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Viruses are obligate intracellular parasites that have co-evolved with their hosts to establish an intricate network of protein–protein interactions. Here, we followed a high-throughput yeast two-hybrid screening to identify 378 novel protein–protein interactions between turnip mosaic virus (TuMV) and its natural host Arabidopsis thaliana. We identified the RNA-dependent RNA polymerase NIb as the viral protein with the largest number of contacts, including key salicylic acid-dependent transcription regulators. We verified a subset of 25 interactions in planta by bimolecular fluorescence complementation assays. We then constructed and analyzed a network comprising 399 TuMV-A. thaliana interactions together with intravirus and intrahost connections. In particular, we found that the host proteins targeted by TuMV are enriched in different aspects of plant responses to infections, are more connected and have an increased capacity to spread information throughout the cell proteome, display higher expression levels, and have been subject to stronger purifying selection than expected by chance. The proviral or antiviral role of ten host proteins was validated by characterizing the infection dynamics in the corresponding mutant plants, supporting a proviral role for the transcriptional regulator TGA1. Comparison with similar studies with animal viruses, highlights shared fundamental features in their mode of action.

It is assumed that host genetic variability for susceptibility to infection conditions virus evolution. Differences in host susceptibility can drive a virus to diversify into strains that track different defense alleles (e.g. antigenic diversity) or to infect only the most susceptible genotypes. Here, we have studied how variability in host defenses determines the evolutionary fate of a plant RNA virus. We performed evolution experiments with Turnip mosaic potyvirus in Arabidopsis thaliana mutants that had disruptions in infection-response signaling pathways or in genes whose products are essential for potyvirus infection. Plant genotypes were classified into five phenogroups according to their response to infection. We found that evolution proceeded faster in more restrictive hosts than in more permissive ones. Most of the phenotypic differences shown by the ancestral virus across host genotypes were removed after evolution, suggesting the combined action of selection and chance. When all evolved viral lineages were tested in all plant genotypes used in the experiments, we found compelling evidences that the most restrictive plant genotypes selected for more generalist viruses, while more permissive genotypes selected for more specialist viruses. Sequencing the genomes of the evolved viral lineages, we found that selection targeted the multifunctional genome-linked protein VPg in most host genotypes. Overall, this work illustrates how different host defenses modulate the rates and extent of virus evolution.

It is assumed that host genetic variability for susceptibility to infection will necessarily condition the evolution of viruses, either by driving them to diversification into strains that track the different host defense alleles (e.g., antigenic diversity), or by canalization to infect only the most susceptible genotypes. Associated to these processes virulence may or may not increase. To tackle these questions, we performed evolution experiments with tobacco etch (TEV) and turnip mosaic (TuMV) potyviruses in different genotypes of arabidopsis. In a first set of experiments, we explored the role of genetic variability for susceptibility in TEV virulence. Results showed a pattern of local adaptation, characterized by a higher virulence on the ecotype(s) encountered during evolution. However, local adaptation not always paid a cost on foreign ecotypes: viral lineages evolved in more susceptible ecotypes evolved as specialists, whereas adaptation to the less susceptible ecotypes resulted in generalists. In a second set of experiments, we explored the effect of the amount of genetic diversity for susceptibility on the rate of TuMV adaptation. In agreement with theoretical expectations, we found that viral lineages maintained in polymorphic host populations evolved slower and were dominated by mild generalists. By contrast, monomorphic host populations rapidly selected for locally-adapted virulent specialists. In a third set of experiments, TuMV evolved in mutant genotypes that differ in mutations in genes involved in resistance pathways and in genes whose products are essential for potyviruses infection. Plant genotypes classified into three categories: hypersusceptible, as wildtype and hyposusceptibles. TuMV induces severer symptoms in hypersusceptible genotypes than those observed in wildtype. By contrast, it induces weaker or no symptoms in hyposusceptible genotypes.

Horizontal gene transfer is common among viruses, while they also have highly compact genomes and tend to lose artificial genomic insertions rapidly. Understanding the stability of genomic insertions in viral genomes is therefore relevant for explaining and predicting their evolutionary patterns. Here, we revisit a large body of experimental research on a plant RNA virus, tobacco etch potyvirus (TEV), to identify the patterns underlying the stability of a range of homologous and heterologous insertions in the viral genome. We obtained a wide range of estimates for the recombination rate—the rate at which deletions removing the insertion occur—and these appeared to be independent of the type of insertion and its location. Of the factors we considered, recombination rate was the best predictor of insertion stability, although we could not identify the specific sequence characteristics that would help predict insertion instability. We also considered experimentally the possibility that functional insertions lead to higher mutational robustness through increased redundancy. However, our observations suggest that both functional and non-functional increases in genome size decreased the mutational robustness. Our results therefore demonstrate the importance of recombination rates for predicting the long-term stability and evolution of viral RNA genomes and suggest that there are unexpected drawbacks to increases in genome size for mutational robustness.