Protease activity of NIa-Pro determines systemic pathogenicity of clover yellow vein virus

Clover yellow vein virus (ClYVV), a potyvirus that infects various dicotyledonous plants, poses a significant threat to the cultivation of legumes. Although potyviral NIa-Pro was extensively studied in viral infection cycle and host antiviral responses, the contribution of NIa-Pro protease activity to virus systemic symptoms has not yet been reported. In this study, we developed infectious clones of a ClYVV isolated from Pisum sativum. The rescued ClYVV showed robust infectivity and induced obvious systemic mosaic and necrosis symptoms in the model host Nicotiana benthamiana and natural hosts Pisum sativum and Vicia faba. Using a potato virus X (PVX) vector to express 11 ClYVV proteins in N. benthamiana ectopically, we identified that NIa-Pro is the key determinant in inducing systemic symptoms and causes higher leaf ROS levels and cell death. Further, we found that the protease-inactive mutant NIa-Pro^C151A causes significantly reduced systemic symptoms when expressed via the PVX vector and does not induce higher cellular ROS levels and cell death when transiently overexpressed compared to wild-type NIa-Pro. Overall, this study provides evidence supporting that the protease activity of a potyvirus protein NIa-Pro directly contributes to the virus symptoms.