Ultradeep Sequencing Analysis of Population Dynamics of Virus Escape Mutants in RNAi-Mediated Resistant Plants

Plant artificial micro-RNAs (amiRs) have been engineered to target viral genomes and induce their degradation. However, the exceptional evolutionary plasticity of RNA viruses threatens the durability of the resistance conferred by these amiRs. It has recently been shown that viral populations not experiencing strong selective pressure from an antiviral amiR may already contain enough genetic variability in the target sequence to escape plant resistance in an almost deterministic manner. Furthermore, it has also been shown that viral populations exposed to subinhibitory concentrations of the antiviral amiR speed up this process. In this article, we have characterized the molecular evolutionary dynamics of an amiR target sequence in a viral genome under both conditions. The use of Illumina ultradeep sequencing has allowed us to identify virus sequence variants at frequencies as low as 2 x 10(-6) and to track their variation in time before and after the viral population was able of successfully infecting plants fully resistant to the ancestral virus. We found that every site in the amiR-target sequence of the viral genome presented variation and that the variant that eventually broke resistance was sampled among the many coexisting ones. In this system, viral evolution in fully susceptible plants results from an equilibrium between mutation and genetic drift, whereas evolution in partially resistant plants originates from more complex dynamics involving mutation, selection, and drift.