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1 INRA, UR407, Station de Pathologie Végétale, BP 94, 84143 Montfavet cedex, France
2 INRA, UR501, Laboratoire de Biologie Cellulaire, 78026 Versailles cedex, France
3 Plant Virology Group, ICGEB Biosafety Outstation, Via Piovega 23, 31056 Ca’ Tron di Roncade, Italy
Correspondence
Mireille Jacquemond
Mireille.Jacquemond{at}avignon.inra.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; García-Arenal et al., 2001, 2003). However, the fact that recombinants have been positively selected in nature is shown by the sequence of plant viral isolates in several genera [e.g. Cucumovirus (Chen et al., 2002; Lin et al., 2004; Bonnet et al., 2005), Tobravirus (MacFarlane, 1997), Luteovirus and Polerovirus (Mayo & Ziegler-Graff, 1996; Moonan et al., 2000), Nepovirus (Vigne et al., 2005) and Potyvirus (Glais et al., 2002; Desbiez & Lecoq, 2004; Tomimura et al., 2003; Glasa et al., 2005)].
Cucumber mosaic virus (CMV) is the type species of the genus Cucumovirus, which also includes Tomato aspermy virus (TAV) and Peanut stunt virus. Based on their serological, biological and sequence properties, two major CMV subgroups (I and II) can be distinguished, and subgroup I can be further divided into subgroups IA and IB (reviewed by Palukaitis & García-Arenal, 2003). The CMV genome consists of three single-stranded, positive RNA segments; RNAs 1 and 2 encode proteins 1a and 2a, respectively, which are constituents of the viral replicase. RNA 3 is bicistronic, encoding the movement protein (MP) and the capsid protein (CP). At least two subgenomic RNAs are also packaged into the virus particles. RNA 4A is derived from RNA 2 and encodes the 2b protein, which acts as a suppressor of post-transcriptional gene silencing and plays an important role in plant antiviral defences (reviewed by Moissiard & Voinnet, 2004; Roth et al., 2004). RNA 4 derives from RNA 3 and encodes the CP, which is essential for movement, encapsidation and aphid transmission (reviewed by Palukaitis & García-Arenal, 2003).
Previous studies showed that rare recombinant RNA 3 molecules can be detected by RT-PCR in a few plants infected with CMV and TAV (Aaziz & Tepfer, 1999; de Wispelaere et al., 2005). Interestingly, a subset of recombinants, including most of those observed in planta, could also be produced directly in vitro by carrying out RT-PCR on a mixture of total RNAs purified from singly infected plants, yielding an easier way to produce recombinant molecules (Fernandez-Delmond et al., 2004). As expected, the recombinants obtained in planta and in vitro also included sequence variants and represented a sampling of the total sequence variability produced by mutation and recombination. However, the possibility for these recombinants to be positively selected in infected plants remained to be evaluated. In this paper, we describe the biological properties of several previously obtained RNA 3 recombinants between I17F-CMV (subgroup IA) and R-CMV (subgroup II), with crossovers in the CP gene or the 3' non-coding region. With certain recombinants, competition experiments with wild-type viruses were also done, in order to provide a preliminary indication of their relative fitness.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The recombinants were named as follows: the first letter designates the strain to which the 5' end of the RNA 3 belongs (I for I17F-CMV and R for R-CMV), followed by a number corresponding to the position of the last nucleotide derived from this strain; the second letter designates the second strain and is followed by a number corresponding to the first nucleotide derived from this strain. For example, I1409/R1381 is composed of the first 1409 nt of I17F-CMV RNA 3 and the last 826 nt of R-CMV RNA 3 (i.e. starting at position 1381). In addition, the two RNA 3 recombinants created in vitro by exchanging the MP and CP genes of I17F- and R-CMV RNAs 3 (Carrère et al., 1999) and used as shuttle vectors were also included in the study.
Production of inoculum and plant inoculation.
Inoculation of tobacco plants (Nicotiana tabacum ‘Xanthi-nc’) by particle bombardment with full-length recombinant RNA 3 cDNA clones and full-length cDNA clones of RNAs 1 and 2 of either I17F- or R-CMV was described previously (Fernandez-Delmond et al., 2004). For convenience, each artificial strain composed of RNAs 1 and 2 of one strain and a recombinant RNA 3 will be termed a recombinant virus and designated by the name of its RNA 3. Virions were purified from bombarded plants according to Lot et al. (1972). Viral RNA was purified by using the two-phase phenol procedure. Viral RNAs at a concentration of 10 µg ml–1 were used to inoculate young tobacco (N. tabacum ‘Xanthi-nc’), pepper (Capsicum annuum ‘Yolo Wonder’) and melon (Cucumis melo ‘Védrantais’) plants. In the case of recombinant virus I1625/R1597, which remained localized in the bombarded leaf, and recombinant virus R1380/I1410, for which virions could not be purified by using the standard protocol, a crude extract of dried infected tissue was used as inoculum. Plants were grown in a greenhouse.
Detection and quantification of viruses.
The presence of virus in the inoculated leaves of pepper and melon, or in asymptomatic plants of all species, was evaluated by ELISA as described previously (Carrère et al., 1999). Total RNA was extracted from inoculated leaves of tobacco plants 6 days post-inoculation (p.i.) and from systemically infected ones 12–14 days p.i. (all experiments) and 30 days p.i. (one experiment; see Results), using TRI Reagent (Molecular Research Center, Inc.). Duplicate samples of 62 ng total RNA were spotted onto a membrane. Membranes were hybridized first with a probe prepared from a mixture of PCR products corresponding to approximately the last 700 nt of the cDNA 3 of both strains, as described previously (Carrère et al., 1999). After dehybridization, the membranes were then hybridized with a probe corresponding to a cDNA of the 25S RNA of pepper. Radioactivity was measured by using a PhosphorImager (Molecular Dynamics). Several total RNA preparations from healthy plants were spotted onto each membrane. The mean 25S value determined for the healthy plant samples was considered to represent 62 ng total RNA. The data obtained with the CMV probe were corrected by using the ratio between the 25S value of infected plants and the mean 25S value of non-infected plants.
Statistical analyses were done by using the mean standardized number of pixels for the two replicates. A mixed-model ANOVA, including a fixed effect factor (‘Virus’) and a random effect factor (‘Experiment’) and their interaction, revealed a significant ‘Virus’ effect (P<10–3), as well as a significant ‘Virus–Experiment’ effect (P<10–3). Because of this highly significant interaction and because, in some experiments, data violated normality or homoscedasticity assumptions, the effect of the ‘Virus’ factor on viral RNA accumulation was ascertained for each experiment by using a non-parametric Kruskal–Wallis test. Then, a Dunnett's means separation test was performed to identify which recombinants differed statistically significantly in their accumulation from the I17F strain.
Competition experiments.
Three young tobacco plants were inoculated simultaneously with a recombinant virus containing recombinant RNA 3 and RNAs 1 and 2 from either I17F- or R-CMV and, as competitor, an equal concentration (5 µg ml–1) of either the reconstituted I17F strain or the reassortant R1-R2-I3 (RNAs 1 and 2 from R-CMV and RNA 3 from I17F-CMV). Total RNA was extracted from inoculated leaves 6 days p.i. and from a systemically infected leaf 12–14 days p.i. The leaves were ground in 0.5 M trisodium citrate, 0.1 % thioglycolic acid (1 g in 2 ml), and RNA was extracted from 200 µl homogenate. At 12–14 days p.i., half a leaf was used for RNA extraction and the other half was ground in 0.03 M disodium phosphate, 0.2 % diethyldithiocarbamate (DIECA; 1 g in 4 ml), and used to inoculate a young tobacco plant. Two or three successive passages were done. Each experiment was repeated twice. RT-PCR products were produced by using a reverse primer corresponding to the 3'-terminal 14 nt of both I17F- and R-CMV RNA 3 (Carrère et al., 1999) and primer I17F982+ (Fernandez-Delmond et al., 2004). RT-PCR was done in two steps, using a murine leukemia virus reverse transcriptase without RNase H activity, in order to minimize the production of recombinant fragments in vitro (de Wispelaere et al., 2005). The products were digested with EcoRI and XbaI, producing a pattern characteristic of either the recombinant or the parental RNA 3 cDNA, and also with both enzymes. RT-PCR/restriction fragment-length polymorphism (RFLP) digests were analysed on 7.5 or 10 % polyacrylamide gels and visualized under UV light by using SYBR green I. Gels were overloaded in order to detect any minor populations.
Analysis of RNA 3 progeny.
RT-PCR products were produced from purified viral RNA and/or total RNA from infected tobacco plants as described above, using the same reverse primer as mentioned previously and the specific forward primers initially used for detecting recombinant regions (Fernandez-Delmond et al., 2004). The RT-PCR products were sequenced directly by using the same primers.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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and their principal features are presented in Table 1. Most of the recombinants are of the precise homologous type, and were obtained in vitro by RT-PCR of a mixture of total RNA from tobacco plants infected with I17F- or R-CMV (Fernandez-Delmond et al., 2004). Recombinants at equivalent sites were also recovered in plants infected with both viruses (I. Fernandez-Delmond & M. Tepfer, unpublished data). Recombinants I2072/R1899 and I2077/R1900, obtained from doubly infected plants, are of the aberrant homologous type and are longer than the parental viral RNAs. Seventeen of the 19 recombinants infected tobacco plants successfully following bombardment (Table 1). The converse recombinants I1583/R1555 and R1554/I1584 were non-infectious (Fernandez-Delmond et al., 2004). Virions of the remaining 17 recombinant viruses, except for R1380/I1410 and R1695/I1725, were purified efficiently by using the standard protocol of Lot et al. (1972). No particles could be purified for virus R1380/I1410. Nevertheless, virus particles were observed clearly in thin sections of infected tissues, suggesting that the difficulty in purifying this virus was due to particle instability rather than lack of encapsidation (data not shown). Dried infected tissue from bombarded plants was used as inoculum for this recombinant. Purified viral RNA of poor quality was obtained only once for virus R1695/I1725, but attempts to infect plants with this virus by bombardment, in order to use dried infected tissue as inoculum as done previously, remained unsuccessful.
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and atypical ones are illustrated in Fig. 2. Two recombinant viruses showed major deficiencies: virus I1625/R1597 remained localized to the inoculated leaves of all hosts (Fig. 2b) and virus R1695/I1725 infected only one tobacco plant of the 21 plants inoculated in six distinct experiments. All other recombinant viruses were systemic on all hosts, except for recombinant R1596/I1626, which was not detected in the upper leaves of inoculated melon plants.
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). All recombinants, except for viruses I1724/R1966 and I1814/R1786, induced symptoms very similar to those of cI17F. In addition, all R/I-type recombinants induced leaf filiformism (Table 2). Viruses I1724/R1966 and I1814/R1786 grouped apart and induced very distinct symptoms, including development of strong yellowing along the veins, yellow spots or patches, crinkling and severe stunting, and even holes in the leaves (Fig. 2c). On melon, most of the I/R-type recombinants induced the same severe symptoms as cI17F (Table 2). In contrast, all of the R/I-type recombinants induced vein yellowing and yellow spotting rather than a true mosaic and, late in infection, plants showed only a few or no yellow spots. Recombinants I1724/R1696 and I1814/R1786 infected melon irregularly, inducing more or less severe symptoms, depending on both the experiment and the plants of the same experiment (Table 2). Interestingly, these recombinants were again very severe on pepper plants, inducing symptoms of yellowing and crinkling like those seen in tobacco, and could thus be distinguished clearly from cI17F and all of the other recombinants. In addition, necrotic symptoms (either vein necrosis or necrotic rings) or leaf filiformism were also observed in pepper infected with certain recombinant viruses (Table 2), but could not be associated with any sequence features. Finally, one recombinant virus, I1409/R1381, differed from all others, as it did not always infect all of the inoculated plants, whatever the host, and induced an infection delayed for 1 week or more (Table 2). However, once infection was well established, plants developed severe symptoms.
Viral RNA accumulation in tobacco plants
Viral RNA accumulation in total RNA prepared from inoculated and systemically infected leaves was evaluated by dot blot. The recombinant viruses were tested at least twice. Dilutions of purified viral RNA (0.5–32 ng) of each parental strain were also blotted onto the membranes. Both were detected at similar levels (data not shown). Fig. 1(b) presents the results obtained in one experiment, including all viruses and involving four plants per virus, where viral RNA amounts are expressed as (no. pixels for each sample)–(mean value determined for healthy controls). Table 3 presents the mean viral RNA accumulation relative to cI17F for all experiments, for each virus, approximately 2 weeks after infection.
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, IL 6 days p.i.). The plants further developed no symptoms and no virus could be detected 15 or 30 days p.i. Plants inoculated with virus I1409/R1381 also showed low virus levels. Three of four plants further developed symptoms and accumulated virus. At 30 days p.i., the mean viral RNA amount was similar to that observed in cI17F-infected plants at 15 days p.i., illustrating the delay in infection with this recombinant virus (Fig. 1b, SIL 30 days p.i.).
According to the mean relative viral RNA accumulation in systemic, fully infected leaves (Table 3), seven recombinants accumulated to a lower level than cI17F, and differences were significant in several experiments. Six recombinants accumulated to levels similar to those of cI17F. Three recombinants accumulated to levels similar to or marginally higher than those of cI17F in most, if not all, experiments. However, differences were generally not significant: this was the case for I1319/R1294 and I1430/R1402, which induced symptoms very similar to those induced by I17F, and I1724/R1696, which induced unusually severe symptoms. Two main features emerge from these data. First, the ability of a recombinant to migrate and accumulate in tobacco plants was not associated with its type, i.e. I/R versus R/I (Fig. 1b, SIL 15 days p.i. and SIL 30 days p.i.; Table 3). Second, converse recombination at the same point could have very different effects. Each pair of converse recombinants in the approximately first half of the CP (I1409/R1381 and R1380/I1410, I1430/R1402 and R1401/I1431, and I1583/R1555 and R1554/I1584) behaved similarly. On the other hand, the converse recombinants of R1596/I1626 and I1724/R1696, which replicated and migrated efficiently, showed important deficiencies.
Competition between recombinant and native RNA 3 molecules
Two recombinants that accumulated to levels equivalent to or greater than those of cI17F (I1430/R1402 and I1724/R1696) were tested in several competition experiments with I17F RNA 3. Three plants per inoculum were inoculated initially in each experiment. Although RT-PCR/RFLP analysis is not quantitative, three patterns could be observed clearly for each RNA 3 progeny: presence, absence or detection to a low level, thanks to the analysis of large amounts of the RT-PCR products (data not shown). In addition, development of yellow spots or vein yellowing was used as a phenotypic marker for the presence of recombinant I1724/R1696. Competition between each recombinant RNA 3 and I17F RNA 3 was first tested when they were associated with RNAs 1 and 2 of I17F-CMV (I1-I2-I3 plus I1-I2-recombinant RNA 3). All of the initially inoculated plants developed symptoms essentially identical to those of I17F-CMV, whatever the inoculum. The I17F RNA 3 restriction pattern was observed in all of the extracts. As shown in Table 4, both recombinants were detected in the inoculated leaves of all but one of the inoculated plants. Recombinant I1430/R1402 was detected easily in one plant during the second passage, and only to a low level in the inoculated leaves during the third passage. Recombinant I1724/R1696 was not detected in any back-inoculated plants. These results indicate that, when associated with I17F-RNAs 1 and 2, the two recombinants were less fit than the native I17F RNA 3. Similar competition experiments were then carried out using RNAs 1 and 2 of R-CMV (R1-R2-I3 plus R1-R2-recombinant RNA 3). An I17F RNA 3 restriction pattern was observed in all plants except for one (see below). Viruses R1-R2-I3 and R1-R2-I1430/R1402 both induced the same mild mosaic on tobacco. The recombinant and I17F RNA 3 were present in similar amounts in all of the co-inoculated plants, and the recombinant was still present during the second passage, at least in the inoculated leaves (Table 4). In one of the two experiments, the recombinant was also present in one plant during the third and fourth passages. In that case, a modified I17F RNA 3 cDNA restriction pattern was observed in the first inoculated plant. Sequencing of the RT-PCR product revealed that it corresponded to I17F RNA 3 modified by recombination in the 3' non-coding region with the conserved counterpart of R genomic RNAs. This new recombinant could still be detected in the inoculated leaves of the first back-inoculated plant and then disappeared. On tobacco, R1-R2-I1724/R1696 induced symptoms typical of the recombinant, although less severe than when the recombinant was associated with I17F RNAs 1 and 2. Yellow spots characteristic of this recombinant could be observed on all plants initially inoculated with the mixture of viral RNAs, although the symptoms that these plants developed were considerably milder than those developed by plants infected with the recombinant alone. A few yellow spots were still observed in one plant during both the second and third passages. The presence of the recombinant was confirmed by RT-PCR/RFLP analyses. Thus, when associated with R-CMV RNAs 1 and 2, both recombinants survived in some plants during serial passages.
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). All encoded a CP of the expected size, except for recombinant I1319/R1294, in which the position of the crossover site resulted in the loss of the amino acid at position 15. Most of the recombinants showed additional sequence changes compared with the reference sequences established previously (GenBank accession numbers Y18137 and Y18138 for strains I17F and R, respectively). Substitution at position 1732, leading to a different amino acid at position 159 in the I17F CP, is part of the natural heterogeneity of the I17F population (Carrère et al., 1999) and, as observed previously, a G at this position is more common than an A. The other substitutions could either represent heterogeneity within the viral populations or be due to misincorporation during RT-PCR. For instance, the substitution at position 1227 in the intergenic region of the cDNA of R-CMV RNA 3 was observed in all R/I-type recombinants synthesized in vitro, but not in the recombinant R1008/I1040, which was constructed in the laboratory, suggesting that this mutation is also part of the natural diversity of R-CMV. Finally, it should also be noted that the sequences of the CP of recombinants R1380/I1410 and R1384/I1431 differed only by the two additional changes that occurred in R1380/I1410 (nt 1612 and 1739), suggesting that the particle instability of R1380/I1410 is likely to be due to these additional changes, rather than to its recombinant nature.
The sequence of the second-generation progeny (produced in plants inoculated with progeny from bombarded plants) was evaluated in total RNA prepared from an inoculated tobacco leaf 6 days p.i. and from a systemically infected leaf of two plants in two distinct experiments, approximately 2 weeks after inoculation. Sequences were in conformity with the cDNA in most cases (Table 1). Two cases of reversion to wild type of one additional substitution were observed (nt 1612 for R1380/I1410, nt 1039 for R1401/I1431). Sequencing of the inocula (first generation) showed that these reversions had already occurred. This suggests strongly that particle instability of R1380/I1410 can be linked to a unique change at position 1739 (aa 161). Sequencing of the second-generation progeny of the aberrant recombinants I2072/R1899 and I2077/R1900 revealed that the sequences were modified considerably, as both became identical to the precise homologous recombinant I2055/R2042. This modification was present in the purified viral RNA used as inoculum and was also observed in the progeny produced in newly bombarded plants 5 days after bombardment.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). A similar distribution of recombinants was selected for the present study.
In CMV, the CP is essential not only for encapsidation and acquisition of viral particles by vector aphids, but also for movement of the virus within infected plants. Thus, it was expected that recombination within the CP could interfere with any of these functions. If cell-to-cell movement is affected, this would result in apparent non-infectivity, which we observed in three of 13 CP recombinants (I1583/R1555, R1554/I1584 and R1695/I1725). If cell-to-cell movement is normal, but systemic movement is affected, then the virus would be limited to the infected leaves, as we observed in one recombinant (I1625/R1597). A different type of defect was observed in R1380/I1410. Here, there were apparent problems with virion assembly and, in addition, attempts to transmit this virus by Myzus persicae, under conditions where controls were transmitted efficiently, remained unsuccessful (data not shown). As virion stability and aphid transmissibility have been associated with a single amino acid change at position 162 in the CP (Ng et al., 2005), it is probable that the defect of R1380/I1410 is due to a non-synonymous mutation that affects the adjacent CP residue (Gln161Arg). All but two of the remaining recombinants induced symptoms that were equivalent to those of the parental I17F-CMV. Surprisingly, the two remaining recombinants (I1724/R1696 and I1814/R1786) induced symptoms that were unlike those of the parental viruses, and could be described as more severe. In these cases, as the additional point mutations were either silent or part of the normal variability of I17 F CMV, their unusual properties should be attributed to their recombinant nature.
Durable survival in nature of any of these recombinants would require that they have fitness at least equivalent to that of the parental wild-type viruses. The recombinants that are defective for an important CP-mediated function described above would clearly be expected to be strongly counter-selected. Of the 19 RNA 3 recombinants, when inoculated with RNAs 1 and 2 of I17F-CMV, only five had levels of viral RNA accumulation in infected plants that were equivalent to wild type, and thus could be considered potentially competitive. The ability to compete with the wild-type RNA 3 of two of the recombinants with the highest accumulation levels (I1430/R1404 and I1724/R1696), of which the latter induced severe symptoms, was evaluated further. However, neither recombinant was able to outcompete the wild type. This suggests that probably none of the recombinants considered here would have been selected in co-infected tobacco plants. This is consistent with the fact that plants expressing the atypically severe symptoms observed with I1724/R1696 and I1814/R1786 recombinants have not yet, to our knowledge, been observed in the field.
Among the various selective forces exerted on the recombinants, this work suggests a role for the replication machinery. The two recombinants that were tested in competition with I17F-CMV RNA 3 appeared to be more competitive when replication was driven by R-CMV RNAs 1 and 2. In one case, the recombinant I1430/R11402 even supplanted the wild-type RNA 3. In this plant, in which I17F RNA 3 was not detected, a recombinant form was observed in the inoculated leaves. Acquisition of the conserved 3' part of R genomic RNAs should have been considered advantageous for I17F RNA 3, for increased replication efficiency. However, this recombination event has probably resulted in a cost for the molecule, as it was supplanted in the upper leaves by the recombinant initially brought in the inoculum. Emergence of a recombinant in an experimental tetrapartite virus composed of genomic segments of CMV and TAV was described by Masuta et al. (1998) although, in that case, both the wild-type and the recombinant genomic RNAs were present during serial passages on a susceptible host, maintaining the tetrapartite nature of the hybrid new virus.
Another essential selective force is the host species. For example, all R/I-type recombinants, which were relatively well adapted to tobacco and pepper, would probably be strongly counter-selected in melon, which developed only limited symptoms 2–3 weeks after infection. Although the recombinants described here were not competitive with wild type in tobacco, this does not predict their relative fitness in other species. In this regard, the results of Chen et al. (2002) are particularly pertinent. They observed that an aberrant recombinant RNA 3 was positively selected in Alstroemeria relative to the corresponding non-recombinant, but was strongly counter-selected in tobacco. Considering the breadth of the CMV host range, it is not possible to formally exclude the possibility that a given recombinant might be positively selected in a CMV host species other than those studied here. In addition, genetic drift could also allow survival of less-fit recombinants.
Nonetheless, there are other, additional reasons to expect that RNA 3 recombinants between subgroups IA and II would be unlikely to emerge. First, co-existence of subgroups IA and II was observed very infrequently in field studies in both Spain (Fraile et al., 1997) and California (Lin et al., 2004), and both subgroups were rarely identified in the same area or season. In the south of France, both subgroups were detected within the same crop, but co-infections were rare, suggesting successive infections by isolates of these subgroups rather than co-existence (Quiot et al., 1979). Second, Takeshita et al. (2004) observed spatial segregation in plants co-infected with CMV IA and II isolates; hence, within doubly infected plants, the viruses would only rarely be present and replicating in the same cells. Thus, the opportunities for CMV IA/II recombinants to be created in the field would be rare.
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ACKNOWLEDGEMENTS |
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Received 11 April 2007;
accepted 14 June 2007.
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