Evolutionary Pathways to Break Down the Resistance of Allelic Versions of the PVY Resistance Gene va.

Emergence of viral genotypes can make control strategies based on resistance genes ineffective. A few years after the deployment of tobacco genotypes carrying alleles of the Potato virus Y (PVY) recessive resistance gene va, virulent PVY isolates have been reported, suggesting the low durability of va. To have a broader view of the evolutionary processes involved in PVY adaptation to va, we studied mutational pathways leading to the emergence of PVY resistance-breaking populations. The viral genome-linked protein (VPg) has been described to be potentially involved in va adaptation. Analyses of the VPg sequence of PVY isolates sampled from susceptible and resistant tobacco allowed us to identify mutations in the central part of the VPg. Analysis of the virulence of wild-type isolates with known VPg sequences and of mutated versions of PVY infectious clones allowed us to (i) validate VPg as the PVY virulence factor corresponding to va, (ii) highlight the fact that virulence gain in PVY occurs rapidly and preferentially by substitution at position AA105 in the VPg, and (iii) show that the 101G substitution in the VPg of a PVYC isolate is responsible for cross-virulence toward two resistance sources. Moreover, it appears that the evolutionary pathway of PVY adaptation to va depends on both virus and host genetic backgrounds.

Specific detection of the PVY(N)-W variant of Potato virus Y.

PVY(N)-W is one of the variant populations of Potato virus Y (PVY). This variant is of concern in seed potato production and requires a specific diagnosis since it induces more or less symptomless infections and is not detectable easily in field inspections. Moreover, this variant is serologically indistinguishable from the common strain PVY(O). This study describes a simple and specific molecular detection test for the PVY(N)-W variant using a PCR protocol based on the recombinant point within the HC-Pro/P3 region of PVY(N) variants (PVY(NTN), PVY(N)-W). To avoid both detection of recombinant PVY(NTN) and PVY(N)-W isolates, a forward PVY(N)-like primer located in the HC-Pro region coupled to a reverse PVY(O)-like primer located in the NIa region was designed to amplify a specific PCR product of 4114 nt from PVY(N)-W isolates. This technique was assessed on 41 PVY reference and field isolates. Only isolates referenced as PVY(N)-W were amplified and gave the expected PCR product of 4114 nt, whereas no band was obtained from PVY(N), PVY(NTN) or PVY(O) isolates. In conclusion, this PVY(N)-W diagnosis tool is rapid, easy-to-use and suitable for large-scale testing in laboratories of seed potato certification.

Genomic variability in Potato potyvirus Y (PVY): evidence that PVY(N)W and PVY(NTN) variants are single to multiple recombinants between PVY(O) and PVY(N) isolates.

Fourteen Potato virus Y (PVY) isolates representative of PVY(O), PVY(N), PVY(NTN) and PVY(N)W groups were characterised at genomic level. Restriction fragment length polymorphism study (RFLP) of each gene of these isolates and sequencing of the first 2700 nucleotides of two PVY(N)W isolates were performed. A mosaic structure was revealed in PVY(N)W and PVY(NTN) genomes, which showed either PVY(O) or PVY(N)-like sequences, depending on the particular gene. Indeed, starting from the 5'-end, these isolates showed a switching, from PVY(N)- to PVY(O)-like sequence, in the HC-Pro C-terminal region. Reversion to PVY(N)-like sequence was also revealed in the NIa N-terminal area of PVY(NTN) isolates, followed by a switching back to a PVY(O)-like sequence in the CP gene. Lastly, some PVY(N)W isolates showed a switching from PVY(O)- to PVY(N)-like sequence in the P1 N-terminal part, thus separating our PVY(N)W isolates into two subgroups. All these apparent recombination events were shown by statistical analysis. Comparison of molecular traits with pathogenic properties of our isolates suggested that the HC-Pro protein is involved in induction of necrosis in tobacco leaves, and the NIa, NIb and/or CP protein in necrosis in potato tubers. Nevertheless, multiple recombination events observed in the PVY(NTN) genome may play a role in the latter phenomenon.

RFLP mapping of the whole genome of ten viral isolates representative of different biological groups of potato virus Y.

Ten PVY isolates representative of four PVY groups (YN, YNTN, YN-W, YO), differing by their ability to induce reactions of vein necrosis on tobacco and tuber necrosis on potato, were studied in order to research the regions of the viral genome involved in these necrosis phenomena. The whole genome of these isolates was amplified in two fragments (4,063 and 5,670 nucleotides) and was subjected to a restriction fragment length polymorphism (RFLP) study. In the first 4,063 nucleotides of the PVY genome, a phenetic analysis of RFLP data resulted in a clustering of our PVY isolates into three groups: PVYN isolates (group A); PVYNTN and PVYN-W isolates (group B) and PVYO isolates (group C). In the last 5,670 nucleotides, two groups were found: PVYN and PVYNTN isolates (group D) and PVYO and PVYN-W isolates (group E). From this clustering and the necrosing properties known for these isolates, the tobacco necrosis determinants seem more likely located in the 5' than in the 3' half part of the viral RNA, whereas it would be the opposite situation for the determinants of the necrosis on potato tubers. Moreover a recombination event seemed to have occurred in the genome of the PVYN-W isolates.