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Short Communication |
1 UR 141, Institut de Recherche pour le Développement (IRD), BP 64501, 34394 Montpellier cedex 5, France
2 UR 121, Institut de Recherche pour le Développement (IRD), BP 64501, 34394 Montpellier cedex 5, France
Correspondence
Eugénie Hébrard
hebrard{at}mpl.ird.fr
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) is present in all rice-growing African countries, where it causes heavy yield losses (Abo et al., 1998; Sy et al., 2001). Very few rice varieties are resistant to RYMV. Recently, the available types of resistance were compared (Sorho et al., 2005). The highest level of resistance was provided by a cultivar of Oryza sativa indica, ‘Gigante’, and a few cultivars of Oryza glaberrima series Tog (Ndjiondjop et al., 1999; Thottappilly & Rossel, 1993). These varieties showed a natural high resistance characterized by a low virus titre and the absence of symptoms (Ndjiondjop et al., 1999). A single recessive gene, rymv-1, localized on chromosome 4, is responsible for this phenotype (Albar et al., 2003).
Examples of recessive resistance against viruses are numerous (reviewed by Kang et al., 2005), but only four other crop genes, all directed against viruses in the family Potyviridae, have been identified at the molecular level (Gao et al., 2004; Kanyuka et al., 2005; Nicaise et al., 2003; Ruffel et al., 2002; Stein et al., 2005). These four genes encode the eukaryotic initiation factor 4E (eIF4E). In addition, artificial mutants in the Arabidopsis thaliana gene eIF(iso)4E, the isoform of eIF4E, exhibit resistance to several potyviruses (Duprat et al., 2002; Lellis et al., 2002). Several resistance-breaking (RB) variants of Potyviridae have been described. Most often, they involve mutations in the genome-linked viral protein (VPg) (Moury et al., 2004), although P3-6K1 and HC-Pro are sometimes involved (Hjulsager et al., 2002; Redondo et al., 2001).
Some field-collected RB isolates of RYMV able to induce symptoms in ‘Tog 5681’ and/or ‘Gigante’ have been found (Konaté et al., 1997; Traoré et al., 2006). Furthermore, RB isolates can emerge experimentally after serial inoculations (Fargette et al., 2002). In this last case, the progeny of the non-resistance-breaking (nRB) isolate CI4 (GenBank accession no. AJ279905
[GenBank]
) induced mottling symptoms 30 days post-infection (p.i.) after four passages in ‘Gigante’ and stunting at the sixth passage (Fargette et al., 2002). The RB variant CI4* was not counterselected in susceptible plants, as it multiplied to a similar level after serial passages, even when mixed with nRB isolate CI4, and it retained its ability to overcome the resistance (Sorho et al., 2005). Nevertheless, the molecular changes from nRB isolate CI4 that led to RB isolate CI4* were not determined. In this study, we identified a candidate mutation in the VPg domain. Nucleotide-specific probes were developed to investigate mutation stability, complementation, reversion and additional mutations during the serial passages. Finally, the involvement of the mutation G1729T in the resistance-breaking phenomenon was validated in an infectious clone.
The full-length sequence of RB isolate CI4*, obtained after six passages in highly resistant plants, was determined by using a method and primers described previously (Fargette et al., 2004). Sequence comparison of the isolates nRB CI4 and RB CI4* showed six differences localized in ORF2 (Table 1; Fig. 1a). Four mutations were synonymous, whilst the other two were non-synonymous, each producing an amino acid change. The polymorphism of these positions was analysed in 16 full-length sequences of nRB isolates described previously, including CI4 (Fargette et al., 2004). Four positions (nt 1544, 1547, 3323 and 3395) of the six mutated sites showed the same nucleotide in CI4* as in several other sequences (7/16, 4/16, 7/16 and 15/16, respectively). The two other positions (nt 1298 and 1729) showed the same nucleotides in all of the isolates as in CI4 (16/16), suggesting that the nucleotide changes observed in CI4* were specific for resistance breaking. The mutation C1298T was synonymous. The mutation G1729T produced a non-synonymous change of Arg374Ile in the polyprotein P2a. The latter mutation was localized close to the conserved motif W(A/G)D (nt 1776–1784) in a D- and E-rich region. This motif is present in analysed and predicted VPg proteins of sobemoviruses (Mäkinen et al., 1995b; Tamm & Truve, 2000). Hence, the mutation G1729T is within the RYMV VPg. This non-synonymous mutation represented a likely candidate for involvement in resistance breaking.
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). Four primers were designed to allow the detection of the RYMV polymorphism at position 1729 (Fig. 1b
). The flanking primers F1 (nt 1469–1483, 5'-CCCGCTCTACCACAA-3') and R1 (nt 2131–2117, 5'-AAAGATGACACCCGA-3') gave a 663 bp fragment, which was used as positive control for PCR in infected plants. Internal primer F2 (nt 1716–1729, 5'-TGGGTGCGTGAGAG-3') was designed to match the sequence of RYMV CI4 to that of isolate CI4*, except for one mismatch (shown in bold) on the 3' end. This primer, together with R1, gave an additional band of 416 bp when a guanine was present at nt 1729. In contrast, the internal primer R2 (nt 1741–1729, 5'-TGGTACCTCTCTA-3') matched the CI4* sequence, giving an additional band of 273 bp when a thymine was present at nt 1729 when used with primer F1. To perform the single-nucleotide polymorphism detection, first-strand synthesis was performed by using Superscript II reverse transcriptase (Gibco-BRL) with primer R1. The cDNA was used as template for PCR amplification using DyNAzyme EXT DNA polymerase (Finnzymes) with the forward primer F1, the reverse primer R1 and a third primer, either F2 or R2. In susceptible plants inoculated with CI4, only the 416 bp fragment was detected, indicating the presence of G1729 and the absence of T1729 (Fig. 1c, lanes 1 and 2). In contrast, after eight passages in resistant plants, only the 273 bp fragment was detected, indicating the absence of G1729 and the presence of T1729 (Fig. 1c, lanes 7 and 8). Interestingly, T1729 was detected as early as the first passage, whilst G1729 remained undetected (Fig. 1c, lanes 3 and 4). T1729 was retained at each subsequent passage (data not shown). Therefore, only the isolates with a thymine at nt 1729 were detected in resistant plants. The non-mutated nucleotide G1729, whether derived from the initial isolate CI4 or from a reversion, was never detected in resistant plants. After the first passage in resistant plants, the isolate was inoculated back into susceptible plants (Fig. 1c, lanes 5 and 6). Again, only T1729 was detected, indicating that an isolate containing the G1729T mutation multiplied in susceptible plants. The same result was observed with the isolate CI4*, obtained after eight passages in resistant plants and inoculated back into susceptible plants after one or four serial passages (Fig. 1c, lanes 9–12). This latter isolate infected resistant plants and presented T1729 alone (Fig. 1c, lanes 13 and 14). Altogether, these results indicated that an isolate containing the G1729T mutation was not counterselected in susceptible plants and that this isolate was still able to break rymv-1 resistance. Again, the non-mutated nucleotide, whether derived from the initial isolate CI4 or from a reversion, was not detected in these susceptible plants. Artificial mixed inoculations with CI4 and CI4* performed by Sorho et al. (2005) were also assessed. The presence of T1729 was detected in both the susceptible and the resistant plants, whilst G1729 was not detected in resistant plants, suggesting that there was no complementation of nRB CI4 by RB CI4* (data not shown).
The mutation G1729T was detected in the first passage in resistant plants, although no symptoms were apparent. To determine whether the increasing intensity of symptoms through subsequent passages was due to additional mutations around G1729T, each CI4* isolate (obtained in resistant plants) was partially sequenced from nt 1230 to 2090 with primers R9 (nt 1506–1525, 5'-ATAGGTGCTGCGGATGGTTA-3') and R10 (nt 1840–1821, 5'-GCTACGGGATGCGATGTCTC-3') after PCR amplification with the primers BS and BAS as described by Fargette et al. (2004). The mutations C1298T, C1547T and G1729T were observed at each passage. The mutation G1544A was not observed in the other CI4* isolates, suggesting that it was not involved in resistance breaking. The only additional mutation detected in the analysed fragment was G1489A at the eighth passage. However, further serial passages in ‘Gigante’ did not reveal this mutation (data not shown), indicating that it was not stable. Moreover, G1489 is conserved in the 16 isolates that have been sequenced fully. No additional mutation around G1729T that could explain the appearance of symptoms was observed through serial passages. However, after inoculation with leaf extracts of all previous passages and a longer period of monitoring symptoms, the appearance of mild symptoms from 45 days p.i. was observed (data not shown). As shown by Sorho et al. (2005), inoculum concentration is a key factor in overcoming resistance, which suggests that, in the previous experiment, the concentration was below the threshold required to produce symptoms during the first passage.
The mutation G1729T was introduced in an nRB infectious cDNA clone to validate its role in resistance breaking. As the clone of CI4 was not available, the infectious cDNA clone of the nRB isolate CIa was used (Brugidou et al., 1995). Two PCR experiments were performed independently with a flanking primer and a primer containing the mutation, FL5R5bis (nt 892–911, 5'-CCCGGGGTTACGTGGTTGAC-3') and RmIIR374I (nt 1738–1717, 5'-TACTTCGTTATCTCACGCACCC-3'), and R14bis (nt 2424–2401, 5'-ACTTCGCCGGTTTCGCAGAGGATT-3') and FmIIR374I (nt 1714–1738, 5'-CCTGGGTGCGTGAGATAACGAAGTA-3'), respectively. The products of the two PCR applications were mixed and used as template for another PCR experiment with the two flanking primers. The PCR fragment was cloned in the infectious cDNA clone CIa after double digestion with AflII and AvrII (Biolabs). The AGG codon at nt 1728–1730 was mutated to ATA, as in RB CI4*, to produce a clone called CIa*. The mutation was confirmed by sequencing the fragment. However, two other mutations, A1414G and G2095A, were introduced randomly during the mutagenesis process. The latter was at the ATG of ORF2b (Fig. 1a). These mutations were not observed in the 16 nRB isolates that have been sequenced (Fargette et al., 2004). The infectivity of the mutated clone CIa* was tested in susceptible plants after in vitro transcription with T7 RNA polymerase (Promega) performed as described previously (Brugidou et al., 1995). Despite the three non-synonymous mutations, in vitro transcripts from the clone CIa* were infectious. This result demonstrated that, as predicted by homology with Cocksfoot mottle virus (Mäkinen et al., 1995a), the ATG codon of ORF2b was not a start codon and ORF2b was translated in fusion with ORF2a from the frameshift signal at nt 1979. Moreover, yellow mottling symptoms appeared on inoculated and uninoculated leaves in susceptible plants. A high content of virus was detected at 21 days p.i. by double antibody-sandwich ELISA, performed as described previously (N'Guessan et al., 2000). No significant difference in the content of virus was observed in susceptible plants infected with progeny of the isolates CIa*, CIa, CI4 and CI4*. Moreover, the isolate CIa* overcame the high resistance of ‘Gigante’. Symptom intensity and virus content at 45 days p.i. in uninoculated leaves were similar to those of CI4*. This experiment was repeated twice on 20 plants, each with different virus preparations. Hence, the mutation G1729T was sufficient to induce symptoms of resistance breaking. After multiplication in resistant plants, isolate CIa* was again fully sequenced and the presence of the mutations introduced by mutagenesis was confirmed. Another mutation, C3541T, which was absent in the original mutated clone CIa*, but present in the 16 full-length sequences, was detected.
It is notable that isolates CI4* and CIa* belong to two different RYMV strains (S1 and S3, respectively) (Pinel et al., 2000) that are geographically, ecologically and genetically distant (Traoré et al., 2006). Moreover, the surrounding context of the mutation G1729T (i.e. Arg374Ile) is different. In particular, the two downstream codons encode amino acids that possess different physicochemical properties (Glu375 Arg376 in CI4* and Thr375 Lys376 in Cla*). We concluded that the mutation G1729T was sufficient to induce resistance breaking in uninoculated leaves in different strains and genetic contexts.
Taken together, the data presented here provide the first evidence that VPg is the virulence factor for recessive resistance in a virus outside the family Potyviridae. The RYMV–rice pathosystem has several specificities. First, no amino acid sequence similarity has been detected between the VPgs of RYMV and members of the family Potyviridae. Secondly, the genome organization of sobemoviruses differs from that of members of the family Potyviridae (Tamm & Truve, 2000): the 3' end of the RNA is not polyadenylated and the VPg is localized downstream of the protease. Finally, the resistance gene rymv-1 appears to be different from the well-studied eIF4E. Recently, we identified rymv-1 as a truncated isoform of the eukaryotic translation initiation factor 4G [eIF(iso)4G] (L. Albar, M. Bangratz-Reiser, E. Hébrard, M.-N. Ndjiondjop, M. Jones & A. Ghesquière, unpublished results). The factors 4E and 4G and their isoforms provide different functions in the translation-initiation complex. Whilst eIF4E binds directly to the cap of mRNA, the high-molecular-mass eIF4G acts as a scaffold for other components of the complex, such as eIF4E. Recently, eIF4G was reported to be involved in recessive resistance against viruses in the families Bromoviridae and Tombusviridae (Yoshii et al., 2004). Whilst direct interactions between eIF4Es and VPgs of members of the family Potyviridae were demonstrated (Léonard et al., 2000, 2004; Schaad et al., 2000; Wittmann et al., 1997), the interactions between eIF4G and these other viruses, which do not possess a VPg, are not known. Further investigations on the molecular properties that enable RYMV to overcome the resistance of O. sativa ‘Gigante’ would contribute to the understanding of recessive resistance mediated by eIF4G genes.
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ACKNOWLEDGEMENTS |
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Received 4 November 2005;
accepted 13 January 2006.
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