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Virology 417 (2011) 400–409

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Virology

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In vivo generated exocortis progeny variants display a range of phenotypes with altered levels of replication, systemic accumulation and pathogenicity

Subhas Hajeri a,1, Chandrika Ramadugu b, Keremane Manjunath c, James Ng a, Richard Lee c, Georgios Vidalakis a,⁎

a Department of and Microbiology, University of California, Riverside CA 92521, USA b Department of Botany and Plant Sciences, University of California, Riverside CA 92521, USA c USDA ARS National Clonal Germplasm Repository for Citrus and Dates, Riverside, CA 92507, USA

article info abstract

Article history: Citrus exocortis viroid (CEVd) exists as populations of heterogeneous variants in infected hosts. In vivo Received 26 January 2011 generated CEVd progeny variants (CEVd-PVs) populations from citrus protoplasts, seedlings and mature Accepted 13 June 2011 , following inoculation with transcripts of a single CEVd cDNA-clone (wild-type, WT), were studied. The Available online 22 July 2011 CEVd-PVs population in protoplasts was heterogeneous and became progressively more homogeneous in seedlings and mature plants. The infectivity and pathogenicity of selected CEVd-PVs was evaluated in citrus Keywords: Viroid RNA evolution and herbaceous experimental hosts. The CEVd-PVs U30C, G128A and U182C were not infectious; G50A and Population composition 108U+ were infectious but reverted back to WT and 62A+, U129A and U278A were infectious, genetically Genomic diversity stable and more severe than WT. The 62A+ and U278A and U129A accumulated at higher levels than WT in Infectivity protoplasts and seedlings respectively. The effect of specific mutations on the predicted secondary structure of RNA secondary structure the CEVd-PVs' RNA coupled with the infectivity and replication studies suggested complex structure-to- Citrus protoplasts function relationships for CEVd. ‘ ’ © 2011 Elsevier Inc. All rights reserved. Gynura Chrysanthemum

Introduction (Semancik and Weathers, 1972). CEVd is the largest of the citrus with 371–475 nt and belongs to the genus of the family Viroids are the smallest known plant infectious agents (246–475 nt) (Flores et al., 1998; Semancik et al., 1994; Semancik and and have a single stranded, circular, highly structured, non-- Duran-Vila, 1999; Szychowski et al., 2005). coding, non-encapsidated RNA . Viroid have approx- All members of Pospiviroidae have a quasirod-like secondary imately equimolar amounts of Watson–Crick base pairs in addition to structure of minimal free energy with five structural-functional non-canonical GU base pairing resulting in a self-complementary rod- domains; the left-terminal (TL); pathogenicity (P); central domain (C) like or quasi-rod like structure (Flores et al., 2005; Semancik et al., 1975; containing a region conserved in many related viroid species; variable Tabler and Tsagris, 2004). (V); and right terminal (TR) (Keese and Symons, 1985; Sano et al., Viroids have been identified as the etiologic agents for a variety of 1992). In addition to these domains, viroid genomes possess additional maladies in agriculturally significant crops. In citrus, exocortis is an structural features such as imperfect palindromes, pre-melting (PM) important viroid disease of the commonly used rootstock, Poncirus regions and thermodynamically metastable structures. The functional- trifoliata and is characterized by bark scaling, leaf epinasty, yellow ity of viroid structural features has been demonstrated in several blotching of twigs, and severe tree stunting (Singh et al., 2003). Citrus members of Pospiviroidae in infectivity (Candresse et al., 2001; exocortis viroid (CEVd), the causal agent of the exocortis disease Hammond and Owens, 1987; Loss et al., 1991; Wassenegger et al., (Fawcett and Klotz, 1948), was the first characterized citrus viroid 1996; Zhu et al., 2002), movement (Hammond, 1994; Zhong et al., 2008), symptom expression (Qi and Ding, 2002, 2003; Wassenegger et al., 1996; Zhu et al., 2002), and replication (Kolonko et al., 2006; Owens ⁎ Corresponding author at: Department of and Microbiology, College et al., 1996; Riesner, 1990; Zhong et al., 2006). of Natural and Agricultural Sciences, University of California, Riverside, 900 University Viroids of the Pospiviroidae family replicate in the nucleus by host Avenue, Riverside CA 92521, USA. Fax: +1 951 827 4294. DNA-dependent RNA polymerase II. They rely on their conserved RNA E-mail address: [email protected] (G. Vidalakis). 1 Present address: University of Florida, IFAS, Citrus Research & Education Center, secondary structures (i.e. hairpin I and loop E) and cellular factors for Lake Alfred, FL 33850, USA. processing the multimeric replication intermediates into unit length

0042-6822/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.virol.2011.06.013 S. Hajeri et al. / Virology 417 (2011) 400–409 401 molecules that are subsequently ligated to form circular infectious CEVd-WT replication in citrus protoplasts at early progeny viroids (Flores and Semancik, 1982; Flores et al., 2004; Gas et Using the methods of Grosser and Gmitter (1990) and Grosser et al., 2007; Mühlbach and Sänger, 1979; Warrilow and Symons, 1999). It al. (2000), we were able to reliably obtain up to 40 million protoplasts is generally accepted that viroids replicate as polymorphic populations from a 40 ml suspension culture of C. amblycarpa (viability over which are described as variations around a master sequence (Ambrós et 90%). In vitro transcripts of a single CEVd-WT cDNA-clone were al., 1998; Codoner et al., 2006; Eigen et al., 1989) in accordance with the transfected into protoplasts and the relative levels of viroid progeny “quasispecies model” proposed by Eigen (1993). The main factors were monitored with a newly developed RT-qPCR assay. responsible for the observed genetic diversity during viroid replication The observed increase of the viroid RNA titer (2.5 fold) between are most likely the absence of proof-reading activity of the plant days 1 and 2 post-transfection (dpt) was not statistically significant polymerase(s), the large population size, and the rapid rate of RNA (p=0.720). Subsequently, the CEVd-WT progeny RNA (positive sense replication (Codoner et al., 2006; Domingo and Holland, 1997; Gandía strands) increased significantly by 80, 100 and 130 folds at 3, 4, and 5 and Duran-Vila, 2004; García-Arenal et al., 2001; Holland et al., 1982). dpt respectively (F=134.116, d.f.=14, pb0.001) (Fig. 1). Similarly, Genomic diversity of CEVd RNA populations at the whole-plant the increase of replicative intermediates (negative sense strands) was level has been studied extensively in the past (Gandía et al., 2005, significant after 2 dpt (F=134.116, d.f.=14, pb0.05 and 2 dpt vs. 1 2007; Semancik et al., 1993; Visvader and Symons, 1985). In an dpt, p=0.159). The ratio of positive to negative sense viroid strands elegant small-scale evolutionary experiment conducted over a period varied with incubation time and peaked at 3 dpt with a ratio of 15:1 of 10 years, Bernad et al. (2009) demonstrated that the CEVd (Fig. 1). The lowest detectable amount of progeny viroid RNA by RT- population profiles changed drastically in susceptible and tolerant qPCR was 20 attograms (20×10− 12 μg) per μg total RNA. The results citrus hosts. However, little is known about the genomic diversity of of the population composition and genomic diversity analysis for the the replicating viroid RNA populations at the cellular level. It is CEVd-WT progeny RNA at the cellular level are presented in Table 1. conceivable that the selection pressure is much greater and more complex at the plant level as compared to the cellular level in protoplasts. An obvious difference in protoplasts, in comparison with CEVd-WT systemic accumulation in citrus seedlings at early infection the whole plant, is the absence of both cell-to-cell and long distance The stem of four-week old tube-grown ‘Etrog’ citron seedlings movement. Thus, protoplasts transfected with viroid RNAs offer a were slash inoculated with in vitro transcripts of a single CEVd-WT synchronous system to study the population composition and cDNA-clone and the systemic accumulation of the viroid progeny RNA genomic diversity at the cellular level (Aoki and Takebe, 1969; was monitored with RT-qPCR assay. Faustmann et al., 1986; Flores et al., 2008; Lin and Semancik, 1985; CEVd-WT progeny RNA was detectable in the stem, at all time Mühlbach, 1982; Mühlbach and Sänger, 1977; Qi and Ding, 2002; points tested, and the titer increased significantly with time from day Zhong et al., 2006, 2008). Such a system is very valuable when it is 1 to day 5 (F=730.334, d.f.=14, pb0.001) (Fig. 2). At the end of coupled with a rapid, sensitive and quantitative method for 5 days post inoculation (dpi), a 35 fold increase in viroid titer was monitoring viroid replication. The number of reports on reverse- recorded at the stem (Fig. 2B). CEVd-WT progeny RNA was first (RT) quantitative polymerase chain reaction (qPCR) detected in the roots by the end of 3 dpi and interestingly, the viroid methods for the quantitative detection of citrus viroids is very small RNA accumulation did not increase significantly with time in the root and limited to the whole-plant level (Rizza et al., 2009). tissue (pN0.05) (Fig. 2B). CEVd-WT progenies were detected in the In the present study, three different citrus experimental systems: leaves of the seedlings only at 5 dpi (H=13.796, d.f.=4, p=0.008) protoplasts, seedlings and mature plants were utilized. Following (Fig. 2B). At five dpi, the CEVd-WT progeny RNA accumulation in inoculation with transcripts of a single CEVd cDNA-clone (wild-type, stems was significantly higher than roots and leaves (F=3776.693, WT), a large number of in vivo generated CEVd progeny variants d.f.=8, pb0.001) while leaves accumulated slightly higher viroid titer (CEVd-PVs) was analyzed for the estimation of the population in comparison to the roots (F=3776.693, d.f.=8, p=0.046) (Fig. 2B). composition and genomic diversity of the CEVd progeny RNAs. The infectivity and pathogenicity of selected CEVd-PVs from the recovered progeny populations were evaluated in citrus and herbaceous experimental hosts. Their replication and systemic accumulation were evaluated using a newly developed RT-qPCR assay. The possible effects of specific mutations on the predicted secondary structure of the CEVd-PVs' RNA and their concomitant effects on the observed biological properties are also discussed.

Results

CEVd-WT

The parental CEVd RNA inoculum, which is referred as CEVd-WT in this study, was obtained from the viroid collection of the Citrus Clonal Protection Program (CCPP) at the University of California, Riverside (UCR). CCPP's sweet (Citrus sinensis) source tree of CEVd isolate E-811 has been repeatedly tested positive for a single disease Fig. 1. Replication of Citrus exocortis viroid wild-type (CEVd-WT) in citrus protoplasts agent for over 25 years. Viroid RNA from the source tree was isolated, (Citrus amblycarpa Ochse.) was monitored by reverse transcription quantitative reverse-transcribed and the amplified full length cDNA was cloned polymerase chain reaction. Relative levels of replicating CEVd progeny RNA (+ sense, and sequenced. The most abundant RNA (83%) identified by the represented by squares) and their replicative intermediates (− sense, represented by sequencing of 30 CEVd E-811 cDNA clones was used as WT inoculum triangles) were monitored over a period of five days post-transfection (dpt). The accumulation levels of CEVd progeny RNA after 1 dpt was arbitrarily set to the value of in the present study. The nucleotide sequence of the CEVd-WT was one. The levels of CEVd progeny RNA from subsequent days were presented as relative deposited in the genetic sequence database at the National Center for levels to this reference value. Data points present the means and standard deviation of Biotechnical Information (NCBI) (GenBank ID: GU295988). triplicate experiments. 402 S. Hajeri et al. / Virology 417 (2011) 400–409

Table 1 The frequencies of the non-variable and variable CEVd progenies in the Population composition and genomic diversity of Citrus exocortis viroid (CEVd) progeny citrus protoplasts and seedlings, were not significantly different (χ2, RNA in different citrus experimental systems. pN0.05). On the other hand, non-variable CEVd progenies recovered Citrus Incubation CEVd progeny sequencesc Genomic from mature plants had significantly higher frequencies in comparison systema periodb diversity χ2 b Total Non- Variable to the variable progenies ( =24.200, p 0.0001, d.f.=1) (Table 1). (%)d analyzed variable The genomic diversity of the CEVd-WT populations was significantly Total Unique altered (χ2 =8.857, pb0.05, d.f. =2) across the different citrus e Protoplasts 5 dpt 80 36 44 32 40.0 experimental systems at early (protoplasts and seedlings) and late Seedlings 5 dpi 80 48 32 21 26.2 Mature plants 10 wpi 80 62 18f 14 17.5e infection stages (mature plants) (Table 1). The diversity of CEVd-WT progenies in protoplasts was significantly higher than the mature plants a Protoplasts: Isolated from suspension cell culture of Amblycarpa mandarin (Citrus (χ2 =8.345,pb0.05, d.f.=1). On the other hand, the diversity of CEVd- amblycarpa Ochse.); Seedlings: Test tube grown citron seedlings (C. medica L.)— 4 weeks old (7–8 cm long 2–4 true leaves); Mature plants: grafted onto Rough WT progenies between protoplasts and seedlings as well as between 2 rootstock (C. jambhiri Lush.)—18 months old. seedlings and mature plants was not significantly different (χ , b dpt: days post transfection; dpi: days post inoculation; wpi: weeks post pN0.05). Overall it was evident that the CEVd-WT progeny population inoculation. was diverse in the protoplast system and became progressively more c Total analyzed: total number of CEVd cDNA clones sequenced; Non-variable and Variable: number of CEVd progeny sequences identical and different from the parental homogeneous in the seedlings and mature plant systems. sequence respectively. d Genomic diversity = (unique/total analyzed)×100. e 2 Statistically significant differences (χ ,pb0.05). CEVd-PVs f Statistically significant differences (χ2,pb0.0001). Over 200 in vivo generated CEVd-PVs in CEVd-WT inoculated citrus protoplasts, seedlings and mature plants were cloned and Replicative intermediates were significantly higher in stems fol- sequenced. The protoplasts system alone contributed 50% of the lowed by leaves and roots at the ratio of 30:10:4 at 5 dpi (F=441.354, mutations generated among the three experimental systems. Eleven d.f.=8, pb0.001; data not shown). The results of the population mutated sites were common between the protoplasts and the composition and genomic diversity analysis for the CEVd-WT progeny seedlings systems, while only one was common between the RNA at the seedling level are presented in Table 1. seedlings and the mature plants. The mutated sites 62, 129, 265 and 278 were common among all three systems (Table 2). CEVd-WT population composition and genomic diversity in mature citrus Mutated sites were distributed unevenly on the five structural plants at late infection and citrus protoplasts and seedlings at early infection domains. Up to 60% of the mutated sites were found on the TL and P The standard citrus viroid host, ‘Etrog’ citron was slash inoculated domains. The V domain had the smallest number of muted sites and the with in vitro transcripts of a single CEVd-WT cDNA-clone. The host C and TR had almost equal number of mutated sites (Table 2).The type of expressed classic exocortis symptoms such as leaf epinasty, rugosity and mutation per site was consistent with two exceptions on sites 10 and stunting at 10 weeks post inoculation (wpi) (Fig. 3A2). The majority 128. Transitions and transversions accounted for 82% of the observed (77.5%) of the recovered CEVd-WT progenies from the symptomatic mutations among the three experimental systems. In the case of citrus citrons was identical to the parental inoculum (non-variable). A high protoplasts and seedlings (early infection stage) transitions and percentage (77.8%) of the variable progenies were unique in the tested transversions accounted for 81.3 and 81.0% respectively while in the population and the overall CEVd genomic diversity was low (Table 1). mature citrus plants (late infection stage) for 90.9% (Table 2).

Fig. 2. Systemic accumulation of Citrus exocortis viroid wild-type (CEVd-WT) in four-weeks old test tube-grown seedlings (7–8 cm long, 2–4 true leaves) of ‘Etrog’ citron (Citrus medica L.) was monitored by reverse transcription quantitative polymerase chain reaction. (A) The seedlings were divided in to three parts namely leaves (L), stem (S) and root (R). The lower part of the S (4–5 cm from the root) was the site of inoculation (inset). (B) Relative accumulation levels of CEVd progeny RNA were monitored in L, S and R over a period of five days post-inoculation (dpi). The level of progeny RNA accumulation in the S at the first dpi was arbitrarily set to the value one. The levels of progeny RNA from L and R from subsequent days were presented as relative value to this reference value. Means and standard deviation of triplicate experiments are presented. S. Hajeri et al. / Virology 417 (2011) 400–409 403

132 A CEVd-WT CEVd-PVs: CEVd-PVs: 62A+ G50A U129A 108U+ A265G 128G- U278A A185U

B CEVd-WT CEVd-PVs: CEVd-PVs: 62A+ G50A U129A 108U+ U278A 128G- A185U A265G

C CEVd-WT CEVd-PVs: CEVd-PVs: 62A+ G50A U129A 108U+ A265G U278A

Fig. 3. Citrus exocortis viroid (CEVd) experimental hosts. (A) ‘Etrog’ citron (Citrus medica L.), (B) gynura (Gynura aurantiaca DC) and (C) chrysanthemum (Dendrathema morifolium)cv Bonnie Jean. Column 1: mock-inoculated controls; Columns 2 and 3: symptom expression after inoculation with in vitro transcripts of CEVd wild-type (WT) and CEVd progeny variants (CEVd-PVs).

CEVd-PVs infectivity, genomic stability and pathogenicity The CEVd-PV U278A (transversion-C domain) was predicted to Based on the results of the sequence analysis, 11 CEVd-PVs with enlarge the loop adjacent to the conserved loop E motif (Supplemen- different types of mutations (i.e., addition, deletion, transition and tary Figures). The CEVd-PV U278A accumulated at significantly higher transversion) in different viroid structural domains were selected for level than the CEVd-WT and all the other CEVd-PVs tested in biological characterization. protoplasts (F=138.021, d.f.=14, pb0.001). Its systemic accumula- In vitro transcripts of selected CEVd-PVs were slash inoculated onto tion in seedlings was similar to CEVd-WT (p=0.182) and 62A+ and citron, gynura and chrysanthemum. The CEVd-PVs displayed distinct A265G (p=0.196 and 0.233, respectively) (Fig. 4B). infectivity and genomic stability properties that placed them into three The CEVd-PV A265G (transition-central conserved region-C domain) categories. I. CEVd-PVs (U30C, G128A and U182C) were not infectious did not affect the predicted Loop E motif of the CEVd-WT counterpart in all three hosts tested both as monomeric and dimeric transcripts. (Supplementary Figures), and the levels of its replication was similar to II. CEVd-PVs (G50A, 108U+, 128G−, and A185U) infected at least two of the CEVd-WT and U129A (p=0.352 and 0.417, respectively). The CEVd- the three experimental hosts but they were genetically not stable, since PV A265G systemic accumulation was similar to the CEVd-WT the progeny RNAs did not retain the original mutation. III. CEVd-PVs (p=0.755) and 62A+and U278A (p=0.685 and 0.233, respectively) (62A+, U129A and U278A) were infectious in all three hosts and the (Fig. 4B). progeny viroid RNAs maintained the original mutation (Table 3). The CEVd-PV U129A (transversion-V domain) affected the predicted Typical CEVd symptoms (i.e., leaf epinasty, rugosity and stunting) thermodynamic RNA secondary structure (Supplementary Figures), and were observed in all experimental hosts at 8–12 wpi for the category II it was accumulated at the CEVd-WT and A265G levels in the protoplasts CEVd-PVs (reverted back to CEVd-WT) (Fig. 3). The CEVd-PVs 62A+, (p=0.740 and 0.417, respectively). The systemic accumulation of the U129A and U278A (category III, genetically stable) exhibited more CEVd-PV U129A was significantly higher than the CEVd-WT and all the severe CEVd symptoms in all experimental hosts in comparison to the other CEVd-PVs tested (F=42.362, d.f.=14, pb0.001) (Fig. 4C). CEVd-WT. Of specific interest was the CEVd-PV A265G that was infectious and genomically stable in all three tested hosts; induced Discussion severe symptoms only in citron and chrysanthemum but not in gynura (Table 3 and Fig. 3). A reliable synchronous infection system with a short incubation period was developed for the study of replication and systemic CEVd-PVs RNA secondary structure, replication, and systemic accumulation accumulation of CEVd. Asymmetry in the rate of synthesis of positive The predicted thermodynamic secondary structure of CEVd-PV to negative sense CEVd-WT RNA strands was observed similarly to the 62A+ (addition-P domain) was altered to a small degree (Fig. 4 and Potato spindle tuber viroid (PSTVd) and positive-strand RNA Supplementary Figures). The CEVd-PV 62A+ RNA accumulation at the (Mandahar, 2006; Qi and Ding, 2002). Systemic accumulation of the cellular level was significantly higher than the CEVd-WT and the CEVd in the young citrus seedlings occurred initially in the roots and A265G and U129A (F=138.021, d.f.=14, pb0.001). The systemic subsequently in the shoots similarly to mature citrus plants (Gafny et accumulation of the CEVd-PV 62A+ in seedlings was similar to that of al., 1995). the CEVd-WT (p=0.747) and A265G (p=0.685) but significantly The three citrus systems utilized in this study captured the effects of lower than U129A (F=138.021, d.f.=14, pb0.001) (Fig. 4B). different biological selection pressures on the viroid genome diversity. 404 S. Hajeri et al. / Virology 417 (2011) 400–409

Table 2 Visvader and Symons, 1985, 1986). In the present study, we used the De novo generated mutations of Citrus exocortis viroid (CEVd) progeny RNA in different novel approach of in vivo generated CEVd-PVs from three citrus citrus experimental systems. systems (natural host of CEVd) that applied different selection CEVd RNA Citrus systema pressures during replication, intra- and inter-cellular movement. fi Domain Nucleotide position Protoplasts Seedlings Mature plant This approach provided a unique insight into the effect of speci c mutations on the predicted secondary RNA structure and its Left terminal 10b −UUNC 12 UNC concomitant effect on infectivity, replication, systemic accumulation, 15 ANG and pathogenicity of naturally occurring CEVd-PVs at early and late 16 GNA infection points. 19 UNC One of the interesting groups of CEVd-PVs was the one recovered 22 UNA from the two early infection systems but not from the late infection one 30b UNCUNC 32 ANG (i.e. U30C, G128A, and U182C). These variants were not infectious in the 42 −G three plant species tested, including citrus, even though they were 335 CNU originally identified in citrus protoplasts and seedlings. It is possible that N 339 A U these variants were generated at the early stages of the CEVd-WT 367b GNAGNA Pathogenic 50b GNAc GNAc infection and replication in the two systems with the lowest selection 53b ANGANG pressure but they were not viable or stable during longer incubation 55 UNA periods in mature plants. It is logical to hypothesize that mature plants 58 ANU applied stronger selection pressures on the viroid than the protoplasts 59 ANG and young seedlings due to more advanced tissue structures and/or 61 −A 61b ANGANG more complex protein and enzymatic activities. The lack of infectivity 62d +A +A +Ac for these variants can be attributed to two factors unrelated to the CEVd 301 UNA RNA secondary conformation since only two out of the three mutations N 302 A G affected the predicted RNA structure (Supplementary Figures). First, 303 CNU 308 UNC they were generated in planta after infection with CEVd-WT thus they 313e CNUCNU did not have to initiate de novo infection for their proliferation. Second, Central 108b +G and +Uc +G and +Uc their infectivity could have been affected by the type of the mutation. 114 UNA The presence of the CEVd-PVs 128G- and G128A supports the later point 263 CNG since a different mutation at the same position resulted into an 264 UNA 265d ANGANGANGc infectious variant. 277 ANU The infectious CEVd-PVs provided interesting clues for the associ- 278d UNAUNAUNAc ation of RNA structures to viroid functions. For example, the mutations b N − Variable 128 G A G G50A and 108U+ are located adjacent to the metastable PM I and in the 129d UNAUNAUNAc 130b ANUANU middle of the HP I structure, respectively. Both HP I and PM I play an 234 ANU important role in viroid biology since they have been associated to Right terminal 159 GNU infectivity and subsequently to disease induction (Hammond and 160 CNG Owens, 1987; Owens et al., 1996) respectively. The G50A mutation 162 UNC resulted in the merging of loops 10 and 11 while the 108U+ introduced 182b UNCUNC 185b ANUc ANU a loop (number 21) in to the CEVd genome (Supplementary Figures). It 194 UNC is possible that these secondary structural changes interfered with the

a Protoplasts: Isolated from suspension cell culture of Amblycarpa mandarin (Citrus formation of PM I and HP I and as a result the mutations that induced amblycarpa Ochse.); Seedlings: Test tube grown citron seedlings (C. medica L.)—4 weeks them were reverted back to wild-type. old (7–8cmlong2–4 true leaves); Mature plants: Citrons grafted onto While CEVd infectivity appeared to be independent of RNA rootstock (C. jambhiri Lush.)—18 months old. secondary structural features, our data indicated the opposite for b Common mutated sites between protoplasts and seedlings. CEVd replication. The CEVd-PV 62A+ accumulated at significantly c Mutation occurred more than once. d Common mutated across all the three systems. higher levels in protoplasts and induced more severe symptoms than e Common mutated sites between seedlings and mature plants. the CEVd-WT. These results emphasized the importance of the polypurine-rich sequence in the modulating-VM region for the regulation of viroid replication (Gross et al., 1982). Bernad et al. (2009) also suggested that the 62A+ mutation may have contributed to The genomic diversity of CEVd-WT in protoplasts was almost double a more relaxed secondary RNA structure leading to altered viroid that of in seedlings and mature plants, most likely due to reduced interactions with host factors that created and proliferated one of the selection pressure at the cellular level. A similar phenomenon has also three principal CEVd haplotypes in two citrus hosts over a period of been observed with the significantly larger and more complex RNA 10 years. The CEVd-PVs A265G and U278A also showed higher levels of genomes of plant viruses like Cucumber mosaic and Tobacco mosaic replication than CEVd-WT. Both mutations were associated with the virus that are not dependent on host RNA polymerases for their loop E motif that has been shown to play a key role in the ligation replication (Li and Roossinck, 2004; Schneider and Roossinck, 2001). process during CEVd and PSTVd replication (Baumstark and Riesner, This indicates that systemic movement plays an important role in 1995; Gas et al., 2007). RNA bases in the viroid genomes are known to determining the population composition and genomic diversity of plant form Hoogsteen base pairs. The Hoogsteen edges of A265/A101 of CEVd- RNA in general. WT, equivalent to A258/A100 of PSTVd, interact to form a symmetric Slight changes in the viroid nucleotide sequence could affect the trans-Hoogsteen/Hoogsteen (Zhong et al., 2006). The overall molecular conformation and concomitantly influencing critical mutation A265G still allowed hydrogen bonding with A101, thus it viroid-host interactions (Ding, 2009; Flores et al., 2005). Previous CEVd did not disrupt the loop E structure. A similar mutant at an equivalent studies on this subject were mostly conducted with chimeras or position in loop 15 of PSTVd (A258/A100) showed no interference with recombinant viroid sequences created in the laboratory using a single, viroid processing activity (Schrader et al., 2003). The U278A mutation is non-woody, annual host (Candresse et al., 2001; Sano et al., 1992; located just adjacent to the loop E motif and maps to a region S. Hajeri et al. / Virology 417 (2011) 400–409 405

Table 3 Infectivity and genetic stability of in vivo generated Citrus exocortis viroid progeny variants (CEVd-PVs).

CEVd-PVs and categories CEVd experimental hostsa

Citron Gynura Chrysanthemum

IGSIGSIGS

I. U30C − n.a. − n.a. − n.a. G128A − n.a. − n.a. − n.a. U182C − n.a. − n.a. − n.a. II. G50A + − + − + − 108U+ + − + − + − 128G− + − + −−n.a. A185U + − + −−n.a. III. 62A+ + + + + + + U129A + + + + + + A265G + + + + + + U278A + + + + + +

I: infectivity; GS: genomic stability. n.a: not applicable. a ‘Etrog’ Citrons (Citrus medica L.) grafted on rough lemon (C. jambhiri Lush.) rootstock; Gynura (Gynura aurantiaca DC); Chrysanthemum (Dendranthema grandiflora Tzvelev.) cv. “Bonnie Jean”. For each progeny variant, three plants were used. comparable to loop13 of PSTVd, which is known to be essential for Inc.). Freshly prepared RNA transcripts (1–8 h), free of DNA template, optimal replication of the viroid (Zhong et al., 2006). were spectrophotometrically quantified and used for inoculation of Viroid structural features, host protein-viroid RNA interaction and/ citrus protoplasts, seedlings and mature plants. or RNA silencing have been implicated in viroid pathogenicity. Four CEVd-PVs with mutations in P, C, and V domains induced more severe CEVd-PVs symptoms than the CEVd-WT. This biological data of in vivo generated progeny variants indicated that the symptom expression is probably In vivo generated CEVd-PVs RNA, following inoculation of citrus not regulated by a single domain in CEVd. It is possible that sequences protoplasts, seedlings and mature plants with a single CEVd-WT cDNA in the P domain may interact with other domains for the symptom clone, was isolated, reverse-transcribed, cloned and sequenced. Based expression regulation. Owens et al. (1996) hypothesized that the VM on the sequence analysis, 11 CEVd-PVs were selected for biological region (P domain) may modulate the accessibility of a second region characterization. For the production of plus-sense CEVd-PV RNA which acts as the primary site of viroid-host interactions. On the other transcripts, the pSTBlue (EMD Chemicals Inc.) containing the hand, we did not detect any stable CEVd variants with mutations in CEVd-PV sequence was linearized with SalIorSnaBI restriction regions predicted to form significant structures like HP I and HP II. In a enzyme (New England Biolabs Inc.) and the linearized plasmid was small RNA entity like the viroid, it is conceivable that different used as template for run-off transcription with T7 or SP6 polymerase domains and structures interact with each other and/or host factors to (MEGAscript® Kits, Ambion Inc.), based on the orientation of the determine virulence. cloned sequence. Freshly prepared RNA transcripts were utilized as Previous studies have shown that the viroids are activators and described above (CEVd-WT section). targets of RNA silencing (Itaya et al., 2001; Markarian et al., 2004; Papaefthimiou et al., 2001; Wang et al., 2004). In addition, the importance of RNA silencing in viroid pathogenicity has been Replication study system: citrus protoplasts demonstrated in non-citrus systems (Diermann et al., 2010; Wang et al., 2004). A BLAST search with the CEVd sequence in expressed Citrus protoplasts were isolated from suspension culture of the sequence tags (ESTs) database revealed numerous sequences from citrus cultivar Amblycarpa mandarin (C. amblycarpa Ochse.) by the citrus and several other plant species that have 18- to 27-nt identities procedures of Grosser and Gmitter (1990) and Grosser et al. (2000). with the CEVd sequence (Supplementary Table). In total, 97% of these An enzyme solution composed of 13% mannitol, 2% Cellulase short sequences corresponded to the P (most of them in the VM region, “Onozuka” RS and 2% Macerozyme R10 (Yakult Pharmaceutical 74%), C, and V domains. Interestingly enough, these same domains are Industry Co. Ltd) was used to digest the suspension cell culture. the ones that accumulated all the mutations that generated the more Following the sucrose/mannitol gradient, the viable protoplasts were virulent CEVd-PVs. It is possible that CEVd siRNAs are acting as miRNAs washed and counted on a haemocytometer by staining the protoplasts and silencing physiologically important citrus producing plant with fluorescein diacetate dye (Widholm, 1972). 5 μg of CEVd-WT in symptoms while the accumulation of mutations on the genome areas vitro transcripts in 50 μl DEPC water (0.1% diethylpyrocarbonate) was involved in the siRNA generation is affecting this mechanism of viroid used to transfect 0.5 million citrus protoplasts using polyethylene pathogenesis. glycol–mediated (PEG 8000) transfection assay (Albiach-Marti et al., 2004; Price et al., 1996). The protoplasts transfected with CEVd-WT Materials and methods transcripts were incubated for 1 to 5 days in the dark without shaking at 28 °C. At every 24 h interval, transfected protoplasts were CEVd-WT harvested from three wells (or replicates). Mock-transfected pro- toplasts and protoplasts transfected in the absence of PEG served as The most abundant RNA identified by the sequencing of CEVd E-811 controls. Based on a pilot experiment conducted with CEVd-WT, 5 dpt cDNA clones was used as WT inoculum (NCBI GenBank ID: GU295988) was selected as the standard time point for analysis of replication of in the present study. For the production of plus-sense CEVd-WT RNA the in vivo generated CEVd-PVs. Total RNA was extracted from the transcripts, the pSTBlue plasmid (EMD chemicals inc.) containing the transfected protoplasts using the Trizol (Invitrogen Corp.) extraction CEVd-WT sequence was linearized with SalI restriction enzyme (New method (Chomczynski and Sacchi, 1987) and treated with RQ1 England Biolabs Inc.). The linearized plasmid was used as template for RNase-Free DNase I (Promega Corp.) following the manufacturer's run-off transcription with T7 polymerase (MEGAscript® Kits, Ambion instructions. The total RNA preparation was aliquoted and stored at 406 S. Hajeri et al. / Virology 417 (2011) 400–409

A

B

Fig. 4. (A) Sequence and predicted secondary structure of the Citrus exocortis viroid (CEVd) wild-type (WT) and mutated sites of the progeny variants (PVs). Five structural domains of CEVd were indicated on the horizontal line above the predicted secondary structure. Terminal conserved region (TCR), hairpins I and II (HP I, HP II), virulence modulating (VM) region, pre-melting regions 1, 2 and 3 (PM 1, 2, 3) are shown. The thick line in the pathogenicity domain represents the conserved polypurine rich sequence. Loop E is shown by underlined bases in central conserved region. Dots and open circles indicate Watson–Crick and non-Watson–Crick base pairs. A wobble pair G–U is shown in Loop E region. Mutated sites and mutations (superscript text) that resulted in: i. non-infectious (*), ii. infectious but genetically unstable (#), and iii. infectious and genetically stable (solid circle) CEVd-PVs are indicated. (B) Replication and systemic accumulation of CEVd-PVs are shown in relation to the CEVd-WT levels. Means and standard deviation of triplicate experiments are presented.

−80 °C. This total RNA was used in the SYBR Green I based RT-qPCR For the analysis of systemic accumulation of CEVd-WT, citron assays and RT-PCR for cloning and sequence analysis. seedlings were divided into three parts; leaves, stem and root, and the viroid titer was monitored at 24 h interval over a period of five days Systemic accumulation study system: test tube-grown citron seedlings using RT-qPCR assay. For each time interval, three biological replicates were sampled. Based on this control experiment, the systemic Seeds from mature citron fruit from disease tested ‘Etrog’ citron (C. accumulation of in vivo generated CEVd-PVs was monitored in the medica L.) selection ‘Arizona 861-S1’, maintained at the quarantine systemically infected leaves of the seedlings at 5 dpi. Total RNA was facilities of the CCPP at the UCR, were peeled, surface sterilized in 10% extracted from the infected leaves using a commercial kit (Spectrum solution of sodium hypochlorite and rinsed with sterile water. The Plant Total RNA Kit-Sigma Aldrich) with slight modifications. Initial sterilized seeds were germinated under aseptic conditions in tissue disruption was performed in Mini-BeadBeater-96® Homoge- 25×150 mm culture tubes containing 25 ml of germination medium nizer (Daintree Scientific) with the Lysis Solution (Sigma Aldrich) and consisted of MS salts with GELRITE® Gellan Gum (CP Kelco β-mercaptoethanol. The rest of the protocol was performed according applications) as a solidifying agent (Navarro, 1981; Navarro et al., to the manufacturer's instructions. Total RNA was treated with RQ1 1975). Seedlings were grown in a plant growth chamber with 16 h RNase-Free DNase I (Promega Corp.) following the manufacturer's photoperiod at 32 °C and 8 h darkness at 28 °C. Under sterile instructions and stored at −80 °C. The total RNA was used in the SYBR conditions, four-week old seedlings, which measured 7–8cm in Green I based RT-qPCR assays and RT-PCR for cloning and sequence length and had 2–4 true leaves, were slash inoculated at the lower analysis. part of the stem (4–5 cm from the root) with an aliquot containing 500 ng of in vitro transcripts of CEVd (WT or PVs) using a sterile razor Infectivity and pathogenicity study system: greenhouse-grown plants blade. Upon inoculation, the seedlings were transferred to 25×150 mm culture tubes containing liquid medium, with 4.33 g/l ‘Etrog’ citrons selection ‘Arizona 861-S1’ propagated on rough lemon MS salts, White's vitamins (1 mg/l nicotinic acid, 1 mg/l pyridoxine, (C. jambhiri Lush.) rootstock were used for the population composition 0.2 mg/l thiamine HCl) (White, 1943, 1963), 100 mg/l inositol, and and diversity studies of the CEVd-WT. For the infectivity, genomic 75 g/l sucrose (Navarro, 1981; Navarro et al., 1975). All seedlings were stability and pathogenicity studies of in vivo generated CEVd-PVs, ‘Etrog’ maintained in a plant growth chamber with 16 h photoperiod at 32 °C citron, and the herbaceous hosts, gynura (Gynura aurantiaca DC) and and 8 h darkness at 28 °C. chrysanthemum (Dendranthema grandiflora Tzvelev.)cv.“Bonnie Jean” S. Hajeri et al. / Virology 417 (2011) 400–409 407 were utilized. Clonal propagations of gynura and chrysanthemum were primers (10 μM) and 1 μlofdNTPs(10mM)weremixedinnuclease-free prepared by rooted cuttings from disease tested sources. All plants were water (total volume of 10 μl). The mixture was incubated at 65 °C for 5 min maintained under standard greenhouse conditions. to denature the RNA template and then it was placed on ice for at least 1 Three plants were used per treatment. Each plant was slash min. To this mixture, 2 μl of 10x RT-buffer, 4 μlof25mMMgCl2,2μlof0.1 inoculated with an aliquot of 10 μg of in vitro generated CEVd M DTT, 1 μl of RNaseOUT (40 U/μl) and 1 μl of SuperScriptTM III reverse transcripts (WT or PVs) using a sterile razor blade (Garnsey and transcriptase (200 U/μl) were added (final volume of 20 μl) and the Whidden, 1973; Rigden and Rezaian, 1992). Following inoculation, reaction mixture was incubated at 50 oC for 50 min. The reaction was plants were maintained at elevated greenhouse temperatures (32– stopped by incubation at 80 °C for 10 min. Serial dilutions of cDNA 38 °C) optimum for CEVd replication and symptom expression. template from each sample were used for qPCR using ankyrin specific Inoculated plants were monitored for symptom expression at weekly reverse (ankyrin-R) and forward (ankyrin-F: 5′-ATCCTCTGCAAGTA- intervals and tested for the presence of CEVd by tissue imprint GATGC-3′) primers. 2 μl of cDNA template, 12.5 μl of RT² SYBR® Green hybridization using full length digoxigenin (DIG) labeled DNA probes qPCR Master Mix, and 0.6 μl of each of the forward and reverse primers (10 (Roche Applied Science) as described by Palacio-Bielsa et al. (1999). μM) were mixed in nuclease-free water (final volume of 25 μl). The Total RNA was extracted from the infected plant tissues following the reaction mix was incubated at 95 °C for 10 min followed by 40 cycles of procedure of Semancik et al. (1975) and enriched for viroid RNA as 95 °C, 15 sec and 60 °C, 60 sec. Melt curve analysis, from 60 °C to 95 °C with described by Semancik (1986). The total RNA was used for RT-PCR, measurement of fluorescence taken at every 0.5 °C, verified the specificity cloning and sequence analysis. of the qPCR products. The cDNA template dilutions from different samples were normalized for the ankyrin fragment using the qPCR cycle RT-PCR, cloning and sequence analysis threshold (Ct) values. The normalized template DNA dilutions were then used for qPCR of CEVd with specific reverse (CEVd-R) and forward (CEVd- The CEVd (WT and PVs) progeny RNA extracted from the citrus F: 5′-GAAGCTTCAACCCCAAACC-3′) primers. CEVd qPCR and melt curve protoplasts, seedlings and mature plants was used as template for RT- analysis was performed as described previously for the ankyrin gene. PCR. The first-strand of cDNA was synthesized using CEVd reverse A standard curve was constructed using serial dilutions of the CEVd- primer (5′-CAAGGATCCCTGAAGGACTTCTTC-3′) by SuperScript™ III WT plasmid over a range of six dilutions from 10−1 to 10−6 starting Reverse Transcriptase (Invitrogen Corp.) following the manufacturer's with 1 ng per μl concentration. The qPCR results were analyzed with instructions. Second-strand synthesis and DNA amplification was MX3005P data analysis software (Stratagene). Quantification of the performed using CEVd reverse and forward (5′-CTCGGATCCCCGGG- CEVd RNA was performed by comparing the Ct values of each sample to GAAACCT-3′) primers by Pfu DNA Polymerase (Biovision Research the Ct values of the standard curve. The mean of three replications per Products) following the manufacturer's instructions. RT-PCR products treatment was used for the protoplasts and seedling CEVd samples. were purified using the High Pure PCR Product Purification Kit (Roche Relative levels of the CEVd-WT accumulation in protoplasts were Applied Science). As Pfu DNA polymerase creates blunt end products, monitored over a period of five days at 24 h interval. The accumulation purified PCR product was used for 3'A-tailing reaction using Taq DNA level of CEVd-WT RNA at 1 dpt was arbitrarily set to a value of one (1) Polymerase (Promega Corp.) at 72 °C for 25 min, which facilitated the and the level of the viroid accumulation on subsequent days was T–A cloning process. Ligation of the A-tailed DNA fragment to the estimated as a relative number to this reference value. pGEM-T easy plasmid vector (Promega Corp.) was performed Relative levels of CEVd-WT accumulation in different parts of the following the manufacturer's instructions. The ligated plasmid vector citron seedling (leaves, stem, and root) were monitored over a period was transformed into Z-Competent™ E. coli Strains—JM109 (Zymo of five days at 24 h interval. In this situation, the accumulation level of Research Corp.) following the method described in Sambrook and the CEVd-WT in the stem tissue at 1 dpi was used as a reference value. Russell (2001). The selected white colonies (from blue/white Comparative analysis was made between the CEVd-WT and the in screening) were grown in a 96-well block containing 1 ml of LB vivo generated CEVd-PVs to evaluate their relative levels of accumula- medium under 50 μg/ml ampicillin selections. Subsequently, the tion in citrus protoplasts and seedlings. For this experiment, accumu- recombinant were sequenced (unidirectional) using SP6 lation of CEVd-WT at 5 dpi was used as a reference value in each case. promoter primer. The CEVd cDNA sequences were organized manually in FASTA format and used for multiple sequence alignment Statistical analysis using ClustalX 1.81 (Thompson et al., 1997). The population composition and genomic diversity of CEVd (WT and PVs) were The CEVd population composition and genomic diversity categorical analyzed. RNAdraw V1.1 (Matzura and Wennborg, 1996) was used to data were analyzed using χ2 contingency analysis. The viroid RNA predict the thermodynamic (temperature ranging 25–42 °C) second- accumulation data (citrus protoplasts and seedlings) were tested for ary structure of CEVd RNAs. normality using the Kolmogorov–Smirnov test at pb0.05 and were analyzed by analysis of variance (ANOVA). One-way ANOVA and the RT-qPCR assays non-parametric Kruskal–Wallis test on ranks were used for the normally and non-normally distributed data respectively. Whenever The relative levels of accumulation of CEVd (WT and PVs) in necessary non-normally distributed data were square root transformed protoplasts and seedling samples were monitored by SYBR Green I before ANOVA. The Student–Newman–Keuls post hoc test at pb0.005 based RT-qPCR assay in a MX3005P qPCR Machine (Stratagene) using RT² was used for all pairwise multiple comparisons among the means. SYBR® Green qPCR Master Mix (SABiosciences Corp.). The nuclear gene of Statistical analysis was performed using the SigmaPlot 11.00 software citrus, phospholipase isoform delta (ankyrin) (GenBank ID: CX074707), (Copyright 2008 Systat Software, Inc.). known to be expressed in equal amounts in different citrus tissues Supplementary materials related to this article can be found online (Tomassini personal communication) was used as an internal control to at doi:10.1016/j.virol.2011.06.013. normalize template quantities prior to the qPCR for CEVd. The RNA extracts were reverse-transcribed using SuperScript™ III Reverse Tran- Acknowledgments scriptase (Invitrogen Corp.). Mixed cDNA was synthesized for each sample using a combination of reverse primers, one specific to the internal control We thank the California Citrus Research Board for the partial support gene, ankyrin (ankyrin-R: 5′-CAACATATCAGGAAGACTCATG-3′)and of this study. We appreciate the generosity of Dr. Jude Grosser (Citrus another specifictoCEVd(CEVd-R:5′-CTTTTTTCTTTTCCTGCCTGCA-3′) Research and Education Center, Lake Alfred, Florida) for providing the with the following protocol. 2 μloftotalRNA,0.5μlofeachofthereverse citrus callus culture. We thank Dr. Robert A. Owens and Dr. Rosemarie 408 S. Hajeri et al. / Virology 417 (2011) 400–409

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