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.

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The rate of evolution of an RNA plant virus has never been estimated using temporally spaced sequence data, by contrast to the information available on an increasing range of animal viruses. Accordingly, the evolution rate of Rice yellow mottle virus (RYMV) was calculated from sequences of the coat protein gene of isolates collected from rice over a 40-year period in different parts of Africa. The evolution rate of RYMV was estimated by pairwise distance linear regression on five phylogeographically defined groups comprising a total of 135 isolates. It was further assessed from 253 isolates collected all over Africa by Bayesian coalescent methods under strict and relaxed molecular clock models and under constant size and skyline population genetic models. Consistent estimates of the evolution rate between 4 ؋ 10 ؊4 and 8 ؋ 10 ؊4 nucleotides (nt)/site/year were obtained whatever method and model were applied. The synonymous evolution rate was between 8 ؋ 10 ؊4 and 11 ؋ 10 ؊4 nt/site/year. The overall and synonymous evolution rates of RYMV were within the range of the rates of 50 RNA animal viruses, below the average but above the distribution median. Experimentally, in host change studies, substitutions accumulated at an even higher rate. The results show that an RNA plant virus such as RYMV evolves as rapidly as most RNA animal viruses. Knowledge of the molecular clock of plant viruses provides methods for testing a wide range of biological hypotheses.The mutation rates of RNA viruses (i.e., the number of nucleotide misincorporations per site and per round of replication) are 10 4 to 10 5 times higher than those of their DNA hosts (8, 9). Such a high mutation rate is attributed to the lack of repair function of the RNA polymerase of these viruses, the short replication times, and the large populations in infected hosts (7). A high mutation rate often results in rapid evolution of RNA animal viruses. This allowed the measure of the evolution rates of an extensive range of animal viruses through the analysis of heterochronous sequences, i.e., sequences of viral genes isolated at different times (11,36). A large variation in the evolution rates of RNA animal viruses was subsequently found and was attributed mostly to differences in replication rates (24,26). Estimates of evolution rates are used increasingly to date the emergence and to reconstruct the population dynamics of major viral epidemics (6).Interestingly, some RNA viruses change little or not at all over time. The best-documented example is an RNA plant virus, Tobacco mild green mosaic virus, which showed no increase in genetic diversity over the 90 years considered, in the longest series of isolates with known isolation times for any virus (20). Indeed, many studies have shown the remarkable genetic stability of RNA plant virus populations from different geographical regions, hosts, and collection times (21). It was claimed that most tobamovirus populations are very stable and do not evolve at a measurable rate (22). It was even observed that populations...

The clock-like diversification of Rice yellow mottle virus (RYMV), a widespread RNA plant virus that infects rice in Africa, was tested following a three-step approach with (i) an exhaustive search of recombinants, (ii) a comprehensive assessment of the selective constraints over lineages, and (iii) a stepwise series of tests of the molecular clock hypothesis. The first evidence of recombination in RYMV was found in East Africa, in the region most favorable to co-infection. RYMV evolved under a pronounced purifying selection, but the selection pressure did vary among lineages. There was no phylogenetic evidence of transient deleterious mutations. ORF2b, which codes for the polymerase and is the most constrained ORF, tends to diversify clock-like. With the other ORFs and the full genome, the departure from the strict clock model was limited. This likely reflects the dominant conservative selection pressure and the clock-like fixation of synonymous mutations.