Molecular evolution of tomato black ring virus and de novo generation of a new type of defective RNAs during long-term passaging in different hosts

Tomato black ring virus (TBRV) is a worldwide-distributed RNA virus infecting a wide range of different host plants, including crop species, trees, shrubs, and weeds. Here, we investigated the molecular evolution of TBRV and its adaptability to different plant species. The TBRV-Pi isolate was used to generate five independent evolution lineages serially passaged in either quinoa, tobacco, or tomato plants. After 15 passages, the genetic variability present in all the lineages was characterized for the movement (MP) and coat (CP) coding cistrons. We addressed two main questions: to what extent does the amount of genetic variability in the TBRV genome depend on the host species, and are there host species-specific adaptive mutations? Overall, 201 different nucleotide substitutions emerged during the evolution experiment, with some of them appearing multiple times in different lineages; two of them (one in CP and one in MP) were unique for a particular host plant. We have shown that the degree of genetic differentiation depends on the host species in which the virus evolved, and that positive selection is operating upon certain residues, particularly in CP. Moreover, we have characterized new types of defective RNAs that arose during the TBRV-Pi evolution in tobacco. Furthermore, this is the first report of a defective RNA from the RNA2 of TBRV.