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-ProC151A 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.

Jiangsu Key Laboratory for Pathogens and Ecosystems Jiangsu Engineering and Technology Research Center for Microbiology College of Life Sciences Nanjing Normal University Nanjing 210023 China

Jiangsu Key Laboratory for Pathogens and Ecosystems Jiangsu Engineering and Technology Research Center for Microbiology College of Life Sciences Nanjing Normal University Nanjing 210023 China; Jiangsu Key Laboratory for Food Quality and Safety State Key Laboratory Cultivation Base of Ministry of Science and Technology Institute of Plant Protection Jiangsu Academy of Agricultural Sciences Nanjing 210014 China

Plant Virus and Vector Interactions Crop Research Institute Drnovská 507 161 06 Prague Czech Republic

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