials (de strooper et al. ) . another important target of aspartic proteases is renin (family a ), which is part of the complex renin-angiotensin-aldosterone system that regulates blood pressure and electrolyte balance. several inhibitors have been designed based on the renin structure and activity, and one of them, aliskiren, is the first non-peptide, orally administered, direct renin inhibitor available on the market for management of hypertension (nguyen et al. ; barrios and escobar ) . the very first protease inhibitor used in humans as a therapeutic agent, namely, an inhibitor of metalloprotease angiotensin-converting enzyme (ace), was also hypertension related. it is an important regulator of the reninangiotensin system, and ace inhibitors are used for treating hypertension, but are also implicated in other cardiovascular and renal diseases (abbenante and fairlie ; ondetti et al. ) . the inhibitors of collagenolytic enzymes, such as cathepsins l and k, prevent bone remodelling and can be useful in treatment of osteoporosis. several synthetic cathepsin k inhibitors are in different stages of clinical trials. in addition to osteoporosis, they are also considered for application in arthritis, atherosclerosis, obesity and cancer (bromme and lecaille ) . administration of cathepsin l inhibitor clik- significantly reduced invasion and metastasis formation in bones (katunuma ) . e d, a methyl ester of e , was tested for treating muscular dystrophy; however, its further development was stopped in phase iii due to insufficient effectiveness and hepatic injury in rats (fukushima et al. ). antipain, leupeptin and pepstatin have also been tested for treatment of muscular dystrophy; however, persuasive benefit was not demonstrated in animal models. specific inhibitors designed to target the serine aminopeptidase dipeptidyl peptidase iv are in clinical trials for management of type diabetes (abbenante and fairlie ; tahrani et al. ) . endogenous plant protease inhibitors constitute one of the plant defence strategies against pathogenic, parasitic and herbivorous organisms. they target the important proteolytic virulence factors of phytopathogenic bacteria, fungi, parasites and viruses, preventing their roles in nutrient acquisition and evasion of host defence. furthermore, they target digestive proteases of herbivorous pests (e.g. insects, mites, slugs), preventing the utilization of food-derived organic nitrogen for their growth and development (ryan ; haq et al. ) . since pests and pathogens depend on utilization of these proteases, there is a strong selection pressure operating to develop resistance to plant endogenous defensive protease inhibitors (haq et al. ; jongsma and beekwilder ) . therefore, the search for novel protease inhibitors with potential protective function is very important for the development of environmentally friendly pest and pathogen management strategies. in addition to investigations aimed at augmenting crop endogenous resistance by conventional breeding, there are several protease inhibitors of plant origin that have been used in the preparation of genetically modified crop plants with superior ability for biotic stress resistance (ferry and gatehouse ) . the use of protease inhibitors for insect pest management has gained most attention. insect pests cause major economic losses annually, of which lepidopteran (butterflies' and moths') larvae are considered the most destructive. agricultural pests causing significant economic impact also belong to orders coleoptera (beetles), diptera (true flies), hemiptera (e.g. aphids), orthoptera (e.g. locust) and thysanoptera (thrips). they cause either direct damage to crops by feeding or indirect damage by transmitting viral diseases or secondary microbial infections. the most notable for their destructive capacity are the migratory locust (locusta migratoria), several beetles, including colorado potato beetle (leptinotarsa decemlineata), boll weevil (anthonomus grandis), japanese beetle (popillia japonica), the western corn rootworm (diabrotica virgifera virgifera) and many species of aphids belonging to all families of the superfamily aphidoidea. different catalytic types of proteases provide the predominant proteolytic activity in different groups of insect pests. while serine proteases are predominant in digestive proteolysis in most insect species (e.g. lepidoptera, diptera), cysteine proteases predominate in hemiptera, coleoptera and thysanoptera. in addition, aspartic and metalloproteases complement protein digestion to different degrees in most insect orders (terra and ferreira ) . therefore, protease inhibitors targeting different groups of proteases have shown variable antinutritional effects when fed to different insect pests. the catalytic, classspecific, small-molecule protease inhibitors of microbial origin have often been used for proof-of-principle feeding experiments, but protein protease inhibitors were then employed to generate insect-resistant transgenic plants. these were predominantly of plant origin, with a few exceptions of animal-derived protease inhibitor genes (haq et al. ; dunaevsky et al. ; malone et al. ) . other important proteins that have been used for constructing pest-resistant transgenic crops are insecticidal proteins derived from bacillus thuringiensis (bt), and genetically modified maize and cotton varieties that express bt toxins have become an important component in agriculture worldwide and have reduced the use of pesticides and lowered production costs (ferry and gatehouse ; kumar et al. ) . the involvement of endogenous protease inhibitors in natural plant resistance against herbivores is probably the basis of the adaptation of lepidopteran and coleopteran species to ingestion of protease inhibitors that has been observed for several species (jongsma and beekwilder ; wu et al. ; bonade-bottino et al. ; lara et al. ) . they circumvent the antinutritional effect of the ingested protease inhibitors either by overexpression of native gut proteases or by production of insensitive proteases; some of which can degrade the ingested protease inhibitors (ferry and gatehouse ; jongsma and beekwilder ) . therefore, the pyramiding or stacking of different families of inhibitors to increase the spectrum of inhibitory activity has been shown to have synergistic effects, as well as combining protease inhibitor genes with genes of other insecticidal proteins, namely, lectins, bt or other bacterial toxins and α-amylase inhibitors (ferry and gatehouse ; schluter et al. ; christou et al. ; malone et al. ) . furthermore, the use of protease inhibitors of microbial and fungal origin could offer superior characteristics, such as stability, resistance to proteolytic degradation and diverse inhibitory patterns, for a more potent antinutritional or insecticidal effect. only a few examples of utilization of microbial small-molecule inhibitors as antinutritional agents are available, e.g. aminopeptidase inhibitors of actinomycetes amastatin and bestatin against the red flour beetle (tribolium castaneum) (oppert et al. ) , aspartic protease inhibitor pepstatin a from actinomycetes against the cowpea bruchid (callosobruchus maculatus) (amirhusin et al. ) , the serine and cysteine protease inhibitor leupeptin from actinomycetes against western corn rootworm (diabrotica virgifera) (kim and mullin ) and cysteine protease inhibitor e- from aspergillus japonicus against colorado potato beetle (leptinotarsa decemlineata) (bolter and latoszekgreen ) . the use of protein protease inhibitors is advantageous since transgenic plants expressing a pest resistance gene in a controlled manner represent a stable and cheap propagation source that would lower the amount of pesticides needed, making plant protection environment friendly. to our knowledge, the only examples of a protein protease inhibitor of microbial or fungal origin as an effective antinutritional agent are the cysteine protease inhibitors macrocypins (family i ) from the edible parasol mushroom (macrolepiota procera), which have been shown to be detrimental to the growth and development of colorado potato beetle larvae (istinič et al. ) . the high capacity for development of resistance to insecticidal proteins in major insect pests drives the search for effective protease inhibitors. novel protein protease inhibitors aimed at serine proteases would be useful for management of major agricultural pests such as beet armyworm (spodoptera exigua), cotton bollworm (helicoverpa armigera and helicoverpa zea) and tobacco hornworm (manduca sexta). a combination of serine and cysteine protease inhibitors would be useful against, e.g. boll weevil (anthonomus grandis), cowpea weevil (callosobruchus maculatus) and red flour beetle (tribolium castaneum), and cysteine protease inhibitors would, for example, be useful against the colorado potato beetle (leptinotarsa decemlineata), western corn rootworm (diabrotica virgifera), banana weevil (cosmopolites sordidus) and pea aphid (acyrtosiphon pisum). microorganisms that are generally recognized as safe and edible mushrooms offer a valuable source of such novel protease inhibitors that would also be acceptable for use in crops for human consumption. in addition to insect pests, other herbivores causing significant crop losses worldwide include mites and slugs. in both cases, cysteine proteases constitute the predominant digestive proteolytic activity. plant cystatins have been shown to be detrimental to mite development and reproductive performance in feeding trials on one of the major mite pests on agricultural crops, the two-spotted spider mite tetranychus urticae (acari: tetranychidae) (carrillo et al. ) . similarly, the growth of juvenile slugs deroceras reticulatum, the important agricultural and horticultural pest, was significantly reduced when fed with leaf tissue overexpressing a plant cysteine protease inhibitor (walker et al. ) . therefore, protease inhibitors of microbial and fungal origin have great potential for protecting plants from important mite pests and suppressing the growth rates of slug populations, in addition to their antinutritional characteristics for different insect pests. another advantage of the application of protease inhibitors in crop protection is that in addition to protection against herbivorous pests, they offer cumulative protection against nematodes and bacterial, fungal and viral pathogens (haq et al. ). the use of protease inhibitors is one of many strategies to protect plants from parasitic nematodes. their targets are intestinal proteases, mostly of the cysteine catalytic class. therefore, heterologously expressed plant cysteine protease inhibitorsphytocystatins (family i )-have been effective to different degrees against beet-cyst nematode (heterodera schachtii), root-knot nematode (meloidogyne incognita), potato cyst nematode (globodera pallida) and burrowing nematode (radopholus similis) (mccarter ; haq et al. ) . a plant-derived serine protease inhibitor offered satisfactory nematode resistance in transgenic wheat against the cereal cyst nematode (heterodera avenae) (vishnudasan et al. ; mccarter ). the antifungal effect of serine protease inhibitors has been determined with plant-derived protease inhibitors that target secreted proteases (mainly families s and s ) of phytopathogenic fungi involved in plant cell wall penetration by hyphae (wong et al. ). in addition, aspartic and cysteine proteases that are also fairly widespread (valueva and mosolov ) represent potential targets for protective protease inhibitors. similarly, phytopathogenic bacteria secrete proteases involved in pathogenesis, mainly aiding plant cell wall degradation. together with pectinases, cellulases and hemicellulases, they contribute to massive degeneration of plant tissue, for example, in wilt (e.g. ralstonia solanacearum) and soft-rot diseases (e.g. erwinia chrysanthemi, erwinia carotovora) (kunkel and chen ) . cysteine proteases belonging to families c , c , c , c and c , which have been implicated in the pathogenicity of many phytopathogens belonging to genera pseudomonas, xantomonas, ralstonia, erwinia and pantoea as effectors of the type iii secretion system, constitute potential targets for protective protease inhibitors (kunkel and chen ; mosolov and valueva ) . the importance of polyprotein processing in replication of viruses, especially those of families potyviridae and comoviridae, indicates that protease inhibitors could mediate resistance to plant viruses by inhibiting target viral proteases. indeed, a rice cystatin (oryzacystatin) expressed in tobacco plants conferred resistance to potato virus y (pvy) and to tobacco etch virus (tev) which correlated to increased inhibition of the target papain-like protease sudarshana et al. ). in addition to their protective role against biotic stress, endogenous plant protease inhibitors have been implicated in the control of proteolytic systems in plants under abiotic stresses with a dehydration component such as drought, increased salt concentration and freezing (vaseva et al. ) . it has been shown that the changes in metabolism triggered by water deficit involve active involvement of regulated proteolysis that assists the protective proteins (dehydrins and chaperones) in the cellular response to the increased levels of denatured, aggregated or oxidatively damaged proteins that accumulate during dehydration stress (brzin and kidrič ; benchabane et al. ; bray ; hoekstra et al. ; feller ) . overexpression of endogenous protease inhibitors increased resistance to drought stress, as shown for overexpression of the cysteine protease inhibitors cystatins atcys and atcys in the model plant arabidopsis thaliana ) and for overexpression of a serine protease inhibitor ocpi (oryza sativa chymotrypsin inhibitor ) in rice, with the latter showing improved drought resistance in terms of yield loss in the field (huang et al. ) . although protease inhibitors have great potential in protecting crops under abiotic stress conditions, detailed knowledge of the specific roles of different proteases in response to abiotic stress is needed before exogenous protease inhibitors can be used to manipulate and improve drought resistance in plants (vaseva et al. ) . small-molecule protease inhibitors are routinely used as buffer additives when preparing protein extracts, in order to prevent proteolytic degradation during protein purification procedures. broad-spectrum inhibitors that cover all the different catalytic classes are generally used, including pepstatin a for aspartic proteases, e- for cysteine proteases, chymostatin for serine proteases, antipain for cysteine and serine proteases, and leupeptin for cysteine, serine and threonine proteases; all of which were originally isolated from actinomycetes. for inhibition of serine proteases, synthetic protease inhibitors such as aebsf (pefabloc; -( -aminoethyl)benzenesulfonyl fluoride hydrochloride), the physiologically more stable derivative of pmsf (phenylmethanesulfonyl fluoride), are used. chelating agents such as edta and , -phenanthroline are generally used to inhibit metalloproteases and, for certain applications, inhibitors of actinomycete origin-phosphoramidon (m and m metallopeptidases inhibited) or bestatin (aminopeptidases inhibited). unwanted proteolytic degradation can cause severe reduction in yield of heterologously expressed proteins in various expression systems. therefore, natural or engineered protease-deficient strains are often used as hosts for bacterial (e.g. escherichia coli bl ), yeast (e.g. pichia pastoris smd , or ) and filamentous fungal (e.g. aspergillus niger prt pep) expression systems. however, these strains may not exhibit optimal bioprocessing characteristics due to lower fitness traits. alternative strategies are therefore used for preventing proteolytic degradation of recombinant proteins, including modification of the recombinant protein sequence to remove protease cleavage sites without affecting protein function, expression with a stabilizing fusion partner, optimization of cultivation conditions (ph, temperature, medium composition, bioprocess strategy) and use of protease inhibitors. for secreted recombinant proteins, small-molecule inhibitors can be added to the culture medium to inhibit the predominant secreted proteolytic activity of the host organism that is often of the serine and aspartic catalytic type. another strategy is coexpression of an appropriate protein protease inhibitor with the recombinant protein, which may, however, influence the yield or complicate the downstream purification procedures (potvin et al. ; sharma et al. ; rozkov and enfors ) . in addition to protection against proteolytic degradation of recombinant protein during the expression process, protease inhibitors as fusion partners have other advantages. one example is the use of the bacterial periplasmic serine protease inhibitor ecotin (family i ) as a vehicle for secretion to the periplasmic space of escherichia coli (paal et al. ). another example is the serine protease inhibitor from oyster mushroom, the pleurotus ostreatus proteinase a inhibitor (poia ) (family i ) that serves as an intramolecular chaperone enabling proper refolding of the fused subtilisin protease from inclusion bodies expressed in a heterologous bacterial expression system (kojima et al. ) . the strategy of using co-expression of protein protease inhibitors for reducing proteolytic degradation of heterologously expressed proteins has been successfully implemented in plant expression systems. preparation of a protease-deficient host is, in this case, not applicable because of the essential roles of proteases in growth and development. plant expression systems offer many advantages over bacterial and yeast expression systems (e.g. posttranslational modification capability) and over animal cell lines (e.g. lower cost and contamination risks) for largescale recombinant protein production, but have not yet been commercialized due to low levels of protein expression and of heterologous protein accumulation. the latter can be influenced by targeting the expression to specific organelles (e.g. endoplasmic reticulum) or tissues (e.g. seeds and tubers) or by co-expression with stabilizing fusion partners or protease inhibitors. co-expression of protective protease inhibitors does not have any adverse effects on plant growth and development, as described previously for examples of protease inhibitor expressing, insect-resistant transgenic plants. furthermore, the heterologously expressed protease inhibitors offer the added advantage of protection against proteolysis also ex planta, especially in the early steps of purification of crude protein extracts, thus minimizing the need for addition of protease inhibitors to the extraction buffers (rivard et al. ; desai et al. ; doran ; benchabane et al. ) . so far, only protease inhibitors of plant and animal origin have been used as co-expression partners; use of microbial or fungal protease inhibitors could offer superior protective characteristics. however, the properties of the protein of interest and of the selected host plant species influence the selection of an appropriate protective protease inhibitor, and the choice still has to be made on a case-by-case basis. another important biotechnological application of protease inhibitors is in protein purification, where they can be used as ligands in affinity chromatography. affinity chromatography is a simple one-step purification method of a molecule from a complex mixture based on specific and high affinity binding to a ligand immobilized on a solid support. reversible protease inhibitors of microbial origin are excellent ligands for purification of their cognate proteases by affinity chromatography. depending on the target protease, inhibitors with broad range or very specific inhibitory spectrum can be selected for a ligand. attention must be paid to the strength of the inhibitor as purification is not possible when the binding is too weak (no binding) or too strong (ineffective elution) (ge ) . the advantage of using microbial and fungal protease inhibitors is that many of them display unique inhibitory profiles and resistance to proteolytic cleavage, as well as high thermal and broad ph range stability, with the latter being very convenient since harsh conditions may be used for immobilization to the matrix as well as for the several cycles of elution steps, usually involving extreme change in ph and/or ionic strength. the broad-range inhibitor of pepsin-like aspartic proteases, pepstatin a, has been used for purification of aspartic proteases from several different sources, including higher fungi (sabotič et al. a) , plants (payie et al. ) and insect recombinant enzyme expressed in a bacterial expression system (volkov et al. ) . several different types of inhibitor have been used for purification of serine proteases, including the synthetic inhibitor benzamidine and plant-and animal-derived protease inhibitors of different families (polanowski et al. ) . differences in the inhibitory spectra of immobilized protease inhibitors can be used in serial affinity chromatography, where, first, a broad-range protease inhibitor can be used to purify proteases from a crude protein extract, followed by the use of a highly specific inhibitor for isolation of a single protease. the microorganisms of prokaryotic domains archaea and bacteria and of the kingdom of fungi, including higher fungi or mushrooms, constitute important sources of protease inhibitors. microbial protease inhibitors are versatile in their structures and mechanisms of inhibition in ways that differ from those of other sources. they have therefore found countless applications in the fields of medicine, agriculture and biotechnology. the diversity of processes in which proteases are key players, together with their multiplicity, drives the search for further novel protease inhibitors that could find applications, directly or as leads in structure-based design. the number and diversity of proteases found in microorganisms (rao et al. ) and higher fungi (sabotič et al. b ) makes them a virtually inexhaustible source of novel protease inhibitors with unique features. protease inhibitors in the clinic the thermolysin family (m ) of enzymes: therapeutic and biotechnological potential ides, a highly specific immunoglobulin g (igg)-cleaving enzyme from streptococcus pyogenes, is inhibited by specific igg antibodies generated during infection quality control of cytoplasmic membrane proteins in escherichia coli penem inhibitors of bacterial signal peptidase protease inhibitors from several classes work synergistically against callosobruchus maculatus viral protease inhibitors alpha -macroglobulin: an evolutionarily conserved arm of the innate immune system the bovine basic pancreatic trypsin inhibitor (kunitz inhibitor): a milestone protein trypsin-specific inhibitors from the basidiomycete clitocybe nebularis with regulatory and defensive functions inhibition of iga proteinases from neisseria gonorrhoeae and hemophilus influenzae by peptide prolyl boronic acids prolyl tripeptidyl peptidase from porphyromonas gingivalis. a novel enzyme with possible pathological implications for the development of periodontitis evaluation of pseudomonas aeruginosa staphylolysin (lasa protease) in the treatment of methicillin-resistant staphylococcus aureus endophthalmitis in a rat model inhibitive effects of alkyl gallates on hyaluronidase and collagenase proteolytic enzymes: nomenclature and classification handbook of proteolytic enzymes aliskiren in the management of hypertension identification of potent and reversible cruzipain inhibitors for the treatment of chagas disease secreted aspergillus fumigatus protease alp degrades human complement proteins c , c , and c preventing unintended proteolysis in plant protein biofactories plant cystatins cathepsin d-many functions of one aspartic protease synthesis and biological evaluation of reversible inhibitors of ides, a bacterial cysteine protease and virulence determinant proteases as anti-cancer targets-molecular and biological basis for development of inhibitor-like drugs against cancer cryptococcus neoformans site- protease is required for virulence and survival in the presence of azole drugs small tripeptide surrogates with low nanomolar affinity as potent inhibitors of the botulinum neurotoxin b metallo-proteolytic activity fungal taxonomy: new developments in medically important fungi effect of chronic ingestion of the cysteine proteinase inhibitor, e- , on colorado potato beetle gut proteinases physiological adaptation explains the insensitivity of baris coerulescens to transgenic oilseed rape expressing oryzacystatin i aspartic protease inhibitors as potential anti-candida albicans drugs: impacts on fungal biology, virulence and pathogenesis nonpeptidic tetrafluorophenoxymethyl ketone cruzain inhibitors as promising new leads for chagas disease chemotherapy molecular responses to water deficit cathepsin k inhibitors for osteoporosis and potential off-target effects proteinases and their inhibitors in plants: role in normal growth and in response to various stress conditions clitocypin, a new type of cysteine proteinase inhibitor from fruit bodies of mushroom clitocybe nebularis substrate analogue inhibitors of the iga proteinases from neisseria gonorrhoeae substrate based peptide aldehyde inhibits bacterial type i signal peptidase expression of a barley cystatin gene in maize enhances resistance against phytophagous mites by altering their cysteine-proteases targeting proteasomes with natural occurring compounds in cancer treatment bortezomib as the first proteasome inhibitor anticancer drug: current status and future perspectives the group b streptococcal c a peptidase is both a specific protease and an invasin evolutionary mechanisms acting on proteinase inhibitor variability recent developments and future prospects in insect pest control in transgenic crops sortase transpeptidases: insights into mechanism, substrate specificity, and inhibition extracellular enzymes with immunomodulating activities: variations on a theme in streptococcus pyogenes matrix metalloproteinase inhibitors and cancer: trials and tribulations inhibition of porphyromonas gingivalis proteinases (gingipains) by chlorhexidine: synergistic effect of zn(ii) combating vancomycin resistance in bacteria: targeting the d-ala-d-ala dipeptidase vanx attenuation of the virulence of porphyromonas gingivalis by using a specific synthetic kgp protease inhibitor interactions of a novel inhibitor from an extremophilic bacillus sp. with hiv- protease: implications for the mechanism of inactivation aspartic peptidase inhibitors: implications in drug development the secretases: enzymes with therapeutic potential in alzheimer disease substrate specificity of the integral membrane protease ompt determined by spatially addressed peptide libraries production of heterologous proteins in plants: strategies for optimal expression bactericidal synergy of lysostaphin in combination with antimicrobial peptides the complete amino acid sequences of two serine proteinase inhibitors from the fruiting bodies of a basidiomycete, pleurotus ostreatus foreign protein degradation and instability in plants and plant tissue cultures matrix metalloproteinase inhibitors: a critical appraisal of design principles and proposed therapeutic utility emerging principles in protease-based drug discovery defense against own arms: staphylococcal cysteine proteases and their inhibitors the staphostatin family of cysteine protease inhibitors in the genus staphylococcus as an example of parallel evolution of protease and inhibitor specificity immunity protein protects colicin e from ompt protease protease inhibitors: use to increase plant tolerance to insects and pathogens cruzipain, the major cysteine protease of trypanosoma cruzi: a sulfated glycoprotein antigen as relevant candidate for vaccine development and drug target. a review the role of ecotin dimerization in protease inhibition plasmepsins as potential targets for new antimalarial therapy influence of parasite encoded inhibitors of serine peptidases in early infection of macrophages with leishmania major proteolysis. in: larry dn (ed) plant cell death processes transgenic crop plants for resistance to biotic stress the gingipains: scissors and glue of the periodontal pathogen, porphyromonas gingivalis inhibitors of cathepsin b an epoxysuccinic acid derivative(loxistatin)-induced hepatic injury in rats and hamsters affinity chromatography: principles and methods computational perspectives into plasmepsins structure-function relationship: implications to inhibitors design ia , an aspartic proteinase inhibitor from saccharomyces cerevisiae, is intrinsically unstructured in solution host cathepsin d response to tumor in the normal and pepstatin-treated mouse isolation and characterization of e- , a new thiol protease inhibitor protein proteinase inhibitor genes in combat against insects, pests, and pathogens: natural and engineered phytoprotection protease inhibitors: a panacea? synthesis and biological evaluation of penem inhibitors of bacterial signal peptidase mechanisms of plant desiccation tolerance co-catalytic metallopeptidases as pharmaceutical targets cysteine protease inhibitors effectively reduce in vivo levels of brain beta-amyloid related to alzheimer's disease inhibition of tumorigenesis in mouse skin by leupeptin, a protease inhibitor from actinomycetes omptin proteins: an expanding family of outer membrane proteases in gram-negative enterobacteriaceae suppressors of bir p (survivin) identify roles for the chromosomal passenger protein pic p (incenp) and the replication initiation factor psf p in chromosome segregation characterization of a stress responsive proteinase inhibitor gene with positive effect in improving drought resistance in rice pmap: databases for analyzing proteolytic events and pathways inhibition of trypsin-like cysteine proteinases (gingipains) from porphyromonas gingivalis by tetracycline and its analogues uporaba makrocipinov kot pesticidnih učinkovin the design of inhibitors for medicinally relevant metalloproteins the role of cathepsin x in the migration and invasiveness of t lymphocytes matrix metalloproteinases in tumor invasion structure-activity relationship studies of a novel series of anthrax lethal factor inhibitors plant protease inhibitors: functional evolution for defense determination of the cytotoxic effect of clostridium histolyticum culture supernatant on hela cells in the presence of protease inhibitors suppression of pathogenicity of porphyromonas gingivalis by newly developed gingipain inhibitors speb-spi: a novel protease-inhibitor pair from streptococcus pyogenes solution structure of marinostatin, a natural ester-linked protein protease inhibitor prokaryote-derived protein inhibitors of peptidases: a sketchy occurrence and mostly unknown function antiviral activity of proteasome inhibitors/cytomegalovirus new aspects on antigen presentation mechanism for immune-responses and allergy expression structure-based development of specific inhibitors for individual cathepsins and their medical applications folding, stability, and secondary structure of a new dimeric cysteine proteinase inhibitor antifeedant effects of proteinase inhibitors on feeding behaviors of adult western corn rootworm (diabrotica virgifera virgifera) anthrax lethal factor: critical virulence factor of pathogenesis of anthrax toxins yeast-hybrid based high-throughput assay for identification of anthrax lethal factor inhibitors proteasome inhibitors: from research tools to drug candidates proteinase inhibitors and helminth parasite infection phage lambda ciii: a protease inhibitor regulating the lysis-lysogeny decision the propeptide of subtilisin bpn' as a temporary inhibitor and effect of an amino acid replacement on its inhibitory activity involvement of the c-terminal region of yeast proteinase b inhibitor in its inhibitory action inhibitor-assisted refolding of protease: a protease inhibitor as an intramolecular chaperone cysteine proteinases and their endogenous inhibitors: target proteins for prognosis, diagnosis and therapy in cancer (review) microbial alkaline proteases: from a bioindustrial viewpoint bacillus thuringiensis (bt) transgenic crop: an environment friendly insect-pest management strategy virulence strategies of plant pathogenic bacteria understanding the mechanism of action of the exfoliative toxins of staphylococcus aureus antiprotease therapy in cancer: hot or not? review article: specifically targeted anti-viral therapy for hepatitis c-a new era in therapy adaptation of spodoptera exigua (lepidoptera: noctuidae) to barley trypsin inhibitor bti-cme expressed in transgenic tobacco an overview of the serpin superfamily subsite specificity of anthrax lethal factor and its implications for inhibitor development proteases: multifunctional enzymes in life and disease staphylococcus aureus infections poriferan survivin exhibits a conserved regulatory role in the interconnected pathways of cell cycle and apoptosis role of microbial proteases in pathogenesis beyond bt: alternative strategies for insect-resistant genetically modified crops purification and characterization of a clostripainlike protease from a recombinant clostridium perfringens culture sortase as a target of antiinfective therapy genetic basis of antifungal drug resistance molecular approaches toward resistance to plantparasitic nematodes modification of the staphylococcus aureus fibronectin binding phenotype by v protease development of protease inhibitors for protozoan infections two approaches to discovering and developing new drugs for chagas disease structure of the carboxypeptidase y inhibitor i c in complex with the cognate proteinase reveals a novel mode of the proteinase-protein inhibitor interaction specific membrane binding of the carboxypeptidase y inhibitor i(c), a phosphatidylethanolamine-binding protein family member microbial metalloproteases and pathogenesis secreted proteases from dermatophytes participation of proteolytic enzymes in the interaction of plants with phytopathogenic microorganisms metalloaminopeptidase inhibitors candida albicans secreted aspartyl proteinases in virulence and pathogenesis candida albicans proteinases and host/pathogen interactions viral proteases and antiviral protease inhibitor therapy design of potent aspartic protease inhibitors to treat various diseases cathepsin x cleaves the c-terminal dipeptide of alpha-and gammaenolase and impairs survival and neuritogenesis of neuronal cells the inhibitory properties and primary structure of a novel serine proteinase inhibitor from the fruiting body of the basidiomycete, lentinus edodes isolation and characterization of a novel elastase inhibitor, aflei from aspergillus flavus biochemical properties and primary structure of elastase inhibitor afuei from aspergillus fumigatus protease inhibitors as prophylaxis against leishmaniasis: new hope from the major surface protease gp design of specific inhibitors of angiotensin-converting enzyme: new class of orally active antihypertensive agents efficacy of bacillus thuringiensis cry aa protoxin and protease inhibitors against coleopteran storage pests towards third generation matrix metalloproteinase inhibitors for cancer therapy a novel ecotin-ubiquitin-tag (ecut) for efficient, soluble peptide production in the periplasm of escherichia coli the structure and mechanism of bacterial type i signal peptidases. a novel antibiotic target evolution of peptidase diversity active and passive intranasal immunizations with streptococcal surface protein c a peptidase prevent infection of murine nasal mucosa-associated lymphoid tissue, a functional homologue of human tonsils purification, n-terminal sequencing and partial characterization of a novel aspartic proteinase from the leaves of medicago sativa l. (alfalfa) natural products from garcinia brasiliensis as leishmania protease inhibitors the potency and specificity of the interaction between the ia inhibitor and its target aspartic proteinase from saccharomyces cerevisiae non-conventional affinity chromatography of serine proteinases and their inhibitors corruption of innate immunity by bacterial proteases titration and mapping of the active site of cysteine proteinases from porphyromonas gingivalis (gingipains) using peptidyl chloromethanes bioprocess engineering aspects of heterologous protein production in pichia pastoris: a review the degradome database: mammalian proteases and diseases of proteolysis molecular and biotechnological aspects of microbial proteases peptidase inhibitors in the merops database merops: the peptidase database evolutionary families of peptidase inhibitors merops: the peptidase database versatile loops in mycocypins inhibit three protease families drug discovery and development for neglected parasitic diseases an in-built proteinase inhibitor system for the protection of recombinant proteins recovered from transgenic plants serpins in unicellular eukarya, archaea, and bacteria: sequence analysis and evolution helminth pathogen cathepsin proteases: it's a family affair antimalarial drug discovery: old and new approaches analysis and control of proteolysis of recombinant proteins in escherichia coli protease inhibitors in plants-genes for improving defenses against insects and pathogens heterogeneity in the cysteine protease inhibitor clitocypin gene family comparison of natural and recombinant clitocypins, the fungal cysteine protease inhibitors basidiomycetes harbour a hidden treasure of proteolytic diversity aspartic proteases from basidiomycete clitocybe nebularis macrocypins, a family of cysteine protease inhibitors from the basidiomycete macrolepiota procera structural basis of trypsin inhibition and entomotoxicity of cospin, a serine protease inhibitor involved in defence of coprinopsis cinerea fruiting bodies cathepsin s inhibitor prevents autoantigen presentation and autoimmunity plant cysteine proteinases: evaluation of the pharmacological activity role of chagasin-like inhibitors as endogenous regulators of cysteine proteases in parasitic protozoa a cut above the rest: the regulatory function of plant proteases aziridinyl peptides as inhibitors of cysteine proteases: effect of a free carboxylic acid function on inhibition recombinant protease inhibitors for herbivore pest control: a multitrophic perspective proteases, matrix degradation and tumor-cell spread role of treponema denticola in periodontal diseases fungal effector protein avr targets diversifying defense-related cys proteases of tomato approaches for refining heterologous protein production in filamentous fungi the role and regulation of the extracellular proteases of staphylococcus aureus anthrax lethal factor inhibition structure and function of plant aspartic proteinases discovery of potent anti-sars-cov m-pro inhibitors alpha-macroglobulins: structure, shape, and mechanism of proteinase complex formation a serpin in the cellulosome of the anaerobic fungus piromyces sp. strain e human gastrointestinal nematode infections: are new control methods required? human cytomegalovirus: drug resistance and new treatment options using natural products methods for engineering resistance to plant viruses bacterial protease inhibitors recent advances towards new antiinfective agents that inhibit cell surface protein anchoring in staphylococcus aureus and other gram-positive pathogens molecular phylogenetic characterization of streptomyces protease inhibitor family management of type diabetes: new and future developments in treatment insect digestive enzymes-properties, compartmentalization and function a kazal-like extracellular serine protease inhibitor from phytophthora infestans targets the tomato pathogenesis-related protease p b new approaches to structure-based discovery of dengue protease inhibitors bacterial proteinases as targets for the development of second-generation antibiotics aminopeptidases of malaria parasites: new targets for chemotherapy inhibition of calpains improves memory and synaptic transmission in a mouse model of alzheimer disease purification and characterization of streptomyces griseus metalloendopeptidases i and ii targeting proteases: successes, failures and future prospects discovery and development of anthrax lethal factor metalloproteinase inhibitors the urokinase plasminogen activator system: a target for anti-cancer therapy pepstatin, a new pepsin inhibitor produced by actinomycetes effects of the protease inhibitor antipain on cell malignant transformation thrombin-activatable fibrinolysis inhibitor is degraded by salmonella enterica and yersinia pestis role of inhibitors of proteolytic enzymes in plant defense against phytopathogenic microorganisms plant proteases: from phenotypes to molecular mechanisms the cladosporium fulvum virulence protein avr inhibits host proteases required for basal defense the response of plants to drought stress-the role of dehydrins, chaperones, proteases and protease inhibitors in maintaining cellular protein function molecular cloning, over expression, and activity studies of a peptidic hiv- protease inhibitor: designed synthetic gene to functional recombinant peptide enzymatic characterization of the streptococcal endopeptidase, ides, reveals that it is a cysteine protease with strict specificity for igg cleavage due to exosite binding assessment of nematode resistance in wheat transgenic plants expressing potato proteinase inhibitor (pin ) gene new aspartic proteinase of ulysses retrotransposon from drosophila virilis transgenic arabidopsis leaf tissue expressing a modified oryzacystatin shows resistance to the field slug deroceras reticulatum (muller) picornaviral c protease inhibitors and the dual c protease/coronaviral c-like protease inhibitors regulation of bacterial protease activity proteins with antifungal properties and other medicinal applications from plants and mushrooms adaptation of helicoverpa armigera (lepidoptera: noctuidae) to a proteinase inhibitor expressed in transgenic tobacco novel inhibitor for prolyl tripeptidyl aminopeptidase from porphyromonas gingivalis and details of substrate-recognition mechanism major surface protease of trypanosomatids: one size fits all? fungal proteases and their pathophysiological effects proteolytic inactivation of tissue factor pathway inhibitor by bacterial omptins two cysteine proteinase inhibitors from arabidopsis thaliana, atcysa and atcysb, increasing the salt, drought, oxidation and cold tolerance current developments in the therapy of protozoan infections acknowledgements we are grateful to dr. jože brzin for numerous discussions and suggestions for the improvement of the review and to professor roger h. pain for critical reading of the manuscript and language editing. we would like to thank dr. miha renko for the assistance with fig. . key: cord- - l gw sk authors: avgoustaki, dafni despoina; xydis, george title: how energy innovation in indoor vertical farming can improve food security, sustainability, and food safety? date: - - journal: nan doi: . /bs.af s. . . sha: doc_id: cord_uid: l gw sk food safety is an important scientific field, but at the same time a discussion topic of modern society that occupies more and more space of our every day time, dealing with the preparation of food, with its nutritious value, and various transportation and storage ways aiming at preventing food-related sickness. this work compares traditional farming with greenhouses and indoor vertical farming focusing on the challenges and the opportunities for each category. the scope of this work was to stress the role of indoor vertical farming towards this direction. indoor vertical farms can produce high quality and virus-free products that can be locally distributed, inside the urban environment that such investments take place, saving annually millions of tons co emissions. beyond that, in this work it was pointed out how energy plays a role in food safety in such systems. it was stressed that indoor vertical farms can act as a demand response aggregator. in large scale units it could play a role to adjust their production according to different electricity prices offered in different time zones throughout the day. this way, the owners under a multi-value business model will create the opportunity to the vertical farm owners not only to improve their production but at the same time absorb inexpensive electricity offered, by creating an additional profit mechanism (multiple revenue streams) under such an approach by entering into contracts with companies in a utility electric region. sustainability of resources and safety in the food production line is a major issue globally. by , it is expected that the global population will reach the . billion people, . billion people more that need to be fed (united nations, department of economic and social affairs, ) . today, agriculture occupies land equal to the size of south america in order to cover the demand of the global population. based on the assumption that the minimum daily demand of a single person is minimum kcal, if we maintain the same agricultural practices, we will need additional land equal advances in food security and sustainability # elsevier inc. issn - all rights reserved. https://doi.org/ . /bs.af s. . . to the size of brazil ( . billion acres) to cover the global food demand (despommier, ). on the other hand, according to lotze-campen et al. ( ) , the land used for agriculture is projected to be transformed for other purposes such as urbanization, energy production, or infrastructure growth. it is worth to mention, that another crucial challenge that will significantly affect agricultural production in the upcoming years is the rapid increase of the global temperature, as per each degree of temperature rise, % of existing agricultural land will be lost (despommier, ) . nowadays, climate change is a huge issue since it is expected that the upcoming years will outstandingly affect the agricultural process. the significant increase of the carbon dioxide emission levels from a global perspective-since it constitutes an important impact factor of agricultural productivity-can influence the global economy via the effects on the agriculture's total production rate. in specific, based on mulatu's et al. ( ) research conducted for ethiopia, indicates that the impact of co emissions will decrease . % to . % the real agricultural gdp since it will lead to lower the agricultural productivity and subsequently reduce the amount of traded and non-traded crops. such population increase certainly indicates a significant rise in the required food production, raising concerns on the deficiency, the quantity, and the quality of future food products. we should also take into account the fact that nowadays food travels daily thousands of miles from the production areas to the urban consumers, in order to meet the demand, releasing huge amounts of co . less developed countries such as ethiopia that were mentioned above, apart from global climate change will have to face and other enlarged problems concerning food safety. for example, human excrements that are used as fertilizers (estimation of % of the global farming) can cause diseases such as cholera, typhoid fever and numerous parasitic infections (despommier, ) . nowadays, even the more developed counties have to face food safety and security problems even if this kind of infectious diseases have been eliminated. it is worth to mention the pandemic of our age, covid- caused by virus sars-cov- that was initially reported in the province of hubei, wuhan in china. the disease is estimated to have originated from a seafood market in wuhan where wild animals were traded such as marmots, bats, snakes and birds (zhou et al., ) . the specific family of viruses, coronaviruses, are known to be transferred from animal to humans. according to zhou et al., , it is mentioned that % of the genetic makeup of covid- is matched with the coronavirus found in bats. the uncertainty that is caused globally via covid- has caused apart from multiple deaths and lockdowns to most of the european countries, will affect significantly the economy and will cost trillions of dollars in the global economy, during and beyond (unctad, ) . food safety is a major issue of our era, as there are multiple reports of cases worldwide over the last years that have caused food recalls due to bacterial infectious diseases leading to loss of billion dollars. why do we seem to have so many outbreaks concerning food production these days? only in the us, despite the attempts to provide a safe food supply, every year are recorded million foodborne illnesses, , hospitalizations and deaths (cdc, ) . in - , e.coli o : h outbreak in the us caused sudden eruption linked to consumption of leafy greens and the romaine lettuce. the pathogen was mainly reported in the regions yuma, az and salinas, california, where greenhouse installations that produce more than % of the leafy vegetables and greens in the united states are based. e.coli contamination in the production line almost all of the times originates from the irrigation water used in the fields. additionally, further risk in the contamination process from various bacteria and pathogens comes from the washing of field-grown products after they are harvested, while this step can spread contamination to the whole production. the most regular technique that outdoor farming applies after harvest is to dunk lettuce heads in water tanks from rainfall or irrigation, while most greenhouses apply triple washes with running water from the local network. vertical farms are a novel type of farming in a controlled-environment with a total replacement of solar radiation with artificial lighting that provides the necessary nanometers of the spectrum for the growth and development of plants. in vertical farms, plants grow in soilless cultivation systems such as hydroponic (roots are immersed in multiple substrates, i.e., perlite, rockwool enriched water with nutrient solution), aeroponic (soilless air/ mist solution) or even aquaponic (co-cultivation of fish and hydroponic plants) systems that allow stacking multiple layers or columns of plants horizontally or vertically. vertical farms are located in completely isolated spaces from outdoor environment with thermally insulated installations (especially when at the top floor of the building) and airtight structures that give the opportunity to the farmers to control the environment in terms of temperature, humidity and co (avgoustaki and xydis, ) . since vertical farms can theoretically be placed anywhere in the urban network, they allow local, nutritious and fresh consumption for consumers. in specific, a study conducted by jill ( ) , mentioned that food sourced from conventional farming uses to times more fuel compared to locally grown food and emits to times more co . meanwhile, vertical farms may be able to increase the productivity rate in highly urbanized areas that can lead to improvements in the food security of the community. the purpose of the following subchapter is to compare the different farming techniques of outdoor farming, greenhouses and vertical farms in between them in terms of input of resources, the final product in terms of safety and the shelve life of the products in terms of nutrient status and freshness. additionally, we will examine the above criteria for lettuce, which is one of the most important cultivated species in vertical farms and will give us access to multiple data. lettuce belongs to the basic daily diet products; its nature is fragile and can be easily contaminated and spread diseases among the population. in order to make more understandable the concept of resource use efficiency, in fig. the essential resources for growing plants under various farming types are shown. the most vital for plant growth is water, co , light, nutrients, electricity (for ventilation purposes) and heating. as shown in fig. , the definition of resource use efficiency (rue) is given by the ratio of the final plants production to the total input. in order to calculate the total input of a system, we have to summarize the input of resources, the environmental pollutants and the production system. in order to evaluate the sustainability and efficiency of a production system in the food industry, we have to assess three key directions of the system. • rue: the amount of necessary resources to produce. • the cost performance: the ratio of the sales amount to the production cost. • the vulnerability of the system, meaning the deviation of the yield production per year and the quality value per product unit. water is absolutely necessary for all food production such as vegetables, fruits, grains, meat etc. based on nederhoff and stanghellini ( ) , the water use for the global food production reaches at km and has a rapid increasing rate. the irrigation water-use efficiency can be researched under different scopes and multiple concepts such as storage, delivery distribution of the water to the farm or out of the farm. additional systems that can affect water use efficiency is the ratio of water that is delivered for irrigation and the water that supplies the system. there are various ways we can calculate water use efficiency as one of the major resource inputs in food production that can be accomplished with agronomic ways, engineering or even economic approaches. more analytically, irrigation efficiency estimates the ratio between the diverted water and the consumed by the cultivation, thus it provides water-use measurements that estimate the performance of the irrigation system. on the other hand, water use efficiency is considered an economic concept that in practice evaluates the farm, as it is calculated by the crop yield unit of water diverted (kg/m ). in terms of energy consumption, it is one of the reasons that causes greenhouse gas emissions (ghgs) contributing at the rising global warming. the main gases released by agricultural production are carbon dioxide (co ), methane (ch ) and nitrous oxide (n o). since the global policy makers, organizations, researchers, retailers and producers try to propose and implement novel techniques that identify and reduce ghgs, it is necessary that we will focus and refer to the status of emissions under each farming type and propose mitigation measures in the sector. in order to describe sustainability in agriculture, it is not enough to relate sustainability with the field only from the resources perspective. understanding and evaluating what constitutes a sustainable farming system, it is of vital importance, to furthermore understand the economic and social terms that influence the contemporary issues, values and perspectives of a unique system. economic efficiency reflects to the value that is relative to the cost. in order a resource to reflect an economic value, has to be rare and difficult to obtain, for the market prices to allocate the use of this resource for competitive purposes. for example, even if air and water are essential resources for life giving them high "intrinsic" value, nevertheless under most circumstances they have no economic value due to their sufficiency levels in the environment (ikert, ) . they only obtain an economic value in cases of scarcity due to, e.g., high levels of pollution or drought. even if is the most ancient way that people use land, over the last decades with the technological breakthroughs and the numerous innovations introduced, outdoor farming has changed. sensors, satellites and advanced machinery allow farmers to apply more targeted (and precision) agriculture to treat the fields individually according to the needs of the crop and the soil, by dividing it in smaller parts in order to take into consideration the variability level of each unit. to complete the whole picture of climate change issues, an additional evolution process that crucially reduce the growth rate of plants is soil degradation due to excessive floods and droughts. traditional food production systems offer food solutions for people from the beginning of human history. over time, additional innovative techniques were applied in traditional farming in order to rise the productivity rate and reduce the cost and the crops overall footprint. in terms of resources, conventional farming seems to have an increase demand for water use (table ) as traditional agriculture uses almost % of the available fresh water globally. furthermore, a very common problem in terms of sustainability in water use efficiency of conventional farming is the limited soil water-holding capacity that results from the limited mulching of the soil and the consistency in the same fertilizers/soil-preparation practices. scientific results (pimentel et al., ) have shown that this maintenance of these practices lead to low soil moisture status and low conservation levels of conventional farming systems. the most used approach for conventional farms is the irrigation efficiency and the water use efficiency. it is worth to mention that the more water applications are applied in a crop, the higher the water delivery losses are. in order to improve the water use efficiency, many farmers apply a combination of hydroponic systems with drip irrigation and smart scheduling of water distribution. hydroponics successfully address the challenge of soil drought and salinity that reduces both yield and crop quality. it should be noted that a decisive factor for the selection of hydroponic systems is the high irrigation water needs that renders the requirement for recirculating water. it becomes apparent that combination of water-saving technologies with limited-water application technologies (such as close-loop hydroponics, drip irrigation, mulching and smart scheduling of water supply) are the most effective solutions for optimizing water use efficiency. regarding land use, growing and producing food to respond to the expanding demand of the world has led agriculture production and food scarcity that can be difficulty bridged. today's farmlands, occupy almost % of the global habitable land (ourworldindata.org). we gathered the footprint of the various resources that meet the demand for lettuce production via traditional farming techniques. worth noticing that deforestation is a major problem, since forests are continuously sacrificed against farmland that leads to climate change acceleration and soil inability to maintain water at lower levels. depending on the cultivated variety, the techniques and the season, traditional farmed lettuce has a cultivation cycle between . and . months. therefore, farmers have the ability to grow multiple successive crops in the same field throughout a yearly cultivation period in order to increase their yield and income. additional techniques that open-field farmers follow in order to increase their yield and income per hectare (ha) of cultivated land is the density of planting, fertigation (combination of fertilization with irrigation) application and the use of healthy transplants grown in nurseries. assuming that romaine lettuce growing in the mediterranean is planted in distances of - cm between the rows and - cm between the plants, then the resulted yield reaches at , - , plants per ha (savvas et al., ) . by increasing the planting distance per row by cm, it can lead to a % reduction of the total production. harvest period vary depending on the type or the variety of the cultivated crop. for the romaine lettuce grown outdoors, the harvest period is between and days with a typical yield of - tons/ha. the energy use in outdoor farming is mainly linked to fossil fuels for operations such as soil plowing, sowing, fertilization, harvesting etc. additionally, further electricity is required for pumping (water irrigation), which in developed countries can reach up to % of the total fossil fuel usage (despommier, ) . conventional farming, unfortunately, is associated to higher emissions in comparison to other types of farming. the majority of the emissions is directly linked to the transportation of the products, also known as food miles. the amount of miles that is required in order for food to travel from the producer to the consumers could release between to kg of co emission depending on the location of the farm (gerecsey, ) . since farmlands are often located many kilometers away from the urban centers, where the majority of the end-user is located. food miles emissions represent on average % of the total emissions released throughout traditional farming. another important source of co emissions that is linked to traditional farming is the significant amounts of food waste. even if food waste is not only linked to traditional farming, maladministration and mismanagement on-farm losses, and non-marketable crops put traditional farming under the spotlight of high shares of carbon footprint. for the estimation and assessment of the economic efficiency of farming, significant role in the calculation, the resources that bear an "economic" value have played a role. in traditional farming, there is limited motivation to protect and evaluate the quality, use and water maintenance, air, solar radiation and in some cases even soil fertility and productivity. the costs of a farm can vary between two main categories: the variable costs (operational expenses-opex) and the fix costs (capital expenses-capex). in the category of variable costs, all the expenses that cover particular farming actions in a specific period of time such as seeds, fertilizers, chemicals, labor are included. on the other hand, in the fix cost category, all the expenses that will be incurred regardless the process and status of production, building expenses (rent, installations, land) and equipment (irrigation system, machinery) are included. thus, the economic efficiency consists from a combination of technical and other components. based on aurangzeb et al. ( ) and a research that conducted to compare the economic efficiency between traditional farming and mechanized farming systems, it is pointed out that the net income in mechanized farms is significantly higher due to the higher yields/ha than the one of traditional farms. this effect of traditional farms could be explained by the longer time periods in soil preparation, limited tillage practices as well as the high cost requirements of labor expenses (specifically in seasonal workers during harvesting and sowing) in comparison to the high technology and mechanization farming systems. last, another factor that highly affects the final quantity of production is biodiversity. for this reason, the selection and maintenance of mono-cropping techniques that provide a uniformity in the applied practices, can reduce the labor costs and make harvesting easier. however, by cultivating only onespecies crops in the entire field, it can highly influence the biodiversity and make crops more susceptible to pathogen infections. to avoid this effect, traditional farmers apply chemicals and genetically modified organisms to maintain a simple farming system. this practice, though, requires a lot of continuous input of resources and energy (cost). the innovative and high quality mechanization and technological innovation can lead to the increase of production and hence income. multiple practices become more and more vital in traditional farming, as they improve the efficiency of resources use in general and can overall enhance sustainability. concerning the water usage, there are several approaches that new farms bring along in the field and can optimize the existing severe water waste situation. common agronomic measures such as improved crop husbandry and changed crop mix driven by the crop selection, can have a huge impact in improvement of water usage. furthermore, there are various cultivation techniques such as modification of the irrigation infrastructure, which can also influence positively the water use efficiency. last, management actions such as optimal irrigation planning and frequent maintenance irrigation system scheduled maintenance can also influence positively the system's efficiency (wheeler et al., ) . due to the growing population, farming has shifted to technologies that enhance significant scale-up of the production via innovative technologies. greenhouses are types of installations, designed to protect and enlarge the cultivation season of various crops. plants growing under greenhouses can grow protected from severe weather conditions such as hail, snow, extreme low temperatures or excessive heat, while at the same time can allow cultivations of out-of-season species. greenhouses first introduced in the th century but only on the th century were commercially applied in the global market. according to their installed area, greenhouses can be presented with various coverage materials such as plastic, glass, polyethylene and rigid that protect crops from the variability of the outdoor conditions, diffuses solar radiation and traps moisture, which contributes to increased plant growth. the coverage system allows farmers to control the cultivation environment according to each crop preference, as they can apply different techniques that will maintain the heating and the cooling requirements to the desired levels. this way, inside the greenhouses, farmers can develop and maintain the desired microclimate and create a more predictable environment that enhances the final plant yield, achieving higher quality and reduced water consumption compared to open field crops. there are different greenhouse systems that are diversified according to the energy flow inside the greenhouse and the resources flow in the production line. in more details, open greenhouses refer to the structure of the irrigation system, meaning that they do not collect the drained water of the crops for reuse (usually have soil-based crops). these systems seem to have low level of water usage efficiency as they are affected by water losses due to soil depletion and constant water drainage, which drains the excess amount of water with fertilizers. this waste of resources cause significant problems to the environment. usually growers can control the amount of drain as part of the management strategy of resources they follow. the percentage of drain can number between % and % of the water supply, but can be improved by reusing this drain in the irrigation system. additionally, open greenhouses use window openings as the only mean of dehumidification and cooling technique. there are also the semi-closed systems of greenhouses that have a smaller cooling capacity and window openings, combined with mechanical ventilation air-cooling systems. the combination use of mechanical systems and window openings depending on the cooling demand. concerning the irrigation systems, semi-closed greenhouses reuse the drained nutrient solution by collecting it to a tank that is constantly topped-up with fresh water. in some cases is followed water disinfection in order the collected drain water to be purified for avoiding diseases spread in the crop. to avoid imbalances in the nutrient solution, farmers use various techniques such as bleeding or dumping. in specific, bleeding techniques remove constantly % of the drain water, while in the dumping technique the mixing tank gets completely emptied and refilled with fresh water enriched with nutrient solution. finally, closed-systems refer to absolute mechanical support of the cooling and dehumidification system by air treatment units. the air treatment unit consists of a heat exchanger that is connected to a ventilator. the purpose of the ventilator is to withdraw air from the interior of the greenhouse, cool it, dehumidify it, and then distribute it back into the greenhouse. furthermore, in closed-systems water usually follows a close loop that allows the collection, recycle and re-distribution of the irrigation water both for irrigation purposes but also for cooling and heating purposes from inside the distribution pipes between the plant lines (qian, ) . concerning the irrigation system in closed-systems of greenhouses, the water does not follow the procedures of bleeding or dumping that are followed in semi-closed systems. on the other hand, the water is constantly recirculated in the mixed tank as it is automatically topped up with the correct and precise amounts of fresh water and each nutrient element. the growers are aware of the status of each nutrient element and are able to adjust it precisely in order not to disrupt the nutrient balance. this process becomes possible because of the high evolvement of automations, sensoring and programming in close greenhouse systems and achieve a - % better water use compared with open greenhouse systems (nederhoff and stanghellini, ) . greenhouses have different techniques for irrigation and water collection and highly depend on if greenhouses use soil based techniques or soilless for crop production. another factor that highly influences the final water use and water use efficiency is the type of the system, meaning it is an open system, a semi-open system or a closed-system. however, as can be retrieved from tables and the big difference in water use efficiency can be explained primarily because of the higher production accomplished in greenhouses compared to traditional farming but also because of the lower transpiration in greenhouses. transpiration is the most important factor that influences the water uptake by %, thus the control and reduction of transpiration rate can have a huge impact on the water use. transpiration is highly affected by the status of humidity and the irradiation levels inside the greenhouse. the higher the humidity inside the greenhouse the lower the transpiration levels are. if growers manage to control these two factors in the optimal levels for each crop, then there is reduced transpiration level per m , which means lower water usage and therefore better water efficiency. the selection of the applied irrigation system, has also a significant influence. drip irrigation is one of the most popular irrigation techniques in greenhouses. water is located at the foot of each plant with the use of a pipe. drip irrigation has the advantage of saving large water amounts and also can control and maintain the humidity levels of the soil or the hydroponic substrate in constant levels. in that way, water stagnation and puddling of the ( ) food miles - km selected substrate mean can easily be avoided. finally, drip irrigation allows the targeted and limited fertilization being dissolved, in the watering system. other irrigation systems are the micro sprinklers that spray water in a range around two meters according to the pressure of the selected nozzle type. this system is mainly used in soil-based greenhouses with sandy soil texture. another very commonly used system is the irrigation with diffusers and is mainly used in narrower areas and the pressure of the diffuser depends on the nozzle that regulates the water supply and flow. finally, other irrigation systems applied in greenhouses are irrigation with hose and underground irrigation mainly found in soil-based greenhouses and present low level of water efficiency. most of the modern greenhouses apply hydroponic solutions that allow plant to grow without soil. in more detail, the word hydroponic comes from the greek words "Ύδωρ + Πονέω" translated as "water + cultivate," meaning that plants do not grow in soil but in mineral nutrient solutions in water solvent. various substrates in the market replace soil such as perlite, rockwool and zeolite. because of the nature of this technology, plants are permitted to dip directly in their roots into the nutrient-rich solution and subsequently plants can absorb faster the nutrients and in an easier way in comparison with soil-based crops. because of this process, plants grown in hydroponics form smaller root system and can divert more energy for growing their leaves and stems. additionally, smaller root allows more plants in the same area to be grown and harvest higher quantities in comparison to the outdoor farming. the above-described capacity of hydroponic systems, boosts the ability of growing food in limited areas as greenhouses can be. hydroponics consist of a total automated system that pumps water, and pipe-system can be completely auto-controlled. under various handlings and monitoring of every aspect that can be practiced in hydroponic systems, the growers can result into optimal food production results. more specifically, this process gives the opportunity to farmers to control the whole irrigation process of the crops according to the demand of each species and the seasonality. in addition, they can have access to data that can optimize the development rate and the resource footprint of the plants such as (a) the quantity of water that is distributed in each plant, and (b) the amount of nutrient solution that was given to the plants. hydroponics offer a big advantage as they are usually installed in close or semi-close loops that return the excessive water with the enriched nutrient solution back to a collective tank in order to re-distribute it back to the cultivation area. in contrast to the hydroponic solutions, traditional farming experiences huge amounts of resource and water waste as farmlands face the negative effects of soil degradation and the harmful effect of eutrophication (when nutrients from agricultural land create massive increase of phytoplankton populations leading to reduction of oxygen and nutrient reduction of from water and suffocation of multicellular water organisms). unfortunately, in traditional agriculture, excess supply of phosphates and nitrates in the soil can cause nutrient run/off and leaches. furthermore, the close or semi-closed loop of hydroponics categorizes them as more efficient in terms of sustainability process for water efficiency in comparison with traditional farming where most of the water is drained to lower levels of soil that plants cannot access. greenhouses consist of air-sealed cultivation rooms where are installed various automations and technologies that can control and provide the optimal environmental conditions for each crop. according to factors such as location, size of installation, height, outdoor climate conditions, greenhouses use different technologies that can properly adjust the indoor environment to the ideal air conditions. heating is one of the most important processes for space heating inside the growing room, when the outdoor conditions and too hostile for the plants' growth. for heating purposes, the technologies that are usually used vary according to the demand of each case. in general, heating systems use the interior hot air of the greenhouse to transfer heat through a heat exchanger to the stored water that is used as a thermal storage medium. a very common and cheap technique is using water heating systems that consist from plastic bags and ground tubes filled with water placed inside and between the rows of the plants. during daytime, this system absorbs and traps the solar irradiation and during nighttime, the stored heat is transferred in the interior of the greenhouse by releasing heat (sethi and sharma, ) . there are electric heaters operated via a thermostat or an automatic timer in order to rise the inside temperature to the desired levels. additional techniques used for heating are rock bed storages, movable insulation and ground air collectors. cooling is a technique of similar importance with heating as it enables to reduce the thermal energy inside a greenhouse and maintain the optimum temperature in each growing stage of the crop. various techniques are used around the world according to the specific climatic conditions, the size and the demand of each case. such techniques can be natural or forced ventilation, fogging and misting, roof cooling and fan-pad systems, as well as shading and reflection systems. the most successful systems are the composite systems since they are giving the opportunity for both heating during the winter period and cooling during the summer period. according to sethi and sharma ( ) , the most promising composite system is the earth-toair-heat exchanger system (eahes) that operates with the underground constant temperature of earth mass and utilize it to transfer or dissipate heat from or to the greenhouse. according to botanists plants are diversified to "long day" plants and "short day" plants based on the photoperiodism needs-meaning on how many hours of light they have to be exposed during the day in order to grow. artificial lighting is a technique that provides greenhouses supplementary lighting in case that the solar radiation does not completely meet the photosynthetic demand of each plant species for optimal growth and development. efficient and proper use of lights in horticulture and with additional boost of reflectors can provide apart from the optimal levels that are required for photosynthesis also can benefit the greenhouses with additional heating (fig. ). heat and energy loss is a common issue in greenhouse and artificial lighting. the latter can become an effective solution that mitigates these losses and add an additional value on the required lighting solutions. the most common types of lamps that are used in greenhouses are high pressure sodium lamps, lighting emitting diodes (led) lamps and ceramic metal halide lamps. energy use into a hydroponic production line is mainly meeting the demand of artificial lighting, heating and cooling loads as well as water pumps. the energy that meets the water pumping needs in a hydroponic system for lettuce is estimated by the average pumping time that is needed to irrigate the plants and the corresponding nominal power of the pump. based on the calculations of kublic et al. ( ) it was estimated that the average irrigation duration for lettuce is four and a half hours of total pumping daily. the energy related to the heating and the cooling loads in a lettuce production greenhouse is estimated by using the following equation the heat transfer coefficient depends on the coverage material of each greenhouse, while the efficiency of cooling and heating systems depends on the height of the greenhouse ceiling. the loss of heat depends on the external climatic conditions and it is a decisive factor of the air technique modification to be used. artificial lighting usage depends on the photoperiod necessary for each species and the active hours of sunlight that plants can absorb for photosynthesis purposes. the active time that lamps have to operate is highly relevant with the location of the greenhouse, meaning that greenhouse areas with limited solar irradiation hours (north part of europe, i.e., netherlands, denmark) have higher demand on artificial lighting in comparison with areas under sunshine (southern part of europe, i.e., spain, greece, italy). furthermore, the duration of the supplementary lighting depends on the nature of the cultivated plants in photoperiodism (if they belong to "long day" or "short day" plants as we mentioned before) . this characteristic can differentiate the need of the plants in total daily radiation and according to the outdoor sunlight, the extra hours that artificial lamps need to operate should be estimated. the ultimate purpose of artificial radiation is to provide to the crop the indispensable photosynthetic active radiation (par) in mol/m /day for optimal yield production. in order to calculate the energy of a mole of photons that reach the canopy the following equation is used: the result value of the above calculation of the energy demand of artificial lighting in the greenhouse is in [kj/kg/year]. food production and consumption is constantly rising, having a significant environmental impact making the implementation of more sustainable practices in food production necessary. in order consumers to satisfy their demand for off-season vegetables and fruits, the necessity of heated greenhouses for production is continually increasing. as it is mentioned in the traditional farming section, food transportation causes huge amounts of ghg emissions. however, this number is lower in comparison to the ghg emissions corresponding to heating hydroponic greenhouses in cold climate areas (ntinas et al., ) that try to meet high yields in order to meet customers demand. when heating of greenhouses is achieved with the use of natural gas, the consumed energy can reach the . mj with . kg of co for the production of kg of tomatoes. since the majority of greenhouses use fossil fuels to meet their heating demand such as natural gas, diesel, fossil fuel and liquid petroleum gas, it is of vital importance to strongly limit the greenhouses heat losses, upgrade the heating systems and to shift in utilization of renewable energy sources . heat losses can be minimized with the use of double glazing coverage material or with the use of multiple screens. the upper goal of these measures is to increase the environmental sustainability of greenhouse production lines. as it has already been mentioned, greenhouses combine different energy technologies, automations and digitalization for plants' monitoring, controlling and harvesting. greenhouses is a type of farming that can provide the option to connect with renewable energy resources in order to increase the sustainability of such systems and the energy efficiency of the various treatments that are necessary for mass food production (manos and xydis, ) . different types of renewable energy sources such as solar, wind, geothermal, hydroelectric, biofuels, biomass etc., are found all over the world bringing the possibility to greenhouse plants to produce yields under a more sustainable, economical and cost-efficient way (xydis, a) . energy policy strategies in a national and a global level, have as a high priority the support of electricity generation and heating from renewable energy and biofuels (xydis, b) . over the last decades significant improvements in a big variety of significant renewable energy systems, which are ground source-based, solar-based energy systems and wind-based energy systems have been made (koroneos et al., (koroneos et al., , . these can be for example electricity-driven heat pumps instead of traditional combustion-based heating systems consumes - % less energy in comparison to a conventional fuel heater (avgoustaki and xydis, ) . another advantage that heat pumps present . - . times higher energy efficiency compared to fossil fuel heaters as also % - % reduced co emissions in the cultivation area in comparison with the conventional. there are also examples of greenhouses that use several solar systems that store energy or other photovoltaic systems (pv) that undertake the conversion of solar energy to electricity that meets the heating and cooling needs of greenhouses. based on research conducted by ntinas et al. ( ) , greenhouses that utilize renewable biofuel (wood pellet) present - times lower global warming potential in comparison with a greenhouse that use fossil fuels for heating purposes ( . - . kg of co per kg of harvested tomatoes), even when the required energy is the same for both cases. greenhouses in the netherlands use complex technology for production of various cultivars that gather multiple operation during the production such are nurseries, growing bedding plants and transplants. these systems are highly automated and occupy land approximately ha or more (kozai et al., ) . even if these machineries occupy a lot of potential cultivated space, they reduce the labor cost and therefore the production cost. without the use of highly automated technology, the average work force required in greenhouses for cultivating purposes is estimated at approximately workers per a m production area. according to penissi et al. ( ) greenhouses produce g of fresh weight of romaine lettuce per m daily while traditional farming produce g of fresh weight of romaine lettuce per m per day. as it can be retrieved from table , the required land use for obtaining kg of fresh romaine lettuce daily is m presenting almost % of decreased land usage in comparison to traditional farming. in greenhouses there are different variables that based on their priority can offer different benefits to the farmers. these could be the location of the greenhouse, the product type, the access to capital, the required work force and other requirements. high significance in the cost efficiency is also the upfront cost and the ongoing growing cost of the greenhouse that can also lead to higher cost depreciation and development rates of the production unit. based on a comparative study conducted by , a greenhouse farm consisting of a semi-closed system of m of growing space in denmark, the opex and capex related with the farm were analyzed. their results showed that by assuming that the wholesale price of greenhouse produced greeneries reached at . €/kg, the annual yield production of harvested products reaches at , kg/year. it is also presented that the capital expenses for the installation of the greenhouses was calculated at , € including the hydroponic system and grow unit racks, natural gas, heating and ventilation system, light connection (for supplementary radiation), and electricity distribution. additionally, for the operational expenses the total amount of expenses rises to an annual cost of , €, including the leasing costs, the electricity demand costs (lighting, ventilation), the natural gas heating cost, the water demand, the labor requirements, the packaging expenses and finally the use of organic material (seeds and nutrients). different greenhouse scenarios were presented and a cash flow analysis in a -year projection, indicated that the cumulative gross profit increased in parallel with the increasing wholesale price of greeneries. more specifically, the payback period was calculated much longer than the operational period of the years resulting in negative prices of the net present value (npv), unless the wholesale price of greens increases to . €/kg or more. indoor vertical farming is an innovative type of closed plant production system that provides the opportunity of a controlled-environment agriculture, which can be controlled according to the crop regardless of the weather conditions. indoor vertical farms use artificial lighting as radiation source in order to cover the demand of plants for growth and development via photosynthesis. vertical farms are based in soilless cultivation techniques such as hydroponics, aeroponics or aquaponics. in addition to the hydroponic systems that recirculate the nutrient solution and benefit greenhouse cultivations, vertical farms use systems that condense and collect the water that is transpired by plants at the cooling panel of the air conditioners and continuously recycle and reuse it for irrigation. some principles concerning the structure elements permeate closedsystems of vertical farms. more specifically, vertical farms are thermally well-insulated and nearly airtight structures that are covered with opaque walls. this characteristic makes the farms capable to totally protect the inside crops from the outdoor climatic conditions and make them able to maintain the indoor conditions to the desired levels without having thermal losses. another characteristic that differentiate vertical farms from greenhouses is the multiple layers of stacked plants in the vertical racks or horizontal columns. this way, the construction provides maximization on the possible yield per unit of land in comparison to both greenhouses and outdoor farming. more specifically, vertical farms, according to the size on the installation, have a multilayer system mostly between and rows or columns with approximately cm of distance between the layers (can slightly vary according to the selected cultivated crop). inside vertical farms airconditioners or heat pumps, which principally are used to reduce the heat generated from the lamps and provide cooling and dehumidification for the crop are installed. furthermore, air-conditioners help to eliminate the water vapor that plants transpire in the cultivation area. fans are installed in order to circulate the air in the culture room; at first to achieve a constant and stable spatial air distribution and secondly to improve the photosynthesis and transpiration status of the plants. key factor in the optimal operation of vertical farms is the co delivery units that stabilize the co levels in the cultivation area at around ppm during photoperiod (when lamps are on) in order to increase the level that plants photosynthesize. an important characteristic of vertical farms is the nutrient solution unit that distributes the nutrients to the crops, the electrical conductivity control unit (ec) and the ph controller that monitors the level of the nutrient solution. last, it is very important to analyze the radiation systems inside vertical farms as part of the total structure essentials. as mentioned above, vertical farms are equipped with artificial lighting due to absolute lack of solar radiation. lighting is a key factor in plants development and depending on the selected lighting solution, plants can present differentiations in morphology, flowering and biomass production. light is electromagnetic energy that includes visible as also invisible wavelengths. sunlight is a free resource input that provides plants the whole spectrum of several wavelengths, % of it is within the range of - nm (kozai et al., ) . however, according to a number of researchers over the last decades (hogewoning et al., ; kim et al., ; lin et al., ; liu et al., ) , it is reported that the most important wavelengths for photosynthesis, morphology of plants and flowering are the wavelengths in the visible ( - nm) and the infrared ( - nm) spectrum. lighting emitting diodes (leds) offer advantages in comparison with other types of lamps such as fluorescent, incandescent, high-pressure sodium or high-intensity discharge (hid) lamps. these advantages are the robustness, they produce, a stable output that is immediately activated after the electric current flow, have long life (approximately , h), the opportunity of controlling the light output etc. for this reason, vertical farms focus on applying lighting recipes that combine different nanometers and can promote plants' growth. apart from the spectrum selection of the lamps crucial factors for plants are the dimensions of light, meaning the intensity of light during photoperiod and the duration that lights operate. what has literally been neglected is the potential of indoor vertical farms to act as a demand response provider (aggregators). it may sound weird, but indoor vertical farms could under a multi-value business models create the opportunity to the vertical farm owners to focus on their crop production and at the same time absorb inexpensive electricity offered. usually plants require some hours daylight and fewer darkness. it has been proven that by selecting the hours throughout the day that are not expensive to give the required light, and "give darkness" when electricity price is expensive, has not a significant impact on plants' growth and development. under a mass deployment scenario of such units in major urban environments (xydis, ) , the owners and operators of the indoor vertical farms could create an additional profit under such an approach by entering into contracts with companies in a utility electric region. the opportunity to earn (or at least save) significant amounts will or course be related to the size of the indoor farms and create multiple revenue streams. indoor vertical farms have thermally insulated walls and high level of airtightness that allows a better cooling by air-conditioners during the time that lights operate. this process is functioning even during cold winter nights, as the interior temperature can be increased due to the operating lamps that constantly generate heat in the cultivation rooms. the ultimate goal of air-conditioners is to maintain the indoor temperature at the desired levels. however, during the cooling process, a lot of the water portion is lost due to evaporation of plants or evapotranspiration. indoor vertical farms have heat pumps with cooling panels, which can condense and collect this water, recycle it and via the close irrigation loop, reuse it for watering the plants. according to kozai et al. ( ) , only a small part of the irrigated mass water is getting lost to the outside because of the high level of airtightness inside the vertical farm. it is also pointed out in this research, that the airtightness level of vertical farms should not exceed the . h À . this is suggested because this level of airtightness helps to reduce the co losses to the outside environment and at the same time to maintain the sanitize level inside the farm by preventing pathogens, bacteria, dust or insects to enter the area of cultivation. greenhouses compared to indoor vertical farms, do not provide the opportunity of collection, reuse and recycle of the water masses that evapotranspired from plants, because the majority of the water is lost via the ventilation process to the outside area and furthermore most of the water vapor of greenhouses is mainly condensed at the inner walls, making impossible its collection process. another remarkable point that influences the resulted transpiration in indoor vertical farms is the operation of the artificial lighting. more specifically, when lamps do not function, the relative humidity of the room can reach up to % (little transpiration in the culture room), and cause physiological and morphological disorders to the plants. in order to solve this issue, farmers operate the lamps in rotation after dividing them in groups (two or three) and each group operates for - h per day. with this action, a constant heat generation during the day from the lamps that aligns with the -h function of the heat pumps that dehumidificate and cool the air in the culture room can be achieved. in order to calculate the water use efficiency in indoor vertical farms the following equation is used: where -wc is the water mass (or weight) that is collected in the cooling panel of the air conditioners for recycling purposes (kg*m À *h À ), -wp is the alteration in the water mass that is detained by plants and hydroponic substrates (kg*m À *h À ) and -ws is the irrigated (or supplied) water mass to the indoor vertical farm. in general, co use efficiency in indoor vertical farms is around . - . (when the level of airtightness is between . and . h À ) and the concertation is around ppm-unlike greenhouses which achieve approximately a . cue with closed ventilation system and airtightness level of . h À and co concentration level at ppm (yoshinaga et al., ) . based on these data we can estimate that the cue of indoor vertical farms is . / . ¼ . times higher compared to the greenhouses that do not operate the ventilators and provide co enrichment in the culture room. this phenomenon can be explained because of the amount of co that is released to the outside area from the culture room and keeps increasing with the level of airtightness but also with the difference between the co levels inside and outside. the fact that the co concentration for enrichment in an indoor vertical farm is usually around - ppm in comparison to the greenhouses that have around - ppm can be explained based on that. in order to calculate the cue the following equation is used: where -cp is the net photosynthetic rate (μmol m- h À ), -cs is the enrichment rate of co (μmol m À h À ) and -cr is the rate of respiration of the workers (if there are) in the culture room (μmol m À h À ) the light energy of the lamps that is send in the canopy aims to provide the necessary energy that plants need to grow and photosynthesize. however, the salable part of plants can only fix maximum - % of the electrical energy as chemical energy. the remaining - % of the electrical energy that is not absorbed by plants is converted to heat energy into the culture room and the remaining is removed by air-conditioners to the outside area (avgoustaki, ). the above-described effect can also explain the negligible heating costs in well thermally insulated indoor vertical farms even in the winter cold nights. nevertheless, indoor vertical farms are based in automations and precision agriculture and all the input resources are measured and validated in order to provide the optimal results in the cultivated crop. for this reason, all farms focus on measurements and optimization of the light energy use efficiency both of the lamps and the plant community. what is important for these measurements in the definition and estimation of the par, which in other words, is the wavelengths of light that are in the visible spectrum of the - nm and are the ones that drive photosynthesis. par is not a measurement of light; rather it defines the type of light that is necessary for plants to photosynthesize. apart from the type of light, farmers need to know and further metrics of light such as the amount and the spectral quality of par. in order to estimate the light energy use efficiency of lamps (lue l ) we use the following equation: where f is the convention factor from dry mass to chemical energy that is fixed in dry mass (around mj kg À ) -d is the increase rate of dry mass of the whole unit of plants or only the salable part of plants in the indoor vertical farm (kg m À h À ) and -par l is the photosynthetic active radiation emitted by the lamps (mj m À h À ) respectively, in order to estimate the light energy use efficiency of the plant community (lue p ) is provided by the following equation: where: -par p is the photosynthetic active radiation that is received at the surface area of the cultivation. based on the calculations and experiments conducted by yokoi et al., , it is shown that indoor vertical farms have . to . times higher lue p in comparison to the greenhouses. only % of the light energy is actually converted into salable portion of plants. nevertheless, there are different techniques which can be applied and can improve the conversion factor to % or a little higher. a simple technique that can be followed is the application of interplant lighting, upward lighting, and use of reflectors (fig. ) . traditional lighting that is located only on top of the crop can cause undesirable shading in dense crops by uneven light distribution and lead to senescence of the leaves that are in lower levels. on the contrary, the application of interplant lighting can provide access of light also in the lower levels of the plants, improve the distribution of light and therefore improve the photosynthetic rate of the crop. according to dueck et al. ( ) , the photosynthetic rate of leaves in low levels is usually negative or nearly zero, but the application of interplant light can increase it in positive values. welldesigned reflectors can significantly enhance the lue l as they can reduce the vertical distance between the canopy and the lamps and increase the distance between the plants or the density, since plants constantly grow. same positive results by interplant lighting have been reported also in greenhouse canopies. the most suitable lamp selection for interplant lighting technique is leds as they have small volume and they perform lower surface temperatures in comparison to fluorescent and other types of light sources. leds have been proven beneficial for reducing the eue l also due to the higher conversion coefficient from electrical energy ( . ) compared to the fluorescent lamps ( . ). although the capital cost of leds is generally higher than the cost of fluorescent lamps, leds have longer operational life and the prices have considerably decreased over the last couple of decades and is expected to continue decreasing. apart from the lighting adjustments, other modifications can improve the lue l such as the control of the environmental conditions. the environment of plants and the ecophysiological status of plants can be enhanced by the optimal selection of air temperature, co concentration, water vapor pressure deficit (vpd), air current speed as well as the combination of ph, electric conductivity (ec) of nutrient solution. these parameters have to be set according to the selected cultivated species. another way to improve the lue l as well as the eue l of the salable part of plants, is to reduce the dry mass of the nonsalable parts of the plants. in indoor vertical farms, the most frequently selected crops for cultivation are leafy vegetables such as lettuce, small fruits and herbs and it is important to limit the percentage of the root mass into less than % of the total mass of the plant (kozai et al., ) . due to of the cultivation technologies used in indoor vertical farms this is an achievable measure only by minimizing the water stress of plants by controlling the water vapor pressure deficit of the room. if the selected crop is root species, then we can significantly increase the salable portion by harvesting earlier than usual in order to have an edible aerial part. finally, other factors that can also help in increasing the relative annual production capacity (per unit land area) of indoor vertical farms are: • limitation of the culture period between transplanting and harvesting by optimal monitoring and controlling of the environmental conditions • increase of the ratio of cultivation area under each farming type (field, tier, floor, culture bed) • increase of the salable part of plants as also the percentage of salable plants. according to kozai et al. ( ) , it is stated that by applying the abovedescribed techniques, the relative production capacity per land area unit in an indoor vertical farm of layers can rise up to - times higher compared to outdoor farming, considering that indoor vertical farms already produce - times more yield than traditional farming (table ). in practice, those techniques could double the efficiency of the whole system. indoor vertical farms use culture beds that are isolated from soil usage and the nutrient solution that enriches the irrigation water is distributed through pumping to the plants. because of the high-automated process of irrigation, the nutrient solution is drained from the culture beds that plants are growing and it follows a close loop by returning to the central nutrient solution tank for recycle and reuse. in order this process to be achieved, nutrient solution is rarely removed to the outside area. this process usually takes place once or twice per year when the level of certain ions such as na + and cl-are not well absorbed by plants and the percentage in the culture beds exceeds the normal levels, requiring discharge. in order this measure to be implemented, the supply of fertilization closes for some days and plants already planted can absorb the nutrient elements existing in the culture beds (kozai et al., ) . on the contrary, the fertilizer use efficiency of greenhouses and of fields in traditional farming is relatively low and occasionally can cause on the soil, surface salt accumulation. in order to calculate the fertilizer use efficiency (fue) the following equation is followed: where: -i u is the absorption rate of plants of ion element i that are in the organic fertilizer and -i s is the supply rate of ion element i into the indoor vertical farm. it is worth to be mentioned that the ion element includes the basic elements of fertilization solutions such as nitrogen (no À and no + ), phosphorus (po À ) and potassium (k + ). artificial lighting apart from a key element in the growth of plants indoor, it does increase the energy consumption of vertical farms. shamshiri et al. ( ) , noted that three major operational expenses in a vertical farm are the electricity cost with - % of the total cost, the operational costs (opex) with % of the total cost and the capital expenditures (capex) with - % of the total cost. indeed, energy consumption is a significant cost of indoor vertical farms and can be used as an measure for their sustainability levels. many research groups and institutes focus on developing innovative technologies and optimizing the lighting recipes in order to reduce the energy footprint of vertical farms and create a more sustainable and cost efficient type of farming. even if the demand for purchased energy is much higher in indoor vertical farms than in greenhouses, the energy efficiency of the former is significantly higher (graamans et al., ) . indoor vertical farms, since are in absolute controlled systems face high efficiency when operating with renewable energy . there are multiple examples of vertical farms that are operating under smart grid systems that generate energy for the demands of the farm via wind turbines or solar panels or even geothermal energy. additional roles in the vertical farm systems towards increasing their efficiency have the connectivity with resourceful batteries that provide the opportunity for smart use of cheap stored electricity from the hours that the electricity prices are lower. an approach gaining constantly more and more attention also under the dynamic pricing concept, where also accurate forecasting plays a crucial role (karabiber and xydis, ) . in order to calculate the energy use efficiency for the lamps (eue l ) is followed the below equation where: h is the conversion coefficient of electrical energy to energy of photosynthetic active radiation that is emitted by lamps. for the latest technology of leds this number reaches the . - . (kozai et al., ) . apart from the energy that is consumed in order to meet the lighting demands, the energy demand of the heat pumps for the cooling (or heating) processes in the indoor vertical farms should be added to the equation. this type of efficiency is often referred in literature as coefficient of performance of heat pumps for cooling purposes. the coefficient of performance of the heat pumps, in a specific room, increases when the outside temperature decreases. the electrical energy use efficiency for cooling by heat pumps (eue c ) is calculated by the following type: where: -h is the heat energy that the heat pumps remove from the cultivation area (mj*m À *h À ) and -a is the consumption of electrical energy by the heat pumps (air conditioners) (mj*m À *h À ). it is worth to mention that the total energy consumption of indoor vertical farms is defined by the sum of the energy consumption of the lamps, the heat pumps/air-conditioners and the electricity demand of other equipment used for the optimal function of the farms such as nutrient solution pumps and air circulation fans. if we focus only in the electricity cost demand of indoor vertical farms, lighting accounts to approx. % of the annual electricity energy use (assuming fluorescent lamps of w), while the electricity cost demand for air conditioning is around % and % the electricity demand of the auxiliary electrical equipment (kozai et al., ) . table presents the estimated representative values of resource use efficiencies in an indoor vertical farm that use artificial lighting. it could be concluded (from table ) in comparison to table , that the relative production capacity per land area unit in an indoor vertical farm of layers is to times higher compared to traditional farming and to times higher compared to greenhouse production. indoor vertical farming is a type of farming which by definition is developed to provide enough production in order to meet the local demand in urban areas with continuous increased demand for fresh and nutritious fruits, vegetables and herbs. in general, the most frequently cultivated species are plants that have higher profitability and have a relatively high price. a significant factor on crop selection is the crop to have a short production cycle in order to reduce the required electricity costs for lighting, heating and cooling of the crop and therefore can be harvested as early as possible. additionally, growers prefer plants that have high harvested yield, meaning a high portion of the crop that can be harvested and sold. for example in crops like lettuce and herbs, growers can harvest and sell the whole unit of the plant, while in tomatoes or peppers they can sell only the harvested fruit but at the same time. therefore the electricity used for the rest of the plant, could be considered as a product waste. another key issue in crop selection is the height of the plants, meaning that it is way more preferable the crop to have a compact status in order to be able to reduce the growing distance between multiple plants and grow more at the same available area. plants are also selected according to the perishability level that they present after harvesting and reaching the market. since indoor vertical farms are mainly located in urban or suburban areas, their goal is to produce crops that can increase their self-life (even of perishable crops), by shortening the harvesting and delivery time to the market. another parameter taken into account when selecting crops is the situation in the local market. if, for instance, tomatoes are missing for some reason from the market, then depending on the price they can get, they could be preferred against of another fruit or herb that is in abundance and its price cannot climb up. finally, most suitable crops are those that have year-round productivity in order to be affordable for the farmers to have a year-round market demand that can be profitable despite the continuous operational expenses. the constant production in a yearly basis of the same crop selection, allows also maintenance of the same, specific engineering settings of the crop, avoiding the modifications in the automations' selection that could cause abnormalities from a horticultural perspective. due to the concept of indoor vertical farming and the technology used in the cultivation areas, growing in an urban environment do not advantage the crops due to possible shading of the building, non-fertile soils or dormant soils. this fact can also be considered as one of the major drawbacks as the land price in urban areas is relatively high. concerning this approach, indoor vertical farms are often installed in large warehouses, industrial factories or even abandoned buildings, where the prices are low. according to kozai et al. ( ) , it is stated that indoor vertical farms can produce the same yield of lettuce heads and other leafy greens in only % of the land required by traditional farming and % compared to a greenhouse construction. based on tables - , it can be retrieved that the land use efficiency of indoor vertical farms ( . m ) required for obtaining kg of fresh romaine lettuce per day is almost % reduced compared to greenhouses and % compared to traditional farming. an indoor vertical farm of layers can produce g of fresh weight of romaine lettuce per m per day ( g fw/m /d for greenhouses and g fw/m /day for traditional farming). adenaeuer ( ) mentions that the increase in yield between indoor vertical farms and traditional farming can be increased by . due to the technology and by due to the technology combined with the stacking ability of the plants. depending on the stacking area and the volume of harvest, cultivation care and crop preparation techniques, the work force can highly vary. , propose that . workers are necessary per , kg of yield, resulting in % of the annual operational expenses of the farm (depending on the labor cost in each country). the same work force is required for a greenhouse production and approximately half of it for an open field farm. more analytically, according to savvas et al. ( ) , in soil-based crops the labor numbers , €/ha while a hydroponic greenhouse or indoor vertical farm requires around , €/ha as production cost. this demand is met by both permanent and by seasonal workers that will be hired for specific labor-intensive operations of the farm (like pruning and harvesting) throughout the year. indoor vertical farms have the advantage that allows them to generate bio-waste as bio-product during the process of edible biomass production. according to the cultivation system that plants grow in (hydroponic, aeroponic or aquaponic), the opportunity to farmers to collect easily all the by-products after the harvest period such as leaves, roots with fibers, stems, or even damaged vegetable and fruits and use it as well waste is offered. based on adenaeuer ( ) , the bio-waste that is collected and used in indoor vertical farms can be metric tons per year and with daily plant wastes that are collected for the indoor farms of roughly . tons. since indoor vertical farms use advanced close loop systems, present also the possibility to convert the daily amounts of biowaste and after careful processing to useful resources material for the crop as liquid fertilizer or biofuel (nikas et al., ) . the are several cases of installation of indoor vertical farms that have designed specific lines of biowaste management in their production line that only serve this specific purpose. it should be stressed that indoor vertical farms have the option to implement high tech equipment for conversion of food waste into energy production via anaerobic digestion. more specifically, this technology is a biogas recovery system that captures methane from food waste and convert it to heat, steam and electricity to meet the energy demands of the farm. this process requires a close-loop system, which creates biogas from organic material by piping it into the turbine generator. the electricity that is finally produced meets the high-energy demand of indoor vertical farms such as the operation of the lamps. anaerobic digestions is also compatible with aquaponic systems by receiving the organic waste of both fish and plants to produce electricity (agstar, ; united states environmental protection agency, ). one of the key factors that influences the selection of the farm system is the selling price of the products. according to tasgal ( ) , traditional farming products are to times cheaper in comparison to greenhouse an indoor vertical farming products. more specifically, traditional farming lettuce price usually costs less than €/head, while greenhouses lettuce and indoor vertical farm lettuce cost - €/head. additionally, based on the same study, the significant upfront capital requirements of indoor vertical farms can highly limit the pool of market participants. this happens because both the land prices, rents and acquisition of high-technology equipment are significantly higher in comparison with the leasing cost of farmland. on the other hand, , by conducting a comparative analysis between indoor urban farms and greenhouses presented slightly different results. in more detail, they assumed an indoor vertical farm with the same growing space and wholesale price of the greeneries as in the greenhouse facility, of m and . €/kg respectively. an interesting point is the massively increased production yield that can be achieved in an indoor vertical farm compared to greenhouses, reaching at , kg of fresh greeneries being annually harvested. the operational expenses of indoor vertical farms according to the examined case reached at , €/year resulting in almost similar numbers with the greenhouse facility. however, the biggest cost of indoor vertical farms noticed were the capital expenditures reaching at , € per grow unit, with the most costly equipment the lamps and integral connection of lamps, installation of growing unit racks and the electric distribution of electricity. subsequently, based on their model and the different cash flow analysis, indoor vertical farms present profitable investment opportunities with a high internal rate of return (irr) and a payback period between and years with a wholesale price equal or more than . €/kg. another research conducted by liaros et al. ( ) , a case scenario of a small iuvf of m growing area inside an apartment was presented, showing profitability to smallholders under various scenarios. worth to mention at this point, micro indoor farming in small growing spaces such as containers, garages or even simple rooms can be profitable depending on the demand and the flexibility to rearrange different cultivation parameters aiming for the optimum result. similar findings were also supported by ucal and xydis ( ) . on the other hand, based on a report conducted by agrilyst ( ) indoor micro-farms can be very costly, nevertheless, there are multiple marketing strategies for optimizing the results. according to the united nations (un) projections, the global population will exceed . billion until , all requiring to meet their food demand. additionally, un estimate that % of the global population will be located in urban areas by that time. in order all this increased food demand to be met, it is necessary to produce % more nutritious and fresh food. however, at the same time, land experts such as agronomists and ecologists, already warn of the growing shortages in agricultural land, necessary for sufficient food production (al-kodmary, ). when it comes to high quality food, the fact that already food prices are climbing high also due to limited agricultural resource inputs such as water and energy is a matter of great concern. over the last decade, the increase demand for more farmland in order to meet global food demand it becomes more and more obvious. as an immediate effect a lot forest areas are substituted by new farmlands in order to supply this demand. at the same time, since cities constantly grow in terms of area they occupy, a lot of farmland is lost due to this expansive urban development. it is important to convert the global production line to a greener form for both human beings but also for the planet. this implies that food production will not sacrifice the attention for the human health against the commercial profit. according to world health organization, more than half of the farms globally, still use for fertilization purposes of their crops raw animal waste that can attract insect as flies or contain weed seeds or even diseases which can contaminate the cultivated crops. subsequently, these techniques can highly affect people's health and can cause diseases. nowadays, the majority of the food is produced in large, industrialized farms and is transported, distributed and sold in supermarkets, grocery stores or multinational food outlets. agronomists, engineers and farmers in order to reduce the production cost and resource footprint of food production and at the same time increase the variety of the available food species for the consumers have developed various techniques. the high centralization level of food supply can allow the possibility of infection from foodborne pathogens and toxins that can poison large numbers of consumers. food usually travels thousands of miles every day leaving huge possibilities for contamination threats as it can be infected in one country and develop pollutant populations in another. because of the high logistic complexity of food supply, it is worth to mention the advantages and drawbacks of each farming type during the whole supply chain. what follows is an exploration of the three subjected farming types, including outdoor farming, greenhouses and indoor vertical farming. we will compare and evaluate products and growing process under the scope of food safety practices. outdoor farming is applied for thousands of years, allowing an unprecedented human development. however, over the past years the continuously increasing demand of the population has led farmers to apply chemical inputs for nourishing of plants, fighting pests, insects and improving soil quality. however, because of its nature, crops growing in the open field are facing all the difficulties from severe weather conditions and the danger of infection from various insects and pathogens. traditional farming is a type of agriculture that allows to multiple plant pathogens, bacteria and insect pests to affect crops, causing scalable losses in global crop production. after heavily tilled farming applications, severe irrigations and monocropped selections, soil has been seriously affected causing depletion of its nutrients, highly requiring additional nutrient solutions that can improve its fertile condition, making it appropriate for cultivation. once crops are harvested, a big after-harvest process and logistic supply has to be followed in order food to be transported from the farmer to customers' table. when we are talking about vegetables and greeneries there is a high level of perishability that needs to be confronted. crops have to keep cool in order to maintain the high fresh and nutritious status. in order farmers to retain a high value for their products, after harvesting, food is transported from the field to processing facilities that are responsible for the cutting, washing of plants in cold water applying centrifugation methods in order to remove the excess water from the products. after removing the roots and fulfilling the described procedure, products begin to decompose. an often procedure that farmers follow is to treat their production with chlorine compounds and/or antioxidants that expand preservation during and after washing. continuously, food is usually packaged and stored in refrigerators and very low temperatures in order to remain in inertia status. however, outdoor farmers are not able to perform refrigeration between harvest and transport of the products for water processing, making it more uncertain in pathogens infection. in order groceries to arrive from the processing facilities to the shelves of the markets, they require on average to km, resulting to - days in transportation. according to kublic et al. ( ) , every three days, products lose % of their nutritious value after being harvested and roots' removal, meaning that consumers finally receive severely influenced vegetables in terms of nutritional value. according to the centers for disease control and prevention, each year, "roughly million people ( in )" are food poisoned in the united states. in terms of food safety what products of outdoor farming face is the severe contamination from improper use of manure, either from human fecal that is used as fertilization mainly in developing countries or from contaminated concentrated animal feeding operations (cafos). even if it has been proven by various researchers as a great nutritious source for the crops after proper compostable process, on the other hand the absence of carefulness, targeted application and lack of sanitation can lead to transmission of various types of parasites. a serious parasite that is worth to be mentioned is geohelminths (hookworm, ascaris and whipworm), that can survive their eggs in soil for years when they find the right climate conditions, causing diarrheal diseases as well as permanent learning deficit to children (hotez and pecoul, ) . e.coli was a foodborne illness that took high publicity after infecting approximately , people and causing about deaths, after severe pollution of agricultural water reservoirs in farms of california. to summarize, even if there are multiple technological automations, innovations and outbreaks in outdoor farming over the last decades, the nature of this agricultural type is very open to foodborne illnesses, illnesses extremely difficult to be traced rising the total risks. because of the importance of food safety and in order to avoid further foodborne illnesses, there are several rules that force outdoor farmers to enhance the safety status of their production for the overall benefit of the population. the strictest and most widely recognized organization of food control audits is the global food safety initiatives (gfsi), which was established in to reduce and control the risks associated with food production as also to streamline and improve the overall food safety while reducing the operating costs. various certifications are provided to farmers including the safe quality food (sqf) and hazard analysis critical control points (haccp) that set the necessary rules and prerequisites of a high-level food safety status. this includes some of the following rules: • it is of significant importance the control and validation of the agricultural water. to be more specific, there are rules that prerequisite the testing of the water quality that is applied via irrigation to the crops, but also the water related to the tangential purposes such as hand-washing of the workers during or after harvest, the ice that refrigerates food and the surfaces that food contacts with. • biological adjustments are often applied in soil for or particular nutritional uses that replace chemical fertilization. it is of vital importance that farmers who follow these techniques to follow specific guidelines for the use of raw manure (such as animal and human feces) as also for the use of stabilization compost in order to maintain a high level of sanitation. • there are rules concerning the compliance of domestic and wild animals either they are working in the farm, invade in the farm or graze. • finally, there are high requirements in workers' health and hygiene that need to be followed in order to prevent the contamination that may source by humans. . . food safety status of greenhouses as has already analyzed, there are greenhouses that are soil-based and the more advanced use hydroponic solutions. in hydroponic greenhouses, plants are transported several times according to the growing stage and are monitored throughout the different growing cycles. that give the opportunity to apply the exact resource requirements in every stage, in comparison with soil-based greenhouses and outdoor farms, where the plants remain in the same position until their harvest. another significant advantage of greenhouses in relation with outdoor farming is the high geographical flexibility of installation as it allows a significant reduction of the transit time of the products from the harvesting and processing point to the final consumers. greenhouse plants in an industry that constantly growing, with today's list accounting half of the tomato production and / of the global pepper production that are distributed in the fresh market (brauther, ) . greenhouses are a significant driver of national economies of the agricultural sector because of the high profit margin as also the opportunities for high added-value products. unlike traditional farming products, greenhouse production is highly protected from dangerous elements and various contaminants. however, the technologies that are applied for monitoring and controlling of the environmental conditions do not guarantee crops free of microbes and pathogens. the management practices applied in greenhouses are these that can conduct to growth, survival and spread of foodborne pathogens. a severe contamination thread could be spread by processing equipment since crates and baskets that are used for transportation of products, from propagation tools or even for surfaces that food contacts with. irrigation water is one of the most important food safety risks even in greenhouses as it can be drawn from a wide variety of uncertain sources such as municipality supply, rainwater, underground aquifer, reservoirs or surface water. greenhouses that use untreated surface water as irrigation source face high contamination risks. for example, in , salmonela saintpaul (cdc, found to have infected cucumber greenhouses in us that caused the infection of individuals across the country as they consumed imported vegetables with questionable irrigation water status. because of the high risk of infection of consumers, even from more controlled agricultural systems (compared to outdoor farming), regulations for food safety have become stricter by establishing new standards for food production (produce rules). the four areas that these standards focus on are the followings: • health and hygiene this practice targets in maintaining hygienic conditions of the personnel that is occupied in the greenhouse factories, involving criteria for personnel cleanliness, handwashing and use of appropriate gloves. even if handwashing is considered one of the simplest and cost-efficient practices, it has been reported that only % of greenhouses practice handwashing before the harvesting process. • irrigation water quality and management since water quality is one of the most crucial and contentious factors, it seems absolutely necessary the mandatory establishment of rules that control the water baseline quality profile. greenhouses withdraw water from a bog variety of sources such as municipality supply, wells, reservoirs and surface ponds. by checking and understanding the quality of the quality of various water sources can provide important information and reduce the risk of contamination. by regulation, greenhouses have to determine frequently microbiological testing on the water sources. furthermore, greenhouses that apply hydroponic solutions in semi-close or closed loops that circulate, recycle and reuse water, have to include filtering treatments that remove possible pathogens before re-applying it. methods that are effective and efficient in water recycling is uv light or disinfectants. • animals and waste significant measure for the protection of crops from foodborne pathogens is also to eliminate the restriction of domestic and wild animals at growing activities inside the greenhouses as well as in the outside area of the buildings. practices that contribute in discouraging animal intrusions can be for example the rapid weeding that will minimize rodents attraction and protection. • sanitation of equipment, tools and greenhouse surfaces foodborne pathogens are usually found all over the greenhouse environment such as harvesting bins and boxes and tarp floor covers of the greenhouse (ilic et al., ) . according to produce rule, all the tools and equipment that used in the production line should be inspected, cleaned, sanitized and maintain in this condition throughout the whole production, harvesting and post-harvesting process, in order to prevent contamination. greenhouses in comparison with traditional farming have the advantage of the three-key elements application that can eliminate contamination risk: innovations, automations and control. in specific, innovations provide to greenhouse farms a more secure food safety support such as water filtration systems, integrated pest management and higher quality control systems. automations can reduce the danger of contamination or cross contamination as they minimize or decrease the introduction of foreign specimens. finally, biometric systems provide to growers the ability to detect tracking information concerning the plants. after harvesting, the produce is set up in a traceability system from the greenhouse plant to the customer delivery service. . . food safety status of indoor vertical farms leafy greens, vegetables and herbs are considered of high-risk crops since they are usually not cooked but eaten raw. the usual process of consumers is to rinse their purchased greeneries after purchasing them from their grocery store and then consume them. this is not a particularly effective and protective procedure, since harmful pathogens need interference of chemicals to be detached from plants. outdoor farming and most of the greenhouses perform triple-wash on the harvested plants in order to mitigate the contamination risk, as a post-harvest process. this process consists of the pre-washing, a saline wash and the final bathing of greeneries in sanitizing, choline base solutions. unfortunately, this method cause quality reduction to greeneries, as is observed loss of flavor and texture along with the concurrent risk of contamination existing and spreading under the possibility of incorrect application. greens that grow outdoors follow the triple-wash procedure as a postharvest measure for increasing their health status. harvested crops are transported in the processing facility and sorted, rinsed, put in spinners, apply a second rinse, spinner again, third rinse, sorted (again), packed, and then at the end they get delivered at the grocery market. crops that follow the above washing method bear usually on the packaging labels such as "triple-washed" or "pre-washed". even if this method can provide sufficient results in harvested outdoor crops, if the water used for the triplewashing process is polluted with pathogens, then this can spread rapidly to the rest of the harvested crop. for this reason, triple washing cannot categorized as the most effective and guaranteed process. indoor vertical farms apply only nutrient elements in the irrigation system and completely avoid the use any chemicals during the growing period of plants, excluding all the types of pesticides, herbicides and chemical spaying for fertilization. the philosophy of indoor farming depends on monitoring and constant controlling of the crops as also of all the resources that come in and out from the farm and they are isolated from mother nature where many threats and contamination sources may appear. for this reason, indoor farmers suggest that their products do not need to be washed before consumption, as they are already clean by a protected and purified growing process and a quick delivery to local grocery stores. hermetically sealed environments, inside highly controlled spaces that are designed to offer the highest possible level of food safety particularly for the growing period, surround the cultivation rooms of indoor vertical farms. since there are no seasons to be followed as in outdoor farming neither humidity, temperature fluctuations nor long gaps on post-harvesting processes and packaging, indoor farmers can dramatically reduce a potential contamination with precise systems. in addition, the hermetically sealed environment protects crops from being exposed to outside elements such as harmful pests, insects, fungi and bacteria. in one of other type of such systems, aquaponics, co-cultivation of fish with plants is done. this method of cultivation uses very innovative water filtration systems, which extract solids from the fish tanks. continuously, solid break down to beneficial bacteria that transforms them into nitrates. then, the nitrate-rich water circulates to the plant culture area where plants absorb the nutrients and purify the water. since the aquaponic system follows a close-loop, the clean water is circulated and reused into the fish tank. plants that grow in soilless systems can travel along their production process giving the opportunity to be inspected for health status. for example, after sowings, seeds are moved to germination rooms with high humidity that boosts their sprouting. then, seedling is moved to propagation room with controlled climatic conditions that promote their development. next, young plants usually located in the main part of the cultivated room in floating rafts, receiving a nutrient-rich water. after finishing their development and reaching their mature stage, they are daily harvested and shipped. between every translocation of plants, there is intensive quality check to prevent crops' contamination. high precision irrigation systems are used in order to monitor the water that travels throughout the crops. innovative hydroponic or aeroponic methods usually draw water from filtered and drinkable sources and distribute it at each crop often without even touching the salable part of the plants. this is achieved either by the use of water in liquid form, mist or fog that sprays it only into the root section of the plants and not in the parts consumed. extensive sterilization and supplier are also applied methodologies of indoor vertical farms that control and assure the input resources of the farms such as seeds, nutrients that need to be absolutely safe and clean. because of control and monitor mechanisms that are carried out indoor, there is clear advantage of indoor farms. they are aware of the cleaning status of plants and maintain it with further regulations during the cultivation period and finally harvest and deliver a healthy and fresh product. even if indoor vertical farms produce food safer to consume than the open field grown products, bottlenecks and hazards can still be introduced during the growing process of crops. such threats can be dirt and bacteria transferred from the workers and dangerous threats in the nutrient medium that include chemical sources, cleanliness and water safety. further risks can also detected at the post-harvest activities such as trimming, sorting and delivery of the products. thus, it is of vital importance even for indoor farmers to perform high status and certified systems for detection, monitoring, testing and evaluation as in outdoor farming and greenhouses. a study conducted by purdue university (wang et al., ) , found that there is also high risk of crops contamination due to pathogen pollution in vegetables grown in hydroponic or aquaponic systems. more specifically, they reported that e.coli o :h was found in fish feces and because of the circulation that close loops systems, it caused water contamination of the plant root surfaces that were in the aquaponic and the hydroponic systems. since fish probably were contaminated by the bacteria, it is important to follow a proper and certified handling, cleaning and sanitizing process in order to reduce the contamination risk in hydroponic and aquaponics. it is a very difficult, time consuming and α costly process to control all the plants even in an indoor vertical farm for having a % safe food product. indoor vertical farms use controlled environment of humidity and temperature in order to provide plants the most suitable conditions. however, in the case that unpredictable production errors occur, e.g., technical malfunctions with the engineering equipment, temperature and humidity can get out bounds to unwanted levels and create fertile environment for bacteria growth. this incident could be possibly avoided in the case of traditional farming, as the constant natural air circulation and the sunlight could smooth out some of these errors. bacteria population are not biased, meaning they do not grow or prefer targeted geographic locations, but they are transported to different locations by human activities as they can be brought by clothes, shoes or skin. furthermore, it should be noted that even if indoor farms consist a safer environment compared to other farming types, if a controlled environment develops for some reason bacterial infection, it will be extremely difficult to eliminate the contamination and protect the rest of the growing crop. for this reason, indoor farms follow high sanity level protocols to avoid the possibility of crops' contamination by human contact that involves all the workers involved with various cultivation processes of the plants. that include strict control by imposing the use of facemasks, hair and beard net, footbaths and clean or single-use suits, which can diminish the risk of contamination. another solution for further risk elimination from potential contamination, is the application of innovative technologies that operate extensive integrated pest monitoring. this can be achieved with the use of ultraviolet light outside of the farms that detect possible threats as also air curtains that are installed in every door and can control air that enters the cultivation room protecting it from the danger of contamination. additional solution that can increase the sanitation levels of indoor crops, is the application of certified hvac filters, in order to perform an extensive pest monitoring. indoor vertical farms belong to a novel type of farming cooperating with innovative technologies in order to provide the safest, higher quality and most fresh and nutritious groceries. both advocates and critics of this technology seem to recognize that indoor vertical farms under suitable circumstances (mainly of the high demand on electricity loads), could offer a solution to the safety and sustainability problems faced in traditional farming. however, consumers seem to be more skeptical and critical on this technology. potential explanation of the consumers' skepticism is the uncertainty and lack of trust in other food innovations such as genetically modified crops, food nanotechnology and artificial irradiation that struggled to find acceptance in the market. nevertheless, the subjective knowledge and awareness level of consumers on the indoor vertical farming is still limited, even with the excessive spread of technology and information globally, it is of vital importance to increase the education of people on this new technology by informing them on the actual growing properties and impugn the unjustified myths and dangers. because of the increasing demand of indoor vertical farms and their establishment in the market, many researchers have focused on designing and addressing customer surveys and other research methodologies in order to define the public opinion on this technology and the status of their trust and preference on already existing agricultural production systems. significant angle on these researches is to explore the existing knowledge and perception of customers between the three different farming systems; traditional farming, greenhouses and indoor vertical farms, in respect of the cultivation techniques, safety, resource sustainability, quality and their willingness to buy products from each category. for this reason, primarily it was of high importance to validate that consumers are able to recognize the different agricultural systems between them in order to provide valid, clarify and comprehensive results. different customer studies that investigated customers' opinion on different agricultural methods show a more skeptical belief concerning novel technologies on food production. more specifically, peoples' perception with technological innovations in agriculture are associated with high risks for food production presenting low expectations on the provided benefits of technology used (sparks et al., ) . in another research (coyle and ellison, ) participants rated higher the greenhouses facilities and the outdoor farms compared to the indoor vertical farms in terms of naturalness in the production process and the final product. concerning the quality status of the final product people also seem to present higher levels of confidence and trust on the greenhouse products and subsequently indoor vertical farms and finally in outdoor farming products. naturalness seems to be a high influencing indicator for consumers' selection globally as also a critical significant factor on the usefulness of the agricultural system. according to j€ urkenbeck et al. ( ) , customers replied that led lighting is not considered a too artificial tool for horticulture and slightly agreed that they do not consider indoor vertical farming too artificial concerning the overall production system. even if consumers in general prefer naturally and traditionally produced food, nevertheless the fact that food of indoor vertical farms grow without chemical additives is highly considered. on the other hand, under a customer research conducted by j€ urkenbeck et al. ( ), it is noticed that consumers seem to present a high acceptance on indoor vertical farming concerning the offering sustainability and the high ecological footprint. people seem to select their purchased food based on their concerns on the naturalness, ethics and environmental status. in more details, % of the respondents in that research declare that they put an extra effort to select and buy locally grown food because of its high level in freshness, nutrition and reduced food mile emissions compared to traditional farming methods. on the other hand, a significant share of the consumers evaluates indoor vertical farming as an artificial agricultural process in order to trust their footprint outcome. for this reason, it is pointed out that knowledge, information and nutritional awareness can become a solid solution for the higher acceptance of indoor farming and irradiated food products. respondents of the specific survey showed a strong willing on buying products that were produced in an indoor vertical farm with . % of the total sample, . % partly agreed on that statement, and finally only . % were not willing to purchase these products. however, it should be noted that the perceived behavioral control does not influence the customers' willingness to buy, but it has some influence on the behavioral intension of willingness to purchase the product. overall, the behavioral intention of customers to purchase products from indoor vertical farms is highly dependent on sustainability. under a different analysis, it has become very clear that perceived sustainability of indoor vertical farming is the main reason of acceptance. it has been observed that the more positive the resulted sustainability status of the system is, the higher and the customers' acceptance and willingness to purchase the product is. furthermore, based on the perceived sustainability level of indoor vertical farms it seems that customers increase and their acceptance of this innovative technological food production system. based on these results, we could indicate that the growing involvement and concern of consumers to select products from agricultural systems that present high environmental performance. indoor vertical farming can be very advantageous in terms of resources sustainability since because of the high technology and the soilless cultivation systems it consumes way less on natural resources (e.g., water and nutrients). additionally, indoor vertical farms significantly decrease the co emissions that are correlated to food transportation from the producers and the processing facilities. in specific, indoor vertical farms can provide times higher productivity per year per unit land area compared to traditional farming due to the zero dependence on weather conditions, seasonality and possible infections from insects, pests and bacteria. due to the evolution of technology it is not anymore a prerequisite holding a large area of land for sufficient fresh food production, but the use of multiple layers, optimally controlled (environmental conditions and physiological parameters of the crops and minimum possible loss from crop threats). significant characteristic of indoor vertical farms in terms of sustainability is the minimization food delivered losses. in addition, significant reductions can be observed in the cooling fuel demand, necessary to cool the production in order to be transported in long distances. this can be achieved since indoor vertical farms are usually installed in the urban or suburban areas in shaded and/or abandoned buildings (or even basements) due to the soilless farming techniques and the artificial lighting, providing access to fresh and nutritious greeneries to citizens. finally, one of the significant benefits that indoor vertical farms provide is the ability after proper processing of the use of waste water, crop wastes and excessive co produced in urban areas, as input resources of water, nutrients and co in the culture area. to summarize some of the basic improvements in resource savings provided by indoor vertical farms compared to the immediately following high technology cultivation system, the greenhouses are the following: • indoor vertical farms save % of the pesticide use in their interior by maintaining the culture area clean and insect-free. • because of the application of close loop irrigation systems and of the collection, recycle and reuse of the water vapor that plant leaves transpire, indoor vertical farms can reduce up to % the water consumption. furthermore, the use of closed loops can decrease up to % the fertilizer usage since it is feasible to recirculate and reuse the nutrient solution. • significant land reduction up to % can be achieved with the application of indoor vertical farming, due to the important increase (more than times) of the annual productivity of crops per unit land area. • yield variation can also reduced by % because of the constant monitoring and control of the crops and the lack of influence from the outdoor environmental conditions. food safety and traceability of products is another important factor highly relevant to indoor vertical farming. even if it does not provide a % safety for consumers, despite the fact that crops grow in a controlled environment protected by wildlife, animals, birds and insects, it upgrades the safety and security feeling of the products than those that grow in open field. the majority of the selected cultivated crops of indoor vertical farms are among the species with the higher contamination risk when they grow outdoors or unprotected, because they grow very close to the ground level. furthermore, one of the most crucial factors that greatly affect the possibility of contamination is the water quality that involves during the whole production process, including the irrigation water as also the washing water at the post-harvest processing techniques. farmers of all categories should follow high standards and criteria for the water sources that channel water into the farms as also frequent control and monitor of the crops for potential threats of contamination. it is now clear, that indoor vertical farms are a high necessity for tackling the challenges concerning the conservation of their resources. nevertheless, in order to enhance the environmental sustainability and improve the efficiency and sufficiency of food production supplies for our society, it is necessary to develop more diverse, effective and ecological agricultural systems including both the traditional farms and the greenhouses. further research and experimentation it is absolutely necessary in order both to improve the efficiency of resources in an indoor vertical farm but also to possibly eliminate the possibilities for contamination threats and constantly provide the outmost safe, fresh and nutritious fresh fruits and vegetables to the human population. notwithstanding the promising benefits that are linked with indoor vertical farming, there are also important challenges in the further implementation of this farming system in the future. it is of vital importance further improvements on the efficiency and effectiveness of the equipment that will lead to a significant decrease of the energy demand of the systems. by achieving the reduction of energy demand, it will add extra value in the environmental sustainability of the system but also it would also make it more appealing for the public, the investors and the industry and will increase the viability and profitability. however, it is pointed out by despommier ( ) that there is the opportunity for energy recovery from the non-salable crops' parts and capture of renewable sources of energy that can create zero energy building for hosting indoor vertical farms. at the same time, the whole system of indoor farming can synchronize and manipulate huge amounts of carbon and simultaneously release into the atmosphere oxygen from plants' respiration. significant is also the start-up costs that are associated with indoor vertical farms as it is clear that it is more expensive to develop a vertical greenhouse than a normal greenhouse (fletcher, ) . as it has been highlighted by many studies one also key barrier that indoor vertical farmers have to confront is the public resistance to these type of products as social masses face difficulty in accepting indoor vertical farms instead if traditional farming ones because of the natural way that food is produced. additionally, as indoor vertical farms serve the concept of local, fresh food production and they are mainly installed in urban or peri urban areas, they have also to salient the issue of affordability because of the expensive land and space use. for this reason, key factor is the productivity rate of indoor vertical farms that can maintain them profitable and keep them prevailed in the future. more specifically, if indoor vertical farms achieve to produce up to times more yield compared to traditional farming, then they can offset the capital expenditures and the expensive land use. previous research conducted by , presented that indoor urban vertical farms regardless the financing scheme, are much more profitable in comparison to greenhouse constructions. in the specific work, different investment scenarios are presented based on the cash flow analyses and show that iuvf can present high irr (investment return rate) as also a payback period between and years. finally, another drawback that is linked with indoor farming production is the limited variety of crops that can be produced with this technology, such as lettuce, herbs, tomatoes and berries. even if theoretically, all types of crops could be cultivated indoors, that would not be economically feasible due to the highly increased energy demand. thus, low-value agricultural crops such as wheat and barley will continue to grow under economically and environmentally unviable conditions. under these circumstances, the indoor vertical farms have to face a limited production compared to the "limitless" hectares of traditional farming and a reconsideration of scaling up would be particularly costly and complicated. the last years that indoor vertical farming gained more recognition and research interest, a plethora of new studies, prototypes and innovation designs have been presented under the academic and industrial scope. indoor vertical farming presents a high interest and potential to play a critical role in the demanded sustainability in food of urban areas. this becomes even more important by the multiple studies that estimate and analyze the significant increased food demand in urban areas. indoor vertical farming presents important advantages compared to traditional farming, concerning the required sustainability in our times by focusing in three main categories: environmental, economic and social. there is a high demand for further development in automation. this will be scaling up the projects in order to create more feasible scenarios both from economic and commercial perspective. future research is necessary towards a holistic approach via the investigation and the analysis of the full life-cycle of indoor vertical farms and the impact to the environment compared to the traditional farms and greenhouses. up, up and away! the economy of vertical farming state of indoor farming agstar data and trends. united states environmental protection agency the vertical farm: a review of developments and implications for the vertical city benefit cost analysis of the maize crop under mechanized and traditional farming systems in the nwfp optimization of photoperiod and quality assessment of basil plantsgrown in a small-scale indoor cultivation system for reduction of energy demand plant factories in the water-food-energy nexus era: a systematic bibliographic review indoor vertical farming in the urban nexus context: business growth and resource savings basil plants grown under intermittent light stress in a small-scale indoor environment: introducing energy demand reduction intelligent technologies comparison of land, water, and energy requirements of lettuce grown using hydroponic vs. conventional agricultural methods multistate outbreak of salmonella saintpaul infections linked to imported cucumbers (final update) will consumers find vertically farmed produce "out of reach"? the rise of vertical farms the vertical farm: controlled environment agriculture carried out in tall buildings would create greater food safety and security for large urban populations the vertical farm: controlled environment agriculture carried out in tall buildings would create greater food safety and security for large urban populations efficiency of light energy used by leaves situated in different levels of a sweet pepper canopy the future of agriculture may be up sustainable vertical farming outperforms other agricultural methods on co outputs plant factories versus greenhouses: comparison of resources use efficiency blue light does-responses of leaf photosynthesis, morphology, and chemical composition of cucumis sativus grown under different combinations of red and blue light manifesto" for advancing the control and elimination of neglected tropical diseases economics of sustainable farming listeria monocytogenes in tomato greenhouses food miles: background and marketing sustainability matters: consumer acceptance of different vertical farming systems electricity price forecasting in danish day-ahead market using tbats green-light supplementation for enhanced lettuce growth under red-and blue-light-emitting diodes solar air conditioning systems impact on the built environment: a thermodynamic approach exergy analysis for a proposed binary geothermal power plant in nisyros island plant factory: an indoor vertical farming system for efficient quality food production comparison of land, water, and energy requirements of lettuce grown using hydroponic vs. conventional agricultural methods technoeconomic evaluation of urban plant factories: the case of basil (ocimum basilicum) the effects of red, blue, and white light-emitting diodes on the growth, development, and edible quality of hydroponically grown lettuce (lactuca sativa l. var. capitata) effects of different spectral lights on oncidium plbs induction, proliferation, and plant regeneration global food demand, productivity growth, and the scarcity of land and water resources: a spatially explicit mathematical programming approach hydroponics: are we moving towards that direction only because of the environment? a discussion on forecasting and a systems review the impact of co emissions on agricultural productivity and household welfare in ethiopia. a computable general equilibrium analysis. environment for development water use efficiency of tomatoes in greenhouses and hydroponics spatial planning of biogas processing facilities in greece: the sunflower's capabilities and the waste-to-bioproducts approach carbon footprint and cumulative energy demand of greenhouse and open-field tomato cultivation systems under southern and central european climatic conditions resource use efficiency of indoor lettuce (lactuca sativa l.) cultivation as affected by red: blue ratio provided by led lighting environmental, energetic, and economic comparisons of organic and conventional farming systems crop growth and development in closed and semi-closed greenhouses. thesis recharging greek youth to revitalize the agriculture and food sector of the greek economy survey and evaluation of heating technologies for worldwide agricultural greenhouse applications advances in greenhouse automation and controlled environment agriculture: a transition to plant factories and urban agriculture gene technology, food production, and public opinion: a uk study the economics of local vertical and greenhouse farming are getting competitive multidirectional relationship between energy resources, climate changes and sustainable development: technoeconomic analysis agstar: biogas recovery in the agriculture sector the occurrence of shiga toxin-producing e. coli in aquaponic and hydroponic systems watering the farm: comparing organic and conventional irrigation water use in the murray-darling basin development of an integrated methodology for the energy needs of a major urban city. the case study of athens wind energy integration through district heating. a wind resource based approach a wind energy integration analysis using wind resource assessment as a decision tool for promoting sustainable energy utilization in agriculture small scale plant factories with artificial lighting and wind energy microgeneration: a multiple revenue stream approach effects of leaf area index of tomato seedling population on energy utilization efficiencies in a close transplant production system energy and mass balance of a closed-type transplant production system (part ): carbon dioxide balance discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin key: cord- -lufg w n authors: schiavon, michela; nardi, serenella; dalla vecchia, francesca; ertani, andrea title: selenium biofortification in the (st) century: status and challenges for healthy human nutrition date: - - journal: plant soil doi: . /s - - - sha: doc_id: cord_uid: lufg w n background: selenium (se) is an essential element for mammals and its deficiency in the diet is a global problem. plants accumulate se and thus represent a major source of se to consumers. agronomic biofortification intends to enrich crops with se in order to secure its adequate supply by people. scope: the goal of this review is to report the present knowledge of the distribution and processes of se in soil and at the plant-soil interface, and of se behaviour inside the plant in terms of biofortification. it aims to unravel the se metabolic pathways that affect the nutritional value of edible plant products, various se biofortification strategies in challenging environments, as well as the impact of se-enriched food on human health. conclusions: agronomic biofortification and breeding are prevalent strategies for battling se deficiency. future research addresses nanosized se biofortification, crop enrichment with multiple micronutrients, microbial-integrated agronomic biofortification, and optimization of se biofortification in adverse conditions. biofortified food of superior nutritional quality may be created, enriched with healthy se-compounds, as well as several other valuable phytochemicals. whether such a food source might be used as nutritional intervention for recently emerged coronavirus infections is a relevant question that deserves investigation. selenium (se) is a semi-metallic element that possesses chemical and physical characteristics of both non-metals and metals (boyd, ) . it is recognized as an indispensable micronutrient for redox biology of many animals and unicellular organisms (kieliszek ). in mammals, se in the form of the unusual amino acid selenocysteine (secys) is incorporated at the catalytic center of selenoproteins via recoding of the opal uga codon into a secys codon (mangiapane et al. ). in humans, at least selenoproteins play important roles in antioxidative systems, hormone balance, immunity, male fertility, resistance to viral infections and cancer prevention (harthill ; rayman rayman , tan et al. ; valea and georgescu ) . however, se exhibits a double-edged behaviour, because it becomes toxic over a threshold concentration established to be µg of se per day (institute of medicine ; vinceti et al. ) . for this reason, similar to boron (b), it has long been termed "an essential poison". selenium has a relatively narrow safety range as compared to other essential trace nutrients, and thus both the deficiency and toxicity of se are of worldwide concern (natasha et al. ; rayman ) . on the global scale, the se deficiency and its negative impact on health have progressively increased over recent decades. so far, it has been estimated that billion people have a scanty se dietary intake (combs ; tan et al. ) . this worrisome scenario is likely to worsen in the future because of projected climate changes that will cause reduction of soil se content, principally in agricultural areas (jones et al. ) . local populations living in low se areas and feeding on low se food crops might suffer from se deficiency disorders because their dietary se consumption does not meet the daily dose ( - µg for adults) recommended to maintain a correct functioning of the metabolism and expression of selenoproteins (usda-ars ; who ) . to reduce se deficiency throughout susceptible regions, the development of high se crops using biofortification interventions has been accomplished in several countries that are low in se, such as finland, united kingdom, new zealand, malawi, parts of china, tibet, and brazil (alfthan et al. ; broadley et al. ; dos reis et al. ; joy et al. ; wu et al. ) . se-biofortified food crops are often enriched in many phytochemicals beneficial to human health, like minerals and antioxidant compounds, which make these foods superior to se supplementation alone via tablets (d'amato et al. ) . the success of biofortification to enrich plants with se depends on several variables such as se species to be used, the mode of se fertilization, and the crop species. in higher plants, no essential metabolic role of se has been established and hitherto no specific mechanisms of secys incorporation into proteins have been documented (schiavon and pilon-smits a) . selenium is ass i m i l a t e d t o s e -a m i n o a c i d s ( s e c y s a n d selenomethionine, semet) by accessing the metabolic pathway of its analog sulphur (s) (white (white , . se-amino acid insertion into proteins in place of s amino acids (cysteine and methionine) can produce malformed proteins causing toxicity (sabbagh and van hoewyk ; van hoewyk ) . on the other hand, at low concentration se can be beneficial to plants. its positive e f f e c t s w e r e o r i g i n a l l y d e s c r i b e d i n s ehyperaccumulator species ). however, further studies with several nonhyperaccumulators have proved that se promotes the growth, accumulation of beneficial phytochemicals, and antioxidant activity (e.g., chauhan et al. ; dall'acqua et al. ; natasha et al. ; schiavon et al. schiavon et al. , d'amato et al. ) . different approaches can be explored to enrich plants with se. some are already widespread or are gaining recognition, while others are limited by in-situ restrictions. the aim of this review is to afford a comprehensive overview of the current knowledge of se biofortification and future research directions. it is focusing on (i) se distribution in soil and soil properties that may affect the efficacy of se biofortification strategies; (ii) se metabolism in plants and possible interferences of se enrichment with metabolic pathways that might influence the nutritional profile of se-enriched food; (iii) se biofortification strategies (conventional and modern agronomic approaches, genetic engineering and plant breeding) and biofortification under challenging conditions; (iv) effects of se biofortification on plant food nutritional traits; (v) impact of se-enriched food on human health with special attention to its role in reducing the risk of and treating viral diseases. the geographical distribution of soil se is very heterogeneous, but se content in most soils is low and ranges from . to mg kg − on average (world mean . mg·kg − ) (fordyce ; oldfield ) . in tropical soils, soil se levels are slightly higher and typically range within - . mg kg − (mehdi et al. ) , while seleniferous soils can contain up to mg se kg − (fordyce ; oldfield ) . selenium in soils is primarily derived from the parental material of soil. its content depends on soil type and texture, origin and geological history, mineralogy, organic matter content, weathering degree and rainfalls, dominant soil geogenic processes, and se deposition (hartikainen ; mehdi et al. ; wen and carignan ) . natural se emissions (crustal weathering, sea spray, volcanic plumes) and anthropogenic se emissions (fossil fuel combustion, non-ferrous metal production and manufacturing) largely contribute to se deposition, which is affected by vicinity to the coast, altitude and wind directions (saha et al. ; wen and carignan ) . low se soils mainly derive from igneous rocks and are settled in areas characterized by limited atmospheric depositions and elevate erosion rates (christophersen et al. ) . volcanic soils and granite are commonly poor in se and lay in the mountainous countries of northern europe (mehdi et al. ) . conversely, high se soils originate from sedimentary rocks, generally cretaceous sediments like black shales, containing selenites and selenides associated with sulphide minerals. black shales are particularly abundant in ireland, china (e.g., enshi in hubei province and ziyang in shaanxi province), and arid areas in the western and southwestern states of the usa (e.g. san joaquin valley in california), and india (punjab) (oldfield ; winkel et al. ) . occasionally, high se contents in soil may depend on the release of toxic levels of geogenicallyderived se in soils and waters caused by human actions (ohlendorf et al. ), on anthropogenic inputs, either industrial or agricultural, or on dust depositions discharged by nearby coal-burning sites (he et al. ) . agricultural activities that increase se levels in soil include irrigation and the long-term use of chemical fertilizers and farmyard manure. irrigation may elevate se in soil by either favoring dissolution of se from minerals rich in se or bringing se loads to soil when se-rich waters are used (bajaj et al. ) , while adding se to chemical fertilizers is a common practice when biofortification is conducted in se deficient areas, where soil se is below . mg kg − , or when prevailing soil conditions hamper se availability to plants (gupta and gupta ) . the specific impact and fate of different se inputs into agricultural soils have been poorly investigated so far. further systematic mass balance studies of se in soils that estimate the amount of se retained in soil and se losses through leaching, volatilization and crop removal, are required to better quantify the contribute of each se source to the content of se in agroecosystems. these studies might be helpful in view of facing with the undergoing global climate-changes predicted to cause reductions of soil se in the future (jones et al. ) . selenium in soils is found under inorganic and organic forms, where it holds different oxidation states ranging from − to + . se solubility and mobility in soil increase with increasing oxidizing conditions (high redox potential). in this regard, selenate (seo − ) is the most water-soluble, mobile and bioavailable inorganic se species in oxic soils, with low adsorption affinity to oxide surfaces (hartikainen ) . under low soil redox potential conditions, selenate can be reduced to selenite (seo − ), whose compounds are typically the most abundant se species in anoxic soils ( fig. ) . selenite is less mobile and bioavailable than selenate owing to its tendency to be adsorbed on oxide surfaces at low ph (hartikainen ) . therefore, available soil se is usually poorly correlated with total soil se. under intense reducing conditions, selenite can be further reduced to elemental se (se ) or selenides (se − ) (hartikainen , winkel et al. . bacteria are reported to produce (nano)-sized se( ) from selenate and selenite ni et al. ) , while se in the − oxidation state can exist as hydrogen selenide or metallic selenides, which are very insoluble forms of se (winkel et al. ) . generally, soil organic forms of se occur as complexes with organic matter and combined with organic or organo-mineral colloids (hartikainen ) . organo-se compounds include methylated or unmethylated seamino acids, and volatile se forms (dimethyl selenide, dmse, and dimethyl diselenide, dmdse), but a large fraction of them is still undisclosed (winkel et al. ) (fig. ) . these compounds can either derive from decomposition processes of plant and microbial biomass or from addition of se-enriched plant material (green manure) to soil when biofortification of crops with se is attained. several factors control se mobility and solubility in soils such as ph, sorption, and desorption reactions, redox potential, organic/inorganic compounds, and dissolution processes in soils and sediments. sorptioninteractions depend on the amount of sorption components and are largely governed by electrostatic forces, which in turn rely on soil ph. when se in soil prevails in anionic form, it can be retain by sorption on oxides and hydroxides (e.g. iron or aluminum oxides/hydroxides), or anionic clays by electrostatic interactions (eich-greatorex et al. ; winkel et al. ) . se adsorption rate increases while decreasing the ph, as electrostatic interactions become sturdier (eich-greatorex et al. ). on the other hand, raising in ph or reducing clay and iron (or aluminum) oxide/hydroxide content in the soil result in higher se mobility and bioavailability. soil organic matter (om) is another critical factor altering soil se availability, as soils rich in om commonly display higher se retention capacity (fordyce, ; winkel et al. ) . om can reduce se available in soil by direct complexing with se. otherwise, indirect complexation of se with om-metal complexes or microbial reduction and incorporation into organic compounds (e.g. amino acids, proteins) may occur in soil (winkel at al. ) . from either organic complexes or compounds, se can be promptly mobilized or retained depending on the type of binding. plants accumulate se in their organs depending on se phytoavailable in soil, with a large variation of shoot se concentration between genera, species and even ecotypes within species (mehdi et al. ; schiavon and pilon-smits a; white white , . plants are distinguished in major ecological groups depending on their capacity to accumulate se in the shoot when growing in their natural environment (white ) . se-hyperaccumulator species thriving on seleniferous soils generally contain and tolerate extremely high se concentrations (over µg se g − d.wt.) that could eventually be toxic to grazers, whereas non-hyperaccumulator species are se-sensitive and thereby accumulate less than µg se g − d.wt. (non-accumulators) or up to µg se g − d.wt. (accumulators or indicators) (schiavon and pilon-smits a, b; white ) . hyperaccumulators store se mainly in the form of methylselenocysteine (mesecys) and selenocystathionine, while semet is the main se organic compound identified in nonhyperaccumulators (pilon-smits, ; schiavon and pilon-smits a, b). plants take up both inorganic (selenate, selenite and elemental se) and organic (e.g. se-amino acids) se species, but not selenides (chauhan et al. ; gupta and gupta ; schiavon and pilon-smits a; white ) or colloidal elemental se (white (white , . their capacity to take up se is apparently higher for organic over inorganic species (kikkert et al. ) . se-amino acids in particular, are likely to enter the plant cells via broad specificity amino acid transporters ( fig. ) . though, selenate is the main form of se taken up by plants, and its transport across cell membranes is an energy-dependent process mediated by the sulphate transport system schiavon and pilon-smits a; white ) . competition events in soil between sulphate and selenate, as well as plant sulphate transporters (sultr) differing in affinity for these two anions, influence the rate of selenate uptake by plants white et al. , white . selenite compounds instead, are conveyed over plant cell membranes via phosphorus (p) and silicon (si) transporters, with differences between selenite anion (seo − ), hydrogenselenite ion (hseo − ), and selenous acid (h seo ) zhang et al. ) . precisely, h seo is transported by si transporters (lsi ) and aquaporins (osnip ; ) zhao (fj) et al. , b) , while hseo − and part of seo − mainly use lowand high-affinity p transporters (ospt ), respectively (zhang et al. ) . once entered the root cells, inorganic se is delivered to the plastids where proceeds along the s assimilation pathway to produce secys and semet (fig. ) (chauhan fig. major pathways of se at the soil-plant-atmosphere interface, se movement and partitioning in the plant, with focus on genetic targets for optimization of se biofortification. transformation processes in soil are indicated in italics at the corresponding arrows. soil microorganisms can degrade se-containing plant litter, thus releasing se into soil. se species can be taken up by soil microorganisms or by plants. in the root structure biomagnification, the main transporters involved in se uptake by plants (sultr ; for selenate, nip ; and pht for selenite, aa tr. for amino acids) are localized at the rhizodermis (rh). se metabolism takes place in the root cortical cells (c), and volatile se species (dmse and dmdse) are released into the soil, while se organic compounds, mainly se-amino acids (se a.a.) are transported into the xylem (x) via amino acid permeases (aa tr.) and delivered to the leaves. dmse and dmdse in soil are then de-methylated by rhizosphere microorganisms. selenate is the main form of se shuttled through the xylem to the leaves, where it is finally assimilated to se-amino acids. selenate is loaded into the xylem by sultr ; localized at the xylem parenchyma cells (xp) and pericycle (p). both sultr ; and sultr ; from the se hyperaccumulator s. pinnata are currently under investigation. organic-se compounds produced in the leaf cells can be loaded into the phloem (ph) and transported throughout the plant. the translocation of semet to the seeds is enhanced by overexpression of the nrt ; b transporter. volatile dmse and dmdse produced in leaves are released into the atmosphere. enzymes in circles are early targets of genetic engineering for biofortification or phytoremediation, while enzymes in hexagonal shaped boxes are new targets to better explore. abbreviations: en: endodermis; e: epidermis; m: mesophyll; cc: companion cells; cgs: se cys γ-synthase. for simplicity, arabidopsis thaliana has been used as a model species in figure et al. ; schiavon and pilon-smits a; sors et al. ; white white , . if selenate is taken up by plants, it must be activated before reduction to selenite. selenate activation is a rate-limiting step mediated by the atp sulphurylase enzyme (aps), which couples selenate to atp to generate adenosine ′phosphoselenate (apse) ). apse reduction to selenite is then conducted by the aps reductase enzyme (apr), via transfer of two electrons from glutathione (gsh). in a parallel pathway, apse can be phosphorylated to produce ′phosphoadenosine ′-phosphoselenate (papse), which is used as a substrate for sulfation of desulfoglucosinolates (fig. ) . selenite can be reduced enzymatically by the activity of the sulfite reductase enzyme (sir) to produce selenide, which is incorporated into secys by the enzyme complex cysteine synthase, containing both serine acetyl transferase (sat) and o-acetylserine (thiol) lyase (oas-tl) enzymes (white ) . alternatively, selenite reduction can be realized through a nonenzymatic two step-reaction, where selenite is initially converted to selenodiglutathione (gs-se-sg) in the presence of gsh. gs-se-sg is further reduced to selenopersulfide/glutathionylselenol (gs-seh), which reacts with o-acetylserine (oas) to generate secys (white (white , . otherwise, secys can be formed directly f r o m s e l e n i t e b y t h e a c t i v i t y o f t h e selenomethyltransferase enzyme (smt) (chauhan et al. ) the synthesis of semet from secys takes place partly in the cytosol and requires selenocystathionine and selenohomocysteine to be formed as intermediates (fig. ). this biochemical transformation involves three enzymes working in series: cystathionine γ-synthase (cgs), which promotes the formation of secystathionine by condensing o-phosphohomoserine (oph) and secys; cystathionine β-lyase (cbl) and methionine synthase (ms) (sors et al. ; white ). incidentally, se-amino acids can be integrated into proteins, thus disrupting their molecular folding and impairing their function (sabbagh and van hoewyk ; van hoewyk ) . the magnitude of this event seems to be more related to the se/s ratio in plant tissues than the se content alone (white et al. ). to prevent toxicity derived from se amino acid misincorporation in proteins, secys and semet can be methylated to produce methyl-secys (mesecys) or methyl-semet (mesemet), respectively, which are further converted into volatile dmse (in non-hyperaccumulators) and a deep knowledge of se biogeochemistry, uptake mechanisms and assimilation by plants is a mandatory prerequisite for se biofortification of food crops. biofortification is the process of adding essential micronutrients and other health-promoting compounds to food crops with the aim to improve the nutritional quality of diets ultimately consumed by people (garg et al. ; jha and warkentin ; schiavon and pilon-smits b; white and broadley ; wu et al. ; zhao and mcgrath ) . it is an innovative and reasonably easy practice to manage, affordable and effective in the long-term to tackling micronutrient malnutrition (ros et al. ; white and broadley ). successful outcomes depend on environmental and economical characteristics of local food systems, but also on their acceptance by farmers and populations that feed on biofortified food. to get acceptance, biofortified crops must be staple, high yielding and worthwhile to be adopted by farmers, and most consumers in target populations must consume the biofortified food in amounts enough to measurably improve their nutritional status (miller and welch ) . several studies have been conducted to generate seenriched functional food and various biofortification interventions have been developed and tentatively optimized using agronomic approaches, genetic engineering and plant breeding (bañuelos et al. ; cakmak, ; d'amato et al. ; ros et al. ; schiavon and pilon-smits b; white ; zhu et al. ). the addition of se compounds to food during its processing (process fortification) carried out by the food industry has also been proposed as an effective practice in alternative to agronomic biofortification performed using se fertilizers (haug et al., ) . biofortification strategies, summarized in fig. , are influenced by several variables such as method of se supplementation, se dose and species, soil se, cropping systems and tillage practices, environmental and weathering conditions, crop species/variety, plant growth stage and joint application with other micronutrients (bañuelos et al. ; d'amato et al. ; dall'acqua et al. ; hawrylak-nowak, ; schiavon et al. schiavon et al. , . so far, most research dealing with se biofortification has targeted at the application of se alone or occasionally combined with only one micronutrient, generally silicon, iodine (i) or zinc (zn) (golubkina et al. ; sattar et al. ; cakmak et al. ; golob et al. ). only few studies have addressed crop biofortification with multiple micronutrients (mao et al. ; zou et al. ) , although deficiency of multiple trace nutrients is an issue of global concern. a recent field experiment managed in six different countries (china, india, mexico, pakistan, south africa, and turkey) varying in soil type and environmental conditions, proved that foliar fertilization of wheat (triticum aestivum l.) plants with a blend of se, fe, i, and zn, was effective in enriching grains with all four elements (zou et al. ) . based on these promising results, future se biofortification research should address the feasibility of supplementing crops with cocktails of micronutrients without unwanted crop yield trade-off. agronomic biofortification is commonly employed in low se areas and mainly consists in se addition to soil and se foliar fertilization, typically realized using selenate-or selenite-based fertilizers (alfthan et al. ; broadley et al. ; wu et al. ) . selenium fertilizers are generally employed in small amounts ( - g se ha − ) to achieve biofortification goals. therefore, to ease their application, they are frequently mixed into other commercial nutrient fertilizers (e.g., mix of nutrients, urea, calcium nitrate) functioning as "carriers" of se (premarathna et al. ; ramkissoon et al. ). the addition of organic acids to se fertilizers can further promote se chelation with organic compounds, thus causing incremental plant uptake of se and efficiency of se fertilizers. the application of se fertilizers to soil commonly results in increased total and bioavailable se , and thus in higher se concentration in crop edible products (chilimba et al. ; curtin et al. ; lyons, ; poblaciones et al. ) . such a practice is apparently not associated with environmental risks of se leaching into groundwater, being this process limited by se binding to soil organic matter and positively charged sites, and se losses via volatilization ). field tests have been successfully conducted by applying se to soil in finland (alfthan et al. ; hartikainen, ) , uk lyons, ) , and new zealand (curtin et al. ) . nevertheless, the efficacy of this approach can be limited by heterogenous distribution of se in soil and by those soil conditions that control se speciation and uptake by plants such as ph, organic matter, oxygenation, content of competing ions, soil age, chemical and biological se transformations (bañuelos et al. ; duncan et al. ; haug et al., ; schiavon and pilon-smits, b; stroud et al. ). it has been estimated that only % of soil-applied se fertilizers is taken up by plants on average, as most of se is fixed and retained in the soil, and thus is not bioavailable . negligible residual se is then available for succeeding crops, and thus se fertilizers must be applied to soil for each growth period. in contrast, foliar se fertilization is up to times more effective than soil se supplementation (ros et al. ) . this is likely because of more rapid se uptake and assimilation processes, no need of se root-to-shoot translocation to the edible parts of crops, and prevention of se losses due to immobilization of se compounds in soil (ramkissoon et al. ) . however, soil amendment with se can determine positive shifts in the amount and diversity of soil microbial communities, by increasing the relative abundance of plant growth promoting rhizobacteria (pgpr) and reducing the occurrence of pathogenic fungi . beneficial rhizosphere microorganisms could enhance soil se phytoavailability and the plant se use efficiency of applied fertilizers (durán et al. ; lindblom et al. ; mora et al. ; yasin et al. a,b) owing to their capacity to reduce oxidized and methylated forms of se (winkel et al. ) and increase the volume of soil explored by the plant roots to acquire se (white and broadley ). the addition of beneficial microorganisms to soil or the inoculation of plants with pgpr might enhance se biofortification of crops. for instance, the inoculation of wheat plants with specific microorganisms, alone or combined with arbuscular mycorrhizal fungi (afm), was effective in increasing se concentration in their edible products (duran et al. ; yasin et al. a,b) . similarly, inoculating shallot bulbs with afm increased selenocystine and selenate contents by % and %, respectively, compared to non-inoculated plants (golubkina et al. ) . crop enrichment with se could also be achieved by combining different types of se phytotechnologies fig. overview of biofortification strategies that can be exploited to enrich crops in selenium. se biofortification can be achieved using genetic tools ( ), via conventional or assisted breeding and genetic engineering, or through fertilization with se-fertilizers via foliar application ( ) or soil amendment ( ). agronomic biofortification can be integrated by the use of rhizosphere or endophytic microorganisms, either inoculated into plants ( ) or applied to soil ( ). alternatively, plant material enriched with se-organic compounds from hyperaccumulators or accumulators used in phytoremediation, or from crops grown in naturally enriched soils can be used as green manure ( ). nanosized biofortification is performed using senps applied to leaves or soil ( ). co-cropping or intercropping practices use sehyperaccumulators to enrich soil with se-organic compounds promptly available for uptake by neighboring plants or succeeding crops ( ) schiavon and pilon-smits b; stonehouse et al. ) . se-laden plant material derived from plants employed in se phytoremediation could be recycled as green fertilizer to increase se content in agricultural soils or as a supplemental fodder for livestock (bañuelos et al. ). in this case, before being introduced into the food web, the plant material must be carefully checked for its content in hazardous elements, as soils intended to be remediated are often contaminated with many metal/loids. alternatively, plant material derived from se-hyperaccumulators thriving on seleniferous soils and containing high levels of se, especially in organic forms (e.g. semet, metsecys), could be used as green manure (bañuelos et al. , wan et al. ). crops to be amended with this material may be selected via breeding or engineered for preferential uptake and accumulation of organic se in edible products. such an approach might have relevant implications for healthy nutrition, because organic se compounds in food may be more quickly used than inorganic se by enzymes that support antioxidant activities in humans and animals (davis ) . se-hyperaccumulators could be otherwise used in co-cropping or intercropping practices. in this case, they will deposit their leaf litter on soil at the end of their growing season, and thus the soil will be enriched with se organic compounds available for the uptake by the neighboring crops. growing crops is soils naturally rich in se or irrigated with waters naturally abundant in se could be another se biofortification option to explore. this practice is known as "natural biofortification" and is gaining interest in certain areas worldwide (wu et al. ) . it has been realized in some regions of china (dinh et al. ) , usa (bañuelos et al. , and india (dhillon and dhillon ), but the number of studies conducted in the field environment is still limited so far, as most research has been performed under controlled and simplified environments, with homogeneous soil properties that do not resemble the real soil conditions of agroecosystems. one drawback of this practice is that agricultural use of seleniferous soils can hasten the release of se in the environment under certain conditions, particularly when the se gets concentrated by evapotranspiration, thus generating possible eco-toxicological hazard. biofortification by using nano-sized selenium in very recent years, emerging cutting-edge technologies have advised the application of se in the form of se nanoparticles (senps) in alternative to conventional se fertilizers for enriching crops with se-organic compounds (babajani et al. ; juárez-maldonado et al., ) . senps vary in shape and size and can be synthesized from se salts precursors, mainly selenite and selenate, in the presence of reducing agents (e.g. proteins, phenols, alcohols and amines) produced by bacteria, fungi and plant extracts (husen and siddiqi ; nayantara and kaur ) . the biogenic conversion of se salts to zerovalent senps by se specialist bacteria has been described in a number of studies (hunter and manter ; hunter ; ni et al. ), but a great task of scientists remains the synthesis of clonable senps. indeed, the effects of nanoparticles (nps) on plants, beyond depending on np concentration and mode of application (foliar, substrate, seeds), are strongly affected by the method handled for their synthesis, which is further responsible for np specific properties (size, shape). recently, pseudomonas moraviensis stanleyae, a bacterium isolated from the roots of the se hyperaccumulator stanleya pinnata, showed to tolerate extremely high se concentrations and produce intracellular senps, and its glutathione reductase enzyme has been proposed as a good candidate for the synthesis of clonable senps (ni et al. ) . harnessing senps as se-nanofertilizers holds the potential for synchronized se management in terms of release and uptake by crops, while preventing se losses in agroecosystems that may occur when commercial se fertilizers containing selenate and selenite salts are employed (babajani et al. ) . senps seem to be less toxic to plants than selenate and selenite salts, as reported in tobacco (nicotiana tabacum l.) (domokos-szabolcsy et al. ), garlic (allium sativum l.) and vigna radiata plants (bărbieru et al. ) . they can also improve the quality traits of vegetables, as described in strawberry (fragaria × ananassa) and tomato (solanum lycopersicon l.), whose fruits were more enriched in organic acids (e.g., malic, citric and succinic acids) and sugars (e.g. glucose, fructose and sucrose) after treatment with senps (morales-espinoza et al. ; zahedi et al. ). however, senps may induce toxicity at high levels (hussein et al. ) . for instance, in groundnut (arachis hypogaea l.) plants, low doses of senps improved yield components and seed oil, but high doses altered the protein profile and fatty acid composition by increasing unsaturated fatty acids and/or decreasing saturated fatty acids compared to untreated plants (hussein et al. ). d e s p i t e t h e a d v a n t a g e s o f f e r e d b y s eagronanotechnologies, insufficient literature exists on senps action in plants and their interactions with plant ecological partners, transformation in the food web and the environment. senps are largely used in pharmaceutical applications to increase the bioavailability of drugs and targeting therapeutic agents to specific organs, and are envisioned as next-generation safe ingredients for dietary supplements because they can gradually release se and better control targeted mechanisms of actions, with only few side effects (constantinescu-aruxandei et al. ) . though, the release of senps and their fate in the environment following biofortification may pose concerns, cause the lack of information about the impact of senps on public safety and their potential toxicity. it must be noted that senps are becoming emerging contaminants in some areas worldwide due to their intense use in electronics and material productions (rashid et al. ). therefore, to avoid any unpredictable health hazard from senps occurrence in the environment, further studies should systematically address senps risk assessment and management when used to agricultural purposes. selenium biofortification through conventional breeding is likely to be the most established, sustainable and long-term method of crop biofortification (white and broadley ). it is termed "genetic biofortification" and aims at selecting plant cultivars of high/moderate capacity to take up and translocate se to the edible parts, or having preferential uptake of organic se (semet and/ or metsecys) (bañuelos et al. ; broadley et al. ; wu et al. ; zhu et al. ). breeding of crops with high ability to accumulate se is a feasible practice because enough inter-and intraspecies genetic variation exists in grain se concentration of several cereal and leguminous crops, as well as in onion bulbs (allium cepa l.), brassicaceae spp., chicory (cichorium intybus l.), lettuce (lactuca sativa l), tomato (solanum lycopersicum l.) and pepper (capsicum annuum l.) fruits, and potato (solanum tuberosum l.) tubers (for specific references see review from white ). in this regard, it has been speculated that soil se phytoavailability and environmental factors are primary factors in controlling se concentration in edible parts, especially in cereal grains, and that genotypic variation of se concentration might become more relevant under high se phytoavailability (zhu et al. ). so far, a number of chromosomal loci (qtls) associated with high se accumulation in grains and leaves of several crops have been identified (ates et al. ; norton et al. ; wang et al. ; white ; zhang et al. ) . selecting plant cultivars of high se concentration in the edible products can be used in marker-assisted breeding (mab) to transfer these high-se qtls to high-yielding low se cultivars (pilon-smits and leduc ; wu et al. ) . one main limitation in the plant breeding, either conventional or marked assisted, is that it must be integrated by agronomic biofortification interventions using se fertilizers when crops are grown in low se areas. the advent of modern molecular tools and analytical technologies has brought to advances in research focusing on se biofortification for designing future and more effective strategies. analytical methods include synchrotron x-ray fluorescence and x-ray absorption near edge structure spectroscopies principally, while molecular technologies benefit from high-speed and low-cost of next generation sequencing (ngs), and encompass oligo-directed mutagenesis, reverse breeding, rnadirected dna methylation, and dna editing (carvalho and vasconcelos ) . such technologies, along with functional genomics gene technology, might be supplementary tools to breeding and genetic engineering (hung et al. ; pilon-smits and leduc ; wang et al. ). however, genetic engineering is far from being widespread and accepted compared to agronomic biofortification and conventional breeding because of imperative restrictions in the use of transgenics yet existing in many countries (zhu et al. ). a few transgenics suitable for biofortification have been generated so far, with increased capacity to acquire and accumulate se, preferentially in organic form, in the edible products (pilon-smits and leduc ; white and broadley ; zhu et al. ). most of these transgenics overexpress sulphate transporters, or enzymes that catalyze rate-limiting steps in se assimilation such as atp-sulphurylase, or are involved in mechanisms that prevent se misincorporation into proteins, like selenocysteine lyase and selenocysteine methyltransferase (zhu et al. ). other genes have been successfully targeted by genetic engineering in the last decade, with positive outcomes for se biofortification. for instance, the overexpression of the selenium binding protein gene sbp in arabidopsis thaliana enhanced the resistance of plants to selenite via a gsh-dependent mechanism (agalou et al. ) . similarly, the loss-of-function mutations in the gene apx coding for a cytosolic ascorbate peroxidase enzyme or the overexpression of the ethylene response factor erf improved se tolerance and accumulation in a. thaliana (jiang et al. (jiang et al. . in the se accumulator brassica juncea l., a novel selenocysteine methyltransferase enzyme has been identified, which is capable to methylate both homocysteine and secys substrates ). the overexpression of this enzyme in tobacco plants increased total se and mesecys accumulation . another potential gene target is the nrt . b transporter, a member of the rice peptide transporter (ptr) family involved in nitrate transport, because it displays semet transport activity and its overexpression in rice leads to higher semet accumulation in grains . new targets for genetic manipulation have also been identified in the se-hyperaccumulator stanleya pinnata, such as selenate/sulphate transporters ) and one isoform of atp-sulphurylase (aps ) ). s. pinnata owns a root high affinity sulphate/selenate transporter, sultr ; , which is constitutively highly expressed and does not undergo the canonical repression operated by high sulphate in nonhyperaccumulators wang et al. ). in addition, this hyperaccumulator shows elevate expression of the low affinity sulphate transporter sultr ; , which plays a role in sulphate/selenate root to shoot shuttling. both sultr transporters could be potential candidates of transgenic technologies for the generation of high se crops. the aps isoform of s. pinnata instead, is a quite unique and fascinating enzyme, as its expression exclusively localizes to the cytosol . normally, aps isoforms work in the plastids for s/se assimilation and the aps isoform from two se non-hyperaccumulators, a. thaliana and stanleya elata, has a dual localization, plastidial and cytosolic (bohrer et al. ; jiang et al. ) . the elevate expression of aps in s. pinnata might be responsible for its se hypertolerance. for this reason, aps function is currently under investigation. early and novel targets of genetic engineering are indicated in fig. . plants often face with adverse environmental constraints, which are mostly the result of anthropogenic activities and global climate changes (mall et al. ) . soil salinity, drought and heat stress in particular, have a negative impact on sustainable agricultural systems and severely impair crop yields, especially in arid and semi-arid regions where rainfalls are scarce. plants growing under such conditions suffer from osmotic stress along with oxidative stress due to increased production of reactive oxygen species (ros) (fahad et al. ) . in this regard, stressed crops might get benefits from se biofortification, se acting as a direct or indirect antioxidant (natasha et al. ) . overall, the beneficial effects triggered by se are primarily associated to its dose and the capacity of plants to accumulate and tolerate se (hawrylak-nowak ; ríos et al. ; schiavon et al. ) . at low doses se promotes the reduction in electrolytic leakage and the recovery of cell integrity under various stress conditions (zembala et al. ; malik et al. ) , and enhances the cell antioxidative capacity by stimulating the activity of antioxidant enzymes (superoxide dismutase, sod, catalase, cat, peroxidase, pod, ascorbate peroxidase, apx, monodehydroascorbate reductase, mdhar, dehydroascorbate reductase, dhar, glutathione peroxidase, gpx, glutathione reductase, gr, and glutathione-stransferase, gst) and the synthesis of antioxidant metabolites (e.g., ascorbic acid, asa, glutathione, gsh), as a mechanism for mitigating the effects of ros jiang et al. ; . indeed, at low levels ros mediate the signal transmission in the defense responses of cells under stress, but at high levels they injure cell components. therefore, the regulation of the antioxidant system by se may lead to the reduction of ros and, in this way, changes their role from detrimental to beneficial. in addition, low se has been reported to delay plant senescence (hajiboland et al. ) , which can be induced prematurely by stress conditions causing reduction of crop yields. in this respect, se modulates the levels of nitric oxide (no) in plants (hajiboland et al. ) , which in turn negatively regulates several elements (receptors, signal transduction proteins and/or transcription factors) involved in ethylene production and senescence processes (freschi ) . nevertheless, at high doses se provokes the synthesis of malformed proteins, reacts with thiol groups and gsh to induce ros over-generation (gosh and biswas ; natasha et al. ; van hoewyk ) , and disrupts reactive nitrogen species (rns) resulting in protein tyrosine nitration kolbert et al. ) . during high se condition, gsh can be possibly depleted because of se/s competition for uptake and assimilation, thereby enhancing ros accumulation (dall'acqua et al. ; van hoewyk ) . typical beneficial effects of se described in food crops growing in salinized environments generally include the improvement of agronomic traits (e.g. shoot length, shoot diameter, fresh and dry biomass) (diao et al. ; hawrylak-nowak ; jiang et al. ) , the increase of net photosynthesis and water contents (diao et al. ; habibi ; hawrylak-nowak ; jiang et al. ) , the stimulation of antioxidant processes and accumulation of non-enzymatic antioxidants (asa gsh, phenolic compounds), accumulation of osmolytes (proline, sugars and k + ), the reduction of lipid peroxidation and ros concentration in cells, and the regulation of na + homeostasis through increased expression of genes encoding na + /h + antiporters (nhx), as reported in maize (zmnhx ) and rice (osnhx ), which results in enhanced sequestration of na + in the root vacuoles to prevent na + delivery to the shoot (ashraf et al. ; elkelish et al. ; habibi ; hawrylak-nowak ; kamran et al. ; jiang et al. ; subramanyam et al. ) . under certain circumstances, the positive action of se in salt stress physiology could be intensified by giving this element along with silicon (si) (sattar et al. ). in plants suffering from drought stress or heat stress, se fertilization may sustain crop yields by alleviating rosinduced damages via activation of cellular antioxidant systems (d'amato et al. a; iqbal et al. ; jóźwiak and politycka ; malerba and cerana ; sajedi et al. ) . increased activity of antioxidant enzymes and reduced lipid peroxidation have been reported in wheat, maize and cucumber plants grown under water deficit (nawaz et al. ; yao et al. ) and in wheat plants subjected to heat stress (iqbal et al. ; jóźwiak and politycka ) . foliar se application increased the relative water content, pigment and free amino acid accumulation, and the agronomic traits (e.g. fodder yield, crude protein, fiber, nitrogen free extract) of water-stressed maize plants (nawaz et al. ) , and promoted gas exchanges, fe uptake and accumulation of osmoprotectants (total soluble sugars and free amino acids) in wheat, thus ultimately increasing grain yield (nawaz et al. (nawaz et al. . recently, high accumulation of osmolytes like proline and k + , and intense n metabolism have been described in maize plants cultivated in soil fertilized with se and poorly water-irrigated (bocchini et al. ) . epigenetic changes in dna methylation in these plants caused by simultaneous water deficit and se fertilization indicated that se could up-regulate the expression of a number of genes implied in the plant tolerance to environmental stresses, like those coding for phytoene synthase (psy), important enzyme for the preservation of cell carotenoids, sorbitol dehydrogenase (sdh), which controls osmolyte biosynthesis under drought stress, and alcohol dehydrogenase (adh), functioning in plant adaptation to abiotic stress. selenium biofortification is also efficient in ameliorating the nutritional quality of vegetable products derived from plants grown under drought stress. for instance, olives produced by olive trees subjected to water shortage hold higher fresh weight and increased the level of phenols and pigments at harvest (d'amato et al. a) . furthermore, the extra virgin olive oil (evoo) obtained from these olives was of superior nutritional value due to the longer shelf life and improved oxidative stability against oxidation processes. in addition to the positive effects elicited by se in plants subjected to abiotic stress, se supplementation has been reported to protect plants from biotic stress caused by pathogenic fungi (kornas et al. ; xu et al. ) and a variety of generalist invertebrates and herbivores (freeman et al. (freeman et al. quinn et al. ; valdez barillas et al. ) , suggesting that crops enriched with se may require less synthetic fungicides and pesticides during their cultivation. therefore, besides being applied to crops to address malnutrition in vulnerable populations, se might be used to attain agroecological plant protection and sustainable use of agrochemicals. alternatively, se could be employed as an ecological insecticide (mechora ) or fungicide (wu et al. ; xu et al. ) . in this respect, wu et al. ( ) proposed the use of se in soil for the control of the postharvest disease of fruits and vegetables caused by penicillium expansum, while liu et al. ( ) and xu et al. ( ) endorsed the use of se for the control of the oilseed rape disease caused by sclerotinia sclerotiorum. effects of se-biofortification on accumulation of se species beyond achieving increases of total se concentration in crops, one main task of se biofortification is to enrich plants with se compounds that provide health benefits to humans and animals (fig. ) (newman et al. ) . secys and semet are important precursors for the synthesis of mammalian selenoproteins, function as direct antioxidants in cells due to their easy oxidation and have a major role in protein repair (rahmanto and davies ) . furthermore, semet and other organic se compounds like methylseleninic acid (msa), methylselenol (mse), γ-glutamyl-selenium-methylselenocysteine (γ-glumesecys) and mesecys, have recognized efficacy in chemoprevention (spallholz ; tarrado-castellarnau et al. ; vinceti et al. ) . crop species and varieties differ in the ability to convert inorganic se into organic forms. cereal crops in particular, can store se in seeds mainly in the form of semet , whose translocation in rice is apparently promoted by the nrt . effects of se-biofortification on se-s crosstalk and accumulation of s-metabolites one common observation from biofortification studies is that the content of inorganic or organic se species tendentially builds up in crops fertilized with se, while the amount of s-containing phytochemicals with functional roles in plant defense mechanisms and human health, such as glucosinolates (gls), glutathione and s amino acids, might undergo variation depending on se dose and species to be used, plant species and genotype, and mode of se fertilization (foliar fertilization or soil amendment with se) (bachiega et al. ; robbins et al. ; schiavon et al. ; schiavon and pilon-smits b) . gls and their hydrolysis products (isothiocyanates, itc) in particular, are recognized key molecules in cancer prevention, and have received great attention in biofortification studies of brassicaceae spp. (melrose ; prieto et al. ) . increased accumulation of itc has been recently observed in kale (brassica oleracea l.) roots in response to selenite and nacl joint application, thus making this food crop superior for chemopreventive effects (kim et al. ) . the impairment of s metabolites has been frequently observed in plants supplemented with high selenate or selenite doses. this is likely because se and s compete for the uptake and metabolic processes in plants (mckenzie et al. ; schiavon and pilon-smits a; tian et al. tian et al. . negative effects of se/s competition on the synthesis of gsh and gls precursor amino acids, and on the expression of genes within the gls pathway have been largely described (dall'acqua et al. ; mckenzie et al. ; schiavon et al. ; tian et al. ) . methionine (met) is the precursor amino acid for the synthesis of aliphatic gls, and the decrease of its content in response to se fertilization has been associated with lower accumulation of metderived-gls in salad rocket (eruca sativa mill.), radish (raphanus sativus l.) and other brassicaceae spp. (dall'acqua et al. ; schiavon et al. ). in broccoli, discrepant results have been reported. barickman et al. ( ) and robbins et al. ( ) noted the decline of aliphatic gls and sulforaphane, a gls hydrolysis sulphur-containing aglycon byproduct, in broccoli supplied with high se. in contrast, sepúlveda et al. ( ) did not find any significant change in gls and sulforaphane content, neither in myrosinase activity, in broccoli treated with high se ( µm selenate). similarly, tian et al. ( ) did not measure any appreciable variation of gls content in broccoli sprouts exposed to µm selenite or selenate, and hypothesized an equilibrium between gls biosynthesis and hydrolysis. differential effects of se application on the synthesis of s-metabolites can be observed between distinct plant species of the same genus, as recently reported in two rocket species, namely salad rocket and wild rocket (diplotaxis tenuifolia) (dall'acqua et al. ). rocket salad plants treated with and µm selenate exhibited reduced capacity to accumulate s amino acids, gsh and gls, mainly because of se/s competition processes and repression of genes involved in s reduction and gls synthesis and hydrolysis. in contrast, wild rocket plants supplied with equal selenate concentrations accumulated more se, without significant effects on s uptake and metabolism. interestingly, wild rocket contained large levels of osmolytes (proline) in response to increasing se application, likely to prevent the oxidative stress that is often generated by high se in tissues. beside s-glucosinolates, brassicaceae spp. supplem e n t e d w i t h s e c a n p r o d u c e (methylseleno)glucosinolates and their se-containing aglycons (matich et al. (matich et al. mckenzie et al. ) , whose bioactivity as anticancer and antimicrobial agents is considered higher compared to gls (emmert et al. ) . the majority of se-gsl identified so far are aliphatic, with se in the position of the s donated by met and not in the sulphate group or on the bond with glucose (matich et al. (matich et al. . the seglucosinolates -(methylseleno)propyl glucosinolate, -( m e t h y l s e l e n o ) b u t y l g l u c o s i n o l a t e , -( m e t h y l s e l e n o ) p e n t y l g l u c o s i n o l a t e , -(methylseleninyl)butyl glucosinolate and seleno- phenylethyl glucosinolate have been described in broccoli, cauliflower and forage rape (matich et al. (matich et al. , while -(methylseleno)but- -enyl glucosinolate a n d r e l a t e d i s o t h i o c y a n a t e s ( i s o m e r s o f -(methylseleno)but- -enyl isothiocyanate) have been tentatively identified in radish plants (mckenzie et al. ). in se-enriched radish and broccoli, the upregulation of the gene coding for aps kinase (apsk), the key branch point enzyme of gsl biosynthesis, was observed and explained as a protective mechanism activated by plants to control s uptake and limit se-gls production (mckenzie et al. (mckenzie et al. . the repression of genes encoding enzymes promoting aliphatic gls biosynthesis and the concomitant up-regulation of genes coding for enzymes involved in indole gls generation were also hypothesized as part of the same protective mechanism that aims to generate indole glss over aliphatic gls to restrict the synthesis of se-gls (mckenzie et al. ). effects of se-biofortification on accumulation of other health beneficial phytochemicals at low doses se is beneficial to plants and typically improves the antioxidative capacity, increases the content of nutrient elements and improves the profile of worthwhile phytochemicals. thus, high se food represents a valuable tool to improve the diet, nutritional quality and health in world population. very recently, d'amato et al. ( ) surveyed the impact of se biofortification on the nutraceutical profile of food crops. the authors grouped them by crop category (arable crops, vegetables, microscale vegetables, and fruit trees) and highlighted the foremost effects of se biofortification on the content of total se, inorganic/ organic se species, and bioactive compounds, which are summarized and implemented in fig. . with respect to arable crops, the enrichment with se mostly leads to higher antioxidant activity of their grains and greater content of nutrients, amino acids, phenols, anthocyanins, sugars, and organo-se compounds (d'amato et al. (d'amato et al. skrypnik et al. ) . also, in upland rice polished grains, se application induced higher concentration of storage proteins like albumin, globulin, prolamin, and glutelin (reis et al. ). in horticultural vegetables, se fertilization is ascertained to augment the antioxidant activity, nitrogen and s assimilation, accumulation of pigments (carotenoids and anthocyanins), soluble phenols, and ascorbic acid (dall'acqua et al. ; hawrylak-nowak et al. mimmo et al. ; schiavon et al. schiavon et al. ríos et al. ) . enhanced production of essential oils, hydroxycinnamic acids, total phenolics, anthocyanin and antioxidant activity has been recently reported by skrypnik et al. ( ) in sweet basil (ocimum basilicum l.) leaves, while increased accumulation of flavonoid compounds, especially naringenin chalcone and kaempferol, and several amino acids except proline, was observed in se-biofortified tomato fruits and radish roots, respectively (schiavon et al. (schiavon et al. . seedlings of edible vegetables supplemented with selenate seem to preferentially accumulate organic se, and their nutritional value is generally ameliorated by se enrichment (d'amato et al. b (d'amato et al. islam et al. ; pannico et al. ). in fact, Ávila et al. ( ) screened se-enriched cultivars from the six largely consumed brassica vegetables and found that all were able to accumulate mesecys and contained higher levels of gls, especially glucoraphanin. increased accumulation of several bioactive compounds (carotenoids, phenolics, flavonoids, ascorbic acid, and anthocyanins) was observed in se-biofortified wheat microgreens (islam et al. ) . similarly, se fertilization induced larger accumulation of total phenolics in coriander (coriandrum sativum l.), green basil, purple basil, and tatsoi (brassica narinosa l.) microgreens grown in soilless cultivation (pannico et al. ) , while seenriched chickpea sprouts were proved to be a valuable source of oil characterized by high oxidative stability index (osi) and cellular antioxidant activity (caa), due to reduced lipase and lipoxygenase (lox) activities and increased content in phenolics, respectively (guardado-félix et al. ) . interestingly, puccinelli et al. ( ) suggested the production of se-enriched sprouts from seeds collected from plants fertilized with se as a novel biofortification approach. little information is available about the effects of se on fruit trees (d'amato et al. ) . in the few studies accomplished in this area of research, spraying leaves or fruits with se was the only approach used to enrich fruits (e.g. pears, peach, apples, olives) with se (d' amato et al. amato et al. a deng et al. ; pezzarossa et al. ) . se foliar fertilization of olive trees increased the accumulation of several nutritional components of olives and evoo, such as mineral elements, pigments (carotenoids and chlorophylls) and phenolic compounds (e.g. oleacein, ligustroside aglycone, and oleocanthal) (d'amato et al. (d'amato et al. a . such results indicate that se fertilization might be used to increase the nutritional value of evoos in order to meet the quality standards required by the european food safety authority (efsa) (d'amato et al. ) . selenium deficiency in humans is associated with a variety of pathological conditions, including a cardiomyopathy endemic in china termed keshan disease, reduced male fertility, mood disorders, and disturbance of thyroid functions (rayman (rayman . the se nutritional status of individuals also impacts on the immune system defenses activated to combat viral infections and on the viral pathogen itself (guillin et al. ; harthill ). if se level in blood is below µm, the metabolic oxidative stress generated in the host cells due to decreased activity of selenoproteins, especially glutathione peroxidases (gpx), may lower the immune system and trigger genome mutations in the parasitic virus that generally change the virus from being benign or mildly pathogenic to become highly virulent (beck et al. ; nelson et al. ) . such rna viral mutations are faster and long-lived in se-deficient than in healthy individuals (harthill ) . se-deficient infected hosts get benefits from se supplementation, which result in decrease of virus mutation frequency and load, and enhanced immune competence with better outcomes for viral infections (beck et al. ; harthill ). this has been corroborated for at least influenza virus type a and coxsackievirus b (cvb ), human immunodeficiency virus/acquired immunodeficiency syndrome (hiv/aids) (beck et al. ; broome et al. ; kupka et al. ; nelson et al. ) . nonetheless, mutated virulent rna viruses persist as pathogenic and infectious parasites in the hosts and can be transferred to individuals with se-sufficient status (harthill ) . very recently, an epidemic caused by a novel coronavirus (covid- or -cov) belonging to the same group of β-coronaviruses as severe acute respiratory syndrome (sars) in and middle east respiratory syndrome (mers) (cohen and normile ) , is spreading worldwide while threating human health and threatening the world economy. so far, no pharmacological treatment or vaccine exist. in any case, pharmaceutical drugs are usually ineffective against rna viruses (lyons ) , while vaccines against coronaviruses carry an unacceptable risk of paradoxical immune enhancement (tirado and yoon ; bolles et al. ; ricke and malone ) therefore, alternative or supplementary natural treatments should be considered to reduce the viral load in the hosts and enhance their immune system. similar to zn, se supplementation to covid- infected people with low se blood levels could be an option to explore as a natural treatment of this virus (zhang and liu ) . in this context, se biofortification programs attain incremental importance, as se enriched food crop is higher in nutritional components compared to se intake via tablets, and thus provides more benefits to infected consumers. if the food is additionally enriched with zn and fe besides se, its potential in contrasting viral infection could be significantly increased. the element se may serve in different agrotechnological applications. among them, agronomic biofortification through se fertilizers and conventional breeding are likely the most widespread and accepted methods to combat the se deficiency worldwide. circular systems where se phytoremediation is combined with biofortification strategies and genetic engineering of crops, although promising in the context of sustainable agriculture, are still poorly developed because of some practical limitations. in contrast, nanosized se biofortification is increasingly gaining attention from scientists. further investigation is needed to understand if the use of senps is safe to consumers, and which chemical modifications they might undergo in the environment and during food processing. studies on crop biofortification with multiple micronutrients and the use of pgpr in agronomic biofortification are only at the beginning, but might represent a future and promising area of se biofortification research. selenium biofortification primarily aims to enrich crops with se-species and antioxidant compounds with positive impact on human (and animal) nutrition and health. the role of se-enriched food in preventing or contrasting viral infections in particular, is of great relevance in the recent scenario of a viral disease pandemic and represents a field of research that deserves a broad investigation. se biofortification could also be used to increase crop yield under sub-optimal conditions, mitigating the negative effects of such environments on plant physiology, while increasing plant antioxidant properties and content in healthy phytochemicals. on this account, studies attempting optimization of se biofortification strategies for improving food crop nutritional under challenging environments are of great interest on a global scale. open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creativecommons.org/licenses/by/ . /. alfthan g, eurola m, ekholm p, venäläinen e-r, root t, korkalainen k, hartikainen h, salminen p, hietaniemi v, aspila p, aro a, for the selenium working group ( ) effects of nationwide addition of selenium to fertilizers on foods, and animal and human health in finland. from deficiency to optimal selenium status of the population. developing selenium-enriched animal feed and biofuel from canola planted for managing se-laden drainage waters in the westside of central california selenium biofortification of broccoli and carrots grown in soil amended with se-enriched hyperaccumulator stanleya pinnata selenium in plants: molecular, physiological, ecological and evolutionary aspects accumulation and speciation of selenium in biofortified vegetables grown under high boron and saline field conditions plant biostimulants based on selenium nanoparticles biosynthesized by trichoderma strains selenium influences glucosinolate and isothiocyanates and increases sulfur uptake in arabidopsis thaliana and rapid-cycling brassica oleracea rapid genomic evolution of a non-virulent coxsackievirus b in selenium-deficient mice results in selection of identical virulent isolates nutritionally induced oxidative stress: effect on viral disease selenium deficiency and viral infection soil selenium (se) biofortification changes the physiological, biochemical and epigenetic responses to water stress in zea mays l. by inducing a higher drought tolerance alternative translational initiation of atp sulfurylase underlying dual localization of sulfate assimilation pathways in plastids and cytosol in arabidopsis thaliana a double-inactivated severe acute respiratory syndrome coronavirus vaccine provides incomplete protection in mice and induces increased eosinophilic plant soil proinflammatory pulmonary response upon challenge selenium stories biofortification of uk food crops with selenium selenium biofortification of highyielding winter wheat (triticum aestivum l.) by liquid or granular se fertilisation an increase in selenium intake improves immune function and poliovirus handling in adults with marginal selenium status enrichment of cereal grains with zinc: agronomic or genetic biofortification? fate and bioaccessibility of iodine in food prepared from agronomically biofortified wheat and rice and impact of cofertilization with zinc and selenium producing more with less: strategies and novel technologies for plant-based food biofortification selenium speciation profiles in selenite-enriched soybean (glycine max) by hplc-icpms and esi-itms understanding selenium metabolism in plants and its role as a beneficial element identification and functional characterization of a novel selenocysteine methyltransferase from brassica juncea l agronomic biofortification of maize with selenium (se) in malawi sources of heavy metals and metalloids in soils. in: alloway bj (ed) heavy metals in soils: trace elements and metalloids in soils and their bioavailability new sars-like virus in china triggers alarm selenium in global food systems selenium analysis and speciation in dietary supplements based on next-generation selenium ingredients butler selenium concentration in wheat (triticum aestivum) grain as influenced by method, rate, and timing of sodium selenate application biofortification (se): does it increase the content of phenolic compounds in virgin olive oil (voo)? the selenium supplementation influences olive tree production and oil stability against oxidation and can alleviate the water deficiency effects selenium biofortification in rice (oryza sativa l.) sprouting: effects on se yield and nutritional traits with focus on phenolic acid profile zea mays l. grain: increase in nutraceutical and antioxidant properties due to se fortification in low and high water regimes current knowledge on selenium biofortification to improve the nutraceutical profile of food: a comprehensive review selenium biofortification differentially affects sulfur metabolism and accumulation of phytochemicals in two rocket species (eruca sativa mill. and diplotaxis tenuifolia) grown in hydroponics selenium supplementation and cancer prevention fate of selenium in soil: a case study in a maize (zea mays l.) field under two irrigation regimes and fertilized with sodium selenite selenium uptake and fruit quality of pear (pyrus communis l.) treated with foliar se application phytoremediation of seleniumcontaminated soils: the efficiency of different cropping plant soil systems selenium promotes the growth and photosynthesis of tomato seedlings under salt stress by enhancing chloroplast antioxidant defense system selenium distribution in the chinese environment and its relationship with human health: a review accumulation of red elemental selenium nanoparticles and their biological effects in nicotiana tabacum overview of selenium deficiency and toxicity worldwide: affected areas, selenium-related health issues, and case studies selenium speciation in wheat grain varies in the presence of nitrogen and sulphur fertilisers enhanced selenium content in wheat grain by co-inoculation of selenobacteria and arbuscular mycorrhizal fungi: a preliminary study as a potential se biofortification strategy en-dophytic bacteria from seleniumsupplemented wheat plants could be useful for plant-growth promotion, biofortification and gaeumanno-mycesgraminis biocontrol in wheat production plant availability of inorganic and organic selenium fertiliser as influenced by soil organic matter content and ph influence of sulfate supply on selenium uptake dynamics and expression of sulfate/ selenate transporters in selenium hyperaccumulator and nonhyperaccumulator brassicaceae selenium protects wheat seedlings against salt stressmediated oxidative damage by up-regulating antioxidants and osmolytes metabolism enhanced nrf -dependent induction of glutathione in mouse embryonic fibroblasts by isoselenocyanate analog of sulforaphane crop production under drought and heat stress: plant responses and management options selenium accumulation protects plants from herbivory by orthoptera via toxicity and deterrence selenium protects the hyperaccumulator stanleya pinnata against black-tailed prairie dog herbivory in native seleniferous habitats selenium deficiency and toxicity in the environment. in: selinus o (ed) essentials of medical geology nitric oxide and phytohormone interactions: current status and perspectives biofortified crops generated by breeding, agronomy, and transgenic approaches are improving lives of millions of people around the world biofortification with selenium and iodine changes morphological properties of brassica oleracea l. var. gongylodes) and increases their contents in tubers effect of selenium biofortification and beneficial microorganism inoculation on yield, quality and antioxidant properties of shallot bulbs yield, quality and antioxidant properties of indian mustard (brassica juncea l.) in response to foliar biofortification with selenium and iodine selenium modulates growth and thiol metabolism in wheat (triticum aestivum l.) during arsenic stress chickpea (cicer arietinum l.) sprouts containing supranutritional levels of selenium decrease tumor growth of colon cancer cells xenografted in immune-suppressed mice selenium, selenoproteins and viral infection plant soil selenium in soils and crops, its deficiencies in livestock and humans: implications for management an overview of selenium uptake, metabolism, and toxicity in plants physiological, photochemical and ionic responses of sunflower seedlings to exogenous selenium supply under salt stress the effects of selenate and sulphate supply on the accumulation and volatilization of se by cabbage, kohlrabi and alfalfa plants grown hydroponically senescence is delayed by selenium in oilseed rape plants review: micronutrient selenium deficiency influences evolution of some viral infectious diseases biogeochemistry of selenium and its impact on food chain quality and human health selenium pretreatment upregulates the antioxidant defense and methylglyoxal detoxification system and confers enhanced tolerance to drought stress in rapeseed seedlings seleniuminduced up-regulation of the antioxidant defense and methylglyoxal detoxification system reduces salinityinduced damage in rapeseed seedlings how to use the world's scarce selenium resources efficiently to increase the selenium concentration in food enhanced selenium content in sweet basil (ocimum basilicum l.) by foliar fertilization beneficial effects of exogenous selenium in cucumber seedlings subjected to salt stress comparative effects of selenite and selenate on growth and selenium accumulation in lettuce plants under hydroponic conditions selenium biofortification enhances the growth and alters the physiological response of lamb's lettuce grown under high temperature stress selenium contamination, consequences and remediation techniques in water and soils: a review characteristics of time-dependent selenium biofortification of rice (oryza sativa l.) identification and characterization of selenate-and selenite-responsive genes in a se-hyperaccumulator astragalus racemosus bio-reduction of selenite to elemental red selenium by tetrathiobacter kashmirensis rhizobium seleniti reducens protein showing selenite reductase activity plants and microbes assisted selenium nanoparticles: characterization and application evaluation of cytotoxicity, biochemical profile and yield components of groundnut plants treated with nano-selenium exogenously applied selenium reduces oxidative stress and induces heat tolerance in spring wheat influence of selenium biofortification on the bioactive compounds and antioxidant activity of wheat microgreen extract biofortification of pulse crops: status and future perspectives effect of exogenous selenium supply on photosynthesis, na + accumulation and antioxidative capacity of maize (zea mays l.) under salinity stress loss-of-function mutations in the apx gene result in enhanced selenium tolerance in arabidopsis thaliana overexpression of ethylene response factor erf gene enhances selenium tolerance in arabidopsis comparison of atp sulfurylase from selenium hyperaccumulator stanleya pinnata and non-accumulator stanleya elata reveals differential intracellular localization and enzyme activity levels selenium deficiency risk predicted to increase under future climate change effect of selenium on alleviating oxidative stress caused by a water deficit in cucumber roots can selenium deficiency in malawi be alleviated through consumption of agro-biofortified maize flour? study protocol for a randomised, double-blind, controlled trial nanoparticles and nanomaterials as plant biostimulants an overview of hazardous impacts of soil salinity in crops, tolerance mechanisms, and amelioration through selenium supplementation selenium-fascinating microelement, properties and sources in food selenium accumulation in durum wheat and spring canola as a function of amending soils with selenite, selenate and or sulphate exposure of kale root to nacl and na seo increases isothiocyanate levels and nrf signalling without reducing plant root growth nitro-oxidative stress correlates with se tolerance of astragalus species foliar application of selenium for protection against the first stages of mycotoxin infection of crop plant leaves selenium status is associated with accelerated hiv disease progression among hiv- -infected pregnant women in tanzania a comparative study on the accumulation, translocation and transformation of selenite, selenate, and senps in a hydroponic-plant system mechanisms of selenium hyperaccumulation in plants: a survey of molecular, biochemical and ecological cues m hyperaccumulator stanleya pinnata and related nonaccumulator stanleya elata with hyperaccumulator rhizosphere fungi-investigation of effects on se accumulation and speciation selenium (se) reduces sclerotinia stem rot disease incidence of oilseed rape by increasing plant se concentration and shifting soil microbial community and functional profiles selenium in cereals: improving the efficiency of agronomic biofortification in the uk selenium and selenoproteins: an overview on different biological systems effect of selenium on the responses induced by heat stress in plant cell cultures selenium antagonizes the toxic effects of arsenic on mungbean (phaseolus aureus roxb.) plants by restricting its uptake and enhancing the antioxidative and detoxification mechanisms effect of climate change on agricultural crops. crop modification, nutrition, and food production using agronomic biofortification to boost zinc, selenium, and iodine concentrations of food crops grown on the loess plateau in china selenoglucosinolates and their metabolites produced in brassica spp. fertilised with sodium selenate distribution of selenoglucosinolates and their metabolites in brassica treated with sodium selenate selenium treatment differentially affects sulfur metabolism in high and low glucosinolate producing cultivars of broccoli selenium application during radish (raphanus sativus) plant development alters glucosinolate metabolic gene expression and results in the production of -(methylseleno)but- -enyl glucosinolate selenium as a protective agent against pests: a review. plants (basel) selenium in the environment, metabolism and involvement in body functions the glucosinolates: a sulphur glucoside family of mustard anti-tumour and antimicrobial phytochemicals of potential therapeutic application food system strategies for preventing micronutrient malnutrition selenium biofortification in fragaria × ananassa: implications on strawberry fruits quality, content of bioactive health beneficial compounds and metabolomic profile improving selenium status in plant nutrition and quality se nanoparticles induce changes in the growth, antioxidant responses, and fruit quality of tomato developed under nacl stress a critical review of selenium biogeochemical behavior in soil-plant system with an inference to human health supplemental selenium improves wheat grain yield and quality through alterations in biochemical processes under normal and water deficit conditions selenium supplementation affects physiological and biochemical processes to improve fodder yield and quality of maize (zea mays l.) under water deficit conditions biosynthesis of nanoparticles using ecofriendly factories and their role in plant pathogenicity: a review host nutritional selenium status as a driving force for influenza virus mutations selenium biofortification of agricultural crops and effects on plant nutrients and bioactive compounds important for human health and disease prevention -a review genetic mapping of the rice ionome in leaves and grain: identification of qtls for elements including arsenic, cadmium, iron and selenium kesterson reservoir: years of selenium risk assessment and management selenium biofortification impacts the nutritive value, polyphenolic content, and bioactive constitution of variable microgreens genotypes effects of foliar and fruit addition of sodium selenate on selenium accumulation and fruit quality phytoremediation of selenium using transgenic plants physiological functions of beneficial elements on the ecology of selenium accumulation in plants. plants (basel) selenium accumulation and speciation in biofortified chickpea (cicer arietinum l.) under mediterranean conditions selenate-enriched urea granules are a highly effective fertilizer for selenium biofortification of paddy rice grain glucosinolates: molecular structure, breakdown, genetic, bioavailability, properties and healthy and adverse effects production of selenium-biofortified microgreens from selenium-enriched seeds of basil selenium hyperaccumulation offers protection from cell disruptor herbivores selenium-containing amino acids as direct and indirect antioxidants improving the efficacy of selenium fertilizers for wheat biofortification evaluation of genotypic variation of broccoli (brassica oleracea var. italic) in response to selenium treatment partitioning of senps in the water soluble and the exchangeable fractions and effects of soil organic matter and incubation time selenium and human health selenium intake, status, and health: a complex relationship dos reis ar ( ) agronomic biofortification with selenium impacts storage proteins in grains of upland rice medical countermeasures analysis of -ncov and vaccine risks for antibody-dependent enhancement (ade). preprints response of nitrogen metabolism in lettuce plants subjected to different doses and forms of selenium cultivation conditions and selenium fertilization alter the phenolic profile, glucosinolate, and sulforaphane content of broccoli selenium fertilization strategies for bio-fortification of food: an agro-ecosystem approach malformed selenoproteins are removed by the ubiquitin-proteasome pathway in stanleya pinnata selenium in the soil-plant environment: a review the effects of selenium and other micronutrients on the antioxidant activities and yield of corn (zea mays l.) under drought stress separate and combined effects of silicon and selenium on salt tolerance of wheat plants selenium fertilization alters the chemical composition and antioxidant constituents of tomato (solanum lycopersicon l.) selenium biofortification in radish enhances nutritional quality via accumulation of methyl-selenocysteine and promotion of transcripts and metabolites related to glucosinolates, phenolics, and amino acids the fascinating facets of plant selenium accumulation -biochemistry, physiology, evolution and ecology selenium biofortification and phytoremediation phytotechnologies: a review changes in semsc, glucosinolates and sulforaphane levels, and in proteome profile in broccoli (brassica oleracea var. italica) fertilized with sodium selenate a critical review of selenium biogeochemical behavior in soil-plant system with an inference to human health the response of broccoli (brassica oleracea convar. italica) varieties on foliar application of selenium: uptake, translocation, and speciation improvement of phenolic compounds, essential oil content and antioxidant properties of sweet basil (ocimum basilicum l.) depending on type and concentration of selenium application selenium uptake, translocation, assimilation and metabolic fate in plants selenomethionine and methioninase: selenium free radical anticancer activity selenium uptake and distribution in chicory (cichorium intybus l.) grown in an aeroponic system selenium metabolism in hemp (cannabis sativa l.)-potential for phytoremediation and biofortification soil factors affecting selenium concentration in wheat grain and the fate and speciation of se fertilisers applied to soil sodium selenate treatment using a combination of seed priming and foliar spray alleviates salinity stress in rice selenium in soil and endemic diseases in china selenium species: current status and potentials in cancer prevention and therapy methylseleninic acid promotes antitumour effects via nuclear foxo a translocation through akt inhibition effect of se treatment on glucosinolate metabolism and health-promoting compounds in the broccoli sprouts of three cultivars effects of selenium supplementation on glucosinolate biosynthesis in broccoli antibody-dependent enhancement of virus infection and disease usda national nutrient database for standard reference, release selenoproteins in human body: focus on thyroid pathophysiology selenium accumulation in plants-phytotechnological applications and ecological implications a tale of two toxicities: malformed selenoproteins and oxidative stress both contribute to selenium stress in plants environmental selenium and human health: an update advances in seleniumenriched foods: from the farm to the fork qtl mapping of selenium content using a ril population in wheat transcriptome-wide comparison of selenium hyperaccumulator and nonaccumulator stanleya species provides new insight into key processes mediating the hyperaccumulation syndrome effect of phosphate and silicate on selenite uptake and phloem-mediated transport in tomato (solanum lycopersicum l.) reviews on atmospheric selenium: emissions, speciation and fate interactions between selenium and sulphur nutrition in arabidopsis thaliana biofortification of crops with seven mineral elements often lacking in human diets-iron, zinc, copper, calcium, magnesium, selenium and iodine selenium accumulation by plants selenium metabolism in plants global health risks: mortality and burden of disease attributable to selected major risks selenium cycling across soil-plant-plant soil atmosphere interfaces: a critical review inhibitory effect of selenium against penicillium expansum and its possible mechanisms of action biofortification and phytoremediation of selenium in china selenium as a potential fungicide could protect oilseed rape leaves from sclerotinia sclerotiorum infection effects of selenium on wheat seedlings under drought stress seleniferous soils as a source for production of selenium-enriched foods and potential of bacteria to enhance plant selenium uptake microbial-enhanced selenium and iron biofortification of wheat (triticum aestivum l.): applications in phytoremediation and biofortification alleviation of the effect of salinity on growth and yield of strawberry by foliar spray of selenium-nanoparticles effect of selenium on macro and microelement distribution and physiological parameters of rape and wheat seedlings exposed to cadmium stress mapping quantitative trait loci associated with selenate tolerance in arabidopsis thaliana ospt , a phosphate transporter, is involved in the active uptake of selenite in rice ) nrt . b improves selenium concentrations in rice grains by facilitating selenomethinone translocation potential interventions for novel coronavirus in china: a systematic review biofortification and phytoremediation the role of the rice aquaporin lsi in arsenite efflux from roots involvement of silicon influx transporter osnip ; in selenite uptake in rice selenium in higher plants: understanding mechanisms for biofortification and phytoremediation simultaneous biofortification of wheat with zinc, iodine, selenium, and iron through foliar treatment of a micronutrient cocktail in six countries acknowledgements the authors thank prof. elizabeth pilon- key: cord- -lv fy e authors: dávalos, alberto; henriques, rossana; latasa, maría jesús; laparra, moisés; coca, maría title: literature review of baseline information on non‐coding rna (ncrna) to support the risk assessment of ncrna‐based genetically modified plants for food and feed date: - - journal: nan doi: . /sp.efsa. .en- sha: doc_id: cord_uid: lv fy e this report is the outcome of an efsa procurement (np/efsa/gmo/ / ) reviewing relevant scientific information on ncrna and on rna interference(rnai) that could support the food and feed risk assessment of ncrna‐based genetically modified (gm) plants. information was retrieved through key words and key questions covering the stability and degradation of ncrnas after oral ingestion, the passage of ncrnas from food and feed to human and animal organs and tissues via the gastrointestinal tract and other barriers, as well as the potential effects on the gastrointestinal tract, the immune system or the entire organism.full description of the strategy used for the literature search and for studies selectionis provided and the number of retrieved publications is reported. this report is divided into four partsdiscussing the kinetics of exogenous ncrnas in humans and animals, with focus on ingested ncrnas (part ); the possible effects of ncrnas on the gastrointestinal tract (part ), systemically(part )and on the immune system (part ). this report suggests that some plant ncrnas (e.g mirnas and sirnas) show higher stability as compared to other ncrnas due to peculiar chemical characteristics ( ’‐o‐methylation at ’ end).however, ingested or administered ncrna must overcome many extracellular and cellular barriers to reach the intended target tissue or functional location in sufficient amount to exert any biological effect. literature data indicate that chemically unmodified and unformulated ncrnas exhibit very low stability in the gastrointestinal tract and in biological fluids and, in general, do not elicit major biological effects.this report also provides an overview of the rna content in plant‐derived foods and diets and discusses the controversies on the presence of dietary exogenous rnas in the biological fluids of humans and animals and their effects. finally, gaps in the scientific literature are highlighted and recommendations provided . introduction this scientific report is the result of a contract awarded by the european food safety authority (efsa) to the madrid institute of advanced studies (imdea)-food, under the contract title: "literature review of baseline information on non-coding rna (ncrna) that could support the food/feed risk assessment of ncrna-based gm plants" (contract number np/efsa/gmo/ / ). for the literature review of the available scientific information on foreign (exogenous) ncrnas that could support the risk assessment of ncrna-based gm plants, the following tasks were defined by efsa. based on available scientific literature (narrative review) to review the kinetics profile of foreign (exogenous) ncrna in humans and animals (experimental animals, livestock and pets). this to be based on information from research and development of ncrnas intended to be used as therapeutics and/or from research on biomarkers in humans (pharmaceuticals/medicine area) and focused primarily on the oral route of administration of ncrna, with detailed information on the absorption, distribution, metabolism and excretion of ncrna molecules. other routes of administration, however, not to be excluded. based on available scientific literature (narrative review), to review the effects (physiological, paraphysiological, and pathological) of foreign (exogenous) ncrnas on the gi tract and annex glands (e.g. liver, pancreas, salivary glands) in humans and animals (experimental animals, livestock and pets). this to be based on information from research and development of ncrna molecules intended to be used as therapeutics and/or from research on biomarkers in humans (pharmaceuticals/medicine area). based on available scientific literature (narrative review), to provide information on the possibility of systemic effects of foreign (exogenous) ncrnas in humans and animals (experimental animals, livestock and pets) with focus on gastrointestinal barrier to absorption, and other barriers as relevant (e.g. placenta). based on available scientific literature (narrative review), to assess the plausibility of effects on the immune system of humans and animals (experimental animals, livestock and pets) of foreign (exogenous) ncrnas. gm plants based on silencing approaches by rnai (through ncrna expression) are developed for food and feed purposes and assessed by efsa within the eu gmo regulatory framework. the team considered that pursuing the four tasks defined by efsa to support the risk assessment of gm plants in this regulatory context required gathering preparatory baseline information on ncrna and refinement of the scope of the tasks. therefore, further elaboration on the terms of reference provided by efsa has been conducted and it is described below. it should be emphasized that the available information on ncrnas which could be possibly relevant to food and feed safety, overlaps with and is barely distinguishable from the broader information on rnas. information on rna in general has therefore been included in the search as warranted; when possible, the relevance of distinguishing peculiar ncrnas features is described. the term "foreign ncrna" or "exogenous ncrna" used in this report refers to ncrna molecules to which humans/animals can be exposed through the diet or via therapeutic treatment. similarly, "foreign rna" or "exogenous rna" refers to rna molecules to which humans/animals can be exposed through the diet or via therapeutic treatment. part : kinetics of exogenous ncrna in humans and animals (efsa task ) efsa task : based on available scientific literature (narrative review) to review the kinetics profile of foreign (exogenous) ncrna in humans and animals (experimental animals, livestock and pets) . this to be based on information from research and development of ncrnas intended to be used as therapeutics and/or from research on biomarkers in humans (pharmaceuticals/medicine area) and to be focused primarily on the oral route of administration of ncrna, with detailed information on the absorption, distribution, metabolism and excretion of ncrna molecules. other routes of administration, however, not to be excluded. to address efsa task , preparatory baseline information is provided i) on general features of plant ncrnas including their function, movement in the plant, biogenesis and degradation (section . . ), ii) on their stability (i.e. for how long ncrna molecules retain their original structure, and how they resist degradation over time and in various conditions, both inside and outside the plant), and turnover, also in comparison with turnover of endogenous ncrna in animals (section . . ). if necessary, comparisons between the ncrnas class and other rna classes are made. general information on rnas used/intended for use as therapeutics (including ncrnas) is provided, expanding on the chemical modifications necessary to avoid degradation and to achieve a target effect after administration (section . . ). lessons from rna-based therapeutics as regards the pharmacokinetics of exogenous rna are discussed, with a focus on non chemically modified (naked)ncrnas. in addition, aspects of the pharmacodynamics of rnas as learnt by therapeutics are addressed (section . . ). a description of cellular uptake of rna (in general and with focus on ncrnas), and of the many barriers represented by human/animal cells after oral ingestion is addressed insection . . . further relevant information on dietary exposure to plant rnas (i.e. exposure to plant rnas following dietary consumption) by different diet types, and presentation of the gaps in the literature on dietary plant rna exposure is provided in section . . . efsa task : based on available scientific literature (narrative review), to review the effects (physiological, paraphysiological, pathological) of foreign (exogenous) ncrna on the gastrointestinal tract and annex glands (e.g. liver, pancreas, salivary gland) to address efsa task preparatory baseline information is provided on i) the gi tract barriers to ingested exogenous rnas, including ncrnas (section . . ); ii) experience in rna-based therapeutics for oral (gi) administration, with focus on delivery of naked (non chemically modified) ncrnas (section . . ). biological effects of dietary exogenous ncrnas (plant and nonplant origin) on the gi tract and its annex glands is presented in section . . . www.efsa.europa.eu/publications efsa supporting publication : en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. to address efsa task , preparatory baseline information is provided on the molecular pathways relevant for the uptake of exogenous ncrnas by human and animal cells and the intracellular trafficking that follows, with description of the effects by exogenous ncrnas once they reach the specific subcellular location (section . . ). to exert systemic biological effects, in most cases the ingested exogenous rna must enter the systemic circulation to reach the target tissues. information is therefore provided on the landscape of exogenous rnas in biological fluids and tissues from humans and animals, with focus on ncrna when possible (section . . ). a description of possible systemic biological effects of dietary exogenous ncrnas is provided in section . . , with details on tissue barriers (i.e. placenta, brain) encountered. a detailed review is made of the contradictory information on the systemic effects exerted by plant-derived exogenous ncrnas. finally, studies on the safety and in general on risk assessment on ncrnas from gmos are reviewed (section . . ). efsa task : based on available scientific literature (narrative review), to assess the plausibility of effects on the immune system of humans and animals (experimental animals, livestock and pets) of foreign (exogenous) ncrna molecules. to address efsa task , preparatory baseline information is provided on ncrna mediated regulation of the immune system in humans and animals. the many pathways of sensing exogenous rnas, both at the endosomal compartment and in the cellular cytosol, and the mechanism of triggering the immune system are reviewed (section . . ). an overview is done of the plausibility of biological effects of exogenous plant ncrnas on the regulation and function of the immune system upon uptake by mammalian cells (section . . ). in addition, since the gut microbiota influences the immune system, a review on the possible effects of exogenous ncrnas as gut microbiota modulators was considered relevant and it is provided (section . . ). in the final section of this report, concluding remarks inform on the gaps existing in the scientific literature, and highlight areas needing further investigations to better understand and support the food and feed risk assessment of ncrna-based gm plants. an extensive search was done to identify as many studies as possible relevant to the literature review questions. to include all possibly relevant information multidisciplinary databases and information resources were explored. unpublished research reports ("grey literature") including dissertations, thesis or other scientific reports were also included, which were retrieved using general search engines (i.e. google). the following information sources, databases or search engines ( table ) were used for the literature review: pubmed (biomedical), world wide science (multidisciplinary), web of science (multidisciplinary), springerlink (multidisciplinary), scopus (multidisciplinary), scielo (multidisciplinary) and biorxiv (preprint biology). finally, the publications reporting the outcome of two efsa procurements aiming respectively at investigating and summarising the state of knowledge on the mode-of-action of dsrna and mirna pathways, the potential for non-target gene regulation by dsrna-derived sirnas or mirnas, the determination of sirna pools in plant tissues and the importance of individual sirnas for silencing ; and reviewing relevant scientific information on rna interference that could serve as baseline information for the environmental risk assessment of rnai-based gm plants ) were also used. www.efsa.europa.eu/publications efsa supporting publication : en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the methodology used for this literature review process and report preparation followed three basic principles: i) methodological rigour and coherence in study retrieval and selection; ii) transparency; and iii) reproducibility. to ensure that these principles were implemented in this literature review process, the search methodology design, study selection and data and results presentation follow, whenever possible, most of the methods and techniques described in the efsa guidelines on application of systematic review methodology for food and feed safety assessments to support decision-making (efsa, ) . the following section describes the general key steps followed in the literature search methodology; detailed descriptions of the literature search methodology for each topic are also provided in this section. to find a comprehensive set of scientific literature related to exogenous ncrnas and their biological effects on a target organism, either local or systemically, key questions were identified and key words were used as search queries for each task section according to a pre-defined review process. to provide a fit-for-purpose literature search, the team defined key questions based on the efsa tender specifications and their interpretation as above described. to support this phase, the team identified key elements of the questions considering the pico approach of efsa ( ). the key elements identified include the population, in this case humans or animals (experimental animals, livestock and pets) as defined by the terms of reference of efsa. the intervention was identified as exogenous rnas (focusing on ncrnas). the comparator was identified as the control or reference group (not exposed to exogenous rnas) in most studies, whenever possible. the comparator could also be identified as the modified (either chemically or biologically) version of the exogenous rnas when referring to the stability or pharmacokinetic of the exogenous rna. the outcome is the biological effect (if any) as a response to the intervention. other specific key questions are determined for every part of the specific tasks defined by efsa. evidence is generally scarce for most of the topics covered by this scientific review, and in some cases the information was available from not directly related studies (i.e. pharmaceutical industry studies). the literature review was helpful in identifying both knowledge gaps and unexpectedly relevant studies or evidence not previously known to exist. these knowledge gaps are used to make informed proposals for future research designs (section . ). the key methodological steps in the literature search, selection and review process are shown in figure . three phases were defined in this process. the preparatory phase started with team discussions of topics to be included in the literature review. during this phase key elements addressing the efsa tender specifications and their interpretation as above described were identified and key questions were defined. the key questions aimed to to assist the literature search in order to produce a comprehensive overview of the topic that best fits tender specifications. the bibliography search and selection phase included several steps. the initial search in bibliographic databases and search engines was done using task-specific key words. the key words used for the search were derived from the efsa tender specifications and their interpretation (tasks - ) and were complemented with other key words identified in the preparatory phase. identified key words are detailed for each task in section . the final phase of review and final selection involved two additional steps, i.e. a detailed review of all relevant previously selected documents and a systematic evaluation of the final selected studies. a detailed review of the selected documents was done for studies with full text availability (either freely available or available for purchase) and, based on this, a final selection was made of full-text studies most relevant to answer the key questions for tasks ( - ) and apt for review in this report. a systematic evaluation of these final documents was done to develop a comprehensive overview of the topic. conflicting or contradictory results were reported in a neutral way and supported by references and the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. citations of all available evidence, both from the published and grey literature. retracted papers were carefully identified and noted. finally, each section of the narrative review was assessed by one reviewer. if more specific data was needed because of missing or novel information, additional bibliographic research was performed. additional key words that could directly address the specific data needed were identified and used to run a new specific search. the search among full-text studies was focused on obtaining only specific missing or required data (e.g. quantitative data on rnas levels in plants, earlier studies on exogenous rna exposure to mammalian cells). this new specific search generally represented less than % of the literature included in this report and it was performed only once for certain topics. finally, to reduce the risk of introducing bias into the literature review process and to assure reproducibility in the review methodology the following general approach was applied: when the search of the identified "key words" in different databases resulted in a myriad of duplicate documents, manual removal of these duplicates was performed in each section by each team expert; when relevant studies that might answer the identified key questions were identified after the first search run, these were used to further refine the literature search, for example refining the keywords; each selected full-text study to be included in the result section was manually examined by the team. to identify literature relevant to the topic, the methodology described in section . . . ( figure ) was applied, with adaptations. a multiple-step approach was followed based on the expertise of the team members who carried out research in the area. following a first search based on identified keywords, a careful selection of retrieved review studies was done, followed by a search of specific publications referred to in these reviews. additional specific key words were identified, and an additional search was run. this search retrieved a relatively small number of studies; therefore, team members reviewed all these to determine which were relevant to the topic. duplicates, irrelevant or erroneously assigned publications were removed. team members also cross-checked each others' findings, sharing discussing the results of their searches. the following areas were investigated. the purpose of this search was to provide general information on rnas function, movement in the plant and aspects of biogenesis and degradation, with a focus on ncrna (either small or long). by comparing animal and plant ncrna pathways, the aim was to identify similarities between animal and plant ncrna processing machineries that would indicate the possibility of ingested plant-derived ncrnas being processed in animals. in addition, this section was organized to avoid details that were outside the scope of this literature review in the topic of plant ncrna biology. this serves as an introductory chapter allowing easier integration of the knowledge presented in later chapters. the following combination of key words or phrases was used in some of the databases indicated above: plant small rnas; plant and sirna; plant and mirnas; plant and lncrnas; plant and circrnas; plant rna and movement. www.efsa.europa.eu/publications efsa supporting publication : en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the initial search using the above-mentioned key words or phrases yielded a large number (≈ ) of documents ( table ) , most of which were not relevant to the topic. particularly, the databse web of science retrieved documents not directly related to the topic. to assist and refine the search, studies were further filtered by their relevance to the key questions identified: -what type of ncrnas are described in plants? -what are the main differences between mammalian and plant ncrnas? -what is the function of plant ncrnas? -what is known about the movement of plant ncrnas? title and abstracts were evaluated, and studies further refined using the key questions. detailed study of the most relevant full-text documents answering the key questions, retrieved documents which were finally selected as relevant to give an overview of plant ncrnas. search was then refined by using other key words including "half-life", "turnover", "fate" or "degradation". the identified studies were then manually evaluated by title and abstract and their number further limited due to their low relevance to the below identified key questions for this specific subchapter: -are plant ncrnas more stable than animal (mammalian) ncrnas? -if so, what makes a plant ncrna more stable than its animal (mammalian) counterpart? -why is it relevant to consider ncrna stability outside the organism of origin (plant)? -are certain types of plant ncrnas more or less stable than others? -what is known about the half-life of ncrnas? documents on the stability of rnas (not only ncrnas) were further screened for relevance to the question. most of the studies in the literature describe the stability of a specific class of intracellular rnas (i.e. mrnas). the stability of rnas other than ncrnas (e.g. mrna, trna), unless are used as model of general ncrna stability, is outside the scope of this literature review, since it is very unlikely they would be used as baseline information for food/feed risk assessment of ncrna-based gm plants. these keywords were specifically selected since they describe the kinetic profile of exogenous ncrnas in human and animals. the word "pharmacokinetics" was included in the search because it is a general term that retrieves data on absorption, distribution, metabolism or excretion of any molecule, particularly those intended for use as therapeutics (pharmaceutical/medicine areas). the keywords "increased intestinal permeability" or "increased plasma clearance" are intended to retrieve any study indicating changes in these parameters. although a large number of publications unrelated to ncrnas were expected, these keywords were chosen to initially explore the general aspects of physiological or pathological conditions associated with modification in these parameters. the search was further refined after this initial exploration. the search of the above-mentioned key words on different databases yielded ≈ . entries ( table ) . a quick exploration of titles and abstracts suggested that (as expected) a number were unrelated to or were not useful to appropriately address the topic. it is worth noting that the search in the previous section (section . . . .) indicated that the chemical modifications generally introduced for rna-based therapeutics are normally absent in nature. scientific data that could serve as a baseline to support risk assessment of ncrna-based gm plants should contain primarily information on ncrnas resembling those present in the nature or in gm plants. therefore, studies on naked exogenous rnas were analysed to prepare this narrative review. to promote comparisons between natural and synthetic rnas, a very few examples of slightly chemically modified rnas were included. heavily chemically modified rnas were not considered due to their unlikely existence in nature. only in vivo studies were considered, both in animals (mammals) or humans. studies were further evaluated for their relevance to answer the key questions identified for this section: -what are the pharmacokinetic properties of rna-based therapeutics? -what routes of administration have been used for rna-based therapeutics? -what is it known about the pharmacodynamic properties of rna-based therapeutics? -do disease conditions modify the pharmacokinetic properties of rnas when administered to mammals? the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. after detailed evaluation of the full-text reports, documents meeting the described criteria were finally selected for review. understanding whether and how exogenous rnas are taken up by cells and what barriers are encountered is relevant to study the possible absorption and systemic exposure to ncrnas introduced by food and feed. this topic is reviewed in this dedicated section and excluded from the section on the pharmacokinetics of exogenous rnas (section . . . . above). to search and select the relevant studies concerning the uptake of exogenous rnas in mammals, the following combination of key words was used in some of the databases listed above: rna and cellular uptake; ncrna and cellular uptake; mirna and cellular uptake; lncrna and cellular uptake. these key words were used to screen for studies where "cellular uptake" was mentioned in the title or abstract for any type of rnas. it was also decided to include specific types of ncrnas including "mirna" or "lncrna" to expand the search to literatures where the more general term "rna" was not necessarily used. the initial search of scientific documents using the above keywords yielded ≈ documents ( table ) . these were explored by their relevance (title and abstract), and only studies addressing naked ncrnas were considered for full text analysis, since highly chemically modified rnas are not common in nature. to address the reference terms for task- of this procurement, and after expert discussion for this section (preparatory phase) key questions were identified and the studies then further filtered for their relevance to the these: -are exogenous rnas taken up by mammalian cells? -how does exogenous rnas uptake in mammalian cells occur? -what are the biological barriers to consumed rnas? -what are the biological barriers for the exogenous rnas after absorption and entering into the mammalian circulatory system? only studies where exogenous rnas were evaluated were included in the literature review. after detailed evaluation of full-text reports and expert judgement on their relevance to efsa task , documents were finally selected for review. to select relevant studies for plant rna exposure, the following combination of keywords were used: plant and rna content; plant and mirna content; plant and sirna content; plant and lncrna content; plant and dsrna content; rna intake and vegetarians; vegetables/fruit intake and vegetarians. these specific keywords were used because the wording "rna (ncrna, mirna, sirna, dsrna, lncrna) content" would yield most of the documents related to quantities of rnas in a specific substrate. these were combined with "plant" to retrieve only those studies where plants were used to evaluate rna content. it was also decided to use "rna intake", "vegetable intake" or "fruit intake" to specifically refer to consumption, and to combine this with "vegetarian" to search only for studies where comparisons are made between vegetarian and other types of diets. alternative wordings including "rna amount" or "vegan" were used to search for alternative information. using the above keywords, ≈ studies were retrieved (table ) during the initial search. to more appropriately cover this section and to refine the search, the following key questions were identified during the preparatory phase: -what is the general amount of rnas naturally occurring in edible plants? -what percentage of total plant rnas are ncrnas, mirnas, sirnas, lncrnas or dsrnas? -does exposure to rnas change in different diet types? -do changes in rnase activity, due to pathologies or human polymorphisms, influence dietary exposure? titles and abstracts were evaluated, and studies further filtered using the key questions above. the search was also refined to focus on those studies where quantitative rnas data were mentioned. only in vivo exposure studies were used (either epidemiological or experimental). common types of diets were only considered (i.e. vegetarian, vegan or omnivorous). for rna exposure by different diet types, studies where comparative analysis of at least two different diets (one of which was the general population diet type) were mainly considered. in all cases, full-text studies were evaluated to screen for relevance to answer the identified key questions. a detailed study of the full-text documents resulted in the identification of studies most relevant to address this specific topic of task- , and which were reviewed for this section of the report. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. to identify literature relevant to the topic, the methodology described in section . . . (figure ) was applied. during the preparatory phase key questions were identified to assist in the literature search. the document search was first done using specific key words and then refined according to the key questions. detailed analysis of the full-text documents was done by team members with expertise in the area. search and analysis results were discussed collectively among team members. the following areas were investigated: in this subsection, there is a general description of the biological barriers within the gi tract that exogenous rnas would need to overcome to exert a biological effect, either locally or systemically. to select relevant studies, the following combination of key words or phrases were used: "gastrointestinal tract anatomy"; "gastrointestinal tract physiology"; intestinal transport pathway; physicochemical properties of rnas. an exploration of titles and abstract of the yielded entries (table ) suggested that a large number of documents were unrelated to the topic. only one key question was identified in the preparatory phase: -what are the biological barriers within the gastrointestinal tract to ingested exogenous rnas? the search for relevant studies addressing this key question was based mainly on team member judgement. detailed study of the most relevant full-text documents answering the key question retrieved documents which were finally selected as relevant to this section of the report. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. this search was intended to collect data from rna-based therapeutics employing oral administration (and other gi routes) which could support the search on the possible biological effects of plant dietary exogenous rna and ncrnas on the gi tract and its annex glands. the initial search was done using the following key words on the different databases: ncrna oral delivery; rna oral delivery; sirna oral delivery; aptamer and oral; sirna gastrointestinal delivery; mirna gastrointestinal delivery; oral exosome rna; extracellular vesicle and gastrointestinal. these key words were selected to search for any documents related to rnas and oral administration. any information on oral administration was considered relevant. to expand the search, different terms referring to different types of ncrnas (mirna, aptamer, sirna or others), and a synonym for "administration", i.e. "delivery", were included. the initial search using the above key words or phrases yielded a small number (≈ ) of documents ( table ). the different databases generated similar number amounts of publications, most of which were duplicates. using the key word "lncrnas" in combination with the other key words did not yield any information relevant to the topic. indeed, just the search using "lncrna exosome" identified fewer than documents, but none was relevant to oral delivery or gi administration. the wording "dsrna and oral" produced ≈ documents, but none of them was relevant to mammalian organisms. the documents were selected by relevance, evaluating title and abstract. in this section, only studies in animals (mammals) or humans were considered. when replacing the word "delivery" with "administration", ≈ additional documents were identified, but almost % were unrelated to the review topic. since most of the documents in the literature describe minimally modified rnas for oral use, all chemically modified rnas were considered in this section. these can provide baseline information to support food/feed risk assessment of ncrna-based gm plants. to assist and refine the search, studies were further filtered for their relevance to the key question identified: is the oral route of administration relevant for rna-based therapeutic development? -what types of formulation (delivery vehicles) are tested for rna-based therapeutics for the oral route of administration or other gastrointestinal tract routes of administration? the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. -do exogenous ncrnas (naked rnas) resist the conditions of the gastrointestinal tract environment? -could ncrnas-based therapeutics -delivered by oral administration or other gi tract routes of administration-exert a biological effect in the gi tract or its annex glands? -are there any natural vehicles for oral administration of exogenous ncrnas? from the initial ≈ entries identified using the above key words and after filtering for relevance to the key question with evaluation of titles and abstracts and detailed study of the full-text reports, documents were selected for review in this section. twenty ( ) of these documents reports selected examples of local effects of nucleic acids-based drugs (mainly exogenous rnas) within the gi tract, using oral administration. since efsa required a literature review also including livestock species, an additional specific search was run for birds and fish. this search was intended to compile information on administration of exogenous ncrnas in either fish or birds, regardless of administration method, exerting any biological effects in vivo. the initial search was done using the following key words on the different databases: exogenous rna and fish; exogenous rna and bird; rna oral delivery and fish; rna oral delivery and bird; ncrna administration and fish; ncrna administration and bird. to expand the search, different terms referring to different types of ncrnas were used (sirna, mirna, circrna, or dsrna). the initial search using the key words or phrases produced a small number (≈ ) of documents (table ) in most of the databases. to assist and refine the search, only one key question was identified during the preparatory phase for this subsection: -are exogenous ncrnas also used to exert rnai effects in fish or birds? from the initial ≈ entries identified using the above key words, and after filtering for relevance to the key question, and evaluating titles and abstracts and detailed study of the full-text reports, documents were selected for the review in this subsection. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. this chapter provides baseline information supporting food/feed risk assessment of ncrnas-based gm plants. in the effort to cover the widest amount of literature containing relevant scientific data on exogenous ncrnas and their possible biological effects, the following key words or phrases were used in the search: dietary plant non coding rnas; food plant non coding rnas; exogenous plant non coding rnas; diet plant micrornas; food plant mirnas; breast milk exosomes; breast milk mirna; breast milk ncrnas; breast milk lncrnas; breast milk rna; dietary exogenous rna. these key words were selected to cover most, if not all, available studies on the topic ( table ) . the search of other common examples of exogenous ncrnas (non-plant origin) consumed by oral intake was focused on breast milk ncrnas, due to their relevance to human nutrition (table ) . almost all documents yielded by the three databases (pubmed, scopus or web of science) were duplicates. in this section, studies reporting local effects (in the gi tract or its annex glands when consuming exogenous ncrnas) were preferentially (but not exclusively) evaluated. other databases including the preprint server for biology (biorxiv), scielo or search engines (i.e. google) were also used. the initial search in some databases using the above key words yielded a number of entries (table and ) not exceeding ≈ documents. because this specific topic is especially salient to future understanding of the possible food/feed risk assessment of ncrnas-based gm plants, all the identified studies were further evaluated by title and abstract and their relevance to answering the identified key questions: does the literature describe the effect of exogenous plant-origin ncrnas when consumed orally? -does the literature describe negative results or contradictory results regarding the possible biological effect of ncrnas when consumed orally? -are ncrna stability properties under gi tract conditions normally evaluated? -are exogenous ncrnas evaluated quantitatively? -are exogenous ncrnas consumed in an amount sufficient to exert a local biological effect?are there other common examples of exogenous ncrnas (other than plant-origin) that could exert a biological effect on the gi tract and its annex glands when ingested orally? local effects (in the gi tract or its annex glands) are mainly described in this section. the possible mechanisms of plant exogenous ncrnas cellular uptake, their intracellular trafficking and systemic biological effects (including plasma levels) are reviewed in another section (section . .). of the initial ≈ documents retrieved using the above key words, and after filtering for relevance to the key questions by evaluating titles and abstracts, and detailed study of the full-text reports, documents were selected for the review in this subsection. from these, documents were selected for plant-origin ncrnas and for non-plant origin (breast milk) ncrnas. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. in line with the methodology described in section . . . . (figure ), a multiple-step approach based on the expertise of the team members responsible for this section was applied to identify literature relevant to the possible systemic effects of exogenous ncrnas. the document search was first done using specific key words in the databases and refined based on the appropriate key questions. detailed analysis of the full-text documents was done by team members and the results discussed collectively. the below areas were investigated. the search objective was to obtain information on basic molecular cellular uptake pathways of exogenous ncrna, including proteins involved in this process (if any). a review was also done on several other aspects of intracellular trafficking, once an exogenous ncrna is internalized, and how it exerts a biological effect. tissue barriers to exogenous ncrna function were also investigated. the content of this section deepens on molecular pathways previously described in section . . . to assist the search the following key words were used in the different databases: exogenous ncrna and mechanism and absorption; ncrna and mechanism and absorption; sidt (sid transmembrane family member ) and ncrna; sidt (sid transmembrane family member ) and ncrna; ncrna and uptake; intracellular trafficking and ncrna. these key words and terms were selected to identify any document related to the mechanisms of uptake and intracellular trafficking of ncrnas exerting a biological effect. the search was expanded by adding and combining different terms for exogenous ncrnas (see table ), including the general term "rna", www.efsa.europa.eu/publications efsa supporting publication : en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. or specific ncrnas such as "mirna". the initial search using the above key words or combinations thereof produced a moderate number of documents (table ). the different databases generated similar amounts of documents, most of which were duplicates. the largest proportion of the references was obtained using the key wording "ncrna and uptake" (≈ ). however, most of these had no relation to this specific section. the search was refined by filtering for relevance to answering the identified key questions: is there any clear transport of exogenous ncrnas into mammalian cells? -are orthologous of environmental rnas transporters in lower organisms (i.e. sid family of proteins) involved in the mammalian exogenous ncrna mechanism of uptake? -what molecular mechanisms are involved in exogenous ncrnas trafficking inside the cell? -what is the cellular fate of exogenous ncrnas once inside the cell? -what are the specific tissue barriers to exogenous ncrna function? very few documents evaluate the molecular mechanism of exogenous ncrnas uptake, intracellular trafficking and function. from the initial ≈ documents retrieved using the above key words, and after evaluation of their titles and abstracts and filtering for relevance to the key question, less than documents initially seemed relevant to the topic and were finally selected for review after detailed study of the full-text reports. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. this subsection is intended to retrieve available information on the presence of exogenous rnas in human and animal biological fluids (i.e. blood, plasma, serum or any other biological fluids) and tissues. the search focused on the presence of dietary plant-derived exogenous ncrnas, due to their relevance to the overall literature review. to cover the largest possible portion of the relevant literature, the following key words were used in the search: exogenous rna and human plasma; exogenous rna and serum; plant ncrna and human tissue; plant lncrna and human plasma; plant mirna and blood; plant mirna and tissue; plant rna and tissue. to expand the search, the above key words were used in different combinations for the same search (i.e. blood, serum or plasma for biological fluids) ( table ). the general wording "rna" was also incorporated into some searches. specific types of ncrnas were also added, including "mirna" or "lncrna", to avoid missing documents possibly related to these specific molecule types. the search using these key words or phrases in different databases yielded more than ≈ . publications (table ) . a quick exploration of the titles and abstracts suggested that only few studies were related to the review topic. only one key question was identified as relevant which was related to the presence or absence of evidence of exogenous ncrnas in biological fluids and tissues. since these findings are important for the risk assessment of ncrna-based gm plants, the key question was presented in its positive and negative forms as follow: are there studies describing the presence of exogenous rnas in the biological fluids and tissues of humans and/or animals? -are there studies contradicting the presence of exogenous rnas in the biological fluids and tissues of humans and/or animals? after a detailed assessment of the full-text, documents were finally selected for review. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. this search primarily aimed to gather information relevant to understand the possibility of systemic effects of dietary exogenous ncrnas after intake (oral administration) by humans and/or animals (mammals). information on the possible passage through specific biological barriers (i.e. placenta or brain) was also searched. the search was guided by the following key words to identify studies providing any data on the possibility of systemic effects of dietary exogenous ncrnas: plant ncrna and diet; plant exogenous rna and diet; plant rna and diet, plant environmental rnai and diet; plant rna and placenta; plant rna and brain; dietary rna and cross kingdom. these key words were used to guarantee that every single document potentially providinginformation on the possibility of systemic effects of exogenous ncrnas was found. to widen the search, other wording such as "environmental rnai", "cross kingdom" or "mobile rnas" were used. although the wording "systemic effects" was not used in the key words during the search, the above key words were used to retrieve any documents in which a biological effect (local or systemic) might be reported. different wording for the term ncrnas was also used (including lncrna, circular rna, mirna, or dsrna). the initial search in some databases using the above key words produced a small number of entries (table ) . the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. approximately ≈ documents were initially identified using the above key words (table ) . after filtering for their relevance to the key question by detailed assessment of the full-text reports, documents were finally selected for review. within this specific subsection, the selected documents are mainly grouped as studies that "support the evidence" or studies that "contradict the evidence" of systemic biological effects of dietary exogenous ncrnas in humans and animals (mammals). although not specifically included in the scope of this literature review, search for toxicological effects of dietary exposure to exogenous ncrnas in humans or animals is relevant to risk assessment. this search was aimed at identifying information on possible toxicological effects of plant-derived exogenous ncrnas following food/feed consumption. the initial search was conducted using the following key words in the different databases: the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. few documents were initially retrieved and after a quick evaluation of titles and abstracts, it became evident that very little information is available in this area. therefore, different terms were used to refer to exogenous ncrnas, including circular rna, lncrna and mirnas. the search using the above key words yielded a small number of documents (table ) . only in vivo studies were considered, both in animals (mammals) and humans (if available), with focus on dietary plant-origin exogenous ncrnas. the key questions identified to refine the literature search for this specific topic were: -is there evidence of any toxicological effects of plant-derived dietary exogenous ncrnas? -are the toxicological effects related to rnai-mediated interaction with the human or animal genome? -are there possible interactions related to unintended rnai-mediated gene regulation? approximately documents were identified using the above key words. after filtering for relevance to the key questions, and assessing the full-text reports, only documents were finally selected for review. a systematic and comprehensive review and collection of the literature relevant to the topic was performed with the aid of the team members' expertise. an extensive search using key words was initially done on multidisciplinary databases to avoid publication bias, followed by a grey literature search using general search engines. the resulting documents were screened by title and abstract to determine relevance to the topic and refined using the identified key questions. after full-text analysis of the documents, irrelevant documents were eliminated. all stages of the screening process were independently assessed and, in case of uncertainty, discussed with other experts to avoid personal biases. the methodology followed the key steps described in section . . . . (figure ) and a total of papers were identified, investigating the areas below described. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the final goal of this search was to identify relevant information on ncrnas-mediated processes associated with innate and adaptive immune responses. this section serves as an introduction to the studies presented later, and it is based on the results of the seach described in the next paragraph. sixty-four ( ) documents were reviewed for this section. the search was aimed primarily at gathering state-of-the-art knowledge on the possible biological effects of exogenous (plant) ncrnas on the regulation and function of the immune system. to cover this specific section, a combination of the following key words was used in the different databases: exogenous rna and immune and human/mice/rat; exogenous plant rna/mirna/sirna/lncrna/dsrna/transgenic rna and immune function/inmmunity and human/mice/rat; genetically modified plants and immune function/immunity and human/mice/rat; exogenous plant rna/mirna/sirna/lncrna/dsrna and primary cells and lymphocyte/monocyte/macrophage; exogenous rna/genetically modified plants and immune function/immunity and zebrafish. the key words were designed to identify all publications that use in vivo, including non-mammalian animal models, and in vitro ncrnas testing to evaluate the influence on tolerance processes as well as immune cell function. the search also included publications on in silico methods. the different key words were entered one by one into the search field 'topic' of the databases ( table ). the search was designed to identify documents including methods that could use non-mammalian animal models (i.e. zebrafish embryos). all search terms within one key word were combined by the operator 'or' and 'and' copied in a second or third search field. 'review' and 'meeting' documents were excluded. a search for grey literature was done in general search engines (i.e. google) using the different key words in the 'search' function of the respective websites. all the information on in vivo studies, using mammalian and/or non-mammalian organisms, and in silico studies was collected. these key words were chosen because this section aimed to describe the possible effects that exogenous ncrnas, specifically of plant dietary origin, might exert on the immune system of humans and animals. the search yielded ≈ entries (table ) . publications were excluded if one of the following criteria was met: the biological effect could not be unambiguosly attributed to ncrnas; the publication addressed immunodeficiency or autoimmunity; the publication reported no immunity (innate or adaptive)-associated endpoint or it is not specific for immune function and/or homeostasis evaluation; evaluation of exposure of and/or effect on cells other than immune cells (e.g. epithelial cells); the study was unable to measure processes related to immune function and/or homeostasis; the study reported data on potentially immunocompetent cells not fully differentiated as mature immune cell; no ncrnabased plant was tested in the publication; and the publication did not address plant-derived exogenous ncrna. the unexpectedly low number of publications retrieved using the key words proved a challenge in the selection process. for example, selection for in vivo studies produced only articles after title/abstract screening. to expand the number of documents, additional key word searches were included and less conservative criteria were set for the title/abstract screening. retrieval of grey literature was also based on information containing "preliminary results" or on-going projects that could possibly answer the key questions. www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. studies were further evaluated for their relevance to answering the key questions. the key questions identified using the above key words were: -which test methods or approaches are available to investigate the possible role of plant-derived exogenous ncrnas on the immune system of humans or animals? -what human or animal immune response(s), innate or adaptive, are possibly associated with administration of plant-derived exogenous ncrnas? -which mediators in humans and animals have been identified and/or ascribed to immune response(s) possibly mediated by administration of plant-derived ncrnas? -which innate immunity mediators (i.e. chemokines, cytokines) have been identified in humans and animals as associated to effects of plant-derived ncrnas? -which human or animal cells (i.e. lymphocytes, macrophages, myeloid) are possibly involved in immune responses mediated by plant-derived ncrnas? after the selection of the full-text documents, publications were excluded if not providing sufficient information (i.e. endpoint, exposure time), if investigating a mechanism other than immune-related processes, or if describing an operational procedure or guideline rather than a research study. from the initial ≈ documents identified using the above key words, and after filtering for the relevance to the key questions and detailed study of the full-text reports, documents were finally selected for review in the three sections of part (efsa task ). of these, documents were selected for review in this section. gut microbiota is important for the function of the immune system, therefore the purpose of this additional search was to provide general information on the effect of dietary exogenous ncrnas on the gut microbiota and the possible consequent secondary modulation of human or animal immune systems. this would serve as an introduction to identifying information gaps and experimental needs for future studies relevant to food/feed risk assessment of ncrna-based plants. selected full-text studies were analysed for their relevance to answering the following key question: -is there evidence for influence of exogenous plant ncrnas on the gut microbiota related to the immune system? five documents were finally selected for review in this section. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. results are presented narratively. when appropriate, tables and figures are included to allow better understanding of the topic covered by the literature review. some sections of this review provide only limited amount of quantitative analysis due to the nature of the available evidence and the key questions identified for each subchapter. at the end of each subchapter, a short summary presents the general points identified in the reviewed literature. gaps in the literature and open questions identified in the field are presented in a subchapter of certain sections. these gaps in the literature may serve as suggestions for further research in each topic. part : kinetics of exogenous ncrnas in humans and animals (efsa task ) ncrnas are transcripts that are not translated into any functional peptides or proteins. these transcripts include structural (transfer, ribosomal, small nuclear, and small nucleolar rnas) and regulatory ncrnas. the latter comprise both small and long ncrnas that can regulate gene expression by acting on chromatin, transcription, rna processing, rna stability and translation. this part of the report introduces general features of regulatory ncrnas including their function and aspects of their biogenesis and degradation. it also establishes possible comparisons between animal and plant ncrna pathways. this can be considered background information to clarify the possibility of plant ncrna biological effect in humans and animals that ingest food/feed from ncrna-expressing gm plants. in addition, ncrna movement inside the plant is also described. specific details on the structural ncrnas and the broad topic of plant ncrna biology are outside the scope of this literature review. following an extensive literature search as described above and based on the team expert decisions on the topic, this section is a review of scientific documents. small ncrnas (srnas) srnas are common to both plants and animals. two major classes of srnas are found in eukaryotes: micrornas (mirnas) and small interfering rnas (sirnas). they function as regulators of endogenous genes or as defenders from invasive nucleic acids. they are characterized by the double-stranded nature of their precursors, and differ from the less abundant piwi-interacting rnas (pirnas) which derive from fragmentation of single-stranded rnas (juliano et al., ) , and are primarily found in animals; they have not been described in plants. pirnas exert their functions in germ and stem cells through interaction with piwi-proteins (juliano et al., ) . by contrast, both mirnas and sirnas are bound to argonaute (ago) proteins, where they identify 'target' rnas by base-pairing interactions, and act in both somatic and germ cells. all srnas in plants are modified at the ' -terminus by '-o-methylation, including mirnas and sirnas, which lack this modification in animals. '-o-methylation is essential to conferring stability and protection from ' uridylation and degradation (borges and martienssen, , ) . mirnas and sirnas are distinguished by their origin and biogenesis (figure ) . mirnas are derived from processing of single-stranded precursors with a hairpin structure, whereas sirnas are generated from long, fully complementary double-stranded rna (dsrna) precursors (carthew and sontheimer, the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. endonucleases (dcls) that excise rna precursors into short double-stranded fragments, - nucleotide (nt) long, with -nt ' overhangs; and argonaute proteins (agos) that engage these duplexes and support their silencing activies based on target complementarity (carthew and sontheimer, ). further details on sirnas and mirnas are provided below. sirnas several different classes of sirnas with specialized biological roles have been identified in plants. the most abundant class consists of heterochromatic sirnas (hetsirnas), which are usually - nt long and originate from the repetitive and intergenic regions in the chromosome and transposable elements. hetsirna are crucial in dna methylation and chromatin modification through a process known as rnadirected dna methylation (daxinger et al., ) . hetsirnas are processed by dcl nuclease and preferentially loaded into ago . mature ago -sirna complex can interact with complementary noncoding nascent rna polymerase triggering the recruitment of dna methyltransferase (domains rearranged methyltransferse ) to silence target loci at the transcriptional level via dna methylation and repressive chromatin modifications (vaucheret, ; lee and carroll, ) . these hetsirnas may account for approximately half of a plant's total mass of srnas. another class of sirnas are naturalantisense transcript sirnas (natsirnas) which can be generated from dsrna precursors through hybridization of independently-transcribed complementary rna strands (vaucheret, ) . in addition, secondary sirnas generated as a "secondary effect" of mirna-mediated target cleavage are found in plants (axtell, ; vaucheret, ) . the mirna-mediated cleaved target is occasionally used by rna-dependent rna polymerase (rdr) to produce secondary sirnas, which can either give rise to a phased set of sirnas (phasirnas) or trans-acting sirnas (tasirnas) with the ability to target genes different from their loci of origin. this secondary pool of sirnas can greatly amplify and sustain a systemic silencing throughout the organism. recognizable rdr-encoding genes are present in the genome of many rnai-competent eukaryotes, with the notable exceptions of insect and vertebrate species in which these secondary sirnas are absent (carthew and sontheimer, ). the lack of these secondary sirnas might have a positive effect on specificity (as sirna amplification can lead to the silencing of multiple transcripts, specifically if they share a highly conserved sequence or a common exon) and a negative effect on the amplification of rna silencing in these species. plants have more diversified and specialized sirna-based pathways than other organisms, which are thought to contribute to plant plasticity . it is generally accepted that these pathways evolved as a cellular defence mechanism against rna viruses and transposable elements, which were later adapted to regulate the expression of endogenous genes. this is consistent with the fact that most small rna classes have a recognized role in defence responses, as well as in epigenetic regulation, and that plants have larger and repetitive genomes (borges and martienssen, ) . the diversification of srna-directed silencing pathways in plants occurred through the expansion of the rdr-polymerases, dcl and ago proteins. rdr genes are found in rna viruses, plants, fungi, protists and some animals, but are absent in flies, mice and humans; this is consistent with the fact that the vast majority of srnas in humans are mirnas (kurzynska-kokorniak et al., ) . absence of rdr activity may also justify the lack of intercellular spreading of rna silencing in vertebrates, whereas systemic silencing is a phenomenon widely reported in plants and nematodes. dicer or dicer-like proteins in plants constitute a four-member gene family, whereas vertebrates have only one member (mukherjee et al., ) . the diversification of agos has resulted in development of distinct gene-silencing processes based on differential ago affinities to small-rna duplexes (borges and martienssen, ) . www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. mirnas mirnas are well-studied srnas deeply conserved over long evolutionary distances, even between the plant and animal kingdoms. however, there are substantial differences between these two kingdoms with regard to mirna biogenesis, and mechanism and scope of mirna-mediated gene regulation (jones-rhoades et al., ) . detailed pathway comparisons and species information are described elsewhere . transcription of mirnas is typically performed by rna polymerase ii, and transcripts are capped and polyadenylated. in animals, most mirnas are derived from longer hairpin transcripts (pri-mirna) by the consecutive processing by the rnase iii-like enzymes drosha and dicer, whereas in plants only dicer (particularly dcl ) is responsible for mirna processing. thus, the biogenesis of most mirnas in plants occurs in the nucleus, whereas in animals it requires the sequential cleavage of pri-mirna in the nucleus and then in the cytoplasm by distinct rnaseiii enzymes (rogers and chen, ) . once processed, one strand of the hairpin duplex is loaded into an ago protein to form the core of the mirna-induced silencing complexes (miriscs). miriscs silence the expression of target genes predominantly at the post-transcriptional level (ptgs). the targets to be silenced are recognised through base-pairing interactions between the loaded mirna and mrna target, which contains a partially or fully complementary sequence (huntzinger and izaurralde, ) . plant mirnas recognize fully or nearly complementary binding sites, which are generally located within the open reading frames (orfs) of the mrna target. of importance is that mirna nt - are usually engaged in base pairing, which allows target cleavage by ago proteins (between nt and ). by contrast, animal mirnas recognize partially complementary binding sites, which are generally located in ' utrs. in both plants and animals, complementarity to the ' end of the mirna (the 'seed' sequence, containing nt - ) is a major determinant in target recognition and is sufficient to trigger silencing. for most mirna-binding sites complementarity is limited to the seed sequence (seed-matched sites) or to the seed sequence plus mirna nucleotide . however, in some rare cases complementarity to the ' region of the mirna might contribute to target recognition, particularly when the mrna has a weak seed match. even for these sites, however, mirna nucleotides - generally bulge out, preventing endonucleolytic cleavage by agos. in both animals and plants the mirna ' terminal nucleotide is buried in the mid domain of agos and is not available for pairing with the target (huntzinger and izaurralde, ) . therefore, there is an important difference in complementarity between plant and animal mirnas and their targets; this is less extensive in animals than in plants. in both animals and plants mirnas can move from cell-tocell. while in mammalian cells mirnas and other types of rnas can be transferred through secretory vesicles (ruvkun, ; valadi et al., ) , in plants they mostly use a different mechanism (kobayashi and zambryski, ) (see section . . . . for details). however, recent evidences also suggest that plants can send small rnas in extracellular vesicles to pathogens to silence virulence genes (cai et al., ) . regarding the silencing mechanism, mirnas were initially thought to inhibit translation in animals and to predominantly promote target endonucleolytic cleavage in plants. however, recent evidence has changed this view by showing that mirnas can trigger translational repression and mrna destabilization in both kingdoms. in both plants and animals, the current evidence suggests that target mrna degradation provides a major contribution to silencing by mirnas (huntzinger and izaurralde, the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. next generation sequencing approaches have revealed a new transcriptional landscape in which many novel lncrnas have been identified. these are transcripts longer than bp without any clear protein coding potential. lncrnas are transcribed either from intergenic regions (lincrnas), introns (incrnas) or the opposite strand to protein-coding genes or other lncrnas; they are thus natural antisense transcripts (nats) (ariel et al., ; chekanova, ; shafiq et al., ; yamada, ) . as a confirmation of their widespread nature and possible biological relevance, lncrnas have been identified in plants, fungi and animals. in fact, lncrnas are involved in diverse biological processes across the eukaryotes, ranging from regulation of mating type in yeast to the pluripotency of embryonic stem cells in mammals (zofall et al., ; flynn and chang, ) . in plants, lncrnas play key roles in flowering time regulation, gene silencing, root organogenesis, seedling photomorphogenesis, and reproduction (ariel et al., ; chekanova, ; shafiq et al., ; yamada, ) . although plant lncrna biological characterization is still limited, detailed analysis of over arabidopsis thaliana transcriptome data sets identified , putative lncrnas, including approximately , nats and over lincrnas. both in plants an mammalians (jin et al., ; liu, j et al., ) these lncrnas do not show association with small rnas and are also expressed at lower levels ( -fold to fold less) than mrnas. plant nat-lncrnas can overlap completely ( %) or have complementary sequences in the ' or ' regions of mrnas. they accumulate in a tissue-specific manner and many are modulated in respond to biotic or abiotic stresses, suggesting fine-tuning regulatory roles wang et al., ; ben amor et al., ; xin et al., ) . nat-lncrnas also accumulate under specific environmental conditions such as light exposure, and their expression overlaps with accumulation of histone acetylation marks, suggesting some transcription regulation effect . in plants there are other types of lncrnas. such is the case of the lncrnas involved in: ) the rna-dependent dna methylation silencing pathway (rddm); and ) the lncrnas generated from phasloci which serve as precursors of -nt and -nt secondary phased phasirnas in many plant genomes (zhai et al., ; fei et al., ; zheng et al., ) . however, the function of most plant lncrnas is still unknown (yamada, ; shafiq et al., ; ariel et al., ) . the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. many lncrnas in mammals and yeast originate from specific genomic locations such as the regions around transcription start sites (tsss), enhancer regions, intron splicing sites, and transcription termination sites. most of the studied lncrnas are expressed around the tss, including exosomesensitive yeast cryptic unstable transcripts (cuts) and stable un-annotated transcripts (suts) (xu et al., ) , and upstream antisense rnas (uarnas) (flynn et al., ) , among others. many mammalian non-polyadenylated lncrnas also correspond to divergently transcribed, exosome-sensitive ernas mapped to enhancer regions (andersson et al., ) ; so far plant ernas have not been reported. although most plant lncrnas are transcribed by rna pol ii, there are two plant-specific rna polymerases, pol iv and pol v, that also produce lncrnas (wierzbicki et al., ; li et al., ; yamada, ) . like many yeast and mammalian lncrnas, most plant lncrnas are polyadenylated, although non-polyadenylated lncrnas do exist (heo and sung, ; shin and chekanova, ; kim and sung, ; andersson et al., ) . in fact, hundreds of non-polyadenylated lncrnas induced by specific abiotic stresses were identified in arabidopsis . the expression of lncrna is regulated at transcriptional level and in combination with the pathways involved in their biogenesis, ' end processing and degradation. the main '- ' exoribonuclease complex may regulate lncrna levels since various groups of polyadenylated ncrnas were originally identified in arabidopsis exosome mutants (chekanova et al., ) . rna exosome is an evolutionary conserved cellular rna processing/degradation complex (lange and gagliardi, ). some of these lncrnas originate from the tsss of protein-coding genes, resembling cuts, or they overlap with the ' ends of protein-coding transcripts and extend into the first intron (chekanova et al., ) . gene expression can be regulated by lncrnas (either in cis or in trans) by sequence complementarity or homology with other rnas or dna, and/or by their structure; lncrnas can thus form specific scaffolds or platforms for assembly of specific complexes (chekanova, ) . most lncrnas regulate transcription. in animals this can be achieved by: ) modulation of transcription factor dna-binding activity; ) control of rna pol ii pausing; or ) recruitment of chromatin modellers, which will ultimately affect chromatin topology and nuclear organization (bonasio and shiekhattar, ) . in plants, there are two lncrnas (hid , apolo) that associate with chromatin, promote loop formation, and modulate transcription ), (ariel et al., (figure ). some lncrnas act at the post-transcriptional and translational level (chekanova, ; jabnoune et al., ) . specifically, lncrnas can act as "decoys" or mimics by blocking certain rnas and/or dnas to access their protein regulators (e.g. ips ) (franco-zorrilla et al., ; huang et al., ) . bioinformatics analyses have also identified other putative mirna target mimics in animals that act similarly to plant mirna sponges (chekanova, ) . another plant lncrna (asco) also acts as a decoy of nuclear speckle rna binding proteins leading to different alternative splicing events and consequent altered root development (bardou et al., ) . other lncrnas with biological function are the enhancerrnas (ernas), described in yeast and mammals, but still unknown in plants. these ernas can act in cis as scaffolds to recruit co-activators and thus promote chromosome looping between enhancer and promoter regions; they can interact with other lncrnas to form specific chromosome structures to control gene expression. ernas are regulated by the exosome which can affect either rna synthesis or degradation in these regions (pefanis et al. ) . the exosome can also protect ernas from genomic instability by resolving r loops, which are stable rna-dna triplexes naturally formed during transcription, which can persist in regions that have divergent transcription, eventually becoming deleterious (skourti-stathaki and proudfoot, the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. action of ernas and exosome can control gene expression and nuclear organization in these enhancer regions. interactions between lncrna and chromatin modifiers can be either dependent or independent of srnas. in animals they are srna-independent and occur via the trithorax complex (h k trimethylation) ; or prc (polycomb repressive complex , that regulates h k methylation) (tsai et al., ) . in plants they are -srna-dependent, with the rna-dependent dna methylation pathway (rddm) (matzke and mosher, ) (see below).further details on specific aspects are provided below. the rddm pathway seems to be a plant-specific pathway that relies on lncrnas transcribed from pol iv that will produce nt sirnas. in parallel, lncrnas produced by pol v act as a scaffold that can then recruit the sirna-ago complex by sequence complementarity (matzke and mosher, ) . however, pol v-dependent lncrnas are difficult to identify probably due to their low accumulation rates. nevertheless, the few identified lncrna are non-polyadenylated and can be either tri-phosphorylated or capped at their ' end (wierzbicki et al., ) . in addition, pol v also seems to collaborate with pol ii to promote rddm (zheng et al., ) . another group of regulatory ncrnas that are pol iv -rdr dependent were identified at intergenic regions overlapping mostly with transposons or sequence repeats. these ncrnas are also non-polyadenylated and at their ' end have a monophosphate instead of ' tri-phosphate or cap (zheng et al., ) . the arabidopsis exosome is involved in metabolism or processing of these lncrnas generated by pol iv, v and also some from pol ii (chekanova, ) . also worth mentioning is that the exosome is constituted by different subunits that are functionally diverse and can affect metabolism of smrnas and dna methylation (chekanova, ) . lncrnas and regulation of flowering some of the best characterized plant lncrnas are those involved in regulation of flowering upon "vernalisation" (the need of plants to experience a period of cold -winter-to enable flowering in spring). therefore, transgenic plants expressing any of these lncrnas may have biotechnological value. to assess the future relevance of these lncrnas it is important to understand the molecular mechanisms behind their function, since they are quite different from those of srnas (sirnas and mirnas).the best studied lncrnas in this process are: ) coldair (heo and sung, ) ; ) coldwrap (kim and sung, ); ) coolair (swiezewski et al., ) and asl (shin and chekanova, ) . all these lncrnas modulate the flclocus, which encodes a transcription factor able to repress flowering (berry and dean, ) . interestingly, these four lncrnas originate from different regions within the flclocus (promoter, first intron, and ' end antisense) suggesting a function in cis. they all contribute to prevent flc transcription by recruiting the prc silencing complex and the accumulation of repressive chromatin marks (crevillén and dean, ; csorba et al., ; rosa et al., ; sun et al., sza; swiezewski et al., ; liu, f et al., ) . of interest is that certain aspects of this regulation are similar to that of the mammalian lncrnas hotair and xist. it has been proposed that some of these lncrnas could directly bind to prc components, but findings showing that mammalian prc can bind very strongly to unrelated rnas (davidovich et al., ) question the need for specific lncrnas in this recruitment step. the study of components of the arabidopsis exosome (rrp l and rrp l ) revealed their role in coolair and asl expression or processing, although this seemed to occur independently of the exosome core complex. both rrp ls could process the ' end of coolair and promote asl accumulation, similarly to xist in humans (shin and chekanova, ; ciaudo et al., the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. regulate the levels of different lncrnas and probably act in different mechanisms to silence flc (chekanova, ) . lncrnas and r-loop formation coolair transcript levels are decreased due to r-loop formation at its promoter (sun et al., b) , which could promote exosome recruitment through the ' end processing pathway (chekanova, ) . this function is also seen in mammals where the exosome also resolves/degrades deleterious r loops (pefanis et al.) . surprisingly, in mutants affected in r loop formation, coolair may accumulate together with flc, suggesting that this regulation is still not fully understood (chekanova, ) . lncrnas and nuclear architecture animal lncrnas are involved in tethering rna, dna and proteins, and thus affect nuclear d structure (engreitz et al., ; quinodoz and guttman, ; hacisuleyman et al., ) . in plants, extensive evidence supports a similar role, either within the rddm pathway (moissiard et al., ) , in the regulation of flowering time (hepworth and dean, ) or in auxin signalling and root development (ariel et al., ; ariel et al., ) . plant and animal lncrnas can be intergenic, intronic or natural antisense transcripts, depending on their location in the genome. although only a few lncrnas have been characterized in detail, they are known to have different modes of action, ranging from chromatin modifications including prc recruitment, to promotion of translation, mirna target mimicry, hijacking splicing factors and formation of chromatin loops. adapted from (ariel et al., ) . not all processes occur simultaneously. different types of lncrnas are shown schematically. circrnas earlier studies of rnas structure proved that viroids (existing as uncoated rna molecules and are known to infect plants) have circular rna (circrna) molecules. the nature of covalently closed circular rna molecules was determined due to: i) the inability to phosphorylate at the '-terminus; ii) resistance to metaperiodate oxidation or borohydride reduction of the '-terminal ribose; or iii) resistance to venom phosphodiesterase degradation (sanger et al., ) the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. al., ), plants (daros and flores, ) , and mammals (nigro et al., ) including humans memczak et al., ) , and across the eukaryotic spectrum. circular rnas' lack of coding potential was identified early on by the inability of circular mrna (kozak, ) or rna (konarska et al., ) to assemble/adhere to eukaryotic ribosomes. circular rnas are also unable to be translated in plants extracts . as with other ncrnas, circrnas in plants exhibit tissue-specific expression, have a considerable number of isoforms, alternative backsplicing (canonical and noncanonical) and alternative circularization patterns (lu et al., ; sun et al., ; wang et al., ; darbani et al., ) . the biogenesis of circrnas is a conserved feature in animal and plant cells. transcribed by rna polymerase ii and backsplicing reactions of pre-messenger rnas, they occur in the spliceosome and require a repeated sequence and rna-binding proteins. spliceosome formation is initiated by sequential assembly of small nuclear ribonucleoproteins onto a specific region of the pre-mrna downstream of the ' donor splice site (dinucleotide gt) and upstream of the ' acceptor site (dnucleotide ag). thus, exonic or intronic circrnas are generated depending on the initial small nuclear ribonucleoprotein binding site (lee et al., ; sun et al., ) . although their mechanisms of action in plants are still unclear, some studies in rice suggest that they do not imply any significant enrichment (as occurring in humans) for mirna target sites, and that circular rna and its linear form may act as a negative regulator of its parental gene (lu et al., ) . other studies have shown that fluctuations in circrnas do not correlate with the levels of their parentalloci encoded linear transcripts (darbani et al., ) . some circrnas have also been shown to contain putative mirna binding sites lu et al., ) and have been identified as mirna sponges (zuo et al., ) . by binding to mirnas, and consequently repress their function, circrnas act as mirna sponge to regulate the response to stress . recent data suggest that circrnas in plants may affect plant response to abiotic and biotic stress. for example, circrnas may be involved in chilling injury in tomato (zuo et al., ) , or may respond to imbalances in iron and zinc (darbani et al., ) . in another study, tan and colleagues (tan et al., ) showed that overexpression of a tomato circrna derived from phytoene synthase (psy ), reduced psy mrna abundance, and lycopene and β-carotene accumulation. this was likely due to the continuous highly expressed circrna and/or the low abundance of linear rna from the overexpression vector. similar results were reported for another circrna derived from the phytoene desaturase gene (tan et al., ) . their role in developmental processes has also been established (cheng et al., ) , including their possible role in different aspects of development and senescence in arabidopsis cheng et al., ) . other biological processes such as photosynthesis (dou et al., ) or mitochondrion function (darbani et al., ) have also been proposed as involving circrnas. circrnas are a popular topic in animal research because of their potential as post-transcriptional regulators (memczak et al., ; piwecka et al., ; guarnerio et al., ; hansen et al., ; ashwal-fluss et al., ) , their recognized function as mirna sponges, as sponges for rna-binding proteins or their competition with linear splicing; and their role as diagnostic markers (zhao et al., a; zhao et al., b) . indeed, circrnas have also been found in biological fluids including saliva (bahn et al., ) , seminal fluid (dong et al., ) and plasma . research in plants is just emerging and functional studies are still lacking. it is unknown if this type of novel ncrnas could resist gastrointestinal tract conditions, due to their expected high stability, and have a biological effect on an organism. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. ncrnas can regulate many biological processes through their impact on gene expression by acting on chromatin structure and affecting transcription, rna processing, rna stability and translation. among these, srnas are produced by cleavage of dsrnas intermediates, either from hairpin precursors (mirnas) or from the synthesis of dsrnas by rdrs (sirnas); most lncrnas regulate gene expression without being processed. despite size and biogenesis differences, most ncrnas share sequence-specific inhibitory functions. important differences are present between plants and other organisms in srnadirected silencing pathways, which are highly diversified and specialized in plants through the expansion of rdr, dcl and ago proteins that mediate srnas biogenesis and function. the vast majority of small regulatory rnas in humans and animals are mirnas. in contrast to plants, no evidence for systemic spreading of rna silencing in humans and vertebrates have been found. although very few plant lncrnas have been studied in detail, they seem to share with their animals' counterparts the ability to recruit chromatin modifiers and thus regulate gene expression. in the same context, very few studies are available on plant circrnas. their functional characterization will bring novel insights into their possible role as a novel class of noncoding regulators. when considering the potential presence of plant ncrnas in food or feed it is important to determine the mobility potential of these molecules inside the source plant (figure ). there are several comprehensive reviews (chitwood and timmermans, ; dunoyer et al., ) addressing sirna/mirna movement in detail. it has been hypothesiezed that srnas may move throughout the plant. although the initial model would suggest non-cell autonomous (one whose action extends beyong the cell producing the signal) rna silencing by srnas, other intermediates could also account for this signal: dsrnas produced by rdr activity (plant specific), or fold back rna acting as silencing trigger (dunoyer et al., ; dunoyer et al., ; himber et al., and smith et al., ) . these intermediates could also diffuse from one cell to another and be processed again by dcl , which has been associated with nt sirna movement (dunoyer et al., ) . in detail, secondary sirnas processed from dsrna by dcl could move cell-tocell to propagate silencing by signal amplification. it has been shown that dcl expression from phloem companion cells is critical to the short-term spread of the silencing effect of a sirna duplex. this was reported in a study in which the viral suppressor p was found to be expressed in phloem companion cells and able to bind to nt small dsrna (vargason et al., ) . dcl -dependent sirna movement has also been shown to occur within certain organs, such as leaves, to create gradients (chitwood et al., ; levine et al., ) . other dcls-dependent sirnas are involved in sirna movement and these can also be sorted into different risc complexes (montgomery et al., ; mi et al., ) . other examples of mobile sirnas could be the ta-sirnas and dcl -dependent nt sirnas, also acting through ago . dcl processing of long dsrna substrates generated by pol iv (a plant-specific rna polymerase) and rdr (another plant-specific protein) leads to production of nt sirnas that are incorporated into ago and will transcriptionally silence transposons (chapman and carrington, ; mi et al., ) . in fact, sirnas derived from transposons or methylated regions in the genome have been shown to be associated with chromatin silencing. these -nt mobile sirnas could then promote epigenetic changes transmissible to following generations. when transgenes are expressed in plants, the generated dsrnas can be processed by dcl and dcl . another dcl, dcl , processes dsrnas into nt sirnas which can induce gene silencing of viral origin or from transgene expression (deleris et al., ) . sirnas may also function as mobile signals able to promote epigenetic modifications. grafting experiments with roots from dcl dcl dcl mutants and shoots expressing gfp sirnas could the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. indicate that sirnas to nt long move throughout the plant, but still unclear if in free or proteinbound form (i.e. bound to ago or other proteins) (molnar et al., ) . within the plant, sirnas are known to move in a source-to-sink direction and into growing meristems (molnar et al., ; palauqui et al., ; schwab et al., ) . their accumulation in reproductive cells could promote epigenetic changes in subsequent generations. in fact, slotkin et al. (slotkin et al., ) showed that dcl dependent sirnas generated by transposon activation in the pollen vegetative nucleus can silence transposons in pollen sperm cells. these cells are then able to transmit genetic material to the following generation. a similar effect may occur if maternally-derived sirnas in the endosperm moved to the embryo and influence genome integrity or even lead to the creation of new epialleles (molnar et al., ) . similar findings of sirnas silencing transposable elements in the animal germ line have also been reported in an animal cell line and tetrahymena (malone and hannon, ). considering that plant development relies more on positional effects than cell lineage, the role of mirnas in the control of several developmental processes has to be strictly regulated at the mobility level (chitwood et al., ; levine et al., ) . for example, as small rnas diffuse into regions of low mrna (targets) expression, it eliminates target molecules therein, but cannot affect regions of high mrna levels (levine et al., ) . as mentioned previously, dcl processes imperfect hairpins into nt mirnas, with a ' u nucleotide incorporated into ago , and cleavage the mrna targets. ago can also incorporate these mirnas (brodersen et al., ) . while sirna movement is better understood, this is not the case for mirna movement. mirnas can be isolated from phloem sap (mir , mir , mir ) but it is unclear if they can leave the phloem (buhtz et al., ; pant et al., ; yoo et al., ) . some reports support both endogenous mirna and artificial mirna movement (chitwood et al., ; schwab et al., ) . it has been shown that mirnas can move from below the shoot apical meristem into the meristematic layers (chitwood et al., ) , and, similarly to sirna movement, from the phloem towards the root meristems (molnar et al., ) . although initial evidence suggested sirna mobility, with only dcl responsible for this, it is now accepted that almost every known rnai pathway in plants has non-cell autonomous activity. movement has been shown for srnas ) of viral origin; ) induced by transgene expression; ) ta-sirnas; ) mirnas and ) repeat-associated sirnas (dunoyer et al., ; carlsbecker et al., ; chitwood et al., ; molnar et al., ; slotkin et al., ) . however, it is still unclear if srnas can move in a free state or associated with specific proteins, and in a single or double strand form. whereas some reports suggest that the srna molecule movement happens in a duplex form, the strand bias found in the srnas present in dcl dcl dcl grafted roots suggests that single strand sirnas can also diffuse (molnar et al., ) . unlike in animals, in which sirnas can move from cell-to-cell through secretory vesicles (ruvkun, ; valadi et al., ) , in plants the size of small rna would allow cellto-cell movement through plasmodesmata channels connecting different plant cells (kobayashi and zambryski, ) . this movement seems to occur in a source-to-sink direction, and could be regulated in some tissues through the formation of sirnas-ago complexes (molnar et al., ; schwach et al., ) . www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. plant small rnas (srnas) can move either cell-to-cell or long distance. cell-to-cell movement occurs through plasmodesmata (green arrows) that allow spreading of srnas from the cell where they are generated to - neighbouring cells (a). this cellto-cell movement can be extended over the surrounding cells by signal amplification (b). in this case, srna targets will be converted into new dsrnas by the combined action of dcls and rdrs. several grafting experiments have demonstrated that - nt sirnas can move over long distances in the plant (c). whether they move as ssrna, dsrna or associated with proteins is still unclear. adapted from (dunoyer, p. et al., ) , (melnyk et al., ) and (molnar et al., ) . in terms of mirna mobility, it is still unclear if all mirnas, or only a subset, can diffuse. different mobility could be associated with different tissues/structures, and/or the size and stability of the passenger strand mirna (a.k.a. mirna*, star strand). in addition, mirna movement regulation could involve subcellular compartmentalization (by nuclear export proteins such as hasty (park et al., ) ) and sequestration via ago proteins (e.g. mir / can be sequestered by ago and affect shoot apical meristem differentiation (liu et al., ; tucker et al., ) ). most of the information is available for srnas since they represent the most studied class of ncrna to date. in addition to the role of srna movement when considering ingestion of transgenic plants expressing altered levels of srnas, it is also important to address the issue of their biogenesis and turnover to assess their temporal availability when ingested. this is especially important since transgenic plants expressing rna interference constructs will accumulate the different srna intermediary forms (pri-mirnas, pre-mirnas, mature mirnas, drnas, sirnas), which may also be processed outside the plant when ingested. several reviews published in high-impact journals (rogers and rogers and chen, ; or scientific reports the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. view of the processes of mirna maturation and degradation in plants. it is important to consider the homology between the mirna biogenesis pathways in plants and animals as well as mirna stability. this knowledge allows understanding ) whether any aspect of animal metabolism or other biological processes could be modulated by plant mirnas; and ) if mirnas can survive for long periods after ingestion. as typical pol ii transcripts, pri-mirnas are stabilized by addition of a ' -methylguanosine cap (xie et al., ) and a ' polyadenylated tail (jones-rhoades and bartel, ; zhang et al., ) . mirna genes are also subjected to transcription factor regulation and can be alternatively spliced. the size of these transcripts varies and their processing normally occurs in a base-to-loop direction (rogers and . in plants, there are multiple dcl endonucleases that possess dexd/h-box rna helicase, duf , paz, tandem rnaseiii, and dsrna-binding domains. dcls cleave the dsrna precursor generating a -nucleotide ' overhang (margis et al., ) . processing of the pri-mirna requires at least two catalytic cycles to free the mirna:mirna* duplex. in animals, this requires sequential cleavage of the pri-mirna in the nucleus first and then in the cytoplasm by different rnase iii enzymes. the biogenesis of most mirnas in arabidopsis requires dcl (park et al., ; reinhart et al., ) , although other dcls may be involved, but are not essential (xie et al., ; gasciolli et al., ) . in contrast to animals, all plant mirna processing steps occur in the nucleus (papp et al., ) . dcl activity will result in mature mirnas of - nucleotides (nt), although variations in size can occur (rogers and chen, ) . confirming its fundamental biological relevance, dcl mutants are lethal (golden et al., ) . other dcl proteins that function in different srna biogenesis pathways might also have minor roles in mirna biogenesis. for instance, several mirna genes are partially processed into - -nucleotide mature mirnas species whose accumulation depends on dcl (vazquez et al., ) . in the absence of dcl , mature -nucleotide mirnas can accumulate due to dcl activity. in contrast, processing of mir , mir , and mir depends primarily on dcl , probably as a consequence of highly complementary fold-backs in these pri-mirnas (ben amor et al., ; rajagopalan et al., ) . dicer cleavage of animal pri-mirnas is facilitated by the action of dsrnabinding domain (dsrbd) proteins (drbs) (jiang et al., ; parker et al., ) . in arabidopsis, there are five drbs that have been shown to bind dsrna in vitro (hiraguri et al., ) . the best studied member of this family is hyl (hyponastic leaves ) which is involved in pri-mirna processing, but also in pri-mirna intron splicing (laubinger et al., ; szarzynska et al., ) . hyl seems to increase the accuracy of dcl processing of most, but not all, pri-mirnas, suggesting that other mechanisms may also be involved (liu, c et al., ) . from the same family, drb and drb are also involved in pri-mirna processing, by associating with dcl (rogers and . another protein involved in mirna biogenesis is serrate (se), which can interact with hyl and dcl (lobbes et al., ; machida et al., ) . like hyl , se also seems to increase dcl pri-mirna cleavage efficiency (dong et al., ) . of interest is that these two proteins (hyl and se) can be regulated by phosphorylation. although hyl phosphorylation can decrease its activity (manavella et al., ) , the biological relevance of this modification in se is not known (rogers and . another regulator, dawdle (ddl), a phosphothreonine-binding forkhead associated (fha) domain protein, seems to stabilize pri-mirnas, possibly by direct binding (yu et al., ) . like se and hyl , ddl can also interact with dcl , but does not seem to be involved in pri-mirna processing. ddl most likely facilitates dcl access or recognition of pri-mirnas. in addition, ddl can recruit phosphorylated se or hyl to the pri-mirna. the human homologue of ddl, snip , seems, among other functions, to be involved in mirna biogenesis and interacts with drosha (yu et al., ) . ddl could therefore be an evolutionarily www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. conserved factor in mirna biogenesis. other examples of conserved srna biogenesis machinery include ago, dicer, hen and exportin (yu et al., ) . two components form the cap structure of rna pol ii transcripts in plants: cap-binding proteins (cbps) and . there is substantial overlap among cbp , abh /cbp , and se mutants in terms of: ) the specific pri-mirnas affected; ) the specific mrna splicing defects observed, and ) a bias for accumulation of first introns (laubinger et al., ) . this connection between pri-mirna processing and pre-mrna splicing seems to converge at se and cbps. in fact, splicing defects are also observed in cbp and abh /cbp in plants. in animals, the cap and its associated cap-binding complex are essential for the correct splicing of the first intron (lewis et al., ) . the connection between pri-mirna processing and splicing also converges in tough (tgh), a g-patch domain rna-binding protein. the tgh mutant accumulates pri-mirnas in vivo and has been shown to affect processing of pri-mirnas into -nucleotide mature mirnas (ren et al., ) . tgh may have either a structural or a regulatory role in pri-mirna processing. tgh binds both pri-and pre-mirnas in vivo, but this probably occurs via association with the loop structure since tgh binds ssrna but not dsrna in vitro (ren et al., ) . tgh paralogs have been described in metazoans (calderon-villalobos et al., ) , but their roles in mirna biogenesis are still unknown. a human tgh paralog is reported in spliceosomal preparations (jurica et al., ) . a conserved connection may exist between tgh and splicing and possibly pri-mirna processing (rogers and chen, ). mirna processing seems to occur in specific subnuclear regions. hyl , se and dcl can make pairwise interactions that occur in certain subnuclear particles (fang and spector, ) . tgh can also interact with dcl , hyl , and se in subnuclear foci (ren et al., ) . these foci seem to be zones of functional pri-mirna processing (fang and spector, ; fujioka et al., ; manavella et al., ) . some of these components (dcl , hyl , se, tgh) also co-localize with components of the splicing machinery, further strengthening the connection between the two processes (calderon-villalobos et al., ; fang and spector, ; fujioka et al., ) . processed mirna:mirna* duplex will leave the nucleus to carry out its function in the cytoplasm. several components have been implicated in this transport and function and are described below. nucleo-cytoplasmic transport hasty (hst) is a member of the importin-ß family of arabidopsis that accumulates in the nuclear periphery (bollman et al., ) . hst is a paralog of human exportin- involved in pre-mirna transport. hst can participate in dcl -dependent mirna:mirna* duplex export (zeng and cullen, ) . but this is probably not the sole mechanism for mirna nucleocytoplasmic transport, since passive diffusion via the nuclear pore may also occur (jacob et al., ; park et al., ) . ema /sad is another importin-ß family protein involved in mirna function. ema is an orthologue of human importin- which facilitates nuclear import of ago and reduces its association with target mrnas (weinmann et al., ) . it is still unclear if ema ) sequesters mirnas; ) is involved in transport of other risc loading factors or; ) affects loading of certain mirnas into risc (rogers and . the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. after leaving the nucleus the mirna:mirna* duplex is loaded into ago, a risc component. the arabidopsis ago family consists of members that associate with small rnas possessing specific ' nucleotides (mi et al., ; takeda et al., ) . in plants, the slicer activity of agos has been shown for ago , ago , ago , ago , and ago (rogers and . ago seems to have a predominant role since it preferably associates with 'u small rnas (most common ' nucleotide in mirnas) (mi et al., ; takeda et al., ; vaucheret et al., ) . however, other agos can also associate with specific mirnas depending on mirna length or even their accumulation in different tissues (ebhardt et al., ; rogers, kestrel and chen, xuemei, ) , suggesting a certain flexibility in this association. in plants, mirna guide strands generally accumulate in higher levels than mirna* strands, although the exact mechanism for strand selection is still not fully understood (rogers and chen, ). hyl (drb) seems to be involved in this process (eamens et al., ; manavella et al., ) . in animals, loading of sirnas into risc requires the cytosolic drb protein loq, but loading of mirnas into risc requires dicer rather than loqs (liu et al., ; liu, x et al., ) , suggesting different mechanisms in plants and animals. hyl is predominantly nuclear, therefore its association with the mirna:mirna* duplex suggests that ) hyl may be transported to the cytoplasm together with the processed mirna duplex; or ) loading may also occur in the nucleus. in addition, other risc loading mechanisms can exist (eamens et al., ). in animals, sirna passenger strand unwinding requires ago slicer activity; mirnas duplexes are unwound by a slicing-independent mechanism (matranga et al., ) . in plants, ago slicer activity removes sirna passenger strand but this function is not required for mirna passenger strand removal (carbonell et al., ; iki et al., ) . as occurs in animals, plant mirnas use an alternative slicerindependent unwinding mechanism. ago loading seems to require heat shock protein (hsp ) too (iki et al., ) . in addition, via hsp , ago is able to interact with squint (sqn) and the phosphatase pp (iki et al., ) . these interactions seem to regulate ago loading and consequently risc activity. ago phosphorylation has been detected in arabidopsis (de la fuente van bentem et al., ) in manner similar to human ago . apparently, phosphorylated ago exhibits reduced srna binding (rüdel et al., ) . phosphoregulation by pp or other proteins may modulate arabidopsis ago loading capacity. unlike in animals, in which mirna biogenesis occurs in the nucleus and cytoplasm, the mirnas in plants are generated in the nucleus in a mostly dcl- -dependent manner. studies confirm that several of the major components are evolutionary conserved, such as the dicer, ago, hen and exportin paralogs involved in nucleocytoplasmic transport of the mirna:mirna* duplex. however, shared pathway components among the different species do not reflect totally identical processes, such as, loading of mirnas into risc which occurs differently in plants and animals. one interesting common aspect remains the pri-mirna processing and pre-mrna splicing which seems to occur both in plants and animals. www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. following a systematic literature search as described in section . . . and following the methodology described in section . . . ., a total of publications were selected as relevant to the stability and turn-over of non-coding rnas. of these, only four analyse ncrnas stability at a genome-wide scale, and one (enuka et al., ) simultaneously addresses the stability of several circrnas. using these studies, the stability of small ncrnas, lncrnas and circrnas can be compared. while earlier studies report the degradation of plant nucleic acids when exposed to hard conditions (i.e. cooking), six recent studies suggest that some ncrnas (i.e. mirnas) from plants can resist these conditions. turnover of ncrna molecules in plants and mammals depends both on their stability and degradation rate and it is described that chemical modifications can increase stability of different types of srnas (e.g. sirnas, mirnas). in mammalian cells, artificially introduced -o-methyl groups can stabilize sirnas without affecting their rna interference activity (czauderna et al., ) . considerable work has been done to address the issue of mirna modifications and how these affect their stability. among these, '-o-methylation has been identified as relevant. plant mirnas have a naturally occurring methyl group on the ' nucleotide ribose ( figure ). in this case, methylation does not require guide rnas, since hen (hua enhancer ) can methylate mirna:mirna* duplexes. this hen -dependent '-omethylation on the ' terminal ribose is a mg + dependent methylation mechanism that will ultimately stabilize mirnas (abe et al., ; molnar et al., ; yu et al., ; yang et al., ) .the hen mg +-dependent methylation mechanism relies on its two dsrbds binding the substrate duplex, and the la motif-containing domain interacting with the ' end of the substrate strand (huang et al., ). this methylation step occurs after dcl processing creates the mirna:mirna* duplex but before duplex www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. unwinding and selection of the mirna guide strand. however, it is still not clear whether hen can methylate free mirnas or can bind hyl , dcl and ago , although it has been shown to interact with hyl and dcl . in animals, the hen homolog has a different structure since it does not possess the dsrbd and la motif-containing domains. therefore, the animal hen homolog methylates singlestranded srnas associated with ago or piwi proteins (saito et al., ; horwich et al., ) . the degradation of ncrna molecules in plants and mammals is mediated by ' nucleotidyl transferases and exoribonucleases. the arabidopsis hen suppressor (heso ) belongs to the dna polymerase β gene family (zhao et al., ; ren et al., ) . in other organisms, heso putative homologs are ribonucleotidyl transferases that are able to add specific nucleotides to the ' end of different rnas (martin and keller, ) . arabidopsis heso can also add nontemplated nucleotides to the ' end of unmethylated mirnas in vitro and exhibits a preference for uridine (ren et al., ; zhao et al., ) . analysis of hen and heso mutants indicates that this ' oligo uridylation triggers degradation of mirnas (ren et al., ; zhao et al., ) . the arabidopsis family of small rna degrading nucleases (sdns) of '- ' exonucleases has similarity to the yeast rex exonucleases. sdn has '- ' exonuclease activity versus short ssrnas but is not active against longer or double-stranded substrates (ramachandran and chen, ) . in plants, ' truncated mirnas can be modified by the addition of ' oligonucleotide tails (see above) (ibrahim, fadia et al., ; li, j et al., ; lu et al., ). in addition, these ' truncated mirnas also seem to associate with ago . this interaction delays their degradation and allow addition of the ' oligonucleotide tail leading to their degradation at a later stage (zhao et al., ) . whereas '-o-methylation may protect against ' oligouridylation by heso (see above) and consequent targeting to degradation, this modification would not protect against sdn . it is possible that sdn can promote mirna ' truncation and then facilitate subsequent ' polyuridylation by heso . however, after ' polyuridylation sdn would be unable to degrade these tailed mirnas, suggesting the existence of other nucleases (ramachandran and chen, ) .for instance, the addition of ' polyuridylated tails in mirnas seems to attract exosome-mediated degradation in algae (ibrahim, f. et al., ) , but in arabidopsis this regulation has not been addressed in detail (rogers and rogers, kestrel and chen, xuemei, ) . another possible mechanism would include loss of ago association and protection of mirnas with long ' tails due to heso activity. in addition, ' polyuridylation may act as a nucleases recruitment platform, thus promoting mirna degradation. nevertheless, ' polyuridylation and nuclease processing seem to be interconnected events that will ultimately result in degradation of the tagged mirnas. the overall turn-over of rnas is mainly described using half-life studies in both plants and animals. most of the available reports assessing ncrna half-life focus on the role of ' truncation, ' uridylation and consequent degradation (see above) as the main processes regulating mirna turnover in plants. however, a recent report identifies the enzymes (sdn and sdn exonucleases) responsible for ' truncation in vivo and its relationship with ' tailing (uridylation). also of importance, this report shows the opposite role of ago and ago in mir / degradation by sdns, specifically sdn . ago promotes mir degradation by sdn , an unexpected role for a plant ago which has been associated with a protecting role in mirna stability (yu et al., ) . this function of ago (enhancing mir degradation) keeps mir out of the stem cell niche in the shoot apical meristem (where ago is not expressed). accumulation of ectopic mir in the shoot apical meristem fails to maintain the stem cell population. this kind of mechanism is thus needed to clear mir since this particular mirna can move between cell layers (see mirna movement section, above). another report evaluates the activity of nucleotidyl transferases in ' uridylation of mirnas and in vitro assays indicated a fast enzymatic process (tu et al., ) . also, xrn - nucleases that affect mir homeostasis in c. elegans do not www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. seem to process mature plant mirs but rather the by-products of pri-mirna processing (ji, lijuan and chen, xuemei, ) . in summary, there are no specific studies on mirna half-life in plants. the reports mentioned here evaluate mirnas levels at steady state in different mutants, for example, when either the exonucleases and/or nucleotidyl transferases involved in mirna turnover are deleted. the half-life of mirnas in mammals has been addressed in studies of dicer ablation in mouse embryonic fibroblasts showing that mirnas are highly stable inside the cell. indeed, turnover assays revealed that the average mirna half-life is h (i.e. ≈ days) (figure ). although some mirnas have a shorter half-life, these data generally indicate that mirnas are much more stable (≈ x more) than messenger rnas. (gantier et al., ) . several features have been described that might explain this high half-life for mirnas. for instance, partial pairing of mirna and the mrna target site generally produces translational repression, while extensive pairing of mirna and mrna produce a mrna cleavage (pasquinelli, ) . in the former case the mirna remains stable, but in the latter it degrades. further studies in mammalian cells have also shown that argonaute proteins stabilize mature mirnas in a slicing-independent manner, increasing mature mirna stability (winter and diederichs, ). for specific mirnas, it has been found that certain rna-binding proteins (i.e. roquin) can enhance mature mirna- stability by reducing its ' end uridylation (srivastava et al., ) . while intracellular levels of mirnas are mostly affected by cell division (ghosh et al., ; gantier et al., ) , mirna activity and turnover can also be controlled by subcellular distribution of microribonucleoproteins; that is, polysome sequestration contributes to an increase of cellular mirna levels, but without an increase in mirna activity (ghosh et al., ) . plant small rna duplexes can be '-o-methylated at their ' terminal ribose by hen , loaded into ago and protected from uridylation and degradation by '- ' exonucleases. ago -bound unprotected small rnas, however, are uridylated by heso and degraded by sdn . adapted from (borges and martienssen, ) and (ji and chen, ) . the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. widespread variation of mirna decay in slow-dividing primary macrophages. bone marrow derived macrophages from when analysing lncrnas stability in mammals at a genome-wide scale, lncrnas half-life generally varies over a wide time range, comparable to, although on average less than, that of mrnas (clark et al., ) . in a study of all transcripts, including mammals, it was found that the range of half-lifes for ncrnas (all types) was similar to that of the mrna half-life (tani et al., ) . indeed, a large number of ncrnas transcripts have a half-life of less than h (tani et al., ) , which agrees with the < h half-life for hundreds of unstable lncrnas (clark et al., ) . however, although there are lncrnas with extreme stability (half-life > h) they are still much less stable than mirnas (gantier et al., ) (see above), but more stable than protein-coding rnas (wang, l et al., ) . circrnas have a covalently closed loop structure with neither '- ' end nor a polyadenylated tail, which confers high resistance to rna exonuclease or rnase r treatment when compared to that of their linear sequence counterparts (memczak et al., ) . indeed, earlier studies of the structure of rnas from viroids, which were found to be circular, suggested that the nature of covalently closed circular rna molecules confers certain properties including resistance to metaperiodate oxidation or borohydride reduction of the '-terminal ribose; the inability to phosphorylate at the '-terminus; or resistance to venom phosphodiesterase degradation (sanger et al., ) . in mammals, some studies have evaluated the stability of circrnas compared to that of their linear isoform derived from the same host gene. for example, circrnas were found to be less abundant and dynamic than their counterparts (enuka et al., ) . calculating the half-life of circrnas and their linear counterparts, enuka et al., showed that the media half-life of circrnas of mammary cells ( . - . h) was at least . times longer than the median half-life of their linear counterparts ( . - . h). this suggests that cirrnas are generally more stable and static compared to linear species (enuka et al., ) (figure frame a). in a stability study on some circrnas it was found that while the associated linear transcripts exhibited a half-life of < h, the circular rna isoforms were highly stable, with transcript half-life exceeding h (jeck et al., ) . in plants, covalently circularized exogenous rna incubated with wheat embryo cell extract in vitro have shown that the circular rnas exhibited considerable stability compared to the linear version (makino et the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. al. , ) . while linear rna degraded within the first few minutes, its circular counterpart was stable for more than min in vitro (figure frame b). by contrast, circularization dramatically reduced the translation capability of the rna, which was restored to some extent after linearization. plant srnas are naturally methylated at their ' nucleotide ribose. this modification depends on hen and protects srna from degradation. unmethylated or truncated srnas can be uridylated by heso and thus be targeted for degradation by '- ' exonucleases of the sdn family. although ago binding to mirnas was believed to protect them from degradation, recent reports suggest that ago in particular may promote mirna degradation by sdns. differently from the plant situation, animal srnas are not methylated. a) mcf a cells were metabolically labelled using su, for or h. the rna was then extracted, biotinylated and purified on streptavidin magnetic beads. flow-through rna was also collected. next, rna was reverse transcribed and quantified using highthroughput real time pcr (fluidigm). the half-lives of circrnas and their corresponding linear counterparts are shown. halflife values were calculated from two samples, labelled with su for or h and then averaged. all data were corrected for any bias introduced due to low uridine (short length) of rna species (see 'materials and methods' section). the circrnas (red dots) and their linear counterparts (blue dots) were sorted from high to low according to the difference between their half-lifes. error bars represent standard errors. source: from (enuka et al., ) . b) stability of circular rnas in the wheat germ cell-free translation system. a denaturing polyacrylamide gel separating the synthesized circular and linear rnas with a (gaaa) sequence after incubation with the wheat embryo extract for the indicated time periods. untreated rna is in lane n. positions of circular, linear, and ribosomal rnas are indicated as c, l, and rrna, respectively, on the right. source: from . compelling evidence supports the significant contribution of srnas to communication between hosts and some eukaryotic pathogens, pests, parasites, or symbiotic microorganisms. (baulcombe, ; (ghag et al., ; koch et al., ; vega-arreguín et al., ; helber et al., ) . srnas or dsrnas, efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. for example, can be transferred from plant to pests such as insects that eat leaves or nematodes that infect roots. in fact, transgenic plants can be created that express dsrna homologous to essential genes of insect pests or nematodes and thus control them (baum et al., ; fairbairn et al., ; mao et al., ) . thus, this silencing transfer mechanism is very relevant for the food and feed risk assessment of ncrna gmo plants, highlighting the need to evaluate the stability of ncrnas outside the plant. earlier studies suggested that food nucleic acid content was hydrolysed when cooked (colling and wolfram, ) . most of ncrnas, including srnas and lncrnas have been discovered in the last years, and thus new data has been added to the body of knowledge. in a study of srna stability under cooking conditions, plant mirnas were detected after cooking although at significantly lower levels when compared to fresh plant tissues (see table ) (zhang, lin et al., ) . differences were observed for mirnas from different food/feed sources (rice, cabbage, wheat or potato), ranging from nearly undetectable values to almost % persistance of mirnas after cooking. levels of mirnas were monitored with a stem-loop quantitative reverse transcription polymerase chain reaction (qrt-pcr) assay using the u snrna for normalization, a commonly used housekeeping gene in mirnas analyses. the same study found that most of the plant mirnas and mammalian mirnas could survive under acidic conditions that mimic the gastric acidic environment. of note is that the degradation rate of mammalian mirnas under acidic conditions was similar to that of their synthetic form (without '-omethylated ' ends), whereas plant mirnas had a much slower degradation rate compared with their synthetic form (without '-o-methylated ' ends), suggesting that methylation had a protective effect on the stability of plant mirnas (table ) . another study reported that endogenous mirnas can be detected by qpcr analysis in rice and soybean plant materials after storage, processing and cooking (philip et al., ) . for processing and cooking, soybeans were soaked in rnase-free water with . % w/v nahco overnight at ºc, then separated from the soaking liquid, rinsed in fresh rnase-free water, and then boiled in rnase-free water for www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. min until they became soft in texture. by constrast, a synthetic mirna showed a significantly higher susceptibility to simulated food processing conditions as compared to plant mirnas, as well as high molecular weight rnas (total rna). the synthetic mirna used for comparisons was the cel-lin- , which is a caenorhabditis elegans mirna. the synthetic mirna was synthesized without '-o-methylated ' ends (philip et al., ) . these results suggest that the methylation and the small size of plant mirnas make them more resistant to degradation than synthetic (not chemically modified) mirnas. in addition, the study showed significant plant mirna stability in a simulated digestion system for min prior to absorption or transport into the blood stream. however, this study provided most of the quantitative data from q-pcr analysis without normalization, and data should be considered as relative compared to the treated control in the experiments. studying the stability of plant srna in vitro, liang and colleagues exposed total rna ( µg), isolated from brassica oleracea, to freshly drawn serum from mice. in serum, a large amount of srnas survived after h of incubation at ºc, but after h only about % of the rna could be detected. the authors also exposed the plant rna molecules to fecal suspensions at ºc. after h incubation significant amounts of srnas were still detectable, but after h rna molecules were undetectable . suggesting that srnas were more resistant to degradation in the presence of serum suspension than in fecal suspensions. these results were produced by gel electrophoresis of srnas, which is known to be less sensitive than qrt-pcr or rna sequencing methods. this study also reports that, when orally ingested, plant mir (chosen because it is the most highly enriched plant mirna from b. oleracea) can pass intact through the gastrointestinal tract in mice (a maximum of . % recovered from the stomach of some individuals), and can be detected in the blood, liver, spleen and kidney. levels of mir were monitored by qrt-pcr using a taqman probe, which can distinguish differences of one nucleotide. since only one mirna was evaluated, the above findings cannot be generalized to other mirnas. other studies have reported mirnas to be stable in watermelon and mixed fruit juices produced by simple extraction without additives . plant mirnas were detected by stem-loop qrt-pcr using taqman probes and some were validated by northern-blot analysis. to reduce the nonspecificity signal of qrt-pcr, no-template controls were used. yang and colleagues studied artificial in vitro digestion systems that simulate mammalian gastric and intestinal conditions, and found that mir was between - fold more stable than other mirnas . this effect was observed for cabbage extracts when compared to either the 'o-methylated or the non-modified form. detection was also done by qrt-pcr. the same research group reported recently that several plant mirnas, including mir , were degraded during storage at ºc in cabbage extracts obtained by mechanical maceration . degradation of these mirnas correlated with that of s rrna, whereas mir accumulation was increased over -fold after hours of storage, and then gradually decreased over a span of days. mirna (mir ) increasing in macerated tissues (rather than degrading) is uncommon for most mirnas. indeed, the authors indicated that, in terms of genesis and amplification, mir is an atypical mirna, which could explain the disparity in serum detection with respect to canonical plant mirnas. thus, mir was shown to be derived from s rrna and processed in a dcl -independent manner, meaning that when s rrna is degraded mir is produced. this study presents the possibility that certain ncrnas can have non canonical features for synthesis or stability, and should be evaluated individually. in another recent study, the in vitro stability of curcuma longa mirna (clo-mir ) was assessed. when exposed to foetal bovine serum (fbs) for up to h, the mir exhibited high resistance . in summary, food nucleic acid content are generally hydrolysed during processing, with some studies suggesting that mirnas from plants may be more resistant to degradation than synthetic or animal www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. mirnas due to endogenous modifications ( '-o-methylated ' ends). however, these findings are not consistent with mirna stability studies in the animal gastrointestinal tract, and further research is required. although there are many studies on plant ncrnas stability (see . . . .), these mainly focus on ncrna stability within the plant itself. mammalian ncrna stability differs from that of plants because lack the natural '-o-methylation at the ' ends occurring in plant ncrnas, mostly in mirnas (yu et al., ) . very few reports were identified in which plant ncrna stability is investigated in a mammalian organism ) (see sections . . and . .) . understanding the molecular basis that confers plant ncrna stability is of vital importance and needs to be further evaluated to better inform on the relevance of plant ncrna for food and feed risk assessment. for instance, modifications plant mirnas not occurring in mammals, such as the methyl group located on the ' nucleotide ribose, could hypothetically have an impact on plant mirnas stability in mammalian systems due to the lack of the appropriate enzymes for their recognition and degradation, or due to increased stability to mammalian rnases . should this be the case, plant mirnas could manifest a much longer than expected half-life in mammalian organisms when compared to mammalian mirna, which could increase the probability of encountering target molecules. the literature contains descriptions of possible in vitro interaction of plant mirnas with mammalian mirna silencing complexes which may trigger target repression chin et al., ) . however, this hypothesis needs to be experimentally validated since there are gaps in the literature. moreover, it needs to be determined if sufficient levels of ingested plant mirnas reach a target cell to determine an effect in mammalian cells. for instance, in mammalian studies it has been suggested that the threshold for target gene regulation is between and copies of mammalian mirna per cell . accumulation of plant exogenous ncrnas has not yet been reported, but recent next generation sequencing studies have reported a plethora of plant mirnas and other rnas in circulating in the human organism (see section . ). the biological significance of their presence is still unknown. it remains unknown if under certain pathological conditions (i.e. compromised intestinal permeability or renal function) exposure to these plant ncrnas could change. all these gaps in the literature would need to be experimentally validated. to determine the half-life of plant ncrnas in mammalian cells would require experiments utilizing labelled plant ncrnas, first transfected into mammalian cells (in vitro studies) and then orally administered to experimental animals (in vivo studies). the markers used for these experiments could be radioactive probes or labelling molecules of very low molecular weight, such as biotin or fluorescein, to prevent distorting the actual size of the plant ncrna under study. due to their small size, this is important when studying mirnas stability. there is evidence that certain plant ncrnas can induce silencing in some eukaryotic pathogens, pests, parasites or symbiotic microorganisms as a defence strategy. in return, various pathogens develop mechanisms to evade this defence system, proving the existence of a two-way sirnas traffic between pathogens and their plant hosts. plant ncrnas stability outside the plant and in animal systems may play a role in cross-kingdom effects but has received limited attention. the resistance of plant ncrna to the gastrointestinal environment or processing has been partly investigated but more research would be needed. efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. following the methodology described in the section . . . and based on a literature search as described in section . . . . , a total of documents were selected as relevant to describing the state of the art in the field of ncrnas used/intended for use as therapeutics in humans (pharmaceuticals/medicine area). this constitutes valuable preparatory material on the pharmacokinetics (and pharmacodynamics) of ncrna, as required by task . of these publications at least indicate that to ensure the biological activity of exogenous rnas for therapeutic requires numerous chemical modifications and delivery methods. although composed mainly of only four basic building blocks, rnas form simple to very complex structures. its intrinsic base-pair property, enhanced by the ability of the extra hydroxyl groups in the ribose sugar to form hydrogen bonds, are the foundations of its diverse structure and functions (lu et al., ) . rna can fold into various secondary structures including stem, loop, bugle, pseudoknots, gquadruplex and kissing hairpin. it can perform a wide range of biological functions, ranging from regulation of gene expression at various levels to catalysing chemical reactions which closely depend on the ribonucleotide chain's spatial structure. other rnas, such as ribozymes (doudna and cech, ) , can form tertiary structures with catalytic activity. certain rnas can also undergo transformation between alternative structures, depending for instance on binding to specific ligands or environmental changes, as is the case of riboswitches (mironov et al., ) . riboswitches are noncoding rna structures located within mrnas that bind endogenous ligands to regulate gene expression or rna splicing events (cheah et al., ) . the sirna mechanism of action involves dicing of endogenous dsrnas from longer rnas transcripts by dicer and loading into the protein complex of the rna-induced silencing complex (risc). argonaute (ago) proteins are the catalytic core of the risc complex in plants and animals (tolia and joshua-tor, ) . one strand (the passenger strand) is discarded and the guide strand is paired to a complementary mrna sequence via the risc complex. gene silencing can be achieved mainly through post-transcriptional gene silencing (ptgs) or transcriptional gene silencing (tgs). in ptgs mechanisms, the mrna undergoing translation can be sequence-specific cleaved by the risc complex and degraded when the target mrna is perfectly complementary to the sirna. when the interaction is partial or only limited complementarity exists, translational repression and rna degradation occurs, which is a mechanism exerted by mirnas. the mechanism of action for mirna differs somewhat. the mirnas are endogenous encoded singlestranded rnas of about nt long that are important post-transcriptional regulators of gene expression by pairing through a sequence-specific complementary binding to the ' utr of the target mrna. this is usually mediated by a sequence of - nucleotides, known as the seed region, at the ' end of the mature small rna (bartel, ). however, mirnas can also bind to other mrna sites including ' utr or protein coding exons (forman and coller, ) . depending on the degree of sequence complementarity, the interaction will result in mrna degradation and/or translational repression (ameres et al., ; bartel, ) , with destabilization of target mrna being the predominant efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. mechanism to reduce protein output exerted by mammalian micrornas (guo et al., ) . most animal mirnas are transcribed by rna polymerase ii into a long primary transcript (pri-mirna) containing a stem-loop structure. the pri-mirna is then processed by a multiprotein complex containing the nuclear rnase iii drosha into a ≈ nt long hairpin-shape precursor (pre-mirna) and exported to the cytoplasm via an exportin- and ran-gtp-dependent mechanism. the cytoplasmic rnase iii dicer further processes the pre-mirna by removing the terminal loop to produce a small rna duplex, containing the functional microrna and the passenger (*) strand (mirna/mirna*). the duplex mirna is then separated and the functional mirna incorporated into ago proteins within the rna-induced silencing complex (risc), guiding it to the target mrna to induce its translational repression or mrna destabilization (krol et al., ; bartel, ). in , zamecnik first reported the use of an exogenous synthetic oligodeoxynucleotide complementary to the rous sarcoma virus s rna as a potent inhibitor of protein translation (stephenson and zamecnik, ) and virus replication (zamecnik and stephenson, ) . since then, the ability to use exogenous (synthetic) agents to control gene expression has revolutionized many aspects of biological research and catalysed the development of a new promising class of exogenous molecules to treat human diseases. however, despite the obvious promise of this approach, progress has been slow because of the need to overcome myriad technical hurdles, particularly those related to the pharmacokinetics, pharmacodynamics and delivery of antisense molecules. indeed, as most antisense-based potential therapies have not yet produced significant clinical results, very few rnabased or dna-based antisense drugs have been approved for clinical use. in , the u.s. food and drug administration (fda) approved the first aso-based therapeutics for the treatment of a human disease. in , fomivirsen (marketed as vitravene) was approved for clinical use by the european medicines agency (ema) for the european market for treatment of cytomegalovirus (cmv) retinitis in immunocompromised patients. fomivirsen is a synthetic -mer aso (dna) that binds complementary to the sequence of the mrna encoding the ie protein from cmv. the first rna-based therapeutic approved for clinical use was pegaptanib (marketed as macugen), which was approved by both the fda (in ) and ema (in ), for treatment of neovascular (wet) age-related macular degeneration. pegaptanib is also the only approved single strand rna aptamer drug, and it targets the vascular endothelial growth factor as an antagonist. in both cases, fomivirsen and pegaptanib, the route of administration is intravitreal injection, and the unique properties of this route provide benefits in terms of pharmacokinetic and immunological response. in , the fda (but not the ema) approved the second rna-based therapeutic, mipomersen (marketed as kynamro) for treatment of homozygous familial hypercholesterolemia. mipomersen is a dna-rna aso that binds to the mrna of apolipoprotein b (apob), thus reducing apob protein and concomitantly ldl cholesterol levels. antisense oligos like fomivirsen (dna) and mipomersen (dna-rna) act by binding to a complementary sequence of a mrna, thus preventing production of its encoded protein. nusinersen (marketed as spinraza) approved by the fda (in ) and ema (in ) as an orphan-drug for treatment of spinal muscular atrophy is a rnabased aso that increases the production of the full-length survival motor neuron (smn ) protein. eteplirsen ( the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. (bobbin and rossi, ) and many others, which suggests that exogenous rnas can be delivered to humans and used to modify gene function, protein accumulation and treat human diseases. the ability of nucleic acids, both dna and rna, to form duplexes by base complementarity, has been used to develop oligonucleotide-based drugs for gene silencing. in principle, an oligonucleotide binds a target rna through watson-crick base pairing and exerts gene silencing through different mechanisms (kole et al., ) . a single strand antisense oligonucleotide (aso) binds to the mrna, which is also single-stranded, and is translated into proteins. different molecular mechanisms to either block gene expression or degrade the rna duplex formed by watson-crick base pairing have been described. cleavage of the rna strand of dna•rna hybrids is predominantly mediated by the enzyme rnase h (walder and walder, ) , which is an abundant enzyme in both the nucleus and cytoplasm of eukaryotic cells (wu et al., ; ten asbroek et al., ) . asos can also bind rna and block ("steric blockers") gene expression rather than facilitating rna cleavage (dias et al., ) . also considered to be within the aso family are peptide nucleic acids (pnas) or phosphorodiamidate morpholino oligomers (pmos or "morpholinos"). their modification usually acts through this mechanism (michel et al., ) , which in some cases exerts high efficacy in vivo (iversen et al., ; kole et al., ) . exogenous rna molecules or asos can alternatively target and bind a splicing site on the target pre-mrna and thus modify its exon content (mayeda et al., ; skordis et al., ) , which results in the production of alternative splicing products with potential applications in exon skipping therapy (goemans et al., ) . because many of these asos contain dna with multiple modifications, they are not discussed here in detail. the present literature review focuses mainly on ncrna and particularly on exogenous ncrnas. the discovery of rna interference (rnai) (fire et al., ) increased the interest in using exogenous chemically-synthesised rnas for silencing genes in mammals, and promoted their potential usage in human therapeutics (song et al., ) . to achieve therapeutic results, rnai can be administered via delivery to the cell of small exogenous rna duplexes -including short interfering rnas (sirnas) (zuckerman and davis, ), mirnas mimics (bouchie, ), short hairpin rnas and dicer substrate rnas (dsirnas) (foster et al., ; kim et al., ) -to the cell for further processing into an rna interfering silencing complex. although double-stranded rnas have been widely used for sirna therapy, single-stranded sirna (ss-sirna) is capable of activating the rnai pathway and exerting rnai effects (holen et al., ; lima et al., ; prakash et al., ) . moreover, singlestranded mirna mimics have also been shown to activate the mirna pathway (chorn et al., ) . in principle, then, both dsrnas and ss-sirnas can trigger gene silencing of complementary messenger rna sequences (holen et al., ) . other small rnas with imperfect matches have also been described to repress mrna translation (saxena et al., ) . for mirna therapeutics, different pharmacological tools have been developed which involved either inhibiting or enhancing mirna function (van rooij and olson, ; janssen et al., ) . approaches to inhibiting mirna function include small-molecule inhibitors; mirna masking; mirna sponges; and aso, such as anti-mirs, locked-nucleic acids (lna), or cholesterol-modified antagomirs (krutzfeldt et al., ) . the latter's complementarity allows them to bind to mirnas, inducing duplex formation or mirna degradation. the lna therapeutic approach successfully led to the first mirna-based clinical trials for the treatment of hepatitis c (miravirsen; santaris pharma, denmark) with promising results (janssen et al., ) . there are also approaches that can be utilized to enhance mirnas function called "mimic". these include small-molecule activators of mirna expression and mirna mimics, which are www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. exogenous mirnas delivered by several methods aiming to repress the function of their endogenous targets. they are also referred to in the literature as "mirna replacement therapy". as proof of concept, a synthetic version of mir- a (mrx , mirna therapeutics), delivered using a liposomal delivery formulation, was the first mirna to enter a clinical trial for cancer (beg et al., ) . the literature also describes other types of rnas that can exert physiological functions similar to that of mirnas and sirnas. for example, a -nt long guide hairpin rna (ghrna), that might not require ago or dicer, has been shown to exert mirna or sirna activity in vivo (ohno et al., ) . this novel rna interference technology may act as a novel platform for rna-based therapy. indeed, systemic or local injection of the ghr-form mirna- a (ghr- a) suppressed tumour growth in a mouse model of ras-induced lung cancer. understanding the manifold mechanisms of rna function requires detailed knowledge of the rna tertiary structure (magnus et al., ) , which can lead to therapeutic development (digiusto et al., ; strobel et al., ) . indeed, different experimental (lu et al., ; weinreb et al., ) and computational methods (magnus et al., ) have been developed to predict rnas structure and interactions. the capacity of rna to fold in various ways can generate unique three dimensional secondary structures capable of specific molecular recognition of their target cognate (zhou and rossi, ; tuerk and gold, ) . aptamers are short single-strand rna molecules ( - nt) with a defined structure that can specifically bind to a molecular target via tertiary structures. aptamers can be rationally designed using selex technology (tuerk et al., ) , and can therefore be incorporated into chemically modified rnas with high nuclease resistance properties suitable for animal and clinical studies (sullenger and nair, ). unlike other ncrnas therapeutics, aptamers can target soluble extracellular proteins or extracellular domains of cell-surface receptors. the latter characteristic is unique and can be used to deliver sirnas, mirnas or other compounds to target tissues, binding them to a cell-specific aptamer (zhou and rossi, ) . aptamer therapeutics have rapidly progressed to clinical studies, and some are already in phase clinical trials (sullenger and nair, ; zhou and rossi, ) . the unique properties of aptamers led to development of the first therapeutic aptamer and the first rna-based drug approved for clinical use, pegaptanib. however, aptamer and other ncrnas therapeutics are not free of adverse effects (lincoff et al., ) . when compared to sirna, dsirna was found to be comparable in potency and duration of effect. however, dsirna was found to be more immunostimulatory when compared to the shorter sirnas (foster et al., ) . preclinical studies suggest that when administered in lipid nanoparticles (lnp) dsirnas can exert biological effects in different mouse models with tumours of diverse origin (ganesh et al., ) . although preliminary data in nonhuman primates suggest acceptable tolerance for this specific lnp formulation (ganesh et al., ) , the dsirna therapeutic arena still needs to demonstrate its efficacy and safety in humans. catalytic rnas, the so-called ribozymes, are another family of rnas for which therapeutics have been actively developed (kobayashi et al., ) , reaching advanced clinical trials (burnett and rossi, ; mitsuyasu et al., ), but this review will not focus on this type of rna molecules. unmodified nucleic acids have been shown to possess limited stability in biological media and are subject to rapid enzymatic-mediated degradation (braasch et al., ; . there are three major classes of intracellular rna-degrading enzymes (houseley and tollervey, ) (a.k.a. ribonucleases or rnases): (i) endonucleases acting within the rna chain, ii) ' exonucleases and iii) ' exonucleases degrading rna from the ' or ' end respectively. very large amounts of nuclear rnas efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. are rapidly degraded (brandhorst and mcconkey, ) , this being considered either as translation noise (struhl, ) or part of the biological function of ncrnas. instead, most cellular rnas are modified to resist to exonuclease degradation (ren et al., ) . most genomes encode a plethora of rnases, often with overlapping activities, making redundancy a general feature of rna degradation systems, presumably with the goal of enhancing the overall efficiency and robustness of these degradation pathways (houseley and tollervey, ). normal physiological degradation of endogenous rna molecules or degradation of endogenous rna molecules with defects in processing, folding or assembly are not addressed in this literature review. abundant rnase activity has been described in different tissues, including human body fluids (blank et al., ) (weickmann and glitz, ) and with differential catalytic properties (leimoni et al., ) . different tissues contribute to body fluid rnases (neuwelt et al., ) , supporting the cellular defence system against, for instance, viruses (barrangou et al, )(gupta et al., . the mammalian ribonuclease a family seems to contribute to this host defence by exerting antimicrobial activity (harder and schroder, ; dyer and rosenberg, ) . rnase , for instance, is a rnase a superfamily member with potent ribonuclease activity that is secreted by epithelial tissues including skin, respiratory tract, genitourinary tract, and the gut (harder and schroder, ) . the abundant rnase a produced in humans very probably functions to reduce rna contamination, whether endogenous or exogenous, preventing entry into unwanted rna-processing pathways (houseley and tollervey, ). as above described, unmodified rnas (naked) are generally unstable in biological systems, due to the large amount of ubiquitously expressed nucleases. thus, several chemical modifications have been tested for exogenous rna developed for in vivo therapies to increase resistance to nucleases, enhance binding affinity, facilitate cellular uptake, improve the pharmacokinetic and pharmacodynamics profiles, and reduce immunological response or toxicity (wan and seth, ; bennett et al., ) . there are several sites of an exogenous ncrna molecule susceptible to chemical modification ( figure ) without interfering with its ability for base-paring or enhancing its drug-like properties. the several sites on rnas that can be modified include the base, the sugars, and the backbone. this allows them to conjugate with a wide variety of molecules. different chemical modifications have been tested in the context of developing rna-based therapeutics aimed at increasing the pharmacological properties of exogenous rnas and allowing their successful use in different pathologies/targeted therapies. the following section briefly reviews the types of rna modifications included in different studies in the available literature. nucleobase modifications can affect base-paring properties, binding affinity or specificity for the target mrna, as well as exert adverse effects due to competition with natural nucleotide pools or interfere with polymerases (wan and seth, ). replacement of adenosine in the sirna guide strand with adenosine analogues has been found to modify immunomodulatory activity. watson-crick face-localized n-ethylpiperidine triazole modification has been found to be less reactive than the hoogsteen edge modification, and the ' end was more effective at blocking cytokine production than those placed at the ' end (valenzuela et al., ) . c- substitution in pyrimidines in the rna guide strand increase thermal stability of sirna duplexes. indeed, smaller -methyl group substitutions resulted in better sirna activity (terrazas and kool, ). major groove modifications have also been found to increase the serum stability of sirnas (terrazas and kool, ). -position purine modifications, where the major groove edge (i.e. the hoogsteen face) is modified, have also been tested. thiazole modifications of guide strand sirna at position is well tolerated in sirnas, but -ethynyl at this location reduces efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. potency (ibarra-soza et al., ) . several other nucleobase modifications have been evaluated to modify thermal stability of the sirna duplex, hydrogen bonding and sterics, or off-target effects (peacock et al., ) . although not exactly a base modification, movement of the nucleobase from c- to another position on the ribose (i.e. isonucleosides) in sirnas has been evaluated (zhang, j et al., ) . while maintaining binding capacity to rna and stability toward nucleases, passenger strand modifications with isonucleoside at the ' or ' terminals can retain the silencing activity and minimize the passenger strand specific off-target effect (zhang, j et al., ) . chiral inversion of the natural d-forms (spiegelmers) of rna has also been tested in aptamers, leading to their clinical evaluation (ludwig et al., ) . increase nuclease resistance modify plasma protein binding prevent rapid renal excretion improve pharmacokinetics enhance rna-binding affinity enhance thermal stability increase nuclease resistance enhance rna-binding affinity enhance cellular uptake reduce renal filtration enhace delivery to certain tissues modulate protein binding enhance specific cellular uptake increase possibility of formulations for specific tissues/mucosal mebranes ability to traverse biological barriers using extracellular vesicles ´to d irection sites susceptible for modification on an rna molecule. exogenous rnas for therapeutic use can be subjected to different chemical modifications without interference on their base-pairing ability and enhancing their drug-like properties. conjugates at the ´-end or the ´-end may have the same objectives. earlier studies showed that the '-oh of the rna ribose sugar is not required for sirna activity (chiu and rana, ) and '-modifications influence the sugar to adopt a '-endo sugar pucker due to the gauche effects between the o '-and '-substitutes, improving affinity properties (egli et al., ) . therefore, the furanose ring structure at the ' position of rnas has been extensively modified to efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. achieve pharmacological properties including increasing nuclease resistance, stability and efficacy (egli et al., ) . the '-o-methyl ( '-ome) or '-o-methoxyethyl ( '-moe) modifications increase resistance to nucleases and duplex melting temperature (lamond and sproat, ; prakash and bhat, ) , and have been widely used to develop mirnas (meister et al., ) , sirnas (judge et al., ; choung et al., ) or aptamer-based (burmeister et al., ) therapies. for example, '-ome in sirna has been shown to selectively protect the particularly vulnerable '-end of the guide strand against exonucleolytic degradation in human blood serum (hoerter and walter, ) . moreover, '-ome is a naturally occurring modification of rna found in trna and other small rnas. '-ome has also been used to modulate rna splicing ( toxicity related to lna modifications has also been described (swayze et al., ) . although sugar-modified oligonucleotides or exogenous rnas, including sirnas or antimirs, are more effective rna inhibitors than their unmodified counterparts, they are still susceptible to serum in addition, the phosphorothioate linkage promotes plasma protein binding (srinivasan et al., ; wan and seth, ; sands et al., ) . this increases pharmacokinetic benefits by reducing rapid renal clearance (sands et al., ) and facilitating tissue delivery, binding to cell surface proteins and cellular uptake (miller et al., ; overhoff and sczakiel, the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. binding affinity (tm) for its target complementary nucleic acids (≈ . ºc/linkage) (freier and altmann, ) including mrnas and mirnas (krutzfeldt et al., ) . thus, the use of a mixture of phosphodiester and phosphorothioate bonds may be preferred over all phosphorothioate bonds for in vivo applications (ghosh et al., ; krutzfeldt et al., ) . also, the phosphorothioathe linkage confers chirality at phosphorus and might be relevant for sirna activity (jahns et al., ) . although initial studies have shown exogenous homologous rna uptake by cell suspension, there was very rapid degradation after uptake (shanmugam and bhargava, ) . duplex rnas may be more stable than single-stranded rnas, even in the absence of phosphorothioate modifications (braasch et al., ) . indeed, '-ome hairpin-designed antimir oligonucleotides do not seem to require phosphorothioate modification to exhibit in vitro activity (lennox and behlke, ). minimally-modified phosphodiester aso has been reported to have less (brigui et al., ) , similar or superior activity than its complete phosphorothioate counterparts (uhlmann et al., ) , but sugar modifications in phosphodiester backbones can compensate the loss of activity (monia et al., ) . some adverse effects related to phosphorothioate modifications, compared to their phosphodiester backbone counterparts, have been described in aso and sirnas, including nonspecific protein binding (brown et al., ) , the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. (dsrna). alpha-tocopherol conjugated sirnas can be delivered to liver tissue (nishina et al., ) or the brain when administered locally (uno et al., ) . other lipid conjugates of rnas have been evaluated including bile acids, long-chain fatty acids, and medium-chain fatty acids (murakami et al., ; lorenz et al., ; wolfrum et al., ) . conjugation with specific peptides has also targeted sirnas to specific tissues (hsu and mitragotri, ; yamayoshi et al., ) . another approach includes conjugation with high-molecular-weight polyethylene glycol (peg) to overcome renal filtration. this strategy was used in rna aptamers either approved (macugen) or in advanced clinical trials for ocular disorders (zhou and rossi, ; drolet et al., ) . the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. rna delivery to a target tissue for appropriate and specific cellular uptake is currently a main hurdle in rna therapeutics. several synthetic non-viral methods for rnas delivery have been developed for local and systemic rna therapeutics ( multiple studies have suggested that exosomes, naturally-released extracellular vesicles, can deliver rnas (mrnas, mirnas, lncrnas) to recipient cells (zhang, l et al., ; . due to their ability to traverse biological barriers, the use of exosomes or exosome-mimetic nanovesicles for rna delivery is an open field for future rna therapeutics (lunavat et al., ; zhou et al., ) . indeed, preclinical studies suggest that exosome-mediated delivery of rnas is feasible in vivo (didiot et al., ; el-andaloussi et al., ) . different rnas have been found in milk exosomes from different species, including humans zhou, q et al., ) , but whether these vesicle-protected exogenous rnas are bioavailable is still in debate (zempleni et al., ) . it is important to note that most of the above described chemical modifications are not found in nature and have been developed generally to increase the drug-like properties of rnas and other nucleotides. to the best of our knowledge there is no evidence of clinical trials using unconjugated naked or unmodified exogenous rna for human use with any administration route. efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. early studies suggested that rnas were poor drug candidates due to their relatively high instability, relatively short half-life in vivo, immunomodulatory effects and the many hurdles to their cellular absorption caused by their negative charge. however, several improvements in stabilization chemistry have been identified in recent decades. indeed, a number of chemical modifications and conjugation strategies have improved their rna-binding affinity, in vivo nuclease stability and pharmacokinetic and pharmacodynamic properties. an exogenous rna can be amenable for chemical modification without interfering with its base-pairing ability. several parts of the exogenous rna molecule can be chemically modified including the nucleobase, the backbone or the ribose (sugar). rnas can also be conjugated with a variety of molecules which can exert different biological properties. in addition to these, chemical and biological approaches can be applied to deliver exogenous rna to cells or specific tissues. although many aspects are still to be fully understood in the field of chemical modification of exogenous rnas for therapeutic use, several exogenous rnas (sirnas, aptamers, mirnas, etc.) have entered advanced clinical trials for human use. adams the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. following a systematic literature search as described in section . . . and . . . ., a total of papers were reviewed on the pharmacokinetics of foreign exogenous ncrnas used/intended to be used as therapeutics (hereafter referred as exogenous ncrnas). this section focuses on the pharmacokinetics of exogenous ncrnas (naked, i.e. nonchemically modified or minimally chemically modified) other than those consumed orally from plants; these will be covered in chapter . . moreover, only in vivo studies, in mammals and humans, are reviewed here. of the documents included here, were used for review of ncrna pharmacokinetics in disease and other conditions, which may be relevant to risk assessment considerations. although not specifically requested by the mandate, information on pharmacodynamics aspects of ncrnas was considered. pharmacodynamics is the study of a drug's effect on an organism, meaning its biological effects and other aspects of its xenobiotic action, (efficacy, potency, and toxicity) and this is considered relevant for ncrnas (see . ). pharmacokinetics is dedicated to determining the fate of substances administered to a living organism. it describes the trajectory of a xenobiotic (in this case exogenous ncrna) after delivery into an organism, and encompasses from the movement of administration, to its movement through the organism, to its complete elimination; in other words, absorption, distribution, metabolism and excretion. a drug's pharmacokinetics depends on an individual's physiology (patient-related factors, i.e. renal function, sex, age) or pathology (i.e. renal failure, hepatic failure), and drug chemical properties. pharmacokinetic relevant parameters include: dose, the amount of drug administered at a single point in time; dosing interval, the time between drug dose administration; c max , the peak plasma concentration of a drug post administration; t max , the time to reach c max ; elimination half-life, the time required for the drug concentration to reach half its original value; area under the curve (auc), the integral of the concentration-time curve (after a single dose or in steady state); and clearance, the volume of plasma from which a substance is completely removed per unit time. pharmacokinetics modelling is done using noncompartmental or compartmental methods; the former estimate exposure to a drug by estimating the auc of a concentration-time graph, and the latter estimate the concentration-time graph using different kinetic models. considering an organism as a homogeneous compartment (monocompartmental model) implies that a drug is distributed equally throughout the entire body (tissues and fluids). therefore, blood plasma drug concentrations are a true reflection of drug concentration in other fluids or tissues, and drug elimination is directly proportional to its concentration in the organism. not all tissues in an organism receive the same blood supply and different drugs have different characteristics (i.e. variation in drug passage through natural barriers, such as the brain blood barrier, due to a drug's biochemical properties), meaning other models are sometimes needed. the two-compartmental model assumes that there is a compartment where distribution is more rapid (central compartment) and another compartment www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. (peripheral compartment) consisting of organs with a lower relative blood flow, which consequently exhibits a slower drug distribution rate. saturation of the enzymes responsible for drug metabolisation, the presence of active transmembrane transport mechanisms independent of drug plasma concentration, and many other factors not taken into account by the two-compartmental model, require the use of multi-compartmental models. very few studies have focused on the pharmacokinetic profile of exogenous rnas (naked or unmodified), with most of the knowledge on the absorption, distribution, metabolism and excretion profile of ncrnas coming from the development of antisense oligonucleotides therapeutics (dirin and winkler, ) . scaling dosing from preclinical animal studies to humans (i.e. based on body weight or surface area) is challenging and apparently depends on the mechanism of clearance. for example, the pharmacokinetics of a naked sirna formulated into polymer-based nanoparticles in mice, rats, monkeys and humans exhibited blood c max shortly after intravenous administration and rapid elimination across all species in correlation with the body weight (zuckerman et al., ) . the pharmacokinetic profile investigated during the development of exogenous rna therapeutics has shown to be influenced by the route of administration, as described below. a comprehensive summary of available information of in vivo studies and related pharmacokinetics of exogenous rnas is provided in table . most studies on exogenous ncrnas have focused on iv administration. studies in mice have shown that naked unconjugated sirna ( µg/kg) injected intravenously disappears from peripheral blood min after administration, restricting the likelihood of accumulation to peripheral organs (gao et al., ). similar results were found following iv administration of naked sirna ( i labelled), which distributed to the kidney and liver within the first minute, peaking in the kidney, liver and bladder within the first minutes (braasch et al., ) . although distribution of sirna ( i labelled) decreased markedly after h, it persisted in the kidney and liver up to h, and lower concentrations were observed in the lung, spleen and heart (braasch et al., ) . in rats, sirnas were distributed to the kidney and excreted into the urine one hour after injection (van de water et al., ) . exogenous mirna iv administration has also been tested in animal models and entered human clinical trials. in this case (mirna mimics), they are either chemically modified dsrnas formulated in different nanoparticles (xue et al., ) to preferentially target a tissue (trang et al., ), or incorporated into naturally circulating extracellular vesicles (bala et al., ) . for example, mirnas mir- and let- b formulated in a neutral lipid emulsion have been iv administered to mice and have shown to exert a biological effect (kasinski et al., ) . the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. administration site for less than one day, while in a chitosan-formulated version it persisted for up to days (ma et al., ) . peptide-modified and chemically modified sirnas applied topically to the skin have been found to exert a local biological effect, as compared to the sirna alone (hsu and mitragotri, ) . mirnas formulated with transfection agents or nanocomplexes have also been injected intradermally (srivastava et al., ) , or subcutaneously (urgard et al., ) , and demonstrated to exert local biological effects. intranasal administration of sirnas in rats for brain targeting has resulted in very low levels of radioactive sirnas in the brain or plasma when naked sirnas were administered compared to a formulated version (perez et al., ) . mirnas administered by intranasal injection using transfection reagents reached the dorsal root ganglia and the olfactory bulb (cheng et al., ) and, using another nanovector delivery system through intranasal administration (as drops), it reached the brain, exerting a local biological effect (zhuang et al., ) . due to ncrnas susceptibility to degradation, very few studies have focused on exogenous ncrna administration in the gi tract. non chemically modified naked sirna administered by oral gavage to female mice ( µg in total), was found intact and at high levels (analyzed by northern blot and by quantitative pcr analysis) and hours after dosing in the stomach, small intestine and colon when formulated with chitosan nanoparticles (ballarin-gonzalez et al., ) . by contrast only low sirnas levels were observed in the stomach, intestine and colon of mice hour after oral administration of the non formulated naked sirna. at this timepoint intact sirna was almost exclusively present in the stomach while partial degradation products were observed in the proximal and distal regions of the small intestines. further reduction of sirna levels was observed hours after administration, with traces only found in the colon (ballarin-gonzalez et al., ) . orally administered sirnas, formulated using specific vehicles including thioketal nanoparticles ( µg kg - d - the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. counterparts were present in all epithelial cells (martirosyan et al., ) . oral administration of exogenous exosomes containing mirnas of bovine origin has been shown to attenuate arthritis in mice models. bovine milk derived extracellular vesicles were administered daily by oral gavage starting at week till week after birth and arthritis was induced by collagen gavage. animals receiving exosomes ameliorated the clinical condition, although no clear mechanisms for this effect have been described . enteral (large intestine) sirna administration for therapeutic gene silencing targeting the liver (via the lymphatic route) has also been described, but using chemically modified and formulated sirnas (murakami et al., ) . enzyme-and ph-responsive microencapsulated nanogels are also being developed for oral sirna delivery, as reported in recent in vitro tests (knipe et al., ) . naked sirna formulated in a water-in-oil microemulsion has been administered via the rectal mucosa to mice along days ( - µg sirna/kg, administrations) and has reached the brain where exerted a biological effect through downregulation of the prion protein expression (lehmann et al., ) . prion infected mice presented an improvement of some neuropathological conditions after receiving the formulated sirna following the rectal mucosa route of administration. being the eye the target for the first rna-based therapy, sirna intraocular administration has been investigated. intravitreally injected naked sirna (slightly modified, dtdt) distributed throughout the eye (vitreous, iris, retina, retinal pigment epithelium and sclera) of rabbits when administered at mg/eye, and the pattern of ocular distribution was similar in male and female rabbits ( . conjugated or formulated sirnas have also been tested in animal models (zhang et al., ; janout et al., ) . intravitreal injection of mirnas has been described in the literature using either naked mirnas in mice , rats (qin et al., ) or using transfection reagent in rats (mcarthur et al., ) or exosomes in rats (mead and tomarev, ) . some studies have evaluated ip administration of naked sirnas (slightly modified, dtdt) in animal models (wilcox et al., ; shimizu et al., ) . the half-life of ip administered naked sirna (dtdt) was found to be ≈ times lower than the formulated version (perepelyuk et al., ) . sirna (dtdt) was also quickly excreted into the urine compared to a formulated version (shimizu et al., ) . in a comparison of a formulated (complexed) and naked sirna after ip administration in mice it was found that after min the formulated version was detectable in muscle, kidney, liver and tumour tissue, while the naked sirna was completely absent (urban-klein et al., ) . while not a common administration option, chemically modified sirnas formulated in ph-responsive nanoparticle complexes have been administered in the submandibular gland of mice (by retroductal injection) and found to exert rnai effects (arany et al., ) . the monitoring of the distribution of the chemically modified sirna h post-administration indicated that the formulated sirna was internalized www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. in the cytoplasm of duct cells via the endosomal pathway. at h post-administration, the signal was concentrated in the acinar compartment as well. after h, ≈ % of the submandibular gland cells were positive for the sirna. the delivery of the sirna formulated in nanoparticles was more efficient compared to that of naked sirna (arany et al., ) . in another study, iv administration of chemically modified sirna ( . mg/kg) was shown to accumulate remarkably in the submandibular gland just min after injection, and had a half-life in this tissue of ≈ . h, but did not have a rnai biological effect . in contrast, direct local injection of liposome-encapsulated sirna ( . mg/kg) into the submandibular gland was shown to have strong gene-silencing effects, while naked sirna injection ( mg/kg) did not . few studies have focused on this administration route. senn and colleagues compared central nervous system administration of sirna vs. aso (senn et al., ) . compared to phosphothioate and 'methoxyethyl protected aso (up to µg), naked (slightly-modified, dtdt) sirna ( µg) administered to rats via icv was not found in any brain region, including the area around the injection site. in contrast, h after aso injection it was distributed in the thalamus and hypothalamus. the naked sirna clearly exhibited very poor distribution and uptake in the brain compared to the aso. although aso exhibited a capacity to reach different brain structures, its administration at a total dose of µg over two days produced no rnai effect. in the same context, icv administration of sirna (up to µg/rat/day) on two consecutive days did not exert rnai effects. however, in another study, a sirna chemically modified to allow for delivery in the absence of transfection reagents (accell sirna) was administered via the icv route at µg/rat. the sirna was incorporated into different types of neurons, although not glia, where it exerted a biological effect in regions like the cortex, the caudate subregion of the striatum and in the pyramidal cells of the hippocampal ca subregion (nakajima et al., ) . in a different study, a mirnas mimic (chemically modified double-stranded rnas) administered by icv ( pmol/g body weight, mixed with cationic lipid dotap) was found to increase forty fold the level of this mirna in the brain (stary et al., ) . several chemically modified mirnas (agomirs) were also found to be active when administered via this route in rats (huang et al., ; ge et al., ) . a double-stranded modified pre-mirnas was also reported as active in rats (davis et al., ), but a naked mirna (double-stranded -bp rna) injected by icv ( pmol) in this study was unable to increase its levels or exert a biological effect, suggesting that the naked mirna had a higher susceptibility to rnase degradation (davis et al., ) . lipid-complexed naked sirna is reported to be absorbed by mouse epithelial and lamina propria cells of the vagina and ectocervix (palliser et al., ) . however, uncomplexed and unmodified sirnas were immediately degraded (< min) when exposed to genital wash fluid (wu et al., ) , suggesting high rnase activity in the cervicovaginal secretion. also, slightly modified but still naked sirna (dtdt) was unable to exert a clear biological effect (zhang et al., ) . in summary, different administration routes have been tested during ncrna therapeutics development. the administration route used for exogenous ncrna delivery clearly influences pharmacokinetic profile of chemically synthesized ncrna. no matter the administration route, it seems that naked and unformulated rnas rapidly degrade and exert very limited or no biological activity. very few studies have evaluated the distribution profile of administered naked oligonucleotides in organs and tissues or compared it to the distribution profile of their chemically modified counterparts. using the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. imaging techniques in baboons (positron emission tomography -pet -and [ f]-labelling) it was shown that naked oligonucleotides are poor drug candidates due to limited distribution into organs and tissues and very rapid elimination (tavitian et al., ) . compared to '-o-methyl or phosphorothioatemodified oligonucleotides, naked oligonucleotides were found in the heart, liver and kidney and, to a lower extent, in other tissues min after iv injection. at that timepoint, naked oligonucleotides were already found in the bladder suggesting that naked oligonucleotides are very rapidly eliminated compared to '-o-methyl or phosphorothioate ones. moreover, one hour after injection, almost all naked oligonucleotides were found in the bladder or residually in the kidney, while the phosphorothioate oligonucleotides were found largely in the liver and kidney, and the '-o-methyl ones were in an intermediate distribution (tavitian et al., ) . kinetics studies with radiolabelled oligonucleotides also showed a dramatic reduction of naked oligonucleotides in the plasma within the first min, while '-o-methyl and phosphorothioate-modified ones showed a much slower reduction during the first min. naked sirna have been reported to distribute to the kidneys and bladder of mice min after iv administration (braasch et al., ; naeye et al., ) . intravenous administration of mg kg - partially-modified unconjugated sirna (partial phosphorothioate backbone and '-o-methyl sugar modification) or cholesterol-modified sirna in mice resulted in a broader tissue distribution (liver, heart, kidney, adipose and lung) of the cholesterol-conjugated sirna h after injection (soutschek et al., ) . in another study, naked sirna (free sirna) iv administered (retro-orbitally) was found in the kidney min after injection (naeye et al., ) . ip administration of synthetic sirna formulated with low molecular weight polyethylenimine resulted in its presence in muscle, liver, kidney and tumour tissues min after injection, while the naked counterpart was completely absent (urban-klein et al., ) . in rats, chemically modified naked sirnas were distributed in a similar manner to those of '-o-methyl or '-f modified sirnas (viel et al., ) , even though the circulation time of the '-f was increased. in other studies with rats, iv injection of sirna (chelated to indium- ) resulted mainly in renal distribution hour after exposure (van de water et al., ) . some liver distribution of sirnas have been also reported in mice, when using either the two phosphodiester rna strands duplexes or one phosphodiester strand and one phosphorotioate strand sirna duplex (braasch et al., ) . orallyadministered naked sirnas ( µg sirna) were found to be present in the stomach, proximal and distal small intestine and colon tissues hour after administration but at very low levels, further lowering hours after administration (ballarin-gonzalez et al., ) . the same sirna formulated with chitosan nanoparticles was found to distribute systemically and reach the liver, spleen and kidney (ballarin-gonzalez et al., ) . in an experimental model of arthritis in mice, iv administered naked sirna ( µg) was found to distribute to arthritic joints immediately after injection, but disappeared within hours, compared to that of specific formulations (komano et al., ) . overall, these finding suggest that naked sirna, although distributing at very low levels and with short residence times compared to other formulated counterpart versions, does distribute to the gi tract when administered orally or other tissues when administered systemically. www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. formulation type (i.e. cationic lipids, neutral lipids, or others) can drive preferential distribution to specific tissues. as described for certain mirnas, a neutral lipid emulsions (nle) formulation has been shown to distribute to the lung after iv injection ( µg) (trang et al., ) . the lack of cationic lipids in the nle seems to bypass some of the shortcomings that can be attributed to the charge, including formation of aggregates in biofluids, filtration by the liver, adherence to the endothelium or uptake by scavenging macrophages. the result is excellent distribution to lung tissues (trang et al., ) . extracellular vesicles (exosomes)-formulated synthetic mir- was found to distribute to liver, adipose, lung, muscle and kidney tissues min after iv injection (bala et al., ) . administration of exosomeloaded mir- resulted in its detection by isolated hepatocytes and liver mononuclear cells, suggesting cellular uptake (bala et al., ) . in summary, naked oligonucleotides, including ncrnas, rapidly distribute to the liver, lung and kidney after iv injection. within the first minutes they are found in the bladder, suggesting rapid renal elimination. chemical modifications or formulations increase their distribution to other tissues or delay clearance. exosome-loaded rnas have recently been shown to be an alternative tool for distribution to different tissues. ncrnas by oral administration seem to distribute to the gi tract or, if formulated, systemically. there are few studies which have evaluated the metabolism of exogenous rnas administered to animals or humans, most of which only investigate their degradation. naked sirnas seem to be rapidly degraded when administered iv (viel et al., ) , decreasing by ≈ % within the first min, although certain chemical modifications (i.e. 'f, 'ome) can slightly increase this time. stability of naked sirna was reduced (< min) compared to that of other chemically modified or formulated sirna when administered by iv injection in mice (gao et al., ) . indeed, incubation of naked sirna with plasma or foetal bovine serum causes a very rapidly degradation of the genetic material (jiang et al., ; braasch et al., ; urban-klein et al., ) . unmodified aptamers in general are also very rapidly degraded (≈ min) (zhou and rossi, ) . when naked sirna is administered as formulated (i.e. nanoparticles) it was shown that the renal filtration barrier can separate the sirna from its carrier (naeye et al., ; zuckerman et al., ) . in the eye, sirnas are degraded by endonucleases without preferences for one side of the duplex (as observed also for chemically modified sirnas) (beverly et al., ) . early in vitro studies also showed that exogenous rnas taken up by cells are rapidly degraded (shanmugam and bhargava, ) , and exogenous rna injected into the blood is rapidly degraded to nucleosides, ribose phosphate and free bases (sved, ). the renal filtration barrier owns an effective size cut-off of ≈ Å (batsford et al., ; bohrer et al., ) , but is also influenced by net molecular charge discrimination (brenner et al., ) . this is supposed to facilitate rapid renal clearance of small molecules and free sirna (zuckerman et al., ; naeye et al., ) since aso, sirnas and aptamers are commonly smaller than this cut-off (huang et al., ) . by comparing naked sirnas and ribose-modified sirnas ( '-o-methyl or 'f) using pet scans in rats and mice, the kidney was identified as the main organ for sirna elimination (viel et al., ) . however, it is worth noting that also the liver participates in sirna elimination. in other studies, sirna appeared to accumulate in the bladder shortly after administration (hatanaka et al., ) . this was also the case for modified sirnas (lna) compared to their versions conjugated with peg administered in mice, in which their levels peaked in the bladder min post injection and they were found in the urine as soon as min after injection (iversen et al., ) . efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. some studies have described rapid elimination of naked sirna and renal excretion after iv injection at doses including µg (jiang et al., ) and µg/kg (gao et al., ) in mice or rats (van de water et al., ) . other studies using dynamic imaging techniques or radiolabelled sirnas have confirmed that most of the naked sirna (free sirna) administered to mice was found in the bladder after just min up to min, faster than the formulated counterparts (naeye et al., ; zuckerman et al., ) . the literature mostly addresses chemically modified rnas (dyke et al., ) in clinical trials (zhou and rossi, ) . the small size of rna aptamers renders them susceptible to rapid clearance by renal filtration which has driven efforts to increase their size by conjugation for therapeutic use (healy et al., ; haruta et al., ) . overall this indicates that naked exogenous small rnas are rapidly cleared from systemic circulation and excreted in the urine. iv injection of a cocktail of four different plant-based small rnas (mir , mir a, mir a, and mir ; pmol each) showed them to be rapidly cleared from circulation (< min) . the exception was mir , which peaked min after injection compared to the other mirnas administered at equal dosages, and then dramatically decreased at min. when compared to other small rnas, anomalous or atypical stability and genesis were proposed for this specific mirna. efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. although scarcely described, there may be several conditions in which, theoretically, the pharmacokinetic properties of exogenous rnas could be modified. increased intestinal permeability has been described in different conditions including consumption of nonsteroidal anti-inflammatory drugs, celiac disease, crohn's disease or other intestinal inflammatory disease (oman et al., ; sundqvist et al., ; michielan and d'inca, ) . for instance, absorption may be altered in cases where vasculature permeability of the gut is increased, as it has been observed in murine models of ageassociated inflammation (jeong et al., ) . increased intestinal permeability ("leaky gut") has been described in alcoholism, contributing to the extra-intestinal tissue damage caused by alcohol (bjarnason et al., ) , including liver damage (keshavarzian et al., ) . similarly, leaky gut has been linked to liver steatosis in obese patients ( theoretically, leaky gut may increase the possibilities of passively absorbing larger amounts of exogenous rnas with consequent release into the circulatory system. however, very few studies are available in which sirnas, aptamers, mirnas or other exogenous ncrnas have been tested under these conditions. absorption of naked sirna formulated into nanoparticles following intrarectal administration was tested in mice models of dextran sulphate sodium (dss) induced colonic inflammation. uptake of sirna (observed mainly in epithelial cells, lamina propria lymphocytes, and cells from the mesenteric lymph nodes including dendritic cells and t cells) was increased in mice suffering from acute colitis compared to uptake (in the same cell types) under healthy conditions (frede et al., ) , suggesting that inflammatory conditions may alter ncrna absorption. although there are no studies using naked unmodified exogenous ncrnas, this topic deserves further attention. acute kidney injury or chronic kidney disease are important public health issues. the kidneys play a key role in elimination of xenobiotics and metabolism products and thus renal clearance should be considered in drug discovery and drug interaction research (feng et al., ) . very few studies have focused on the clearance of exogenous ncrnas in pathological renal conditions. for example, plasma clearance of chemically modified sirna administered by iv injection ( mg/kg) was slightly reduced in renally impaired rats than in normal animals (thompson et al., ) . although sirna concentration in residual kidney tissue of partially nephrectomised animals was slightly greater than the mean in normal animals, the differences did not result in appreciably greater sirna distribution into other organs (thompson et al., ) . in general, similar results, but using asos, were observed in rats after cisplatin-induced renal injury (masarjian et al., ) . also in a study of an aso against mirnas, it was recently reported that in end stage renal disease patients, only relatively modest increases in aso plasma levels were observed when compared to control subjects (http://regulusrx.com/wpcontent/uploads/ / / -aasld-rg- -pk-and-safety-in-esrd-vs-normal.pdf). also using asos, distribution within the kidney was found to be altered in a mice model with chronic kidney disease (ckd) compared to controls (donner et al., ) . moreover, concentration of `-moe asos in the liver of mice with ckd was elevated as compared to mice without ckd, suggesting that a reduction of renal function and aso excretion can potentially influence the systemic delivery of asos (donner et al., ). www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. liver disease may also lead to alterations in drug pharmacokinetics, and thus the potential effects of hepatic impairment are considered in drug development (allen et al., ) . although exogenous ncrnas have not been analysed quantitatively, one study showed that chemically modified and formulated sirnas in lipid nanoparticles were found to be distributed and similarly accumulated in pathological or normal liver tissue from mice (yu et al., ) . other physiological, pathological or lifestyle factors, such as aging (cohen, ) and exercise activity (van baak, ) , may influence the pharmacokinetics of exogenous molecules. however, no information is yet available regarding exogenous rnas and their behaviour under these conditions. pharmacodynamics refers to the relationship between the concentration of a drug at the site of action and the resulting effect, including the time course and intensity of therapeutic and adverse effects. there is scarce data on the pharmacodynamics of naked unmodified exogenous ncrnas. some information is available on chemically modified rnas (or nuclease-stabilized rnas, the latter being outside the scope of this literature review). for example, in a mouse model of viral hepatitis the administration (hydrodynamic injection) of naked or chemically modified (methyl-modified) sirnas was associated with clear dose-related effects of various rnai constructs (peng et al., ) . in this study the naked sirnas had the highest inhibitory effects, but these rapidly declined (shorter-lasting inhibitory effects); in contrast, the methyl-modified sirna duplexes were more stable inside cells and exerted their effect over a longer time period (peng et al., ) . non-compartmental analysis of rna therapeutics is used in most preclinical studies when analysing pharmacokinetic and pharmacodynamic parameters. for example, a non-compartmental model was used to estimate the properties of intravitreal injection of slightly chemically modified sirna (dtdt) (dejneka et al., ) . also, in a mirna mimic therapy by iv administration of a liposomal formulation, a non-compartmental model was used to evaluate different parameters (beg et al., ) . systemic delivery of naked sirna has generally failed to produce significant biological effects (gene silencing). no biological effects have been reported when naked sirna ( µg/eye) was administered by intravitreous injection in rats . few studies report gene silencing effects following local administration, although some of the tested rnas were slightly chemically modified (i.e. dtdt overhangs). intrathracheal administration of naked (but slightly modified, dtdt) sirna was effective through gene silencing effects in a mouse model of haemorrhagic shock and sepsis (perl et al., ) . in non-human primates, naked (but slightly modified, dtdt) and unformulated sirna administered intranasally was effective in severe acute respiratory syndrome sars coronavirus infection reducing viral load and host symptoms (li et al., ) . it looks that naked ncrnas require proper formulation to attain efficacy. naked sirna administered intranasally to mice was effective in reducing viral replication when complexed with lipofectamine (fulton et al., ) . naked sirna formulated in ph-triggered nanoparticles exerted gene silencing effects when administered by iv injection in mice ( mg/kg) (kolli et al., ) . a formulation of naked sirnas using cyclodextrin-containing polycations and transferrin, as a targeting ligand for delivery to transferrin receptor-expressing tumor cells, also showed gene silencing effects when administered by iv injection ( . mg/kg) in a mouse model (hu-lieskovan et al., ) . when administered orally efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. ( µg/kg) to a mice model of thioglycollate-elicited inflammation, a formulated sirna reached macrophages in the peritoneum, spleen, liver and lung, exerting a sirna effect (aouadi et al., ) . a liposome-formulated naked mirna iv administered twice a week was efficacious in clinical trials (beg et al., ) . a cationic liposome formulated dicer-substrate sirna (dsirna) (a solid dsirna core in an envelope of cationic lipids and cholesterol) was found to be efficacious in mice when administered by iv injection ( mg/kg) (ganesh et al., ) . preliminary results within this formulation demonstrated a tolerability profile in non-human primates upon repeated administration (ganesh et al., ) . when comparing the naked sirna and to a highly formulated sirna in cyclodextrin-containing polycations with surface peg and transferrin as the targeting ligand, only the formulated version ( . mg/kg iv) was effective in reducing the expression of the ews-fli gene and tumour engraftment, while the naked version exerted no biological effect (hu-lieskovan et al., ) . similarly, in a mouse model of concanavalin a-induced hepatitis a reduction of hepatic injury by liver-specific induction of rna interference was observed upon iv administration a of galactose-conjugated liposome nanoparticles (gal-liponp) bearing naked sirna, while no effects were noted following administration of the unformulated naked sirna (jiang et al., ) . in a collagen-induced arthritis mouse model and using systemic administration, naked sirna (dtdt) was used against tnf-α, and was completely unable to repress tnf-α mrna expression, whereas a formulated version repressed it (komano et al., ) . in a mouse tumour model, intraperitoneal injection of naked sirna exerted no rnai effects, while its complex-formulated version did (urban-klein et al., ) . while developing a model to study sirna pharmacodynamics, intramuscular administration ( µg) of naked sirna (chemically stable) in mice showed no effects without use of electroporation (stevenson et al., ) . there are several ongoing clinical trials using sirnas, rna aptamers, mirnas and other noncoding rnas intended for use in humans to treat diseases including cancer, cardiovascular disease, inflammation, ocular disease and others (zuckerman and davis, ; li and rana, ; bobbin and rossi, ; zhou and rossi, ; sullenger and nair, ) . most of these ncrnas are chemically modified or formulated. adverse effects were identified in some of these studies. the delivery component of the formulation (i.e. liposomes), rather than the naked sirna component, was primarily responsible for the adverse effects observed in different preclinical toxicity studies (zuckerman et al., ) . for the mir- a mimic, adverse effects in humans have been reported including acute renal injury, back pain and fatigue, all likely attributed to the liposomal carrier rather than the mirna (beg et al., ) . liposomerelated toxicities are considered due to activation of the complement and have been well characterized with the most frequent symptoms observed being flushing, rash, dyspnoea, chest pain, back pain and subjective distress (szebeni et al., ) . in non-human primates, repeated iv administration of escalating doses of a non chemically modified sirna led to increased il- and inf-gamma levels, as well as kidney and liver toxicity at the highest dose tested ( mg/kg) (heidel et al., ) . in several clinical trials on rna therapeutics, dexamethasone premedication was required to reduce infusion-related adverse effects or reactions (i.e. hypersensitivity, flushing, oedema, etc.) (beg et al., ) . for some sirnas used in the clinic, a predosing hydration protocol has been used to protect the kidneys (zuckerman et al., ), thus reducing in humans the toxicities observed in animals. there are also animal studies in which no toxic effects are reported. for instance, when highly formulated naked sirnas were administered by iv route at . mg/kg, no apparent liver or kidney toxicity, immunogenicity-related toxicity or organ damage were detected in a murine model of metastatic ewing's sarcoma (hu-lieskovan et al., ) . no histological toxic effects were seen in liver, kidney, lung, heart and spleen in the same model following repeated treatment with the same formulation the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. (twice weekly for weeks) (hu-lieskovan et al., ) . the same results were obtained for the naked sirna administered at the same dose and for the same duration (hu-lieskovan et al., ) . in addition, when administered daily via oral gavage ( µg sirna/kg) for days in mice, sirna nanoparticles in galactose modified trimethyl-cysteine conjugates were reported to have no histological toxicity in major organs (brain, heart, kidney, lung, liver or spleen) (han et al., ) . since there are so few studies in the current literature on the pharmacokinetics of naked/unmodified rna molecules, this section included studies of other, minimally-modified, exogenous rnas to compare their pharmacokinetic and pharmacodynamic properties to that of naked rnas, when possible. the findings suggest that naked or unmodified rnas are rapidly cleared from circulation and have been reported in the kidney and urine immediately after administration (generally intravenously). in general, no major gene silencing effects have been observed for naked rnas when compared to chemically modified or formulated ones. this lack of efficacy has probably discouraged studies investigating the toxicity and safety of naked unmodified rnas. while limited studies have reported minor biological effects of certain naked rnas in animal models (very much dependent on the route of administration), the lack of studies in humans prevents scientists from understanding the kinetic profile of exogenous rnas and developing effective therapeutics. finally, scientific literature on toxicological or safety studies provides no evidence for concern about the systemic toxicity of naked exogenous small rnas under normal physiological conditions. allen the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. following a systematic literature search as described in section . . . . and following the methodology described in the section . . . , a total of papers were selected as relevant to reviewing the topic of rnas uptake. information on naked (non chemically modified) exogenous rnas is mainly presented in this section. of these publications, support the uptake of exogenous ncrnas. one study reports no intestinal absorption of rna (baintner and toth, ) , and three others suggest that rna is degraded before absorption (loretz et al., ; o'neill et al., ; sonoda and tatibana, ) . uptake of plant exogenous rnas, after oral intake is not included here, and will be reviewed in chapter . ; and uptake of exogenous rnas from the pharmaceutical/medicinal area is described in section . . . herein, uptake refers to passage of the molecule (in this case a ncrna) through the cellular membrane as a part of the absorption process. an important feature for the possible biological effect that exogenous rnas, including ncrnas, may have within an organism is their cellular uptake. because of their size and negative charges, exogenous rnas cannot easily passively cross cell membranes (see below). however, earlier studies suggested that exogenous rnas, either homologous (shanmugam and bhargava, ; galand and ledoux, ; sirdeshmukh and bhargava, ) or heterologous (i.e. from e. coli, yeast or others) (natural academy of sciences, ; herrera et al., ; galand et al., ) , could be taken up by mammalian cells (bensch and king, ) . intact, rna may be present inside the cell but is reported to degrade rapidly in some studies (herrera et al., ; shanmugam and bhargava, ) . extracellular ribonuclease activity seems to inhibit uptake (shanmugam and bhargava, ) or function (niu et al., ) of exogenous rnas. uptake of exogenous rna is apparently not due to altered membrane permeability, impaired cell viability, or ribonuclease degradation of the macromolecule during incubation experiments (herrera et al., ) . radioautographic evidences (radioactive assays) have shown the uptake of macromolecular rnas by normal and neoplastic mouse cells, but degradation was also shown to occur prior to or after rna uptake (schwarz and rieke, ) . although intracellular degradation may occur, early studies also described biological actions in response to uptake of exogenous rnas (niu et al., ; niu et al., ; ; esposito, ) , suggesting that once taken up by a cell, rna could eventually exert a biological effect. studies on other types of cellular uptake of exogenous rnas, such as transfer rnas, either homologous or heterologous (depending on donor rna and recipient cell type) have been conducted (gallagher et al., ; sakai and cohen, ; crooke et al., ) . however, as noted in rna therapeutics development (see section . . . ), the large number of barriers encountered by rnas from intake to cell uptake needs to be considered when evaluating possible exogenous rna bioavailability (figure ). the gi tract offers a large surface area potentially relevant for ncrnas cellular uptake and following systemic release, since it contains a large variety of cell types at different maturational levels (e.g. stem cells in the crypts of lieberkühn) located only a few microns from an extensive capillary network (lozier et al., ) . however several barriers exist that could prevent ncrnas uptake by mammalian cells (o'neill et al., ) . for example, in a study conducted in neonatal pigs, no intestinal absorption of rna was observed (baintner and toth, ), suggesting possible rna degradation or an inability to cross the intestinal barrier. gastrointestinal barriers can be distinguished in extracellular and cellular barriers and are presented below. efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. orally ingested exogenous ncrna would have to pass different barriers in the human gi tract. for clarity, only the general barriers are shown. gi barriers to the uptake of orally ingested rnas. the first environment encountered by any orally ingested rna is the oral cavity, where lytic enzymes such as amylase or lipase are secreted in the saliva, and the ph ranges from . to . . the stomach is a more acidic environment (ph . - . ) (dressman et al., ) , and nucleic acids are known to be denatured and depurinated (hydrolysis of nucleic acids to release purine bases) over time in acidic gastric fluid (loretz et al., ) . the gastroenteric fluid flow and peristaltic activity in the gi tract could reduce the contact time between ncrnas and the epithelial layer, therefore diminishing the opportunities for cellular uptake, while nuclease enzymes present in the gi lumen could degrade nucleic acids before any cellular uptake (o'neill et al., ) . indeed, purines and pyrimidines in nucleic acids are believed to be absorbed mainly in the form of nucleosides (sonoda and tatibana, ) . the gut flora, predominant in the distal ileum and in the large intestine, produces a range of enzymes that could degrade ncrnas as well. the gi tract is lined by a viscous sticky layer of mucus entrapping foreign particles before reaching the underlying epithelium (figure ) . this "trapping" effect could be mediated by mucus composition, which confers a net negative charge to the mucous layer. since nucleic acids present a net negative charge as well, this mucus could represent an electrostatic barrier difficult to overcome by rnas, and particularly ncrna, if not properly delivered or chemically modified. furthermore, mucus turnover efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. (secretion, shedding and discard) happens in a relatively short time period ( - min) (lehr, ), preventing rnas from traversing the mucous layer and to reach the underlying epithelium. in conditions such as ulcerative colitis this mucus layer can be reduced or missing in areas of acute inflammation (pullan et al., ) , which would allow nucleic acids to reach the epithelial cell layer. another barrier is the glycocalyx, a glycoprotein and polysaccharide layer ( - nm thick) associated with the apical cellular membrane of enterocytes (frey et al., ) . this layer prevents the access of certain viruses, bacteria and particles (such as rnas) to the underlying plasma membrane by acting as a size-selective diffusional barrier. the intestinal mucosa is structured as a three-layer barrier: a single layer of epithelial cells, the lamina propria ( furthermore, the epithelial cells (enterocytes), which represent approximately % of the epithelium, have a short lifetime of - days, and are continuously shed and replaced (quastler and sherman, ; jung et al., ) . the enterocyte apical membranes are characterized by a high number of microvilli, structures approximately mm long and nm wide (johnson, ) . since endocytosis occurs primarily at the base of microvilli (jung et al., ) , particles with a diameter greater than nm could not be efficiently endocytosed (uduehi et al., ) . m-cells (cells dedicated to trans-cellular transport and associated to intestinal lymphoid follicles) are fewer in number than enterocytes, but, due to their role in the transport of foreign material from the lumen to the immune cells of the lamina propria, are characterized by a high endocytic activity. moreover, m-cells have microfolds in their apical membrane, and are covered by a reduced glycocalyx and mucous layer. this determines a low breakdown capacity (e.g. low alkaline phosphatase activity, and a lower number of lysosomes) so intracellular degradation of ncrnas could be spared. conversely the capillary network underlying the m-cells is less dense and less permeable (o'neill et al., ) . although the literature has described these cellular barriers when studying several molecules, there is little specific data (o'neill et al., ) on the relevance of these to exogenous rna molecules. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. assuming that ncrnas could overcome the gi barriers and reach the circulatory system, they would still encounter other biological barriers before reaching the appropriate machinery to exert any effects. significant degradation occurs of sirna duplexes (non chemically modified) in min when they are incubated in foetal calf or human sera (haupenthal et al., ; urban-klein et al., ) . sirna in human plasma is rapidly degraded, with nearly % degraded within min (layzer et al., ) . nuclease activity in plasma and tissues is relevant to degrading rnas, including ncrnas. the major activity in plasma is a ' exonuclease, but cleavage of internucleotide bonds can also take place (juliano et al., ) . encapsulation methods, which protect rnas from degradation, include exosomes, microvesicles and apoptotic bodies, all of which are extracellular vesicles distinguished by size, biogenesis and cargos (ela et al., ; van niel et al., ) . exosomes, for instance, contain various species of rna, conferring them protection against degradation and providing them a vehicle for cellular uptake of rnas by endocytosis (cui et al., ). the reticuloendothelial system (res) is comprised of phagocytic cells, including circulating monocytes and tissue macrophages, whose physiological function is to clear the body of foreign pathogens, remove cellular debris generated by tissue remodelling, and clear cells that have undergone apoptosis. additionally, the res plays an important role in uptake and clearance of individual "free" oligonucleotides. phagocytic cells of the res, particularly the abundant kupffer cells in the liver and splenic macrophages, express a number of cell surface receptors, including integrins and scavenger receptors, that are potentially involved in uptake, although the role of scavenger receptors in uptake of free oligonucleotides is somewhat controversial (juliano, ). following internalization of an oligonucleotide, the phagosome fuses with lysosomal compartments, where the contents are subjected to enzymatic degradation by proteases and hydrolases that operate efficiently in the low-ph lysosomal environment. tissues macrophages are most abundant in the liver and spleen, and both these tissues receive high blood flow (juliano et al., ) . the capillary lumen is surrounded by a layer of endothelial cells interlinked by adherence junctions (vecadherin containing) and by tight junctions (occluding and claudin-containing) and tightly adherent to the underlying extracellular matrix. this structure forms a barrier between blood and the parenchymal space. molecules in the blood can be transported across the endothelial barrier by two routes. paracellular transport occurs through the junctions between cells and is limited to molecules of approximately nm diameter or less; only micrornas would be able to transit this barrier by this method. caveolar-mediated transcytosis carries large proteins across the endothelium within vesicles of about nm. in some tissues, such as liver and spleen, there are gaps or fenestrations of - nm diameter between the endothelial cells, which allow egress of larger molecules (juliano, ). endothelial permeability is also increased in sites of inflammation and in some tumours, in which increased permeability is associated with fenestrations between the endothelial cells that result from rapid and disorganized angiogenesis in the tumour. this is known as the epr effect (enhanced permeability and retention), although some argue that this is mostly due to poor lymph flow in the area the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. and many minutes to several hours for the elimination phase; (ii) oligonucleotides are accumulated in most tissues, particularly in kidney and liver, but not in the central nervous system (probably due to the blood brain barrier); (iii) the major route of elimination is via the kidneys; and (iv) although the most detailed studies have been performed in rodents, the pharmacokinetic behaviour of the modified oligonucleotides in humans is similar to that observed in lower animals (juliano et al., ) . the pharmacokinetics and biodistribution of sirna duplexes is similar to that of single-stranded antisense molecules, with the highest uptake in the kidney followed by the liver (juliano et al., ). sirna and uncharged oligonucleotides do not bind extensively to plasma proteins, and are thus cleared by the kidney much more readily than modified oligonucleotides and tend to accumulate at lower levels in tissues (juliano, ) . typically, molecules with sizes of - nm or less can be ultrafiltered by the kidney; micrornas could therefore be rapidly excreted by the renal route, unlike long ncrnas. in fact, sirna molecules intravenously injected can be visualized in renal filtrate within seconds of injection (molitoris et al., ) . plasma sirna concentrations are reported to decline by more than % within min (relative to levels at min post dose) and decline by more than % within h (thompson et al., ) . only about - % of the iv administered dose is absorbed by tissues and the majority of this uptake occurs in the kidney (thompson et al., ) , most of it being cleared from tissues within h of administration . to reach their target, ncrnas must enter mammalian cells and arrive at the appropriate subcellular location (the nucleus or the cytoplasm, depending where the target molecules are located). like other large, polar and charged biological macromolecules, ncrnas are internalized by endocytosis and then trafficked through multiple membrane-bound intracellular compartments. the ability of ncrnas to reach their targets depends on both cellular internalisation (uptake) and intracellular trafficking. (figure ). there are five major classes of endocytosis: (i) the clathrin-coated pit pathway; (ii) the caveolar pathway; (iii) the noncaveolar, clathrin-independent pathways (clic pathways); (iv) phagocytosis (mainly taking place in "professional phagocytes" such as macrophages and granulocytes); and (v) macropinocytosis (in which internalized macromolecules are simply dissolved in the ambient medium) (juliano et al., ). non-coding rnas (ncrnas) can enter cells via different endocytic pathways, including micropinocytosis. internalized ncrnas can traffic from early endosomes (ee) to late endosomes (le) and to lysosomes. ncrnas must escape endosomal organelles to reach the cytosol and nucleus and act on the target molecules. some proteins may direct ncrna to non-productive pathways. the micropinocytosis pathway may represent a non-productive pathway for naked ncrnas. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the first four classes often involve a cell surface receptor and are collectively termed receptor-mediated endocytosis. these pathways are mainly utilized in cellular delivery of oligonucleotides. in general, receptor-mediated endocytosis includes three major steps. (i) receptor binding and internalization represents the primary barrier for oligonucleotide transport; the ligand-receptor binding determines which target cells and tissues oligonucleotides are delivered to. (ii) sequential intracellular trafficking leads oligonucleotides into a variety of low ph endomembrane compartments, including early/sorting endosomes, late endosomes/multivesicular bodies, and lysosomes. in some cases, receptors/ligands can traffic to the golgi complex. in many instances, receptor and ligand are dissociated in the low ph endosome environment. vesicular trafficking can prevent ncrnas from reaching their targets, for example, by sorting to secretory or lysosomal vesicles which may lead to export of ncrnas out of cells or degradation in the lysosomes. (iii) ncrnas must exit from the endosome to reach the site of action in the cytoplasm or nucleus. endosomal trapping represents an important barrier for ncrnas final functional objective. on the other hand, oligonucleotides are able to continuously shuttle between the nucleus and the cytoplasm mediated by nuclear pore structures, and do not require classical nuclear localization signals (juliano et al., ). the available data clearly describe several barriers to be considered when evaluating exogenous rna absorption, circulation, cellular uptake or intracellular trafficking. exogenous rna must overcome numerous biological barriers to reach its intended target within mammalian cells. considering oral administration as the most relevant for exogenous plant ncrnas, the first major limiting step to exogenous rna bioavailability is the gi tract itself. the gi tract encompasses both extracellular and cellular barriers. the extracellular barriers include the presence of enzymes in the lumen, such as amylase or nucleases, a harsh environment with ph ranging from . to . , and a net negative charged mucous layer with a very rapid turnover ( - min). the cellular barriers include a single layer of epithelial cells, the lamina propria and the muscularis mucosa, constituting the threelayer intestinal mucosa barrier. ncrnas could cross the epithelium between cells but this is limited by the presence of tight junctions. pore size in the human intestine would prevent passage of all ncrnas other than mirnas, the smallest ncrnas. ncrnas could also cross this epithelium through transcytosis by traversing the cells; this mechanism implies exposure to intracellular nucleases, recycling of ncrnas back to the lumen and nuclear uptake. many biological barriers and environmental conditions between the gi epithelium and the target tissue are then encountered by ingested rnas. ncrnas would be exposed to nucleases both in the plasma and in the target tissues. once inside the circulatory system, these rnas would be subjected to distribution and elimination, accumulating mostly in the liver and kidney. due to their small size, mirnas are especially rapidly cleared by the kidney, unlike long ncrnas. therefore, a very small percentage of rnas could actually be absorbed by tissues. furthermore, ncrnas would need to escape the reticuloendothelial system, the function of which is to clear the organism of foreign molecules. then rnas must cross the vascular endothelial barrier to reach the target tissue. for ncrnas to exert their functions they must enter the target cells. rna cellular uptake is achieved by endocytosis after which they would enter the intracellular trafficking system through multiple membrane-bound compartments. ncrnas would need to exit these compartments to reach their functional sublocation within the cell, be it the cytosol or the nucleus, while simultaneously escape degradation in the lysosomes. efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. seeds, corn kernels and rice grains, all of which are common food and feed, and numerous endogenous plant srnas are reported to have perfect complementarity to genes in humans, and other animals . however studies in mammals and humans with sirna indicates that the exposure levels required to produce regulatory effects exceed the levels achieved following ingestion . all animal and plant-related foods and feeds contain naturally occurring coding (i.e. mrnas) and ncrnas. early (srivastava, ; lassek and montag, ) and more recent studies ) have estimated that the total rna content in plants is about mg/g plant tissue. plants contain an array of ncrnas, including highly abundant transfer rnas (trnas) and ribosomal rnas (rrnas), single-stranded antisense rnas, and the mirnas and sirnas that trigger rnai and their precursor dsrnas . long dsrnas from non-plant, exogenous sources are particularly common in plants, including plants used for food and feed, due to infection from rna-containing viruses. animalderived foods are generally richer in rna than plant-derived foods, and are likely to contribute significantly to overall rna consumption (jonas et al., ) . of the mg/g tissue of total rna content in plants, relative percentages (by weight) of the major rna forms are approximately % rrna, - % mrna, and - % trna, with srnas accounting for less than % of total rna in plants . srnas are present at levels of up to . µg/g of conventional soybean grain (average = . µg/g grain) and comparable amounts are present in the grains of conventional corn and rice . in tobacco plants engineered to overexpress a dsrna under the control of a constitutive promoter, sirnas levels in leaves reached about . % of total rna (chau, b. l. and lee, k. a., ) . total exposure to construct-derived srnas from a putative biotech soybean product was estimated to be µg/kg/day in the general population and µg/kg/day for children aged six and under . using these same assumptions for rna expression levels in sweet corn, consumption of corn at the highest possible rate would result in an estimated intake of µg/kg/day in the general population and µg/kg/day for children aged in the absence of encapsulation or chemical stabilization to prevent degradation or without the addition of penetration enhancers, the absorption of rna -including sirnas -across the gi tract is described as negligible (akhtar, ; jain, ) . one study suggests that activity could be possible for certain highly expressed plant mirnas following food intake (zhang et al., a) . this study reports that in mice fed a diet consisting entirely of cooked rice (i.e. human equivalent of about kg/day of cooked rice) , several rice mirnas were detectable in mouse serum and liver. some of the results presented in this study were contested in later studies (chen et al., ) . petrick et al. clearly stated the unlikelihood of achieving such high concentrations of mature plant mirnas in the serum, plasma and organs of humans or animals via food intake since the high levels of rice administered the experimental animals did not reflect anticipated dietary exposure levels. in another study, it was concluded that plant mirnas identified in animal srna sequencing data can originate from artefacts of the sequencing process (zhang et al., b) . even in the case of -mer oligonucleotides modified to increase their stability, oral bioavailability in rats was only . % (nicklin et al., ) and intestinal absorption was less than % in a model that bypasses the gastric acidic environment. most of the labelled oligonucleotide was associated with the luminal epithelial cell membrane and very little efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. was localized intracellularly (khatsenko et al., ) . regarding the possibility of dietary sirnas and other dsrnas effects on gastrointestinal tissues, studies with radiolabelled or immunostained dna oligonucleotides demonstrate that these are primarily located extracellularly within the intestinal tract (lumen and luminal wall) (khatsenko et al., ; nicklin et al., ) , and are thus presumed to be minimally absorbed (negligible bioavailability, < %). it has also been observed that absorption decreased as the oligonucleotide length increased (from to nt) (khatsenko et al., ) . the only published quantitative data on srnas content in plant-derived foods ) (detailed above) indicate that . µg of srna is obtained per gram of soybean, rice seeds or corn kernels. as total rna content in plant-derived foods appears to be around mg/g tissue (lassek and montag, ), srnas could therefore represent . - . % of plant seed materials. the amount of sirnas in plants genetically modified to carry hairpin transgenes is estimated at about ng sirna/ µg total rna/g tobacco leaf tissue (chau, bess l. and lee, kevin aw, ) . the resulting estimated human dietary exposure to plant small rnas from rna-based biotech crops was derived from this (table ). . (b) assuming % of the specific food is consumed from a biotech crop, with total rna levels of ≈ mg/g grain and . % of these small rnas derived from the transgene. for details see . these doses are significantly lower than those required to elicit adverse effects in rodents or monkeys ( or mg/kg of chemically-stabilized sirna, respectively) by iv administration (thompson et al., ) . moreover, high mirna concentration is required to target suppression, and levels below copies/cell mirnas have little regulatory capacity (mullokandov et al., ) . according to snow et al., dietary plant mirnas are present at less than one copy per cell in target organs assuming reliable quantification . in another study the calculated dietary exposure in humans of a specific dsrna (dvsnf ) was estimated to be around . x - µg/kg/day in the us population (petrick et al., b) . the same study reported that mg dvsnf /kg/day in a -day repeated dose oral (gavage) toxicity study in mice did not produce any toxic effects (petrick et al., a) . levels for this specific dsrna in this biotech crop were reported to be ≈ . x - µg/g grain tissue on a dry weight basis for corn food, and ≈ . x - µg/g in the whole plant on a dry weight basis . of note is that there is little information on the impact of intended rnai in gm plants on the coding and non-conding plant rna. knowledge of which other rnas are modified, or how other rnas are compensated or desregulated in the gm plant needs to be experimentally evaluated for each specific case. different studies have evaluated the amount of plant-derived foods consumed by the general population. in a study of cohort of women in the uk, the difference between the mean intake of overall fruit and vegetables consumption was three times greater in high consumers vs. low consumers (pollard et al., ) ; vegans and vegetarians were the highest consumers of fruit and vegetables. in an efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. evaluation of the flemish population it was observed that vegans, while having the lowest total energy intake compared to omnivores, have the highest total fruit and total vegetables consumption. the intake of fruit, nuts and/or legume about twice in vegans, using either the healthy eating index (clarys et al., ) or total mean intake (clarys et al., ) . in the epic cohort study, which includes ten european countries, mean daily consumption was between and g/day vegetables and between and g/day fruit (agudo et al., ) . although not homogeneous among countries and centres, the ratio between the highest and lowest consumers was around . for vegetables and for fruit (agudo et al., ) . countries in southern europe had the highest vegetables and fruit consumption. for example, consumption of vegetables and fruit in healthy adults in spain (epic data), considered higher than most european countries and the usa, was g ( . servings) of vegetables and g ( . servings) of fruit (agudo et al., ) , equal to ≈ g/d vegetables and fruit. in the united states (us) the total vegetable and fruit intake has increased slightly (up to ≈ . servings/day) in all consumer categories from the late s (krebs-smith and kantor, ) and remained stable over the last decade (rehm et al., ) . actual quantities, however, are around . servings/day (rehm et al., ) , that is . for fruit and . for vegetables (eaton et al., ) . in cup-equivalents, this is . cup equivalents for vegetables and . cup equivalent for fruit (moore and thompson, ) . mean total vegetable intake in the us for vegetarians is reported to be around g/day and that for non-vegetarians as g/day. total fruit intake was around g/day for vegetarians and g/day for non-vegetarians (haddad and tanzman, ) . in other words, there is less than two-fold difference in the amount of fruit and vegetables consumed between vegetarians and non-vegetarians in the general us population. in a specific population (i.e. the adventist health study) in the us, daily mean consumption of fruits was higher (≈ g/day) for vegans than for other types of vegetarians or non-vegetarians (≈ g/day). the same trend was observed for daily mean consumption of fruit which was higher in vegans (≈ g/day) than other vegetarians or non-vegetarians (≈ g/day) (orlich et al., ). based on these data, dietary exposure to rnas from plants can in general be assumed to be higher in vegans and vegetarians than omnivores. although total rna content in plant-derived foods varies (lassek and montag, ), a person consuming an estimated daily dose of total fruit and vegetables ≈ g/day would theoretically ingest mg of total rnas assuming about mg/g of plant tissue. this value agrees with the dietary rna intake, which typically ranges from . - g/person/day (jain, ) . considering that srnas represent up to . % of plant seed materials ), estimated daily intake in the general population would be around . mg srnas. since vegans and vegetarians normally consume higher amounts of vegetables and fruits (see above), they could be exposed to an average of three times more plant exogenous rna ( . mg/day). no pharmacokinetics studies were identified in these groups. changes in rnase activity can occur in physiological or pathological conditions, this possibly impacting exposure after dietary intake. for instance, increased serum rnase activity has been observed in pancreatic carcinoma, pancreatitis, and renal failure (peterson, the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. et al., ) . human argonaute (ago- ) is a catalytic core component of rnai machinery. ago- mediated gene silencing has been proposed to be linked to the mitogen activated proteine kinase signalling pathway (zeng et al., ) , which is activated in response to cellular stress. since various kinds of stress on human cells induce formation of stress granules, ago- can be recruited to these granules, modifying its intracellular localization and the efficacy of rnai activity (detzer et al., ) . ago- is thus regulated at both the transcriptional and post-translational levels, suggesting that ago- levels can influence cellular mirna activity (adams et al., ). it has also been proposed that inactivation of mitochondria could lead to a strong decrease in mirna-mediated rnai efficiency, and, to a lesser extent, to sirna-mediated rnai (huang et al., ) . due to the interaction of p-bodies with mitochondria, reduced ago- activity could be due to changes in location of endogenous ago- from pbodies (huang et al., ) . association of ago- with cytoplasmic rna granules is known to regulate the translational repression activity of the protein, but phosphorylation of certain residues may prevent this inhibition, allowing the protein to remain active in the cytoplasm (lopez-orozco et al., ) . overall, it seems clear that ago- activity can be modified by cellular stress conditions. however, whether this can influence the function or the exposure to dietary ncrnas remains unknown and would benefit from further research. most studies have been focused on mirnas since these ncrnas were the first to be described. these studies have consequently extended to transgenic dsrnas as these have increasingly used to induce gene silencing. as mentioned previously, little is known about dietary exposure to other ncrnas. given the role of lncrnas in plant physiology, it is important to better understand human and animal exposure to these ncrnas. further research is needed on how transgenic ncrnas affect expression levels of other ncrnas, and other rnas such as mrnas. alteration of the expression levels of certain ncrnas within the plant may modify the levels of other ncrnas due to putative compensatory circuits and codifying rnas. this in turn could lead to changes in protein and enzymatic content, with putative consequent alteration of the modified plant's nutritional value. controlling for these changes would require complete transcriptome sequencing, and comparison of rna levels in unmodified to those of the altered plant . another point to consider is whether special diets (e.g. vegetarian or vegan) modulate ncrnas uptake throughout the gi tract, possibly leading to increased exposure. humans and animals have been continuously exposed to naturally-occurring plant rnas. estimated amounts of ingested plant ncrnas contrast with the much higher doses administered to experimental animals and in clinical trials to humans. although some reports on the presence of plant mirnas in serum, plasma and organs of humans or animals do not agree, the extremely low reported concentrations prevent them from being functional, even in the case of rnas modified artificially to augment their bioavailability. humans following special diets, such as vegetarians and vegans, have high intake of plant rnas, but their increased estimated exposure to such rnas is barely on average fold greater than in omnivorous diets. there are no specific studies determining the effects of increased dietary exposure to plant ncrnas. adams bd, claffey kp, white ba. . argonaute- expression is regulated by epidermal growth factor receptor and mitogen-activated protein kinase signaling and correlates with a transformed phenotype in breast cancer cells. endocrinology , - . efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. to exert a biological effect, exogenous ncrnas need to overcome the physical and biological barriers present in both the gi tract and the circulatory system, reach the target tissue, and enter the appropriate intracellular pathway at sufficient dose. this section describes the obstacles and challenges encountered by exogenous ncrnas following oral intake. following an extensive literature search, and based on expert judgement in the topic, publications were reviewed for this section. rna molecules are large, hydrophilic and negatively charged. even the smallest rna molecules (mirnas) have a high molecular weight (∼ kd) and their net charge is negative (akhtar and benter, a; akhtar and benter, b) . due to these physicochemical properties, rna molecules are highly water soluble with low membrane permeability, thus falling into class iii of the biopharmaceutical classification system (fda, ; amidon et al., ) . like many other class iii compounds, they are not absorbed at all or only to a very small degree. being highly charged species, rna molecules resist partitioning across lipid bilayers, and higher molecular weights effectively restrict their movement by function of the tight junctions present in the gi tract epithelium (tillman et al., ) . in vitro and in vivo preclinical studies have assessed the use of medium chain fatty acids ( - carbon atoms), as well as bile salts, as absorption enhancers to cross the intestinal mucosa (gonzalez ferreiro et al., ; tillman et al., ; raoof et al., ) . orally introduced rna molecules encounter relevant obstacles in the gi tract, these precluding their absorption and activity. in general oligonucleotides are rapidly degraded in the harsh biological milieu of the acidic stomach and enzyme-rich gi tract, as described previously, and show poor transcytosis across the gut (akhtar, ) . in humans the ph in the gi lumen can vary from in the stomach to in the intestine (gamboa and leong, ) . exposure to these ph values can cause ph-induced oxidation or de-amination of rna, resulting in loss of activity (pridgen et al., ) . in addition, nucleases present in the gi lumen for digestion of biological molecules enzymatically degrade rna molecules too (gamboa and leong, ; kriegel et al., ) . the presence of a rich bacterial population, mostly in the intestines (the human microbiome project consortium, ) may also contribute to degradation of these molecules, as bacterial degradation of ribonucleic acids through the secretion of extracellular nucleases has been early described (nishimura, ; eaves and jeffries, ) . finally, if the above-mentioned obstacles are overcome, rna molecules still face the intestinal extrinsic and intrinsic barriers (see also . . ). the extrinsic barrier consists of the mucus layer covering the epithelial cells. this is a complex hydrogel material composed of proteins, carbohydrates, lipids, salts, antibodies, bacteria, and cellular debris (o'neill et al., ; pridgen et al., ) . it consists of loosely and firmly adherent layers that vary in thickness along the gi tract and can fluctuate based on diet. the mucus is secreted by cells presenting a rapid cell turnover, approximately - days in the small intestine in humans (gamboa and leong, ; o'neill et al., ; atuma et al., ) . among other functions, the mucus layer protects www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. epithelial surfaces by trapping pathogens and foreign particulates, and rapidly clearing them. penetration of this mucus barrier is necessary to reach the absorptive epithelial cells. several mucuspenetrating and mucoadhesive materials can be used to enable penetration of molecule to the mucus layers, increasing residence time and contact of the delivered molecules to the epithelium (pridgen et al., ) . the intrinsic barrier consists of the epithelial cell monolayer, constituting the greatest barrier to material from the intestinal lumen to the lamina propria and bloodstream. cells maintain this barrier by forming tight junctions, whose permeability can be modulated through specific combinations of different proteins (o'neill et al., ) . there are several possible pathways across the intrinsic (epithelial) barrier (figure ).  the transcellular pathway passes through the apical and basolateral cell membranes, as well as the cytoplasm. this pathway is very restrictive to the passive flow of hydrophilic solutes such as rna molecules, because of the lipid bilayer membrane and its impermeability to large molecules. transport mechanisms for this pathway can be passive for hydrophobic molecules, or active with membrane pumps for specific molecules such as ions.  the paracellular pathway is the major passive permeation pathway and allows diffusion of small molecules in the space between epithelial cells. the tight junctions regulate permeability of this pathway based on molecules' size and charge.  finally, transcytosis is an active transport pathway that relies on molecule-specific receptors guiding the molecule through the cell without entering degradation pathways. because of their large hydrodynamic size, macromolecules such as rnas are restricted to this pathway (pridgen et al., ) . the m cell transcytosis pathway is the most extensively studied for oral delivery of rnas. this pathway, which is used to transport antigens across the epithelium for immune surveillance, is attractive because m cells lack mucus secretion and have a sparse glycocalyx, among other features. however m cells are closely associated with immune cells in the lamina propria and peyer's patches, such as dendritic cells and macrophages, this from one side limiting the ability of the delivered molecule to reach the bloodstream and on the other side increasing the risk of triggering an immune response (florence, ; kriegel et al., ; pridgen et al., ) . moreover, m cells only make up a small percentage ( - %) of the non-absorptive epithelium in humans. the surface properties of m cells, as well as the number of peyer's patches vary among species, which could make difficult extrapolating animal model data to humans (pridgen et al., ) . an additional obstacle is represented by the immune system, which is intimately associated with the epithelium. numerous types of immune cells patrol the lamina propria, including t cells, macrophages and dendritic cells. if the ingested rna molecules reach the bloodstream, they must also evade the mononuclear phagocytic system (reischl and zimmer, ; pridgen et al., ) . the presence of endogenous and luminal nucleases, together with gi ph conditions, in general determine rapid degradation and low rna molecules' biological half-life (bhavsar and amiji, ) the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the paracellular pathway allows diffusion of molecules in the space between epithelial cells and is regulated by intercellular tight junctions. the transcellular pathway passes through the apical and basolateral cell membranes and cytoplasm. it is restricted to hydrophobic molecules or molecules transported by membrane pumps. the transcytosis pathway is an active transport system that relies on molecule-specific receptors guiding the molecule through the cell escaping degradation pathways. transcytosis pathways are found in both epithelial and m cells. from pridgen et al, . transepithelial transport: paracellular, transcellular and transcytosis pathways. first-pass elimination occurs when a compound is metabolised between its site of administration and the site of sampling for drug concentration measurement. this greatly affects the bioavailability of orally administered compounds since the concentration of the active substance reaching systemic circulation is decreased. the liver is usually assumed to be the major site of first-pass metabolism of an orally administered drug, but other sites are the gi tract, blood, vascular endothelium and lungs (pond and tozer, ) . in fact, the obstacles previously described for rna molecule absorption in the gi tract can be considered a first-pass effect. rna molecules reaching the circulatory system must avoid filtration by the kidneys, accumulation in non-target reticuloendothelial system (res) of the liver, kidney, lungs and spleen, degradation by endogenous nucleases, aggregation with proteins in the serum, and uptake by non-targeted cells such as phagocytes (kriegel et al., ; reischl and zimmer, ). once cellular uptake has taken place in the potential target tissue (surmounting the obstacle of crossing the vascular walls and the lipid bilayer cellular membranes), nucleic acids have to undergo efficient intracellular trafficking to ultimately produce the intended pharmacological effects. this includes endosomal transportation (escaping endolysosomal degradation), cytoplasm release, and efficient incorporation into the relevant intracellular pathways such as risc loading (for sirna, mrna, and mirna) or nuclear uptake (for lncrna) kriegel et al., ; pouton and seymour, ) . www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. rna molecules are large, hydrophilic and negatively-charged molecules. to achieve effect via the oral route, these molecules need to overcome the physical and biological barriers present in both the gi tract, where they encounter variable and harsh conditions along with degrading nucleases, and the circulatory system. rna molecules must reach the target tissue, escaping endo-lysosomal degradation, and enter the appropriate intracellular pathway in sufficient doses to exert their effects. www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. following a literature search as described in section . . . . and based on the methodology described in section . . . , a total of documents were selected as relevant to a general review of rna-based therapeutics administered by gi route. of these documents, at least report the use of specific vehicles for gi administration of exogenous ncrnas (i.e. nanoparticles). six of these documents simultaneously evaluated formulated exogenous rnas and their naked version, and reported either rapid degradation under gi conditions, or reduced or absence of biological effect of the naked version compared to the formulated one (table ) . twenty ( ) of these documents provide specific examples of local effects of rna-based drugs administered orally. in addition, papers were selected as relevant to inform on administration of exogenous ncrnas in either fish or birds and possibly exerting biological effects. orally delivered rna has been used as a dietary supplement or empirical therapeutic agent since s (jain, ). for example, yeast rna supplementation as a source of dietary ribonucleic acids has been used in different animals (choudhury et al., ; jha et al., ) including mammals (sukumar et al., ; heaf and davies, ) and in human studies (gianotti et al., ; tepaske et al., ) . in human studies, up to g per day (in divided doses) of exogenous rnas have been administered (for - days), producing an increase in plasmatic uric acid levels (zollner and grobner, ; zollner, ) . the absence of attempts to study the gi absorption of pure rna in these studies suggests that its effects, if any, could be related to rna molecules as a source of nucleotides. indeed, nucleotide supplementation in rats has been shown to affect the healing of ulcerative conditions, ameliorating indomethacin-induced ileitis and aggravating the severity of dextran sulfate sodium (dss)-induced colitis (sukumar et al., , sukumar et al., . also, a study of body fluid composition of rats orally administered yeast rna, mixtures of its constituent nucleosides or its constituent bases and ribose was performed (heaf and davies, ) . the authors showed that ingestion of rna increased intestinal levels of ribose, inorganic phosphate, uridine, pseudouridine, uracil, inosine or uric acid. the effect of orally administered mixed nucleosides on blood and urine composition was similar to that of rna, while some differences were noted for some equivalent mixture of free bases (heaf and davies, ) . this study showed that the dietary rna-phosphate passed to the urine from the gut, suggesting that most of the rna-ribose was probably metabolized (heaf and davies, ) . it has been shown in a murine model of staphylococcus aureus infection that the oral administration of rna was ineffective while the intraperitoneal nucleoside-nucleotide mixture was more effective in maintaining host resistance against bacterial infection (adjei et al., ) . supplementation with nucleotides has also been shown to exert biological effects, including expression regulation of certain genes (sanchez-pozo and gil, ; gil, ) , although the mechanisms of action are still unknown. it is generally assumed that nucleotides are not essential nutrients, but under certain conditions (i.e. low dietary intake, tissue needs increased or stress) dietary nucleotides may play a role as conditionally essential nutrients (jung and batal, ; carver and walker, ) . in this context, dietary supplementation with yeast rna promoted ulcer healing in a rat model of indomethacin-induced ulcerative ileitis (sukumar et al., ) . similar effects were observed when nucleosides and nucleotides were administered intravenously (veerabagu et al., ) . in both cases, the possible mechanism of action was partly due to increased cell proliferation in the damaged tissue (sukumar et al., ; veerabagu et al., ) . similar effects were observed when rna was administered orally or its constituents (nucleotides) were injected iv, suggesting that the effects are due to the absorbed nucleotides. dietary nucleotide supplementation in formula-fed infants efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. has been shown to improve gut microbiota composition (singhal et al., ) . in studies on certain fish model, supplementation with dietary rna at a range of concentrations ( . - . % of nucleic acids in the diet compared to the non-suplemented control group) enhances immunological response (choudhury et al., ; jha et al., ) . in human studies, postoperative enteral diet supplementation with rna, arginine and omega- fatty acids has been shown to modulate the postoperative immune response after surgery for upper gastrointestinal cancer (senkal et al., ) , overcoming more rapidly the immunologic depression after surgical trauma (kemen et al., ) . also in humans, oral supplementation with rna (from yeast) during days before surgery improved host defence in patients undergoing cardiac surgery (tepaske et al., ) or gi cancer surgery (gianotti et al., ) . in both cases, rna supplementation was also accompanied with arginine and omega- fatty acids administration. in summary, the specific contribution of orally administered rna to these effects cannot be ascertained. the instability of orally administered rna molecules and the presence of a large array of rnaases within the gi tract (see sections . . and . . ) suggest low absorption, if any. exposure to a highly active enzymatic environment in the gi tract, extreme ph conditions, and the existence of a mucosal epithelial barrier are the main challenges for oral delivery of rna therapeutics (martirosyan et al., ) . although a considerable amount of food rna is ingested, which varies widely between individuals but is typically in the range . - g/person/day, it is assumed it is degraded and absorbed in minimal amount (jain, ) . however, further data are required to document the extent to which this conclusion pertains to rna molecules with complex structures. to overcome the multiple barriers encountered by exogenous rnas for oral delivery, several strategies in the laboratory or clinical trials have been developed (see . . . below). the oral administration route is advantageous because it increases patient compliance and comfort over injection, provides for simple, repeatable administration, and offers a large surface area for absorption (forbes and peppas, ) . rna delivery may also benefit from advances in the oral delivery of aso for animal (raoof et al., ; raoof et al., ; . indeed, polymeric nanoparticles containing alginate, dextran, chitosan, polyethylenimine, polyethylene glycol, polylactide, yeast derived β-glucans and/or several other natural or chemically synthesized molecules have been tested by the pharmaceutical/medicine field. the literature describes several strategies for the oral delivery of rna oligonucleotides. the target is to stabilize and protect rnas during gi transit, enable cellular uptake in the intestine, and in some cases, promote endosomal escape and increase biological action. thioketal nanoparticles (tkns) were formulated from a polymer, poly-( , -phenyleneacetone dimethylene thioketal) that degrades selectively in response to reactive oxygen species ( the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. (plga) nanoparticles were also tested in the same study, but with no efficacy in diminishing tnfα expression. to obtain efficient mucus transportation, targeted cellular uptake and endosomal/lysosomal escape, different nanoparticles have been tested ( nanoparticles of galactosylated trimethyl chitosan-cysteine containing sirna against map k were tested and found to target activated macrophages in colonic tissue (zhang et al., b) . compared to nanoparticle-protected sirnas, naked sirnas were completely degraded by the intestinal fluids. when administered orally ( µg sirna/kg) for days, nanoparticles improved the dss-induced model of colitis in mice (zhang et al., b) . other ternary polymeric nanoparticles formed by thiolated trimethyl chitosan with tripolyphosphate have been tested in mice and found to efficiently deliver sirnas to the intestine and other tissues when administered orally (zhang et al., a the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. ( %, % and %) were also evaluated in trimethyl chitosan-cysteine conjugate nanoparticles . in a mouse model of ulcerative colitis, the mannose density of % was the most effective for sirna knockdown of tnfα following oral administration ( µg/kg) of '-o-methyl-modified tnfαspecific sirna duplex . the use of supramolecular self-assembly nanoparticles (ssnps) -which contain oleyl trimethyl chitosan, poly(γ-( -(((piperidin- -yl)ethyl)amino)methyl)benzyl-lglutamate), oleyl-peg-mannose, oleyl-peg-cysteamine, and sodium tripolyphosphate -with specific functions of mucoadhesion, transepithelial permeation, membrane penetration and active targeting have also been tested ( however, not all nanoparticle types can efficiently deliver rnas. in a study evaluating the ability of unassisted epithelial entry of nucleic acids as a consequence of nanocarrier contact with mucus, antisense oligonucleotides but not sirna (naked) were shown to be delivered to mouse intestine when formulated with nanocarriers composed by chitosan (martirosyan et al., ). multi-compartmental formulations -consisting of one or more internal compartments surrounded by protective external compartments -have been developed to surmount the many barriers to oral rna delivery (kriegel et al., ; . one example is the "nanoparticles in the microspheres oral system" (nimos), a solid-in-solid multi-compartmental system (kriegel et al., ) . gelatin nanoparticles entrapped in poly(epsilon-caprolactone) microspheres were prepared carrying tnfα sirna (slightly chemically modified dtdt) to treat dss-induced colitis. oral administration of sirna ( . mg/kg) to female balb/c mice decreased colonic tnfα expression and supressed the expression of proinflammatory cytokines (kriegel, c and amiji, m, ) . when combined with sirna against cyclin d (naked sirna, also at . mg/kg/day), the silencing effects were more potent than with tnfα sirna alone (kriegel c and amiji mm, ) . dectin- recognizes beta- , and beta- , linked glucans rich particles and intact yeast and is particularly expressed on the monocyte/macrophage and neutrophil lineage (herre et al., ) . β- , -d-glucan has been used to deliver exogenous rna targeting macrophages (aouadi et al., ). delivery of β- , -d-glucan sirna particles containing µg/kg unmodified sirna by oral gavage to mice for days reduced map k expression in different macrophages from different tissues and protected them from lipopolysaccharide-induced lethality (aouadi et al., the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the cd gene was the target of the antigen-presenting dendritic cells and oral administration of the recombinant yeast. short hairpin rna delivered in lactobacillus casei have also been tested to target the intestine (kuwahara et al., ) . other organisms, including attenuated salmonella typhi, were used as vectors to deliver rnas, even ribozymes (bai et al., ) or sirna (jiang et al., ) , using oral administration in mice to target either local or systemic effects (bai et al., ; jiang et al., ) . the the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. extracellular vesicles are a heterogeneous family of vesicles delimited by membranes. these can be classified by their size as i) exosomes ( - nm in diameter), which originate from the endosomal system, ii) microvesicles ( - nm) formed by budding out of the plasma membrane, and iii) apoptotic bodies (> nm) formed by blebbing of the plasma membrane during apoptosis (reviewed in yanez-mo et al., ; colombo et al., ) . increasing evidence suggests that extracellular vesiclemediated communication can take place in vivo, but the nature of the extracellular vesicles involved in these effects remains to be clarified (reviewed in tkach and thery, ; pitt et al., ) . exosomes, which are formed in multivesicular compartments, are secreted when these compartments fuse with the plasma membrane (kowal et al., ; abels and breakefield, ) . exosomes have been shown to contain or transport a myriad of molecules including proteins, carbohydrates, lipids, and a variety of genetic material including dna, mrna and ncrnas (reviewed in choi et al., ; abels and breakefield, ) . exosomes are also described as participating in intercellular communication (costa-silva et al., ; pironti et al., ; nojima et al., ) , delivering their parental cell-derived molecular cargo to a recipient cell. some of these processes are thought to be mediated by ncrnas, including mirnas thomou et al., ) . detailed information on how all these biological processes occur is outside the scope of this review. given the above characteristics of exosomes, these vesicles hold promise as delivery vehicles for therapeutics (munagala et al., ; van der meel et al., ) . indeed, exosomes have been used to deliver sirna to different tissues when administered systemically, including the brain (alvarez-erviti et al., ; cooper et al., ; didiot et al., ) . mirnas have also been delivered via exosomes to different tissues including xenograft breast cancer tissue (ohno et al., ), brain and other tissues when administered systemically or locally (zhang, d et al., ) . interestingly, oral administration of exosomes or extracellular vesicles has also been described in animal models agrawal et al., ; oliveira et al., ; oliveira et al., ) . however, their biological effects were not related to transport of ncrnas. some extracellular vesicles have been shown to resist digestion under in vitro simulated gi conditions (benmoussa et al., ; , particularly those from bovine milk (vashisht et al., ) . moreover, milk exosomes seem to exhibit cross-species tolerance and are not described to induce adverse immune and inflammatory responses (munagala et al., ) . however, whether exosomes or other extracellular vesicles resist the harsh in vivo conditions of the gi tract and the digestive process remains poorly described and needs to be studied (tomé-carneiro et al., ) . exosome-like nanoparticles from plants have been isolated from different species including ginger, grape, grapefruit and carrot (mu et al., ; ju et al., ; zhang et al., ) . some authors indicated that the approximate amount of exosome-like nanoparticles in these edible plants were reported to be ≈ mg/ g edible plant (mu et al., ) , while other authors reported only ≈ mg/kg ginger . most of these studies reported the presence of rnas, either small or large, including ncrnas (mu et al., ; zhang et al., ; ju et al., ) . the amount of rna present in these exosome-like nanoparticles was reported to be ≈ µg rna/ mg exosomelike nanoparticles for grape and grapefruit, and ≈ µg rna/ mg exosome-like nanoparticles for ginger and carrot (mu et al., ) . the in vivo biological effects of these nanovesicles have been assessed. oral delivery of ginger nanovesicles during days at a dose of . mg/day was found to target the colon, to be taken up by epithelial cells and macrophages, and to reduce dss-induced colitis in mice . treatment for weeks with the same dose also exhibited antiinflammatory activity in a mouse model of chronic colitis . ginger-derived lipid vehicles have also been generated from ginger lipids and loaded with sirna-cd (zhang m et al., www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. ). oral administration of these ginger-derived lipid vehicles targets the colon tissue and they are effective in treatment of induced ulcerative colitis in mice. grapefruit exosomes at a dose of mg/kg per day for days also ameliorated the dss-induced colitis targeting intestinal macrophages . when administered orally at a dose of mg of nanovesicles/day, grape exosomes-like nanoparticles were also found to protect mice from a dssinduced model of colitis by induction of intestinal stem cells (ju et al., ) . local intestinal macrophages and stem cells from the small and large intestine were also found to be the target of these exosome-like nanoparticles when administered by oral gavage ( mg/day) (mu et al., ) . even though mirnas were reported to be present in these exosome-like nanovesicles (ju et al., ; mu et al., ; zhang et al., ) , the biological effects of these ncrnas were not evaluated. whether these mirnas resist the gi tract conditions remains unknown. a main hurdle in rna-based therapeutics is the successful delivery of naked rna (non-chemically modified) molecules to specific (target) tissues in the gi tract. the literature contains few studies in which exogenous rnas are administered in vivo using routes of administration other than oral, and most are in the field of inflammatory bowel disease. an amphiphilic cationic cyclodextrin (cd) vector was developed for a complex sirna against tnfα, where the complex was concentrated to µg/ µl in % glucose (mccarthy et al., ) . cd.tnfα.sirna was found to be efficient against dss-induced colitis in c bl/ mice treated twice (day and post-dss) by intrarectal administration with the test solution ( µl). cd.sirna administration reduced tnfα and il- expression as compared to the non-silencing sirna control or the naked tnfα sirna (not delivered in formulated cyclodextrin). enteral delivery of sirnas for systemic effect was also tested. a nuclease resistance and chemically modified sirna against apolipoprotein b was conjugated with α-tocopherol and was administered ( mg/kg) as lipid nanoparticles in the large intestine of mice. the lipid nanoparticle was composed of mixed micelles comprised of linoleic acid and peg- hydrogenated castor oil. the sirna efficiently reached the liver and other tissues using the chylomicron-mediated pathway via the lymphatic route (murakami et al., ) . delivery of sirna into hepatocytes was markedly reduced when the sirna was not bound to α-tocopherol, indicating that conjugation with α-tocopherol was essential to this delivery system using this route. a calcium phosphate (cap) core coated with sirnas and encapsulated in poly(d,l-lactide-co-glycolide acid) (plga) nanoparticles containing an outer layer of polyethyleneimine (pei) was developed for intrarectal delivery (frede et al., ) . in a dss-induced model of colonic inflammation, intrarectal administration ( µg/day) of nanoparticles containing ng sirna against either tnfα, ip- or kc from day to after % dss treatment, ameliorated the colitis. indeed, while intestinal epithelial cells, dendritic cells, macrophages and t cells could uptake the tnfα sirna nanoparticles, reduction of tnfα was only detected in epithelial cells and t cells (frede et al., ) . the stability of sirna within the cap/plga/pei nanoparticles against enzymatic nucleases under colonic conditions was evaluated. while the sirna-loaded cap/plga/pei nanoparticles were detectable after h incubation at ºc with colonic fluid or homogenate, the same sirnas not formulated in nanoparticles was not detected and was assumed to be degraded. even after exposure to colonic homogenate for h at ºc, the nanoparticles retained biological activity. since the supernatant of the homogenate subsequently incubated with a cell line, still reduced the expression of tnfα by % (frede et al., ) , suggesting that sirna-loaded nanoparticles retain its biological activity compared to that of nacked sirnas (not formulated). www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. other alternatives for rna delivery have been tested. ultrasound-mediated rna delivery has been efficiently achieved in mice (schoellhammer et al., ) . chemically modified sirna against tnfα ( ng sirna in µl water) was delivered in the rectum using ultrasound exposure to khz for . s and after a -day treatment tnfα was efficiently repressed in colonic tissue of a mouse model of induced colitis. in the same study, a mrna (≈ kda) complexed in lipid nanoparticles (lnps) was administered in the colon ( µl lnps solution) with ultrasound and found to be successfully translated locally in the colonic tissue (schoellhammer et al., ) . oral administration of exogenous rnas to exert a biological effect, either locally or systemically, is successful if combined with specific delivery technologies, such as vehicles developed for pharmaceutical/medical purposes. nanoparticles have been designed to release their cargo in specific regions of the gi tract (i.e. where inflammation occurs) or to resist the gi conditions. in contrast to other routes of administration, where normally highly chemically modified rnas are used, oral delivery normally involves use of naked rna or minimally modified rnas formulated with complex delivery vehicles. achieving the local desired effect (i.e. targeting inflammation in the colon) has contributed to the development of delivery technologies and the usage of other routes of administration different than the oral as described above. however, the available literature also suggests that naked or unmodified exogenous rnas are rapidly degraded when exposed to the gi conditions without incorporation into delivery vehicles. extracellular vesicles are natural lipid particles released by many cell types with the potential to mediate cell-to-cell communication. these extracellular vesicles have a tremendous potential as delivery vehicles for therapeutics. however, there are still very few studies evaluating their resistance to the harsh conditions of the gi tract following oral administration. knowledge of their biological effects as transporters of exogenous ncrnas is still very limited. nucleic acid-based therapeutics have the potential to treat numerous diseases by correcting abnormal expression of specific genes. as indicated previously, the oral route of drug administration poses serious delivery challenges due to the gi degrading environment, as well as the need to overcome other barriers preventing nucleic acid delivery. because of these obstacles, efforts in developing oligonucleotide-based therapeutics using the oral route have focused on local gastrointestinal delivery, i.e. directly delivering the drug to the target tissue for localized effects, and, therefore, increasing local bioavailability and maintaining doses low while diminishing possible effects to non-target tissues kriegel et al., ) . crohn's disease and ulcerative colitis are the two principal forms of ibd, characterised by being a chronic relapsing inflammatory condition of the gi tract. the pathogenesis of ibd is dependent on the interaction between local immune and environmental factors in genetically-susceptible individuals (o'neill et al., ) . inflammation in crohn's disease is characterised by high production of the cytokines interferon (ifn)-gamma, il- and tnf-alpha, representing a t helper (th )-th response. in ulcerative colitis, the immune response is characterised by th and an atypical th , with high production of il- , il- and il- (o'neill et al., ) . conventional treatment consists of antiinflammatory and immune-suppressive drugs but, while some medications are effective to combat inflammation in the acute phase, they are ineffective in maintaining remission due to toxicity, dependency and higher relapse rates (kriegel, c and amiji, m, ) . the goal for ibd treatment is local delivery of therapeutics to intestinal immune cells. initial approaches involved il- supplementation www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. due to its huge potential in blocking proinflammatory cytokines and inflammatory tissue damage. but human clinical trials have not proven that systemic il- supplementation can help prevent or improve the ibd symptoms, probably due to il- low mucosal levels . separate studies have used gene therapy to increase il- mucosal availability by engineering il- plasmid dna, and achieving more success in rodent models (barbara et al., ; steidler et al., ; lindsay et al., ; bhavsar and amiji, ) . deregulation of tumour necrosis factor (tnf)-alpha production, a proinflammatory cytokine primarily produced by activated macrophages along with other cell types, is associated with the onset and progression of a number of inflammatory diseases (such as ibd, rheumatoid arthritis), alzheimer's disease and psoriasis . the harmful effects of tnf-alpha are thought to be mainly mediated by activation of nf-kappab, therefore regulating expression of over genes. systemic blockage of tnf-alpha is accompanied by several side effects that can be avoided if local inhibition occurs. rnai-mediated local suppression of tnf-alpha can be beneficial for overcoming many of these side effects. different groups have developed various delivery systems for sirna molecules targeting tnf-alpha expression in the gi tract, assaying them both on cells and several rodent models for ibd (wilson et al., ; kriegel, c and amiji, m, ; kriegel, c and amiji, mm, ; laroui et al., ; yin et al., ; he et al., b, a; he et al., ) . even though therapeutic rnai development is in its infancy, current approaches and tools for delivery of these biomolecules via the oral route are improving. in fact, recent reports describe the effects of an oral smad antisense oligonucleotide in a randomized, placebo-controlled, double-blind, phase clinical trial in patients with crohn's disease (monteleone et al., ; monteleone et al., ) . although in this instance the biomolecule used is an antisense oligonucleotide and not a sirna, the design of the oral formulation could be very similar. iron is an essential metal required for numerous physiological functions, but in excess it is a well-defined risk factor in the pathogenesis of several diseases, including cardiovascular and neurodegenerative diseases. since there is no recognised active pathway of iron excretion, disposal of excess iron from the body is the primary therapeutic goal of treating patients with iron overload. use of iron chelators is limited due to nonspecific distribution in non-target tissues, which results in a number of serious side effects and toxicity . another way to treat iron excess is by limiting absorption of exogenous iron. the divalent metal transporter (dmt ) protein plays a well-established role in iron absorption. the primary site of dmt activity is the intestinal epithelium. dmt expression is regulated in response to body iron levels, with expression enhanced when iron stores are low and, conversely, reduced when iron stores are high. oral delivery of sirna-encapsulated nimos to selectively suppress intestinal dmt could decrease intestinal uptake of dietary iron, thereby mitigating iron overload and preventing iron-mediated toxicity . celiac disease is caused by a t-cell mediated immune response in the small intestine against deamidated cereal gluten peptides modified by the enzyme transglutaminase (tg ). the only way to prevent celiac symptoms is strict adherence to a gluten-free diet. the pathophysiology of celiac disease involves a combination of environmental, genetic and immunological factors. among several immune mediators, enzyme tissue tg and the proinflammatory cytokine interleukin- (il- ) have emerged as key players in promoting inflammatory responses against dietary gluten. given their important role in the pathophysiology of celiac disease, sirna mediated silencing of intestinal tg and il- (i.e. by oral administration using nimos) may result in neutralizing proinflammatory effects and could therefore alleviate disease symptoms . efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. colorectal cancer is one of the most common forms of cancer, and rnai therapy has great potential in the treatment of local intestinal cancers. however, rnai therapy faces major challenges since the genotypic footprints of the various cancers differ widely, even from individual to individual. depending on the tumour, several different sets of genes may be misexpressed, making it extremely difficult to determine a common/unique "culprit" gene. moreover, mice and rats do not generally spontaneously develop colon cancer, so induction of tumour growth is required. but these developed animal models do not reflect the genetic changes observed in human tumours (o'neill et al., ) . genetic mouse models that spontaneously develop intestinal cancer have also been developed. mutations in the apc gene are associated with development of colon cancer as well as a condition called familial adenomatous polyposis (fap). fap is an inherited condition characterised by development of numerous polyps in the colon. some of these polyps can subsequently become malignant adenocarcinomas (o'neill et al., ) . regarding fap and human gene therapy, one study explored oral delivery of escherichia coli expressing shrna against beta-catenin, and found significant silencing activity in healthy mice (xiang et al., ) . the gi tract provides the possibility of acting at systemic level by targeting local resident cells, such as m cells. m cells can take up encapsulated biomolecules to be phagocytosed by macrophages. in fact, aouadi et al. reported that glucan encapsulated sirna particles (gerps) loaded with sirna were phagocytosed by the underlying gut-associated lymphatic tissue (galt) macrophages and then translocated to distal organs of the reticuloendothelial system such as the liver, spleen and lung (aouadi et al., ) . another report claims functional cd shrna expression delivered into dendritic cells in mice by oral administration of recombinant yeast (xu et al., ) . in this manner, the targeted cells located in the intestine can undergo sirna-mediated gene silencing and migrate into tissues throughout the body. when successfully administered, rnai is a very useful therapeutic strategy for efficiently modulating gene expression. orally administered local rnai therapeutics have great potential due to the increasing bioavailability of the therapeutic biomolecule at the targeted tissue or cell type, coupled with simultaneous evasion of systemic side effects and immunogenic reactions. most advanced studies are focused on inflammatory bowel disease, although other potential diseases are being explored. the current main challenge is development of suitable encapsulation methods to protect the rna molecules from the harsh and varying conditions of the gi tract. the literature on rna delivery in fish and birds is scarce, with some examples of dietary delivery of exogenous rnas. in birds, most of the studies have been done using central nervous system administration of sirnas. for example, in the zebra finch bird, sirnas (three - nt long sequences against tyrosine kinase b, trkb) administration via direct injection into the brain was effective in reducing the volume of the high vocal centre by diminishing the robust nucleus of the arcopallium and the relative number of cells within it (beach et al., ) . in another study, direct administration of sirnas in the brain of white-crowned sparrows reduced resting time, spontaneous production of complex vocalizations, and stimulated brief agonistic vocalizations (ubuka et al., ; ubuka et al., ) . in the japanese quail, central administration of the same sirnas against gonadotropin-inhibitory hormone the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. sirnas for rna interference in chicken embryos has also been used to target marek's disease virus in vivo (chen et al., ). in fish models, systemic exosome-mediated sirna delivery to zebrafish resulted in effective crossing of the brain blood barrier and localization in the brain in vivo (yang t et al., ) . exosome-delivered sirnas decreased fluorescence-labelled tumour cells in the brain more than fourfold by inhibiting vascular endothelial growth factor (vegf) in a xenograft zebra fish brain tumour model (yang t et al., ) . these results suggested that brain endothelial exosomes could be used to deliver exogenous sirnas to the target site in the brain for treatment of brain cancer. the proposed higher delivery efficiency, lower immunogenicity, and better compatibility than existing foreign rna carriers have promoted the possible use of exosomes for exogenous rna delivery (mei et al., ) . several other studies have evaluated systemic delivery of sirnas to fish animal models, including delivery to the heart using peg-pla nanoparticles (diao et al., ) or using a neutralized non-charged polyethyleniminebased system . the literature describes other examples of sirna delivery into zebrafish embryos to inhibit specific gene function (gruber et al., ) . in the context of other types of ncrnas, direct injection of mir- a mimic rnas at the one-cell stage of zebrafish embryos resulted in inhibition of formation of the caudal vein plexus by means of targeting the endothelial cell bone morphogenic protein smad signalling (icli et al., ) . also in the zebrafish model, injection of a mir- mimic into embryos at the one-cell stage to deliver mir- ubiquitously resulted in protection against sterile inflammation (hsu et al., ) . dsrna microinjection administration to zebrafish embryos ( - cell stage) at a concentration of - pg rna/embryo resulted in rna interference, an effect that was diminished when ssrna was used (wargelius et al., ) . in cultured european sea bass, intramuscular injection ( weeks, µg of dsrna per dose) followed by in vivo electrically mediated dsrna delivery resulted in a reduction of myostatin gene expression (terova et al., ) . delivery of exogenous rnas has also been tested in lower aquatic organisms. in the crustacean red claw crayfish cherax quadricarinatus, systemic injection of sequence specific dsrnas against viral protein b of macrobrachium rosenbergii nodavirus (mrnv) was conducive to rnai effects that were able to functionally prevent and reduce mortality in infected individuals (hayakijkosol and owens, ) . dsrnas (intramuscular injection) have been used to induce antiviral immunity in invertebrates as they can also recognize dsrna as a virus-associated molecular pattern, resulting in activation of an innate antiviral response (robalino et al., ) . oral administration of encapsulated dsrnas (inactivated bacteria expressing dsrna) in the prawn macrobrachium rosenbergii fed twice daily at a rate of % total biomass for days resulted in their protection against white tail diseases caused by mrnv (naveen kumar and karunasagar, ) . dsrnas designed against the capsid and b genes of mrnv reduced the mortality of prawns by ≈ % due to sequence specific-mediated silencing of the viral genes (naveen kumar and karunasagar, ) . injection of viral protein -targeted dsrna (≥ µg dsrna/g shrimp body weight) into shrimp resulted in a high level of nt sirnas mapping across the entire white spot syndrome virus genome after virus challenge, which resulted in protection against the virus (nilsen et al., ) . in planarians, ingestion of bacterially-expressed dsrnas or direct microinjection into the animals can inhibit gene expression through rna interference (newmark et al., ) . efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. mongersen, an oral smad antisense oligonucleotide, and crohn's disease. n engl j med , - . mu j, zhuang x, wang q, jiang h, deng zb, wang b, et al. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. - . zollner n. . purine and pyrimidine metabolism. proc nutr soc , - . zollner n, grobner w. . influence of oral ribonucleic acid on orotaciduria due to allopurinol administration. z gesamte exp med , - . following a literature search as described in section . . . and based on the methodology described in section . . . , a total of documents were selected as relevant to reviewing the effect of dietary exogenous ncrnas on the gi tract and annex glands. very few studies have focused on the gi tract and their annex glands. four documents specifically evaluated the resistance of plant-derived srnas to simulated in vitro digestion system, ex vivo digestion system or in vivo digestion samples (philip et al., ; yang, et al., a; and reported that the percentage of unaltered mirnas after digestion might be below . %. due to their relevance in early human nutrition, literature on breast milk ncrnas was chosen to describe the biological effects of dietary exogenous ncrnas of non-plant origin, for which full-text reports were studied and reviewed. zhang et al. were the first to report on the transfer of mirnas from food plants to the mammalian circulatory system causing effects on recipient cells . this study reported the detection by high-throughput sequencing of plant srnas in human serum, as well as in tissues of the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. humans, mice and calves. the plant mirnas were distinguished from human mirnas by their '-omethyl modification, which made them resistant to periodate whereas the human mirnas having free ' hydroxyl were periodate sensitive. it was then assumed that the identified plant mirnas were coming from the food uptake. two rice mirnas named osa-mir a and osa-mir a were particularly abundant in human serum samples, since rice is common in human diets. additional in vitro experiments showed that these plant mirnas can endure conditions that mimic the acidic environment of the gut, suggesting that plant mirnas can survive being eaten and digested, pass through the mammalian gi tract and reach target organs. the authors also showed that diet-derived plant mir a can target the low density lipoprotein receptor adapter protein (ldlrap ) mrna based on sequence complementarity. this mirna was able to reduce the ldlrap protein level in the blood and liver of mice fed rice, and eventually increase their plasma low-density lipoproteins (ldl) content. this publication raised questions about whether food-derived srnas could play an active role in human/animal health. however, it was controversial and its results were questioned due to nutritional imbalances in the test-diet studies or lack of rnai-mediated modulation of ldlrap protein levels in mouse liver (witwer and hirschi, ; chen et al., ; . scientists from the monsanto and miragen companies were unable to reproduce these findings . measuring ldlrap protein levels in the liver of mice fed with mir a-containing rice diets by elisa instead of western-blot analysis, dickinson and colleagues did not detect any protein change modulated by mirna a-diets. although changes in the ldl plasma levels were indeed detected, the authors concluded that ldl increases resulted from nutritional imbalances rather than from a mir consumption mediated effect. additionally, plant mirnas including the highly abundant and stable osa-mir a were not detected in liver and plasma samples of mice fed under different rice-based regimes by srna sequencing with the hiseq illumina system . other independent research groups failed to detect dietary plant mirnas in plasma and tissues when conducting controlled feeding studies in humans and in animal models using different detection methods, such as real time quantitative pcr (rt-qpcr), droplet digital pcr (ddpcr) and next generation sequencing (ngs) analysis . these studies provide contradicting evidence of mirnas dietary absorption. in all the attempts, plant mirnas were detected at substantial levels in the diets but were undetectable in animal fluids and tissues. snow and colleagues studied three plant mirna species (mir a, mir a, and mir a) highly abundant in fruits routinely ingested by a group of ten healthy athletes. examination of the athletes' plasma by rt-qpcr analysis using taqman probes revealed high levels of endogenous mirnas but not the three dietderived plant mirnas . the same results were obtained with mice fed vegetarian diets rich in these mirnas. furthermore, using a mir null mutant mouse they showed that none of the dietary mirna including mirna was detected in blood and body tissues, suggesting that dietderived mirnas were not absorbed or maintained in mouse body fluids or organs. another group reported negative results on dietary mirna absorption in non-human primates . using qrt-pcr analysis and the same timeframe of as in zhang's report a, plant-specific mirnas were undetectable in tissues from macaques given a vegetarian diet. another feeding study in humans showed no significant differences in the levels of the plant mir a and mir in the plasma after consumption of broccoli sprouts highly rich in these two mirnas (baier et al., ) . more recently, a different research group using by ngs analysis failed to detect plant mirnas in the plasma of healthy volunteers that normally consume olive oil . the possibility that the plant rnas detected in human tissues and body fluids in some studies were contaminants remains a serious concern . by searching the composition of srna-seq data generated by the zhang's team from amphioxus animals, www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. it was found that the amphioxus data contained rice mirnas as previously reported for human serum. given that amphibia have an exclusive algae-based diet, this strongly suggests that the rice mirnas in both studies are the result of samples contamination . spurious detection of such sequences could arise due to contamination during sample handling, library preparation or sequencing, or result from errors in data analysis. since next-generation sequencing is very sensitive, just a few molecules could cause a false positive detection. more recently a comprehensive meta-study surveying for the presence of cross-species mirnas in sequencing data sets from various human tissues and body fluids indicated that xenomirs (cross-species mirnas) are technical artefacts rather than the results of dietary intake . a bioinformatics study identified plant mirnas in human and porcine milk exosomes . by searching publicly available ngs datasets, the authors identified several plant mirna species, most of them belonging to evolutionarily conserved and highly abundant mirna families. the target prediction suggests that these mirnas may interact with mrnas coding several transcription factors, protein receptors, transporters and immune-related proteins, thus potentially influencing biological functions in humans. of note is that the plant mirna profiles found in mammalian breast milk were similar to the composition in human blood presented by zhang and colleagues . liang and colleagues reported positive results on the absorption of food-derived mirnas from feeding studies in mice using cabbage (brassica oleracea) . they showed that mir , the most abundant mirna in cabbage, was detected in blood and various organs after cabbage consumption. however, there was no absolute quantification of mir or a description of an endogenous control for normalization of qrt-pcr data. a more recent study by zhang's group described how plant mirna from honeysuckle (lonicera japonica) inhibited influenza a virus replication in mice when entering through the gi tract, and they hypothesized that this mirna might be the active component in the traditional chinese medicine based on honeysuckle herb to treat influenza infection . the authors demonstrated that mirna is highly stable in honeysuckle decoction by solexa sequencing and northern blot analysis. they also showed that mir levels increased in mouse peripheral blood and lung following continuous honeysuckle decoction intake (drinking) or gavage feeding, as measured by qrt-pcr using taqman mirna probes. additionally, luciferase reporter assays and in vivo experiments showed that mir was able to target the influenza a virus, and consequently inhibited its replication and reduced mice mortality. the same authors reported that plant mirnas can be found in human umbilical cord blood and amniotic fluid, transferred from mother to the foetus, and that these influence foetal development and health . they showed that mir content increased in the maternal plasma and foetal liver of mice fed honeysuckle. a fluorescently labelled sirna was used to trace the transplacental transmission through feeding. furthermore, they showed that after feeding mice with different amounts of sirnas targeting the alphafetoprotein mrnas the levels of this protein were down regulated in foetal tissues. these results suggest that dietary sirnas delivered from the mother to the foetus could regulate foetal gene expression. more recently, the anti-proliferative effect of a plant mirnas on breast tumours was demonstrated in feeding experiments in mice (chin et al., ) . the plant mir , particularly abundant in broccoli, was found in sera from women, and its levels were inversely correlated with breast cancer morbidity and progression. in human sera, mir was detected in extracellular vesicles by qrt-pcr using a taqman probe. transfection of breast cancer cells with a synthetic mimic mir was shown to reduce their proliferative growth by targeting the transcription factor mrnas. more importantly, oral administration of synthetic mir significantly inhibited the growth of xerograph breast tumours in mice. these results agree with a previous report in which oral administration of a cocktail of three www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. tumour suppressor mirnas that were '-o-methylated to mimic plant mirnas reduced colon tumour burden . additional research is needed to address discrepancies among different studies and verify whether plant srnas derived from diet are absorbed at functional levels in animal species. most of the available data suggests either that gastrointestinal absorption of dietary plant mirnas does not occur in healthy consumers or mirnas levels in blood and tissues of consumers are too low to have a biological impact. however, the biological activity of food mirnas may be more dependent on their accumulation in exosomes than on their abundance per se, because of their reported low concentration. development of sensitive sensors to help to detect the functionality of low-level absorbed mirnas would facilitate assessment of the effects of dietary mirnas in consumers. future studies are needed to establish how exogenous ncrnas absorption and tissue distribution occurs, as well as to address their bioavailability and their biological function. reported that increased mir was detected in sera and urine after consumption of particular foods (i.e. honeysuckle), and disappeared h after the honeysuckle was removed from the diet; this supports the idea that these small rnas were of dietary origin. the role of kidney damage in mirna retention in the animals was also evaluated. using two models of acute renal failure (baliga et al., ) , i.e. the cisplatin (a chemotherapeutic agent) and the glycerol-induced models, it was observed that only mice receiving cisplatin exhibited measurable levels of dietary mirnas in sera and urine. this suggests that kidney damage alone did not lead to enhanced mirna retention in the mouse circulatory system . of interest is that cisplatin treatment, but not glycerol treatment or honeysuckle feeding, disrupted the organization of small intestine epithelial cells as shown by histological analysis. in this study, yang et al. used droplet pcr to demonstrate that the amplified products were likely to be specific and not due, for example, to nonspecific amplification of endogenous rnas . whether particular foods and/or alterations in intestinal permeability could improve the capacity to absorb small rnas from the diet or influence the biological effect within the gi tract, annex glands and systemically is poorly described, but still relevant for risk assessment of exogenous ncrnas in a subset of population. no studies specifically report the biological effects of dietary exogenous plant ncrna on the gi tract. only a few studies (described above) report the possible biological effects in their annex glands, including the liver . for gastrointestinal tissues or the liver, liang et al. reported that icr mice (a strain of albino mice) fed plant total rna ( - µg) extracted from brassica oleracea showed detectable levels of mir in the stomach (up to ≈ copies), intestinal (up to ≈ copies) and faecal content (up to ≈ copies), with the maximum amount present at to h after feeding . estimation of the proportion of orally administered rna that survived digestion suggested that the stomach contained . - . %, the intestines . - . % and the faeces . - . %, while the blood contained about . - . % and the spleen about . - . % . the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. which was similar to that of their synthetic form (without '-o-methylated ' ends) . whereas plant mirnas had a much slower degradation rate compared to their synthetic form without '-o-methylated ' ends. it is important to note that extreme ph conditions are not the only physicochemical or biological condition within the gi tract (see section . . . . for details). using a simulated human digestion system in vitro, philip et al. evaluated the resistance of soybean and rice mirnas to simulated gastric fluid early stage digestion (philip et al., ) . the study found continual survivability of plant mirnas in an in vitro simulated digestion system for over min without any significant decrease in their levels (philip et al., ) . mirna levels have been analysed qualitatively by real-time pcr using taqman mirna assays. stability of the plant-derived small rna mirna was also assessed using an artificial in vitro digestion system that simulates mammalian gastric and intestinal conditions . yang et al. compared three samples which contained a) ml cabbage extract with pmol plant-derived mir (measured by qrt-pcr), and pmol spiked-in synthetic '-o-methylated mir a and artificial mirna termed c ; b) ml phosphate buffered saline (pbs) with pmol each of synthetic '-o-methylated mir , mir a and c ; and c) ml pbs with pmol each of synthetic mir , mir a and c without the '-o-methylation. time course analysis during both gastric and intestinal phase digestion showed that most mirnas displayed modest resistance in the acidic gastric environment (up to min), while in the intestinal phase the levels of all mirnas were drastically reduced after five minutes (figure ) . regardless of synthesis origin and '-o-methylation, the digestive stability of mir was up to -fold higher than that of the other mirnas. the most stable form of mir was the plant-derived form in the cabbage extract, since . % survived, compared to . % for the '-o-methylated form, and . % for the non-modified form. similar results were observed when pmol of synthetic '-o-methylated mir , mir a and c were digested in vitro using the ex vivo intestinal fluids ( figure ) . indeed, mir appeared to be more stable after -hour digestion than the other tested mirnas. finally, by directly measuring mir levels in vivo in the small intestines of mice fed the plant-based diet (daily dietary mir intake pmol) it was observed that this particular mirna reached level more than -fold higher than those observed in the animals consuming a single pmol dose of the synthetic mir . mir a, a mirna also present in the plant-based diets, was also detected in the small intestines at a level times lower than that of mir . using transgenic arabidopsis lines expressing the artificial mirna amir-rice, the murine mirna mmu-mir- a or their controls, yang et al. studied gastrointestinal digestion and bioavailability of transgenic mirnas (yang et al., b) . levels of transgenic mirnas in plants (qrt-pcr quantification) were similar ( . and . fmol/g of fresh weight for amir-rice and mmu-mir- a, respectively) to that of srna mir ( - fmol/g in fresh shoot tissue). however, in diets stored at room temperature, the abundance of amir-rice and mmu-mir- a decreased gradually by ≈ -fold (≈ fmol/g diet) while that of the srna mir increased ≈ fold after h. in vitro gastric and intestinal simulated digestion of the synthetic forms of the plantbased transgenic mirnas showed that amir-rice and mir had similar digestive stability, while mmu-mir- a was significantly less stable. the surviving percentage in the intestinal phase after min was . % for mmu-mir- a compared to ≈ . % for mir or amir-rice (figure , frame a). the surviving percentage in the in vivo assays (gavage-fed pmol of synthetic srnas) further decreased the stability of the mirnas, reducing their level to . % for amir-rice and . % for mir (figure , frame b) . however, the levels of mirnas in the small intestines were higher for mir ( fmol/mouse) than amir-rice ( . fmol/mouse), while mmu-mir- a levels were undistinguishable from the host mouse mirna (figure , frame c). amir-rice was not found in serum (neither are mirnas reported in previous studies and present in transgenic plants) while mir was found only in the serum ( . fm, or . x copies per mouse) of mice fed the plant transgenic diets (yang, j. et al., b) www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. altogether, these results suggest that the plant-derived mir might be more stable than other plantbased small rnas. whether this exceptional stability could be generalized to other ncrnas is unknown. however, recent evidence, including some studies evaluating mir and mir , suggests that there may be some misidentified alternative mirnas . indeed, several other rarer but consistently mapped plant mirnas also have % or near % matches to human transcripts or genomic sequences (i.e. rrna), and some do not map to plant genomes at all, suggesting artefactual results of plant mirnas in mammalian sequencing datasets. this emphasizes the need for rigorous filtering strategies when assessing possible dietary exogenous mirnas . indeed, the small rna mir is derived from s rrna and does not undergo canonical mirna processing, as it does not depend on dcl (yang, j. et al., a) . mir is also inefficiently assembled into the risc complex in human cells and modestly regulates gene expression. this suggests that this exogenous srna is different from the canonical plant-based mirnas (yang, et al., b) . these results highlight the need to evaluate other ncrnas as they could originate from precise processing at the ' or ' end of mature or precursor rnas generating other abundant small rnas (lee et al., ) . also, the rna degradome is a crucial component of the total cellular rna pool of plants (nowacka et al., ) . they can be stable degradation intermediates present in a high copy number, or they can derive from various rna species including trna, rrna, mrna and snrna (addo-quaye et al., ; nowacka et al., ) . among the few examples in the literature of possible biological effects of exogenous non-plant origin ncrnas, milk has been studied because it constitutes a rich source of secreted mirnas. human breast milk is an essential source of nutrition for new-borns. in addition to its nutritional function, breast milk contains myriad biologically-active components that influence the development of the the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. more than % of the unique mirnas identified in porcine milk exosomes (gu et al., ) or % in other studies (chen et al., ) . in human exosomes, the ten most abundant mirnas account for % of the unique mirnas identified . lncrnas previously reported as being important for developmental processes were found in human breast milk extracellular vesicles (karlsson et al., ) . maternal factors such as the mother's diet seem to influence mirna expression in the breast milk fat globule (munch et al., ) or exosomes (sun j, ) . maternal weight (xi et al., ) also seems to influence mirna expression. certain mirnas found in breast milk have also been found in placenta tissue (munch et al., ) , although their physiological role is unknown. other studies have shown that colostrum mirnas (early lactation period) are more abundant than in the later lactation period (gu et al., ; xi et al., ; sun et al., ) . bovine milk mirnas isolated from the colostrum whey fraction were more abundant than in the mature milk whey fraction (izumi et al., ) . immunerelated mirnas expression is reported to be higher in colostrum than mature milk (izumi et al., ; na et al., ) . in vitro studies of human milk mirnas using either rnase digestion, freeze-thaw cycles, low ph or other gi conditions have shown that these mirnas are relatively highly stable (kosaka et al., ; liao et al., ) . similarly high stability of mirnas was found when using one or all of the above mentioned hard conditions for milk from cow (hata et al., ; izumi et al., ), pig (gu et al., or panda . indeed, it has been proposed that rna in milk is present in microvesicles and is protected from rnases and the other gi conditions by surrounding membranes (hata et al., ; gu et al., ; . however, technological conditions such as pasteurization (golan-gerstl et al., ) have been shown to reduce mirnas abundance in cow and goat milk by up to ≈ %, or up to ≈ % for certain mirnas (howard et al., ) , and in human exosome (personal communication, faseb conference ). microwave heating also reduces the amount of certain mirnas (howard et al., ) , while ultrasonication influences exosome membrane integrity and thus increases cargo instability (sun et al., ) . among dairy products, mirnas concentrations varied considerably, but were generally lower than the concentration in pasteurized whole milk (howard et al., ) . the exception was fresh cheese ("queso fresco") dip, which contained higher concentrations of mirnas than observed in pasteurized milk (table ). in vitro studies using mirnas isolated from breast milk exosomes, either human or bovine, were found (liao et al., ) , and crl , k or lim (golan-gerstl et al., ) . when subjected to proteinase k treatment or when the vesicle structure was destroyed, uptake or transport of these exosomal mirnas was compromised (kusuma et al., ; wolf et al., ; sun et al., ) . different studies have attempted to quantify the amount of rnas present in breast milk. rna content in microvesicles isolated from cow milk has been estimated to be ≈ ng for colostrum and ≈ for mature milk when isolated from ml sample (hata et al., ) . in human milk, total rna was estimated to be ≈ ng/ cells of the milk cell fraction and ≈ ng/µl fat from milk fat (alsaweed et al., c) . exogenous plant mirnas have also been found in panda milk exosomes , the whole milk or exosomes of human breast milk the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. et al., ). however, levels of mirnas or total rnas were very low (golan-gerstl et al., ; alsaweed et al., c) compared to that of breast milk. indeed, alsaweed et al. reported that the total amount of rna found in bovine milk-based infant formula was ≈ . ng/µl formula ( human mirnas detected) as compared to . ng/µl for a soy-based formula ( human mirnas detected) (alsaweed et al., c). in terms of possible breast milk mirna absorption, plasma levels of mirnas were found to be higher in colostrum-fed piglets compared to mature milk-fed piglets (gu et al., ) . in humans, plasma mirnas (mir- b and mir- c) increased when different doses of cow milk were consumed (baier et al., ) . in contrast to the studies above, the literature also describes several studies that show either different or contradictory results regarding uptake of exogenous rnas from breast milk. auerbach et al. re-analysed a study in which mir- b- p and mir- c- p were found in plasma after bovine milk ingestion by healthy humans (baier et al., ) and found no significant altered mirna levels after milk ingestion (auerbach et al., ) . these results were confirmed by qpcr and rna-sequencing. several technical issues including low mir- b expression, use of control mirnas (normalization), and variation level may have contributed to these discrepancies (auerbach et al., ) . piglets fed cow milk for weeks showed very low levels of cow-specific mirnas in the bloodstream, which were compared to the levels measured from animals fed maize, which also showed similar counts of cow-specific sequences , suggesting a lack of transfer of exogenous small rnas. using a transgenic mouse model that overexpresses mir- b precursor in the mammary epithelial cells, and thus increases levels of mir- b in milk (≈ -fold), no differences were found in blood, liver, small intestine, kidney or lung tissues of pups from these dams when compared to pups from wild type dams (laubier et al., ) . interestingly, the level of mir- b was ≈ -fold higher in the stomach of mice consuming the transgenic milk. oral ingestion of . µg and . µg of total rna from porcine milk exosomes administered daily during three weeks to mice, significalty increased villus height and crypt depth of the duodenum and jejunum relative to the control group, suggesting an effect improving the development of the gi tract in mice . using genetic knock-out (ko) mouse models for mirnas mir- and mir- c/ , title et al., evaluated the uptake of maternal-milk derived mirnas using the foster mother offspring exchange model to prevent the confounding effects of mirnas derived from tissues of suckling offspring . wild type (wt) offspring consuming mir- ko milk at day exhibited basal levels of mir- in milk from the stomach, which likely comes from stomach epithelial cells. at day of suckling, it was confirmed that most mir- /mir- c came from the milk itself rather than the offspring www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. genotype, because there was no significant difference between wt pups and ko pups receiving wt milk. evaluating different parts of the gi tract (jejunum, ileum and colon), no evidence of uptake was observed because there was no measurable increase in mir- levels in enterocytes of ko pups receiving wt milk compared to ko milk. moreover, no changes in plasma, liver or spleen levels of mir- were observed in ko pups receiving wt or ko milk . similar effects were observed at day of treatment and upon evaluating mir- c on day . when the fate of milkderived mirnas downstream of the stomach was analysed, the levels of mir- in the intestinal content of ko pups receiving wt milk was seen to dramatically decrease compared to that of the stomach contents. these data suggest that milk mirnas are being degraded by the digestive system (since no evidence of uptake was observed). indeed, when the spike in cell-mir- was tested, it was degraded more rapidly than milk mir- , suggesting that milk mirnas exhibited a certain degree of resistance (perhaps because of their exosome content). but less than % of mir- copies remained after h incubation of intestinal content, suggesting a possible digestive enzymatic degradation of the milk-derived (exosomes) mirnas . observational studies also suggest that human breast milk mirnas are not related to preventing atopic dermatitis in infancy months postpartum . in general, rna contamination has been proposed as the main source of controversy in mirnas studies reported in the field of breast milk (bagci and allmer, ) . the literature contains very few studies on the biological effects of dietary exogenous ncrnas in the gi tract and its annex glands. indeed, these few studies only report a biological effect in the liver. the available literature suggests that dietary exogenous plant small ncrnas seem to resist the harsh conditions of the gi tract. however, the stability of plant mirnas under gi conditions in vitro, ex vivo or in vivo is low. moreover, these studies suggest that the percentage of surviving mirnas in the gi tract in the best-case scenario is around ≈ %, although this depends on the specific small ncrnas evaluated. several studies report the presence of dietary exogenous mirnas in breast milk of different mammals, including humans. some protected mirnas (i.e. transported in extracelluar vesicles) have been found to be stable under in vitro degradative conditions. while some evidence suggests a possible absortion through oral feeding, other studies indicates a very limited biovailability or lack of accumulation within the gi tract or its annex glands. recent evidence supports the significant contribution of srnas to communication between hosts and some eukaryotic pathogens, pests, parasites, or symbiotic microorganisms. this silencing transfer phenomenon is very relevant to food and feed risk assessment of ncrna gm plants. although this section (efsa task ) will focus on studies related to the possible trafficking of ncrnas between plants and humans and animals, studies supporting the movement of rna-silencing signals between plants and pathogens are also reviewed. virus-induced gene silencing represents the model of gene silencing in the host by exogenous sirnas. in this case, the sirna specificity determinant is derived from viral rnas. typically, with a discrete length of nt they form perfect duplexes which are produced in plants by dcl and dcl . virusinduced gene silencing is a very effective defence system, and, consistently with the normal dynamics of host-pathogen interactions, all viruses encode silencing suppressors as a counter defence (baulcombe, ) . www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. cross-kingdom rnai implies that a translocation of gene silencing signals occurs between hosts and these organisms. this implies a two-way traffic of sirnas between pathogens and their plant hosts. several studies report that rnas produced in a host plant can be transferred to a fungus or oomycete, symbiont or pathogen, to induce rna silencing in the interacting microorganism (ghag et al., ; koch et al., ; vega-arreguín et al., ; helber et al., ) . transgenic plants carrying a rnai construct, usually with a sense-intron-antisense palindromic structure that produces dsrnas and sirnas, can induce silencing of the targeted transcripts in the interacting organism. furthermore, treatment of fungal conidia with dsrnas of essential genes led to fungal growth inhibition, demonstrating rna interference from environmental silencing signals (koch et al., ) . conversely, the rna produced in a fungus can affect a host plant's defence system (weiberg et al., ) . remarkably, scientists have developed an effective disease control strategy, called host-induced gene silencing (higs), by generating transgenic plants that express exogenous rnai triggers to successfully silence essential genes in pathogens and pests (weiberg et al., ) . in this context, transgenic plants expressing dsrnas or hairpin rnas targeting vital fungal genes of fusarium sp. developed resistance to these important phytopathogens (ghag et al., ; koch et al., ) . small rnas or dsrnas can also be transferred from plant to pests, such as insects eating leaves or nematodes infecting roots. indeed, transgenic plants that express dsrna homologous to essential genes of insect pests or nematodes became resistant to these specific parasites through the silencing activity produced within the target organism when srnas expressed from the plant transgenes is consumed (baum et al., ; fairbairn et al., ; mao et al., ) . many aspects of these cross-kingdom rna interference phenomena are still poorly understood. one of them is how these rna molecules 'travel,' sometimes over long distances through diverse cellular boundaries between plants and interacting organisms. these silencing signals may utilize conserved cell-to-cell as well as systemic rnai pathways present in plants and animals, and may also use organismspecific pathways. rna-protective factors such as agos, other rna-binding proteins, or encapsulation into extracellular vesicles likely play important roles in protecting mobile rnas against degradation during transport (weiberg et al., ; lefebvre and lecuyer, ) . another important question is how these srnas use the target cell rna interference machinery to convey the silencing effect. based on current knowledge, rna-mediated gene silencing seems to be an ubiquitous phenomenon that exists in almost all eukaryotes, and which always follows the principle of complementary nucleotide base-pairing between regulatory srna and mrna sequences (weiberg et al., ) . despite the tremendous differences present in the structural features of regulatory rnas, and the completely unrelated or highly divergent rna gene-silencing mechanisms and pathways that have evolved in diverse organisms, complementary sequence matches seem to be sufficient enough to trigger cross-kingdom gene silencing, as exemplified by plants-parasites, bacteria-to-worms and other lower organism interactions (weiberg et al., ) . following a literature search as described in section . . . and based on the methodology described in the section . . . , a total of documents were selected for review of the topic molecular mechanisms of exogenous ncrna uptake and function. the uptake of exogenous ncrnas is described from the molecular mechanisms point of view (i.e. the presence of a specific transporter, if any). efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. although plant-derived exogenous ncrnas are the main topic of this subsection, some other examples of general molecular mechanisms of exogenous ncrnas uptake, intracellular trafficking and function are included as relevant to understanding the possible fate of plant-derived exogenous ncrnas. other general aspects of rnas uptake are reviewed in chapter . . , and the uptake of exogenous rnas from the pharmaceutical/medicinal area is covered in section . . . as previously mentioned in section . . , cellular uptake of ncrnas presents great challenges due to the physicochemical properties of these molecules. their size and negative charges prevent their easy entry into mammalian cells. cell membranes pose a major barrier to ncrnas entrance since entry into cells is tightly controlled and regulated (mcerlean et al., ) . the anionic lipophilic bilayer prevents entry of macromolecular anionic nucleic acids in their naked form, both restricting their binding to and passive diffusion across these lipophilic cell membranes . furthermore, even though their internalization may depend on the endocytic pathway, endosomal entrapment and lysosomal degradation can be major issues because of the reduced accessibility of ncrnas to their sites of action (nucleus, cytoplasm or mitochondria) (won et al., ; . many different carrier molecules have been developed to achieve the entrance of ncrnas (mostly mirnas and dsrnas) into target cells (bolhassani, ; lindgren and langel, ; mao et al., ; ragelle et al., ; rudzinski and aminabhavi, ; shum and rossi, ) . exogenous ncrnas must be carried by polymers, synthetic cationic lipids or cell-penetrating peptides neutralizing the negative charges of the nucleic acids. most of these cationic lipids tend to form liposomes when dispersed in an aqueous phase, such as blood. endocytosis has been suggested as the main pathway for this nucleic acids-cationic lipid complex internalization by the cell (el ouahabi et al., ) . most reports suggest that so-called cell-penetrating peptides (cpps) bind initially to negatively-charged proteoglycans at the cell surface and are internalized into endosomes (juliano et al., ) . there are multiple pathways of endocytosis (see also section . . ). i. the clathrin-coated pit pathway is the archetype of endocytosis pathways. cell surface receptors and their associated ligands interact with adapter proteins and accessory factors, clustering the receptors into specialized membrane areas subtended by a network of clathrin triskelions. the clathrin network is invaginated by means of membrane curvature promoting specialized proteins, giving rise to a clathrin-coated endosome. this endosome quickly uncoats, generating an uncoated vesicle that will begin its intracellular journey (juliano et al., ) . ii. the caveolar pathway implies the presence of small cell membrane invaginations rich in cholesterol and sphingolipids containing caveolin . cavins, which coat proteins helping to stabilize caveolar structures, are present in these invaginations. there is controversy as to whether caveolae generate independent intracellular vesicles or whether they remain as tubular structures linked to the plasma membrane; however, some data suggest that caveolae generate vesicles able to participate in intracellular traffic, generally presenting a smaller size than other forms of endocytotic vesicles (juliano et al., ) . iii. a number of clathrin-and caveolin-independent pathways have been described. these pathways are often defined in terms of the morphologies of the vesicles they generate or in terms of the cargo that is preferentially internalized. for instance, the flotillin pathway involves the presence of flotillin-rich membrane microdomains, where flotillins are membrane-inserted proteins that may be involved in ordering lipid domains and subsequent endocytosis, similar efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. to caveolin (juliano et al., ) . another such clathrin-and caveolin-independent pathway is the clic/geec pathway, which seems to be particularly important for fluid phase endocytosis. clic/geec stands for clathrin and dynamin independent carriers/gpi-ap enriched early endosomal compartments. this pathway gives rise to high-volume tubular endosomes rich in gpi-proteins and that typically contain fluid phase markers such as dextrans (juliano et al., ) . additional clathrin-and caveolin-independent pathways exist, some involving dynaminmediated disjunction of vesicles from the plasma membrane (juliano et al., ) . iv. macropinocytosis is a process by which cell protrusions pinch off large volumes of extracellular fluid and is therefore an important pathway in fluid phase endocytosis. it is also involved in internalization of clustered, activated receptor tyrosine kinases. v. phagocytosis and entosis are large-volume internalization mechanisms as well. they come into play in specialized cells or unusual circumstances but do not play much of a role in oligonucleotides processing in most cell types (juliano et al., ) . vi. the actin cytoskeleton plays an important role in most of the endocytotic processes described above, although certain arenaviruses enter cells by a pathway independent of clathrin, caveolin, dynamin and actin. it has been reported that phosphorothioate antisense oligonucleotides seem to enter the cells by this pathway as well (juliano et al., ) . there have been many attempts to identify endogenous receptors for antisense or sirna molecules. however, no direct evidence for their involvement in oligonucleotide trafficking has been provided (juliano et al., ) . integrins of the beta- subclass as well as scavenger receptors have been suggested as candidates, but this is controversial. toll-like receptor (tlr) family members seem to be the most convincing examples of cellular receptors for oligonucleotides; tlr binds dna having cpg motifs, tlrs / binds single-stranded rna, while tlr binds double-stranded rna. although these tlrs are usually found within endosomes rather than at the cell surface, in some cases they seem to be able to assist in accumulation of oligonucleotides by cells (juliano et al., ). one interesting candidate as a receptor for oligonucleotides is the mammalian homolog of the doublestranded rna (dsrna) transport protein sid- found in caenorhabditis elegans (feinberg and hunter, ) . the human homolog of this protein, sidt , has been described as facilitating rapid contactdependent intercellular small rna transfer (elhassan et al., ) and as selectively binding long doublestranded rna (li, w et al., ) . another member of the sid transmembrane family, sidt , has been shown to take up extracellular double-stranded rna in drosophila s cells (mcewan et al., ) via endocytosis, although in mammalian cells this protein has been located in lysosomal membranes (jialin et al., ) . in fact, some authors have proposed that sidt could mediate cellular rna degradation inside lysosomes through a novel type of autophagy called rnautophagy (aizawa et al., ) . another research group recently reported that sidt is required to transport internalized dsrna from endocytic compartments into the cytoplasm for immune activation (nguyen et al., ), while others have described that this protein mediates "gymnosis", facilitating uptake of naked single-stranded oligonucleotides into living cells (takahashi et al., ) . a more recent report indicates that both sidt and sidt not only do not transport rna, but are involved in cholesterol transport (mendez-acevedo et al., ) . once an oligonucleotide has entered a cell in an endosome, it encounters a complex maze of intracellular pathways leading to multiple destinations and regulated by an intricate protein machinery. key subcellular membrane bound compartments include early and recycling endosomes, late www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. endosomes/multi-vesicular bodies, lysosomes, the golgi apparatus and the endoplasmic reticulum. intracellular trafficking is not a random process, but rather a carefully orchestrated choreography that allows the cells to transport endogenous and exogenous materials to the most appropriate places. for nucleotides to exert their function, they need to leave the membrane bound compartments and access the cytosol and/or nucleus (juliano et al., ) . knowledge as to how endocytotic cargos are delivered to subcellular compartments is still partial. many of the internalization pathways previously described converge at the stage of early endosomes, raising the question of how the differentially internalized components traffic to different destinations. certain evidences suggest that membrane domains originating from different internalization pathways maintain their identity within early endosomes, thus providing the means for specific sorting and trafficking to distinct downstream destinations (juliano et al., ) . all membrane traffic proceeds through the same basic steps: i) a coated vesicle is pinched off from a larger donor membrane compartment; ii) the vesicle uncoats, allowing display of the tethering and fusion proteins; iii) the vesicle is carried to its destination along "tracks" provided by actin-or tubulin-based cytoskeletal structures; iv) the vesicle recognizes its target membrane compartment using tethering proteins and then utilizes snare proteins to complete the fusion process and deliver membrane and contents to the target compartment (juliano et al., ) (figure ). ncrnas must escape from the endomembrane compartments and reach the cytosol to exert their function. as mentioned above, intracellular trafficking involves a highly dynamic flux of membrane vesicles engaged in a multitude of fusion and disjunction events. there are a few key points in these processes to be considered when designing non-viral oligonucleotide delivery strategies: ) fusion involves localized stress on the fusion partners, including formation of non-bilayer lipid domains; ) non-bilayer regions of membranes can be much leakier than bilayer regions; ) many enveloped viruses fuse with cells via specialized membrane interacting proteins that, while differing in sequence, act in a manner similar to cellular snare proteins; in many cases these proteins can also induce increments in membrane permeability. therefore, there is an intrinsic relationship between the fusion events inherent to intracellular trafficking and transient leakage of vesicular contents. thus, the innate activity of oligonucleotides taken up by cells is likely due to a modest amount of continuous leakage from endomembrane compartments spontaneously occurring during intracellular trafficking (juliano et al., ) . some ncrnas function is exerted in the nucleus, although nuclear entry may not be the rate-limiting step for oligonucleotide action. studies have shown that oligonucleotides, particularly those with phosphorothioate backbones, are able to continuously shuttle between the nucleus and cytoplasm. this is an active process mediated by nuclear pore structures but does not require classic nuclear localization signals. for phosphodiester oligonucleotides, both passive diffusion and active transport have been described as nuclear entry mechanisms (juliano et al., ) . in terms of the uptake and trafficking of "free" or "naked" oligonucleotides, the co-existence of both productive and non-productive uptake routes has been suggested. the non-productive pathway seems to involve trafficking to lysosomes, while the pathway that results in rnase h-dependent antisense effects eventually leads to interaction with cellular pre-mrna (koller et al., ) . other studies have involved so-called "gymnotic" uptake of antisense oligonucleotides modified with lna (locked nucleic acid) moieties (stein et al., ; zhang et al., ) . subcellular distribution studies surprisingly suggested that the deoxy lna compounds became associated with p-bodies that are usually thought to be sirna action sites (stein et al., ) . unlike the study performed with phosphorothioate www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. oligonucleotides in which the concentrations used were in the nanomolar range, the antisense effects of "naked" lna required micromolar concentrations (juliano et al., ). as described in section . . , exogenous ncrnas must overcome a series of barriers to be functional at systemic level. in addition to the extracellular barriers specifically related to oral absorption, defensive humoral and cellular barriers efficiently prevent the intrusion of exogenous entities into the organism (jeong et al., ) . systemically-administered nucleic acids are rapidly degraded by nucleases in a few minutes and, even in nucleic acid drug therapy using positively-charged polyplexes, they readily interact with serum components to form larger aggregates, resulting in rapid clearance by res or phagocytes (jeong et al., ) . if the nucleic acids survive in the blood stream, they must still reach the appropriate tissues and enter the target cells. in systemic nucleic acid therapy, continuous endothelial walls in the microvasculatures are one of the main barriers limiting the access of oligonucleotides to target cells. in certain pathological cases, such as cancer, the presence of leaky vasculatures in the vicinity of highly penetrating solid tumours allows nanoparticulates to penetrate and accumulate in tumours due to the enhanced permeation and retention effect (jeong et al., ) . caveolae-mediated transcytosis has been considered one of the important mechanisms of macromolecules transport across the endothelium (schnitzer, ) . another possible limiting barrier is the extracellular matrix consisting of various proteoglycans, which are proteins covalently cross-linked with carboxylic or sulphated glycosaminoglycans (gags) (ruponen et al., ) . since gags, such as heparin sulphate, chondroitin sulphate and hyaluronic acid, are polyanions, they may repel the negatively-charged oligonucleotides, affecting mobility of the nucleic acids in the tissue extracellular matrix and therefore limiting their access to target cells. in the case that oligonucleotides reach the target cell membrane, they must enter the cell. the first barrier is the anionic plasma membrane itself, and the second is cellular uptake via endocytosis (see section . . ). without a specific ligand-receptor interaction mechanism, the entrance of ncrnas results in a lack of cell specificity (jeong et al., ) . once taken up by cells, the nucleic acids are localized within the endosomal compartments, where the ph rapidly drops to about by the action of membrane-bound atp-driven proton pumps. the endosomes mature to lysosomes where the oligonucleotides can be degraded by various enzymes (jeong et al., ) . as previously indicated, any surviving nucleic acids must then escape from the endosome to exert their functional effects. in nucleic acid drug therapy, certain endosomal disruptive agents, such as lysomotropic chloroquine, fusogenic peptides and ph-sensitive neutral lipid, are used to facilitate or promote endosomal escape through membrane disruption (jeong et al., ) . after endosomal escape, ncrnas should move through the cytoplasm to encounter their molecular targets, or to the peri-nuclear space where nuclear translocation takes place if the nucleus is the target compartment. passive diffusion of high-molecular weight macromolecules is limited in the cytoplasm. a complex network of microfilaments and microtubules, highly concentrated proteins and various subcellular organelles provide the cytoplasm with a high fluid phase viscosity and mesh-like structure with to Å pores from, making the diffusion process size-dependent (jeong et al., ) . in fact, microinjection of plasmid dna into the cytoplasm showed that normal dna was practically immobile in the cytoplasmic space (dowty et al., ) . however, dna molecules with less than base pairs seem to undergo free diffusion in the cytoplasm (lukacs et al., ) , suggesting that small oligonucleotides (i.e. mirnas) do not face limitations regarding their intracellular trafficking, contrary to large ncrnas (i.e. lncrnas) which might encounter more restrictions reaching the peri-nuclear space. finally, some ncrnas need to enter the nucleus to be functional. the nuclear envelope consists of a double-layer membrane with nuclear pores. the nuclear pore complex allows free diffusion of ions and small molecules, but restricts passage of macromolecules with molecular efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. weights greater than kda, unless accompanied by a nuclear localization signal peptide (nls) (jeong et al., ) , which is not the case with lncrnas oligonucleotides are initially accumulated in an endomembrane compartment (the donor compartment, e.g. early endosomes) and are then trafficked by shuttle vesicles to various other endomembrane compartments (the recipient compartment, e.g. the trans-golgi). the first step ( ) involves disjunction ('pinching off') of a shuttle vesicle under the influence of a coat protein as well as other accessory proteins. at this stage there are non-bilayer regions at the junction between the membranes of the donor compartment and the shuttle vesicle. this provides an opportunity for some oligonucleotides to escape into the cytosol. step involves uncoating of the vesicle; and rab proteins can contribute to this step. step comprises movement of the shuttle vesicle toward its destination along cytoskeletal tracks. motor proteins such as various myosins (for the actin system) or dyneins or kinesins (for the microtubular system) propel the vesicle. rab proteins are involved in forming the appropriate linkages to the cytoskeleton. step entails recognition of the recipient ('target') compartment by the shuttle vesicle. tether proteins work with rab proteins to provide interaction specificity while v-snare proteins in the vesicle membrane interact with t-snare proteins in the recipient compartment membrane to provide firm bridging, as well as contributing to specificity. in step the snare proteins undergo major conformational changes, and, with the assistance of accessory proteins, trigger fusion of the shuttle vesicle membrane with the membrane of the recipient compartment. at this stage non-bilayer regions exist at the junction between shuttle and recipient membranes potentially allowing oligonucleotide escape. reprint with permission from (r l juliano, x ming, o nakagawa. cellular uptake and intracellular trafficking of antisense and sirna oligonucleotides. bioconjugate chemistry : - ). copyrigth ( ) american chemical society. figure : proposed mechanism of vesicular trafficking of oligonucleotides some research has been done on cellular uptake, trafficking and tissue distribution of oligonucleotides without specific targeting or carrier mechanisms. in terms of tissue distribution, certain cell types exhibit a preferential in vivo uptake, particularly kidney proximal tubule cells and liver kupffer cells for phosphorothioate (ps) antisense compounds and sirna (juliano et al., ) . however, only one report has studied systemic level uptake and function of orally administered mirnas . these authors studied intestinal uptake of maternal milk-derived mirnas by new-born mice employing genetically-modified models to distinguish endogenous mirnas from milk-derived exogenous mirnas. their analysis of the intestinal epithelium, blood, liver and spleen revealed no evidence for mirna www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. uptake, even though milk in lactating mothers has been shown to be a particularly rich source of secreted mirnas. sequencing and microarray analysis of mirnas in the milk of various mammalian species has led to the discovery of hundreds of mirnas in cow, pig, rat and human milk, which are derived from exosomes and cellular components. furthermore, their study revealed rapid degradation of milk mirna in intestinal fluid, indicating that mirnas in the milk play a nutritional rather than a generegulatory role in new-born mice . there are certain tissues containing special physical structures that can function as additional barriers to ncrnas entry. such is the case of the central nervous system (cns), where delivery of nucleic acids is particularly challenging because of its anatomical and physiological complexities. the cns is protected by the blood-brain barrier (bbb), which consists of tightly joined capillary endothelial cells, restricting access of large molecules into the brain. the cns extends to include the spinal cord, which is protected by the blood-cerebrospinal fluid barrier (bcsfb), made up of choroid plexus epithelial cells limiting the free diffusion of molecules into the cerebrospinal fluid (csf). the bbb and the bcsfb express numerous transporters and receptors which contribute to the transfer of essential nutrients such as glucose and amino acids into the cns. within the cns, different cell types exist including neurons, astrocytes and other glial cells. entry of nucleic acids into neurons is notoriously difficult for unclear reasons, although it is likely related to their post-mitotic nature, as well as the complex structures and intricacies of neuronal networking (o'mahony et al., ) . for instance, variances in the uptake of sirna lipoplexes (liposome and nucleic acid complexes) were reported at different parts of the neuron structure, with greater uptake efficiency at the cell soma compared to the neuritis, which may occur because of different membrane compositions in these domains (o'mahony et al., ) . circumventing the bbb is only possible by direct administration of ncrnas to a target region of the brain or by administration to the spinal cord where trans-synaptic retrograde transport to the brain is possible. some delivery systems exploit the receptor-mediated uptake of molecules such as transferrin, lactoferrin and insulin receptors by attaching a ligand for the receptor to the non-viral delivery system, or promoting a transient mechanical disruption of the bbb (o' mahony et al., ) . in certain neurological diseases, and in particular in brain cancers, anatomical and physiological changes occur that can impact bbb integrity and the cns accessibility. delivery to the csf to bypass the bbb presents its own obstacles, including rapid clearance from the csf, the bcsfb and limited diffusion from the csf into the brain parenchyma (o'mahony et al., ) (figure ) . another specialized barrier is the placenta. the placenta serves as the interface between the maternal and foetal circulatory systems, and regulates the transfer of oxygen, nutrients and waste products. when exogenous substances are present in the maternal bloodstream, the extent to which these affect the foetus is determined by transport and biotransformation processes in the placental barrier. this barrier is formed by fusion of the outer blastocyst trophoblast cells facing the uterine epithelium into multinucleated syncytiotrophoblast. proliferation of syncytiotrophoblast is mediated by the fusion of precursor cytotrophoblast cells. maternal blood in the intervillous space and foetal blood in the villous capillaries are separated by a continuous layer of syncytiotrophoblast, a discontinuous layer of cytotrophoblast cells, basal lamina, connective tissue, and foetal endothelial cells (al-enazy et al., ) . the placenta undergoes several changes as pregnancy progresses. overall, the placental barrier becomes thinner over time, the distance separating the maternal circulation from the foetal circulation decreasing from - µm in the second month to only - µm at term. passive diffusion across the placenta is favoured for lipophilic substances, allowing them to cross the phospholipid bilayer. smaller compounds tend to cross the placenta more readily, with compounds having a molecular weight less than da crossing it most easily (al-enazy et al., ) . since nucleic acids are polarized, negatively charged molecules, it is unlikely they would be able to cross the placenta by passive diffusion. nevertheless, the placental syncytiotrophoblast is capable of efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. endocytosis (al-enazy et al., ) . no reports refer to exogenous ncrnas entering the placental barrier. however, the presence of placental mirnas has been described in the blood circulation of pregnant women. these mirnas have been reported to be actively secreted into microvesicles and exosomes. these specific exosomal mirnas may be important for embryo implantation, playing a significant role in the intercellular communication pathways that potentially contribute to placentation and development of maternal-foetal vascular exchange (mitchell et al., ) . since exosomes can cross the blood brain barrier, meaning they can cross several layers of cells, it has been speculated that exosomes could directly move from maternal to foetal tissues, and vice-versa, during pregnancy (record, ). the the molecular mechanisms of exogenous ncrna cellular uptake have been inferred from studies performed when developing strategies for nucleic acid delivery in therapeutics. few studies have been done with "naked" oligonucleotides, and even in these cases, the oligonucleotides were chemically modified. these molecular mechanisms of cellular uptake include the different types of endocytosis mechanisms known to date. the existence of receptor-mediated uptake of oligonucleotides through the sidt and sidt proteins has also been described, although their function has been contradicted since they have been described as cholesterol transporters. among the numerous challenges ncrnas must overcome once taken-up by the cell, escaping the endosomal compartments is the most relevant and significant, although reaching their site of action is also crucial to exerting their function. specialized barriers such as the blood-brain barrier or the placental barrier present further obstacles to overcome. www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. zhang y, qu z, kim s, shi v, liao b, kraft p, et al. . down-modulation of cancer targets using locked nucleic acid (lna)-based antisense oligonucleotides without transfection. gene ther , - . zhou g, zhou y, chen x. following a literature search as described in section . . . . and based on the methodology in section . . . ., a total documents were selected for reviewing the landscape of exogenous rnas in biological fluids of human and animals. using public databases of rna sequencing datasets, several documents report the application of in silico bioinformatics analysis (table ) clearly describing the widespread presence of exogenous ncrnas in biological fluids(from multiple sources, including diet) wang et al., ; beatty et al., ; yeri et al., ; . however, four documents suggest that exogenous ncrnas present in the public datasets may represent contamination from the laboratory environment since their abundance is extremely low, or they are of non-dietary origin, or could originate from technical artefacts or contamination bagci and allmer, ) . five documents report the development of bioinformatics resources to either predict transportable mirnas, predict functional analysis, or list and compare the exogenous mirnas found in samples chiang et al., ; zhang et al., ; zheng et al., ) . the generalized use of highly parallelized next generation (nexgen) sequencing technologies has promoted the use of sequencing to study complex biological systems, including human and animal biological fluids and tissues. initial studies evaluating the possible presence of exogenous ncrnas in biological fluids were done in silico using public databases of rna-seq results. several studies report the presence of exogenous rnas (i.e. dietary plant ncrnas) in the biological fluids of humans and animals. initial studies of the srnas spectra in human plasma showed that a small fraction of sequence reads from plasma mapped to human mirnas (≈ . %) or human transcripts and human genome sequences (≈ %) . this fraction increased by up to ≈ % when a higher sequence mismatch tolerance was allowed (under two mismatch allowance). surprisingly, a significant number of the unmapped reads aligned with various bacterial and fungal sequences (i.e. ribosomal rnas and trnas). also, many processed reads mapped to common food items, the most abundant being rna sequences from corn (zea mays) and rice (oryza sativa). compared to data from a serum sample from a chinese individual, the sequence abundance between corn and rice was reversed , suggesting the influence of dietary habits. other sequences from other foods in human plasma samples included soybean (glycine max), tomato (solanum lycopersicum), grape (vitis vinifera) and others. including exogenous mirnas, various common household insects were identified . treating plasma samples with dnase, protease, triton x- , and additional rnase reduced the total number of reads compared to the addition of rnase alone, suggesting that some of the exogenous rna molecules are probably associated to circulating protein and/or lipid complexes . in the same study, a lower percentage of exogenous sequences was observed in mouse plasma samples compared to human samples . another study evaluating a larger number of human plasma samples (n= young male athletes) reported exogenous srnas from different bacteria, but very few srnas were consistently detected in all samples (yeri et al., ) . in a study on public srna datasets from various tissues from mammals, chicken and insects, plant mirnas were found in out of datasets analysed, with mir- being extremely over-represented efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. . in datasets ( humans, mice, pig and others) the plant mirnas reads ranged from ≈ . - . % of the total animal mirna reads. insects fed with plant foods containing mir- , did not show accumulation of mir- when evaluated by northern-blot. however, in a very few samples sequencing data detected plant mirnas not present in the food source, including mir- , in addition to mir- at a moderate read, suggesting that plant mirnas observed in some public srna databases may be artefactual and due to sequencing methodology . plant mir- a from monocots was also found to be the most abundant exogenous plant mirna, in most cases, in other srna sequencing datasets studies with levels typically < % of human mirnas . but when analysis was performed in the absence of any known sources of plant contamination, plant mirnas were not detected. this suggests that precaution should be taken to prevent cross-contamination of the samples due to the high sensitivity of next-generation sequencing methods . other human public srna sequencing datasets have also been questioned for the presence of exogenous plant mirnas . of analysed human plasma samples, plant mirnas were found when compared to the genome of different plant model organisms (arabidopsis thaliana, triticum aestivum, oryza sativa, zea mays, and brachypodium distachyon). at least one read was present in selected samples. peu-mir- , found in all samples, is a singular mirna found in selected samples with more than counts. mir- is found in zea mays and conserved in fruits and vegetables including melon, sorghum, tomato, tea and oil palm. other highly-expressed plant mirnas found in up to hundreds of copies in selected samples include peu-mir- (found in samples), peu-mir- (found in samples), and tae-mir- (found in samples) . plant mir- (found in samples) and mir- a (found in samples) were detected in relatively small amounts (less than copies). using bioinformatics tools, the same study showed that seed sequences of mir- and mir- are similar to hsa-mir- and mir- - p for the former and has-mir- - p for the latter, and thus potentially target several gene targets of their human mirna counterparts . other studies have also reported in mouse plasma samples the presence of exogenous rnas matching exogenous species including bacteria, fungi, viridiplantae and food items . these included mir- d and mir- a (corn or rice as the most likely source), and mirnas from worms and insects . abundant non-human rnas were found in other healthy individuals human plasma samples (n= ) assigned to metazoan, bacteria or fungi (beatty et al., ) . several exogenous srnas were found that could potentially derive from food items including fragaria vesca, medicago truncatula, lotus japonicus, glycine max, arabidopsis thaliana, and solanum lycopersicum (beatty et al., ) . twelve public srna sequencing databases were analysed for the presence of plant exogenous mirnas. breast milk exosomes from humans ( samples, . million reads) and pigs ( samples, . million reads) were analysed. although plant mirna species belonging to mirna families ( samples) were found, they showed low abundance (< read counts for the highest expressed mirna). zma-mir- a, zma-mir- a, ath-mir- a, ath-mir- b and ptc-mir- d showed the highest abundance levels, while in humans only samples contained plant mirnas from mirna families (< read counts, for the highest expressed mirna, average of samples). ath-mir- a, pab-mir- , pc-mir- a, bdi-mir- , aly-mir- d, osa-mir- b. and zma-mir- a showed the highest abundance level in human samples . however, bagci and allmer reanalysed the data from lukasik and zielenkiewicz study and found that exogenous plant-derived mirnas in milk are likely to be contamination (bagci and allmer, ) . in a later observational study, lukasik et al. evaluated plant mirnas mir- a, mir- a, mir- a, mir- a and mir- a and reported the presence of plant mirnas in human (n= ) breast milk, both in whole milk and exosomes . out of mirnas, only were found in exosomes (mir- a and mir- a). while the human has-mir- a was found in all samples in a concentration range of . - pm for whole milk efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. and . - . pm in exosomes fractions, plant-derived mirna mir a was found in all samples at a concentration of - fm (≈ -fold higher than other plant mirnas) . compared to endogenous mirnas, the levels of exogenous plant mirnas were rather low and varied significantly between the samples . kang et al. analysed public sequencing srnas datasets from various human tissues and body fluids (n= ), including from serum and plasma samples, from exosomes, from cerebrospinal fluids, from liver, from blood cells and from brain . of note is that exogenous mirnas (xenomirs) were only found in % of the tissue samples (from total samples), with several studies being completely void of any xenomirs. xenomirs were absent from most human tissues samples, or present at very low abundances (with a median of . read counts). no enrichment of xenomirs was found in tissues relevant to dietary intake (i.e. hepatocytes and blood cells). by contrast, out of human body fluids datasets, xenomirs were found in % of samples, although at very low levels (median of read counts). even though the blood brain barrier separates the cerebrospinal fluid from the bloodstream, xenomirs were found in comparable quantities in both , suggesting no depletion (as expected by the presence of the barrier) of xenomirs in body fluids separated from the main bloodstream. moreover, the lack of co-occurrence of xenomirs in cerebrospinal fluid and serum of the same individuals, do not support that xenomirs detected in cerebrospinal fluid samples have entered through the bloodstream, and thus do not support a dietary origin of xenomirs. the most common xenomirs in body fluid samples belonged to the clades for rodents ( %), dicots ( %), insects ( %) and euphyllophytes ( %), suggesting that xenomirs composition does not reflect human food sources. from billion sequence reads (all samples), xenomirs were in low abundance, comprising only . % of the total reads ( , ). the same study reported a controlled feeding trial in rats and piglets (see section . . below for details). shu et al. ( ) used bioinformatics tools to characterize the structural characteristics of exogenous mirnas that can contribute to cross-species transportation and thus be transferred into human circulation . thirty-four thousand ( . ) mirnas sequences were compiled from species (from five kingdoms including animal, plantae, fungi, protista and viruses), including dietary mirnas ( mirnas) from types of common food species such as cow milk, breast milk, tomato, grape, and apple. using a support vector machine (smv)-based feature elimination strategy, features based on sequence, structure, and physicochemical properties were analysed to distinguish human circulating mirnas from the remaining sequences. predictions were made for features (categorized into groups) to better predict transportable mirnas in human circulation. known human plasma mirnas were ranked among the top of the list (dominated by animalia origin). only dietary mirnas were ranked among the top mirnas, five of which have a sequence identical to that in humans (bta-mir- b, bta-mir- , bta-mir- , gga-mir- a- p, gga-mir- b- p) . while only one mirna from plantae ranked among the top mirnas, plantae mirnas ranked in the top as possibly transportable , suggesting that animal-borne mirnas may be subject to more significant absorption in humans than in plant mirnas . it is important to note that this study of evaluated , animalia mirnas and plantae mirnas, whether this discrepancy in the original number of evaluated mirnas may have biased the smv-based classifiers to mammalian transportable mirnas was not addressed. in the same context, a database called "dietary mirna database" that describes different dietary mirna parameters and functional analysis has been described and is freely available. additional databases for exogenous mirna discovery using rna-seq data are also available in the scientific community . using bioinformatics tools (i.e. rnahybrid database), other studies have also predicted the potential function of dietary mirnas in the human body . in a bioinformatics study of plant mirnas, some similar functions between human and plant target genes were identified, such as ion transport and stress response . efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. analysing public srna sequencing datasets from humans ( ), mice ( ), pig ( ), chicken ( ), fly ( ), c. elegans ( ) and many other nematodes, protozoa or prokaryota, zheng et al. developed a database (exo-mirexplorer) for exploring and comparatively analysing exogenous mirnas (zheng et al., ) . two hundred and thirty-seven ( ) plant-derived mirnas were detected in sequencing samples. common mirna families included mir- , mir- , mir- , mir- and mir- . mir- a was the most frequently found mirna, and was detected in human samples from different studies (zheng et al., ) . the average abundance of mir- a was , . reads per million. in total, mir- a was found in samples from multiple species. mir- a is another frequently observed exogenous mirna from plants, and was detected in samples with an average abundance of . read per million (zheng et al., ) . regarding other biological fluids, exogenous rnas of bacterial origin were found in (urine) and (saliva) samples from young male athletes analysed for srna-seq (yeri et al., ) . plant small rna mir- was detected in urine of mice fed honeysuckle decoction (yang et al., b) . mir- a has been also detected in serum when honeysuckle was supplemented with pmol synthetic mir- a. mir- and mir- a were detected in the urine of mice that had received cisplatin treatment (cisplatin administration produces acute renal failure) and consumed a honeysuckle decoction alone or supplemented with synthetic mir- a. the increased detection of plant small rnas was attributed to disruption of the organization of small intestine epithelial cells produced by cisplatin treatment or longterm consumption (pre-fed) of honeysuckle (yang et al., b) , by mechanisms as yet unknown. mir- levels in urine reached ≈ fm. in tissues, the overall percentage of exogenous sequences in human lung tissue (normal lung rna samples), was found to be less than % . in spermatozoa samples, using public srna sequences datasets, tosar et al. found high amounts of exogenous sequences in three samples . however, in their own analysis of three spermatozoa samples they found no detectable traces of any plant mirnas, suggesting that exogenous mirnas present in the public datasets may have been caused by laboratory environment contamination . in terms of the possible passage of plant mirnas through the brain-blood barrier, kang et al. analysed samples from cerebrospinal fluid (csf) (from one study), which is separated from the bloodstream by the brain-blood barrier, and brain samples (from studies) . in general, xenomirs were absent from most of the human tissue samples, and, when present, they were found at very low abundance ( % of analysed tissues). although the brain-blood barrier would relatively protect the brain from dietary molecules, xenomirs were present in similar fractions in the exposed liver and in the relatively protected brain samples , suggesting, according to the authors, a nondietary origin . xenomirs were present in more body fluids samples (≈ % of samples) than tissue samples (≈ % of samples) and were identified in comparable proportions in the serum and cfs. this study does not provide support for the dietary origin of xenomirs (including plant exogenous rnas) meaning it may originate from technical artefacts . efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. in addition to mirnas reported by the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. very few studies have reported the influence of dietary or physiological factors on levels of exogenous ncrnas in biological fluids. in a small study (n= per group), wang and colleagues compared some abundant exogenous mirnas species identified in plasma samples from healthy subjects, colorectal cancer and ulcerative colitis patients . although ulcerative colitis patients apparently had increased levels of circulating mirnas of certain species (i.e. from a bantam family), a large variability of mirnas levels were observed among subjects of the three groups , suggesting that health/disease status may influence the presence of circulating mirnas in human plasma. tissue injury produced by excessive consumption of common pharmaceutical drugs can also influence exogenous rnas levels in plasma . wang et al. showed that acetaminophen overdose in a mouse model produced liver injury, and an increase of rna concentration in plasma compared to the control group was observed (from ≈ to ≈ ng/ml of total rna h after treatment). plasma ncrnas levels were affected by acetaminophen overdose, both endogenous and exogenous. plantderived exogenous mirnas levels were reduced, and this drop in plant rna was related to the observation that mice lose appetite after acetaminophen injection , suggesting the influence of diet on plasma plant-derived mirna levels. levels of exogenous rnas can be affected in case of drug-induced kidney damage. sera and urine from mice treated with cisplatin, a chemotherapeutic agent also producing small intestines epithelial cells disruption, showed increased plant exogenous ncrnas (i.e. plant mirnas), not observed in untreated mice (yang et al., b) . the effect was not observed in a glycerol-induced model of acute renal failure (which does not affect small intestine organization). detection levels of exogenous plant mirnas, including mir- and mir- a, were below background levels in mice pre-fed with chow diet devoid of honeysuckle. however, when mice were pre-fed with honeysuckle and honeysuckle was administered (alone or mir- a-supplemented) dose-dependent levels of mir- a was detected in serum and elevated serum levels of mir- were observed (yang et al., b) . the study shows that long-term honeysuckle feeding potentiates absorption of dietary mirnas through a mechanism that likely differs from cisplatin treatment, suggesting that particular diets can modify the capacity to absorb small rnas, and that altered or damaged guts resulting from illness and/or therapeutic treatment may enhance dietary rna uptake (yang et al., b) . while comparing the presence of exogenous srnas in three human plasma samples, beatty et al. reported that the largest proportion of reads matching plant sequences were found from one individual who reported following a vegetarian diet (beatty et al., ) , suggesting that the diet can influence levels of exogenous srnas of food origin. in another study comparing human breast milk for mirnas derived from plants, no significant differences were observed for exosomal breast milk plant-derived mirnas (mir- a and mir- a) between vegetarian or non-vegetarian volunteers . in the whole milk an increase in plant mirna mir- a was observed in non-vegetarian versus vegetarian diet volunteers . although some studies (see above) have reported the presence of exogenous rnas in human and animal circulation and tissues, very few have focused on quantitative data relevant to risk assessment. wang et al. estimated the concentration of rna in plasma to be < . pm based on an average of ng/ml of rna in plasma with an average of nt in length and a selected rna (i.e. mirna) sequence representing less than . % of the total rna population. after days feeding of herbals, yang et al. reported an increase of mir- in the sera of mice from fm ( -fold higher than control chow-fed animals) following ginseng intake to fm ( -fold higher than control chow-fed animals) following honeysuckle intake . the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the lack of enrichment of plant-derived small ncrnas in samples to be analysed by rna-sequencing (i.e. through periodate oxidation) might also influence the low enrichment of these exogenous sequences in biological fluids. however, although '-o-methyl modification of synthetic rna oligonucleotides '-ends can affect ligation activity of the t ligase in srna library preparation and may potentially result in srna sequencing bias and under-representation of srna with '-o -methyl '-ends in quantification experiments (munafo and robb, ) , others suggest a broad capability to detect plant mirnas during the sequencing procedure of small rna library construction . the available literature clearly suggests the widespread presence of exogenous rnas from multiple species, including plants, microbiota and many others, in human and animal biological fluids. evidence for plant-origin ncrnas, possibly relevant to risk assessment of ncrna-based gm, suggests that plantderived mirnas can be found in the biological fluids of humans and animals. whether these sequences are directly derived from dietary sources has not been clearly demonstrated. their overall low abundance, lack of enrichment in tissues most exposed to dietary changes, and their high variation between samples suggest that some may have originated as technical artefacts or contamination, as indicated by some authors. although few reports were available in this area, recent evidence also suggests that the levels of exogenous plant ncrnas in biological fluids may be influenced by diet, subject physiological or pathological conditions, or certain therapeutic treatments. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. following a literature search as described in section . . . . and based on the methodology described in the section . . . ., a total of papers were selected as being relevant to review of the systemic effects of dietary exogenous ncrnas. very few of these studies ( ) provide supporting evidence (in favour) that dietary exogenous ncrnas can be detected in biological samples and/or exert a biological effect after dietary ingestion. by contrast, studies provide contradicting evidence (against) that dietary exogenous ncrnas are either bioavailable or exert a systemic biological effect. one study provides evidence of the possible transfer of small ncrnas through the mammalian placenta . in addition, following a literature search as described in section . . . . and based on the methodology described in the section . . . , papers were selected as relevant to review toxicological effects of dietary exposure to exogenous ncrnas in humans or animals. the first report of systemic effects of dietary exogenous mirna in animals was published in by . plant mirnas were detected in human chinese healthy donors, among which mir- a and mir- a were highly expressed. these mirnas were also detected in calves and mice. plant mirna levels were relatively lower, but at a similar concentration range as endogenous mammalian mirnas present in serum. periodate oxidation, which oxidize mirnas but not plant mirnas (which are '-o-methyl modified), confirmed that these mirnas were genuine plant mirnas as most mammalian mirnas in human sera were oxidized and failed to be sequenced by solexa . the plant mirnas mir- a and mir- a were also detected in different mice tissues, while plant mir- a was not, although it was present in the sera. both mir- a and mir- a were primarily detected in microvesicles in c bl/ j mouse plasma. foods were found to contain the plant mirnas mir- a, mir- a and mir- a, including a chow mice diet at a concentration of . , . and . fmol/g, respectively. fresh rice contained between to -fold higher amounts than the chow diet. plant mirnas were detected in other foods including chinese cabbage, wheat and potato. plant mirnas were found to be stable in cooked foods. plant mir- a and mir- a were higher in the sera and liver of mice after h feeding with fresh rice. mir- a also increased in the stomach and small intestines, but not in the kidney, of mice fed a chow diet or fresh rice. mice fed with total rna extracted from rice or synthetic mir- a (methylated) showed an increased level of mir- a in serum and liver h after feeding. in vitro stability assays suggested that plant mirnas had a much slower degradation rate than their synthetic form (without '-o-methylated 'ends), suggesting that methylation has a protective effect on the stability of plant mirna . of note efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. is that mir- a targets the liver-enriched gene low-density lipoprotein receptor adapter protein (ldlrap ), which plays a role in facilitating the removal of ldl from the circulatory system (garcia et al., ) . direct binding of mir- a to exon of ldlrap , which is in the orf (open reading frame), was demonstrated by luciferase reported assays. transfection of both pre-mir- a and mature mir- a resulted in decreased ldlrap protein expression (but not that of mrna) in hepg cells. moreover, transfection of the intestinal epithelial caco- cell line with mir- a produced microvesicles with plant mir- a which can be taken up by hepg and reduce ldlrap protein expression. mir- a seemed to be associated to ago protein and other cells like t cells which were also able to package mir- a in microvesicles and then deliver mir- a into hepg . injection (iv) of antisense oligonucleotide against mir- a into mice during fresh-rice feeding reduced mir- a levels in the liver and reversed ldlrap repression. mice were finally fed with fresh rice for days to evaluate the physiological function of food-enriched in plant mir- a. serum and liver mir- a levels were induced already at day of feeding and the liver ldlrap protein was repressed after , and days. consequently, ldl levels in mouse plasma were significantly elevated on days and after fresh rice feeding. administration of an antisense oligonucleotide against mir- a reduced plasma levels of mir- a, repressed reduction of ldlrap in the liver and blocked the rice-induced elevation of plasma ldl levels, suggesting that elevation of fresh-rice derived mir- a in the mouse liver specifically decreased liver ldlrap expression and that this caused elevated ldl levels in mouse plasma (figure ). this study provides the first overall evidence of cross-kingdom regulation of gene expression through dietary plant mirnas. the authors further tested a mature mammalian mir- added to the chow diet for mice, and reported an increase in serum and liver levels of mir- after three days , leading to downregulation of liver c-myc expression . the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. by analysing western human sera samples from female stage ii-iii breast cancer patients, chin et al. detected multiple plant mirnas from arabidopsis thaliana and soybean (glycine max), but with sparse counts (chin et al., ) . among the detected plant mirnas, ath-mir- a, gma-mir- a- p and gma-mir- e- p had the highest counts (≤ read counts) and were over times more abundant than other plant mirnas. circulating mir- was more abundant in female healthy donors (n= ) than breast cancer patients (n= ) as analysed by qrt-pcr. moreover, circulating mir- was less abundant in patients who relapsed with metastatic disease, suggesting that human serum mir- levels inversely correlate with breast cancer incidence and progression (chin et al., ) . mir- in human serum was detected in an extracellular vesicle-enriched serum fraction obtained by ultracentrifugation and was resistant to sodium periodate oxidation, suggesting its plant origin due to '-o-methylation of the ' end (which makes plant mirnas more resistant to periodate oxidation). in situ hybridization confirmed the presence of mir- in tumour samples, suggesting that mir- in human serum is capable of entering breast tissue (chin et al., ) . the authors analysed several plant food items for the presence of mir- and found that broccoli is particularly rich in this mirna and most of this mirna was still present after cooking (boiling for min, > % remained stable, compared to fresh). in vitro studies showed that synthetic mir- (both double-stranded and '-o-methylated) reduced cell proliferation in breast cancer cell lines but not the non-cancerous breast cell line. extracellular vesicles from healthy human donors also have a similar effect. luciferase assays determined that mir- targets transcription factor (tcf ) and binds to two different sites within the ' utr. tcf is a transcription factor of the wnt signalling pathway and is upregulated in breast cancer. in vitro assays also showed that mir- inhibits breast cancer cell growth by targeting tcf . oral gavage of synthetic '-o-methylated mir- ( mg/kg mir- or scramble control oligos, daily) reduced tumour growth from day of treatment until the end of xenograph experiments ( weeks) (figure ) , an effect that was lost when tumour cells stably overexpress tcf without the ' utr (chin et al., ) . mir- was detected in xenografts of tumours by in situ hybridization analysis. overall, these experiments suggest that plant mirnas can inhibit cancer growth in mammals. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. liang et al. evaluated and quantified plant mirnas in human plasma after consuming a single dose of watermelon juice or a mixture of fruits (liang et al., b) . using taqman probe-based qrt-pcr assays and appropriate non-template controls, sixteen plant mirnas including ath-mir- a, mir- a, mir- a, mir- a, mir- a, mir- a, mir- a, mir- a, mir- a, mir- a, mir- a, mir- a, mir- a, mir- , mir- and mir- were evaluated and a standard curve generated for quantification. mirnas concentration ranged from . pm for mir- a to , . pm for mir- in watermelon juice, and from . pmol/kg for mir- a to , . pmol/kg for mir- in the fruit mixture. watermelon juice was orally administered to nine healthy male adult volunteers and plasma samples were taken . , , , and h after drinking . l of juice. different mirnas followed different peak time or appearance kinetics in plasma after administration (liang et al., b) . using six plant mirnas ( table ) that showed typical kinetic absorption curves, the total amount of plant mirnas absorbed by the body was calculated. using the plant mirnas concentration in watermelon juice and the areas under the plasma concentration-vs-time curves (auc) of plant mirnas, the total uptake amount, total absorption amount and absorption rate for each plant mirna was calculated (table ) . absorption rate ranged from . % to . %. mir- a, mir- a and mir- a were found almost exclusively encapsulated in microvesicles. mirnas were validated in watermelon juice or plasma by northern blotting. using the same protocol, a fruit eating study was performed. each subject consumed an equal portion of watermelon, apple, banana, orange, grape, mango and cantaloupe ( . kg in total). while mirnas concentration varied, the kinetics of plant mirnas after fruit administration appeared similar to the kinetics after juice administration (liang et al., b) . the authors also suggest that the real concentrations of plant mirnas may be underestimated and, for instance, the real concentration of mir- a in the watermelon juice might be six times higher and its plasma levels four times the previously calculated (table ) concentration (liang et al., b) . adapted from (liang et al., b) in a comment (correspondence) to the editor, the results of above article were criticized as being either a result of technical artefacts or a contamination problem, due to the presence of mir- in monocots and not in the dicot watermelon (witwer, ) . in a reply letter, the original authors indicated that mir- also exists in dicots and that the mirnas display a dynamic physiological kinetic absorption curve in human plasma, supporting the concept of uptake of food-derived mirnas by humans and animals (liang et al., a) . efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. analysing mir- by qrt-pcr in dried flowers and herbs used in chinese medicines or as tea, yang et al. reported different levels in different products including: sophora (≈ fmol/g), honeysuckle (≈ fmol/g), chamomile (≈ fmol/g), blue mallow (≈ fmol/g), ginseng flowers (≈ fmol/g) and as low as . fmol/g in hibiscus . herbal feeding of male mice for days increased plasma circulating levels of mir- compared to that of chow-fed animals. honeysuckle increased -fold ( fm) in sera levels, while chamomile increased -fold ( fm), sophora -fold ( fm), lavender -fold ( fm), blue mallow increased -fold ( fm), ginseng increased -fold ( fm) and no significant changes were detected in hibiscus ( fm). post-feeding urine levels of mir- ranged from -fold higher ( fm) for honeysuckle, and -fold higher ( fm) for chamomile and to -fold higher ( fm) for lavender . however, when chemically synthesized mir- ( '-o-methylated, plant-specific) was administered after single dose feeding of pmols, mice serum levels were elevated roughly -fold within min after gavage but decreased to background levels ≈ h after gavage . mir- was not associated with ago in sera and remained in the unbound fraction. clearance of mir- and other plant-derived synthetic mirnas (mir a, mir a and mir ) were analysed in mice by tail vein injection of fmols. sera collected from mice ( min, min, h, h and h post injection) showed that most mirnas were rapidly cleared from circulation. compared to the other mirnas administered at equal doses, mir- levels were substantially higher at min after injection but after h the apparent clearance of all srnas tested was complete . in the same study, gut microbiota did not seem to influence mir- absorption despite its -fold increase in their microbiome titre after honeysuckle consumption. honeysuckle feeding of mice was also found not to influence gut permeability, discarding its possible absorption due to changes in gut permeability . rapeseed (brassica campestris) bee pollen was evaluated for the possible absorption of plant mirnas in male mice after pollen oral feeding ( g/kg). mirnas were analysed by srna sequencing after or h in mice serum ( µl). from ≈ m clean reads, only reads were plant mirnas, which belonged to mirnas. of the plant mirnas, bna-mir- a ( reads) and bna-mir- ( reads) showed the highest levels in mouse blood and were mapped to the rapeseed genome. the rest of the plant mirnas had ≤ reads, of which mirnas had ≤ reads. both mirnas, mir- a and mir- , were also detected in the rapeseed bee pollen as evaluated by qrt-pcr, with higher abundance for mir- a. serum levels of mir- a after h of rapeseed bee pollen ingestion versus a control were ≈ pm for treated animals while in chow diet fed animals it was ≈ . pm, suggesting that plant mirnas from rapeseed bee pollen can be absorbed by mice by oral ingestion and detected in systemic circulation . evaluated herbal medicine honeysuckle mirnas before and after decoction (boiling for min, ≈ . g/ml water). from ≈ m reads, ≈ . m reads of mirnas were obtained, from which osa-mir- g, peu-mir- and peu-mir- contained more than . copy numbers. most mirnas were degraded by decoction (i.e. mir- g degraded from . to reads), while mir- remained high ( . reads) reaching % of total mirnas in the final honeysuckle decoction. concentration analysis of mir- showed a reduction from . pmol/g in honeysuckle to . pmol/ml in decoction when analysed by qrt-pcr, or from ≈ . pmol/g to . pmol/ml when assessed by northern-blot . compared to other plant mirnas, the apparent high resistance to boiling of mir- was also supported by its high resistance to rnase treatment, which was dependent on its unique sequence and high gc content. oral administration (n= per group) of a single dose ( µl) of honeysuckle decoction (mir- concentration ≈ . pmol/ml) resulted in a maximum peak level in mice plasma and lung tissues h after ingestion (i.e. plasma levels increased from . to . fm). most plasma mir- was found in cell-derived microvesicles and associated efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. to the ago complex. continuous drinking of decoction during three days ( . pmol/ml, ≈ ml/day) increased plasma levels from . to fm, while no changes were observed in the control mice (from . to . fm). levels of mir- in lung and liver tissues increased from to -fold, while the intestines and kidneys remained unchanged. synthetic mir- administration ( nmol/ml, µl given once per mouse, n= per group) resulted in an increase at peak ( h) from . to . fm in plasma and also an increase at peak ( h) in lung tissue, suggesting overall that atypical dietary mirna mir- is absorbed and delivered to tissues . studying its possible biological function, zhou et al. found influenza virus h n encoded mrnas targets of mir- , i.e. virus-encoded proteins pb and ns , which are relevant for viral replication (zanin et al., ) . luciferase activity assays confirmed that mir- binds to pb and ns , which is lost by point-mutation of mir- binding sites of the individual viral gene sequences. in vitro h n replication was reduced by synthetic mir- or total rna extracted from honeysuckle decoction. the inhibitory effect was abolished by co-transfection with anti-mir- antagomir. moreover, no effects in viral replication were observed when pb and ns mutant virus were used with synthetic mir- or honeysuckle decoction rnas . in vivo administration of honeysuckle decoction or synthetic mir- ( . nmol per day) by gavage one day before and during days after inoculation reduced weight loss and viral titre in lung tissues. these results were abolished when the anti-mir- antagomir was used concomitantly. moreover, weight loss and high viral titre were not affected when pb and ns mutant virus were used, and synthetic mir- or honeysuckle decoction administered . the authors also showed similar effects for synthetic mir- or honeysuckle decoction with other influenza viruses, including h n and h n , tested in vitro and in vivo (figure ) . the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. regarding this particular mirna, yang et al. observed a different bioavailability of plant-derived mir- vs. its synthetic '-o-methylated counterpart . oral administration of cabbage extract containing . pmol of mir- per µl gavage volume was compared with a -fold higher dose of synthetic '-o-methylated mir- ( pmoles per mouse). after gavage, mice plasma levels of cabbage mir- reached an approximately -fold increase (≈ fm) versus that of synthetic mir- (≈ fm), suggesting that mir- derived from plants is more bioavailable than synthetic rnas . whether this observation can be generalized to other dietary plant mirnas is unknown. the authors also showed that mir- was not present in exosome vesicles that were proteinase-k resistant in circulation . du et al. evaluated the presence of srnas in hong jing tian (hjt, rhodiola crenulata), which is a chinese herbal medicine. male c bl/ j mice were fed with hjt rnas by gavage for three days ( µg/kg per day) and lung tissue collected at , , or h (n= mice per time). the srna sequences of lung tissues (top eight ranked) that were also present in the plant hjt had ≈ k read counts . one of the small rna (hjt-srna-m ) was effective in reducing in vitro transfected gene expression vectors for α-sma, fibronectin and collagen type i α (col a ) in a tgf-β induced fibrosis model. although the type of srna is not clearly described, hjt-srna-m targets the ' utr of these three genes. using the hjt-srna-m agomir (cholesterol-conjugated srna), by intratracheal administration ( mg/kg in µl of saline) (n= mice per group), the authors showed that fibrosis was reduced in a bleomycin-induced fibrosis mouse model . due to the relevance of liposomes in delivering rna molecules, the authors evaluated the possible formation of liposomes during decoction (boiling for min) with plant lipids. they found that phosphocholine lipid molecules were also present in the decoction and suggested that liposomes may be formed that might facilitate the entry of rnas from plants to human cells and tissues; this was not tested . in another study, mlotshwa et al., described oral administration of a cocktail of tumour suppressor mirnas (normally downregulated in colon cancer) and a reduction of tumour burden in the apc min/+ mouse (c bl/ j) model of colon cancer . mir- a, mir- and mir- were chemically synthesized with the same mice sequence, but with a methyl group on the ' position of the ribose at the ' end, which is a chemical characteristic of plant mirnas (see section . . ). for days (starting at weeks of age), mice (n= per group) received either total plant rna spiked with the mix of the three mirnas, total plant rna alone (from a. thaliana) or water. oral dose was µg of total plant rna alone or plus . to . µg (corresponding to . to . nmole) of each species of tumour suppressor mirnas. tumour burden was dramatically reduced in mice receiving the mirna mixture. moreover, mice receiving the plant rna alone also showed a reduced tumour burden, although it was not statistically significant. after periodate oxidation, intestinal levels of mir- a were -fold higher than that of the group receiving water alone . mir- and mir- intestinal levels failed to be detected due to high background of endogenous mice mirnas after periodate oxidation. although the study does not directly evaluate the mirnas produced by the plant, mirnas designed to mimic small rnas produced in plants seemed to be taken up by the digestive tract of apc min/+ mice upon ingestion and exert a biological effect . oral ingestion of total rna isolated from brassica oleracea ( µg) resulted in the detection of plant mir- (the most highly enriched exogenous plant mirna in b. oleracea) in blood of icr mice between h and h after feeding . mir- was also detected in the spleen ( . - . % recovered of orally administered mir- ), and merely detected in the liver or kidney. no evidence of sex differences was reported . at lower doses of total rna ( µg or µg), no mir- was detected in the blood of mice . efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. using pigs as experimental animals, luo et al. evaluated the detection of plant-derived mirnas in the serum and tissues after feeding fresh maize to pigs (sus scrofa). jinhua female pigs consumed a fresh maize diet and water ad libitum for days (n= ), and blood and different tissue samples were obtained for plant-derived mirna analysis. eighteen maize mirnas were evaluated ( were detected) in serum and tissues, and exhibited relatively low abundance in the pancreas and longissimus dorsi muscle tissue . periodate oxidation treatment of the samples confirmed the detection of zma-mir- a- p, zma-mir- e- p, zma-mir- a- p, zma-mir- a- p, and zma-mir- a- p (while the rest were completely degraded), suggesting that these mirnas are bona fide plant mirnas (figure ). in a separate experiment, female pigs were fed one meal with fresh maize ( kg per pig) after overnight fasting. serum was collected at different times (i.e. , , , , , and h). after h, pigs were provided with fresh maize diet ad libitum, and serum collected at , , and days and tissue collected after sacrifice at days. the serum levels of some of these mirnas (mir- a- p, mir- a- p, mir- e- p, mir- a- p and mir- a- p) gradually increased after one feeding of fresh meal, reaching peak values at or hours within the first hours and maintaining stable detectable levels (by qrt-pcr) in the serum following days of access to fresh maize. the changes in mirna expression at all measured time-points in most cases were modest (≈ to -fold increase). the five mirnas were primarily present in serum exosomes (≈ . % of concentration in the serum) ( figure ). using bioinformatics, potential targets were determined within the porcine mrnas for zma-mir- a- p. using in vitro luciferase assays with porcine kidney cells, the authors showed that zma-mir- a- p binds to three of these potential target genes including cspg , otx and plagl , an interaction lost when their mutant constructions were used, suggesting overall that dietetically-absorbed mirnas can target endogenous porcine mrnas . the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. several of the above-mentioned studies have reported the presence of dietary plant mirnas in circulation associated to extracellular vesicles or exosomes (chin et al., ; liang et al., b; . this could be relevant for their possible biological effects, as extracellular vesicles can deliver a cargo to several tissues, including crossing the blood-brain barrier, and other biological barriers such as the placenta (matsumoto et al., ; yang, t et al., ; jayabalan et al., ) . their presence in microvesicles could modify their function, for example, it has been described that in some cases delivered naked mirnas cannot exert the same effects as those exerted by delivered exosomal mirnas (thomou et al., ) . also, exosomes as therapeutic drug carriers and delivery vehicles are gaining relevance in therapeutics (aryani and denecke, ; ha et al., ) . recently, it has been described that edible plant-derived exosome-like nanoparticles can be isolated from these plants (ju et al., ) , can transport ncrnas (i.e. mirnas) (mu et al., ) and can exert a biological effect (raimondo et al., ) , highlighting their role as therapeutic nanoparticles . it is still unknown if these exosome-like nanoparticles contribute to the resistance of ncrnas in the harsh conditions of the gi tract and to the absorption of plant ncrnas or the formation of exosomes circulating in biological fluids. regarding other biological barriers, only one study has reported on the transfer of small ncrnas through the placenta. li et al. found that exogenous plant mirnas were consistently detected in the umbilical cord blood and amniotic fluid of healthy chinese pregnant woman . this belongs to foetal circulation system, suggesting that mirnas may transfer through the placenta to the foetal side. three hours after gavage feeding of synthesized mature mirnas of the influenza virus to pregnant mice (at last -day pregnant and with mature placenta) increased mirnas levels were detected in the maternal plasma. an increase of exogenous mirnas levels in the foetal liver was also observed . to determine if mature plant mirnas can pass through the placenta and efficiently enter foetal organs, the traditional chinese herbal honeysuckle was tested. mice were gavage fed with . ml honeysuckle decoction ( . pmol/mice of mir- ) and maternal plasma and foetal liver collected h after gavage feeding. plant mir- levels were elevated ≈ . -fold in the maternal plasma (reaching fm), ≈ -fold in the placenta and ≈ . -fold in the foetuses, suggesting that plant mirna mir- in honeysuckle can efficiently transfer through the placenta to enter the foetal liver (figure ) . the amount of circulating mir- increased only in microvesicles, suggesting that circulating mir- was primarily present in microvesicles and that transplacental transmission may involve a microvesiclesmediated pathway . the same study also tested gavage fed synthetic sirnas ( nmol), which were found in the plasma and foetal liver. protein targeting of the sirna was dramatically reduced, suggesting that exogenous sirnas delivered from the mother to the foetus by trasnplacental transmission may regulate foetal gene expression. direct injection of microvesicles loaded with sirnas also reduced their mrna target in the foetus liver, suggesting overall that exogenous small ncrnas can be transplacentally transmitted from the mother to the foetus. although outside the scope of this review, other non-plant derived exogenous ncrnas have also been reported to exert systemic effects in animals. bovine milk-derived extracellular vesicles (bmevs) were orally administered to il- ra -/mice on the balb/c background, which spontaneously develop polyarticular arthritis, from week to week . drinking water was also supplemented and administered to a collagen-induced arthritis (cia) dba/ j mice model starting at week before immunization to day . mice receiving the higher dose of x bmevs (≈ micrograms/ml) showed a considerable delay in disease onset in the il- ra -/mice model. moreover, in the cia model the higher dose administered in drinking water . x /ml (≈ microgram/ml) reduced arthritis incidence and severity, accompanied by reduced expression of inflammatory cytokine il- and mcp- . although bmevs contained mirnas mir- a, mir- and mir- a, it is unclear if they are efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. responsible for the observed biological effects. in a similar study, oliveira et al. administered bmevs (particle concentration of . x /ml or . x /ml) in the drinking water to female dba/ j mice for weeks and observed an increase in osteocyte numbers. moreover, the highest concentration of bmevs increased the percentage of woven bone compared to the pbs group, and a reduction of the adipocyte area in the bone marrow (oliveira et al., ) . although the bmevs also contained mir- a (oliveira et al., ) , its implication in the biological effects is unknown. exogenous plant-derived ncrnas can pass the placenta and reach the foetus. by analysing small rnas components in royal jelly, honey, beebread and pollen collected during the cabbage (brassica campestris) flowering stage, zhu et al. found that honeybee srnas were present at a far higher level in royal jelly than in beebread and pollen, while the abundance of cabbage small rnas gradually increased from royal jelly to honey, beebread and pollen . several mirnas were detected in the samples, the plant mirnas occurring at far higher concentrations. moreover, beebread and polled mirnas were at a much higher concentration and their composition of individual mirnas was highly similar. the plant representative mirnas with the highest concentration were mir- a, mir- a, mir- a, mir- a, mir- a, mir- a, mir- g, mir- a, mir- a, mir- a, mir- c, mir- a, mir- a, mir- , mir- and mir- a. the same analysis in samples collected during the camellia (camellia japonica) flowering stage showed similar results and out of the enriched mirnas were similar, suggesting that plant mirnas in beebread and pollen may not be very diverse between different sources . absolute concentration of the selected plant mirnas ranged from < . fmol/µg of total rna (almost undetectable) in royal jelly and honey, to a maximum of ≈ fmol/µg total rna in pollen and beebread. feeding experiments to larvae with either rna purified from cabbage pollen (levels of mirnas similar to natural beebread mirnas levels) or a efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. synthetic mix of the plant mirnas, increased whole body accumulation of the representative plant mirnas and developed worker bee-like characteristics (reduced weight and size, extended pre-adult development time and decreased ovary size). ninety-six honeybee genes were predicted to be targeted by the plant mirnas, some of which are known to influence the developmental fate of honeybees (i.e. apis millifera tor, amtor). luciferase assays showed that mir- a directly targets aptor mrna. dietary supplementation of larvae food with synthetic mir- a decreased amtor mrna levels in honeybees and showed reduced body weight, size and ovary size . similar experiments were performed in drosophila melanogaster (dm) with total pollen rna, a synthetic mirna pool or mir- a alone, and in all cases similar phenotypes were obtained (delayed drosophila development, reduced final size, ovary size and fecundity). dmtor was also a target of mir- a. overall, this study implies that plant rnas can be transmitted between species of different kingdoms. the literature also describes some in vitro studies of plant mirnas and their possible effects on mammalian cells. for example, exposure ( h) of plant mirnas extracted from a glycyrrhiza uralensis decoction to human pbmcs produced apparent cell aggregation and increased cell number and hla-dr+ cell proportion (shao et al., ) . in silico studies have also attempted to predict possible plant mirnas with potential targets in humans . as described before (see section . ), in contrast to mammalian mirnas, plant mirnas possess a '-omethylation at the '-end that stabilizes and provides resistance to rnases and oxidation. this characteristic has been used to improve the quantification method for mature exogenous mirnas from plants in biological mammalian matrices . thus, periodate oxidation and βelimination were introduced to evaluate the origin of exogenous mirnas. male c bl/ mice were fed with corn/soy-based rodent chow diet for weeks. plant mir a, mir a and mir a were evaluated. these mirnas were detected in abundant amounts in a corn/soy-based chow diet, but after periodate oxidation and β-elimination . %, . % and . % of mir a, mir a and mir a were determined to be in methylated forms ( . , . and . fmol/g of diet, respectively). the control diet showed no differences between the diet and non-template control ct values after periodate oxidation/β-elimination. this study also suggests the use of exogenous rnas (spike-in), both methylated and un-methylated forms of mirnas, to ensure appropriate periodate oxidation/β-elimination . no detectable levels of plant mirnas were found in plasma, liver or faeces samples from the mice fed the corn/soy-based chow diet, since no differences occurred between the diet and the non-template control after periodate oxidation/β-elimination . this suggests that false positive results can be resolved using treatment with periodate oxidation/β-elimination. plant mirnas from extra virgin olive oil (evoo) and beer have also been evaluated by small rna sequencing. however, very few sequences (≤ reads) were identified, and most aligned to soybean and orange . a feeding study of a single dose ingestion of ml of evoo in healthy adult volunteers (n= ) was performed. mirnas analysis at baseline and after h ingestion was performed. no plant mirnas were detected in plasma after single ingestion of evoo . only one read count was found for other species mirna vvi-mir - p from grape, while two read counts were found from medicago trunculata, grape or brachypodium distachyon when mismatches were accepted . this suggests that beer or evoo do not contain mirnas that contribute to dietary intake, even though mirnas were reported in the pulp of olive (olea europaea) (donaire et al., ) . efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. dickinson et al. evaluated a controlled mouse feeding study with rice-containing chow diets (modified ain -g with . % of rice or rice-based with % of rice) or with a purified synthetic chow devoid of plant grain or forage for , and days (figure ) . the "synthetic chow" had negligible levels of osa-mir- a, the "balance rice chow" ( . % of rice) contained reads, while the rice grain contained reads. the concentration of osa-mir- a in each diet was . , and fmol/g of diet for synthetic chow, balance rice chow and rice chow, respectively. plasma and liver analysis of mice fed the diets did not reveal measurable uptake of any rice grain mirnas, including osa-mir- a. indeed, fewer than ten reads were detected in five out of eight samples from mice fed on rice-containing chow and four out of five samples from mice fed on synthetic chow . since the synthetic chow contained no grain or forage from plants, this low number for rice mirnas-mappable reads can only be explained by sequence errors or cross-contamination. in agreement with , the authors indicated that animals fed a chow containing high levels of uncooked rice (%) had significantly increased plasma low-density lipoproteins (ldl) levels at and days after treatment initiation. however, this increase in ldl was not observed in the balanced rice/chow group ( . % rice), which contained fmol/g osa-mir- a. the increase of ldl was attributed to nutritional imbalances between the test and control groups . ldlrap protein was also evaluated by elisa in mouse liver samples, and no changes in levels were observed in any group at any time, suggesting that dietary intake of osa-mir- a (from to fmol/g) does not produce rnai-mediated modulation of ldlrap protein levels in mouse liver . efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. a commercially available plant-based, plant mirna-rich substance containing soy and fruit materials, but not animal products, was administered by gastric gavage (≈ % of estimated blood volume for each animal) to male macaques (macaca nemestrina). relative abundance of plant mirnas mir- , mir- , mir- , mir- , mir- and mir- were analysed by qrt-pcr in the plant-based dietary substance. for instance, for mir- a consistent and efficient amplification through at least pcr cycles was detected. plant mirnas were analysed in plasma samples of macaques (n= ) before and after , and h oral gavage of plant rich mirnas. qrt-pcr results indicated late amplifications of some plant mirnas, but results were highly variable. mir- did not amplify before cycles, and mirs- , - , and - had a median cq greater than . mir- non-template controls also showed regular amplification within the same cq range. mir- exhibited a tendency to increase after ingestion, but was highly variable, and cq cycles were greater than . this was not the case for endogenous animal mirnas or the spike-in control, supporting the case that the late apparent amplification of plant mirnas in plasma was non-specific . moreover, droplet digital pcr analysis showed that counts from plasma were generally very low and of variable intensity, as occurs in non-specific amplifications. this even occurred in mir- (plant mirna with relatively high apparent count by qrt-pcr), which showed large number of counts but with variable intensity plots ("rain" appearance) (figure ) , and is consistent with non-specific amplification . overall, the study (though only subjects were included) suggests that the detection of plant mirnas in plasma does not support a general and consistent uptake of dietary plant mirnas . : substantial levels of mirna in oral diets consumed by macaque but negligible steady-state presence of diet-derived mirnas in plasma or organ tissues. snow et al. ( ) analysed the presence of exogenous plant mirnas after regular intake of a western diet containing fruits in healthy human subjects, and mir- in plasma or organ tissues of mir- -/mice . the conserved plant mirnas mir- a, mir- a and mir- a were highly the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. expressed in many fruits common in the human diet such as ripened avocado, apple, banana, orange and cantaloupe. mir- a and mir- reached up to , copy numbers/mg food. the conserved animal mirna mir- was expressed at substantial levels (≈ , copies/mg food) in meat but not in fruits. mouse vegetarian and soy-enriched diets also contained high quantities (up to , copy numbers/mg food) of mir- a and mir- a, but not mir- . in contrast, mouse diets enriched with animal products contained elevated levels of mir- , suggesting that conserved plant and animal mirnas are commonly present in oral diets for various animal organisms. although the dietary record of of the healthy adults reported the intake of fruits replete in mir- a, mir- a and mir- a the day prior to plasma harvest, these plant-derived mirnas were undetectable in plasma samples. following -week feeding with a custom animal lard diet containing mir- no detectable levels of mir- were found in plasma samples of mir- -/mice, while high quantities of this mirna in plasma were seen in control wild type mice fed the same diet . in tissues (liver, lung, kidney and stomach) mir- was either not detected or detected at exceedingly low levels corresponding to less than one copy of mir- per cell (assuming that the mammalian cell expresses roughly pg of total rna). wild type-mice showed only a non-significant trend toward increased plasma levels of mir- a after consuming either a vegetarian or soy-enriched diet when compared to mice fed a lard diet for the same period ( -week) ( figure ) ; however, levels were very low. indeed, mir- a was detected in organ tissues (liver, lung, kidney and stomach) but at levels of less than one copy of mirna per cell and regardless of diets. mir- a and mir- a were not detected at all. fresh avocado containing three specific plant mirnas was also fed to mice ( . g per mouse over h). even though the three mirnas were found in the stomach content of mice at high levels, very low levels were found in plasma and organ tissues, reaching levels lower than one copy mirna per cell in tissues. this provides no evidence of substantial steady-state presence of these plant mirnas in organ tissues after dietary intake . assuming that, on average, at least copies per cell (> copies/pg srna per cell) are necessary to achieve canonical target gene repression (brown et al., ) , at least copies of a given plant mirna must be ingested and delivered for widespread biological activity in humans (the average human body contains ≈ cells). based on the concentration in ripe cantaloupe (≈ x copies mir- a/mg fruit), an individual would need to consume , kg of cantaloupe just to release this number of copies in the gi tract . in terms of srnas, the literature describes the threshold for target gene regulation to be between and copies of mammalian mirna per cell . in an analysis of activity for hundreds of mirnas, only the most abundant mirnas in cells were found to mediate targeted repression and a large amount of mirnas had no discernible activity (mullokandov et al., ) . this suggests that only a minimum amount of mirnas need be reached to exert a biological activity, although this will also depend on the amount of target transcripts. a study mentioned above the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. analyses of other common plant mirnas reported by others to be bioavailable were not successful. this implies that although the transgenic mirnas had resistance to degradation comparable to that of mir- and high expression levels in plants, they were not readily bioavailable . huang (huang et al., ) analysed the possible bioavailability of orally-ingested corn mirnas in mice when incorporated into a rodent diet and consumed for days. in an initial experiment, male c bl/ mice (n= per group) received either water (control), random nucleotides ( µg, equivalent to the same amount of small rnas) or purified small rnas isolated from corn kernel ( µg, equivalent to the same amout of random nucleotides). in the second experiment, mice received either a control diet (ain- m), ain- + % autoclaved corn kernel powder or ain- + % fresh corn kernel powder. mirna levels in the isolated rna contained . pg mir- a, . pg mir- a, and . pg mir- a per µg corn srna isolates. the diet supplement with fresh corn powder contained pg mir- a, pg mir- a, and pg mir- a per gram of diet. autoclaving the corn powder at ºc for min reduced the amount of initial mirnas by more than %. in the first study, liver and whole blood sample mirnas (analysed by qrt-pcr), showed no differences in ct values between the three groups. after periodate oxidation followed by β-elimination, no differences were detected between the no-template control group and the experimental groups. similar observations were made in the liver and whole blood in the second study using fresh corn powder. also, after periodate oxidation followed by β-elimination, no differences were detected between the no-template control group and the experimental groups in the liver and blood samples. analysis of corn mirnas in cecal and faecal samples www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. showed similar results after periodate oxidation, although certain minor differences remained between the no-template control group and the experimental groups. in all cases, less than . % of total the mirna tested in both studies was recovered from the faecal samples. moreover, analysis of the mirnas in the content collected from different parts of the mouse gi tract from the animal studies administered via gavage or dietary intake showed that calculated recovery accounted for less than . % of the content originally ingested in the stomach, less than . % of that originally ingested in the intestine and faeces, and less than . % of that originally ingested in the colon and cecum (huang et al., ) . further experiments using in vitro digestion system suggested that after the gastric phase, over % of corn mirna mir- a from the diet or from the extract were degraded, suggesting overall that significant degradation of corn mrnas occurred during digestion, which resulted in minimal uptake of corn mirnas after oral intake (huang et al., ) . witwer re-analysed the plasma data evaluated by liu et al., and found that only one putative plant mirna mapped above a median cut-off ( reads), which was the plant mirna peu-mir- . this implies that all rnas -including previously reported exogenous mirnas (xenomirs) such as mir- a, mir- a and the plant ribosomal degradation fragment mir- -are below the level of background noise. it is not clear if mir- is a real mirna as it is found in the highly conserved and expressed subunit rrna of plants . moreover, the mir- sequence is not only a fragment of plant rrna, but also has a % coverage and % identity match with human s rrna, while others do not map to plant genomes at all . perhaps more analyses are needed when assessing possible xenomirs. after in silico xenomirs analysis using public sequencing datasets (see section . . for details), kang et al. performed controlled feeding studies to detect the transfer of exogenous plant mirnas to rat blood or from bovine milk sequences into piglet blood . adult rats (n= per group) followed a -day controlled feeding study with supplementation in three different diets: monocot plant material (rice); dicot material (potatoes); and husbandry chow containing a defined mixture of grains, cereals, vitamins, minerals and fats. small rnas were isolated from serum and sequenced. most samples showed either a lack of plant xenomirs in rat serum, or three or less read counts in certain samples . these extremely low numbers suggested a probable false positive result. in the same study, piglets were fed either cow milk or soy milk for weeks followed by weeks of feeding with maize. few cow-specific sequences were found ( reads) in the piglets fed cow milk, while piglets fed maize as well did show cow-specific sequences ( reads). this suggests likely false positive results. both studies provide no evidence of dietary transfer of xenomirs . the abovementioned studies are summarized in table . although outside the scope of this literature review, pollen plant mirnas ingested as part of a typical diet of adult honey bees were not found to be robustly transferred across the epithelial barrier under normal conditions (masood et al., ) . for example, despite the high levels of plant mir- a in pollen or its detected levels in the bee digestive tract ( . copies per pg total rna in the midgut), less than . copy per pg total rna was found in abdominal tissue (masood et al., ) . www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. large amount of plant mirnas mir- a, mir- a and mir- a detected in vegetarian, soy-enriched, or avocado diets (i.e. ≈ x copies for vegetarian soy/diets or ≈ . x copies for avocado diet of mir- a after h), but very low levels found in plasma and less than copy per/cell in tissues. after weeks mir- rich diet in mir- ko mice, no plasma levels and less than one copy of mir- per cell detected in tissues. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. after periodate oxidation, no major differences observed in mir- a, mir- a, or mir- a among treatment, control groups or no-template control in liver and blood. in cecal and fecal samples no major differences observed among control and treated groups. mirna analysis in the gi content collected from different parts of mouse showed that the recovery (calculated) account for less than . % of originally ingested in the stomach, less than . % of originally ingested in the intestine and feces, and less than . % of originally ingested in colon and cecum. in vitro digestion experiments suggested that after the gastric phase, over % of corn mirna mir- a from the diet or from the extract is degraded. (huang et al., ) the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. exogenous plant mirnas found in umbilical cord blood and amniotic fluid of healthy chinese pregnant women. plant mir- increased in plasma of the mothers, placenta and foetus liver h after oral gavage ( . nmol/mice). exogenous sirna gavage ( nmol) increased plasma levels of mother and foetus liver. protein target in foetus repressed. direct injection of microvesicles loaded with sirnas also increased its levels in foetal liver and reduced mrna expression of its target. exogenous small ncrnas can be transplacentally transmitted from mother to foetus. n.d., not determined. ko, knock out; wt, wild type; mirna, microrna; srna, small rna; dsrna, double-stranded rna; col a , collagen type i α ; α-sma, alpha smooth muscle actin; hjt, hong jing tian; www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). the available evidence presents several examples of systemic effects of plant-derived exogenous ncrnas when administered orally. however, there are also several reports that do not support these findings and contradict the hypothesis of cross-kingdom regulation. in either case, the essential question concerning the existence of this possibility is still heavily debated. important aspects such as the precise mechanism of transport of plant ncrnas from food to the circulatory system, the amount of exogenous ncrnas reaching tissues or the molecular mechanism of cellular uptake need to be understood. it remains unknown if such a transfer could be modulated in particular context (i.e. specific diet, disease status, medication). the available evidence also suggests that plant ncrnas can directly target mammalian genes through rnai effects when they are exposed in in vitro systems. without considering other physiological parameters, if a synthetic plant mirna is exposed to its mammalian target, it can effectively bind by base complementarity and exert a biological effect (i.e. gene repression). however, whether the exogenous plant ncrna can bypass all the biological barriers to reach and interact with the possible target remains to be clarified. apparently, exogenous plant-derived ncrnas are found in exosomes or macrovesicles. it is not yet known how they reach these types of structure in biological fluids, or if novel plant exosome-like nanoparticles participate in any of these processes. www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). immune cells were mainly from studies in human and mouse isolated lymphocytes. however, these studies were descriptive/not comprehensive, reporting specific effects of ncrnas in proinflammatory cytokine production using human cells, and would not allow identifying specific endpoints that can be used to evaluate changes in the regulation of immune function and homeostasis by ncrnas. retrieval of grey literature using the key words 'thesis plant rna immune' provided numerous results (i.e. google = . ), mostly regarding the function of mirnas and rna silencing in plant immunity. for instance, a document could be chosen based on title selection because it included the terms 'plant, double-stranded rna and systemic immunity' (https://uknowledge.uky.edu/plantpath_etds/ ). however, this study was not further considered since it refers to plant immunity. after analysing the documents and refining by key questions, a large number of screened publications were discarded because they refer to the role of endogenous ncrna (human and animals) as well as viral infections in relation to the immune function, which is outside the scope of this literature review. as an example, within original in vivo publications related to the key words, only studies were considered relevant for the proposed review questions. in summary, from the literature search, papers were finally selected as relevant to review of the effects of ncrnas on immunity. very few studies analyse the direct relationship of exogenous plant ncrna with immune function and homeostasis of humans or animals. albeit controversial, exogenous ncrnas derived from plants and microorganisms have also been described in human blood (detailed in section . . .). overall, the results indicate that there is scarce information on the specific impact of plant ncrnas on activation of specific immunocompetent cells. of these references, documents were used for reviewing the general features of ncrnas in immune function and immunity in humans and animals (section . . .), were used for the effect of exogenous plant ncrnas on immune system (section . . .) and for the additional section on the effects of exogenous ncrnas on gut microbial composition (section . . .) ncrnas have been shown to play important roles in immune cell development and function in normal and disease conditions (ansel, ) . human and mouse studies demonstrate that mirnas play a critical role in the regulation of immune cell development and their function. there is also evidence of the relevance of neonatal mirna-mediated immune activation (kosaka et al., ) . here, mirna molecules of maternal origin appear to be stable in the infant's gut conditions, allowing dietary intake and transfer of mirna (kosaka et al., ) . ncrna have also been identified as regulators of metabolic shifts within microbial communities (paul et al., ) . thereby, ncrnas may significantly influence the critical roles of gut microbiota on health and disease. this part of the literature review (efsa task ) presents the current knowledge on the role of exogenous ncrna molecules on the immune system of humans and animals and aims to provide information on the possible effets of dietary ncrnas on the regulation and function of the immune system. in addition, since the gut microbiota influences the immune system, a review on the possible effects of exogenous ncrnas as gut microbiota modulators is provided this chapter provides background information on the modulatory effects of ncrna on the immune system of humans and animals and serves as a general introduction to explore the possibility of dietary plant ncrnas effect on the immune system after dietary intake. sixty-four ( ) documents were finally used to review this section. when activated, the immune system tightly regulates innate and adaptive response(s), aiming at restoring homeostasis, and preventing autoimmunity. the 'amplitude' (i.e. intensity and duration) of the www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). interaction with the t-(tcr) and b-(bcr) cell receptors determines cell fate decisions. in the periphery, regulatory t cells (tregs) play a key role in restraining the activity of mature b and t (th , th and th ) cells, and preserving tolerance mechanism(s). these responses depend on transcriptional and epigenetic regulation including regulation by mirnas. emerging systems for measuring mirna activity during immune activation revealed the complex network of genes that may be simultaneously targeted by mirnas to tune distinct cell fate decisions (wells et al., ) . rnas exhibit an intrinsic property of base pairs that is crucial in defining their structure and, thereby, their physiological role (lu et al., ) . plant mirnas are '-o-methyl modified on their terminal nucleotide, making them more difficult to be ligated to the cloning adapter. the inhibitory effect of '-o-methyl modification at the ' end of the rna substrate for the mammalian nuclease family member nef-sp has been described (silva et al., ) . as described elsewhere, most mammalian mirnas require 'pre-processing steps' to become physiologically active. nucleotide sequence complementarity appears as a critical feature driving the predominant result exerted by mirnas. notably, it has been reported that a modification pattern involving alternating '-o-methyl rna bases improves dsirna in vitro stability and evades activation of the innate immune system (collingwood et al., ) . these data were obtained with isolated peripheral blood mononuclear cells (pbmcs) as a mixed 'lymphocyte' population of immune receptors that are encountered in vivo. however, the authors do not provide any molecular mechanism for the observed effects. sirnas have been shown to be potent stimuli of interferon- production by plasmocytoid dendritic cells (hornung et al., ) . this sirna technology induced systemic immune responses in the same range as the toll-like receptor (tlr)- ligand 'cpg', including activation of t cells and dendritic cells in spleen. notably, immunostimulation by sirna was absent in tlr -deficient mice. there is a lack of data related to the effects of plant ncrnas on pge production, implicated in immunosuppression by tlr via cox . several distinct classes of lncrnas are transcribed from different dna elements or are derived from long primary transcripts with noncanonical rna processing pathways, generating new rna species with unexpected formats. these lncrnas can be processed by several mechanisms, including ribonuclease p (rnase p) cleavage to generate mature ' ends, capped by small nucleolar rna (snorna)-protein (snornp) complexes at their ends, or the formation of circular structures. recently, it was reported that several lncrnas mediate their regulatory effects through binding to specific rna-binding proteins . in addition, expression of lncrnas has been defined as highly cell-type specific (guttman et al., ; washietl et al., ) . this high specificity has been reported to be critical for the development and activation of immune lineages. however, lncrnas-mediated regulation of innate immune activation (i.e. il- production) has not been tested for function in vivo, but only evaluated in vitro (roux et al., ) . the regulation of inflammatory mediators or cytokines by lncrnas is still poorly understood. synthetic nucleic acids, such as dsrnas, have been reported to be recognized by toll-like receptor (tlr)- and can stimulate the innate immune system and trigger a type i interferon response (marques et al., ; mian et al., ) . in plants, it has been described that dsrna with ' overhangs contributes to endogenous and antiviral rna silencing pathways (fukunaga and doudna, ) . earlier reports defined a structural basis (i.e. ' overhangs) to cause nonspecific effects on the ds-rna-activated signalling pathways through the interferon responsive factor (irf)- (marques et al., ) . similarly, immune activation by the subcutaneous route with a viral mimic dsrna (poly i:c) of c bl/ mice during neonatal early or late brain development differentially affects depression-related behaviours developed in adolescent and adult mice (majidi-zolbanin et al., ) . the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). specific molecular interactions of the exogenous rna triggering immune responses have been reported. several studies have demonstrated the role of components of innate immunity, including toll-like receptors (tlrs), the retinoic acid-inducible gene i/melanoma-differentiation factor (rig-i/mda ) and the interferon response effectors protein kinase r (pkr) and rnase l, as well as mirnas in the recognition of viral or synthetic dsrna (malathi et al., ; urcuqui-inchima et al., ) . tlrs are a family of proteins recognizing different pathogen and damage-associated molecular patterns, pamps and dmaps, respectively. rig-i/mda , pkr and rnase l constitute cytosolic rna sensing proteins. the pathways responsible for these defined signalling processes can be largely separated (figure ) , despite the existence of some degree of crosstalk between them where the engagement of interferon regulatory factors is a common feature. tlrs play an essential role in innate immune responses in mammals. in humans, ten different tlrs recognize distinct molecular patterns, where tlr- , - , - and - stimulate innate immune responses upon interaction with nucleic acids weber et al., ) . tlr is located on the plasmatic cell membrane, while tlr- , - and - display endosomal localization. it has been reported that tlr recognizes dsrna, viral or the synthetic poly i:c, and sirna has also been identified (in the presence or absence of delivery systems) as a relevant ligand . notwithstanding, rna interference is considered an unlikely mechanism to be engaged for viral sensing (cullen, ) . gurich rna from viruses or synthetic single-stranded (ss) oligoribonucleotides displays an apparent preference for stimulating human tlr / -mediated immune effects (heil et al., ; forsbach et al., ) . a minimum of four nucleotides, uugu, found in the gu-rich region are reported as necessary to stimulate cytokine responses. endogenous or exogenous rnas are internalized into the endosomal compartment. the rna sensor system is composed of i) several innate immune 'toll-like' receptors (tlrs) located at the endosomal compartment that trigger different signalling pathways engaging adaptor distinct molecules (myd and trif), and ii) a cytosolic system (rig-i/mda- ) that coordinates its signalling with tlrs. antigen processing will lead to rearrangement of mhc molecules to activate the adaptive immune response(s). type i interferons (ifn-α/β) are expressed rapidly after infection and serve as a link between the innate and adaptive immune responses. : schematic representation of the eukaryotic rna sensor (single-stranded rna and short/long double-stranded rna) and its relation to innate immunity. the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). two classes of ssrna motifs have been described that either preferentially activate tlr -mediated or tlr / -mediated immune responses. au-rich oligoribonucleotides stimulate tlr -but not tlr mediated immune activation (forsbach et al., ) . rna motifs activating tlr -associated signalling fail to induce ifn- from tlr -expressing plasmacytoid dendritic cells but induce the secretion of th like and proinflammatory cytokines from monocytes or myeloid dendritic cells. in addition, rna motifs activating the tlr / -associated signalling pathway stimulate cytokine secretion from both tlr -and tlr -positive immunocytes (forsbach et al., ) . differences in the protein sequence of tlr- between different mammals (human, monkey, chimpanzee, cattle, porcine, mouse, and rat) can be found, and species-specific recognition of ssrna via tlr / had been described (heil et al., ) . the tlr -specific rna sequences are unable to trigger cytokine responses from mouse, rat, and porcine immune cells (forsbach et al., ) . a dual function has also been suggested for the murine coreceptor cd to enhance tlr- , - and - signalling (baumann et al., ; lee et al., ) . however, further studies showed evidenced that human cd did not appear to function as a co-receptor for tlr- or tlr (weber et al., ) .the innate immune system detects rna lacking nucleoside methylation (m c, m a and m u) or otherwise (i.e. pseudouridine or '-o-methyl-u) modified as a mechanism to selectively trigger immune responses to nucleic acids from necrotic tissues or of exogenous origin. a recent systematic study on transcriptional and, especially, post-transcriptional regulation of stressresponsive lncrnas in oryza sativa showed that hundreds of lncrnas with down-regulated polyadenylation are highly conserved in stresses . in the ascidian ciona intestinalis a novel alternative polyadenylation signal activated by the prototypical tlr agonist (i.e. lps) has been described (vizzini et al., ) . moreover, alternative polyadenylation has been identified as a regulatory mechanism during the innate antiviral immune response in macrophages (jia et al., ) . these authors showed an enrichment of polyadenylated genes and mrna abundance change in tlrs as well as rig-i-like receptor, jak-stat and apoptosis-related signalling pathways. studies conducted on the crystal structure of the sensing tlr- ectodomain allowed definition of dsrna recognition of at least bp (liu et al., ; leonard et al., ) . it was concluded that tlr assembles on dsrna as stable dimers and that the minimal signalling unit is one tlr dimer. recognition by secondary structure 'alone' has been reported, independently of the sequence motif, by tlr . this dsrna length is larger than the typical helix occurring in normal mirnas and sirnas. tlr- and - have been found to recognize single-stranded rna (ssrna). the existence of a similar double horseshoe structure for tlr- , - and - upon activation has been proposed, although their binding modes to their ligands remain unclear . often, in a partial view, tlr is considered as the ssrna-sensing tlr and as complementary to tlr . however, higher structural features have been identified as important aspects in tlr rna sensing (jöckel et al., ) . in addition, small molecules that may be viewed as nucleoside analogues are able to activate tlr and/or tlr (hemmi et al., ; lee et al., ) . it has not been defined if the recognition mode for small molecules, including srnas, resembles that of tlr for rna recognition. besides tlrs, rig-i is a cytosolic multi-domain protein sensing viral rna associated to n-terminal caspase activation and recruitment domains (cards) to transmit signalling but preventing it in the absence of rna. the identification of rig-i/mda as cytoplasmic sensors for dsrna, associated to facilitators such as pkr and '- '-olygoadenylate synthetase, suggests that additional 'aspects' (i.e. dependence on atp) other than dsrna structure or short size influence innate immune responses to srnas (i.e. sirnas and mirnas). these aspects remain unclear. the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). current information on mirnas points to clear differences in cellular recognition of endogenous and exogenous dsrna. here, '-triphosphate and dsrna represent molecular patterns enabling rig-i to discriminate exogenous from endogenous rna (myong et al., ) . dsrna products as short as - bp are sensed by rig-i independently from the '-termini (marques et al., ) . similarly, the ifninducible '- '-olygoadenylate synthetase (oas) requires dsrna that must be at least nucleotides long for activity, and no modification of the ′-hydroxyl group is tolerated (sarkar et al., ) . the oas family requires synthesizing di-, tri-, and tetrameric '- '-oligoadenylates ( - a), which in turn bind and activate rnase activation of rnase l together with pkr, irf and c-jun-n-terminal kinases, constituting pro-apoptotic mediators in response to dsrna. moreover, human oas may also enhance rig-i mediated signalling by mimicking polyubiquitin . further structural and mechanistic studies identify a key role of domain duplication in the oas family, thus revealing different functions of oas- and oas- in sensing dsrna (donovan et al., ; . rna interference (rnai) as well as antisense oligonucleotides and aptamers have been postulated to exert immune stimulatory effects in mammals (agrawal and kandimalla, ; cullen, ; mustonen et al., ) . however, at present, there is still an important open debate about agents exerting physiologically relevant functions at systems level biology (cullen, ; kleinman et al., ) . moreover, clinical trials of sirna demonstrated that the only population carrying the ff coding variant for tlr was protected from sirna-induced cytotoxicity (kleinman et al., ) . this study concluded that, because multiple cells express surface tlr , the therapeutic approach with sirna might induce unanticipated vascular or immune effects. here, anti-angiogenic innate immunity triggered by sirna was found to occur without the induction of inf-α/β via trif activation being biased toward nf-ĸb rather than irf- . side effects, for example, the immunosupressive effects of t regulatory cell function are inhibited by ifn-α- β (yu et al., ) , are still only vaguely understood in association with exogenous ncrnas administration. naturally occurring mammalian rnas do not display a stimulatory potential on innate immune responses through tlr- , - , - and - in dendritic cells and tlr-expressing cells (karikó et al., ) . by contrast, it has been shown that major innate immune responses to chemically synthesized sirnas are mediated by tlr and/or tlr . notably, replacement of uridines with their '-modified counterparts in the sirnas, reduced immune activation (sioud, ; barik and lu, ) . studies conducted on synthetic sirnas have shown results directly dependent on the nucleotide sequence (judge et al., (judge et al., , hornung et al., ) , and not to silencing effects of a target gene. putative immunostimulatory motifs allowing definition of ifn-α induction by β-gal control sirna or its constituent sense (gu-rich) and antisense ssrna oligonucleotides (judge et al., ) have been identified. these studies were performed in pbmcs, and therefore lack a clear demonstration of these effects at the organism level. intravenous administration of encapsulated sirna induced substantial dose-dependent ( - g sirna) ifn-α responses in outbred institute for cancer research (icr) inbred mice. in this line, reports also exist of increased hepatotoxicity of asos containing locked nucleic acid modifications (swayze et al., ) . in addition, asos carrying '-o-methoxyethylribose modifications showed no cytotoxic effects but were able to reduce targeted mrna. by analysis of sequence variants of either ss or ds sirna, uridine-rich immunostimulatory motifs within sirna were identified in human peripheral blood cells via tlr (sioud, ; jurk et al., ; judge et al., ) . there is a report of sequence-and targetindependent angiogenesis suppression induced by sirna via tlr (kleinman et al., ) . a minimum length for dsrna of about -nucleotide or -nucleotide luc sirna, but not truncated versions the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). suppressed by choroidal neovascularization have been observed in wild-type mice (kleinman et al., ) . recently, it has been established that nuclease-resistant aptamers can play a 'dual' role in immune signalling pathways (gefen et al., ; rajagopalan et al., ) . these aptamers seem to possess a unique feature acting as both agonist and antagonist depending on their degree of oligomerization (nozari and berezovski, ) . aptamers are ss oligonucleotides ( - nucleotides) capable of recognizing, in a specific manner and with high affinity, several types of target molecules by means of a three-dimensional folding of their chain. preclinical studies in murine models have highlighted distinct regulatory points that may be influenced by oligonucleotide aptamers, including clusters of differentiation (cd) (pastor et al., ) , cell-to-cell communication such as immunoglobulins (gefen et al., ) and interleukin (il) signalling (rajagopalan et al., ) . the cd molecules can act in several ways, often acting as receptors or ligands important to the cell initiating signal cascades or serving as adhesion molecules. here, cd is one of the main co-stimulatory receptors responsible for proper activation of t lymphocytes that could be manipulated to modulate the immune response (pastor et al., ) . further studies reported enhancement of t cell functions by specific t cell immunoglobulin- aptamers through negative regulation of ifn- secretion by cd + and cd + cells (gefen et al., ) . positive effects attributed to oligonucleotide aptamer have also been associated to attenuation of il- signalling in cd + cells (rajagopalan et al., ) . while sirna and dsrna in cultured cells have been shown to trigger an interferon response (alexopoulou et al., ; sledz et al., ) , the administration of naked (dtdt) synthetic sirnas ( . mg/kg) to mice (either intraperitoneally or iv via low pressure or high pressure injection) induced no interferon response (heidel et al., ) . this suggests that sirna administration in vivo may elicit little or no immune response. also, modification of nucleotides within rna molecules was shown to reduce immune response (durbin et al., ) , which is relevant when applying nucleotide modifications to rna therapeutics. for example, n- -methyladenosine (m a) and pseudouridine seems to reduce the retinoic acid inducible gene i triggering of the innate immune signalling (durbin et al., ) . thus, rna modification seems to influence recognition of exogenous rnas, for example, ribose '-o-methylation of mrna at the '-end provides a molecular signature for the discrimination of self and non-self rna (züst et al., ) . prior to immune cells performing their immunological action on target cells, they need to receive a suitable 'maturation' signal to enhance clonal expansion and acquire the effector function. activation signals occur in peripheral lymphoid organs through a concerted interaction with mhc molecules. the major histocompatibility complex (mhc) regulates the cell-mediated immune response(s). there are recent reports on the regulatory role of mirnas on mhc class i and ii molecules (wongfieng et al., ; xie et al., ) . for instance, mhc class i chain related protein b present in natural killer (nk) cells was associated to cell-mediated antitumor immune response through engagement with the nkg d receptor (xie et al., ) . the expression level of this receptor has been shown to be regulated by exogenous mir- c mimics in nk cells . based on the temporal and spatial regulation which determines the expression patterns of mirnas, that suggestion was made for the participation of complex regulatory networks composed of several transcription factors influencing the expression level of 'each' mirna. the mhc class ii complex has been identified as a target in mammalian mirnas regulation (wongfieng et al., ) . in addition, some lncrnas have been identified as candidates to be translated into peptide fragments (guttman et al., ) suggesting, as several other 'indirect' lines of evidence, the the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). participation of ncrnas-derived peptides on mhc complexes (yewdell, ; mellman et al., ) . peptides bound to mhc molecules serve as recognizing fragments of antigen for the tcr. here, it has been shown that mirnas can influence the amplitude of tcr signalling modulating t cell fate decisions towards survival and maturation wells et al., ) . here it is worth noting the recently defined dual role of mirna in maintenance of the naive phenotype of cd t cells or clonal expansion, where modulation of let- mirnas levels is required (wells et al., ) . in a similar way to mhc class ii for t cells, bcr determines the fate of developing b cells allowing their adequate maturation or the development of self-reactive b cells leading to immunodeficiency of b cell malignancy. the mirnas set threshold for lymphocyte development targets several genes in the pi k pathway (simpson and ansel, ) . it is also important to highlight that pi k displays an inhibitory interaction on tlrs-induced production of il- , therefore limiting excessive th polarization and suggesting a potential immunoregulatory role for mirnas. a few relatively recent studies address the potential regulatory role of 'specific' mirnas in treg functions (jeker et al., ; warth et al., ) . these studies have shown that inhibition of mirna- a in c bl/ j mice led to reduced foxp expression dependent on the levels of mediators such as tgf and/or retinoic acid (ra) (jeker et al., ) . another study reported induction of treg cell differentiation regulated by a mirna 'network' influencing the promoting factor mtor (warth et al., ) . these studies revealed that the underlying molecular mediators responsible for the mirna regulation of t cell signalling pathways remain unknown. unlike animals, plant mirnas have not been associated to tgf nor ra. intersecting the rapidly emerging field of treg function, it has been discovered that ra controls both the homing and differentiation of treg. in addition, computational and systems biology approaches have identified mtor as a potential pathway to explain the cross-kingdom mirnas-mediated regulatory potential of camptotheca acuminata ). although many reports can be found regarding the immunomodulatory role of endogenous and viral ncrnas, very few evaluated the impact of plant ncrnas on immune function and homeostasis when ingested/absorbed by animals and/or humans. thus, this section was reviewed using scientific documents. however, no studies were found that directly evaluate the effect of plant exogenous ncrna and their effects on the immune system following dietary intervention trials. recent reports highlight the shortcomings of existing mirna measuring systems (i.e. qpcr) in comparison to sequencing procedures to explore mirnas in biological fluids chen et al., a) . these studies point out the considerable number of unmapped ncrnas as a major factor limiting the use of qpcr techniques. notably, the abundance level of identified exogenous rna sequences in biological fluids (i.e. human plasma and breast milk, and serum from mice) was rather low. in addition, the role of food matrix on ncrna delivery to their cellular or tissue targets has not been defined. although the identification of exogenous ncrnas in biological samples led to considerations on their role as cross-kingdom regulators of immune-related processes, the impact (i.e. influence and regulatory role) of exogenous plant ncrnas on immune function and homeostasis is inferential. exogenous rnas sequences have been found in plasma , serum (chen et al., b) and breast milk ) from humans and/or animals. these studies reported the presence of ncrnas in biological fluids, although their impact on immune function remains to be fully clarified. the recent discovery of blood circulating rnas originating from foods seems to indicate that certain dietary plant mirnas can be absorbed at significant levels . however, the potential contribution of distinct 'plant food matrices' on ncrnas the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). absorption has not been addressed as it has been described for viral rnas (seljelid et al., ; zou et al., ) . the existing studies show that adequate antigen-processing processes at the intestinal level can be avoided by ncrnas bound to coadjuvant molecules (i.e. polycations, cationic lipids, phospholipids) (seljelid et al., ; zou et al., ; vertzoni et al., ) . notably, phospholipid homeostasis is central in the response to toll like receptors (tlrs) activation through a type i interferon 'autocrine-paracrine' loop (song et al., ) . in biological samples, rna sequences have been reported from most common gm food matrices, including corn (zea mays), rice (oryza sativa japonica group), soybeans (glycine max), tomato (solanum lycopersicum) and grape (vitis vinifera) . the identification of rice mir- a at fm concentration level in various mouse tissues may indicate the possibility of a cellular active uptake system for circulating rna . these data are in agreement with the role of defined food matrices as effective delivery systems protecting ncrnas from interaction with macrophages, dendritic cells, b-lymphocytes, and t-lymphocytes found in peyer's patches and other sites of gut-associated lymphoid tissue. however, several other studies contradict these results (see section . . . for details). enrichment of mirnas with targets related to immune response has been observed in colostral milk . colostrum represents a primordial food driving the first immunization of offspring and provides the nutritional needs of their immature organs in the earliest stages of life. the facts that immune-related mirnas from host, and exogenous plant mirnas (from bamboo diet to milk exosomes of giant panda) targeting synapse organization, neuron migration or axon guidance are enriched in giant breast milk of panda (restricted to a diet primarily comprising bamboo) indicate that breast milk may facilitate dietary intake of plant mirnas by infants for possible regulation of postnatal development ). very few studies address feeding plant ncrnas to animals (i.e. weaning, adult and old age) to evaluate induction of specific adaptive immune response(s) (i.e. th , th or th ). similarly, there is a lack of data on the impact of exogenous plant ncrnas on expression of mhc-ii molecules to assess the potential allergenicity of these rnas. additionally, the different immune response(s) induced by the administration of distinct exogenous plant ncrnas and well-known weak or strong immune inducers by different routes (intraperitoneal or intragastric) have not been tested. understanding of the allergenic functions of exogenous plant ncrnas is comparatively meagre in relation to proteins. the homology of distinct ncrnas could elicit different immune response(s) as proteins showing homology values up to % on their amino acid sequences induce different igg and igg responses (adel-patient et al., ) . for example, the literature search identified one study on safety assessment of the possible effects of gm-triticale on the immune system using a murine c bl/ model (krzyżowska et al., ) . immunoblotting analysis in serum showed significantly increased il- levels, but not those of ifn-γ. it was concluded that multigenerational use of feeds for rodents containing the gm-triticale leads to expansion of the b cell compartment in the secondary lymphoid organs not associated to allergic response(s) (krzyżowska et al., ) . however, this study did not report the ncrna content in gmtriticale. efforts over the last decade have focused on elucidating the role of ncrnas in immune function. although no immunogenic potential has been found, ncrnas implications in pathways regulating the production of inflammatory mediators (witwer et al., ) , the development of hematopoietic cells (satpathy and chang, ) , regulation of tlrs signalling (carpenter et al., ) and changes in t cells needed to support clonal expansion and growth have been highlighted. papers on ncrnas-mediated regulation of the amplitude and quality of immune response(s) could not be found. the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). at the moment, knowledge on the role of ncrnas in immune responses at systems-level remains incomplete. earlier reports defined significant differences in the effects of dsrna (poly i:c) in innate immune responses as a function of 'length' ( . - . kb and . - . kb), depending on cell type (i.e. splenocytes, pbmcs, raw . and thp- ) (mian et al., ) . notably, it was reported that the 'shorter' dsrna caused higher immunomodulatory effects. poly i:c, a synthetic analogue of viral dsrna, has long been known as a strong inducer of innate immune responses by interacting with tlr in the endosome. following ligand recognition, specific ligand cascades involving interferon regulatory proteins and nuclear factor kappa b (nf-b) are activated to produce inflammatory mediators (démoulins et al., ) . similarly, signal-specific lncrnas have been reported in dendritic cells (dcs) stimulated with tlr agonists, % of which clustered with nf-b signalling components (guttman et al., ) . further approaches also identified upregulated responses in a long intergenic noncoding rna (lincrna) in response to tlr- , - , - and - , but not tlr- in macrophages (carpenter et al., ) . specifically, lincrna-cox was found to mediate both activation and repression of immune response genes dependent on interactions of lincrna-cox with heterogeneous nuclear ribonucleoprotein a/b and a /b . a further study determined that lincrna-cox plays a key role as a coactivator of nf-b for the transcription of late-primary inflammatory response genes in macrophages through modulation of epigenetic chromatin remodelling (hu et al., ) . due to the relevance of gut microbiota in immune system development and homeostasis, the possibility of modulating the gut microbiota through dietary exogenous ncrnas was reviewed. as described in section . , during the preparatory phase this novel topic was identified as relevant to this literature review. this additional section serves as a general introduction to emphasize the relevance of the possible modulation of the immune system by dietary exogenous ncrnas through the gut microbiota, and to identify gaps pointing to needs for future studies relevant to food/feed risk assessment of ncrnabased gm plants. specific details of the possible mechanisms of action, if any, are outside the scope of this review. due to the novelty of this topic and the lack of information on the scientific literature, only documents were reviewed for this section. the intestinal tract harbours a complex community (microbiota), which has an enormous impact not only on the nutritional, but also immune status of the host. a balanced gut microbiota composition confers benefits to the host, while microbial imbalances are associated with metainflammatory disorders. the dynamic processes initiated upon birth define the microbiota composition that co-evolves with the host and its environment. thus, colonization of the intestine in early life seems particularly important as it represents one of the major environmental stimuli for immune system maturation. particularly, breast feeding is crucial to influencing early intestinal colonization. as previously indicated, development of emerging 'next generation sequencing technologies' has allowed identification of a significant fraction of the circulating rna in human plasma that originated from exogenous bacterial and fungi species . this study, based on microarray profiling results, reported that plasma can contain ncrnas from common foods influencing the expression profile of a number of genes in the cells. however, several other studies suggest that the quantitative amount of ncrnas are irrelevant for influencing gene expression (see section . . . for details). though the interaction between humans and their environments, particularly microbes, raises the possibility of some sort of feedback signalling process, it is still not fully understood. the literature search showed how few publications address the role of ncrnas in shaping gut microbiota. emerging the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). evidences point out interactions between environmental factors and inter-species gene regulation facilitating host control of the gut microbiota choi et al., ) . however, the impact of exogenous plant ncrnas on gut microbiota modulation remains elusive. increasing evidence demonstrates that a significant number of ncrnas are found in the extracellular media, and it is apparent that mammalian cells release srna that might carry functions which transcend the confines of single cells (valadi et al., ) . few studies have identified changes in mirna expression in human stool. overall, they provide an inconsistent association between their level and dietary habits and lifestyle (tarallo et al., ; . in this context, the survival of exogenous (orally administered) plant mirnas in faeces from mice has been observed . although the amount of particular mirnas such as mir- that survived the gi tract reached a maximum of . %, this study does not support consistent survival rates for distinct ncrnas. a recent study identified mirnas secreted by intestinal epithelial cells in intestinal contents and supports their role in modulating gut microbiota composition . this study reports the potential of host mirnas to enter bacteria and co-localize with microbial nucleic acids. similarly, exogenous plant mirnas that were present in animal faeces were indicated as being primarily acquired orally . recently, bacterial rnas comparable in size to mirnas have been identified that could serve as signalling molecules mediating bacteria-to-human interaction (choi et al., ) . these data were obtained from outer membrane vesicles produced by periodontopathogens and were able to exert 'slight' immunosuppressive effects on t cells cytokine production (il- , il- and il- ). mirna expression levels and amount in stool appear to be modulated by diet (micro-and macronutrients, phytochemicals) without excluding the possibility that the results were affected by other lifestyle factors; however, the influence of gm plant exogenous ncrnas has not yet been exhaustively studied in animals or humans. distinct innate immune mediators participating in the recognition and discrimination between the distinct nucleic acids have been identified; thus, additional definition of the dynamic interplay between the different 'sensors' (i.e. tlrs, rig-i/mda , the interferon response effectors protein kinase r and rnase l) in response to ncrnas have to be clarified. the chemical modifications observed in plant ncrnas highlight the need to address the detection and measurement of exogenous plant ncrnas in body fluids, as well as the sensitivity and specificity of these rnas to cellular and tissue targets. although many reports focusing on synthetic ss-and dsrna mimics can be found in the literature, scientifically assessed direct hazardous impacts of food and feed on fauna and flora are scarce and conflicting. notably, there is a need to evaluate the effects of exogenous plant ncrnas at the system biology level and in human trials. current knowledge on the immune functions of exogenous ncrnas is comparatively lacking. most studies use cell models based on isolated cells that could not mimick the whole set of players that determine immune response(s), which are necessary for mechanistic studies, but need approach cell-to-cell communication in target tissues. additionally, these studies should focus on the relationship of immune responses to production of ifns since tlrs signalling converge on the production of this immune regulatory factor. to better understand the impact of exogenous ncrnas in host microbiota composition and diversity, further studies are needed to establish the microbial targets and their effect on physiological conditions. understanding the basic biological aspects of plant-derived exogenous ncrna (including their half-life and stability), gathering information on human and animal exposure by diet to these molecules, their subsequent bioavailability and possibility to exert local or systemic effects, and reviewing the experience from the development of rna-based therapeutics are relevant aspects for the risk assessment of ncrnabased gm food and feed. the literature includes very few studies evaluating the half-life of plant-derived ncrnas. while no specific studies on plant mirnas and lncrnas half-life were found in this review, the studies on plant circrnas half-life suggest that they exhibit considerable stability as compared to linear rnas (section . . . ). in mammals, the few reports related to ncrnas half-life suggest that mirnas and circrnas are generally much more stable than mrna, while lncrnas have a half-life like that of mrna. however, when assessing the stability of plant ncrnas outside the plant (section . . . .), compelling evidence exists that plant mirnas are highly stable under different conditions including food storage, processing, cooking, or simulated digestion. moreover, they seem to survive after long incubation in serum, or are detected in the gastric content of mice, suggesting that plant mirnas are more resistant to degradation than synthetic or animal mirnas. this high resistance has been attributed to the unique characteristics of plant mirnas (and sirnas), in contrast to mirnas from mammalian and other organisms; the '-omethylation at their ' end which confers plant mirnas more resistance to degradation (section . . . ). efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). the experience from development of ncrna therapeutics (section . . ) shows that unmodified nucleic acids (i.e. rnas) exhibit very low stability in biological media and are subjected to rapid nuclease mediated degradation. indeed, a plethora of rnases are encoded within most genomes, often with overlapping activities, making redundancy a general feature of rna degradation systems. consequently, different chemical modifications or conjugations have been introduced to potential rna-based therapeutics (section . . . ) to improve their rna-binding affinity, their in vivo nuclease stability and their pharmacokinetic and pharmacodynamic properties. the available literature also suggests that naked or unmodified rnas parenterally administered (e.g. iv) are rapidly cleared from circulation and their presence in the kidney and urine has been reported immediately after administration (section . . ). similarly, following oral administration naked or unmodified exogenous rnas are rapidly degraded when exposed to the gi harsh conditions, and different delivery vehicles for exogenous rnas have been developed. in general, and in contrast to chemically modified or formulated rnas, no major biological effects have been observed for naked rnas, which has probably limited further evaluation in humans (section . . . ). although certain disease conditions may influence the pharmacokinetics of exogenous rnas, there are no studies that specifically evaluate this for naked unmodified exogenous ncrnas. to exert a biological effect, an exogenous ncrna first needs to reach the intended target tissue in sufficient quantity levels, this implying the necessity to overcome many biological barriers (section . . ). considering the oral intake as the main possible entry point of exogenous plant ncrnas into both animals and humans, the first major barrier is the mammalian gi tract, which encompasses a group of extracellular and cellular barriers. extracellular barriers include the presence of different enzymes (i.e. nucleases), the harsh environment and a net negative charged mucous layer. the cellular barriers include a three-layer (epithelial cells or enterocytes, the lamina propria and the muscularis mucosa) barrier known as the intestinal mucosa. possible ncrnas passage between cells is limited by the presence of tight junctions and the pore size in the human intestine would prevent the passage of all but mirnas, which are the smallest ncrnas. although crossing the cells by transcytosis may be possible, this mechanism implies facing new intracellular barriers, such as nucleases, recycling of ncrnas back to the lumen and nuclear uptake. however, m-cells, present in the gut epithelium interspersed between enterocytes, present certain characteristics that would make them more amenable for rna uptake. a second barrier is represented by a set of obstacles during the passage from the intestine to the target tissue, which encompasses plasma and tissue nucleases. once in the circulatory system, exogenous ncrnas are subjected to distribution and elimination. for example, small rnas would be rapidly cleared by the kidney due to their small size. furthermore, ncrnas need to escape the reticuloendothelial system, the function of which is to clear foreign pathogens. finally, they would have to cross the vascular endothelial barrier to reach the target tissue, facing hurdles like those in the enterocytes. packaging or incorporation of ncrnas into extracellular transport systems (i.e. exosomes/microvesicles or inclusion in ribonucleoprotein complexes) would confer certain resistance to nucleases and other barriers. ncrnas must enter the target cells to exert their functions. the molecular mechanisms of exogenous ncrna cellular uptake have been inferred from studies performed when developing strategies for nucleic acids delivery as therapeutics and is also largely derived from invertebrates, with little data reported in mammals. descriptions exist of receptor-mediated uptake of oligonucleotides through the sidt and sidt proteins, although this is now in doubt since they have been described as cholesterol transporters. due to the charged nature of rnas, cellular uptake could be achieved by endocytosis after which they would enter the system of intracellular trafficking through multiple membrane-bound compartments. ncrnas must exit these compartments to reach their functional sublocation within the efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). cell, either the cytosol or the nucleus, while simultaneously escaping degradation in the lysosomes. specialized barriers such as the blood-brain barrier or the placental barrier represent additional obstacles to overcome. while rna content in plant-derived foods varies (≈ mg/g of tissue), it could be estimated that when consuming a daily dose of total fruit and vegetables of ≈ g/day, humans theoretically ingest mg of total rnas (dietary intake of plant rna may typically range from . - g/person/day) (section . . . ). humans following special diets, such as vegetarians and vegans, have higher intake of plant origin rnas, but estimated exposure to such rnas implies only a -fold increase on average (section . . . ). it has been estimated that small rnas make up far less than % of total rna in plants, suggesting a very low exposure to plant ncrnas under normal conditions. whether sufficient levels of exogenous plant ncrnas can be consumed in the diet to exert a biological effect is also another aspect that needs to be evaluated. some examples in the literature suggest that for certain plant mirnas (i.e. mir- a) a person would need to consume kg of cantaloupe to reach the copies of mirna per cell possibly determining an effect. these aspects should be evaluated for each plant ncrna (on a case basis), considering that some plant-derived mirnas have shown unexpectedly high resistance to degradation and greater bioavailability. the available literature indicates a widespread presence of exogenous rnas (including plant derived ncrnas, i.e. small rnas) in the biological fluids of humans and animals (section . . ). whether these rnas are derived from dietary intake is unclear. their generally low abundance and lack of enrichment in tissues mostly exposed to dietary changes (i.e. liver) suggest that some of may have be originated as technical artefacts or through contamination. very few studies address biological effects of dietary exogenous ncrnas in the gi tract and its annex glands (e.g. liver) (section . can directly target mammalian genes through rnai effects, but still clarification is needed if exogenous plant ncrnas can overcome all the biological barriers to reach and interact with their possible targets. exogenous plant-derived ncrnas have been found in exosomes or macrovesicles. how they reach these types of structures in biological fluids is unknown. in summary, supporting and contradicting evidence concerning the existence of systemic effects of dietary plant-derived exogenous ncrnas is heavily debated. important aspects such as the precise mechanism/s of transport of plant ncrnas from food into the systemic circulation, the amount of exogenous ncrnas reaching tissues or the molecular mechanisms of cellular uptake need to be determined. while several in vitro studies cover the participation of exogenous ncrnas in the dynamic network to modulate innate immunity responses, few tackle the immunomodulatory effects of plant ncrnas in adaptive immune response (section . ). none of these studies have considered the potential impact of exogenous dietary plant-derived ncrnas at the systemic level. since the first publication suggesting a possible cross-kingdom effect by dietary ncrna (through rice mir- a) the knowledge on exogenous ncrnas, and particularly plant-derived exogenous ncrnas, on efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). their fate when ingested has increased. however, the knowledge useful to support the assessment of dietary ncrna such as ncrna-based gm food and feed still faces several gaps. an important aspect needing further investigation is the stability of plant ncrnas. the meagre available literature contains a very few studies of the half-life of plant ncrnas (i.e. mirnas, lncrnas and circrnas) both inside the plant cell and outside the plant. specifically, the stability of circrnas should be assessed due to their apparently high stability. moreover, very few reports focus on assessment of plant ncrnas stability in mammalian systems, when ingested. understanding the molecular basis that confers plant ncrnas stability is of great importance and needs to be further evaluated. for instance, plant mirnas and sirnas carry modifications that do not occur in mammals. these modifications, such as the presence of a methyl group located on the ' nucleotide ribose, could hypothetically have an impact on the plant mirnas stability in mammalian systems/cells due to either the lack of the appropriate enzymes for recognition and degradation of plant mirnas by mammalian cells, or the increased stability to degradation by mammalian rnases. the circularized structure of plant circrnas would hypothetically confer them increased stability to mammalian rnases. if this is the case, plant mirnas and circrnas could exhibit a much longer half-life in mammalian systems than expected, especially when compared to mammalian mirna or circrnas. this would increase their chances of reaching the appropriate target tissue and encountering suitable target molecules within the cells. the half-life of plant ncrnas within mammalian cells is also an important aspect to consider. experiments are needed with labelled plant ncrnas, first in transfected mammalian cells (in vitro studies) and then orally administered to experimental animals (in vivo studies). radioactive probes or molecules of very low molecular weight, such as biotin or fluorescein, should be used, in order to prevent distorting the actual size of the studied plant ncrna; this is important when studying mirnas stability due to their small size. another aspect possibly relevant for risk assessment is to understand whether and how ingested gm plant-derived ncrnas can affect the expression levels of other ncrnas and other rnas, due to putative compensatory circuits and codifying rnas. whole transcriptome sequencing, comparing the rna levels of unmodified (wild type) to those of modified (transgenic) plants coul be useful to this aim. although available information suggests that many biological barriers exist in mammalian system to ingested plant exogenous ncrnas preventing these to exert a possible local or systemic effect, several gaps are still present. putative pathways of dietary exogenous ncrnas after ingestion and the many transporters potentially involved (i.e, in cellular uptake, or barrier passage transport pathways) in the transit from the gut to the target tissues should be characterized using standard molecular biology gainof-function and loss-of-function approaches. also, the many routes of mammalian intracellular trafficking to the specific intracellular target should be studied. to this aim, ncrnas isolated from human diets should be used preferentially, since most of the incomplete current knowledge comes from the rna-therapeutics field or is derived from the invertebrate world. which molecular mechanisms support the possible passage through specialized barriers (i.e. blood-brain barrier or the placental barrier), is another critical aspect requiring further studies. while there are in vitro studies describing the interaction between plant mirnas and mammalian mirna silencing complexes possibly leading to target repression, these findings need to be experimentally validated in vivo. therefore, more studies are needed to understand the possibility of processing plant ncrnas in mammalian cells. it is also unknown if ingested plant mirnas reaches or needs to reach a necessary level to exert a biological effect. for instance, studies in mammals suggest that the threshold for target gene regulation is > copies per cell for a certain study; however, another study suggests around to . copies of mammalian mirna per cell. clearly, a minimum amount of small rnas efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). would be required to achieve biologically relevant effects on gene expression. on a case by case basis it would be useful to evaluate the amount of consumption of a given ncrna in a normal diet. it is also relevant to study if exposure to these plant ncrnas could change under certain pathological conditions (i.e. compromised intestinal permeability or renal function) or dietary patterns (i.e. vegetarian or vegan diets). although tissue accumulation of dietary plant exogenous ncrnas has not been reported to date, it seems clear that plant mirnas and other exogenous rnas are present in biological fluids from humans and animals. the biological significance of their presence is still unknown and needs to be addressed. methods that do not require amplification for detection should be used. more quantitative approaches to determine the levels of plant-derived exogenous ncrnas should be determined both in biological fluids and tissues to understand the magnitude of presence. for example, this could be partially obtained from studies reporting the presence of exogenous rnas in biological fluids using public small rna-seq databases. in the case of plant-derived mirnas, the use of sodium periodate oxidation of samples could be a good start to verifying the presence of genuine plant-derived mirnas from diet in human and animal biological fluids. the immunomodulatory effects of exogenous rnas have been widely described in the literature. however, there is no in vivo information regarding the immunomodulatory effects of dietary exogenous ncrnas. these types of studies should be performed both in vitro and in vivo to determine if the different types of ncrnas consumed in the diet can affect the immune system. in the case of target gene repression by plant ncrnas investigations in specific animal models could be considered. expression levels of other ncrnas and rnas, which may be modified due to putative compensatory circuits could be evaluated in these models, as well as information on the stability and half-life of the specific ncrna in specific cells and systemically following ingestion. genome-wide high-resolution mapping of exosome substrates reveals hidden features in the arabidopsis transcriptome genome-wide identification of circular rnas in arabidopsis thaliana a lariat-derived circular rna is required for plant development in arabidopsis pattern formation via small rna mobility small rnas are on the move literature review of baseline information on rnai to support the environmental risk assessment of rnai-based gm plants identification of circular rnas from the parental genes involved in multiple aspects of cellular metabolism in barley a group i plant intron accumulates as circular rna forms with extensive ' deletions in vivo toward a consensus on the binding specificity and promiscuity of prc for rna a stepwise pathway for biogenesis of -nt secundary sirnas and spreading of dna methylation site-specific phosphorylation profiling of arabidopsis proteins by mass spectrometry and peptide chip analysis hierarchical action and inhibition of plant dicer-like proteins in antiviral defense characterization of stress-responsive lncrnas in arabidopsis thaliana by integrating expression, epigenetic and structural features identification and characterization of human testis derived circular rnas and their existence in seminal plasma the rna-binding proteins hyl and se promote accurate in vitro processing of pri-mirna by dcl genome-wide discovery of circular rnas in the leaf and seedling tissues of arabidopsis thaliana intra-and intercellular rna interference in arabidopsis thaliana requires components of the microrna and heterochromatic silencing pathways dicer-like is required for rna interference and produces the -nucleotide small interfering rna component of the plant cell-to-cell silencing signal plant mobile small rnas the arabidopsis thaliana doublestranded rna binding protein drb directs guide strand selection from microrna duplexes naturally occurring variations in sequence length creates microrna isoforms that differ in argonaute effector complex specificity the xist lncrna exploits three-dimensional genome architecture to spread across the x chromosome identification of nuclear dicing bodies containing proteins for microrna biogenesis in living arabidopsis plants phased, secondary, small interfering rnas in posttranscriptional regulatory networks antisense rna polymerase ii divergent transcripts are p-tefb dependent and substrates for the rna exosome long noncoding rnas in cell-fate programming and reprogramming target mimicry provides a new mechanism for regulation of microrna activity location of a possible mirna processing site in smd /smb nuclear bodies in arabidopsis partially redundant functions of arabidopsis dicer-like enzymes and a role for dcl in producing trans-acting sirnas short integuments /suspensor /carpel factory, a dicer homolog, is a maternal effect gene required for embryo development in arabidopsis oncogenic role of fusion-circrnas derived from cancer-associated chromosomal translocations topological organization of multichromosomal regions by the long intergenic noncoding rna firre natural rna circles function as efficient microrna sponges vernalization-mediated epigenetic silencing by a long intronic noncoding rna flowering locus c's lessons: conserved chromatin switches underpinning developmental timing and adaptation transitivity-dependent andindependent cell-to-cell movement of rna silencing specific interactions between dicer-like proteins and hyl /drb-family dsrna-binding proteins in arabidopsis thaliana phosphate utilization efficiency correlates with expression of low-affinity phosphate transporters and noncoding rna, ips , in barley gene silencing by micrornas: contributions of translational repression and mrna decay cyclophilin facilitates hsp -mediated risc assembly in plants in vitro assembly of plant rna-induced silencing complexes facilitated by molecular chaperone hsp a rice cis-natural antisense rna acts as a translational enhancer for its cognate mrna and contributes to phosphate homeostasis and plant fitness the nuclear pore protein attpr is required for rna homeostasis, flowering time, and auxin signaling dicer- and r d -l catalyze microrna maturation in drosophila molecular insights into mirna processing by arabidopsis thaliana serrate covalent circularization of exogenous rna during incubation with a wheat embryo cell extract small rnas as guardians of the genome fast-forward genetics identifies plant cpl phosphatases as regulators of mirna processing factor hyl the evolution and diversification of dicers in plants passenger-strand cleavage facilitates assembly of sirna into ago -containing rnai enzyme complexes circular single-stranded rna replicon in saccharomyces cerevisiae rna-directed dna methylation: an epigenetic pathway of increasing complexity intercellular and systemic movement of rna silencing signals circular rnas are a large class of animal rnas with regulatory potency sorting of small rnas into arabidopsis argonaute complexes is directed by the ' terminal nucleotide morc family atpases required for heterochromatin condensation and gene silencing small silencing rnas in plants are mobile and direct epigenetic modification in recipient cells specificity of argonaute -mir interaction and dual functionality in tas trans-acting sirna formation evolution of animal and plant dicers: early parallel duplications and recurrent adaptation of antiviral rna binding in plants literature review of baseline information to support the risk assessment of rnai-based gm plants systemic acquired silencing: transgene-specific post-transcriptional silencing is transmitted by grafting from silenced stocks to non-silenced scions microrna is a long-distance signal for the regulation of plant phosphate homeostasis evidence for nuclear processing of plant micro rna and short interfering rna precursors nuclear processing and export of micrornas in arabidopsis carpel factory, a dicer homolog, and hen , a novel protein, act in microrna metabolism in arabidopsis thaliana rde- preferentially binds long dsrna and its dimerization is necessary for cleavage of dsrna to sirna rna exosome-regulated long non-coding rna transcription controls super-enhancer activity prediction of trans-antisense transcripts in arabidopsis thaliana genome-wide identification of long noncoding natural antisense transcripts and their responses to light in arabidopsis identification and characterization of circrnas in pyrus betulifolia bunge under drought stress analysis of noncoding transcriptome in rice and maize uncovers roles of conserved lncrnas associated with agriculture traits a long noncoding rna maintains active chromatin to coordinate homeotic gene expression arabidopsis noncoding rna mediates control of photomorphogenesis by red light identification of circular rnas and their targets in leaves of triticum aestivum l. under dehydration stress importin is a gene silencing factor that targets argonaute proteins to distinct mrnas noncoding transcription by rna polymerase pol ivb/pol v mediates transcriptional silencing of overlapping and adjacent genes expression of arabidopsis mirna genes genetic and functional diversification of small rna pathways in plants identification and characterization of wheat long non-protein coding rnas responsive to powdery mildew infection and heat stress by using microarray analysis and sbs sequencing bidirectional promoters generate pervasive transcription in yeast functions of long intergenic non-coding (linc) rnas in plants widespread noncoding circular rnas in plants a systemic small rna signaling system in plants wavy leaf , an ortholog of arabidopsis hen , regulates shoot development by maintaining microrna and trans-acting small interfering rna accumulation in rice vigs, higs and figs: small rna silencing in the interactions of viruses or filamentous organisms with their plant hosts control of coleopteran insect pests through rna interference the expanding world of small rnas in plants genome-wide analysis of long noncoding rna stability structural variations and stabilising modifications of synthetic sirnas in mammalian cells circular rnas are long-lived and display only minimal early alterations in response to a growth factor host-delivered rnai: an effective strategy to silence genes in plant parasitic nematodes analysis of microrna turnover in mammalian cells following dicer ablation polysome arrest restricts mirna turnover by preventing exosomal export of mirna in growth-retarded mammalian cells a versatile monosaccharide transporter that operates in the arbuscular mycorrhizal fungus glomus sp is crucial for the symbiotic relationship with plants the drosophila rna methyltransferase, dmhen , modifies germline pirnas and single-stranded sirnas in risc uridylation of mature mirnas and sirnas by the mut nucleotidyltransferase promotes their degradation in chlamydomonas circular rnas are abundant, conserved, and associated with alu repeats regulation of small rna stability: methylation and beyond methylation protects mirnas and sirnas from a ' end uridylation activity in arabidopsis assessing the survival of exogenous plant microrna in mice effective detection and quantification of dietetically absorbed plant micrornas in human plasma adenylation of plant mirnas covalent circularization of exogenous rna during incubation with a wheat embryo cell extract silencing a cotton bollworm p monooxygenase gene by plant-mediated rnai impairs larval tolerance of gossypol rna-specific ribonucleotidyl transferases oral delivery of sirna and antisense oligonucleotides toxicogenomics of non-viral drug delivery systems for rnai: potential impact on sirna-mediated gene silencing activity and specificity nonviral delivery of synthetic sirnas in vivo a theoretical basis for a biopharmaceutic drug classification: the correlation of in vitro drug product dissolution and in vivo bioavailability oral nucleic acid therapy using multicompartmental delivery systems the adherent gastrointestinal mucus gel layer: thickness and physical state in vivo polymeric nano-and microparticle technologies for oral gene delivery isolation and properties of an extracellular nucleases of serratia marcescens guidance for industry on waiver of in vivo bioavailability and bioequivalence studies for immediate-release solid oral dosage forms based on a biopharmaceutics classification system issues in oral nanoparticle drug carrier uptake and targeting in vitro and in vivo models for the study of oral delivery of nanoparticles alginate/poly-l-lysine microparticles for the intestinal delivery of antisense oligonucleotides multi-compartmental oral delivery systems for nucleic acid therapy in the gastrointestinal tract extracellular ribonucleases from bacillus subtilis. i. crystallization and specificity intestinal delivery of non-viral gene therapeutics: physiological barriers and preclinical models first-pass elimination. basic concepts and clinical consequences key issues in non-viral gene delivery polymeric nanoparticle drug delivery technologies for oral delivery applications effect of sodium caprate on the intestinal absorption of two modified antisense oligonucleotides in pigs drug delivery of sirna therapeutics: potentials and limits of nanosystems oral delivery of antisense oligonucleotides in man the human microbiome project consortium. structure, function and diversity of the healthy human microbiome milk-derived exosomes for oral delivery of paclitaxel delivery of sirna to the mouse brain by systemic injection of targeted exosomes orally delivered sirna targeting macrophage map k suppresses systemic inflammation oral administration of bovine milk derived extracellular vesicles attenuates arthritis in two mouse models oral nucleic acid therapy using multicompartmental delivery systems oral delivery of rnase p ribozymes by salmonella inhibits viral infection in mice protection and systemic translocation of sirna following oral administration of chitosan/sirna nanoparticles interleukin gene transfer prevents experimental colitis in rats inhibition of trkb limits development of the zebra finch song system commercial dairy cow milk micrornas resist digestion under simulated gastrointestinal tract conditions oral il- gene delivery in a microsphere-based formulation for local transfection and therapeutic efficacy in inflammatory bowel disease the role of nucleotides in human nutrition retroviral delivery of rna interference against marek's disease virus in vivo biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles systemic exosomal sirna delivery reduced alpha-synuclein aggregates in brains of transgenic mice pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver proteomics of extracellular vesicles: exosomes and ectosomes dietary yeast rna supplementation reduces mortality by aeromonas hydrophila in rohu (labeo rohita l.) juveniles effects of mannose density on in vitro and in vivo cellular uptake and rnai efficiency of polymeric nanoparticles peg-pla nanoparticles facilitate sirna knockdown in adult zebrafish heart exosome-mediated delivery of hydrophobically modified sirna for huntingtin mrna silencing oral delivery of small rna and dna colonic gene silencing using sirna-loaded calcium phosphate/plga nanoparticles ameliorates intestinal inflammation in vivo a randomized controlled trial of preoperative oral supplementation with a specialized diet in patients with gastrointestinal cancer a novel class of small rnas: trna-derived rna fragments (trfs) small non-coding rnas transfer through mammalian placenta and directly regulate fetal gene expression assessing the survival of exogenous plant microrna in mice effective detection and quantification of dietetically absorbed plant micrornas in human plasma human milk exosomes and their micrornas survive digestion in vitro and are taken up by human intestinal cells detection of plant mirnas abundance in human breast milk in silico identification of plant mirnas in mammalian breast milk exosomes--a small step forward? detection of dietetically absorbed maizederived micrornas in pigs exosomal micrornas in giant panda (ailuropoda melanoleuca) breast milk: potential maternal regulators for the development of newborn cubs unsuccessful detection of plant micrornas in beer, extra virgin olive oil and human plasma after an acute ingestion of extra virgin olive oil a novel chemopreventive strategy based on therapeutic micrornas produced in plants annotation of the goat genome using next generation sequencing of microrna expressed by the lactating mammary gland: comparison of three approaches expressional analysis of immune-related mirnas in breast milk identification of stable, high copy number, medium-sized rna degradation intermediates that accumulate in plants under non-stress conditions determination of the potential bioavailability of plant micrornas using a simulated human digestion process human breast milk mirna, maternal probiotic supplementation and atopic dermatitis in offspring the effect of dietary ginseng polysaccharide supplementation on the immune responses involved in porcine milk-derived esrnas immune modulatory function of abundant immunerelated micrornas in microvesicles from bovine colostrum uptake and function studies of maternal milk-derived micrornas mining of public sequencing databases supports a nondietary origin for putative foreign mirnas: underestimated effects of contamination in ngs literature review of information supporting food/feed risk assessment of rnai-based gm plants www.efsa.europa this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document alternative mirnas: human sequences misidentified as plant mirnas in plant studies and in human plasma transfer and functional consequences of dietary micrornas in vertebrates: concepts in search of corroboration: negative results challenge the hypothesis that dietary xenomirs cross the gut and regulate genes in ingesting vertebrates, but important questions persist real-time quantitative pcr and droplet digital pcr for plant mirnas in mammalian blood provide little evidence for general uptake of dietary mirnas: limited evidence for general uptake of dietary plant xenomirs the intestinal transport of bovine milk exosomes is mediated by endocytosis in human colon carcinoma caco- cells and rat small intestinal iec- cells the levels of human milk micrornas and their association with maternal weight characteristics detection of dietary plant-based small rnas in animals anomalous uptake and circulatory characteristics of the plant-based small rna mir the atypical genesis and bioavailability of the plant-based small rna mir : bulking up while breaking down bioavailability of transgenic micrornas in genetically modified plants exogenous plant mir a specifically targets mammalian ldlrap : evidence of cross-kingdom regulation by microrna analysis of plant-derived mirnas in animal small rna datasets immune-related micrornas are abundant in breast milk exosomes honeysuckle-encoded atypical microrna directly targets influenza a viruses one step forward, two steps back; xeno-micrornas reported in breast milk are artifacts small rnas from plants, bacteria and fungi within the order hypocreales are ubiquitous in human plasma dietary microrna database (dmd): an archive database and analytic tool for food-borne micrornas lack of detectable oral bioavailability of plant micrornas after feeding in mice survey of + data sets from human tissue and body fluid reveals xenomirs are likely artifacts plant mirnas found in human circulating system provide evidences of cross kingdom rnai detection of plant mirnas abundance in human breast milk in silico identification of plant mirnas in mammalian breast milk exosomes--a small step forward? optimization of enzymatic reaction conditions for generating representative pools of cdna from small rna computational characterization of exogenous micrornas that can be transferred into human circulation mining of public sequencing databases supports a nondietary origin for putative foreign mirnas: underestimated effects of contamination in ngs the complex exogenous rna spectra in human plasma: an interface with human gut biota? the spectrum of circulating rna: a window into systems toxicology detection of an abundant plant-based small rna in healthy consumers oral administration of bovine milk derived extracellular vesicles attenuates arthritis in two mouse models exosomes as a nanodelivery system: a key to the future of neuromedicine? endogenous microrna can be broadly exploited to regulate transgene expression according to tissue, lineage and differentiation state identification of dietetically absorbed rapeseed (brassica campestris l.) bee pollen micrornas in serum of mice lack of detectable oral bioavailability of plant micrornas after feeding in mice high-throughput sequencing of rna silencingassociated small rnas in olive (olea europaea l.) plant-derived phosphocholine facilitates cellular uptake of anti-pulmonary fibrotic hjt-srna-m autosomal recessive hypercholesterolemia caused by mutations in a putative ldl receptor adaptor protein exosomes as therapeutic drug carriers and delivery vehicles across biological membranes: current perspectives and future challenges extensive degradation and low bioavailability of orally consumed corn mirnas in mice an improved method to quantitate mature plant microrna in biological matrices using modified periodate treatment and inclusion of internal controls this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document cross talk between adipose tissue and placenta in obese and gestational diabetes mellitus pregnancies via exosomes grape exosome-like nanoparticles induce intestinal stem cells and protect mice from dss-induced colitis survey of + data sets from human tissue and body fluid reveals xenomirs are likely artifacts cross-kingdom regulation of putative mirnas derived from happy tree in cancer pathway: a systems biology approach small non-coding rnas transfer through mammalian placenta and directly regulate fetal gene expression assessing the survival of exogenous plant microrna in mice reply to dr. witwer's letter to the editor effective detection and quantification of dietetically absorbed plant micrornas in human plasma plant mirnas found in human circulating system provide evidences of cross kingdom rnai detection of dietetically absorbed maizederived micrornas in pigs negligible uptake and transfer of diet-derived pollen micrornas in adult honey bees the transport mechanism of extracellular vesicles at the blood-brain barrier unsuccessful detection of plant micrornas in beer, extra virgin olive oil and human plasma after an acute ingestion of extra virgin olive oil a novel chemopreventive strategy based on therapeutic micrornas produced in plants interspecies communication between plant and mouse gut host cells through edible plant derived exosome-like nanoparticles high-throughput assessment of microrna activity and function using microrna sensor and decoy libraries milk extracellular vesicles accelerate osteoblastogenesis but impair bone matrix formation corn rootwormactive rna dvsnf : repeat dose oral toxicology assessment in support of human and mammalian safety a -day oral toxicity evaluation of small interfering rnas and a long double-stranded rna targeting vacuolar atpase in mice citrus limon-derived nanovesicles inhibit cancer cell proliferation and suppress cml xenograft growth by inducing trail-mediated cell death this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document ineffective delivery of diet-derived micrornas to recipient animal organisms adipose-derived circulating mirnas regulate gene expression in other tissues uptake and function studies of maternal milk-derived micrornas contamination or artifacts may explain reports of plant mirnas in humans alternative mirnas: human sequences misidentified as plant mirnas in plant studies and in human plasma real-time quantitative pcr and droplet digital pcr for plant mirnas in mammalian blood provide little evidence for general uptake of dietary mirnas: limited evidence for general uptake of dietary plant xenomirs detection of an abundant plant-based small rna in healthy consumers anomalous uptake and circulatory characteristics of the plant-based small rna mir bioavailability of trasngenic micrornas in genetically modified plants exosome delivered anticancer drugs across the blood-brain barrier for brain cancer therapy in danio rerio molecular basis of mammalian transmissibility of avian h n influenza viruses and their pandemic potential exogenous plant mir a specifically targets mammalian ldlrap : evidence of cross-kingdom regulation by microrna honeysuckle-encoded atypical microrna directly targets influenza a viruses plant micrornas in larval food regulate honeybee caste development to evaluate oral toxicity of exogenous ncrnas in mice, petrick et al. administered for days a repeated oral dose of sirnas and dsrna and evaluated several parameters including body weight, food consumption, clinical observations, clinical chemistry, haematology, gross pathology, or histopathology endpoints it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). signs (mortality, abnormalities, and sign of pain and distress), body weight and food consumption, haematology, organ weight, or pathology results. the minor differences observed in certain parameters were limited to single intervals, were not dose-related, and were attributed to interanimal variability torula yeast rna (rna negative control) at mg/kg/day or a control vehicle were used. ten animals per sex and per group were used. no treatment-related effects were observed on clinical signs (mortality, or sign of pain and distress), body weight and food consumption, haematology, organ weight, or pathology results. minor changes in selected parameters in selected groups were observed, but these were attributed by the authors to normal variability and not treatment-related effects this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document ecological risk assessment for dvsnf rna: a plant-incorporated protectant with targeted activity against western corn rootworm literature review of baseline information on rnai to support the environmental risk assessment of rnai-based gm plants a comparative evaluation of the regulation of gm crops or products containing dsrna and suggested improvements to risk assessments endogenous small rnas in grain: semi-quantification and sequence homology to human and animal genes literature review of baseline information to support the risk assessment of rnai-based gm plants corn rootwormactive rna dvsnf : repeat dose oral toxicology assessment in support of human and mammalian safety a -day oral toxicity evaluation of small interfering rnas and a long double-stranded rna targeting vacuolar atpase in mice rnai technologies in agricultural biotechnology: the toxicology forum th annual summer meeting no impact of dvsnf rna on honey bee (apis mellifera l.) adults and larvae in dietary feeding tests microstructure and ultrastructure of highamylose rice resistant starch granules modified by antisense rna inhibition of starch branching enzyme a -day toxicology study of high-amylose transgenic rice grain in sprague-dawley rats a three generation reproduction study with sprague-dawley rats consuming high-amylose transgenic rice high-amylose rice improves indices of animal health in normal and diabetic rats studies performed with human cells were mainly carried out ex vivo using lymphocytes and, to a significant extent, macrophages. from the studies retrieved during the screening phase on immune cells in relation to exogenous ncrna %) from rat and ( . %) from mouse. and within the studies on immune cells other than lymphocytes (i.e. macrophages), the studies were allocated into ( . %) from human, ( . %) from rat, and ( . %) from mouse. all these data clearly show that studies retrieved in relation to ncrnas and www this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document immunological and metabolomic impacts of administration of cry ab protein and mon maize in mouse role of toll-like receptors in antisense and sirna recognition of double-stranded rna and activation of nf-kappab by toll-like receptor rna regulation of the immune system therapy of respiratory viral infections with intranasal sirnas cd is a coreceptor of toll-like receptors and a long noncoding rna mediates both activation and repression of immune response genes identification of dietetically absorbed rapeseed (brassica campestris l.) bee pollen micrornas in serum of mice identification of dietetically absorbed rapeseed (brassica campestris l.) bee pollen mirnas in serum of mice chemical modification patterns compatible with high potency dicer-substrate small interfering rnas is rna interference involved in intrinsic antiviral immunity in mammals? poly (i:c) induced immune response in lymphoid tissues involves three sequential waves of type i ifn expression structural basis for cytosolic double-stranded rna surveillance by human oligoadenylate synthetase structural mechanism of sensing long dsrna via a noncatalytic domain in human oligoadenylate synthetase rnas containing modified nucleotides fail to trigger rig-i conformational changes for innate immune signaling this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document identification of rna sequence motifs stimulating sequence-specific tlr -dependent immune responses dsrna with ′ overhangs contributes to endogenous and antiviral rna silencing pathways in plants a tim- oligonucleotide aptamer enhances t cell functions and potentiates tumor immunity in mice ab initio reconstruction of cell type-specific transcriptomes in mouse reveals the conserved multi-exonic structure of lincrnas ribosome profiling provides evidence that large noncoding rnas do not encode proteins chromatin signature reveals over a thousand highly conserved large noncoding rnas in mammals lack of interferon response in animals to naked sirnas species-specific recognition of single-stranded rna via toll-like receptor and small anti-viral compounds activate immune cells via the tlr myd -dependent signaling pathway sequencespecific potent induction of ifn-alpha by short interfering rna in plasmacytoid dendritic cells through tlr lincrna-cox promotes late inflammatory gene transcription in macrophages through modulating swi/snf-mediated chromatin remodeling microrna a marks regulatory t cells the role of alternative polyadenylation in the antiviral innate immune response the '-o-methylation status of a single guanosine controls transfer rna-mediated toll-like receptor activation or inhibition design of noninflammatory synthetic sirna mediating potent gene silencing in vivo sequence-dependent stimulation of the mammalian innate immune response by synthetic sirna immunostimulatory potential of silencing rnas can be mediated by a non-uridine-rich toll-like receptor motif suppression of rna recognition by toll-like receptors: the impact of nucleoside modification and the evolutionary origin of rna sequence-and target-independent angiogenesis suppression by sirna via tlr microrna as a new immune-regulatory agent in breast milk this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document the effect of multigenerational diet containing genetically modified triticale on immune system in mice cross-kingdom regulation of putative mirnas derived from happy tree in cancer pathway: a systems biology approach double-stranded rna-mediated tlr activation is enhanced by cd molecular basis for the immunostimulatory activity of guanine nucleoside analogs: activation of toll-like receptor the tlr signaling complex forms by cooperative receptor dimerization mir- a is an intrinsic modulator of t cell sensitivity and selection assessing the survival of exogenous plant microrna in mice structural basis of toll-like receptor signaling with double-stranded rna the host shapes the gut microbiota via fecal microrna in silico identification of plant mirnas in mammalian breast milk exosomes--a small step forward? exosomal micrornas in giant panda (ailuropoda melanoleuca) breast milk: potential maternal regulators for the development of newborn cubs microrna- c promotes natural killer cell cytotoxicity via up-regulating the expression level of nkg d neonatal immune activation during early and late postnatal brain development differently influences depressionrelated behaviors in adolescent and adult c bl/ mice small self-rna generated by rnase l amplifies antiviral innate immunity a structural basis for discriminating between self and nonself double-stranded rnas in mammalian cells cancer immunotherapy comes of age length of dsrna (poly i:c) drives distinct innate immune responses, depending on the cell type oligonucleotide-based pharmaceuticals: non-clinical and clinical safety signals and non-clinical testing strategies cytosolic viral sensor rig-i is a '-triphosphate-dependent translocase on double-stranded rna aptamers for cd antigens: from cell profiling to activity modulation cd aptamers as powerful immune response modulators influences of diet and the gut microbiome on epigenetic modulation in cancer and other diseases this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document aptamer-targeted attenuation of il- signaling in cd + t cells enhances antitumor immunity catalog of differentially expressed long non-coding rna following activation of human and mouse innate immune response. front immunol , long noncoding rna in hematopoiesis and immunity the effect of poly-l-lysine on the uptake of reovirus doublestranded rna in macrophages in vitro characterization of the mammalian rna exonuclease /nef-sp as a testis-specific nuclear ' → ' exoribonuclease microrna regulation of lymphocyte tolerance and autoimmunity advances in rna sensing by the immune system: separation of sirna unwanted effects from rna interference activation of the interferon system by short-interfering rnas type i interferons function as autocrine and paracrine factors to induce autotaxin in response to tlr activation antisense oligonucleotides containing locked nucleic acid improve potency but cause significant hepatotoxicity in animals microrna expression in relation to different dietary habits: a comparison in stool and plasma samples interplay between dengue virus and toll-like receptors, rig-i/mda and micrornas: implications for pathogenesis exosome-mediated transfer of mrnas and micrornas is a novel mechanism of genetic exchange between cells dissolution media simulating the intralumenal composition of the small intestine: physiological issues and practical aspects lps injection reprograms the expression and the ' utr of a cap gene by alternative polyadenylation and the formation of a gait element in ciona intestinalis the complex exogenous rna spectra in human plasma: an interface with human gut biota? induced mir- a expression represses mtor cooperatively with mir- to promote regulatory t-cell differentiation evolutionary dynamics and tissue specificity of human long noncoding rnas in six mammals toll-like receptor (tlr) immune modulation by unformulated small interfering rna or dna and the role of cd (in tlrmediated effects) homology modeling of human tolllike receptors tlr , , and ligand-binding domains modulation of let- mirnas controls the differentiation of effector cd t cells this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document microrna regulation of ifn-beta protein expression: rapid and sensitive modulation of the innate immune response . '-utr and '-utr regulation of micb expression in human cancer cells by novel micrornas microrna- is downregulated by histone deacetylase inhibitors and confers resistance to natural killer cytotoxicity in hepatocellular carcinoma cells ncrna-and pc methylation-dependent gene relocation between nuclear structures mediates gene activation programs plumbing the sources of endogenous mhc class i peptide ligands infalpha- b inhibitory effects on cd (+)cd (+)foxp (+) regulatory t cells in the tumor microenvironment of c bl/ j mice with melanoma xenografts stress-responsive regulation of long noncoding rnas' polyadenylation in oryza sativa exogenous plant mir a specifically targets mammalian ldlrap : evidence of cross-kingdom regulation by microrna antiviral activity of human oligoadenylate synthetases-like (oasl) is mediated by enhancing retinoic acid-inducible gene i (rig-i) signaling lipid-mediated delivery of rna is more efficient than delivery of dna in non-dividing cells ribose '-omethylation provides a molecular signature for the distinction of self and non-self mrna dependent on the rna sensor mda the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). (zhou et al., ) . twenty female and ten male rats per group in the first generation (f ) were fed the different diets. their pups, weanling/female/group and weanling/male/group were randomly selected as an f generation. the f generation was acquired by using the same procedure described above. at weaning, f generation rats were chosen, and rats/sex/group provided the corresponding diets and observed for weeks. no major differences in animal survival, health status, behaviour, body weight, food consumption, or reproductive capacity were observed with the different diets. some statistically significant differences were observed in animals given the transgenic rice diets compared to those receiving the standard diet for certain clinical chemistry parameters, in certain generations (alt, alkp, ldh, cholesterol, hdlc, ldlc). these changes were not considered adverse or biologically significant (zhou et al., ) . moreover, no evidence of altered incidence or altered severity of background changes was observed in any organ or tissues of the rats fed the three diets (zhou et al., ) . although minor changes in the mean relative weight of epididimides in male f adults was observed in rats fed the rice diets compared to the control group, no difference was observed between the transgenic rice fed rats and their near-isogenic line controls, suggesting overall that the transgenic line was unlikely to cause any risk to rat health in reproduction or development, even when consumed for up to three generations (zhou et al., ) . however, these studies did not evaluate any aspect related to rnai.heinemann et al. compared the history of risk assessment of gmos producing dsrnas in australia, new zealand and brazil, with a focus on regulatory context (heinemann et al., ) . the authors suggested some processes to properly assess the safety of dsrna-producing gm plants before their release or marketing. these include i) bioinformatics analysis to identify any likely, unintended targets of the dsrna in human and animals; ii) experimental procedures that would identify all new intended and unintended dsrna molecules in the gm product; iii) testing animal and human cells in tissue cultures for a response to intended and unintended effects of dsrnas from the product; iv) long-term testing on animals; and possibly v) clinical trials on human volunteers (heinemann et al., ) . in response to this article, the regulatory agency food standards australia new zealand (fsanz) published a document addressing regulation of gm crops and foods developed using gene silencing (http://www.foodstandards.gov.au/consumer/gmfood/pages/response-to-heinemann-et-al-on-theregulation-of-gm-crops-and-foods-developed-using-gene-silencing.aspx). this document criticised some of the comments of the above risk assessment review. some key points included the lack of weight of scientific evidence (published up to ) to support the view that small dsrnas in foods are likely to have adverse consequences in humans; the lack of a scientific basis for suggesting that small dsrnas present in gm foods have different properties than naturally-occurring ones; or the adequate acknowledge of many barriers (in the uptake, distribution and targeting) during oral development of small dsrna therapies, among others. the overall suggestion was that there was no need to consider additional studies as proposed by heinemann et al. ( ) .commentary documents reviewing scientific meetings in the context of rnai technologies in agricultural biotechnology discuss some of the aspects reviewed in this document .additional relevant documents of baseline information to support the risk assessment and environmental risk assessment of rnai-based gm plants can be obtained from efsa external scientific reports the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s).