A Single Nonsynonymous Substitution in the RNA-Dependent RNA Polymerase of Potato virus Y Allows the Simultaneous Breakdown of Two Different Forms of Antiviral Resistance in Capsicum annuum.

The dominant Pvr4 gene in pepper (Capsicum annuum) confers resistance to members of six potyvirus species, all of which belong to the Potato virus Y (PVY) phylogenetic group. The corresponding avirulence factor in the PVY genome is the NIb cistron (i.e., RNA-dependent RNA polymerase). Here, we describe a new source of potyvirus resistance in the Guatemalan accession C. annuum cv. PM949. PM949 is resistant to members of at least three potyvirus species, a subset of those controlled by Pvr4. The F1 progeny between PM949 and the susceptible cultivar Yolo Wonder was susceptible to PVY, indicating that the resistance is recessive. The segregation ratio between resistant and susceptible plants observed in the F2 progeny matched preferably with resistance being determined by two unlinked recessive genes independently conferring resistance to PVY. Inoculations by grafting resulted in the selection of PVY mutants breaking PM949 resistance and, less efficiently, Pvr4-mediated resistance. The codon substitution E472K in the NIb cistron of PVY, which was shown previously to be sufficient to break Pvr4 resistance, was also sufficient to break PM949 resistance, a rare example of cross-pathogenicity effect. In contrast, the other selected NIb mutants showed specific infectivity in PM949 or Pvr4 plants. Comparison of Pvr4 and PM949 resistance, which share the same target in PVY, provides interesting insights into the determinants of resistance durability.

dN/dS-based methods detect positive selection linked to trade-offs between different fitness traits in the coat protein of potato virus Y.

The dN/dS ratio between nonsynonymous and synonymous substitution rates has been used extensively to identify codon positions involved in adaptive processes. However, the accuracy of this approach has been questioned, and very few studies have attempted to validate experimentally its predictions. Using the coat protein (CP) of Potato virus Y (PVY; genus Potyvirus, family Potyviridae) as a case study, we identified several candidate positively selected codon positions that differed between clades. In the CP of the N clade of PVY, positive selection was detected at codon positions 25 and 68 by both the softwares PAML and HyPhy. We introduced nonsynonymous substitutions at these positions in an infectious cDNA clone of PVY and measured the effect of these mutations on virus accumulation in its two major cultivated hosts, tobacco and potato, and on its efficiency of transmission from plant to plant by aphid vectors. The mutation at codon position 25 significantly modified the virus accumulation in the two hosts, whereas the mutation at codon position 68 significantly modified the virus accumulation in one of its hosts and its transmissibility by aphids. Both mutations were involved in adaptive trade-offs. We suggest that our study was particularly favorable to the detection of adaptive mutations using dN/dS estimates because, as obligate parasites, viruses undergo a continuous and dynamic interaction with their hosts that favors the recurrent selection of adaptive mutations and because trade-offs between different fitness traits impede (or at least slow down) the fixation of these mutations and maintain polymorphism within populations.

The helper component proteinase cistron of Potato virus Y induces hypersensitivity and resistance in Potato genotypes carrying dominant resistance genes on chromosome IV.

The Nc(tbr) and Ny(tbr) genes in Solanum tuberosum determine hypersensitive reactions, characterized by necrotic reactions and restriction of the virus systemic movement, toward isolates belonging to clade C and clade O of Potato virus Y (PVY), respectively. We describe a new resistance from S. sparsipilum which possesses the same phenotype and specificity as Nc(tbr) and is controlled by a dominant gene designated Nc(spl). Nc(spl) maps on potato chromosome IV close or allelic to Ny(tbr). The helper component proteinase (HC-Pro) cistron of PVY was shown to control necrotic reactions and resistance elicitation in plants carrying Nc(spl), Nc(tbr), and Ny(tbr). However, inductions of necrosis and of resistance to the systemic virus movement in plants carrying Nc(spl) reside in different regions of the HC-Pro cistron. Also, genomic determinants outside the HC-Pro cistron are involved in the systemic movement of PVY after induction of necroses on inoculated leaves of plants carrying Ny(tbr). These results suggest that the Ny(tbr) resistance may have been involved in the recent emergence of PVY isolates with a recombination breakpoint near the junction of HC-Pro and P3 cistrons in potato crops. Therefore, this emergence could constitute one of the rare examples of resistance breakdown by a virus which was caused by recombination instead of by successive accumulation of nucleotide substitutions.

Estimation of the number of virus particles transmitted by an insect vector.

Plant viruses are submitted to narrow population bottlenecks both during infection of their hosts and during horizontal transmission between host individuals. The size of bottlenecks exerted on virus populations during plant invasion has been estimated in a few pathosystems but is not addressed yet for horizontal transmission. Using competition for aphid transmission between two Potato virus Y variants, one of them being noninfectious but equally transmissible, we obtained estimates of the size of bottlenecks exerted on an insect-borne virus during its horizontal transmission. We found that an aphid transmitted on average 0.5-3.2 virus particles, which is extremely low compared with the census viral population into a plant. Such narrow bottlenecks emphasize the strength of stochastic events acting on virus populations, and we illustrate, in modeling virus emergence, why estimating this parameter is important.

Molecular dissection of the potato virus Y VPg virulence factor reveals complex adaptations to the pvr2 resistance allelic series in pepper.

The virulence properties of potato virus Y (PVY) towards an allelic series at the pvr2 locus in pepper genotypes are related to variations in the genome-linked viral protein (VPg). Eleven amino acid substitutions in the central part of the VPg were identified in strains differing by their virulence properties and were introduced, either singly or in combination, in an infectious PVY clone to get an in-depth genetic analysis of the virulence determinant. The virulence spectrum of these mutants was evaluated by inoculation of four pepper genotypes carrying different alleles at the pvr2 locus. The mutations introduced had complex effects on virulence, including antagonistic epistasis and trade-offs for virulence towards different pvr2 alleles. In addition, several mutants showed new virulence properties that were unknown in the natural environment. Such complex effects of mutations on plant virus virulence are unprecedented. They provide a better understanding of the variable levels of durability of the resistance conferred by the different pvr2 alleles, and have important consequences for a durable management of the resistances.

Phylogenetic analysis of isolates of Beet necrotic yellow vein virus collected worldwide.

A study of molecular diversity was carried out on 136 sugar beets infected with Beet necrotic yellow vein virus (BNYVV, Benyvirus) collected worldwide. The nucleotide sequences of the RNA-2-encoded CP, RNA-3-encoded p25 and RNA-5-encoded p26 proteins were analysed. The resulting phylogenetic trees allowed BNYVV to be classified into groups that show correlations between the virus clusters and geographic origins. The selective constraints on these three sequences were measured by estimating the ratio between synonymous and non-synonymous substitution rates (omega) with maximum-likelihood models. The results suggest that selective constraints are exerted differently on the proteins. CP was the most conserved, with mean omega values ranging from 0.12 to 0.15, while p26 was less constrained, with mean omega values ranging from 0.20 to 0.33. Selection was detected in three amino acid positions of p26, with omega values of about 5.0. The p25 sequences presented the highest mean omega values (0.36-1.10), with strong positive selection (omega=4.7-54.7) acting on 14 amino acids, and particularly on amino acid 68, where the omega value was the highest so far encountered in plant viruses.