A citrus exocortis viroid variant from broad bean (Vicia faba L.): infectivity and pathogenesis

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1 Journal of General Virology (1995), 76, Printed in Great Britain 227l A citrus exocortis viroid variant from broad bean (Vicia faba L.): infectivity and pathogenesis C. Fagoaga, 1 J. S. Semancik 2 and N. Duran-Vila ~* 11nstituto Valenciano de Investigaciones Agrarias, Apartado Oficial, Moncada, Valencia, Spain and 2 Department of Plant Pathology, University of California, Riverside, CA 92521, USA A viroid present in very low titres was isolated from symptomless field broad bean plants. It was identified as a variant of citrus exocortis viroid in the T2, V and C domains. Infection of several hosts resulted in a change in the composition of the viroid population. Serial passage through tomato and back to the host of origin, broad bean, resulted in major changes in replication efficiency, host range and pathogenicity. The unique nucleotide sequence differences identified in the original broad bean variant were not conserved after passage through alternative hosts. The effects of these sequence variations on viroid secondary structure result in nonpathogenic viroid variants which can remain unnoticed in certain plant species but may act as reservoirs of viroid disease. Introduction Viroids are not commonly associated with epidemics in vegetable crops but a number of viroid-induced diseases have occasionally been reported in potato (Diener & Raymer, 1969), cucumber (Van Dorst & Peters, 1974) and tomato (Walter et al., 1980; Galindo et al., 1982; Mishra et al., 1991). The results of a recent survey demonstrated the presence of a new viroid in eggplant (Fagoaga et al., 1994) and several variants of the citrus exocortis viroid (CEVd) in field-grown tomato, eggplant, carrot and turnip (C. Fagoaga, unpublished results). The inoculation of sensitive hosts has not only been used for the detection of CEVd variants from field-grown vegetable crop species but is a common practice in the routine indexing of viroid-induced diseases (Fernow, 1967; Weathers et al., 1967). Moreover, the characterization of most viroids, including CEVd, has been accomplished after their purification from alternative herbaceous hosts which provided high viroid titres (Semancik & Weathers, 1972; Gross et al., 1982; Visvader et al., 1982). Since the viroid population of a given isolate can be modified as a result of host and tissue selection (Semancik et al., 1994) its biological properties may also vary depending on the host chosen for its characterization (Semancik et al., 1993). Here we report a naturally occurring CEVd variant from broad bean (Vicia faba L.) and the alterations in * Author for correspondence. Fax nuria@ivia-inia.es molecular and biological properties introduced as a result of host selection pressures on the viroid population. Methods Plant material sources. The original samples were collected from symptomless broad bean plants (Vicia faba L.) growing in the field plots of the' Servicio de Plantas de Vivero', Spain, during the growing season (March, 1992). The samples were kept at - 20 C until they were processed. Nucleic acid extraction. Tissue samples consisting of leaves and stems were homogenized in extraction medium containing water-saturated phenol as described by Semancik et al. (1975). The total nucleic acid preparations were partitioned in 2 M-LiC1 and the soluble fraction was concentrated by ethanol precipitation and resuspended in TKM buffer (10 mm-tris HC1, 10 mm-kc1, 0.1 mm-mgclz ph 7.4). These nucleic acid preparations were analysed for the presence of viroids and/or used as inocula for infectivity analysis and host range studies. Viroid analysis. Aliquots of the nucleic acid preparations (20 I~t, equivalent to 300 mg of fresh tissue) were subjected to spage as described by Rivera-Bustamante et al. (1986) and the circular forms of the viroids were viewed following silver staining (Igloi, 1983). Alternatively, the nucleic acids were electrotransferred, at 80 V for 1 h, to PVDF membranes (Immobilon N; Millipore) for Northern blot analysis. The CEVd crna probes were synthesized by a transcription reaction with T7 RNA polymerase in the presence of [~-32p]UTP ( > 400 Ci/~tmol) using the plasmid pcevd (kindly provided by Prof H. L. S~,nger, Max-Planck-Institut, Miinchen) which contains a dimeric cdna clone of CEVd as described by Puchta & Sanger (1988). Prehybridization and hybridization were performed in 50 % formamide and 5 x SSPE, at 68 C for 24 h as described by Maniatis et al (1989). RT-PCR analysis. The LiC1 supernatant extracts were subjected to Cf-ll cellulose chromatography (Semancik, 1986) and extensively SGM

2 2272 C. Fagoaga, J. Semanick and N. Duran-Vila washed with 30% ethanol prior to cdna synthesis.the cdna was synthesized as described by Yang et al. (1992) using a CEVd-specific synthetic oligonucleotide complementary to residues 80 to 98 of CEVd- C (Gross et al., 1982) using Moloney murine leukaemia virus reverse transcriptase (Promega). Second strand synthesis and amplification of dsdna were performed with synthetic oligonucleotides homologous and complementary to residues 99 to 117 of the CEVd-C using Taq DNA polymerase in PCR buffer containing 1 mm-mgclz. The thermocycling regime was 92 C for 30 s, 45 C for 1 min and 72 C for 2 min for 30 cycles, with a final extension at 72 C for 15 min and 30 C for 1 min. The RTPCR products from multiple reactions were combined and analysed by electrophoresis in 2 % agarose. Sequencing procedure. DNA obtained after RT-PCR was purified by electrophoresis in 2 % low melting point agarose, and sequenced using the 5' end-labelled primer extension method (fmol sequencing system; Promega). This sequencing protocol was employed to determine the predominant sequence variant replicated in different hosts. Any significant sequence difference selected and/or derived from a particular host should be readily detected in this manner. Although the sequence represents a summation of a random sampling rather than an actual molecular species, the sequencing of some undefined yet large number of clones is obviated. The DNA was denatured at 95 C for 2 rain and the thermocycling regime was 95 C for 30 s, 45 C for 30 s and 70 C for 1 min for 30 cycles. At least two sequencing reactions, one with the radiolabelled complementary primer and the other with the radiolabelled homologous primer, were performed to produce a full viroid sequence. For further verification of the sequence, dsdna was synthesized using as primers the synthetic oligonucleotides complementary to residues 307 to 330 and identical to 331 to 354 of CEVd- A (Visvader et al., 1982) and sequenced as described. In this case, the annealing temperature for the PCR reaction was 55 C instead of 45 C. The sequences were analysed for their minimum free energy secondary structure using the FOLD program (Zuker, 1989). Infectivity assays. Aliquots of nucleic acid preparations from broad bean were mechanically inoculated to broad bean 'Muchamiel' seedlings by stem puncture. Inoculated plants were grown at 1855 C to approximate field temperature conditions and observed for symptom development. Host range studies. Aliquots of nucleic acid preparations were inoculated onto tomato (Lycopersicon esculentum Mill.) 'Rutgers', cucumber (Cucumis sativus L.) 'Suyo' (kindly donated by Shikata Seed Company), chrysanthemum (Chrysanthemum morifolium Ramat.) 'Bonnie Jean', Gynura aurantiaca DC and Arizona 86l-S1 citron (Citrus medica L.). Tomato and cucumber were grown from seeds and inoculated as seedlings by stem puncture. Chrysanthemum and Gynura were propagated as rooted cuttings and inoculated by razor blade slash. Citron was propagated by bud grafting on Rough lemon (Citrus jambhiri Lush.) rootstock and inoculated by razor blade slash. The inoculated plants were kept at C and artificial light was provided during the winter to achieve a photoperiod of 16 h. Tomato, cucumber, chrysanthemum and Gynura were pruned every 3~4 weeks to control growth, and analysed for viroid content after 6-8 weeks. Citron was pruned 3 months after inoculation and the new flush of tissue analysed for viroid content. Results Identification of a CEVd variant in broad bean Viroid RNAs were not detected by spage analysis of nucleic acid preparations from broad bean (Fig. 1 a). When the same preparations were electrotransferred to CEVd (a) (b) Fig. 1. (a) Analysis by spage and silver staining of nucleic acid extracts of broad bean plants. (b) An identical gel was electrotransferred to a PVDF membrane and hybridized at 68 C with a CEVd-cRNA probe (1 x 10 6 c.p.m./ml). Extracts: lane 1, tomato infected with CEVd included as a positive control; lane 2, uninoculated broad bean seedling included as a negative control and lane 3, field grown broad bean. PVDF membranes and subjected to Northern blot analysis with a CEVd crna probe, the two bands with mobilities characteristic of the circular and linear forms of viroids were detected (Fig. 1 b). This nucleic acid preparation was subjected to RT- PCR. Analysis of the product by agarose gel electrophoresis showed a DNA band with the expected mobility. Sequence analysis demonstrated that the nucleic acid detected in broad bean was a variant of CEVd containing 373 nucleotides with ten differences as compared with CEVd-C (Fig. 2a, b) (Gross et al., 1982). Six of these variations (see asterisks in Fig. 2b), four occurring in the C domain, had not been described previously. The change C-+ U at nucleotide 232 (see double asterisk in Fig. 2b) has only been found in two unusual isolates recovered from grapevine (Garcia-Arenal et al., 1987) and tomato (Mishra et al., 1991). Changes U~A, A+ C and G-+ A at nucleotides 234, 263 and 303, respectively have been reported in several other variants (Visvader et at., 1982; Visvader & Symons, 1983, 1985; Semancik et al., 1993). As illustrated by the minimum free energy secondary structure (Fig. 2 b), this broad bean viroid variant presents structural modifications as compared with other CEVd sequences.

3 CEV variant from broad bean 2273 (a),'loocoo ooooo,l,?%oo,oo,... 21;, ooo,oo.ou o+ ;!oo ooo.,o oo JL o olu %o'oi:o,o oo,o.,,oo'l c TAAC, UC A uagt, UGGC C l AG UCUcUUFG A nc cgcuc CGcU cgcoaagu GrU? AGC('UA (- A('(" U 6GUCG ('~CUUU C uuc('ucu G go e ~uev uc, ~'c, mmcc ~ ~c- ucaecva"%cc u %.c.c,a.~"~ac.a ^ ~J~,c~ ~.ce.a e ~u-a~'~u'vceuucag c.a Aucc.ee~c,~o CCU~e~,G.e.~A~.U. I g C UcecUe, e,c-m~c,~c-cc-~,e~ e ucc.c~a~a~a~a. ~ ueauc ev ucvauat't.cvtnpcc, ccaaaeecc~oav- ce,~ A ~ua % ~cc% c G~/CVCuCtrOC%,@C'CC'GCUCqCCe'Gc u ce'~'~'vc%uqc~eccva-c.aecvc'e'~ve~'vc=clnmc uucc~ u ~ _S V I I /A_ LXI ' 3vs z~o ~0 I ~ "u c~v / / / ~ ] c c ' ' ~'~ ~=~, ",!0 / I I /I ~.~/.0 I,,o )'~1,~0 I,0o I ~ ~o 40 eo so too l~o 14o eo ~? ~77 ~.=.ut~? Y?7 ~,Y~Y-.~..- ~ ~ ~ ~ - -~...Y. 77~? 77~77~" -77.~..~Yy... i ~coa~,c.~.r~c.,n,c.cc.~.,,cc.c~'w~ ~.~ e~c.~r ~ucouc~.ucv~ vuc'v ~c~cc ~cccc~ct~a ~.~uc~a~= ue.,ccc. ~'CUCuCU~C~A@C.CC, ~=CU~CCCCo C]~-CC.,~.OUC~c=tra c t~ccwlc.~ccoc-o~vco~c cu~sc~uuccacu~ ,,, ~, t.,, ~, ~,,~,, 'I ~ ~o ~.!o_,~... x_i~ c '... c i i" " -= "~ "~", "1 " ] i / 1 "]\ -<'i,~o I:=o- I,~ol I,;o , &,,okk 'k Fig. 2. Sequence of CEVd isolated from field grown broad beans (b) as compared to the CEVd-C sequence (a) (Gross et al., 1982). Sequences of CEVd variants from inoculated tomatoes (broad bean -+ tomato ;c) and from broad beans inoculated with tomato extracts (broad bean-~ tomato-+ broad bean;d) as compared to the CEVd variant isolated from field grown broad beans (b). Nucleic acid changes and inserts are identified in boxes with arrows pointing at the specific nucleotides. The structural domains (Keese & Symons, 1985) are also indicated: T~ and T~, terminal domains; P, pathogen sis; V, variable; C, central conserved region. When the nucleic acid preparations from broad bean were inoculated on broad bean seedlings, no symptoms were observed but the viroid was detected by RT-PCR analysis. The infected plants remained symptomless throughout the growing cycle (Fig. 3 a, b). Properties of the CEVd variant recovered from broad bean Nucleic acid preparations from broad bean were used to inoculate the CEVd hosts Gynura, tomato, chrysanthemum, cucumber and citron. Infection was demonstrated on tomato, chrysanthemum and cucumber but none of these hosts displayed symptoms (Table 1). Tomato and cucumber yielded the highest viroid titre. Unexpectedly, Gynura, the host from which CEVd was originally isolated (Semancik & Weathers, 1972), and citron, the citrus indicator used for the biological indexing of the exocortis disease, remained uninfected. The viroid titres in chrysanthemum were very low and infection could only be demonstrated by RT-PCR analysis. RT-PCR products obtained from tomato, chrysanthemum and cucumber were subjected to sequence analysis. The tomato and chrysanthemum sequences displayed 97% identity to the CEVd sequence from

4 2274 C. Fagoaga, J. Semanick and N. Duran-Vila (a) (b) (c) (d) Fig. 3. Broad bean plants infected with the original CEVd field variant (a) as compared with uninoculated controls (b). Broad bean plants infected with the CEVd variant after passage through tomato (broad bean -, tomato -> broad bean) (c) as compared with uninoculated controls (co. Table 1. Infectivity of the broad bean variant of CEVd in different hosts Source/passage Detection by history Host Symptoms spage Broad bean (field Gynura - - source) Tomato - + Chrysanthemum - - * Cucumber - + Citron - - Gynura + + Tomato (Broad bean-+tomato) Broad bean (Broad bean+tomato~ broad bean) Tomato + + Chrysanthemum + + Cucumber - + Citron - ~ Gynura + + Tomato + + Chrysanthemum + + Cucumber - + Citron + + * RT-PCR analysis of chrysanthemum extracts was positive. t RT PCR analysis of citron extracts was also negative. broad bean and had a molecular size of 371 nucleotides. The cucumber sequence with a molecular size of 372 nucleotides had an identity of 96 %. The differences in these sequences as compared with the broad bean variant (Fig. 2 b) used as inoculum, are summarized in Table 2. Other changes identified in Table 2 imply that different hosts produced different selection pressures on the viroid population from in the original broad bean isolate. The sequence of the viroid from tomato revealed a characteristic change in nucleotide 50, also identified in other sequences of CEVd from tomato (Visvader & Symons, 1983, 1985; Semancik et at., 1993), deletions in nucleotides 163, 217 and 265, and the insertion of an A residue between residues The sequences from chrysanthemum and cucumber also contained characteristic changes in nucleotides 6, 316 and 279 (chrysanthemum) and in 6, 147, 316 and 331 (cucumber). Effect of host on the biological properties of the broad bean CEVd isolate To identify any changes in biological properties of CEVd as a result of host selection, the extracts from inoculated tomato were subjected to infectivity assays and host range studies. Broad bean seedlings inoculated with nucleic acid preparations after passage through tomato (field broad bean + tomato-~ broad bean) were stunted and had shorter internodes than the uninoculated controls (Fig. 3 c, d). These plants had high viroid titres

5 CEV variant from broad bean 2275 Table 2. Nucleotide changes from the broad bean CEVd variant detected following selection in various hosts* Nucleotide change Domain Residue Tomato Chrysanthemum Cucumber T U U~A C>A 352 C~U C-+U C~U P 50 G>A A-+G A~G U-+A U>G C A C-~A C-+A 265 -G U - -U A U~A U-+A U~-A 291 G+C G-+C G~C V A C~U 217 -A U~C U-+C U C A-+U A-+U Tz 162 A-~U A-+U A-~U 163 -C U-+A U-+A U-+A * Tomato, chrysanthemum and cucumber had been inoculated with extracts from the original broad bean isolate. Table 3. Nucleotide changes from the broad bean CEVd variant following serial transmission through tomato to broad bean of these represented reversions from the source field bean sequence to the sequence of Gross et al. (1982) and two (130 and 218) could be traced to the tomato passage. As supported by the biological evidence, the viroids isolated from broad bean before and after passage through tomato must be viewed as characteristically distinct variants. The variations identified in residues 50, ,262, and 350 in the sequence from inoculated tomato (broad bean-+ tomato) were not retained in the sequence from the derived broad bean isolate after passage through tomato (broad bean~+ tomato-+ broad bean) (Table 3). This variability illustrates how different hosts select viroid structures from the available population of molecules. These changes in the CEVd population as a result of host selection also resulted in changes of their biological properties. The results of host range studies using as inocula nucleic acid preparations from inoculated tomatos (broad bean--tomato) and from inoculated broad bean after passage through tomato (broad bean tomato -+ broad bean) are summarized in Table 1. Passage through tomato resulted in an expansion of the host range and the severity of the isolate (Table 1) as compared to the properties of the original field broad bean source (Table 1). Tomato Broad bean Discussion Nucleotide Nucleotide Domain Residue change Residue change T C + U - - P 50 G-~A - - C A G G U A 287 U-~A 287 U~A 289 G -+ C 289 G -~ C V 130 +A 130 +A A A 231 U-+C 233 U-+C T A-~U 163 A+U C U-~A 180 U~-A comparable to those recovered from inoculated tomatoes and distinctly different from the titres observed in the source field broad bean. Viroid sequence analysis of inoculated broad bean extracts (broad bean-~ tomato ~ broad bean) demonstrated that the nucleotide sequence differed from those of the original field source and the inoculated tomato (broad bean-+ tomato) (Fig. 2c, d). From a total of 13 changes identified in the CEVd sequence from inoculated tomato, eight were retained in the inoculated broad bean after passage through tomato (Table 3). However, seven It is not possible here to assert definitively whether selection has ocurred from a population of sequence variants and/or new sequences have arisen during the passage through different hosts. Nevertheless, the experimental approach of sequencing a random RT-PCR product provides an overall indication of the predominant sequence favoured in a particular host. The CEVd found in field broad beans contains six changes unique to this isolate. The differences found in the V and T 2 domains suggest a more relaxed secondary structure than typical of CEVd strains. The CEVd broad bean variant is characterized by very low titres in symptomless broad beans which was consistent with reinoculation to broad bean seedlings. The CEVd-broad bean displayed an unusually narrow host range for a CEVd variant as well as an inability to induce symptoms in species recognized as very sensitive to CEVd infection. Given the very small non-translated genomes of viroids, subtle variations in nucleotide sequence may impact more critically on molecular conformation and resultant biological properties (Visvader & Symons, 1986; Owens et al., 1986; Hammond & Owens, 1987; Ishikawa et al., 1985). The results presented here illustrate how a shift in the composition of the viroid population, owing to host selection, results in major

6 2276 C. Fagoaga, J. Semanick and N. Duran-Vila changes in biological properties (infectivity and symptoms) as postulated by Semancik et al. (1993). Although several changes were identified in the C domain, none was located in upper part (nucleotides ) which can form the hairpin loop I involved in the processing of viroid intermediates (Branch et al., 1981; Owens & Diener, 1982). In fact point mutation studies with infectious cdna viroid clones demonstrated that this region, which contains two inverted repeats, is strictly conserved and that nucleotide changes in this region of the C domain resulted in total impairment of viroid infectivity (Visvader & Symons, 1986; Hammond & Owens, 1987). Data reported here indicate that sequence differences in the lower part of the domain (nucleotides ) did not suppress viroid replication in broad bean but the CEVd variant containing these changes replicated and/ or accumulated inefficiently. Four of the five nucleotides (265, 280, 289 and 291) are unique for the broad bean source variant. These were not conserved after passage through alternate hosts resulting in the recovery of a distinctly different variant in broad bean from the original broad bean source. This observation is especially striking in the comparison of the field bean source isolate with the derived bean variant after passage through tomato. All of the characteristic changes noted in the field source reverted to the sequence of Gross et al. (1982) along with the retention of only two additional changes (130 and 218) introduced in the tomato passage. As illustrated by the minimum free energy secondary structure (Fig. 2 b), these changes may actually affect the viroid secondary structure and interfere with the premelting region 2 located in the C domain (Schn61zer et al., 1985). The variation in the nucleotide sequence of the source broad bean CEVd suggests a more relaxed secondary structure with larger single-stranded regions in the V domain and the Tz loop. The loss of infectivity of PSTVd when the terminal loop of the T 2 domain of PSTV was affected by mutations, insertions and deletions suggests a critical role for this domain (Hammond & Owens, 1987). The unique variations at nucleotides 162 and 179 in the T 2 domain of the source broad bean variant did not prevent viroid infectivity but did affect replication and/or acumulation in broad bean. Construction of enlarged terminal loops in the T~ domain of PSTVd also resulted in loss of infectivity (Hammond & Owens, 1987). The A ~-U change identified in nucleotide 179 of the original broad bean isolate affects the viroid secondary structure resulting in the formation of a nine nucleotide terminal loop in the T 2 domain (Fig. 2 b). The viroid variants (introduced by passage through alternate hosts resulting in the recovery of variants after passage through tomato) replicated, accumulated efficiently and were highly pathogenic in broad bean, a distinctly different property from the source field bean variant. This observation would caution against the random choice and use of alternate hosts. Although molecular studies may be facilitated by enhanced replication and accumulation, fidelity to the unique properties of an unusual source isolate may be sacrificed. The lack of symptom expression of the original broad bean variant in broad bean may be correlated with the low viroid titres, which may in turn be associated with the unique nucteotide exchanges in the C and T 2 domains and their effect on viroid secondary structure, replication efficiency and accumulation. It has been demonstrated (Semancik & Szychowski, 1994) that the different symptoms of the syndrome are associated with the accumulation of avocado sunblotch viroid (ASBVd) variants containing significant differences in the right terminal loops of the T 2 domain. It has been already demostrated that CEVd can persist in a number of sequence variants in a single infected plant (Visvader & Symons, 1985), whereas potato spindle tuber viroid (PSTVd) is thought to be highly stable and single point mutations result in the impairment of infectivity and replication efficiency (Hammond & Owens, 1987; Owens et al., 1986).The unique sequence differences of the source broad bean isolate of CEVd do not cause an impairment of viroid survival in broad bean. Other CEVd variants have also been identified in four vegetable crop species (C. Fagoaga, unpublished results). These can be perpetuated as undetected nonpathogenic CEVd populations in certain hosts. 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