Repeated loss of the ability of a wild pepper disease resistance gene to function at high temperatures suggests that thermoresistance is a costly trait.

Specificity in plant-pathogen gene-for-gene (GFG) interactions is determined by the recognition of pathogen proteins by the products of plant resistance (R) genes. The evolutionary dynamics of R genes in plant-virus systems is poorly understood. We analyse the evolution of the L resistance locus to tobamoviruses in the wild pepper Capsicum annuum var. glabriusculum (chiltepin), a crop relative undergoing incipient domestication. The frequency, and the genetic and phenotypic diversity, of the L locus was analysed in 41 chiltepin populations under different levels of human management over its distribution range in Mexico. The frequency of resistance was lower in Cultivated than in Wild populations. L-locus genetic diversity showed a strong spatial structure with no isolation-by-distance pattern, suggesting environment-specific selection, possibly associated with infection by the highly virulent tobamoviruses found in the surveyed regions. L alleles differed in recognition specificity and in the expression of resistance at different temperatures, broad-spectrum recognition of P0 + P1 pathotypes and expression above 32°C being ancestral traits that were repeatedly lost along L-locus evolution. Overall, loss of resistance co-occurs with incipient domestication and broad-spectrum resistance expressed at high temperatures has apparent fitness costs. These findings contribute to understand the role of fitness trade-offs in plant-virus coevolution.

Dynamics of the rice yellow mottle disease in western Burkina Faso: Epidemic monitoring, spatio-temporal variation of viral diversity, and pathogenicity in a disease hotspot.

The rice yellow mottle virus (RYMV) is a model in plant virus molecular epidemiology, with the reconstruction of historical introduction routes at the scale of the African continent. However, information on patterns of viral prevalence and viral diversity over multiple years at a local scale remains scarce, in spite of potential implications for crop protection. Here, we describe a 5-year (2015-9) monitoring of RYMV prevalence in six sites from western Burkina Faso (geographic areas of Bama, Banzon, and Karfiguela). It confirmed one irrigated site as a disease hotspot and also found one rainfed lowland (RL) site with occasional high prevalence levels. Within the studied fields, a pattern of disease aggregation was evidenced at a 5-m distance, as expected for a mechanically transmitted virus. Next, we monitored RYMV genetic diversity in the irrigated disease hotspot site, revealing a high viral diversity, with the current coexistence of various distinct genetic groups at the site scale (ca. 520 ha) and also within various specific fields (25 m side). One genetic lineage, named S1bzn, is the most recently emerged group and increased in frequency over the studied period (from 20 per cent or less in 2015-6 to more than 65 per cent in 2019). Its genome results from a recombination between two other lineages (S1wa and S1ca). Finally, experimental work revealed that three rice varieties commonly cultivated in Burkina Faso were not different in terms of resistance level, and we also found no significant effect of RYMV genetic groups on symptom expression and viral load. We found, however, that infection outcome depended on the specific RYMV isolate, with two isolates from the lineage S1bzn accumulating at the highest level at early infections. Overall, this study documents a case of high viral prevalence, high viral diversity, and co-occurrence of divergent genetic lineages at a small geographic scale. A recently emerged lineage, which comprises viral isolates inducing severe symptoms and high accumulation under controlled conditions, could be recently rising through natural selection. Following up the monitoring of RYMV diversity is required to confirm this trend and further understand the factors driving the local maintenance of viral diversity.

Molecular Tools to Infer Resistance-Breaking Abilities of Rice Yellow Mottle Virus Isolates.

Rice yellow mottle virus (RYMV) is a major biotic constraint to rice cultivation in Africa. RYMV shows a high genetic diversity. Viral lineages were defined according to the coat protein (CP) phylogeny. Varietal selection is considered as the most efficient way to manage RYMV. Sources of high resistance were identified mostly in accessions of the African rice species, Oryza glaberrima. Emergence of resistance-breaking (RB) genotypes was observed in controlled conditions. The RB ability was highly contrasted, depending on the resistance sources and on the RYMV lineages. A molecular marker linked to the adaptation to susceptible and resistant O. glaberrima was identified in the viral protein genome-linked (VPg). By contrast, as no molecular method was available to identify the hypervirulent lineage able to overcome all known resistance sources, plant inoculation assays were still required. Here, we designed specific RT-PCR primers to infer the RB abilities of RYMV isolates without greenhouse experiments or sequencing steps. These primers were tested and validated on 52 isolates, representative of RYMV genetic diversity. The molecular tools described in this study will contribute to optimizing the deployment strategy of resistant lines, considering the RYMV lineages identified in fields and their potential adaptability.

Genetic diversity and epidemic histories of rice yellow mottle virus in Ghana.

Rice yellow mottle virus (RYMV) has persisted as a major biotic constraint to rice production in Africa. However, no data on RYMV epidemics were available in Ghana, although it is an intensive rice-producing country. Surveys were performed from 2010 to 2020 in eleven rice-growing regions of Ghana. Symptom observations and serological detections confirmed that RYMV is circulating in most of these regions. Coat protein gene and complete genome sequencings revealed that RYMV in Ghana almost exclusively belongs to the strain S2, one of the strains covering the largest area in West Africa. We also detected the presence of the S1ca strain which is being reported for the first time outside its area of origin. These results suggested a complex epidemiological history of RYMV in Ghana and a recent expansion of S1ca to West Africa. Phylogeographic analyses reconstructed at least five independent RYMV introductions in Ghana for the last 40 years, probably due to rice cultivation intensification in West Africa leading to a better circulation of RYMV. In addition to identifying some routes of RYMV dispersion in Ghana, this study contributes to the epidemiological surveillance of RYMV and helps to design disease management strategies, especially through breeding for rice disease resistance.

Rapid evolution of an RNA virus to escape recognition by a rice nucleotide-binding and leucine-rich repeat domain immune receptor.

Viral diseases are a major limitation for crop production, and their control is crucial for sustainable food supply. We investigated by a combination of functional genetics and experimental evolution the resistance of rice to the rice yellow mottle virus (RYMV), which is among the most devastating rice pathogens in Africa, and the mechanisms underlying the extremely fast adaptation of the virus to its host. We found that the RYMV3 gene that protects rice against the virus codes for a nucleotide-binding and leucine-rich repeat domain immune receptor (NLRs) from the Mla-like clade of NLRs. RYMV3 detects the virus by forming a recognition complex with the viral coat protein (CP). The virus escapes efficiently from detection by mutations in its CP, some of which interfere with the formation of the recognition complex. This study establishes that NLRs also confer in monocotyledonous plants immunity to viruses, and reveals an unexpected functional diversity for NLRs of the Mla clade that were previously only known as fungal disease resistance proteins. In addition, it provides precise insight into the mechanisms by which viruses adapt to plant immunity and gives important knowledge for the development of sustainable resistance against viral diseases of cereals.

Characterization of Grapevine Fanleaf Virus Isolates in 'Chardonnay' Vines Exhibiting Severe and Mild Symptoms in Two Vineyards.

Fanleaf degeneration is a complex viral disease of Vitis spp. that detrimentally impacts fruit yield and reduces the productive lifespan of most vineyards worldwide. In France, its main causal agent is grapevine fanleaf virus (GFLV). In the past, field experiments were conducted to explore cross-protection as a management strategy of fanleaf degeneration, but results were unsatisfactory because the mild virus strain negatively impacted fruit yield. In order to select new mild GFLV isolates, we examined two old 'Chardonnay' parcels harbouring vines with distinct phenotypes. Symptoms and agronomic performances were monitored over the four-year study on 21 individual vines that were classified into three categories: asymptomatic GFLV-free vines, GFLV-infected vines severely diseased and GFLV-infected vines displaying mild symptoms. The complete coding genomic sequences of GFLV isolates in infected vines was determined by high-throughput sequencing. Most grapevines were infected with multiple genetically divergent variants. While no specific molecular features were apparent for GFLV isolates from vines displaying mild symptoms, a genetic differentiation of GFLV populations depending on the vineyard parcel was observed. The mild symptomatic grapevines identified during this study were established in a greenhouse to recover GFLV variants of potential interest for cross-protection studies.

Evidence of differential spreading events of grapevine pinot Gris virus in Italy using datamining as a tool.

Since its identification in 2003, grapevine Pinot gris virus (GPGV, Trichovirus) has now been detected in most grape-growing countries. So far, little is known about the epidemiology of this newly emerging virus. In this work, we used datamining as a tool to monitor in-silico the sanitary status of three vineyards in Italy. All data used in the study were recovered from a work that was already published and for which data were publicly available as SRA (Sequence Read Archive, NCBI) files. While incomplete, knowledge gathered from this work was still important, with evidence of differential accumulation of the virus in grapevine according to year, location, and variety-rootstock association. Additional data regarding GPGV genetic diversity were collected. Some advantages and pitfalls of datamining are discussed.

Genetic Diversity of Rice stripe necrosis virus and New Insights into Evolution of the Genus Benyvirus.

The rice stripe necrosis virus (RSNV) has been reported to infect rice in several countries in Africa and South America, but limited genomic data are currently publicly available. Here, eleven RSNV genomes were entirely sequenced, including the first corpus of RSNV genomes of African isolates. The genetic variability was differently distributed along the two genomic segments. The segment RNA1, within which clusters of polymorphisms were identified, showed a higher nucleotidic variability than did the beet necrotic yellow vein virus (BNYVV) RNA1 segment. The diversity patterns of both viruses were similar in the RNA2 segment, except for an in-frame insertion of 243 nucleotides located in the RSNV tgbp1 gene. Recombination events were detected into RNA1 and RNA2 segments, in particular in the two most divergent RSNV isolates from Colombia and Sierra Leone. In contrast to BNYVV, the RSNV molecular diversity had a geographical structure with two main RSNV lineages distributed in America and in Africa. Our data on the genetic diversity of RSNV revealed unexpected differences with BNYVV suggesting a complex evolutionary history of the genus Benyvirus.

Full-Length Genome Sequences of Recombinant and Nonrecombinant Sympatric Strains of Rice yellow mottle virus from Western Kenya.

Five isolates of Rice yellow mottle virus from western Kenya were fully sequenced. One isolate of strain S4lv had been collected in 1966. Two isolates belonged to the emerging strain S4ug recently described in Uganda. Two isolates collected in 2012 are putative recombinants between the S4lv and S4ug strains.

Metagenomic-based impact study of transgenic grapevine rootstock on its associated virome and soil bacteriome.

For some crops, the only possible approach to gain a specific trait requires genome modification. The development of virus-resistant transgenic plants based on the pathogen-derived resistance strategy has been a success story for over three decades. However, potential risks associated with the technology, such as horizontal gene transfer (HGT) of any part of the transgene to an existing gene pool, have been raised. Here, we report no evidence of any undesirable impacts of genetically modified (GM) grapevine rootstock on its biotic environment. Using state of the art metagenomics, we analysed two compartments in depth, the targeted Grapevine fanleaf virus (GFLV) populations and nontargeted root-associated microbiota. Our results reveal no statistically significant differences in the genetic diversity of bacteria that can be linked to the GM trait. In addition, no novel virus or bacteria recombinants of biosafety concern can be associated with transgenic grapevine rootstocks cultivated in commercial vineyard soil under greenhouse conditions for over 6 years.

Identification of a Hypervirulent Pathotype of Rice yellow mottle virus: A Threat to Genetic Resistance Deployment in West-Central Africa.

Rice yellow mottle virus (RYMV) causes high losses to rice production in Africa. Several sources of varietal high resistance are available but the emergence of virulent pathotypes that are able to overcome one or two resistance alleles can sometimes occur. Both resistance spectra and viral adaptability have to be taken into account to develop sustainable rice breeding strategies against RYMV. In this study, we extended previous resistance spectrum analyses by testing the rymv1-4 and rymv1-5 alleles that are carried by the rice accessions Tog5438 and Tog5674, respectively, against isolates that are representative of RYMV genetic and pathogenic diversity. Our study revealed a hypervirulent pathotype, named thereafter pathotype T', that is able to overcome all known sources of high resistance. This pathotype, which is spatially localized in West-Central Africa, appears to be more abundant than previously suspected. To better understand the adaptive processes of pathotype T', molecular determinants of resistance breakdown were identified via Sanger sequencing and validated through directed mutagenesis of an infectious clone. These analyses confirmed the key role of convergent nonsynonymous substitutions in the central part of the viral genome-linked protein to overcome RYMV1-mediated resistance. In addition, deep-sequencing analyses revealed that resistance breakdown does not always coincide with fixed mutations. Actually, virulence mutations that are present in a small proportion of the virus population can be sufficient for resistance breakdown. Considering the spatial distribution of RYMV strains in Africa and their ability to overcome the RYMV resistance genes and alleles, we established a resistance-breaking risk map to optimize strategies for the deployment of sustainable and resistant rice lines in Africa.

Human Management of a Wild Plant Modulates the Evolutionary Dynamics of a Gene Determining Recessive Resistance to Virus Infection.

This work analyses the genetic variation and evolutionary patterns of recessive resistance loci involved in matching-allele (MA) host-pathogen interactions, focusing on the pvr2 resistance gene to potyviruses of the wild pepper Capsicum annuum glabriusculum (chiltepin). Chiltepin grows in a variety of wild habitats in Mexico, and its cultivation in home gardens started about 25 years ago. Potyvirus infection of Capsicum plants requires the physical interaction of the viral VPg with the pvr2 product, the translation initiation factor eIF4E1. Mutations impairing this interaction result in resistance, according to the MA model. The diversity of pvr2/eIF4E1 in wild and cultivated chiltepin populations from six biogeographical provinces in Mexico was analysed in 109 full-length coding sequences from 97 plants. Eleven alleles were found, and their interaction with potyvirus VPg in yeast-two-hybrid assays, plus infection assays of plants, identified six resistance alleles. Mapping resistance mutations on a pvr2/eIF4E1 model structure showed that most were around the cap-binding pocket and strongly altered its surface electrostatic potential, suggesting resistance-associated costs due to functional constraints. The pvr2/eIF4E1 phylogeny established that susceptibility was ancestral and resistance was derived. The spatial structure of pvr2/eIF4E1 diversity differed from that of neutral markers, but no evidence of selection for resistance was found in wild populations. In contrast, the resistance alleles were much more frequent, and positive selection stronger, in cultivated chiltepin populations, where diversification of pvr2/eIF4E1 was higher. This analysis of the genetic variation of a recessive resistance gene involved in MA host-pathogen interactions in populations of a wild plant show that evolutionary patterns differ according to the plant habitat, wild or cultivated. It also demonstrates that human management of the plant population has profound effects on the diversity and the evolution of the resistance gene, resulting in the selection of resistance alleles.

Environment and host genotype determine the outcome of a plant-virus interaction: from antagonism to mutualism.

It has been hypothesized that plant-virus interactions vary between antagonism and conditional mutualism according to environmental conditions. This hypothesis is based on scant experimental evidence, and to test it we examined the effect of abiotic factors on the Arabidopsis thaliana-Cucumber mosaic virus (CMV) interaction. Four Arabidopsis genotypes clustering into two allometric groups were grown under six environments defined by three temperature and two light-intensity conditions. Plants were either CMV-infected or mock-inoculated, and the effects of environment and infection on temporal and resource allocation life-history traits were quantified. Life-history traits significantly differed between allometric groups over all environments, with group 1 plants tolerating abiotic stress better than those of group 2. The effect of CMV infection on host fitness (virulence) differed between genotypes, being lower in group 1 genotypes. Tolerance to abiotic stress and to infection was similarly achieved through life-history trait responses, which resulted in resource reallocation from growth to reproduction. Effects of infection varied according to plant genotype and environment from detrimental to beneficial for host fitness. These results are highly relevant and demonstrate that plant viruses can be pleiotropic parasites along the antagonism-mutualism continuum, which should be considered in analyses of the evolution of plant-virus interactions.

Alternative mutational pathways, outside the VPg, of rice yellow mottle virus to overcome eIF(iso)4G-mediated rice resistance under strong genetic constraints.

The adaptation of rice yellow mottle virus (RYMV) to rymv1-mediated resistance has been reported to involve mutations in the viral genome-linked protein (VPg). In this study, we analysed several cases of rymv1-2 resistance breakdown by an isolate with low adaptability. Surprisingly, in these rarely occurring resistance-breaking (RB) genotypes, mutations were detected outside the VPg, in the ORF2a/ORF2b overlapping region. The causal role of three mutations associated with rymv1-2 resistance breakdown was validated via directed mutagenesis of an infectious clone. In resistant plants, these mutations increased viral accumulation as efficiently as suboptimal RB mutations in the VPg. Interestingly, these mutations are located in a highly conserved, but unfolded, domain. Altogether, our results indicate that under strong genetic constraints, a priori unfit genotypes can follow alternative mutational pathways, i.e. outside the VPg, to overcome rymv1-2 resistance.

Historical contingencies modulate the adaptability of Rice yellow mottle virus.

The rymv1-2 and rymv1-3 alleles of the RYMV1 resistance to Rice yellow mottle virus (RYMV), coded by an eIF(iso)4G1 gene, occur in a few cultivars of the Asiatic (Oryza sativa) and African (O. glaberrima) rice species, respectively. The most salient feature of the resistance breaking (RB) process is the converse genetic barrier to rymv1-2 and rymv1-3 resistance breakdown. This specificity is modulated by the amino acid (glutamic acid vs. threonine) at codon 49 of the Viral Protein genome-linked (VPg), a position which is adjacent to the virulence codons 48 and 52. Isolates with a glutamic acid (E) do not overcome rymv1-3 whereas those with a threonine (T) rarely overcome rymv1-2. We found that isolates with T49 had a strong selective advantage over isolates with E49 in O. glaberrima susceptible cultivars. This explains the fixation of the mutation T49 during RYMV evolution and accounts for the diversifying selection estimated at codon 49. Better adapted to O. glaberrima, isolates with T49 are also more prone than isolates with E49 to fix rymv1-3 RB mutations at codon 52 in resistant O. glaberrima cultivars. However, subsequent genetic constraints impaired the ability of isolates with T49 to fix rymv1-2 RB mutations at codons 48 and 52 in resistant O. sativa cultivars. The origin and role of the amino acid at codon 49 of the VPg exemplifies the importance of historical contingencies in the ability of RYMV to overcome RYMV1 resistance.

The adaptation of Rice yellow mottle virus to the eIF(iso)4G-mediated rice resistance.

The rymv1-3 allele of the eIF(iso)4G-mediated resistance to Rice yellow mottle virus (RYMV) is found in a few Oryza glaberrima cultivars. The same resistance-breaking (RB) mutations emerged in the central domain of the VPg after inoculation of isolates of different strains. The RB mutations were fixed, often sequentially, at codons 41 and 52 which paralleled an increase in virus accumulation. RB mutations also emerged after inoculation of an avirulent infectious clone, indicating that they were generated de novo in resistant plants. Only virus isolates with a threonine at codon 49 of the VPg broke rymv1-3 resistance, those with a glutamic acid did not. A small subset of these isolates overcame rymv1-2 resistance, but following a specific pathway. Comparison with the RB process of rymv1-2, a resistance allele found in a few Oryza sativa cultivars, showed similarities in the mode of adaptation but revealed converse virulence specificity of the isolates.

Direct interaction between the Rice yellow mottle virus (RYMV) VPg and the central domain of the rice eIF(iso)4G1 factor correlates with rice susceptibility and RYMV virulence.

The adaptation of Rice yellow mottle virus (RYMV) to recessive resistance mediated by the rymv1-2 allele has been reported as a model to study the emergence and evolution of virulent variants. The resistance and virulence factors have been identified as eukaryotic translation initiation factor eIF(iso)4G1 and viral genome-linked protein (VPg), respectively, but the molecular mechanisms involved in their interaction are still unknown. In this study, we demonstrated a direct interaction between RYMV VPg and the central domain of rice eIF(iso)4G1 both in vitro, using recombinant proteins, and in vivo, using a yeast two-hybrid assay. Insertion of the E309K mutation in eIF(iso)4G1, conferring resistance in planta, strongly diminished the interaction with avirulent VPg. The efficiency of the major virulence mutations at restoring the interaction with the resistance protein was assessed. Our results explain the prevalence of virulence mutations fixed during experimental evolution studies and are consistent with the respective viral RNA accumulation levels of avirulent and virulent isolates. Our results also explain the origin of the residual multiplication of wild-type isolates in rymv1-2-resistant plants and the role of genetic context in the poor adaptability of the S2/S3 strain. Finally, the strategies of RYMV and members of family Potyviridae to overcome recessive resistance were compared.

Why Rice yellow mottle virus, a rapidly evolving RNA plant virus, is not efficient at breaking rymv1-2 resistance.

Rice yellow mottle virus (RYMV) reaches a high virus content in rice, is genetically highly variable and evolves rapidly. Nevertheless, only a small proportion of isolates overcome rymv1-2 rice resistance by mutations in the VPg (viral protein genome-linked). The accumulation rates of wild-type (WT) and resistance-breaking (RB) genotypes of the E- and T-pathotypes of RYMV, with average and low virulence, respectively, were assessed. By quantitative reverse transcriptase-polymerase chain reaction, it was shown that: (i) in resistant plants, both WT genotypes reached a level of 10(5)-10(7) viral copies per milligram of fresh leaf; (ii) the accumulation of RB genotypes was variable, but was always much higher than the WT, with an RB/WT accumulation ratio of up to 10(6); (iii) in susceptible plants, the RB genotypes were counter-selected to a similar level. In competition experiments, there was a straightforward exclusion of WT by RB genotypes in resistant hosts. The mutation rate in VPg was more than 1 x 10(-3) mutations per site per year. Overall, a steady supply of highly adaptive RB genotypes was expected in resistant plants. However, the use of the few possible mutational pathways to virulence is tightly regulated by pathotype-specific genetic constraints: codon usage, mutational bias and sign epistasis. In addition, genetic drift may restrict the fixation of RB mutants. Altogether, both genetic and demographic constraints contribute to the low ability of RYMV to break rymv1-2 resistance.

Theme and variations in the evolutionary pathways to virulence of an RNA plant virus species.

The diversity of a highly variable RNA plant virus was considered to determine the range of virulence substitutions, the evolutionary pathways to virulence, and whether intraspecific diversity modulates virulence pathways and propensity. In all, 114 isolates representative of the genetic and geographic diversity of Rice yellow mottle virus (RYMV) in Africa were inoculated to several cultivars with eIF(iso)4G-mediated Rymv1-2 resistance. Altogether, 41 virulent variants generated from ten wild isolates were analyzed. Nonconservative amino acid replacements at five positions located within a stretch of 15 codons in the central region of the 79-aa-long protein VPg were associated with virulence. Virulence substitutions were fixed predominantly at codon 48 in most strains, whatever the host genetic background or the experimental conditions. There were one major and two isolate-specific mutational pathways conferring virulence at codon 48. In the prevalent mutational pathway I, arginine (AGA) was successively displaced by glycine (GGA) and glutamic acid (GAA). Substitutions in the other virulence codons were displaced when E48 was fixed. In the isolate-specific mutational pathway II, isoleucine (ATA) emerged and often later coexisted with valine (GTA). In mutational pathway III, arginine, with the specific S2/S3 strain codon usage AGG, was displaced by tryptophane (TGG). Mutational pathway I never arose in the widely spread West African S2/S3 strain because G48 was not infectious in the S2/S3 genetic context. Strain S2/S3 least frequently overcame resistance, whereas two geographically localized variants of the strain S4 had a high propensity to virulence. Codons 49 and 26 of the VPg, under diversifying selection, are candidate positions in modulating the genetic barriers to virulence. The theme and variations in the evolutionary pathways to virulence of RYMV illustrates the extent of parallel evolution within a highly variable RNA plant virus species.