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.