Benoît Moury

In spite of their widespread occurrence, only few host jumps by plant viruses have been evidenced and the molecular bases of even fewer have been determined. A combination of three independent approaches, 1) experimental evolution followed by reverse genetics analysis, 2) positive selection analysis, and 3) locus-by-locus analysis of molecular variance (AMOVA) allowed reconstructing the Potato virus Y (PVY; genus Potyvirus, family Potyviridae) jump to pepper (Capsicum annuum), probably from other solanaceous plants. Synthetic chimeras between infectious cDNA clones of two PVY isolates with contrasted levels of adaptation to C. annuum showed that the P3 and, to a lower extent, the CI cistron played important roles in infectivity toward C. annuum. The three analytical approaches pinpointed a single nonsynonymous substitution in the P3 and P3N-PIPO cistrons that evolved several times independently and conferred adaptation to C. annuum. In addition to increasing our knowledge of host jumps in plant viruses, this study illustrates also the efficiency of locus-by-locus AMOVA and combined approaches to identify adaptive mutations in the genome of RNA viruses.

The effective size of populations (Ne) determines whether selection or genetic drift is the predominant force shaping their genetic structure and evolution. Populations having high Ne adapt faster, as selection acts more intensely, than populations having low Ne, where random effects of genetic drift dominate. Estimating Ne for various steps of plant virus life cycle has been the focus of several studies in the last decade, but no estimates are available for the vertical transmission of plant viruses, although virus seed transmission is economically significant in at least 18% of plant viruses in at least one plant species. Here we study the co-dynamics of two variants of Pea seedborne mosaic virus (PSbMV) colonizing leaves of pea plants (Pisum sativum L.) during the whole flowering period, and their subsequent transmission to plant progeny through seeds. Whereas classical estimators of Ne could be used for leaf infection at the systemic level, as virus variants were equally competitive, dedicated stochastic models were needed to estimate Ne during vertical transmission. Very little genetic drift was observed during the infection of apical leaves, with Ne values ranging from 59 to 216. In contrast, a very drastic genetic drift was observed during vertical transmission, with an average number of infectious virus particles contributing to the infection of a seedling from an infected mother plant close to one. A simple model of vertical transmission, assuming a cumulative action of virus infectious particles and a virus density threshold required for vertical transmission to occur fitted the experimental data very satisfactorily. This study reveals that vertically-transmitted viruses endure bottlenecks as narrow as those imposed by horizontal transmission. These bottlenecks are likely to slow down virus adaptation and could decrease virus fitness and virulence. Short generation times and high mutation rates are the hallmarks of virus. They favor their fast adaptation as illustrated by their ability to overcome natural as well as man-made barriers such as host resistance or drug treatments. However, such a fast adaptation could be slowed down when genetic drift, which introduces random sampling effects in the evolution of virus populations, is important. Whether genetic drift or selection dominates depends on the effective size of populations (Ne). Ne has been estimated for several steps of plant virus infectious cycle, such as horizontal transmission by insects and the colonization of plant cells and tissues. However, although economically important, no estimate of Ne during vertical transmission of viruses, i.e. the infection of plant progenies from parental plants, is available. Here, we report that Pea seedborne mosaic virus (PSbMV), a seed transmitted virus infecting pea crops, undergoes very drastic genetic drift during vertical transmission, with an average number of infectious virus particles contributing to the infection of a seedling from an infected mother plant close to one. Such bottlenecks, as narrow as those imposed by horizontal transmission, could slow down virus adaptation and should be taken into account to improve plant protection strategies.

Summary: Genetic resistance provides efficient control of crop diseases, but is limited by pathogen evolution capacities which often result in resistance breakdown. It has been demonstrated recently, in three different pathosystems, that polygenic resistances combining a major‐effect gene and quantitative resistance controlled by the genetic background are more durable than monogenic resistances (with the same major gene in a susceptible genetic background), but the underlying mechanisms are unknown. Using the pepper– P otato virus Y system, we examined three mechanisms that could account for the greater durability of the polygenic resistances: (i) the additional quantitative resistance conferred by the genetic background; (ii) the increase in the number of mutations required for resistance breakdown; and (iii) the slower selection of adapted resistance‐breaking mutants within the viral population. The three mechanisms were experimentally validated. The first explained a large part of the variation in resistance breakdown frequency and is therefore expected to be a major determinant of resistance durability. Quantitative resistance factors also had an influence on the second mechanism by modifying the virus mutational pathways towards resistance breakdown and could also have an influence on the third mechanism by increasing genetic drift effects on the viral population. The relevance of these results for other plant–pathogen systems and their importance in plant breeding are discussed.

The pathogenic properties of Potato virus Y (PVY; genus Potyvirus , family Potyviridae ) isolates collected in naturally infected pepper ( Capsicum annuum ) fields in Tunisia were evaluated against recessive resistance alleles at the pvr2 locus of pepper. Two pathotypes were observed. Pathotype (0,1,3) isolates were able to infect plants carrying the susceptibility allele pvr2 ⁺ , together with pvr2 ¹ / pvr2 ¹ and pvr2 ³ / pvr2 ³ plants, but not pvr2 ² / pvr2 ² plants. Pathotype (0) isolates were only able to infect pvr2 ⁺ / pvr2 ⁺ plants. On the other hand, sequence data and phylogenetic analyses revealed three major groups of isolates, each characterized by particular amino acid residues in the central part of the VPg, the pathogenicity factor towards pvr2 . Correspondence between pathogenic properties and phylogeny suggested a single evolutionary step for pathogenicity towards the pvr2 ¹ and pvr2 ³ resistances, possibly under the selective pressure of pvr2 ¹ . Indeed, 23% of the pepper plants in this area were shown to carry the pvr2 ¹ resistance, while pvr2 ³ was not detected. The data suggested that pathogenicity towards pvr2 ¹ and pvr2 ³ were not costly for PVY to infect susceptible pepper genotypes and supported the matching allele model for pepper–PVY interactions.

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.

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.

Tomato spotted wilt virus (TSWV; genus Tospovirus, family Bunyaviridae) genetic diversity was evaluated by sequencing parts of the three RNA genome segments of 224 isolates, mostly from pepper and tomato crops in southern Europe. Eighty-three per cent of the isolates showed consistent clustering into three clades, corresponding to their geographical origin, Spain, France or the USA, for the three RNA segments. In contrast, the remaining 17 % of isolates did not belong to the same clade for the three RNA segments and were shown to be reassortants. Among them, eight different reassortment patterns were observed. Further phylogenetic analyses provided insights into the dynamic processes of the worldwide resurgence of TSWV that, since the 1980s, has followed the worldwide dispersal of the western flower thrips (Frankliniella occidentalis) tospovirus vector. For two clades composed essentially of Old World (OW) isolates, tree topology suggested a local re-emergence of indigenous TSWV populations following F. occidentalis introductions, while it could not be excluded that the ancestors of two other OW clades were introduced from North America contemporarily with F. occidentalis. Finally, estimation of the selection intensity that has affected the evolution of the NSs and nucleocapsid proteins encoded by RNA S of TSWV suggests that the former could be involved in the breakdown of resistance conferred by the Tsw gene in pepper.

SUMMARY: Potato virus Y (PVY) is one of the rare plant viruses for which some biological traits (host range and symptomatology) are highly correlated with phylogeny, allowing the reconstruction of the evolutionary history of these traits. In this article, a new lineage of PVY isolates from Chile is described, showing unique genomic and biological properties. This lineage was found to be the sister group of all other PVY isolates and helped in the reconstruction of the ancestral traits and evolutionary history of PVY, suggesting that veinal necrosis in tobacco is an ancestral state and that adaptation to pepper ( Capsicum spp.) and potato ( Solanum tuberosum ) has been modified several times during PVY history.

The dominant Pvr4 gene identified in Capsicum annuum cv. Criollo de Morelos 334 (CM334) is frequently used in pepper cultivars because it possesses one of the largest spectra of action among plant virus resistance genes. This gene was previously shown to confer efficient resistance to all known Potato virus Y isolates, to Pepper mottle virus , to Pepper yellow mosaic virus and to Ecuadorian rocoto virus. This study showed that the W4 line, derived from CM334 and carrying Pvr4 , was also resistant to Peru tomato mosaic virus and Pepper severe mosaic virus , but not to Pepper veinal mottle virus , Chilli veinal mottle virus or Tobacco etch virus . It was noticed that the phenotype of the resistance was atypical since, in the W4 line, hypersensitive reaction or extreme resistance could be observed, depending on virus isolates and inoculated organs. Despite the large deployment of Pvr4 in hybrid cultivars, the numerous tests performed in controlled conditions and the use of W4 serial back‐inoculations with potyvirus isolates controlled by this line, no virulent variant isolates were obtained. However, it was shown that the use of graft inoculation experiments allow PVY virulent variants to be selected.

Sequencing 2,951 nucleotides of the 3′ proximal region of the genome of a potyvirus isolate collected from Capsicum pubescens (rocoto) pepper in Ecuador revealed that this was the first representative of a new species tentatively named Ecuadorian rocoto virus (ERV). Phylogeny reconstruction showed that this isolate clustered with potato virus V (PVV), Peru tomato virus and wild potato mosaic virus into a monophyletic group, and was closest to PVV. The isolate was shown to be infectious in tobacco, tomato and, contrary to PVV, in pepper. The pvr2 ¹ , pvr2 ² , and Pvr4 genes present in many pepper cultivars conferred resistance toward this isolate and could help control ERV.