Monitoring the directed evolution to a tripartite genome from a bipartite torradovirus genome

We have previously shown that tomato apex necrosis virus that cannot express the RNA2-ORF1 protein (P21) is not able to systemically infect plant hosts but is not affected in cell autonomous aspects of virus replication/accumulation. Here we attempted to provide P21 in trans by co-agroinfiltrating the RNA2-ORF1 null constructs (a stop codon mutant and a deletion mutant) with a P21-expressing construct under control of the 35S promoter and containing the 5’ and 3’ UTRs of wild type (WT) RNA2. Such construct when co-agroinfiltrated with the stop codon mutant originates a WT bipartite virus through homologous recombination. More surprisingly, when co-agroinfiltrated with the P21 deletion mutant it cannot immediately complement the mutant, but it serendipitously originates a tripartite virus with an actively replicating P21-expressing RNA3 only after this replicating RNA3 accumulates deletions in a small region inside the original 3’-UTR provided by the cDNA clone. Such virus can be transmitted mechanically and by whiteflies, is competent for virion formation, and its RNA3 is encapsidated. The tripartite virus can be mechanically transferred for eleven generations without losing its infectivity or show major genomic rearrangements. Furthermore, mixing equal amounts of WT and tripartite virus inocula in the same leaf originated plants systemically infected only with the WT virus, showing that the tripartite virus has lower fitness than the WT. To our knowledge this is the first example of a stable virus evolving in vitro from bipartite to tripartite genomic structure from a synthetic construct in a plant virus.