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

Abstract

Summary 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‐5bL33P/K319E/R927A and Sw‐5bL33P/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.

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

“…Although rTSWV R124A did not cause any disease symptoms, it still accumulated in systemic leaves of Sw-5b-transgenic N. benthamiana plants (Figure 3d). These data suggest that TSWV D122A and We recently obtained two artificially improved versions of Sw-5b, namely, Sw-5b L33P/K319E/R927A and Sw-5b L33P/K319E/R927Q (Huang et al, 2021), which are effective against RB TSWV isolates carrying the NSm C118Y or NSm T120N mutation, as well as other American-type tospoviruses. We examined whether these two improved Sw-5b mutants were also effective at recognizing the above NSm21 alanine substitution mutants.…”

“…To engineer Sw-5b and develop an immune receptor capable of conferring effective resistance against RB TSWV isolates, we recently conducted a stepwise artificial evolution of Sw-5b based on the two-step recognition mechanism. We obtained Sw-5b L33P/K319E/R927A and Sw-5b L33P/K319E/R927Q mutants, which provide effective resistance to RB TSWV isolates carrying the NSm C118Y or NSm T120N mutation (Huang et al, 2021).…”

“…TSWV carrying the NSm D122A or NSm R124A mutation was able to break Sw-5b-mediated resistance and establish systemic infection on Sw-5b-transgenic N. benthamiana plants. The two improved versions of the resistance gene, Sw-5b L33P/K319E/R927A and Sw-5b L33P/K319E/R927Q (Huang et al, 2021), which were engineered by a two-step artificial evolution strategy, recognize all alanine substitution NSm mutants and confer effective resistance against new experimental RB isolates of TSWV with the NSm D122A or NSm R124A mutation. Hence, the artificially improved Sw-5b resistance genes could provide effective resistance to both field RB TSWV isolates and also potential RB isolates.…”

“…, M (-)opt T120N , Sw-5b L33P/K319E/R927A , and Sw-5b L33P/K319E/R927Q were described by Huang et al (2021). All…”

Natural Resources Resistance to Tomato Spotted Wilt Virus (TSWV) in Tomato (Solanum lycopersicum)

“…Additionally, substantial breakthroughs have been achieved in plant engineering of NLRs to defend against pathogens [148]. Huang et al [149] conducted a stepwise artificial evolution strategy to select Sw-5b mutants, which are effective against resistance-breaking isolates of TSWV, in order to provide a new vision and ideas that resist TSWV by artificial evolution.…”