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1 Plant Virology Group, ICGEB Biosafety Outstation, Via Piovega 23, 31056 Ca' Tron di Roncade, Italy
2 INRA, Laboratoire de Biologie Cellulaire, UR501, 78006 Versailles cedex, France
3 INRA, Station de Pathologie Végétale, UR407, BP 94, 84143 Montfavet cedex, France
Correspondence
Mark Tepfer
tepfer{at}icgeb.org
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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These authors contributed equally to this work. 
Present address: Laboratoire Interactions Plantes–Pathogènes, UMR INRA/INAPG/UnivP6, 16 rue Claude Bernard, 75005 Paris, France.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and, since the early 1990s, various concerns have been expressed regarding the potential impact of such plants, particularly those expressing viral genes (Tepfer, 2002). One of the most intractable questions has been whether viral sequences expressed from the transgene could recombine with the genome of a non-target virus, and thus lead to emergence of a novel virus. As has been reviewed previously (Tepfer, 2002), mRNA/viral RNA recombination has been clearly shown to occur in transgenic plants expressing sequences from several viruses (cauliflower mosaic virus, cowpea chlorotic mottle virus, plum pox virus, tobacco mosaic virus and tomato bushy stunt virus) when they are inoculated with viruses that bear deletions in the sequence expressed from the transgene, but there is no clear evidence that this phenomenon constitutes a risk of virus emergence.
As mRNA/viral RNA recombination can occur in transgenic plants, the critical question is whether this can be the source of recombinants different from those that would be produced in non-transgenic plants infected with more than one virus, a situation that occurs frequently in the field. Attempts to answer this question should be based on a comparative study of the populations of recombinants that occur under conditions of the lowest possible selection pressure in favour of the recombinants (Tepfer, 2002). This requires development of highly sensitive RT-PCR techniques for specifically amplifying recombinant viral RNAs that occur as a very minor component of the total viral RNA population. This has been shown to be possible with two cucumoviruses, cucumber mosaic virus (CMV) and tomato aspermy virus (TAV) (Aaziz & Tepfer, 1999; de Wispelaere et al., 2005), but was not possible with three other virus groups (Dietrich et al., 2007; Koenig & Buttner, 2004; Meier & Truve, 2006). For this reason, we have developed the cucumovirus recombination system further.
To do this, we have improved the RT-PCR detection of recombinant cucumoviral RNAs and have compared the populations of recombinant RNA3 molecules that appear in transgenic and non-transgenic plants, both between TAV and CMV and also between two isolates representing the major CMV subgroups. In order to increase the possibility of finding differences, these studies were carried out under worst-case conditions, i.e. with transgenic plants that express, in addition to the coat protein (CP), the 3' non-coding region (NCR) of RNA3, as this can serve as an initiation site for the viral replicase (Teycheney et al., 2000) and thus increase the frequency of appearance of recombinants. Our results show that similar populations of recombinant RNA3 molecules appeared in both experimental systems and suggest that novel recombinants should not be expected in plants expressing this type of CP gene.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) (subgroup II) and I17F-CMV (Jacquemond & Lot, 1981) (subgroup I) were isolated in the south of France, and P-TAV was isolated in Hungary (Salánki et al., 1994). Cloned cDNAs of R-CMV, I17F-CMV and P-TAV were used initially to establish the viruses (Carrère et al., 1999; Salánki et al., 1997; Fernandez-Delmond et al., 2004), after which they were maintained by regular inoculation of new plants.
All transgenic tobacco plants were derived from Nicotiana tabacum ‘Xanthi XHFD8’. The transgenic lines were transformed with a vector described previously (Jacquemond et al., 2001), but the transgene included the entire 3' NCR of RNA3 in addition to the CP coding sequence of R-CMV. The transgene is under the control of the cauliflower mosaic virus (CaMV) 35S promoter and the pea E9 RuBisCO small-subunit polyadenylation signal. None of the plant lines studied was resistant to P-TAV or I17F-CMV (M. Jacquemond & M. Tepfer, unpublished).
Plants at the three- to four-leaf stage were dusted lightly with carborundum and rub-inoculated with 20 µl viral crude sap, prepared by grinding symptom-expressing leaves in 0.1 M sodium phosphate buffer, pH 7.0. For recombination experiments, co-infection of non-transgenic plants with P-TAV and R-CMV was carried out by inoculating first with P-TAV, followed by inoculation with R-CMV 5 days post-inoculation (p.i.), as described previously (de Wispelaere et al., 2005). Co-infection with I17F-CMV and R-CMV was done concomitantly, using purified viral RNAs (5 µg each virus ml–1). Homozygous T2 progeny of transgenic plants were inoculated with either P-TAV or I17F-CMV.
RNA extraction.
Total RNA was purified from either inoculated or systemically infected leaves. It has been shown previously that equivalent populations of recombinants are observed in inoculated and systemically infected leaves (de Wispelaere et al., 2005). For non-transgenic plants inoculated with P-TAV and R-CMV, total RNA was extracted from samples collected 7 days after the second inoculation (i.e. 12 days after the first inoculation) by using a two-phase phenol procedure (Aaziz & Tepfer, 1999). For non-transgenic plants inoculated with I17F-CMV and R-CMV, total RNA was extracted from samples harvested 7 days p.i. as described previously (Fernandez-Delmond et al., 2004). Samples of transgenic plants were harvested at 10 days p.i. and RNA was purified by a silica-capture procedure (Rott & Jelkmann, 2001). The integrity of all extracted RNA was checked by electrophoresis in a 6.7 % formaldehyde, 1 % agarose gel buffered with 0.02 M MOPS, pH 7.0.
RT-PCR and cDNA cloning.
Primers were given names consisting of three parts: virus strain for which the sequence was specific (R, I or T for R-CMV, I17F-CMV or P-TAV, respectively), nucleotide position on the viral genome of the 5' end of the primer, and sense (+ or –) depending on whether the primer sequence corresponded to the positive or negative viral strand. The presence of locked nucleic acids is indicated by the suffix LNA, and in the primer sequence by upper-case letters.
Primers containing locked nucleic acids were designed by using the tools at two sites (http://lnatools.com/ and http://www.genelink.com/newsite/products/LNALockedNucleicAcidDESIGN.asp). These sites were particularly useful for determining the proper number and placement of locked nucleic acids within the primers. First-strand cDNAs were synthesized at 52 °C as recommended by the manufacturer of the reverse transcriptase (Promega) with minor modifications, in a 20 µl reaction mixture containing 1 µg total RNA, 140 U Moloney murine leukemia virus reverse transcriptase RNase H– point mutant (Promega) and 0.6 µM primer R2191– (5'-aacctgtggaaaaccaca-3') for I17F-CMV/R-CMV experiments, and R2058–LNA2 (5'-CatCggcgtgtgaaaga-3') for P-TAV/R-CMV ones. The cDNA mixture (3 µl) was submitted to PCR in a 25 µl reaction volume (after 2 min at 94 °C, 35 cycles of 30 s at 94 °C, 30 s at optimal annealing temperature, 1 min at 68 °C) with 1.5 U Platinum Taq (Invitrogen) according to the manufacturer's instructions. Optimal annealing temperatures for primer pairs I1188+LNA3 (5'-atTgtCtaCtgactatataga-3') plus R2183– (5'-ggaaaaccacaggcgatac-3'), and T1279+ (5'-cgtaataataacaacaacaactcg-3') plus R2058–LNA2, were 55 and 58 °C, respectively. Because of the variability observed between batches of reverse transcriptase and reverse transcriptase buffer, all experiments included appropriate positive and negative controls.
Positive controls for the detection of recombinant RNA3 molecules consisted of in vitro transcripts generated from engineered viral chimeric RNA3 cDNAs. Plasmid pTR3 (Salánki et al., 1997), which contains the 5' part of P-TAV and the 3' part or R-CMV, was used in the P-TAV/R-CMV experiments, and pIR3 (Carrère et al., 1999), which contains the 5' part of I17F-CMV and the 3' part or R-CMV, was used in the I17F-CMV/R-CMV ones. Transcription was carried out by using T7 RNA polymerase (Promega) according to the manufacturer's instructions. Template DNA was subsequently degraded with TURBO DNase (Ambion) and its absence was verified by PCR. RNA was purified by phenol : chloroform extraction and ethanol-precipitated (Maniatis et al., 1982), then quantified spectrophotometrically. The essential negative controls were mixtures of total RNA extracted from transgenic plants or ones infected with only one virus. The absence of amplification from mixtures made either before or after reverse transcription demonstrated the absence of recombination in vitro by the reverse transcriptase or Taq polymerase, respectively.
Following electrophoresis in a 1 % agarose/TAE (40 mM Tris/acetate, 1 mM EDTA) gel, PCR products were purified on MicroSpin S-400 columns (GE Healthcare), cloned in the pGEM-T Easy vector (Promega) and sequenced by BMR Genomics (Padua, Italy). Sequence analyses were carried out by using the Vector NTI Advance 9 software package (Invitrogen). The target was to analyse ten clones from each RT-PCR-positive sample. In some cases, fewer than ten clones were obtained and, for a few samples, more than ten clones were analysed.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Svarovskaia et al., 2000) and in vitro (Fernandez-Delmond et al., 2004); (ii) the optimization of RT-PCR conditions, in particular the incorporation of locked nucleic acids in reverse transcription and PCR primers (Tolstrup et al., 2003; Wang et al., 1999); and (iii) inclusion in every assay of a robust set of controls. In particular, these included negative controls using combinations of independently extracted plant total RNA containing parental viral or transgene mRNA, in order to eliminate the possibility that the recombinants observed were created in vitro. In addition, all plant samples testing positive for recombinants were retested at least once to ensure repeatability of the RT-PCR profile prior to sequence analysis.
Once RNase H and reverse transcriptase(s) had been assessed for efficacy, primers were designed based on standard criteria and, in addition, we also tested primers containing locked nucleic acids for their performance with regards to both specificity and sensitivity. Primers containing locked nucleic acids exhibit enhanced thermal stability when hybridized with their DNA or RNA targets, allowing them to function under more stringent conditions than their counterparts without locked nucleic acids, thereby improving the possibility of discrimination between highly similar RNAs whilst at the same time increasing sensitivity (Tolstrup et al., 2003; Valoczi et al., 2004; Wang et al., 1999).
In order to assess the sensitivity of the RT-PCR assay in the detection of recombinant virus molecules, tenfold serial dilutions of artificial chimeric RNA3 transcribed in vitro were diluted in total plant RNA extracted from non-infected tobacco leaves. A comparative RT-PCR assay, carried out with primers with or without locked nucleic acids, showed that an increase in sensitivity was achieved with primers containing locked nucleic acids in both recombinant systems, with a greater yield of amplification product at all dilutions tested (Fig. 1). The limits of detection were 800 and 15 fg for the I17F-CMV/R-CMV and P-TAV/R-CMV systems, respectively. By using these primers, no discernible fragment was amplified when tested against negative-control RNA extracted from transgenic plants infected singly with P-TAV, R-CMV or I17F-CMV, or from combinations of independently extracted plant total RNA containing parental viral or transgenic RNA (Fig. 2). In some experimental samples, occasional additional bands were observed. However, they never appeared in the negative controls and, when sequenced, were found to be non-recombinants derived from one of the parental RNAs or from non-viral sequences (data not shown). For example, this was the case for the upper bands in Fig. 2(b) lanes 13 and 14, and similar non-recombinants also occurred occasionally in the I17F-CMV/R-CMV system described below.
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). The proportion of plants yielding putative recombinants (8.9 %) was comparable with that found in a previous study (9.6 %) in which recombinant molecules were detected in the converse direction, i.e. by using a P-TAV specific primer at the 3' NCR and an internal R-CMV primer (de Wispelaere et al., 2005). Putative recombinant RT-PCR products from six plants were cloned and sequenced, which in all cases confirmed their recombinant nature, with crossovers at seven different recombination sites (Table 1). In each plant, one to three crossover sites were observed. Of the 61 cloned recombinant molecules analysed, 60 arose by precise homologous recombination at six sites with 100 % identity, with a predominance of crossovers in larger blocks of sequence identity (Fig. 3a). As reflected in both the number of plants in which a given recombination site was observed (Table 1) and the proportion of clones at that site (Fig. 4b), three recombination sites predominated (nt 1940–1965, 1998–2018 and 2020–2038), with a scattering of less frequent crossovers elsewhere. All of the major sites had been identified previously as crossover sites for precise homologous recombination in the converse direction (de Wispelaere et al., 2005), with just one minor site observed in this study that was not observed in the converse, and vice versa. One of the recombination sites that was not in the 3' NCR (one clone at nt 1441–1442) was also the only site at which an imprecise homologous recombinant, in which there was a 2 nt substitution (TC
AG) at the crossover site, was observed.
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). RT-PCR products from 12 randomly chosen positive plants were cloned and sequenced, which in all cases confirmed their recombinant nature (Table 1). Of the 126 recombinant clones analysed, all arose by precise homologous recombination at seven sites with 100 % sequence identity, with the major sites of recombination in the larger blocks of sequence identity (Table 1; Fig. 3a). As in the non-transgenic plants, there was good agreement between the number of plants in which a given recombination site was observed (Table 1) and the proportion of clones observed at that site (Fig. 4b). In the transgenic plants, four major sites of recombination predominated (nt 1902–1922, 1940–1965, 1998–2018 and 2020–2038), with minor sites in other blocks of sequence identity. The recombination sites identified were the same as those identified in doubly infected plants, except for two minor crossover sites at nt 1757–1764 and 1977–1981, with one and two clones, respectively. Overall, similar populations of RNA3 recombinants were observed in transgenic and non-transgenic plants (Fig. 4b), and the most important sites were in large blocks of sequence identity (Fig. 3a) and were equivalent to those observed in non-transgenic plants screened for the converse recombinants (de Wispelaere et al., 2005).
Comparison of recombination sites between I17F-CMV and R-CMV in non-transgenic and transgenic plants
Sixty non-transgenic plants co-infected with R- and I17F-CMV were screened by RT-PCR for the presence of recombinants. Five were identified as containing putative recombinant molecules, which was subsequently verified by cloning and sequencing (Table 1). The proportion of plants containing recombinants (8.3 %) was similar to that found in the P-TAV/R-CMV system (8.9 %) described above. In each plant, one to six crossover sites were observed (nine in total). Of the 51 cloned recombinant molecules, 47 were of a precise homologous type at eight sites of 100 % identity, with one strongly predominant site at nt 1523–1554, as 59 % of the total crossovers occurred at this site (Fig. 4c). Imprecise homologous recombination was observed at one site (four clones) located at nt 1680–1695, which included an insert of 25 or 26 nt, corresponding to a duplication of sequence from either parental virus.
Of the 144 transgenic plants inoculated with I17F-CMV screened for recombinant RNA3 by RT-PCR, 38 (26 %) yielded putative recombinants. This is similar to what was observed in the P-TAV/R-CMV system (32 %). Eight of the positive samples were selected randomly for further characterization; as in the rest of this study, cloning and sequencing confirmed the recombinants. All 80 clones studied were produced by precise homologous recombination in the block of sequence identity at nt 1523–1554 (Table 1; Fig. 3b). This is the same site that predominated among the recombinants observed in non-transgenic plants (Fig. 4c). As in the P-TAV/R-CMV system, the overall pattern of the distribution of recombination sites was equivalent in transgenic and non-transgenic plants. However, it is noteworthy that, although fewer plants and clones were analysed, additional rare recombination sites were observed in non-transgenic plants that were absent in the transgenic ones (Fig. 4c).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The overall pattern of P-TAV/R-CMV recombinants observed, with a predominance of homologous precise recombination in the larger blocks of sequence identity, is similar to that observed previously in the converse direction (de Wispelaere et al., 2005). However, there were two salient differences between the results of the two studies. For R-CMV/P-TAV recombinants, an extremely strong hot spot for recombination was observed by de Wispelaere et al. (2005) at the GC dinucleotide at positions 1899–1900, which was not a hot spot for P-TAV/R-CMV recombination in either the transgenic or non-transgenic plants studied in the present work. In addition, more than one-third of the R-CMV/P-TAV recombinants were of an aberrant type, resulting in a 160–180 nt duplication in the 3' NCR (de Wispelaere et al., 2005). As similar recombinants have also been observed in certain natural CMV and TAV isolates (Chen et al., 2002; Moreno et al., 1997), it is surprising that this type of recombinant was not detected in the present study, but this may be due to the instability in tobacco plants of I17F-CMV/R-CMV recombinants of this type (Pierrugues et al., 2007).
The results presented here were obtained under conditions of low selection pressure, using wild-type virus inocula that are not targeted by the virus-resistance transgene, in order to observe the maximum number of types of recombinant produced. The recombination sites were extremely different for I17F-CMV/R-CMV and P-TAV/R-CMV, with, in the latter case, a single predominant hot spot for recombination in a large block of sequence identity within the CP gene (nt 1523–1554). As no crossovers were observed in nearby blocks of sequence identity of similar length, this hot spot may be determined by the presence of strong RNA secondary-structural features. However, with both experimental systems, we have observed equivalent populations of recombinant cucumoviruses in transgenic and non-transgenic plants, nearly all of which were of the precise homologous type.
As the recombinants described here have never been observed in the field, many of them may have reduced fitness. In a parallel study, the fitness of certain I17F-CMV/R-CMV and R-CMV/I17F-CMV recombinants was evaluated (Pierrugues et al., 2007). It is particularly interesting to note that the predominant I17F-CMV/R-CMV recombinant observed here under conditions of low selection pressure was in fact non-viable when inoculated alone into several CMV host plants, including tobacco.
In both experimental systems, recombinants were detected in a higher proportion of transgenic plants than in non-transgenic ones. In addition, there was a tendency to observe stronger RT-PCR amplification from samples from infected transgenic plants. Neither of these features is surprising because, as has been pointed out previously (Tepfer, 2002), recombinants can only occur in the small proportion of cells of non-transgenic plants in which both viruses are replicating, whereas the transgene mRNA and viral RNAs will be present in all infected cells. As explained in the Introduction, these experiments were carried out with transgenic plants designed to favour recombination, as the transgene includes the viral 3' NCR. These results give further support to the broadly supported suggestion that virus-resistant transgenic plants for commercial release should not include the 3' NCR, in order to prevent initiation on the transgene mRNA by the viral replicase and thus strongly reduce the appearance of recombinants; this has been shown to be highly effective in two other viral systems (Tepfer, 2002).
It is important to consider the limits to impose on the interpretation of the results of this type of study. First, as there are no equivalent results with other virus groups, it is impossible to know to what extent the conclusions can be generalized. Second, these experiments were carried out under highly controlled conditions, so it would be of great interest to determine whether they can be validated under the more variable conditions encountered in the field. Third, as it is impossible to sample the molecules present exhaustively, we cannot formally exclude the possibility that extremely rare recombinants could be present in transgenic plants, but absent in non-transgenic ones. Here, it should be noted that we have observed the opposite tendency, as we detected I17F-CMV/R-CMV recombinants in non-transgenic plants that were absent in the transgenic ones. In any case, in addition to exclusion from the transgene of the 3' NCR, there is an additional feature of the virus cycle that would further reduce the likelihood of emergence of rare recombinants, even if their intrinsic fitness were high. This is the presence of at least two narrow population bottlenecks, one at the stage of systemic movement of the virus and the other at vector transmission, which will strongly favour the most abundant RNAs (Ali et al., 2006; Li & Roossinck, 2004).
We have observed equivalent populations of recombinant cucumoviral RNAs in transgenic and non-transgenic plants, which argues strongly in favour of the same mechanism(s) of recombination being at work in both types of plant. Thus, our results are clearly not compatible with the hypothesis that novel recombinant viral genomes would be produced in transgenic plants, and thus are contrary to the hypothesis that these plants would increase the likelihood of emergence of new viruses, which would in addition require that any novel recombinant be able to compete favourably with the parental viruses.
Transgenes expressing a viral CP sequence can confer resistance via two types of mechanisms (reviewed by Goldbach et al., 2003). In some systems, including CMV-resistant tobacco (Jacquemond et al., 2001), the CP itself is involved in resistance, in which case, high levels of transgene mRNA accumulate. In other systems, resistance is mediated by post-transcriptional gene silencing (PTGS) and, in this case, only low levels of mRNA are observed. Clearly, the issue of potential impacts of mRNA/viral RNA recombination is more pertinent in the former cases and, indeed, it has been proposed that it would be preferable to use PTGS-mediated transgenic resistance in order to reduce the probability of appearance of recombinants (Tepfer, 2002). However, when PTGS-mediated resistance breaks down, this may create circumstances particularly favourable to recombination in transgenic plants (Jakab et al., 1997).
As our results showed no appreciable risk due to recombination in plants that accumulated high levels of transgene mRNA, we suggest that this should not be a major criterion for selecting a virus-resistance strategy and that far more important criteria would include the efficacy, breadth and stability of the resistance obtained.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 27 July 2007;
accepted 18 September 2007.
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