Stepwise artificial evolution of an Sw-5b immune receptor extends its resistance spectrum against resistance-breaking isolates of Tomato spotted wilt virus

Plants use intracellular nucleotide-binding leucine-rich repeat immune receptors (NLRs) to recognize pathogen-encoded effectors and initiate immune responses. Tomato spotted wilt virus (TSWV), which has been found to infect >1000 plant species, is among the most destructive plant viruses worldwide. The Sw-5b is the most effective and widely used resistance gene in tomato breeding to control TSWV. However, broad application of tomato cultivars carrying Sw-5b has resulted in an emergence of resistance-breaking (RB) TSWV. Therefore, new effective genes are urgently needed to prevent further RB TSWV outbreaks. In this study, we conducted artificial evolution to select Sw-5b mutants that could extend the resistance spectrum against TSWV RB isolates. Unlike regular NLRs, Sw-5b detects viral elicitor NSm using both the N-terminal Solanaceae-specific domain (SD) and the C-terminal LRR domain in a two-step recognition process. Our attempts to select gain-of-function mutants by random mutagenesis involving either the SD or the LRR of Sw-5b failed; therefore, we adopted a stepwise strategy, first introducing a NSmRB-responsive mutation at the R927 residue in the LRR, followed by random mutagenesis involving the Sw-5b SD domain. Using this strategy, we obtained Sw-5b(L33P/K319E/R927A) and Sw-5b(L33P/K319E/R927Q) mutants, which are effective against TSWV RB carrying the NSmC118Y or NSmT120N mutation, and against other American-type tospoviruses. Thus, we were able to extend the resistance spectrum of Sw-5b; the selected Sw-5b mutants will provide new gene resources to control RB TSWV.