Induction of cell death by tospoviral protein NSs and the motif critical for cell death does not control RNA silencing suppression activity

Groundnut bud necrosis virus induces necrotic symptoms in different hosts. Previous studies showed reactive oxygen species-mediated programmed cell death (PCD) resulted in necrotic symptoms. Transgenic expression of viral protein NSs mimics viral symptoms. Here, we showed a role for NSs in influencing oxidative burst in the cell, by analyzing H2O2 accumulation, activities of antioxidant enzymes and expression levels of vacuolar processing enzymes, H2O2-responsive microRNA 319a.2 plus its possible target metacaspase-8. The role of NSs in PCD, was shown using two NSs mutants: one in the Trp/GH3 motif (a homologue of pro-apototic domain) (NSs(s189R)) and the other in a non-Trp/GH3 motif (NSs(L172R)). Tobacco rattle virus (TRV) expressing NSs(S189R) enhanced the PCD response, but not TRV-NSs(L172R), while RNA silencing suppression activity was lost in TRV-NSs(L172R), but not in TRV-NSs(S189R). Therefore, we propose dual roles of NSs in RNA silencing suppression and induction of cell death, controlled by different motifs.