AbstractBACKGROUNDTobacco curly shoot virus (TbCSV, Begomovirus, Geminiviridae) is a viral agent that poses a serious threat to economically important crops like tomato and tobacco in Southwest China. The geminiviral V2 protein has been demonstrated to play multifunctional roles essential for viral infection and pathogenicity. This study comprehensively investigated the biological importance of five cysteines within the conserved ‘WCCH’ motif of the TbCSV V2 protein, and evaluated the effects of cysteine mutations on viral pathogenicity and the efficacy for cross‐protection against wild‐type severe TbCSV train.RESULTSSite‐directed mutagenesis revealed that among the five cysteines in the V2 protein, C84, C86 and C89 residues are critical for its RNA silencing suppression activity, with C84 being the only residue whose mutation impaired its ability to induce tissue necrosis when expressed by potato virus X (PVX). Mutations at C84, C86 or C89 attenuated viral pathogenicity, and the mild symptoms were accompanied by reduced viral accumulations in Nicotiana benthamiana plants. Notably, pre‐inoculation with the attenuated TbCSV mutant C84A in N. benthamiana and tomato plants conferred effective cross‐protection against subsequent challenge infection by wild‐type TbCSV.CONCLUSIONThe cysteine residues within the ‘WCCH’ motif of TbCSV V2 protein play distinct roles in its functions and viral pathogenicity. Importantly, the attenuated C84A mutant generated in this study not only offers insights into the pathogenic mechanisms of TbCSV but also holds promise for application in crop protection through cross‐protection strategy in the field. © 2025 Society of Chemical Industry.