A progeny virus from a cucumovirus pseudorecombinant evolved to gain the ability to accumulate Its RNA-silencing suppressor leading to systemic infection in tobacco.

Two isolates of Tomato aspermy virus (TAV), V-TAV and C-TAV, can systemically infect Nicotiana benthamiana but only C-TAV can move systemically in N. tabacum. Any pseudorecombinants between the two strains could not move systemically in tobacco as efficiently as C-TAV. However, a pseudorecombinant consisting of RNAs 1 and 3 of V-TAV and RNA 2 of C-TAV (V1C2V3), which cannot infect tobacco systemically, generated progeny with a mutation in V1 and a recombination in C2 (V1(m)C2(r)V3), enabling the virus to move systemically. To avoid further mutation and recombination in the virus, we used Cucumber mosaic virus RNA3 (Y3) for subsequent experiments. Northern blot analyses showed that RNA4A, which encodes the 2b protein (2b), and RNA5 abundantly accumulated in V1(m)C2(r)Y3-infected tobacco. V1(m)C2(r)Y3 actually caused higher accumulation of 2b than did V1C2Y3 in Western blots, and overexpression of 2b by the PVX vector enabled V1C2Y3 to move systemically in tobacco, suggesting that 2b accumulation promotes viral systemic movement. Because RNA-silencing suppressor (RSS) activity of 2b was thought to be involved in systemic movement, we compared the RSS activity of 2b for the two TAV isolates; C-TAV 2b had stronger activity than did V-TAV 2b in tobacco in a transient protoplast assay. Our data also demonstrated that 2b and RNA5 play an important role in the evolution of members of genus Cucumovirus by generating mutant/recombinant viruses and viral systemic movement over RNA silencing.