key: cord-1008540-nokd5kmx
authors: Yang, Guang; Che, Xibing; Gofman, Rose; Ben-Shalom, Yossi; Piestun, Dan; Gafny, Ron; Mawassi, Munir; Bar-Joseph, Moshe
title: D-RNA Molecules Associated with Subisolates of the VT Strain of Citrus Tristeza Virus which Induce Different Seedling-Yellows Reactions
date: 1999
journal: Virus Genes
DOI: 10.1023/a:1008105004407
sha: 718da02d090ae7ab9874cf89ad0c8ac673231a0a
doc_id: 1008540
cord_uid: nokd5kmx

Citrus tristeza virus (CTV) strains were previously catalogued as seedling-yellows (SY) and non-SY (nSY) types, according to their yellowing and stunting effects on indicator seedlings. Among subisolates of the VT strain, which were selected from chronically infected Alemow plants, there was a correlation between the presence of 2.4-, 2.7- and 4.5-kb D-RNAs, and SY and nSY reactions, respectively. Similarly, plants infected with Mor-T subisolates, which cause SY, contained D-RNAs of 2.6 to 2.8 kb, while nSY subisolates from recovered sour orange tissue contained a major D-RNA of 5.1 kb. Plants harboring the 2.7-kb D-RNA were protected against challenge inoculation with a subisolate harboring the 4.5-kb D-RNA. This study suggests that the nSY reaction results either from the absence of SY gene(s) in the genomes of certain CTV strains or through the suppression of the effects of SY gene(s) by D-RNAs with 5′ parts larger than 4000 nt.

Citrus tristeza virus (CTV) (1, 2) , a member of the closterovirus group and the Closteroviridae family (3±7) is an important pathogen, causing considerable economic losses to citrus industries worldwide. Citrus trees infected with CTV display two main types of disease: (i) quick decline of sweet oranges (SwO) (Citrus sinensis L.) and of some other species grafted on the sour orange (C. aurantium) rootstock (8) ; and (ii) stem pitting of grapefruit (C. paradisi) and pummelo (C. grandis) (9) . Other manifestations of infection with CTV include the seedling-yellows (SY) reaction (9±12) which is primarily a disease of experimentally inoculated plants but which might also be encountered in the ®eld in top-grafted plants. Seedlings of sour orange, lemon (C. limon) and grapefruit become chlorotic and stunted when inoculated with CTV-SY isolates, but no symptoms are elicited when SwO or mandarin (C. reticulata) is inoculated (1, 13) . The CTV-SY phenomenon is one of the long-standing enigmas in citrus virology. The early studies of McClean & van der Planck (9), Fraser (10) and Wallace (11) all suggested a complex aetiology of the CTV-SY disease. There have been reports of spontaneous recovery from SY infection by sour orange plants which initially showed SY symptoms, and of the elimination of the SY causal agent by the passage of SY-inducing CTV subisolates through SY-sensitive citrus hosts such as grapefruit and sour orange (12) , which has led to the emergence of non-SY (nSY) isolates. These phenomena have given rise to the hypothesis that the CTV-SY reaction is caused by two separate components: the CTV agent, capable of autonomous replication and responsible for the quick decline and the lime reaction; and a second component, responsible for the SY reaction and able to replicate only in plants harboring the CTV component.

The CTV particles contain a single-component positive-stranded genomic RNA of 19296 nt for the Florida isolate, T36 (14) and of 19226 nt for the VT strain from Israel (15) . The genomes of these CTV strains showed considerable sequence deviation within the 5 H half, but were found to have similar organization and to encompass 12 ORFs which potentially code for at least 17 protein products. In addition to the large replicative form (RF) RNA molecule, the infected plants contain a nested set of at least nine smaller species of 3 H -co-terminal single-and double-stranded subgenomic RNAs (sgRNAs). These sgRNAs correspond to the 3 H -terminal ORFs (16, 17) . Cloning of the VT strain of CTV revealed the presence of several defective (D) RNAs of various sizes, composed of the 5 H and 3 H termini of the genomic RNA with extensive internal deletions, along with the full-length virus. The sizes of the termini varied among species, with minimal lengths of 442 nt and 858 nt from the 3 H and the 5 H termini, respectively, resulting in different sizes of D-RNAs with different junction sites (18, 19) .

Inoculation of VT on the sour orange indicator resulted in SY symptoms (20) . Later infections of sour orange seedlings by grafting with CTV-VT infected Alemow budwood resulted in inconsistent SY reactions; and not all plants showed the SY symptoms. Recently, we selected subisolates of two CTV strains, VT and Mor-T (21) , which differed in their SY reactions on sour orange seedlings. The present paper reports the association of D-RNAs with 5 H termini larger then 4000 nt, with VT and Mor-T subisolates which do not elicit the SY reaction. D-RNAs may be involved in the long-standing enigma of the complex etiology of the SY-CTV reaction.

The VT strain was originally isolated in 1970 from a SwO cv. Valencia tree grafted on sour orange. The tree showed advanced quick-decline symptoms. Inoculation of sour orange plants with the VT inoculum maintained in sour lime caused typical SY symptoms (20) . Later passages of the VT strain from sour lime and Alemow plants to sour orange often resulted in inconsistent SY reactions: not all sour orange seedlings showed the SY symptoms, even when inoculum from a single Alemow plant was used to infect groups of plants from a single seed source (Bar-Joseph, unpublished). Subisolates of CTV-VT (Table 2) were randomly selected in 1994 from chronically infected Alemow plants which had been graft inoculated several years earlier (1988 to 1994) with different passages of this strain. The VT subisolates were maintained in a propagation glasshouse with temperatures ranging between 15 and 35 C. The SY reaction was assayed by grafting chip buds from infected Alemow stems onto sour orange seedlings grown in a temperature-controlled glasshouse facility with incandescent illumination to complete 20 h of light, and two temperature regimes (TR) of 26/18 C or 29/21 C for the normal and the semi-warm TR, respectively. In both TRs the high and low temperatures were maintained for 8 and 12 h, respectively, and the adjustment from the high daytime to the low night time level and vice versa took 2 h. The SY reactions were recorded 8 and 16 weeks after inoculation, for the normal and semiwarm TR, respectively. The Mor-T isolate originated from a declining Minneola tangelo tree (21) . The virus was propagated in Alemow and was used to inoculate a group of sour orange seedlings, some of which were inarched with the CTV-tolerant rootstock Go-Tou. Sour orange twigs and leaves showing SY and SY recovery, respectively, were used to infect sour orange and Alemow seedlings.

Double-stranded (ds) RNAs were isolated from 5±7 g of Alemow or sour orange tissues, according to Dodds and Bar-Joseph (22) . The RNAs were separated by electrophoresis in formamide-formaldehyde denaturating, 1.1% agarose gels, prepared in MOPS buffer, transferred to Hybond N membranes. The hybridization probes consisted of a 611-bp and a 762-bp cDNA fragment from the 3 H and 5 H ends of CTV-VT genome, respectively (15) . The DNA probes were either non-radioactively labeled using the Gene Images Random Prime Labeling Module Kit from Amersham or radioactively labeled with 32 P according to Mawassi et al. (17) . RNA probes labeled with 32 32P-UTP were synthesized, with the Riboprobe System-T7 kit (Promega) according to the manufacturer's instructions, from cDNA fragments of 611 bp and 762 bp of the CTV-VT 3 H and 5 H ends, respectively, cloned in pGEM (Promega).

Antibodies for ELISA capture were prepared in sheep primed with recombinant CTV coat protein (rCTV-CP) antigen and boosted with a partially puri®ed CTV preparation. The second antibodies were obtained from egg yolks of chickens immunized with rCTV-CP. The ELISA procedure for CTV viral antigen quanti®cation in different tissues, which were soaked overnight in the antibody-coated ELISA wells, was according to Bar-Joseph et al. (23) .

The cDNAs were prepared from dsRNA templates of VT5 and VT12, with primers P1 and P2 for the ®rststrand synthesis, and primers P3±P4 and P5±P6 for nested and direct PCR ampli®cation ( Table 1 ). The cDNA fragments were separated by electrophoresis on 1% agarose gel. The bands were excised from the gel and tested with the restriction enzymes, Sac I and Nsi I (Promega). For sequence analysis we used primers P7 and P9; P10 and P11; P10 and P8 to obtain three cDNA fragments located at ORF1 (1300±2486), ORFs 9 10(17260±17857) and ORFs 9 10 11(17260±18397), respectively. The cDNA fragments were cloned into the pUC 57/T (Fermentas) and sequenced from both sides by using Sequenase Version 2 from USB. Sequences of at least 150 bases were read from the 5 H and 3 H termini of each of the cDNA fragments. The dsRNAs from Alemow plants infected with two Mor-T subisolates, desig-nated #a and #b for SY-recovered and SY-reacting plants, respectively, were poly-A tailed and used for ®rst-strand cDNA synthesis with primer dT14V (Table 1 ) and for second-strand synthesis with primers P9 and P8, for nested PCR ampli®cation of the viral 3 H and with primers P12 and AD for the viral 5 H . The cDNA fragments were separated by electrophoresis on 1% agarose gel, cloned into pUC 57/T (Fermentas). Sequencing from both sides of the 3 H fragments, was performed by using Sequenase Version 2 from USB and the 5 H sequence was determined with the aid of an automatic sequencing machine.

Two groups of 9 month old Alemow seedlings were graft inoculated at heights of 25±30 and 30 cm, with two chip buds from Alemow plants infected with VT5 or VT12, respectively. Two weeks post-infection (wpi), the plants were pruned and allowed to develop two side branches. Tests for the presence of the speci®c D-RNAs were conducted after 10 wpi. The plants where challenged, 20 wpi by top grafting with stems infected with the reciprocal subisolates. Two lateral buds were allowed to sprout from each of the protected plants and leaf and stem bark tissue were tested for the presence of D-RNAs by Northern blotting. 

Biological Characterization of VT and Mor-T Subisolates 

Hybridization with an approximately 0.7-kb cDNA probe or riboprobe from the 5 H end of the VT genome with dsRNA extracts from Alemow plants, revealed the presence of the large RF and the low-molecularweight tristeza 5 H -corresponding RNA molecules (LMT) (18) and D-RNAs. VT-subisolates 6±8 and 13, and 1, 5, 9, 10, with apparently similar SY reactions, showed the presence of two types of D-RNAs, of 2.4 kb and 2.7 kb, respectively. The three nSY subisolates (3,4 and 12) showed the presence of a 4.5-kb D-RNA ( Fig. 1A and Table 2 ). The hybridization patterns of dsRNAs extracted from sour orange seedlings infected with VT subisolates VT12 (nSY) and VT5 (SY) are shown in Fig. 2B . Only weak or no hybridization signals of genomic and/or defective RNA could be located in bark and leaves from the sour orange plant which showed severe SY compared with those from the nSY plant. Hybridization of dsRNAs from Alemow plants inoculated with Mor-T subisolates #a1 (nSY) and #b1 (SY), showed the presence of major large (ca. 5.1 kb) and small (ca. 2.6 kb) D-RNAs respectively (Fig. 1C) . One of the SY Mor-T subisolates #c1 showed only weak bands of D-RNA molecules compared with the nSY subisolate #e1, which showed the major D-RNA of ca. 5.1 kb (Fig. 1D, lane 1) . Sequence analyses revealed that SY subisolate #b1 contained two D-RNAs of 2634 and 2815 nt with junctions of their 5 H termini located at positions 1772 and 1521, whereas the nSY subisolate #a1, contained a major D-RNA of 5125 nt, with the junction of the 5 H terminus located at position 4376 (Fig. 3B) .

The hybridization with the VT 5 H probe with different VT and Mor-T subisolates suggested a close relationship between their genomic RNAs. In order to examine the genomic composition of the VT5 (SY) and the VT12 (nSY) subisolates, we compared the sequences of termini of their genomes by means of nested RT-PCR and sequencing analyses. Primers P1 and P2 were used for ®rst-strand cDNA synthesis and primers P3, P4, P5 and P6 (Table 1) ampli®cation. The resulting cDNA fragments for both subisolates gave the expected lengths for the 5 H (8-709) and 3 H (18611±19227) ends of their genome. Restriction analysis of these products with SacI and NsiI gave restriction fragments of identical size (not shown). Sequence analyses of internal regions, at least 150 nt in length, of three cDNA fragments positioned at different regions of the VT genome ( positions 1300±2486, 17260±17857 and 17260±18397) did not reveal any sequence deviation between the products obtained from the dsRNAs of the VT5 (SY) and the VT12 (nSY) subisolates (not shown).

The possibility of interference between two VT subisolates, VT5 and VT12, harboring the 2.7-and the 4.5-kb D-RNAs, respectively, was tested in Alemow plants. The dsRNAs from plants which had ®rst received a protective inoculation with either the VT5 or the VT12 subisolate and were later challenged by top grafting with the reciprocal subisolate, were hybridized with the 5 H -speci®c probe. At 18 weeks post challenge inoculation (wpci), the basal parts of each combination had predominantly the D-RNAs of the protective isolate (not shown). Later tests at 41 wpci showed only the 2.7-kb D-RNA in the basal parts of plants protected with VT5 (Fig. 2, lanes 3, 4  and 6 ). Plants protected with VT12 showed the presence of either a conspicuous or a weak band of the challenging 2.7-kb D-RNA in addition to the 4.5kb D-RNA (Fig. 2, lanes 5 and 7, respectively) .

Sour orange seedlings infected with Alemow tissues from the interference experiments, which harbored both the 2.7-and the 4. stronger ELISA titers and higher dsRNAs concentrations (Fig. 2B, lane 3) .

Biological and molecular characterization of 11 VT subisolates, which were randomly selected from chronically infected Alemow plants, revealed the presence of eight SY and three nSY subisolates. The VT subisolates caused similar symptoms and comparable ELISA reactions in Alemow plants (not shown). The virus titers were considerably higher in sour orange plants infected with nSY than in those infected with SY subisolates. These differences were consistent among plants which were maintained under different TRs (Table 2 ). Low virus titers or the absence of virus (indicated by negative reactions on indicator plants) in sour orange leaves and roots showing severe SY symptoms, suggest the possibility that the SY isolates emit a long-distance signal for a hypersensitive reaction. A similar situation has been previously observed in mature trees infected with CTV-Mor-T, where the collapse of the sweet orange/ sour orange combination often preceded the spread and redistribution of the virus towards the upper parts of the infected trees (21) .

The profound differences among the sour orange reactions to the various VT-subisolates were associated with the presence of different major D-RNAs. The nSY subisolates, 3, 4 and 12, showed the presence of a major band of 4.5-kb D-RNA, whereas the eight SY subisolates, 1, 5±10 and 13, showed the presence of two smaller D-RNAs of 2.4 and 2.7 kb, with no apparent difference in the intensity of the SY reaction to subisolates which contained either of the smaller D-RNAs. Infection of sour orange with tissues from Alemow plants concomitantly infected with mixtures of VT5 and VT12 resulted in reactions ranging from SY to nSY, with virus titers depending on the relative concentrations of the 2.7-and 4.5-D-RNAs in the inoculum source.

Previously, we showed variations in the presence of the 2.4-, 2.7-and 4.5-kb D-RNAs in Alemow plants infected with budwood from a single VT-infected source plant (18) . Differences in D-RNA populations might have accounted for the previously noticed inconsistencies in the SY reaction of sour orange plants infected with VT strain (Bar-Joseph, unpublished). The selection of VT subisolates which show a more consistent SY reaction was correlated with the presence of a major type of D-RNA (Table 2) . One probable reason for obtaining apparently stable subisolates was their selection from chronically infected plants ( 4 2±3 years after inoculation) at a time when a single type of D-RNA had become dominant. (Fig. 3) . A 16nt sequence, 5 H -GAAAACTAATTTATCA, with no homology to other regions of the CTV genome was found at the junction site (Fig. 3) . A different short sequence, probably of host origin had previously been observed at the junction site of the 2.4-kb D-RNA (19) .

The CTV-SY phenomenon is one of the longstanding enigmas in citrus virology. The ®nding that both the CTV and the CTV-SY diseases could be transferred by mechanical inoculation of preparations of CTV particles (26,27) raised the question (28) of the dual-component theory of the causal agent of the CTV-SY disease (12) . Dodds et al. (29) noted an association between two dsRNAs of about 0.8 and 2.7 kb and SwO trees infected with SY subisolates. Molecular characterization associated the 0.8-kbp dsRNA with the replicative subgenomic RNA coding for ORF11 (940 nt) (16, 17, 30, 31) and hybridization with a 3 H -speci®c probe did not reveal quantitative differences in the amounts of the 0.8kbp dsRNAs from SY and nSY plants (not shown). Moreover, low-molecular-weight D-RNAs of 2.4 kb were located in Alemow infected with nSY isolates Mik-T and Ach-T (32) (not shown).

CTV isolates were previously classi®ed by a variety of criteria into subisolates which differed in host reactions, vector transmissibility and dsRNAs patterns (29,33±37). The variability among subisolates was considered as an indication of the high frequency of mixed CTV infections. D-RNAs were previously implicated in the variability between the dsRNA patterns of parental isolates and their subisolates (5, 35) and the present ®ndings indicate a correlation between certain D-RNAs and host reactions, and support a working hypothesis that the nSY reaction results either from the absence of SY gene(s) or through the suppression of their effects by D-RNAs with 5 H parts larger than 4000 nt.

The genomic and D-RNA fragments of the two differentially reacting VT subisolates were found to show a complete sequence identity. Nevertheless, the possibility that a minimal sequence deviation between other parts of their genomes is involved in these biological differences cannot at the present be completely ruled out. Moreover, the question of the mechanism that causes SY symptoms in sour orange tissues, which contain only low concentrations of viruses or D-RNA remains to be answered. D-RNAs have been isolated from a broad spectrum of animal viruses and, more recently, also from a large number of plant viruses (for recent reviews, see (38) ). Different D-RNAs have previously been reported to have different effects on disease expression: while D-RNAs of tombusviruses had attenuating effects on infection (39, 40) , the D-RNAs associated with the turnip crinkle virus tended to increase the severity of symptoms (41) and the D-RNAs associated with broad bean mottle virus had no effect on some host plants but intensi®ed the severity of symptoms in others (42) . The correlation between the SY reactions of sour orange seedlings and the genomic composition of the D-RNAs in the Alemow inoculum, support the notion that the host type is a major determinant of the biological effects of D-RNAs (43) .

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Sem Virol 7