Evolution of a novel engineered tripartite viral genome of a torradovirus

Viruses in the Secoviridae include monopartite and bipartite genomes, suggesting the possibility to study members of this family to experimentally address evolutionary transitions resulting in multipartitism. Torradoviruses are bipartite members of the family Secoviridae characterized by a genus-specific 5ʹ open reading frame (ORF), named P21, encoded by RNA2. Here, in a study originally intended to verify if P21 can function in trans, we attempted to provide P21 from a third P21-expressing construct under control of the 35S promoter and containing the 5ʹ- and 3ʹ- untranslated regions (UTRs) of wild type (WT) RNA2. When this construct was combined with an RNA2 with a complete deletion of the P21 coding region we verified that the P21 provided in trans cannot immediately complement the mutant, but occasional systemic infections in a limited number of the inoculated plants display the presence of a tripartite virus with an actively replicating P21-expressing RNA3. Furthermore, in all the systemically infected plants investigated in six distinct experiments, this replicating RNA3 accumulates deletions in a small region inside the original 3ʹ-UTR provided by the cDNA clone. Such tripartite virus, which we obtained through deconstructing the coding potential of the RNA2 in two distinct RNAs, can be transmitted mechanically and by whiteflies, is competent for virion formation, and its RNA3 is encapsidated. It can be mechanically transferred for eleven serial passages without losing its infectivity or showing major genomic rearrangements. Furthermore, mixing equal amounts of WT and tripartite virus inocula in the same leaf resulted in 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 an engineered tripartite viral genome becoming stable through artificial evolution in vivo, in plants. This tripartite system was also used to derive a stable viral vector to express GFP systemically in the context of viral infection.