Determination of key residues in tospoviral NSm required for Sw-5b recognition,their potential ability to overcome resistance,and the effective resistance provided by improved Sw-5b mutants

Sw-5b is an effective resistance gene used widely in tomato to control tomato spotted wilt virus (TSWV), which causes severe losses in crops worldwide. Sw-5b confers resistance by recognizing a 21-amino-acid peptide region of the viral movement protein NSm (NSm21, amino acids 115–135). However, C118Y or T120N mutation within this peptide region of NSm has given rise to field resistance-breaking (RB) TSWV isolates. To investigate the potential ability of TSWV to break Sw-5b-mediated resistance, we mutagenized each amino acid on NSm21 and determined which amino acid mutations would evade Sw-5b recognition. Among all alanine-scan mutants, NSm^P119A, NSm^W121A, NSm^D122A, NSm^R124A, and NSm^Q126A failed to induce a hypersensitive response (HR) when coexpressed with Sw-5b in Nicotiana benthamiana leaves. TSWV with the NSm^P119A, NSm^W121A, or NSm^Q126A mutation was defective in viral cell-to-cell movement and systemic infection, while TSWV carrying the NSm^D122A or NSm^R124A mutation was not only able to infect wild-type N. benthamiana plants systemically but also able to break Sw-5b-mediated resistance and establish systemic infection on Sw-5b-transgenic N. benthamiana plants. Two improved mutants, Sw-5b^{L33P/K319E/R927A} and Sw-5b^{L33P/K319E/R927Q}, which we recently engineered and which provide effective resistance against field RB isolates carrying NSm^C118Y or NSm^T120N mutations, recognized all NSm21 alanine-substitution mutants and conferred effective resistance against new experimental RB TSWV with the NSm^D122A or NSm^R124A mutation. Collectively, we determined the key residues of NSm for Sw-5b recognition, investigated their potential RB ability, and demonstrated that the improved Sw-5b mutants could provide effective resistance to both field and potential RB TSWV isolates.