Competition among co-infecting potyviral chimeras with adaptive, single-nucleotide differences in HCPro: Properties, and influence of environment factors on outcomes.

We previously showed that two natural, single-amino acid mutations in the HCPro of a potyviral chimera, separately increased virulence by enhancing either its silencing-suppression or proteolytic functions. To investigate how beneficial variants generated during infection compete with non-mutant forms, or among each other, we co-expressed them in pairs, in Nicotiana benthamiana. We assessed how variants co-exist and influence each other in the cell, and whether altered abiotic (elevated temperature) or biotic (a silencing-impaired plant) environmental factors, affected local and systemic infection outcomes. In local tissues, our results are consistent with a model in which variants replicate and accumulate independently in co-infected cells: observed variant titers and ratios correlated with those that could be deduced from single infections. Trans-acting effects of one variant over the other were not observed. By contrast, environment parameters influenced not only local titers but also ratios, likely because of their differential effects on functions affected by the specific mutation of each variant. Additional mutations in HCPro appeared separately in systemic infections in specific plants, and induced either new substitutions in the protein, or in a putative small reverse reading frame. Thus, further adaptation could proceed through separate paths via the incorporation of distinct, novel mutations in the HCPro sequence in different plants.