key: cord-009743-2rntyccn
authors: Wang, Ke‐Ming; Kumar, Senthil; Cheng, Yi‐Sheng; Venkatagiri, Shripathi; Yang, Ai‐Hwa; Yeh, Kai‐Wun
title: Characterization of inhibitory mechanism and antifungal activity between group‐1 and group‐2 phytocystatins from taro (Colocasia esculenta)
date: 2008-09-10
journal: FEBS J
DOI: 10.1111/j.1742-4658.2008.06631.x
sha: 
doc_id: 9743
cord_uid: 2rntyccn

Tarocystatin from Colocasia esculenta, a group‐2 phytocystatin, is a defense protein against phytopathogenic nematodes and fungi. It is composed of a highly conserved N‐terminal region, which is homological to group‐1 cystatin, and a repetitive peptide at the C‐terminus. The purified recombinant proteins of tarocystatin, such as full‐length (FL), N‐terminus (Nt) and C‐terminus (Ct) peptides, were produced and their inhibitory activities against papain as well as their antifungal effects were investigated. Kinetic analysis revealed that FL peptide exhibited mixed type inhibition (K (ia) = 0.098 μm and K (ib) = 0.252 μm) and Nt peptide showed competitive inhibition (K (i) = 0.057 μm), whereas Ct peptide possessed weak papain activation properties. A shift in the inhibitory pattern from competitive inhibition of Nt peptide alone to mixed type inhibition of FL peptide implied that the Ct peptide has an regulatory effect on the function of FL peptide. Based on the inhibitory kinetics of FL (group‐2) and Nt (group‐1) peptides on papain activity, an inhibitory mechanism of group‐2 phytocystatins and a regulatory mechanism of extended Ct peptide have each been proposed. By contrast, the antifungal activity of Nt peptide appeared to be greater than that of FL peptide, and the Ct peptide showed no effect on antifungal activity, indicating that the antifungal effect is not related to proteinase inhibitory activity. The results are valid for most phytocystatins with respect to the inhibitory mechanism against cysteine proteinase.

Tarocystatin from Colocasia esculenta, a group-2 phytocystatin, is a defense protein against phytopathogenic nematodes and fungi. It is composed of a highly conserved N-terminal region, which is homological to group-1 cystatin, and a repetitive peptide at the C-terminus. The purified recombinant proteins of tarocystatin, such as full-length (FL), N-terminus (Nt) and C-terminus (Ct) peptides, were produced and their inhibitory activities against papain as well as their antifungal effects were investigated. Kinetic analysis revealed that FL peptide exhibited mixed type inhibition (K ia = 0.098 lm and K ib = 0.252 lm) and Nt peptide showed competitive inhibition (K i = 0.057 lm), whereas Ct peptide possessed weak papain activation properties. A shift in the inhibitory pattern from competitive inhibition of Nt peptide alone to mixed type inhibition of FL peptide implied that the Ct peptide has an regulatory effect on the function of FL peptide. Based on the inhibitory kinetics of FL (group-2) and Nt (group-1) peptides on papain activity, an inhibitory mechanism of group-2 phytocystatins and a regulatory mechanism of extended Ct peptide have each been proposed. By contrast, the antifungal activity of Nt peptide appeared to be greater than that of FL peptide, and the Ct peptide showed no effect on antifungal activity, indicating that the antifungal effect is not related to proteinase inhibitory activity. The results are valid for most phytocystatins with respect to the inhibitory mechanism against cysteine proteinase. they are usually 12-16 kDa in size and show high homology with chicken egg white cystatin [4] . The group-2 phytocystatins are approximately or greater than 23 kDa, such as those found in cabbage [5] , soybean [6] , taro [7] , sesame [8] and strawberry [9] . They have a highly conserved N-terminal region, which is similar to that in group-1, and are tailed by a repetitive peptide at the C-terminus, in which variation is possibly caused by gene duplication [10] . The third group of phytocystatins, group-3, is found in potato [11] and tomato [12] , and includes an 80 kDa multi-cystatin with eight cystatin domains. Phytocystatins show variable expression patterns during plant development and defense responses to biotic and abiotic stresses [13] [14] [15] . Although at least two functions have been assigned to phytocystatins, such as regulation of protein turnover and protection of plants against insects and pathogens [16] , their physiological functions remain obscure.

The taro, Colocasia esculenta, is an important staple food of Taiwan aborigines, and is widely cultivated in local mountainous farms. This crop, especially Kaohsiung No. 1, is popular for its high productivity and lower susceptibility to pathogens. It might be expected that such taro corms display the characteristic mechanisms regulating protein turnover, as well as defense barriers towards pathogens. In a preliminary survey of proteinase inhibitors from taro tuber, copious amount of a cysteine proteinase inhibitor were discovered [7] . Recently, we isolated a group-2 phytocystatin from taro corms, named CeCPI, and demonstrated its anti-papain activity as well as anti-fungal activity [7] . In the alignment data, we also found that the group-2 phytocystatin is like a group-1 phytocystatin with the addition of a C-terminal extension. Moreover, the C-terminal region of the group-2 phytocystatin shares a high consensus sequence among the discovered species [7] . The C-terminal part is probably responsible for regulating inhibitory activity and target specificity. To obtain a better understanding of the structure and biochemical function of tarocystatin CeCPI, we amplified separately the intact full length (FL), N-terminal region (Nt) and C-terminal region (Ct) peptides by PCR and studied their relationship by in-gel anti-papain activity, inhibitory patterns and anti-fungal activity. Based on a comparative study of group-1 (Nt peptide) and group-2 (FL peptide), we discuss the inhibitory mechanism of group-2 phytocystatins and their evolutionary significance. In addition, both the inhibitory characteristics of the 'noncanonical' binding mode of group-2 phytocystatins towards papain and the 'canonical' binding mode of group-1 phytocystatins are addressed.

Purification of recombinant proteins from Escherichia coli and in-gel inhibitory activity assay

The FL peptide comprises of 205 amino acids, including 98 amino acids of Nt peptide and 107 amino acids of Ct peptide. Expressed recombinant FL, Nt and Ct peptides were further purified from the E. coli and analyzed by 12.5% SDS ⁄ PAGE. Purified proteins of both FL and Nt peptides showed two bands, each with the lower band corresponding to a 27 kDa glutathione S-transferase (GST) protein, with the upper band corresponding to 56 kDa for GST-FL and 40 kDa for GST-Nt peptide fusion proteins (Fig. 1A) . The Ct peptide showed only one band corresponding to 42 kDa (GST-Ct). The free recombinant proteins of the three peptides ( Fig. 1B) were obtained by digesting off GST peptide and performing chromatography [1] for further biochemical analysis. The inhibitory activity of recombinant proteins was assessed by an in-gel activity assay and can be visualized by the clear zone of hydrolysis (Fig. 1C ). By contrast, increasing the concentration of recombinant Ct peptide acting on papain confirmed that the Ct peptide enhanced its capacity (Fig. 1D ).

A previous study showed that tarocystatin (i.e. FL peptide) has effective activity on hyphal growth inhibition against several phytopathogenic fungi [7] . In an attempt to compare the antifungal effect of different peptides of tarocystatin, a bioassay on mycelial growth of Sclerotium rolfsii was carried out. FL (group-2) and Nt (group-1) peptides exhibited apparent antifungal activity at a concentration > 3.4 nm, but no antifungal activity was observed in the Ct peptide bioassay ( Fig. 2A) . It appeared that Nt peptide (group-1) was more effective than the FL peptide (group-2) (Fig. 2B) . Although antifungal activity of phytocystatins from taro, strawberry and chestnut has been reported previously [7, 9, 17] , the mechanism of inhibitory activity of phytocystatins against phytopathogenic fungi remains unclear. The presence of the Ct peptide in the FL peptide appears to be the cause of the reduction in antifungal activity. The hyphal morphology was also observed under low and high magnification microscopy. The growth-retarded mycelium exhibited swelling, less branches and blunt tips at an Nt peptide concentration of 3.4 nm (Fig. 2C) , and displayed swelling, no branches, very short tips and fragmentation at a concentration of 5.1 nm.

Before inhibition analysis, the recombinant protein was purified by being passed through affinity columns and subsequently cleaved by thrombin and identified by SDS ⁄ PAGE analysis. Electrophoresis of free recombinant protein of FL, Nt and Ct peptides, showed maximum purity (Fig. 1B) . To determine the inhibition constant, N a -benzoyl-d,l-arginine b-naphthylamide hydrochloride (BANA) was used as a substrate at a concentration range of 20-260 lm for the assay with equimolar (25 nmol) papain and inhibitor concentrations (Fig. 3A) . The Ct peptide curve appeared above the control (Ck), indicating that the Ct peptide enhances the enzyme activity, which is consistent with the anti-papain activity determined by the in-gel assay (Fig. 1C,D) . Both FL and Nt peptides could inhibit papain activity by 55% and 39%, respectively, whereas Ct peptide activated papain by 18% (Table 1) . Therefore, FL peptide exhibited mixed inhibition, Nt peptide exhibited competitive inhibition and Ct peptide exhibited allosteric activation (Fig. 3B ).

Further verification of the inhibition characteristics was performed by repeating the experiment after making a slight modification, with BANA at a concen-tration in the range 60-240 lm, as well as varying the inhibitor level in the assay. A Lineweaver-Burk plot of the reactions with varied inhibitor levels again showed competitive inhibition for Nt peptide and mixed inhibition for FL peptide (Fig. 4A,B) . Thus, the presence of two inhibition types was confirmed. The inhibition constants (K i values) could be calculated from the apparent K m and V max changes ( Table 1 ). The K i value of Nt peptide (group-1) inhibition on papain was found to be 5.7 · 10 )8 m. This value is very close to the K i of rice OC-I (3.0 · 10 )8 m) [18] . In addition, comparison of inhibitory activity with other group-1 species showed that K i for Nt peptide of tarocystatin is lower than those for rice OC-II (8.3 · 10 )7 m) [18] , Job's tears cystatin (1.9 · 10 )7 m) [19] and soybean cystatin L1 (1.9 · 10 )5 m) [20] , but higher than those for sesame (2.7 · 10 )8 m) [8] and maize CCI (2.3 · 10 )8 m) [21] . Nt peptide inhibitory activity appears to be intermediate among the group-1 phytocystatin family.

In mixed inhibition, the K i value is separated into K ia and K ib . K ia is described as the dissociation of inhibitor from enzyme, whereas K ib is for that between the inhibitor and enzyme-substrate complex. A prominent characteristic of mixed inhibition compared to competitive inhibition is that the mixed inhibitors bind to enzymes as well as enzyme-substrate complexes, but competitive inhibitors bind only enzymes. Thus, the Ct peptide of tarocystatin may be able to dock onto the papain structure when the active site is occupied by a substrate. Furthermore, the occurrence of the K ib value is always tailed with an unknown regulatory effect, indicating that the Ct peptide functions to alter the target protein conformation and prevent product formation. The Nt peptide functions like the entire OC-I and confers tarocystatin with an affinity for the competing active site. The 3D structural model of tarocystatin was predicted to infer the interaction between group-2 tarocystatin and papain. The Ct peptide sequence shares 48% identity and 68% similarity with taro Nt (1-97 amino acids), as solved by NMR spectroscopy [22] . Although there was no established template for Ct peptide 3D structure prediction, it shares 13% identity and 38% similarity to group-1 OC-I (Fig. 5 ). Therefore, the Ct peptide structure was predicted using secondary structure estimation and a folding pattern simulation program with pseudo-energy minimization. Subsequently, the entire tarocystatin 3D structure was obtained by combining the structures of two segments. Its conformation resembled an earphone comprising two solid masses and a linear structure (Fig. 6) . A highly structural similarity between the Nt and Ct peptides was found and, presumably, the Ct peptide compete with the Nt peptide for binding to the active site (Fig. 6) . However, the assay using varied concentrations in the range 0-10 000 lm of Ct peptide to compete with the Nt peptide at a concentration of 62.5 lm did not demonstrate that the Ct peptide reduced the inhibitory capacity of the Nt peptide (Fig. 7A) . Instead, it revealed that the Ct peptide does not act competitively.

To determine whether the connection between the Nt and Ct peptides is important for inhibitory capacity of the FL peptide, equal amounts of Nt and Ct peptides were mixed and the inhibitory capacity of the mixture was then compared with that of only the Nt or FL peptides. The curve of the mixture of Nt and Ct peptides did not tend to that of the FL peptide in the retrieve test (Fig. 7B) . The pattern of competitive inhibition against papain by the Nt peptide of group-1 is consistent with the previous findings obtained for many other group-1 phytocystatins [18, 19] , whereas the mixed type inhibition against papain by FL peptides of group-2 has not been reported to date.

Information about mixed inhibition by other phytocystatins is scarce. A similar inhibition model, noncompetitive inhibition, was reported in strawberry FaCPI-1 [9] and in soybean L1 and R1 [20] . Of these, only FaCPI-1 belongs to the group-2 phytocystatins and demonstrates a strong inhibitory activity (1.9 · 10 )9 m). The FaCPI-1 amino acid sequence is highly homologous with tarocystatin, but its mechanism cannot show mixed inhibition. To unravel the mechanism, a detailed investigation of the 3D structural interaction between group-2 phytocystatins and papain is necessary.

In the present study we are the first to show the inhibition difference between group-1 and group-2 phytocystatins, and to examine the importance of the extended C-terminal region (Ct peptide of tarocystatin) with respect to interaction with anti-papain activity.

In the analysis of the primary structure of tarocystatin, we found that the group-2 tarocystatin (FL peptide) is a group-1 phytocystatin (Nt peptide) possessing an additional Ct peptide. Moreover, the Ct peptide of the group-2 phytocystatin shares a high consensus sequence among the discovered species [7] . Both the FL and Nt peptides exhibit a good inhibitory property on papain activity, whereas the Ct peptide exhibited papain activation that was also evident in an in-gel inhibitory assay (Fig. 1C) . The inhibition constant demonstrated that the FL peptide exhibited mixed inhibition, and the Nt peptide exhibited competitive inhibition, suggesting a canonical binding mode as with many other group-1 phytocystatin species previously reported ( Table 2 ). The enhancement of the proteinase activity by 18% (Table 1) implicates that the interaction between papain and the Ct peptide possesses refolding in the conformation change of the papain protein. The mixed type inhibition against papain by the FL peptide might be due to the presence of the Ct peptide, which plays an activation role on papain when used alone. Therefore, the mechanism of the Ct peptide with respect to enhancing papain activity presumably involves allosterically binding adjacent to the active ⁄ substrate binding site and altering the papain conformation to be more accessible for the substrate, which is defined as the 'noncanonical' binding mode, where these inhibitory characteristics are quite different from the 'canonical' binding mode of group-1 phytocystatins, as noted previously [23] . This change may also shift the orientation of the Nt peptide to bind with competitive inhibition and result in blocking the substrate from the approaching catalytic site. When the Ct peptide was bound to papain and linked with the Nt peptide, substrates still had the chance to bind to the active cleft. Nevertheless, the Nt peptide was so close to active cleft that allows Nt peptide binding prior to any approaching substrates. Thus, the enhancing effect of the Ct peptide was followed by an immediate binding of the Nt peptide. In this case, substrates still could reach the active cleft and be trapped by some inner pulling force, but could not be fixed in the catalytic site that the Nt peptide blocked. This mechanism was like a noncompetitive inhibition, where substrates could bind to the enzyme-inhibitor complex but not to be turned to products. However, if the Nt peptide bound to papain before the Ct peptide, tarocystatin would simply exhibit competitive inhibition. The alternative binding pattern strongly supports the idea that tarocystatin is a mixed type inhibitor, and provides evidence for the difference between group-1 and group-2 phytocystatins. Phytocystatin has been known for its defense function against attack by insects and pathogens. These proteins have received much attention from researchers due to their potential utilization as bioinsectides in agrobiotechnology [3, 4] . To extend our previous study on antifungal activity [7] , a bioassay on mycelial growth of S. rolfsii was performed, and revealed that the FL and Nt peptides exhibited apparent antifungal activity at a concentration above 3.4 nm, but no significant antifungal activity was observed for the Ct peptide ( Fig. 2A,B) . Microscopic observations indicated that the Nt peptide appeared to be stronger than FL peptide (Fig. 2B) . Because the Ct peptide alone does not show any antifungal effect, this implies that the antifungal activity might be connected to the Nt peptide conformation. A reduction of antifungal activity in vitro by FL peptide may be due to a molecular mass difference. It has been speculated that the FL peptide is larger than the Nt peptide, making it more inefficient to diffuse inside hyphal cells. The true mechanism responsible for the antifungal activity of tarocystatin still requires further investigation.

To date, the physiological significance of the Ct peptide remains unknown. Accumulating evidence shows that this repeated domain may originate from gene duplication and be exploited for other functions [10] . Recent evidence also demonstrated that carboxy terminus-extended PhyCys have the capacity to inhibit human legumanin peptide due to the presence of the conserved motif SNSL and act as a bifunctional inhibitor [24] . Our findings focused on the Ct peptide on cysteine protease, which may function with three roles: (a) to endow the N-terminal domain with more specificity and inhibition to papain; (b) to prevent the N-terminal domain from rapid digestion by endogenous or exogenous peptidase; and (c) to enrich its molecular size as an ideal storage protein. Due to these beneficial characteristics, the Ct peptide could be reserved under evolutionary selection. Further studies, including mutagenesis and structural studies, are required to better understand the molecular mechanisms involved in the tarocystatin binding to papain and to identify the regulatory cleft involved in the inhibition process [15] .

Based on the characterization of inhibitory function of group-1 and group-2 phytocystatins, we suggest that Fig. 7 . Competition and retrieve test of the Ct peptide to Nt peptide. (A) Competition test: the y-axis is catalytic velocity of papain, which was measured as the change in optical density over time. Each reaction had the indicated amount of Ct and Nt peptides that reacted with papain. An increasing Ct level did not reduce the inhibitory capacity of the Nt peptide, but instead maintained a steady intensity. (B) Retrieve test: the mixture of Nt and Ct peptides of 625 lM was compared with an equal amount of only FL or Nt peptides in the reaction with varied substrate concentrations. The curve of Nt plus Ct peptides highly overlapped that of the Nt peptide, indicating that the Nt peptide cannot retrieve the inhibition efficacy of the FL peptide when it disconnects from the Ct peptide. group-1 and group-2 both evolved from a common ancestor. The evolutionary direction from group-1 toward group-2 by gene duplication appears to be an adaptation resulting from an evolutionary 'arms race' of rapid change in both interacting proteins.

Construction of three DNA regions of the CeCPI gene The underlined bases in the primers indicate restriction sites for BamHI (GGATCC) or EcoRI (GAATTC). The primer combination of F1 and R1 was used for amplification of the FL peptide; F1 and R2 was for the Nt peptide, and F2 and R1 was for the Ct peptide. The PCR products were digested with BamHI and EcoRI, and ligated to pGEX-2TK vector (Amersham Biosciences, Piscataway, NJ, USA) at the corresponding restriction sites.

Expression, purification and characterization of recombinant tarocystatin E. coli BL21 (DE3) cells containing the appropriate construct were grown at 37°C in 2YTA liquid medium until D 600 of 1.5 was reached. The recombinant CeCPI expres-sion was induced by addition of 1 mm isopropyl-b-d-thiogalactopyranoside. Two hours after induction, the recombinant proteins were extracted from 250 mL of bacterial culture by using B-PERÒ GST-fusion protein purification kit (Pierce No. 78400; Pierce Biotechnology, Rockford, IL, USA). For the assay of inhibitory kinetics of CeCPI fragments, the GST fusion protein was cleaved with 20 units of thrombin for 16 h at room temperature. Finally, the recombinant proteins were collected by passing the extract through a glutathione Sepharose 4B affinity column (Amersham Biosciences). The protein was quantitated with a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA) using BSA as a standard.

Qualitative analysis of CeCPI protein was performed according to Michaud et al. [25] on 12.5% SDS ⁄ PAGE containing 1% gelatin. A mixture of CeCPI proteins and papain was first incubated at 37°C for 15 min in a mildly denaturing buffer (62.5 mm Tris-HCl, pH 6.8; 2% SDS, 2% sucrose; 0.01% bromophenol blue), and then subjected to electrophoresis using a Hoefer SE250 system (Hoefer, Inc., Holliston, MA, USA). After migration, the gels were transferred to a 2.5% v ⁄ v aqueous solution of Triton X-100 for 30 min at room temperature to allow renaturation followed by incubating in reactive buffer (100 mm sodium phosphate, pH 6.8, containing 8 mm EDTA, 10 mm l-cysteine and 0.2% Triton X-100) for 75 min at 37°C. Subsequently, the gels were rinsed with water and stained with Coomassie Brilliant Blue. Proteinase inhibitor activity was visualized as clear zones on a blue background and the intensity of the clear band is inversely related to the inhibition level.

K i values of papain inhibition were determined from Lineweaver-Burk plots, a double reciprocal plot of substrate concentration versus velocity. The velocity was determined by measuring the A 540 of the chromophore, as described by Pernas et al. [17] . Briefly, an appropriate amount of inhibitor was pre-incubated with 1 lm of papain in 100 lL of reaction mixture containing 0.1 m sodium phosphate buffer (pH 6.5), 10 mm EDTA and 10 mm 2-mercaptoethanol at 37°C for 10 min. The reaction was started by the addition of 100 lL of a varied concentration in the range 20-260 lm of BANA (Sigma, St Louis, MO, USA) as substrate. The reaction mixture was incubated at room temperature for 20 min and 300 lL of 2% HCl in ethanol (w ⁄ v) was added to stop the reaction. The chromophore was then developed by addition of 300 lL of 0.06% p-dimethylaminocinnamaldehyde in ethanol followed by incubation at room temperature for 15 min and measurement of A 540 . Corre-Menguy et al. [33] The inhibitory activity was recorded as the inhibition percentage (%) and the inhibition percentage (I%) of papain was calculated using the equation:

where, T and T* are the velocities in the absence and presence of the inhibitor from reactions, respectively. The average inhibitory activity was calculated from I% values of varied substrate concentrations.

The fungal activity assay was performed as described previously [7] . Five pieces of sclerotia of phytopathogenic fungus S. rolfsii were cultured in 1 mL of half strength potato dextrose broth, which contained purified GST-tarocystatin segment fusion proteins at concentrations of 1.7, 3.4, 5.1 and 6.8 nm in four separate sets. The fungi were cultured at 28°C under continuous shaking (200 r.p.m.) on an orbital shaker for 72 h. Hyphal growth inhibition by tarocystatin segment proteins was observed directly, as well as under a microscope.

The tarocystatin primary sequence (AAM88397) was subjected to NCBI psi-blast with a threshold of 0.0001 for searching for homologous sequences from various plants.

The sequence similarities of 18 amino acid sequences were distributed with the highest identities of 66% and a positive of 83% for soybean to the lowest identities of 55% and a positive of 77% for tomato, excluding nonplant and multidomain cystatin homologs. These 18 sequences were aligned using clustalw [26] and shaded with genedoc [27] software. The secondary structure of these 18 sequences was analyzed by two programs, psi-pred [28] and yaspin [29] . The results obtained by the two programs were consistent with each other and showed that both Nt and Ct peptide secondary structures were arranged in a similar pattern. This was also verified by aligning the OC-I with the tarocystatin Nt and Ct peptide regions. Therefore, the stereofolding pattern of OC-I [22] can be taken as a template for the CeCPI folding prediction by modeler 8.1 [30] . The two structural conformations were merged after analysis by the automatic docking system, zdock 2.3 [31] , and then remodeled by modeler 8.1 [30] .

Structural and phylogenetic relationships among plant and animal cystatins

Mechanism of inhibition of papain by chicken egg white cystatin

Papain-inhibitory activity of oryzacystatin, a rice seed cysteine proteinase inhibitor, depends on the central Gln-Val-Val-Ala-Gly region conserved among cystatin superfamily members

Molecular cloning of a cysteine proteinase inhibitor of rice (oryzacystatin). Homology with animal cystatins and transient expression in the ripening process of rice seeds

Characterization of a cDNA encoding a cysteine proteinase inhibitor from chinese cabbage (Brassica campestris L. ssp. pekinensis) flower buds

Soyacystatin, a novel cysteine proteinase inhibitor in soybean, is distinct in protein structure and gene organization from other cystatins of animal and plant origin

Molecular cloning, recombinant gene expression, and antifungal activity of cystatin from taro (Colocasia esculenta cv

Cloning, functional expression, and characterization of cystatin in sesame seed

The strawberry gene Cyf1 encodes a phytocystatin with antifungal properties

Evolutionary mechanism acting on proteinase inhibitor variability

Characterization of a genomic sequence coding for potato multicystatin, an eight-domain cysteine proteinase inhibitor

Purification and characterization of a cystatin form the leaves of methyl jasmonate-treated tomato plants

Trade-offs between pathogen and herbivore resistance

Differential expression of soybean cysteine proteinase inhibitor genes during development and in response to wounding and jasmonate

The involvement of cysteine proteases and protease inhibitor genes in the regulation of programmed cell death in plants

The cystatins: protein inhibitors of cysteine proteinase

A chestnut seed cystatin differentially effective against cysteine proteinases from closely related pests

Two distinct cystatin species in rice seeds with different specificities against cysteine proteinases

Molecular cloning and functional expression of cDNA encoding a cysteine proteinase inhibitor, cystatin, from Job's tears (Coix lacryma-jobi L. var Ma-yuen Stapf)

Two woundinducible soybean cysteine proteinases have greater insect digestive proteinase inhibitory activities than a constitutive homolog

Corn cystatin I expressed in Escherichia coli: investigation of its inhibitory profile and occurrence in corn kernels

Three dimensional solution structure of oryza cystatin-I, a cysteine proteinase of the rice, Oryza sativa L japonica

Structural basis of the endoproteinase-protein inhibitor interaction

Carboxy terminal extended phytocystation are bifunctional inhibitors of papain and legumanin cysteines proteinases

Assessing the stability of cystatin ⁄ cysteine proteinase complexes using mildly-denaturing gelatin polyacrylamide gel electrophoresis

CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice

GeneDoc: analysis and visualization of genetic variation

The PSI-PRED protein structure prediction server

A simple and fast secondary structure prediction algorithm using hidden neural networks

Comparative protein modelling by satisfaction of spatial restraints

ZDOCK: an initialstage protein-docking algorithm

A cold inducible multidomain cystatin from winter wheat inhibits growth of the snow mold fungus, Microdochium nivale

Characterization of the expression of a wheat cystatin gene during caryopsis development

The present study was supported by the National Science Council, Taiwan, under project NSC-95-2317-B-002-005 to Kai-Wun Yeh. We thank Dr Michael Conrad (University of North Carolina at Chapel Hill) for critically reading the manuscript and for his helpful suggestions.