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.

Complete genome sequence of a novel marafivirus infecting pearl millet in Burkina Faso.

Pearl millet (Pennisetum glaucum (L.) R. Br.) is a staple food that is widely cultivated in sub-Saharan Africa. In August 2018, a survey was conducted in the main producing regions of Burkina Faso, and leaf samples were analyzed using virion-associated nucleic acid (VANA)-based metagenomic approach and Illumina sequencing. A new virus, tentatively named "Pennisetum glaucum marafivirus" (PGMV), was detected, and its complete nucleotide sequence of 6364 nucleotides was determined. The sequence contains a large open reading frame (ORF) encoding a polyprotein of 224.2 kDa with five domains (methyltransferase, papain-like protease, helicase, RNA-dependent RNA polymerase, and coat proteins), typical of marafiviruses. Additionally, a characteristic conserved marafibox domain was detected in the genome. The nucleotide sequence of the complete PGMV genome shares 68.5% identity with that of sorghum bicolor marafivirus, and its coat protein shares 58.5% identity with that of oat blue dwarf virus. Phylogenetic analysis confirmed that the pearl millet virus is unambiguously grouped with members of the genus Marafivirus in the family Tymoviridae. This is the first report on the occurrence of a marafivirus in pearl millet.

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.

Rivers and landscape ecology of a plant virus, Rice yellow mottle virus along the Niger Valley.

To investigate the spread of Rice yellow mottle virus (RYMV) along the Niger River, regular sampling of virus isolates was conducted along 500 km of the Niger Valley in the Republic of Niger and was complemented by additional sampling in neighbouring countries in West Africa and Central Africa. The spread of RYMV into and within the Republic of Niger was inferred as a continuous process using a Bayesian statistical framework applied previously to reconstruct its dispersal history in West Africa, East Africa, and Madagascar. The spatial resolution along this section of the Niger River was the highest implemented for RYMV and possibly for any plant virus. We benefited from the results of early field surveys of the disease for the validation of the phylogeographic reconstruction and from the well-documented history of rice cultivation changes along the Niger River for their interpretation. As a prerequisite, the temporal signal of the RYMV data sets was revisited in the light of recent methodological advances. The role of the hydrographic network of the Niger Basin in RYMV spread was examined, and the link between virus population dynamics and the extent of irrigated rice was assessed. RYMV was introduced along the Niger River in the Republic of Niger in the early 1980s from areas to the southwest of the country where rice was increasingly grown. Viral spread was triggered by a major irrigation scheme made of a set of rice perimeters along the river valley. The subsequent spatial and temporal host continuity and the inoculum build-up allowed for a rapid spread of RYMV along the Niger River, upstream and downstream, over hundreds of kilometres, and led to the development of severe epidemics. There was no evidence of long-distance dissemination of the virus through natural water. Floating rice in the main meanders of the Middle Niger did not contribute to virus dispersal from West Africa to Central Africa. RYMV along the Niger River is an insightful example of how agricultural intensification favours pathogen emergence and spread.

Comparing patterns and scales of plant virus phylogeography: Rice yellow mottle virus in Madagascar and in continental Africa.

Rice yellow mottle virus (RYMV) in Madagascar Island provides an opportunity to study the spread of a plant virus disease after a relatively recent introduction in a large and isolated country with a heterogeneous host landscape ecology. Here, we take advantage of field survey data on the occurrence of RYMV disease throughout Madagascar dating back to the 1970s, and of virus genetic data from ninety-four isolates collected since 1989 in most regions of the country to reconstruct the epidemic history. We find that the Malagasy isolates belong to a unique recombinant strain that most likely entered Madagascar through a long-distance introduction from the most eastern part of mainland Africa. We infer the spread of RYMV as a continuous process using a Bayesian statistical framework. In order to calibrate the time scale in calendar time units in this analysis, we pool the information about the RYMV evolutionary rate from several geographical partitions. Whereas the field surveys and the phylogeographic reconstructions both point to a rapid southward invasion across hundreds of kilometers throughout Madagascar within three to four decades, they differ on the inferred origin location and time of the epidemic. The phylogeographic reconstructions suggest a lineage displacement and unveil a re-invasion of the northern regions that may have remained unnoticed otherwise. Despite ecological differences that could affect the transmission potential of RYMV in Madagascar and in mainland Africa, we estimate similar invasion and dispersal rates. We could not identify environmental factors that have a relevant impact on the lineage dispersal velocity of RYMV in Madagascar. This study highlights the value and complementarity of (historical) nongenetic and (more contemporaneous) genetic surveillance data for reconstructing the history of spread of plant viruses.

Complete Genome Sequences of Rice Yellow Mottle Virus Isolates from the Federal Democratic Republic of Ethiopia.

The full-length genomes of two isolates of Rice yellow mottle virus from Ethiopia were sequenced. A comparison with 28 sequences from East Africa showed that they clustered within a new strain named S4et, related to the S4mg and S4ug strains found in the Lake Victoria Basin and Madagascar, respectively.

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.

Protocol for RYMV Inoculation and Resistance Evaluation in Rice Seedlings.

Rice yellow mottle virus (RYMV), a mechanically transmitted virus that causes serious damage to cultivated rice plants, is endemic to Africa. Varietal selection for resistance is considered to be the most effective and sustainable management strategy. Standardized resistance evaluation procedures are required for the identification and characterization of resistance sources. This paper describes a protocol for mechanical inoculation of rice seedlings with RYMV and two methods of resistance evaluation - one based on a symptom severity index and the other on virus detection through double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA).

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.

Complete Genome Sequence of a New Strain of Rice yellow mottle virus from Malawi, Characterized by a Recombinant VPg Protein.

The complete sequence of the isolate Mw10 of Rice yellow mottle virus was determined. Sequence comparisons revealed 8.4% to 10.7% nucleotide divergence from the published sequences, resulting in the definition of the strain S7. Importantly, a putative recombination event was identified encompassing the viral genome-linked protein involved in host adaptation.

Mutations in Rice yellow mottle virus Polyprotein P2a Involved in RYMV2 Gene Resistance Breakdown.

Rice yellow mottle virus (RYMV) is one of the major diseases of rice in Africa. The high resistance of the Oryza glaberrima Tog7291 accession involves a null allele of the RYMV2 gene, whose ortholog in Arabidopsis, CPR5, is a transmembrane nucleoporin involved in effector-triggered immunity. To optimize field deployment of the RYMV2 gene and improve its durability, which is often a weak point in varietal resistance, we analyzed its efficiency toward RYMV isolates representing the genetic diversity of the virus and the molecular basis of resistance breakdown. Tog7291 resistance efficiency was highly variable depending on the isolate used, with infection rates ranging from 0 to 98% of plants. Back-inoculation experiments indicated that infection cases were not due to an incomplete resistance phenotype but to the emergence of resistance-breaking (RB) variants. Interestingly, the capacity of the virus to overcome Tog7291 resistance is associated with a polymorphism at amino-acid 49 of the VPg protein which also affects capacity to overcome the previously studied RYMV1 resistance gene. This polymorphism appeared to be a main determinant of the emergence of RB variants. It acts independently of the resistance gene and rather reflects inter-species adaptation with potential consequences for the durability of resistance. RB mutations were identified by full-length or partial sequencing of the RYMV genome in infected Tog7291 plants and were validated by directed mutagenesis of an infectious viral clone. We found that Tog7291 resistance breakdown involved mutations in the putative membrane anchor domain of the polyprotein P2a. Although the precise effect of these mutations on rice/RYMV interaction is still unknown, our results offer a new perspective for the understanding of RYMV2 mediated resistance mechanisms. Interestingly, in the susceptible IR64 variety, RB variants showed low infectivity and frequent reversion to the wild-type genotype, suggesting that Tog7291 resistance breakdown is associated with a major loss of viral fitness in normally susceptible O. sativa varieties. Despite the high frequency of resistance breakdown in controlled conditions, this loss of fitness is an encouraging element with regards to RYMV2 resistance durability.

The biogeography of viral emergence: rice yellow mottle virus as a case study.

Rice yellow mottle virus (RYMV) exemplifies the key role in plant virus emergence of the early steps of crop extension and intensification in traditional agriculture. In East Africa, RYMV emerged in the 19(th) century after rice intensification along the Indian Ocean coast, and later spread inland concomitantly with rice introduction. In West Africa, the contrasted history of rice cultivation among regions differently shaped RYMV populations. A biogeographical approach - which jointly considers the spatial distribution of the virus and its hosts over time - was applied to reach these conclusions. We linked the evolution of RYMV over the past two centuries to a geographical map of the history of rice cultivation in Africa.

Emergence of rice yellow mottle virus in eastern Uganda: Recent and singular interplay between strains in East Africa and in Madagascar.

Epidemics of rice yellow mottle virus (RYMV) have developed recently in eastern Uganda, close to Lake Victoria in East Africa. Unexpectedly, all isolates from the affected area belonged to a single strain (named S4ug), a strain that is different from the S4lv strain that has been prevalent in the Lake Victoria basin for the past five decades. Interestingly, the S4ug strain is most closely related at the genomic level (except ORF1) to the strain present in Madagascar (S4mg), 2000km away. The minor parent of the S4mg recombinant strain could not be detected. Molecular clock dating analysis indicated that the singular sequence of events - that associated the emergence of a new strain (S4ug), a modular recombination between closely related strains (S4mg and S4ug) and a long distance transmission (S4mg) - occurred recently, within the past few decades. This finding is at variance with the process of gradual strain dispersal and diversification over two centuries throughout Africa that was previously established.

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.

Sites under positive selection modulate the RNA silencing suppressor activity of rice yellow mottle virus movement protein P1.

RNA silencing is a eukaryotic mechanism for RNA-based gene regulation that plays an essential role in diverse biological processes, such as defence against viral infections. The P1 of rice yellow mottle virus (RYMV) is a movement protein and displays RNA silencing suppression activity with variable efficiency, depending on the origin of the isolates. In this study, the positive selection pressure acting on the P1 protein gene was assessed. A site-by-site analysis of the dN/dS ratio was performed and 18 positively selected sites were identified. Four of these were mutated, and the ability to suppress RNA silencing was evaluated for the resulting mutants in a transient expression assay. All mutations affected quantitatively RNA silencing suppression, one caused a significant decrease in the activity and three significantly increased it. This work demonstrates, for what is to the best of our knowledge the first time, that the RYMV gene encoding the P1 RNA silencing suppressor is under adaptive evolution.

Rice yellow mottle virus in Madagascar and in the Zanzibar Archipelago; island systems and evolutionary time scale to study virus emergence.

Rice yellow mottle virus (RYMV), of the genus Sobemovirus, is a major threat to rice cultivation in Africa. Long range transmission of RYMV, difficult to study experimentally, is inferred from a detailed analysis of the molecular diversity of the virus in Madagascar and in the Zanzibar Archipelago (Zanzibar and Pemba Islands; Tanzania) compared with that found elsewhere in Africa. A unique successful introduction of RYMV to Madagascar, which is ca. 400 km from mainland Africa, contrasted with recurrent introductions of the virus to the Zanzibar Archipelago, ca. 40 km from the East African coast. Accordingly, RYMV dispersal over distances of hundreds of kilometers is rare whereas spread of the virus over distances of tens of kilometers is relatively frequent. The dates of introduction of RYMV to Madagascar and to Pemba Island were estimated from three sets of ORF4 sequences of virus isolates collected between 1966 and 2011. They were compared with the dates of the first field detection in Madagascar (1989) and in Pemba Island (1990). The estimates did not depend substantially on the data set used or on the evolutionary model applied and their credible intervals were narrow. The estimated dates are recent - 1978 (1969-1986) and 1985 (1977-1993) in Madagascar and in Pemba Island, respectively - compared to the early diversification of RYMV in East Africa ca. 200 years ago. They predated by 5-10 years the first field detections in these islands. The interplay between virus sources, rice cultivation and long range dispersal which led to RYMV emergence and spread is enlightened.

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.