Within-plant genetic drift to control virus adaptation to host-resistance genes.

Manipulating evolutionary forces imposed by hosts on pathogens like genetic drift and selection could avoid the emergence of virulent pathogens. For instance, increasing genetic drift could decrease the risk of pathogen adaptation through the random fixation of deleterious mutations or the elimination of favorable ones in the pathogen population. However, no experimental proof of this approach is available for a plant-pathogen system. We studied the impact of pepper (Capsicum annuum) lines carrying the same major resistance gene but contrasted genetic backgrounds on the evolution of Potato virus Y (PVY). The pepper lines were chosen for the contrasted levels of genetic drift (inversely related to Ne, the effective population size) they exert on PVY populations, as well as for their contrasted resistance efficiency (inversely related to the initial replicative fitness, Wi, of PVY in these lines). Experimental evolution was performed by serially passaging 64 PVY populations every month on six contrasted pepper lines during seven months. These lines exhibited highly divergent evolutionary trajectories, ranging from viral extinctions to replicative fitness gains. The sequencing of the PVY VPg cistron, where adaptive mutations are likely to occur, allowed linking these replicative fitness gains to parallel adaptive nonsynonymous mutations. Evolutionary trajectories were well explained by the genetic drift imposed by the host. More specifically, Ne, Wi and their synergistic interaction played a major role in the fate of PVY populations. When Ne was low (i.e. strong genetic drift), the final PVY replicative fitness remained close to the initial replicative fitness, whereas when Ne was high (i.e. low genetic drift), the final PVY replicative fitness was high independently of the replicative fitness of the initially inoculated virus. We show that combining a high resistance efficiency (low Wi) and a strong genetic drift (low Ne) is the best solution to increase resistance durability, that is, to avoid virus adaptation on the long term.

Quantitative trait loci in pepper control the effective population size of two RNA viruses at inoculation.

Infection of plants by viruses is a complex process involving several steps: inoculation into plant cells, replication in inoculated cells and plant colonization. The success of the different steps depends, in part, on the viral effective population size (Ne), defined as the number of individuals passing their genes to the next generation. During infection, the virus population will undergo bottlenecks, leading to drastic reductions in Ne and, potentially, to the loss of the fittest variants. Therefore, it is crucial to better understand how plants affect Ne. We aimed to (i) identify the plant genetic factors controlling Ne during inoculation, (ii) understand the mechanisms used by the plant to control Ne and (iii) compare these genetic factors with the genes controlling plant resistance to viruses. Ne was measured in a doubled-haploid population of Capsicum annuum. Plants were inoculated with either a Potato virus Y (PVY) construct expressing the green fluorescent protein or a necrotic variant of Cucumber mosaic virus (CMV). Newas assessed by counting the number of primary infection foci on cotyledons for PVY or the number of necrotic local lesions on leaves for CMV. The number of foci and lesions was correlated (r=0.57) and showed a high heritability (h2=0.93 for PVY and h2=0.98 for CMV). The Ne of the two viruses was controlled by both common quantitative trait loci (QTLs) and virus-specific QTLs, indicating the contribution of general and specific mechanisms. The PVY-specific QTL colocalizes with a QTL that reduces PVY accumulation and the capacity to break down a major-effect resistance gene.

Absence of Seed-Mediated Transmission of Cucumber Mosaic Virus in Espelette Pepper Crops despite Widespread and Recurrent Epidemics.

In the past decade, severe epidemics of cucumber mosaic virus (CMV) have caused significant damage to Espelette pepper crops. This virus threatens the production of Espelette pepper, which plays a significant role in the local economy and touristic attractiveness of the French Basque Country, located in southwestern France. In 2021 and 2022, CMV was detected via double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) in Gorria pepper seed lots harvested from naturally infected fields scattered throughout the entire Espelette pepper production area. These seed lots were used in greenhouse grow-out tests to determine whether CMV could be transmitted to seedlings from contaminated seeds, using visual symptom assessment, DAS-ELISAs, and reverse transcription-polymerase chain reaction (RT-PCR). Despite the widespread occurrence of CMV in seeds of field samples, the grow-out experiments on a total of over 5000 seedlings yielded no evidence of seed transmission of local CMV isolates in Gorria pepper. Therefore, rather than seeds from infected pepper plants, sources of CMV inoculum in Espelette are more likely to be alternative hosts present in and around pepper fields that can allow for the survival of CMV during the off-season. These results have important epidemiological implications and will guide the choice of effective measures to control current epidemics.

Concurrent evolution of resistance and tolerance to potato virus Y in Capsicum annuum revealed by genome-wide association.

We performed a genome-wide association study of pepper (Capsicum annuum) tolerance to potato virus Y (PVY). For 254 pepper accessions, we estimated the tolerance to PVY as the coefficient of regression of the fresh weight (or height) of PVY-infected and mock-inoculated plants against within-plant virus load. Small (strongly negative) coefficients of regression indicate low tolerance because plant biomass or growth decreases sharply as virus load increases. The tolerance level varied largely, with some pepper accessions showing no symptoms or fairly mild mosaics, whereas about half (48%) of the accessions showed necrotic symptoms. We found two adjacent single-nucleotide polymorphisms (SNPs) at one extremity of chromosome 9 that were significantly associated with tolerance to PVY. Similarly, in three biparental pepper progenies, we showed that the induction of necrosis on PVY systemic infection segregated as a monogenic trait determined by a locus on chromosome 9. Our results also demonstrate the existence of a negative correlation between resistance and tolerance among the cultivated pepper accessions at both the phenotypic and genetic levels. By comparing the distributions of the tolerance-associated SNP alleles and previously identified PVY resistance-associated SNP alleles, we showed that cultivated pepper accessions possess favourable alleles for both resistance and tolerance less frequently than expected under random associations, while the minority of wild pepper accessions frequently combined resistance and tolerance alleles. This divergent evolution of PVY resistance and tolerance could be related to pepper domestication or farmer's selection.

Knock-out mutation of eukaryotic initiation factor 4E2 (eIF4E2) confers resistance to pepper veinal mottle virus in tomato.

Translation initiation factors 4E (eIF4E) are the main source of resistance to potyvirus. We systematically assessed tomato single and double knock-out (KO) mutants of members of the eIF4E-coding gene family for resistance to Pepper veinal mottle virus (PVMV), a major constraint to tomato production. We show that the KO mutant of eIF4E2 has partial resistance to PVMV isolate IC, with plants harboring weak symptoms and low virus loads at the systemic level. The causal effect of eIF4E2 loss-of-function on resistance was confirmed on a progeny segregating for the KO mutation. The eIF4E2 KO mutant was resistant to six of the eight PVMV isolates tested and no resistance to other potyviruses was observed. This is the first evidence that mutation of eIF4E2 is in itself conferring resistance to a potyvirus and 3D protein modelling suggests that the eIF4E2 gene could be converted into a functional resistance allele.

Genome-wide association mapping of QTLs implied in potato virus Y population sizes in pepper: evidence for widespread resistance QTL pyramiding.

In this study, we looked for genetic factors in the pepper (Capsicum annuum) germplasm that control the number of potato virus Y (PVY) particles entering the plant (i.e. effective population size at inoculation) and the PVY accumulation at the systemic level (i.e. census population size). Using genotyping-by-sequencing (GBS) in a core collection of 256 pepper accessions, we obtained 10 307 single nucleotide polymorphisms (SNPs) covering the whole genome. Genome-wide association studies (GWAS) detected seven SNPs significantly associated with the virus population size at inoculation and/or systemic level on chromosomes 4, 6, 9 and 12. Two SNPs on chromosome 4 associated with both PVY population sizes map closely to the major resistance gene pvr2 encoding the eukaryotic initiation factor 4E. No obvious candidates for resistance were identified in the confidence intervals for the other chromosomes. SNPs detected on chromosomes 6 and 12 colocalized with resistance quantitative trait loci (QTLs) previously identified with a biparental population. These results show the efficiency of GBS and GWAS in C. annuum, indicate highly consistent results between GWAS and classical QTL mapping, and suggest that resistance QTLs identified with a biparental population are representative of a much larger collection of pepper accessions. Moreover, the resistance alleles at these different loci were more frequently combined than expected by chance in the core collection, indicating widespread pyramiding of resistance QTLs and widespread combination of resistance QTLs and major effect genes. Such pyramiding may increase resistance efficiency and/or durability.

Impact of genetic drift, selection and accumulation level on virus adaptation to its host plants.

The efficiency of plant major resistance genes is limited by the emergence and spread of resistance-breaking mutants. Modulation of the evolutionary forces acting on pathogen populations constitutes a promising way to increase the durability of these genes. We studied the effect of four plant traits affecting these evolutionary forces on the rate of resistance breakdown (RB) by a virus. Two of these traits correspond to virus effective population sizes (Ne ) at either plant inoculation or during infection. The third trait corresponds to differential selection exerted by the plant on the virus population. Finally, the fourth trait corresponds to within-plant virus accumulation (VA). These traits were measured experimentally on Potato virus Y (PVY) inoculated to a set of 84 pepper doubled-haploid lines, all carrying the same pvr23 resistance gene, but having contrasting genetic backgrounds. The lines showed extensive variation for the rate of pvr23 RB by PVY and for the four other traits of interest. A generalized linear model showed that three of these four traits, with the exception of Ne at inoculation, and several pairwise interactions between them had significant effects on RB. RB increased with increasing values of Ne during plant infection or VA. The effect of differential selection was more complex because of a strong interaction with VA. When VA was high, RB increased as the differential selection increased. An opposite relationship between RB and differential selection was observed when VA was low. This study provides a framework to select plants with appropriate virus evolution-related traits to avoid or delay RB.