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. 2021 Jun 7;19(11):2164–2176. doi: 10.1111/pbi.13641

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

Haining Huang 1,2, Shen Huang 1,2, Jia Li 1,2, Huiyuan Wang 1,2, Yaqian Zhao 1,2, Mingfeng Feng 1,2, Jing Dai 1,2, Tongkai Wang 1,2, Min Zhu 1,2, Xiaorong Tao 1,2,✉
PMCID: PMC8541788  PMID: 34036713

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.

Keywords: NLR, immune receptor, artificial evolution, Sw‐5b, Tomato spotted wilt virus, resistance breaking

Introduction

Plants have evolved various types of resistance (R) proteins to combat pathogen attacks (Kourelis and Hoorn, 2018; Maekawa et al., 2011). Intracellular nucleotide‐binding leucine‐rich repeat (NLR) immune receptors represent the largest group of R proteins in plants; they play critical roles in pathogen perception and host immune response activation (Dodds and Rathjen, 2010; Jones and Dangl, 2006; Jones et al., 2016). Plant NLRs typically contain an N‐terminal domain, a central NB‐ARC domain (nucleotide‐binding adaptor, APAF‐1, R proteins and CED‐4) (van der Biezen and Jones, 1998) and a C‐terminal leucine‐rich repeat (LRR) domain (Kapos et al., 2019; Meier et al., 2019; van Wersch et al., 2020). There are two types of N‐terminal domains in plant NLRs: Toll/interleukin‐1 receptor (TIR) and coiled‐coil (CC). Non‐canonical domains are also frequently integrated into plant NLRs at the N‐terminal end, in the middle region or at the C‐terminal end (Aravind, 2000; Kroj et al., 2016; Meyers et al., 2003; Sarris et al., 2016). Plant NLRs can recognize pathogen effectors either directly or indirectly (Cesari, 2018). In the absence of pathogens, plant NLRs are maintained in an auto‐inhibited or OFF state via intramolecular interactions involving the N‐terminal TIR/CC, NB‐ARC and C‐terminal LRR domains. Upon recognition of pathogen‐encoded effectors, these intramolecular interactions are disrupted and plant NLRs switch to an activated or ON state (Takken and Tameling, 2009; Wang et al., 2019a; Wang et al., 2019b). The activated NLRs then induce effector‐triggered immunity (ETI) and result in hypersensitive response (HR), which is typically associated with a rapid and localized cell death at the infection site (Dodds and Rathjen, 2010; Jones and Dangl, 2006; Jones et al., 2016; van Ooijen et al., 2007; Win et al., 2012).

Tomato spotted wilt virus (TSWV), which infects more than 1000 plant species, is among the most devastating plant viruses worldwide (Kormelink et al., 2011; Oliver and Whitfield, 2016; Scholthof et al., 2011). TSWV is transmitted by thrips species in a persistent propagative manner (Gilbertson et al., 2015; Hogenhout et al., 2008; Oliver and Whitfield, 2016; Whitfield et al., 2005). Due to the rapid global spread of western flower thrips (Frankliniella occidentalis), TSWV has caused serious annual economic losses of agronomically important crops (e.g., tomato, peppers, potato, lettuce and flowers) in many parts of the world (Kormelink et al., 2011; Oliver and Whitfield, 2016; Pappu et al., 2009; Turina et al., 2016). The most effective control measure for TSWV is the use of resistant cultivars (Zhu et al., 2019). The tomato Sw‐5b gene, which was originally derived from Solanum peruvianum, is the most effective and widely used resistance gene in tomato resistance breeding (Bendahmane et al., 2002; Boiteux and Giordano, 1993; Brommonschenkel et al., 2000; Spassova et al., 2001) and has been introduced into numerous tomato cultivars to control tospoviruses. However, the wide application of tomato cultivars carrying the Sw‐5b resistance gene has resulted in the emergence of resistance‐breaking (RB) TSWV. Sw‐5b RB isolates have now been reported in Spain, Italy and California (USA) (Aramburu and Marti, 2003; Batuman et al., 2017; Ciuffo et al., 2005). RB TSWV has caused substantial problems in some areas of California (Batuman et al., 2017), where tomato cultivars containing the Sw‐5b resistance gene have been widely grown over the past decade. The TSWV movement protein NSm is an avirulent protein (elicitor) that is recognized by Sw‐5b (Hallwass et al., 2014; Lopez et al., 2011; Peiro et al., 2014). Co‐expression of Sw‐5b and TSWV NSm has been shown to induce strong HR in Nicotiana benthamiana and tomato leaves. An analysis of TSWV RB isolates determined that RB TSWV isolates carry the C118Y or T120N mutation in NSm (Lopez et al., 2011). The co‐expression of Sw‐5b with NSmC118Y or NSmT120N is no longer sufficient to induce HR in plants (Zhao et al., 2016). There is currently no effective strategy to control Sw‐5 RB TSWV. Because other resistance genes are less effective than Sw‐5b in tomato resistance breeding against TSWV, new effective resistant genes are urgently needed for introduction into tomato cultivars to control the newly emerging RB TSWV.

Substantial breakthroughs have been made in engineering plant NLRs (Tamborski and Krasileva, 2020). The bacteria effector AvrPphB targets and cleaves the host decoy protein PBS1, which is monitored by Arabidopsis RPS5 NLR, the cleavage behaviour of PBS1 causes RPS5 activation, which leads to a robust host defence response (Shao et al., 2003). By engineering the cleavage site of PBS1 targeted by a bacterial effector to a cleavage site targeted by other pathogen proteases a plant virus‐encoded effector, RPS5 was activated by these proteases and expanded its recognition to new pathogens (Kim et al., 2016). Random mutagenesis has also been used to perform artificial evolution of plant NLR immune receptors. The potato R3a immune receptor cannot confer resistance to Phytophthora infestans strains producing the AVR3aEM effector. Random mutagenesis of full‐length R3a has generated R3a mutants with expanded response, from AVR3aEM to other variants of AVR3a (Eugenia Segretin et al., 2014). Random mutagenesis involving the LRR domain of Rx has been applied to successfully select Rx mutants with broad‐spectrum resistance that includes resistance to potato virus X and the distantly related Poplar mosaic virus (Farnham and Baulcombe, 2006). However, one of the selected Rx mutants with broad‐spectrum resistance developed a trailing necrosis that killed the plant. Stepwise artificial evolution of Rx through random mutagenesis involving the N‐terminal domains was conducted to select Rx mutants that could eliminate this cost of broad‐spectrum resistance. The selected Rx mutants enhanced activation sensitivity and had a stronger response to Poplar mosaic virus (Harris et al., 2013). Therefore, short‐term artificial evolution of NLR receptors can be used to select mutants that offer broader resistance and modify activation phases to eliminate the negative aspects of disease resistance.

In the present study, we performed artificial evolution to select Sw‐5b mutants that could extend the spectrum of resistance against RB isolates of TSWV, including TSWVC118Y and TSWVT120N. Unlike traditional plant NLRs, Sw‐5b contains an extended N‐terminal Solanaceae domain (SD) and the classical CC, NB‐ARC and LRR domains (Lukasik‐Shreepaathy et al., 2012; Chen et al., 2016). Our initial screening of the mutant library involving the C‐terminal LRR or N‐terminal SD domain failed to select any Sw‐5b mutants that induce the HR response in the presence of NSmC118Y and NSmT120N from RB TSWV. Our recent studies showed that both the SD and the LRR of Sw‐5b are involved in the two‐step recognition of the NSm (Li et al., 2019; Zhu et al., 2017). In the first step, the Sw‐5b SD interacts with NSm and relieves the inhibitory effects of SD and CC on NB‐ARC‐LRR. In the second step, the LRR domain interacts with NSm and fully activates the receptor, presumably by disrupting the intramolecular interaction between LRR and the NB‐ARC domains. Based on our knowledge of the two‐step recognition mechanism of Sw‐5b NLR (Li et al., 2019; Zhu et al., 2017), we adopted here a stepwise strategy for artificial evolution of Sw‐5b NLR, combining site‐directed and random mutagenesis of the LRR and SD domains. Using this stepwise strategy, we obtained two Sw‐5b mutants, which are effective against TSWV RB carrying the NSmC118Y or NSmT120N mutation. The results of this study will lead to the development of a new resistance gene resource to control RB TSWV.

Results

Random mutagenesis of the SD domain of Sw‐5bR927A to select mutants with HR to NSmC118Y and NSmT120N from TSWV RB isolates

To select Sw‐5b NLR mutants that could extend the spectrum of resistance to RB isolates of TSWV, we performed artificial evolution of the Sw‐5b gene using random mutagenesis. Sw‐5b mutants were generated using Mn2+ and dITP‐mediated two‐round polymerase chain reaction (PCR) amplification (see Materials &Methods) and cloned into a binary vector for agro‐infiltration together with RB NSm constructs. Some NLR proteins were found to recognize pathogen effectors via either by the LRR domain or by indirectly the N‐terminal domain (Collier and Moffett, 2009; Dodds et al., 2006; Dodds et al., 2001; Ellis et al., 1999; Jones et al., 2016; Li et al., 2019; Padmanabhan et al., 2009; Rairdan and Moffett, 2006; Ueda et al., 2006). The LRR and the N‐terminal SD domains of the Sw‐5b gene were initially subjected to random mutagenesis, respectively. In total, 1637 clones were screened for mutagenesis involving the LRR domain of Sw‐5b and 1489 clones were screened for mutagenesis involving the SD domain of Sw‐5b; however, no Sw‐5b mutagenized clones triggered HR through co‐expression with NSmC118Y or NSmT120N in N. benthamiana leaves.

We recently discovered that Sw‐5b NLR exhibited a two‐step recognition process to detect NSm using both the LRR and SD domains (Li et al., 2019), suggesting that mutagenesis involving either the LRR or SD domain alone may be insufficient to obtain Sw‐5b mutants with a cell death response to NSmC118Y or NSmT120N from TSWV RB isolates. Therefore, we adopted a new stepwise strategy to select gain‐of‐function Sw‐5b mutants with responses to NSmC118Y or NSmT120N. We previously identified a NSmRB‐responsive mutation at the R927 residue in the LRR domain (Zhu et al., 2017). Sw‐5b NB‐ARC‐LRRR927A triggered an auto‐active HR in the absence of NSm. Co‐expression of NB‐ARC‐LRRR927A with NSm, NSmC118Y or NSmT120N in N. benthamiana leaves induced a stronger cell death response for Sw‐5b NB‐ARC‐LRRR927A, compared with the response caused by Sw‐5b NB‐ARC‐LRRR927A alone (Figure S1; the data adapted from Zhu et al., 2017). We also found that the introduction of R927A into full‐length Sw‐5b did not cause auto‐active HR, because the SD‐CC suppressed the auto‐activity of Sw‐5b NB‐ARC‐LRRR927A. When the Sw‐5bR927A mutant was co‐expressed with NSm, it continued to induced HR in N. benthamiana leaves. However, the Sw‐5bR927A mutant was unable to induce HR when co‐expressing with either NSmC118Y or NSmT120N (Figure S1; the data were adapted from Li et al., 2019). Based on these knowledge, we conducted random mutagenesis involving the SD domain in the backbone of full‐length Sw‐5b with the R927A mutation to select Sw‐5bR927A mutants that exhibited a cell death response to NSmC118Y or NSmT120N. The Sw‐5bR927A mutagenesis library construction and screening schematics are illustrated in Figure 1. We hypothesized that random mutagenesis involving the SD domain might select the SD mutants that are able to gain the ability to recognize NSmC118Y or NSmT120N and this first recognition would activate SD to relieve CC inhibition from Sw‐5b NB‐ARC‐LRRR927A. In the second recognition step, Sw‐5b NB‐ARC‐LRRR927A was already a mutant that showed enhanced HR induction activity in the presence of NSmC118Y or NSmT120N (Figure S1).

Figure 1.

Figure 1

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Schematics depicting construction of the random mutagenized Sw‐5bR927A library and selection of mutants that gained hypersensitive response (HR) to NSmC118Y or NSmT120N from Tomato spotted wilt virus (TSWV) resistance‐breaking (RB) isolates. Random mutagenesis of the Solanaceae‐specific domain (SD) in full‐length Sw‐5bR927A 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‐5bR927A. The recombination reaction mixture was directly transformed into A. tumefaciens strain GV3101 using electroporation to generate the Sw‐5bR927A mutagenized library. The library was used to screen Sw‐5bR927A mutants that could trigger HR in N. benthamiana leaves in the presence of NSmC118Y or NSmT120N. HR phenotypes were monitored at 3–7 days post‐infiltration (dpi).

In total, 2036 clones were screened to select mutants for the appearance of HR in the presence of NSmC118Y or NSmT120N in N. benthamiana leaves, two of these clones, Sw‐5bmutant‐137 and Sw‐5bmutant‐1665, triggered HR when co‐expressed with NSmC118Y or NSmT120N (Figure 2a). The Sw‐5bmutant‐137 and Sw‐5bmutant‐1665 clones did not induce HR in the absence of NSm. When two clones were co‐expressed with the wild‐type (WT) NSm, they were fully functional (Figure 2a). These results indicated that the Sw‐5bmutant‐137 and Sw‐5bmutant‐1665 clones gained the ability to induce HR in response to NSmC118Y or NSmT120N, and that the mutants did not lose their original abilities to recognize WT NSm and induce NSm‐dependent HR in N. benthamiana plants.

Figure 2.

Figure 2

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Screening of the Sw‐5bR927A mutagenesis library led to the selection of two candidates, Sw‐5bmutant‐137 and Sw‐5bmutant‐1665, that gained HR to NSmC118Y and NSmT120N from TSWV RB isolates. (a) Sw‐5bmutant‐137 and Sw‐5bmutant‐1665 were co‐expressed with or without the WT NSm, NSmC118Y or NSmT120N 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‐5bmutant‐137 and Sw‐5bmutant‐1665. The amino acid sequences of the SD domain of WT Sw‐5b, Sw‐5bmutant‐137 and Sw‐5bmutant‐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‐5bmutant‐137 and Sw‐5bmutant‐1665 are boxed and indicated in red.

Next, we sequenced the SD region in the Sw‐5bmutant‐137 and Sw‐5bmutant‐1665 clones. We found that both clones contained two mis‐sense mutations, resulting in two amino acid changes, L33 to P and K319 to E, in the Sw‐5b SD domain (Figure 2b). Both the Sw‐5bmutant‐137 and Sw‐5bmutant‐1665 clones had identical amino acid mutations in the SD domain. We also sequenced the full lengths of the Sw‐5bmutant‐137 and Sw‐5bmutant‐1665 clones and found no other mutations in the remaining domains of Sw‐5bR927A. These results confirmed that Sw‐5b SD plays a critical role in the detection of NSm and that L33P/K319E double amino acid changes provided the Sw‐5b SD domain with the ability to recognize NSmC118Y and NSmT120N.

Double mutations of L33P and K319E in the SD domain together with the R927A or R927Q mutation in the LRR domain are required for Sw‐5b to gain HR function in response to NSmC118Y or NSmT120N

To determine which amino acid change in L33P and K319E is important for the recognition of NSmC118Y or NSmT120N by Sw‐5bL33P/K319E/R927A, we introduced L33P and K319E separately or simultaneously into Sw‐5bR927A (or YFP‐Sw‐5bR927A), generating Sw‐5bL33P/R927A (or YFP‐Sw‐5bL33P/R927A), Sw‐5bK319E/R927A (or YFP‐Sw‐5bK319E/R927A) and Sw‐5bL33P/K319E/R927A (or YFP‐Sw‐5bL33P/K319E/R927A). These constructs were then co‐expressed with or without NSm, NSmC118Y and NSmT120N for HR induction in N. benthamiana leaves. The results showed that none of the three mutants induced HR in the absence of NSm (Figure 3a‐c), which suggested that these amino acid mutations in the SD domain of Sw‐5bR927A did not cause constitutive activity. All three mutants induced HR in N. benthamiana leaves co‐expressing the WT NSm (Figures 3a and S2), which suggested that L33P and K319E did not affect the HR induction ability of Sw‐5bR927A in response to the WT NSm. When these mutants were co‐expressed with NSmC118Y and NSmT120N, respectively, Sw‐5bL33P/R927A (or YFP‐Sw‐5bL33P/R927A) and Sw‐5bK319E/R927A (or YFP‐Sw‐5bK319E/R927A) did not induce HR in N. benthamiana leaves, whereas Sw‐5bL33P/K319E/R927A (or YFP‐Sw‐5bL33P/K319E/R927A) induced strong HR in the presence of either NSmC118Y or NSmT120N (Figures 3a and S2). Western blotting confirmed that the YFP‐Sw‐5bL33P/R927A, YFP‐Sw‐5bK319E/R927A and YFP‐Sw‐5bL33P/K319E/R927A mutant proteins accumulated in a manner similar to the accumulation of WT YFP‐Sw‐5b in the agro‐infiltrated plant leaves (Figure 3b). These results suggested that both L33P and K319E mutations in the SD domain are required for Sw‐5bL33P/K319E/R927A to gain the abilities to recognize NSmC118Y and NSmT120N.

Figure 3.

Figure 3

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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 NSmC118Y or NSmT120N mutants in N. benthamiana leaves. (a) (YFP)‐Sw‐5bL33P/R927A, (YFP)‐Sw‐5bK319E/R927A, (YFP)‐Sw‐5bL33P/K319E/R927A, (YFP)‐Sw‐5bL33P, (YFP)‐ (YFP)‐Sw‐5bK319E, (YFP)‐Sw‐5bL33P/K319E, (YFP)‐Sw‐5bL33P/K319E/D642E, (YFP)‐Sw‐5bL33P/K319E/R927Q and (YFP)‐Sw‐5bL33P/K319E/R927L were co‐expressed with and without NSm, NSmC118Y and NSmT120N, 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‐5bL33P/R927A, Sw‐5bK319E/R927A, Sw‐5bL33P/K319E/R927A, Sw‐5bL33P, Sw‐5bK319E, Sw‐5bL33P/K319E, (YFP)‐Sw‐5bL33P/K319E/D642E, (YFP)‐Sw‐5bL33P/K319E/R927Q and (YFP)‐Sw‐5bL33P/K319E/R927L co‐expressed with NSm, NSmC118Y and NSmT120N 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.

Next, we introduced L33P and K319E mutations, alone or in combination, into the SD domain of WT Sw‐5b (or YFP‐Sw‐5b), generating Sw‐5bL33P (or YFP‐Sw‐5bL33P), Sw‐5bK319E (or YFP‐Sw‐5bK319E) and Sw‐5bL33P/K319E (or YFP‐Sw‐5bL33P/K319E). Sw‐5bL33P (or YFP‐Sw‐5bL33P), Sw‐5bK319E (or YFP‐Sw‐5bK319E) and Sw‐5bL33P/K319E (or YFP‐Sw‐5bL33P/K319E) induced strong HR cell death in N. benthamiana leaves co‐expressing the WT NSm. However, none of the three Sw‐5b mutants induced HR cell death in N. benthamiana leaves with NSmC118Y or NSmT120N (Figures 3a and S2). Western blotting showed that proteins of all three YFP‐tagged Sw‐5b mutants accumulated in a manner similar to the accumulation of WT YFP‐Sw‐5b in N. benthamiana leaves (Figure 3b). These results suggested that the L33P/K319E double mutations in the SD domain alone were insufficient for Sw‐5b to gain HR function in response to NSmC118Y or NSmT120N.

Sw‐5b exhibits two‐step recognition of WT NSm, first by the SD domain and then by the LRR domain (Li et al., 2019). To further select Sw‐5b variants for RB isolates of TSWV, we conducted a second round of artificial evolution on Sw‐5bL33P/K319E through random mutagenesis involving the LRR domain. A random mutagenesis library involving the LRR domain of Sw‐5bL33P/K319E was generated in Agrobacterium. Although we screened 1247 mutagenized clones, none triggered HR cell death in N. benthamiana leaves co‐expressing NSmC118Y or NSmT120N.

Following the failure of random mutagenesis selection involving Sw‐5bL33P/K319E, we focused on Sw‐5bL33P/K319E/R927A. For Sw‐5bL33P/K319E/R927A, both L33P/K319E mutations in the SD domain and the R927A mutation in the LRR domain were required to gain the ability to recognize NSmC118Y and NSmT120N. The R927A mutation in Sw‐5b NB‐ARC‐LRR caused auto‐activity, independent of NSm. According to the two‐step recognition model, when the SDL33P/K319E domain recognizes NSmC118Y or NSmT120N, SD‐CC domains are activated and relieve their repression of NB‐ARC‐LRRR927A. Because NB‐ARC‐LRRR927A triggered constitutive cell death activity, we next examined whether other auto‐active amino acid mutations involving NB‐ARC‐LRR could allow Sw‐5bL33P/K319E to gain HR in the presence of NSmC118Y or NSmT120N. Our previous study determined that Sw‐5b NB‐ARC‐LRR with the D642E mutation in the NB domain causes auto‐active cell death, independent of NSm (Chen et al., 2016). To examine whether introduction of the D642E mutations into the NB domain of Sw‐5bL33P/K319E would allow the receptor to gain HR in the presence of NSmC118Y or NSmT120N, we generated Sw‐5bL33P/K319E/D642E (or YFP‐Sw‐5bL33P/K319E/D642E) constructs and then co‐expressed the mutant with NSm, NSmC118Y or NSmT120N in N. benthamiana leaves. The results showed that Sw‐5bL33P/K319E/D642E (or YFP‐Sw‐5bL33P/K319E/D642E) recognized only WT NSm. However, the mutant did not induce HR in N. benthamiana leaves with NSmC118Y or NSmT120N (Figures 3a, b and S2). These results suggested that L33P/K319E mutations in the SD domain, combined with another constitutive mutant in the NB–ARC‐LRR, are not functional in the recognition of NSmC118Y or NSmT120N. Sw‐5bL33P/K319E recognized NSmC118Y or NSmT120N only when the R927A mutation was introduced into the LRR domain of Sw‐5bL33P/K319E.

Because the R927 residue in the LRR domain is critically important, we also introduced separate R927Q and R927L mutations into the LRR domain of Sw‐5bL33P/K319E. Sw‐5bL33P/K319E/R927Q (or YFP‐Sw‐5bL33P/K319E/R927Q) and Sw‐5bL33P/K319E/R927L (or YFP‐Sw‐5bL33P/K319E/R927L) constructs were generated. Sw‐5bL33P/K319E/R927Q (or YFP‐Sw‐5bL33P/K319E/R927Q) was able to induce HR in N. benthamiana leaves with either NSm, NSmC118Y or NSmT120N (Figures 3a, b and S2), whereas Sw‐5bL33P/K319E/R927L (or YFP‐Sw‐5bL33P/K319E/R927L) induced cell death only when co‐expressed with WT NSm, not with mutant NSmC118Y or NSmT120N, in N. benthamiana (Figures 3a, b and S2). Sw‐5bL33P/K319E/R927Q did not cause auto‐active cell death in the absence of NSm. These results suggested that an R927 to Q mutation in the LRR domain of Sw‐5bL33P/K319E/R927Q also conferred the ability to trigger HR in the presence of NSmC118Y or NSmT120N.

Sw‐5bL33P/K319E/R927A and Sw‐5bL33P/K319E/R927Q induced a defence response against the accumulation of TSWVC118Y and TSWVT120N eGFP replicon reporters transiently expressed in N. benthamiana leaves

Next, we examined whether Sw‐5bL33P/K319E/R927A and Sw‐5bL33P/K319E/R927Q were able to induce a defence response upon recognition of TSWVC118Y and TSWVT120N RB mutants. Recently, we established a reverse genetics system for TSWV (Feng et al., 2020), which represents the first reverse genetics system for a plant‐segmented negative‐stranded RNA virus and allows the generation of mutant viruses to study the role of viral gene functions in disease pathology, as well as the molecular mechanisms underlying virus–plant interactions. In the present study, we used this reverse genetics system to examine the activities of Sw‐5bL33P/K319E/R927A and Sw‐5bL33P/K319E/R927Q constructs against the TSWVC118Y and TSWVT120N RB mutants. We introduced the C118Y and T120N mutations into NSm of M(–)opt, the optimized full‐length infectious clone of the TSWV M segment, and generated the RB mutant constructs M(‐)opt C118Y and M(‐)opt T120N, respectively (Figure 4a). The WT TSWV and RB mutant eGFP replicon reporter infectious clones L(+)opt + M(–)opt + SR(+)eGFP, L(+)opt + M(–)opt C118Y + SR(+)eGFP and L(+)opt + M(–)opt T120N + SR(+)eGFP, respectively, were agro‐infiltrated into N. benthamiana leaves in combination with three viral suppressor of RNA silencing (VSRs), P19‐Hc‐Pro‐γb. The results showed that the TSWV, TSWVC118Y and TSWVT120N replicons carrying eGFP reporters were rescued from these infectious clones and expressed eGFP fluorescence in agro‐infiltrated leaves at 3 days post‐infiltration (dpi). Significantly more eGFP fluorescence accumulated at 5 dpi, and those fluoresced cells were clustered together (Figure 4b), which suggested that the TSWV, TSWVC118Y and TSWVT120N replicons carrying eGFP reporters had moved among cells. The eGFP accumulation levels of the rTSWVC118Y and rTSWVT120N replicons were similar to the level of WT TSWV replicons at 3 dpi and 5 dpi (Figure 4b, c). The eGFP replicon reporter infectious clones of WT TSWV, C118Y or T120N mutant were also co‐expressed with the WT Sw‐5b in N. benthamiana leaves by agro‐infiltration. A few single cells were detected with eGFP fluorescence from leaves co‐expressing Sw‐5b with eGFP replicon reporter infectious clones of WT TSWV at 3 dpi, but this fluorescence disappeared at 5 dpi. However, significantly more cells and stronger eGFP fluorescence were detected in leaves co‐expressing Sw‐5b with eGFP replicon reporter infectious clones of C118Y or T120N mutant in N. benthamiana leaves at 3 dpi than in leaves co‐expressing WT TSWV replicon reporter infectious clones with Sw‐5b (Figure 4b). eGFP fluorescence and protein levels were increasingly accumulated in these leaves at 5 dpi (Figure 4b, c). These results suggested that Sw‐5b induces a defence response against WT TSWV, leading to reduced GFP expression; however, the TSWVC118Y and TSWVT120N were able to break Sw‐5b‐mediated resistance.

Figure 4.

Figure 4

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Analysis of Sw‐5bL33P/K319E/R927A and Sw‐5bL33P/K319E/R927Q induced resistance against mini‐replicons of TSWV, TSWVC118Y and TSWVT120N 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‐5bL33P/K319E/R927A and Sw‐5bL33P/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.

Next, Sw‐5bL33P/K319E/R927A and Sw‐5bL33P/K319E/R927Q constructs were co‐expressed with the eGFP replicon reporter infectious clones of WT TSWV, C118Y or T120N mutant in N. benthamiana leaves by agro‐infiltration. The results showed minimal eGFP in leaves co‐expressing Sw‐5bL33P/K319E/R927A or Sw‐5bL33P/K319E/R927Q with clones of WT, C118Y or T120N mutant at 3 and 5 dpi (Figure 4b, c), which suggested that Sw‐5bL33P/K319E/R927A and Sw‐5bL33P/K319E/R927Q induced a strong defence response against the accumulation of TSWVC118Y and TSWVT120N in N. benthamiana leaves. The results also suggested that Sw‐5bL33P/K319E/R927A and Sw‐5bL33P/K319E/R927Q maintained the ability to induce resistance against WT TSWV in N. benthamiana leaves.

Transgenic N. benthamiana plants carrying Sw‐5bL33P/K319E/R927A and Sw‐5bL33P/K319E/R927Q provided systemic resistance against TSWVC118Y and TSWVT120N infection

To investigate whether Sw‐5bL33P/K319E/R927A‐ or Sw‐5bL33P/K319E/R927Q‐mediated resistance also prevents the WT or RB TSWV from moving systemically in plants, we generated transgenic N. benthamiana plants stably transformed with Sw‐5bL33P/K319E/R927A and Sw‐5bL33P/K319E/R927Q constructs. Transgenic N. benthamiana plants carrying empty vector (EV) and WT Sw‐5b (Chen et al., 2016) were used as controls. We utilized the full‐length TSWV (Feng et al., 2020) and RB mutant infectious clones L(+)opt + M(–)opt + S(+), L(+)opt + M(–)opt C118Y + S(+) and L(+)opt + M(–)opt T120N + S(+) for a systemic infection/resistance assay. Agrobacterium carrying the TSWV full‐length infectious clones was agro‐infiltrated into leaves of EV control N. benthamiana plants together with three viral suppressors of RNAi (VSRs; P19, Hc‐Pro and γb). Typical TSWV symptoms including leaf curling and stunting were observed in the EV control N. benthamiana systemic leaves of the rescued (r) TSWV, rTSWVC118Y and rTSWVT120N mutants at 10‐15 dpi (Figures 5a and S3). Western blotting analysis confirmed the accumulation of viral N proteins from the WT, C118Y and T120N mutants in systemic leaves of the EV control N. benthamiana plants (Figure 5b).

Figure 5.

Figure 5

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Analysis of Sw‐5bL33P/K319E/R927A and Sw‐5bL33P/K319E/R927Q induced resistance against TSWVC118Y and TSWVT120N 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‐5bL33P/K319E/R927A and Sw‐5bL33P/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.

When Sw‐5b‐transgenic N. benthamiana plants were agro‐inoculated with the full‐length infectious clones of WT TSWV, no rTSWV systemic infection was detected at 7‐21 dpi (Figure 5a, b), which suggested that Sw‐5b conferred resistance to WT rTSWV. As expected, when Sw‐5b transgenic N. benthamiana plants were agro‐inoculated with the infectious clones of C118Y and T120N mutants, respectively, both TSWVC118Y and TSWVT120N were rescued from these infectious clones, and viral symptoms including leaf curling and wilting were found in systemic leaves of the Sw‐5b transgenic plants (Figures 5a and S3). The presence of both rTSWVC118Y and rTSWVT120N was confirmed in systemic leaves of transgenic N. benthamiana plants carrying Sw‐5b, whereas no WT rTSWV was detected in these plants (Figure 5b). These results suggested that the rTSWVC118Y and rTSWVT120N mutants broke Sw‐5b‐mediated resistance and caused the systemic infection of Sw‐5b transgenic plants.

Transgenic N. benthamiana plants carrying Sw‐5bL33P/K319E/R927A and Sw‐5bL33P/K319E/R927Q constructs were then agro‐inoculated with the full‐length infectious clones of WT TSWV, C118Y and T120N mutant, respectively. No viral symptoms were observed in systemic leaves in any treatments at 10–21 dpi (Figures 5a and S3). Western blotting results confirmed that no viral N protein from rTSWV, rTSWVC118Y or rTSWVT120N had accumulated in systemic leaves of transgenic N. benthamiana plants carrying Sw‐5bL33P/K319E/R927A and Sw‐5bL33P/K319E/R927Q (Figure 5b). Together, these results suggested that Sw‐5bL33P/K319E/R927A and Sw‐5bL33P/K319E/R927Q transgenic N. benthamiana plants provide resistance to both WT TSWV and to RB TSWVC118Y and TSWVT120N mutants.

Sw‐5bL33P/K319E/R927A and Sw‐5bL33P/K319E/R927Q confer resistance to other American‐type tospoviruses

WT Sw‐5b confers resistance to all American tospoviruses, but not to European or Asian tospoviruses (Turina et al., 2016; Zhu et al., 2017). To determine whether Sw‐5bL33P/K319E/R927A and Sw‐5L33P/K319E/R927Q confer resistance to other members of Orthotospovirus, we examined their resistance to Tomato zonate spot virus (TZSV), a representative European/Asian‐type tospovirus, and to Impatiens necrotic spot virus (INSV), another representative American‐type tospovirus. TZSV NSm and INSV NSm were co‐expressed with Sw‐5b, Sw‐5bL33P/K319E/R927A and Sw‐5L33P/K319E/R927Q, respectively, in N. benthamiana leaves. Sw‐5bL33P/K319E/R927A and Sw‐5L33P/K319E/R927Q strongly induced cell death in the presence of INSV NSm‐FLAG. However, no cell death was induced when Sw‐5bL33P/K319E/R927A and Sw‐5L33P/K319E/R927Q were co‐expressed with TZSV NSm‐FLAG (Figure 6a). Western blotting results confirmed that the absence of HR was not caused by the protein levels of the Sw‐5b mutants or TZSV NSm‐FLAG in infiltrated tobacco leaves (Figure 6a).

Figure 6.

Figure 6

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Analysis of Sw‐5bL33P/K319E/R927A and Sw‐5bL33P/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‐5bL33P/K319E/R927A and (YFP)‐Sw‐5bL33P/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‐5bL33P/K319E/R927A and Sw‐5bL33P/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.

To further characterize Sw‐5bL33P/K319E/R927A‐ and Sw‐5L33P/K319E/R927Q‐mediated resistance to other members of Orthotospovirus, transgenic N. benthamiana plants carrying EV, Sw‐5b, Sw‐5bL33P/K319E/R927A and Sw‐5bL33P/K319E/R927Q constructs were inoculated with TZSV and INSV, respectively. TZSV symptoms including leaf curl and stunting were observed in the systemic leaves of all types of transgenic plants at 10‐21 dpi, which suggested that Sw‐5bL33P/K319E/R927A and Sw‐5L33P/K319E/R927Q were ineffective against TZSV. INSV also caused severe symptoms in EV control plants, but not in systemic leaves of the Sw‐5b, Sw‐5bL33P/K319E/R927A and Sw‐5L33P/K319E/R927Q transgenic N. benthamiana plants (Figure 6b). Western blotting confirmed the presence of TZSV, but the absence of INSV, in systemic leaves of Sw‐5b, Sw‐5bL33P/K319E/R927A and Sw‐5L33P/K319E/R927Q transgenic N. benthamiana plants (Figure 6c). These results suggested that the selected Sw‐5bL33P/K319E/R927A and Sw‐5L33P/K319E/R927Q mutants also conferred resistance to INSV, but not to TZSV.

Discussion

Tospoviruses cause global annual losses of approximately 1 billion USD, posing a serious threat to global food security. Sw‐5b is the only resistance gene that is effective and widely used to control tospoviruses in tomatoes; however, due to the emergence of RB TSWV, this resistance gene is no longer effective. In the present study, based on our knowledge of the two‐step recognition mechanism of Sw‐5b NLR, we performed artificial evolution and adopted a stepwise strategy to select two mutants, Sw‐5bL33P/K319E/R927A and Sw‐5bL33P/K319E/R927Q, that were demonstrated to extend the spectrum against TSWV RB isolates. We found that Sw‐5bL33P/K319E/R927A and Sw‐5bL33P/K319E/R927Q induced HR in the presence of NSmC118Y and NSmT120N from RB isolates (Figures 3). Sw‐5bL33P/K319E/R927A and Sw‐5bL33P/K319E/R927Q induced a marked defence response against the local viral accumulation of TSWVC118Y and TSWVT120N mini‐replicons, indicated by eGFP fluorescence (Figure 4). Transgenic N. benthamiana plants stably expressing the selected mutants also showed that Sw‐5bL33P/K319E/R927A and Sw‐5bL33P/K319E/R927Q conferred systemic resistance to TSWVC118Y and TSWVT120N infection (Figure 5). Sw‐5bL33P/K319E/R927A and Sw‐5bL33P/K319E/R927Q were also effective against other American‐type tospoviruses (Figure 6). The Sw‐5b mutants selected in this study will provide new gene resources for tomato resistance breeding to control RB TSWV and other American‐type tospoviruses.

Based on the known mechanisms of NLR immune receptors in the recognition of pathogen‐encoded effectors, NLR proteins have been successfully engineered and/or artificially evolved to expand the spectrum of resistance against both new and distantly related pathogens (Eugenia Segretin et al., 2014; Farnham and Baulcombe, 2006; Harris et al., 2013; Kim et al., 2016). Sw‐5b contains an extended N‐terminal SD domain and the CC, NB‐ARC and LRR domains. Our initial screening of the mutant library involving the C‐terminal LRR domain or N‐terminal SD domain (approximately 1500 clones per domain) did not identify any Sw‐5b mutants that gained the cell death response to NSmC118Y and NSmT120N from RB isolates. Our recent studies showed that both the N‐terminal SD domain and the C‐terminal LRR domain of Sw‐5b are involved in recognition of the viral elicitor NSm (Li et al., 2019). Therefore, random mutagenesis involving the LRR domain to select a gain‐of‐function mutant for other traditional plant NLRs cannot be simply applied to genes like Sw‐5b. The R927A mutation in NB‐ARC‐LRR induced auto‐activity, whereas SD‐CC suppressed the auto‐activity of NB‐ARC‐LRRR927A. Therefore, we adopted a stepwise strategy, by first introducing an R927A mutation in the LRR domain, followed by random mutagenesis involving the Sw‐5b SD domain. The selected Sw‐5bL33P/K319E/R927A construct conferred HR induction in the presence of NSmC118Y and NSmT120N. Single L33P or K319E mutation in Sw‐5bR927A did not confer HR function to either NSmC118Y or NSmT120N. Mutations of both L33P and K319E in the SD domain were required to gain HR function. The generation of a gain‐of‐function mutant through simultaneous mutation of both amino acids explains why the screening of more than 2,000 colonies in the random mutagenesis library led to the selection of very few Sw‐5bR927A mutants that could expand the response to NSmC118Y and NSmT120N. The second round of artificial evolution on Sw‐5bL33P/K319E through random mutagenesis involving the LRR domain did not obtain any gain‐of‐function mutant. This may be because the random mutagenesis library is not large enough to screen a functional mutant in response to NSmC118Y or NSmT120N. Our previous studies identified an M8 polymorphic site (amino acids 136–147) in the SD domain that is critical for the recognition of NSm (Li et al., 2019). L33P and K319E mutations did not occur at the M8 polymorphic site. We speculate that L33P and K319E mutations may have caused a slight conformational change in the SD domain, which enabled it to recognize NSmC118Y and NSmT120N mutants; this conformation would also have retained the ability to recognize WT NSm. Sw‐5bL33P/K319E/R927A presumably still function through a two‐step recognition mechanism. In the first step, SDL33P/K319E recognizes NSmC118Y or NSmT120N, and the activation of SDL33P/K319E relieves the repression of SD‐CC on NB‐ARC‐LRRR927A. In the second step, NB‐ARC‐LRRR927A either automatically activates or interacts with NSmC118Y and NSmT120N to achieve activation.

We also found that double mutations of L33P and K319E in the SD domain and the R927A mutation in the LRR domain were required for the selection of Sw‐5b mutants with responses to NSmC118Y and NSmT120N. The R927A mutation in the Sw‐5b NB‐ARC‐LRR causes constitutive cell death (Chen et al., 2016). The D642E mutations in the NB domain of Sw‐5b NB‐ARC‐LRR also cause auto‐activity (Chen et al., 2016); however, Sw‐5bL33P/K319E/D642E did not trigger HR in the presence of NSmC118Y and NSmT120N. This finding suggested that in the second recognition step, constitutive activity involving NB‐ARC‐LRR is not the main reason for Sw‐5bL33P/K319E to gain function in response to NSmC118Y and NSmT120N. Our recent study showed that the WT NB‐ARC‐LRR region did not induce HR to NSmC118Y and NSmT120N in N. benthamiana leaves (Zhu et al., 2017). However, an NB‐ARC‐LRRR927A mutant induced stronger HR in the presence of the non‐elicitors NSmC118Y and NSmT120N. This finding suggested that an R927A residue mutation in the LRR domain likely directly involved in interaction with the C118Y or T120N residue on NSm. In our previous study, we found that Sw‐5b recognized a 21‐amino acid (residues 115–135) peptide region in viral NSm. Polymorphic sites 3–6 in the LRR domain of Sw‐5b were critical for the recognition of this peptide region (Zhu et al., 2017). The modelled three‐dimensional structure of Sw‐5b NB‐ARC‐LRR shows that R927 is structurally located next to polymorphic sites 3–6. We previously proposed that polymorphic sites 3–6 in the LRR domain are NSm binding sites and that the R927 residue is an activation site. Binding of the NSm21 peptide region to polymorphic sites 3–6 disturbs residue R927, leading to receptor activation (Zhu et al., 2017). Because NB‐ARC‐LRRR927A induced stronger HR when co‐expressed with C118Y and T120N mutants in either the NSm21 peptide region or full‐length NSm, we propose that C118Y or T120N in the NSm21 peptide region and/or full‐length NSm may interact directly with the R927A residue of Sw‐5b in the LRR domain, which would disrupt the intramolecular interaction between NB‐ARC and the LRR domain, leading to Sw‐5b NB‐ARC‐LRR switching from an auto‐inhibited state to an activated state. Without R927A mutation in the LRR domain, Sw‐5bL33P/K319E was unable to switch to an activated state in response to NSmC118Y and NSmT120N. We also found that an R927Q mutation in the LRR domain of Sw‐5bL33P/K319E was functional in response to RB isolates, further supporting the conclusion that mutation of the R927 residue is critical for Sw‐5bL33P/K319E to gain HR function in response to NSmC118Y and NSmT120N.

Our recent study reported that Sw‐5b SD‐CC enhanced the sensitivity of NB‐ARC‐LRR in the detection of NSm (Li et al., 2019). At low levels of NSm, NB‐ARC‐LRR was unable to induce HR. However, the addition of SD‐CC allowed NB‐ARC‐LRR to trigger HR at low levels of NSm. In the presence of SD‐CC, NB‐ARC‐LRR interacted more strongly with NSm, which suggested that the SD domain can enhance NB‐AR‐LRR in NSm recognition. In the present study, double mutation of L33P and K319E in the SD domain functioned only in concert with R927A mutation in the LRR domain, but not with D642E in the NB domain; these findings suggested that the SDL33P/K319E domain may also interact with the LRRR927A domain, which help NB‐ARC‐LRRR927A to interact with NSmC118Y and NSmT120N.

Recent cryo‐electron microscopy has enabled resolution of the three‐dimensional structure of a CNL‐type NLR (ZAR1) and two TNL‐type NLRs (RPP1 and ROQ1) (Wang et al., 2019a; Wang et al., 2019b). The inactive ZAR1 binds to ADP, while the activated ZAR1 assembles into a resistosome that binds to ATP. The ZAR1 resistosome assembled into a pentameric structure and mediates plant defence activation (Wang et al., 2019a; Wang et al., 2019b). RPP1 and ROQ1 assemble into a tetrameric resistosome, and the TIR domain of the resistosome forms a holoenzyme for NAD+ (Ma et al., 2020; Martin et al., 2020). The dissected three‐dimensional structures of the plant resistosome improve our understanding of the activation mechanisms of plant NLR proteins and will help to design the new resistance gene. Recent advances in CRISPR/Cas9 technologies have revolutionized genomic engineering and plant breeding projects (Zhu et al., 2020). Because Sw‐5bL33P/K319E/R927A and Sw‐5bL33P/K319E/R927Q are effective against TSWV RB isolates, future studies will likely involve site‐directed editing of the triple mutation into the Sw‐5b gene within the genome sequences of susceptible tomato cultivars to facilitate tomato resistance breeding for the control of RB TSWV and other tospoviruses.

Materials and methods

Plasmid construction

The plasmids L(+)opt, M(–)opt, S(+), SR(+)eGFP and P19‐Hc‐Pro‐γb were described previously (Feng et al., 2020). To generate M(–)opt C118Y and M(–)opt T120N, NSmC118Y and NSmT120N were amplified from p2300S‐NSm using Phanta Super‐Fidelity DNA Polymerase (Vazyme Biotech, Nanjing, China) and used to replace the NSm gene in pCB301‐2×35S‐HH‐M(–)opt‐RZ‐NOS (M(–)opt) using the ClonExpress II One Step Cloning Kit (Vazyme Biotech). The Sw‐5bL33P, Sw‐5bK319E, Sw‐5bL33P/K319E, Sw‐5bL33P/R927A, Sw‐5bK319E/R927A and Sw‐5bL33P/K319E/R927A mutants were generated using site‐directed mutagenesis overlap PCR, as described previously (Feng et al., 2016). INSV NSm and TZSV NSm were amplified from p2300S‐INSV NSm and p2300S‐TZSV NSm, respectively (Zhu et al., 2017), and then cloned into p1307‐3×FLAG to generate TZSV NSm‐3×FLAG and INSV NSm‐3×FLAG. All constructs were confirmed by DNA sequencing. The primers used in this study are listed in Table S1.

Construction and screening of the Sw‐5b random mutagenesis library

Sw‐5b mutants were generated by performing Mn2+ and dITP‐mediated two‐round PCR amplification (Xu et al., 1999). In the first round of PCR, DNA fragments were amplified in 50 μL PCR mixtures containing 40 μm Mn2+, 1 mm dNTP, 2 mm MgCl2, 100 μg DNA template and 5 U Taq DNA polymerase (Sangon Biotech, Shanghai, China). The PCR cycle was 94  for 3 min; 20 cycles of 94  for 1 min, 51  for 1 min and 72  for 1 min 15 s; followed by extension for 10 min at 72 . In the second round of PCR, DNA mutagenesis was generated in a 50 μL reaction mixture containing 40 μm dITP, 2 μL first‐round PCR product, 1 mm dNTP, 2 mm MgCl2 and 5 U Taq DNA polymerase. Second‐round PCR was performed for 33 cycles with parameters identical to those of first‐round PCR. The amplified products were purified from the second PCR and recombined into the linearized binary vector carrying Sw‐5b using the ClonExpress II One Step Cloning Kit. The reaction mixtures were directly transformed into Agrobacterium tumefaciens strain GV3101 using electroporation (Ma et al., 2017). Screening of the Sw‐5b random mutagenesis mutant library was conducted through Agrobacterium‐mediated transient expression in N. benthamiana leaves in the presence of NSmC118Y or NSmT120N.

Agro‐infiltration and plant growth

Constructs used in this study were individually transformed into A. tumefaciens strain GV3101 through electroporation. Six‐week‐old WT, Sw‐5b and Sw‐5b mutant transgenic N. benthamiana plants were used for all Agrobacterium‐mediated infiltration analyses and infection assays, as previously described (Ma et al., 2015; Wang et al., 2018). Agrobacterium cultures were pelleted and resuspended in induction medium (10 mm MES pH 5.6, 10 mm MgCl2 and 150 mm acetosyringone). After three hours of incubation at room temperature, the culture was infiltrated into the abaxial side of an N. benthamiana leaf using a needleless syringe. Agro‐infiltrated N. benthamiana plants were grown in a growth chamber (Model GXZ500D; Jiangnan Motor Factory, Ningbo, Zhejiang, China) at day/night temperatures of 23°/25 °C, with a photoperiod of 16‐h light/8‐h darkness. The HR phenotype was monitored at 3‐7 dpi. All experiments were repeated at least three times.

Fluorescence microscopy

Nicotiana benthamiana leaves were harvested at 24–72 h post‐agro‐infiltration, and GFP fluorescence was examined by using an inverted fluorescence microscope (IX‐71‐F22 FL/DIC; Olympus, Tokyo, Japan). The GFP was excited at 488 nm, and fluorescence was collected using a GFP barrier filter (Olympus). Images were captured and analysed using Image‐Pro software (Olympus).

Transformation of N. benthamiana plants

A standard leaf disc transformation protocol was used to generate transgenic N. benthamiana as previously described (Chen et al., 2016), with slight modifications. Leaf discs from N. benthamiana leaves were incubated with A. tumefaciens cultures containing Sw‐5bL33P/K319E/R927A or Sw‐5bL33P/K319E/R927Q with 150 mm acetosyringone for 5–10 min. The leaf discs were then placed on co‐cultivation medium for 2 days at 25  in the dark. After co‐cultivation, the leaf discs were transferred to shoot regeneration medium and incubated at 25 . When the shoots had grown to a length of 1–2 cm, they were cut from the callus and transferred to rooting medium, and incubated at 25 . When the roots reached a length of 3–5 cm, the plants were transferred into soil.

Infectious clones and virus inoculation

Agrobacterium‐mediated expression/inoculation of mini‐replicons or full‐length infectious clones of TSWV and derivatives was performed as described (Feng et al., 2020), with slight modifications. Cultures of A. tumefaciens strains carrying mini‐replicons or infectious full‐length clones were mixed at equal volumes and infiltrated into 4‐ to 6‐stage leaves of N. benthamiana, together with three VSRs (P19, Hc‐Pro and γb). The OD600 of L(+)opt was adjusted to 1.5, which substantially improved its virus rescue efficiency up to 85‐100%. Inoculation of TZSV and INSV was performed as previously described (Huang et al., 2020). The inoculated tobacco plants were grown in a growth chamber as described above.

Western blotting analysis

Total protein from 1.0 g agro‐infiltrated or systemically infected leaves of WT or transgenic N. benthamiana plants was extracted in 2 mL extraction buffer as previously described (Wang et al., 2011), with slight modifications. Protein samples were centrifuged at 12 000 g for 15 min; the supernatants were heated at 95 °C for 10 min and then subjected to 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis. The proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (GE Healthcare, UK) through electroblotting, blocked with 5% skim milk solution for 1 h and incubated with anti‐TSWV N (1:5000, produced in our laboratory), anti‐FLAG‐HRP (1:10 000, Sigma‐Aldrich), anti‐YFP (1:5000, produced in our laboratory), anti‐INSV N (1:5000, produced in our laboratory) or anti‐TZSV N (1:5000, produced in our laboratory) primary antibodies for 1.5 h at room temperature. Following incubation with HRP‐conjugated goat anti‐rabbit secondary antibody (1:10 000, Sigma‐Aldrich) for 1 h, the signal on the blots was developed using the ECL Substrate Kit (Thermo Scientific, Hudson, NH, USA) and visualized using the ChemiDoc Touch Imaging System (Bio‐Rad). Blots were also stained with Ponceau S to show sample loading.

Conflict of interests

The authors declare that they have no competing interest.

Author contributions

H. H., J. L. and X. T. planned and designed the research. H. H., S. H., H. W., Z. Y., F. F., J. D., T. W. and M. Z. performed the experiments. H. H., J. L. and X. T. analysed data and drafted the manuscript. All authors read and approved the final manuscript.

Supporting information

Figure S1 The HR phenotype of Sw‐5b NB‐ARC‐LRR (i), NB‐ARC‐LRRR927A (ii), full‐length Sw‐5b (iii), and full‐length Sw‐5bR927A (vi) co‐expressing with or without wild‐type (WT) NSm, NSmC118Y, or NSmT120N in Nicotiana benthamiana leaves. –, no HR; +++ and +++++ indicate HR strength.

Figure S2 HR indexes of Sw‐5bL33P/R927A, Sw‐5bK319E/R927A, and Sw‐5bL33P/K319E/R927A, or Sw‐5bL33P, Sw‐5bK319E, and Sw‐5bL33P/K319E, or Sw‐5bL33P/K319E/D642E, Sw‐5bL33P/K319E/R927Q and Sw‐5bL33P/K319E/R927L, corresponding to the experiment shown in Figure 3a.

Figure S3 Enlarged images showing the viral infection and symptoms in systemic leaves of various agro‐infiltrated plants from Figure 5a.

PBI-19-2164-s001.pdf (1.4MB, pdf)

Table S1 Primers used in the study.

PBI-19-2164-s002.docx (26.9KB, docx)

Acknowledgements

This work was supported by the National Natural Science Foundation of China (31630062, 31925032 and 31870143 to XT, 31801705 to JL), the Fundamental Research Funds for the Central Universities (JCQY202104 and KYXK202012), Youth Science and Technology Innovation Program to XT and the Natural Science Foundation of Jiangsu Province (Grant No. BK20180532) to JL.

Huang, H. , Huang, S. , Li, J. , Wang, H. , Zhao, Y. , Feng, M. , Dai, J. , Wang, T. , Zhu, M. and Tao, X. (2021) Stepwise artificial evolution of an Sw‐5b immune receptor extends its resistance spectrum against resistance‐breaking isolates of Tomato spotted wilt virus . Plant Biotechnol J, 10.1111/pbi.13641

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1 The HR phenotype of Sw‐5b NB‐ARC‐LRR (i), NB‐ARC‐LRRR927A (ii), full‐length Sw‐5b (iii), and full‐length Sw‐5bR927A (vi) co‐expressing with or without wild‐type (WT) NSm, NSmC118Y, or NSmT120N in Nicotiana benthamiana leaves. –, no HR; +++ and +++++ indicate HR strength.

Figure S2 HR indexes of Sw‐5bL33P/R927A, Sw‐5bK319E/R927A, and Sw‐5bL33P/K319E/R927A, or Sw‐5bL33P, Sw‐5bK319E, and Sw‐5bL33P/K319E, or Sw‐5bL33P/K319E/D642E, Sw‐5bL33P/K319E/R927Q and Sw‐5bL33P/K319E/R927L, corresponding to the experiment shown in Figure 3a.

Figure S3 Enlarged images showing the viral infection and symptoms in systemic leaves of various agro‐infiltrated plants from Figure 5a.

PBI-19-2164-s001.pdf (1.4MB, pdf)

Table S1 Primers used in the study.

PBI-19-2164-s002.docx (26.9KB, docx)

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