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

Abstract

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 NSm^RB -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 NSm^C118Y or NSm^T120N 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.

Keywords: Tomato spotted wilt virus; NLR; Sw-5b; artificial evolution; immune receptor; resistance breaking.

Figure 1

Schematics depicting construction of the random mutagenized Sw‐5b^R927A library and selection of mutants that gained hypersensitive response (HR) to NSm^C118Y or NSm^T120N from Tomato spotted wilt virus (TSWV) resistance‐breaking (RB) isolates. Random mutagenesis of the Solanaceae‐specific domain (SD) in full‐length Sw‐5b^R927A was performed using Mn²⁺ and dITP‐mediated two‐round polymerase chain reaction (PCR) amplification. The second‐round PCR products of the Sw‐5b SD variants were recombined into linearized binary vector p2300S‐Sw‐5b^R927A. The recombination reaction mixture was directly transformed into A. tumefaciens strain GV3101 using electroporation to generate the Sw‐5b^R927A mutagenized library. The library was used to screen Sw‐5b^R927A mutants that could trigger HR in N. benthamiana leaves in the presence of NSm^C118Y or NSm^T120N. HR phenotypes were monitored at 3–7 days post‐infiltration (dpi).

Figure 2

Screening of the Sw‐5b^R927A mutagenesis library led to the selection of two candidates, Sw‐5b^mutant‐137 and Sw‐5b^mutant‐1665, that gained HR to NSm^C118Y and NSm^T120N from TSWV RB isolates. (a) Sw‐5b^mutant‐137 and Sw‐5b^mutant‐1665 were co‐expressed with or without the WT NSm, NSm^C118Y or NSm^T120N in N. benthamiana leaves. Co‐expression of WT Sw‐5b and NSm was used as a positive control. The HR phenotypes were monitored at 3‐10 dpi and photographed at 7 dpi. (b) Amino acid mutations in the SD domain of Sw‐5b^mutant‐137 and Sw‐5b^mutant‐1665. The amino acid sequences of the SD domain of WT Sw‐5b, Sw‐5b^mutant‐137 and Sw‐5b^mutant‐1665 were aligned using the ClustalW program. Amino acid differences at residues 33 (L substituted to P) and 319 (K to L) among the WT Sw‐5b, Sw‐5b^mutant‐137 and Sw‐5b^mutant‐1665 are boxed and indicated in red.

Figure 3

Analysis of single, double and triple mutations of L33P, K319E and R927A in the SD and LRR domains of Sw‐5b for their ability to induce HR to WT NSm or NSm^C118Y or NSm^T120N mutants in N. benthamiana leaves. (a) (YFP)‐Sw‐5b^L33P/R927A, (YFP)‐Sw‐5b^K319E/R927A, (YFP)‐Sw‐5b^{L33P/K319E/R927A}, (YFP)‐Sw‐5b^L33P, (YFP)‐ (YFP)‐Sw‐5b^K319E, (YFP)‐Sw‐5b^L33P/K319E, (YFP)‐Sw‐5b^{L33P/K319E/D642E}, (YFP)‐Sw‐5b^{L33P/K319E/R927Q} and (YFP)‐Sw‐5b^{L33P/K319E/R927L} were co‐expressed with and without NSm, NSm^C118Y and NSm^T120N, respectively, in N. benthamiana leaves via agro‐infiltration. HR phenotypes were monitored at 3–10 dpi and photographed at 7 dpi. (b) Western blotting analysis of YFP‐tagged Sw‐5b, Sw‐5b^L33P/R927A, Sw‐5b^K319E/R927A, Sw‐5b^{L33P/K319E/R927A}, Sw‐5b^L33P, Sw‐5b^K319E, Sw‐5b^L33P/K319E, (YFP)‐Sw‐5b^{L33P/K319E/D642E}, (YFP)‐Sw‐5b^{L33P/K319E/R927Q} and (YFP)‐Sw‐5b^{L33P/K319E/R927L} co‐expressed with NSm, NSm^C118Y and NSm^T120N in N. benthamiana leaves using YFP‐specific and NSm‐specific antibodies. Plant leaves agro‐infiltrated with empty vector (EV) were used as a negative control. The rubisco large subunit was stained with Ponceau S to indicate sample loading. Protein size is indicated on the left.

Figure 4

Analysis of Sw‐5b^{L33P/K319E/R927A} and Sw‐5b^{L33P/K319E/R927Q} induced resistance against mini‐replicons of TSWV, TSWV^C118Y and TSWV^T120N transiently expressed in N. benthamiana leaves. (a) Schematic diagram of DNA constructs harbouring L_(+)opt, M_(–)opt, M_(–)opt ^C118Y, M_(‐)opt ^T120N, S₍₊₎ and SR_(+)eGFP. M_(–)opt ^C118Y and M_(–)opt ^T120N were generated by introducing C118Y and T120N mutations into NSm of M_(–)opt. Opt, optimized sequence; (–), viral strand of TSWV genomic RNA; (+), viral complementary strand of TSWV genomic RNA. (b) Infectious mini‐replicon clones of TSWV L_(+)opt + M_(–)opt + SR_(+)eGFP, L_(+)opt + M_(–)opt ^C118Y + SR_(+)eGFP or L_(+)opt + M_(–)opt ^T120N + SR_(+)eGFP, in combination with three VSRs (P19, Hc‐Pro and γb), were co‐expressed with the p2300S EV, WT Sw‐5b, Sw‐5b^{L33P/K319E/R927A} and Sw‐5b^{L33P/K319E/R927Q} in N. benthamiana leaves by agro‐infiltration. eGFP fluorescence foci in agro‐infiltrated leaves were photographed at 3 and 5 dpi using inverted fluorescence microscopy. Scale bars = 800 μm. (c) Western blotting analysis of eGFP protein accumulation for various recombination treatments shown in panel (b) at 3 and 5 dpi using GFP‐specific antibody. The rubisco large subunit was stained with Ponceau S to indicate sample loading. Protein size is indicated on the left.

Figure 5

Analysis of Sw‐5b^{L33P/K319E/R927A} and Sw‐5b^{L33P/K319E/R927Q} induced resistance against TSWV^C118Y and TSWV^T120N RB mutant infection in transgenic N. benthamiana plants. (a) Full‐length infectious clones of TSWV L_(+)opt + M_(–)opt + S₍₊₎, L_(+)opt + M_(–)opt ^C118Y + S₍₊₎ and L_(+)opt + M_(–)opt ^T120N + S₍₊₎, together with three VSRs (P19, Hc‐Pro and γb), were agro‐infiltrated into p2300S EV control transgenic N. benthamiana plants and Sw‐5b, Sw‐5b^{L33P/K319E/R927A} and Sw‐5b^{L33P/K319E/R927Q} transgenic plants. Viral infection and symptoms in systemic leaves of various agro‐infiltrated plants were monitored from 7–21 dpi and photographed at 15 dpi. (b) Accumulation of TSWV in systemic leaves of various treated plants shown in panel (a) was analysed by Western blotting analysis using TSWV N‐specific antibody. The rubisco large subunit was stained with Ponceau S to indicate sample loading. Protein size is indicated on the left.

Figure 6

Analysis of Sw‐5b^{L33P/K319E/R927A} and Sw‐5b^{L33P/K319E/R927Q} induced resistance against Tomato zonate spot virus (TZSV), a representative European/Asian‐type tospovirus, and Impatiens necrotic spot virus (INSV), a representative American‐type tospovirus. (a) (YFP)‐Sw‐5b, (YFP)‐Sw‐5b^{L33P/K319E/R927A} and (YFP)‐Sw‐5b^{L33P/K319E/R927Q} were co‐expressed with TZSV NSm‐3×FLAG or INSV NSm‐3×FLAG in N. benthamiana leaves. Pictures were taken at 7 dpi. Protein accumulation of YFP‐tagged Sw‐5b mutants, TZSV NSm‐3×FLAG and INSV NSm‐3×FLAG in N. benthamiana leaves was analysed by Western blotting using YFP‐ and FLAG‐specific antibodies. Plant leaves agro‐infiltrated with EV were used as negative controls. The rubisco large subunit was stained with Ponceau S to indicate sample loading. Protein size is indicated on the left. (b) p2300S EV control transgenic N. benthamiana plants and Sw‐5b, Sw‐5b^{L33P/K319E/R927A} and Sw‐5b^{L33P/K319E/R927Q} transgenic plants were inoculated with crude extracts of TZSV and INSV from freshly infected tissue. Viral infection and symptoms in systemic leaves of various agro‐infiltrated plants were monitored from 7‐21 dpi and photographed at 15 dpi. (c) The accumulation of TZSV or INSV in systemic leaves of various treated plants shown in panel (b) was analysed by Western blotting using TZSV N‐ or INSV N‐specific antibodies. The rubisco large subunit was stained with Ponceau S to indicate sample loading. Protein size is indicated on the left.