Emergence of a resistance breaking isolate of Rice yellow mottle virus during serial inoculations is due to a single substitution in the genome-linked viral protein VPg

International audienceThe recessive gene rymv-1 responsible for the high resistance of Oryza sativa cultivar Gigante to Rice yellow mottle virus (Sobemovirus) was overcome by the variant CI4* which emerged after serial inoculations of the non-resistance-breaking (nRB) isolate CI4. By comparison of the full-length sequences of CI4 and CI4*, we identified a non-synonymous mutation at position 1729 localised in the putative VPg domain and developed an assay based on this single nucleotide polymorphism. The mutation G1729T was detected as early as the first passage in resistant plants and was found in all subsequent passages. Neither reversion nor additional mutation were observed. The substitution G1729T introduced by mutagenesis in the VPg of an nRB infectious clone is sufficient to induce symptoms in non-inoculated leaves of cultivar Gigante. This is the first evidence that VPg is a virulence factor in plants with recessive resistance against viruses outside the family Potyviridae

Diversification of Rice Yellow Mottle Virus and Related Viruses Spans the History of Agriculture from the Neolithic to the Present

The mechanisms of evolution of plant viruses are being unraveled, yet the timescale of their evolution remains an enigma. To address this critical issue, the divergence time of plant viruses at the intra- and inter-specific levels was assessed. The time of the most recent common ancestor (TMRCA) of Rice yellow mottle virus (RYMV; genus Sobemovirus) was calculated by a Bayesian coalescent analysis of the coat protein sequences of 253 isolates collected between 1966 and 2006 from all over Africa. It is inferred that RYMV diversified approximately 200 years ago in Africa, i.e., centuries after rice was domesticated or introduced, and decades before epidemics were reported. The divergence time of sobemoviruses and viruses of related genera was subsequently assessed using the age of RYMV under a relaxed molecular clock for calibration. The divergence time between sobemoviruses and related viruses was estimated to be approximately 9,000 years, that between sobemoviruses and poleroviruses approximately 5,000 years, and that among sobemoviruses approximately 3,000 years. The TMRCA of closely related pairs of sobemoviruses, poleroviruses, and luteoviruses was approximately 500 years, which is a measure of the time associated with plant virus speciation. It is concluded that the diversification of RYMV and related viruses has spanned the history of agriculture, from the Neolithic age to the present

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

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

Virulence domain of the RYMV genome-linked viral protein VPg towards rice rymv1-2-mediated resistance

International audienceVirulent variants of Rice yellow mottle virus (genus Sobemovirus) can emerge on the highly resistant rice cultivars Gigante and Bekarosaka. Non-synonymous mutations responsible of the breakdown of the recessive resistant gene rymv1-2 were located in the VPg after determination of its termini in the polyprotein P2a. The secondary structure of this protein was predicted to include a central -helix. The two major amino acids related to virulence are located in the same side of this helix. The 3D-topology and the biochemical properties of virulence mutations both suggested a direct site-to-site interaction between RYMV VPg and rice eIF(iso)4G encoded by rymv1. Rice yellow mottle virus (RYMV) of the genus Sobemovirus [7] is present in all rice-growing African countries where it causes high yield losses [9]. Two Oryza sativa indica cultivars Gigante and Bekarosaka and a few O. glaberrima cultivars Togs expressed high resistance towards RYMV [1]. However, several RYMV isolates of different strains were able to overcome the high resistance [13]. Mutations associated with virulence towards cvs. Gigante and Bekarosaka were restricted to five sites in the RYMV genome [13]. They are located in a stretch of 15 amino acids in the polyprotein P2a which contains sequence motifs of the serine protease and of the VPg (Viral Protein genome-linked). The sites and the kinetics of RYMV polyprotein maturation are still unknown. However, the proximity of the virulence sites to the triplet WAD which is conserved in VPg sobemoviruses [11] suggested that they belong to the putative VPg domain. Moreover, VPgs are involved in potyvirus virulenc

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

P>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 105-107 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 106; (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

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

International audienceThe 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