New insights into CNL‐mediated immunity through recognition of Ralstonia solanacearum RipP1 by NbZAR1

Nicotiana benthamiana requires the coiled-coil nucleotide-binding leucine-rich repeat receptor protein NbZAR1 to recognize the type III effector RipP1 from Ralstonia solanacearum. Moreover, RipP1-induced cell death and immunity relies on EDS1 and NRG1, two core components of the Toll-interleukin 1-like receptor nucleotide-binding leucine-rich repeat receptor signaling pathway Some plants use intracellular immune receptors, mainly from the nucleotide-binding leucine-rich repeat receptors (NLRs) super-family, to detect pathogen effectors and restrict pathogen infection. NLRs are categorized as TIR-NLR (TNL) or CC-NLR (CNL) based on their N-terminal Toll-interleukin 1-like receptor (TIR) or coiled-coil (CC) domain. Additionally, there are helper (h) NLRs that aid in translating signals from pathogen-sensing NLRs into effector-triggered immunity responses. NLRs can directly detect effectors or indirectly monitor their host targets, leading to a robust immune response and programmed cell death, known as the hypersensitive response (HR). Certain plants have developed NLRs that recognize Ralstonia solanacearum effectors. Notably, the deletion of two type Ⅲ effectors, RipP1 and RipAA, markedly attenuated R. solanacearum GMI1000-induced HR in Nicotiana benthamiana (Poueymiro et al., 2009). This demonstrates the crucial role of these two effectors in plant-R. solanacearum recognition. However, the specific NLRs responsible for recognizing RipP1 and RipAA, along with their action mechanisms, remain unknown. To identify NLRs recognizing R. solanacearum RipP1 and RipAA, we screened NbNLRs in N. benthamiana using the virus-induced gene silencing (VIGS) method. Silencing NbZAR1 prevented RipP1-induced cell death but not that by other avirulent effectors (Figure 1A–C), indicating its potential role in RipP1 perception. The co-expression of NbZAR1 enhanced RipP1-induced cell death in wild-type (WT) plants (Figure S1). A NbZAR1 deficient mutant Nbzar1 (Figure S2) confirmed this observation, with restored cell death upon NbZAR1 complementation (Figure 1D, E). Homologs of NbZAR1 could not restore RipP1-induced cell death in the Nbzar1 mutant (Figure 1F, G), indicating the specific recognition of RipP1 by NbZAR1. These results highlight the role of NbZAR1 as a key NLR in RipP1 recognition. The Ralstonia solanacearum effector RipP1 is recognized by NbZAR1 and the perception is dependent on EDS1 and NRG1 (A) RipP1 did not induce cell death in NbZAR1-silenced Nicotiana benthamiana. Photos were taken at 48 h post-infiltration (hpi). Three known avirulence effectors, RipE1, RipAA and RipAW, were used as controls. (B) Silencing efficiency of NbZAR1 by RT-qPCR. Values represent means ± SEs (n = 3, ***P < 0.001, Student's t-test). (C) Detection of RipP1 protein expression in (A). Coomassie brilliant blue (CBB) staining shows equal loading. (D) RipP1-induced HR was abolished in Nbzar1 mutant and restored by NbZAR1 complementation. Photos were taken at 48 hpi. (E) Western blot showing successful protein expression of RipP1 and NbZAR1 in (D). CBB staining shows equal loading. (F) NbZAR1 homologs could not restore RipP1-induced cell death in Nbzar1. Photos were taken at 48 hpi. AtZAR1, CaZAR1, StZAR1, and SlZAR1 are NbZAR1 homologs in Arabidopsis thaliana, Capsicum annuum, Solanum tuberosum, and Solanum lycopersicum, respectively. (G) Western blot showing successful protein accumulation in (F). CBB staining shows equal loading. (H) Bacterial population in leaves of wild-type N. benthamiana (WT) and Nbzar1 mutant at 5 d post-inoculation (dpi) with GMI1000 or its RipP1 deletion mutant (ΔripP1) (OD600) = 0.00001. Different lowercase letters indicate significant differences (P < 0.01, Duncan's tests). (I) NbZAR1 and RipP1 are essential for N. benthamiana-GMI1000 recognition. Photos were taken at 48 hpi. (J) Mutations at the putative acetyltransferase active site of RipP1 resulted in its failure to induce cell death in wild type N. benthamiana. RipP1C229A, the conserved amino acid cysteine in putative acetyltransferase domain of RipP1 was mutated to alanine. Photos were taken at 48 hpi. (K) Cell death in (J) evaluated by the degree of ion leakage. Values represent means ± SEs (n = 3). (L) Time course curve of ROS production in leaves of Nbzar1 mutants transiently expressing RipP1 and RipP1C229A in response to 100 nmol/L flg22. RLU, relative luminescence units. Data were collected using a GLOMAX96 microplate luminometer and shown as means ± SEs (n = 14). (M) Bacterial growth in Nbzar1 plants expressing RipP1, RipP1C229A. LTI6b, a known small intrinsic protein unrelated to plant immunity, was expressed as a control. Bacterial population was determined at 5 dpi with GMI1000 (OD600 = 0.00001) or 3 dpi with DC3000 ΔhopQ1-1 (OD600 = 0.001). DC3000 ΔhopQ1-1, a mutant of Pseudomonas syringae DC3000, is virulent to N. benthamiana. Values are means ± SEs (n = 6). Different lowercase letters indicate significant differences (P < 0.01, Duncan's tests). (N) RipP1 failed to induce cell death in NbJIM2-silenced plant (TRV:NbJIM2) and regained this ability when co-expressed with NbJIM2syn (synthesized NbJIM2 with modified codons). Photos were taken at 3 dpi. NbPBS1, a cytoplasmic receptor-like kinase unrelated to ZAR1-mediated signaling, was a negative control. (O) Expression of corresponding proteins in (N). CBB staining shows equal loading. (P) Protein associations detected by firefly luciferase complementation imaging assay. BIK1 + XLG2 served as a positive control, while BIK1 + CPR5 was a negative control. (Q) Co-immunoprecipitation assay showing interaction among RipP1, NbJIM2, and NbZAR1. Red triangles indicate bands of interest. (R) RipP1-triggered HR was compromised in Nbeds1 and Nbnrg1 mutants but not in Nbnrc2/3/4. RipAA was used as a control. Photos were taken at 48 hpi. (S) Western blot showing RipP1-GFP expression in (R). CBB staining shows equal loading. (T) GM1000-induced HR was attenuated in Nbeds1 and Nbnrg1 mutants. Photos were taken at 48 hpi. All experiments were repeated at least twice with similar results. The bacterial population of the RipP1 deletion mutant (ΔripP1, Figure S3) was larger than that of WT GMI1000 in WT plants but not in the Nbzar1 mutant (Figure 1H). Furthermore, NbZAR1 deficiency reduced plant resistance to WT GMI1000 but had no impact on ΔripP1 (Figure 1H). It was also demonstrated that RipP1 deletion, as well as NbZAR1 deficiency, hindered the recognition of GMI1000 by N. benthamiana (Figure 1I), confirming NbZAR1-RipP1 recognition. RipP1, containing a putative acetyltransferase domain similar to the acetyltransferase effector HopZ1 recognized by ZAR1 (Lewis et al., 2013), failed to induce cell death in N. benthamiana when the conserved amino acid was changed to alanine (RipP1C229A) (Figure 1J, K). Expression of RipP1 suppressed the flg22-induced reactive oxygen species (ROS) burst, whereas the mutant RipP1C229A lost the ability to inhibit the flg22-induced ROS burst (Figure 1L). Additionally, RipP1C229A failed to promote bacterial growth of GMI1000 and DC3000 ΔhopQ1-1 in the Nbzar1 mutant background (Figure 1M). These results underscore the indispensable role of the putative acetyltransferase domain for both the avirulence and virulence functions of RipP1. Given the necessity of pseudokinase NbJIM2, a member of the receptor-like cytoplasmic kinase (RLCK) subfamily XII, for NbZAR1's perception of Xanthomonas perforans XopJ4 (a putative acetyltransferase) and Pseudomonas syringae pv. actinidiae HopZ5 (Schultink et al., 2019; Zheng et al., 2022; Ahn et al., 2023), we speculated that NbJIM2 may play a role in RipP1 recognition. Indeed, RipP1 failed to induced cell death in NbJIM2-silenced plants, but this ability was regained upon complementation with NbJIM2syn (synthesized NbJIM2 with modified codons) (Figure 1N, O). In contrast, another RLCK, NbPBS1a (MK140809.1), could not restore RipP1-induced cell death in NbJIM2-silenced plants, indicating the specific requirement of NbJIM2 for RipP1 perception. RipP1 did not associate with NbZAR1 but indirectly associated with NbJIM2 (Figures 1P, Q, S5, and S6). Additionally, NbJIM2 interacted with NbZAR1 (Figure 1P, Q), indicating that NbJIM2 functions as a key regulator in NbZAR1 signaling. The increasing number of RLCK members guarded by NLRs suggests that RLCKs, especially RLCK XII members, are central hubs targeted by various pathogen effectors (Diplock et al., 2024). TNLs typically signal dependent of EDS1 (Enhanced Disease Susceptibility 1) and helper NLR NRG1 (N Requirement Gene 1) (Qi et al., 2018). Notably, RipP1-induced cell death was abolished in both Nbeds1 and Nbnrg1 mutants (Figure 1R, S), but this phenotype was rescued through genetic complementation (Figures S7, S8). Meanwhile, HR induced by ΔripP1 was attenuated in WT N. benthamiana, and HR triggered by WT GM1000 was similarly diminished in the Nbeds1 and Nbnrg1 mutants, but remained unaffected in the Nbnrc2/3/4 mutant (Figures 1T, S9). These findings indicate that EDS1 and NRG1 are essential for RipP1-triggered immune signaling. Similarly, the recognition of XopJ4 by NbZAR1 also requires EDS1 and NRG1 (Figure S10), suggesting a role for these proteins in ZAR1 recognition of diverse effectors. However, NbZAR1 encodes a CNL, in which signaling is usually independent of EDS1 and NRG1. Our results suggest the likely involvement of a TNL that coordinates with NbZAR1 for RipP1 recognition. Further investigation into the interplay between TNLs and CNLs in this context could provide valuable insights into the intricate mechanisms underlying plant immune responses. Notably, AtZAR1 recognition of HopZ1a does not necessitate EDS1 (Lewis et al., 2010; Macho et al., 2010), and we observed that AtZAR1 was unable to rescue RipP1-induced cell death in the Nbzar1 mutant (Figure 1F, G). Moreover, Ahn et al. (2023) demonstrated that the NbZAR1 signaling pathway is associated with the solanaceous-specific Ptr1 resistance gene, which is absent in Arabidopsis. Harant et al. (2022) showed that the mutation of the MHD motif in ZAR1 results in a distinct outcome between N. benthamiana and Arabidopsis. These results suggest functional differences between ZAR1 in N. benthamiana and Arabidopsis. In summary, our study established that the ancient and conserved CNL, ZAR1, is responsible for recognizing the R. solanacearum effector RipP1 in N. benthamiana. The participation of EDS1 and NRG1 in NbZAR1 signaling offers new insights into CNL-mediated immunity. We thank Professor Brian Staskawicz for providing the Nbeds1 and Nbnrg1 seeds, and Professor Sophien Kamoun for the Nbnrc2/3/4 seeds. This work was supported by grants from the National Natural Science Foundation of China (32372483, 32302296, 32272641) and the Fundamental Research Funds for the Central Universities (GK202201017). The authors declare no conflict of interest. Y. A., J. L., X. Z. and M. Z. conceived the study. J. L., X. Z. and B. F. conducted the experiments. Y. A., J. L. and M. Z. wrote the manuscript. All authors read and approved this manuscript. Additional Supporting Information may be found online in the supporting information tab for this article: http://onlinelibrary.wiley.com/doi/10.1111/jipb.13855/suppinfo Figure S1. Overexpression of NbZAR1 enhanced RipP1-induced hypersensitive response (HR) Figure S2. Generation of the Nbzar1 mutant using clustered regularly interspaced small palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene editing Figure S3. Generation of the ΔripP1 mutant Figure S4. Sequence alignment of RipP1, XopJ4, and HopZ1 Figure S5. Yeast transformants expressing JIM2 and RipP1 were assayed for their interaction Figure S6. RipP1 does not interact with JIM2 in vitro Figure S7. RipP1-induced hypersensitive response (HR) is restored by NbEDS1 (Nicotiana benthamiana Enhanced Disease Susceptibility 1) complementation in the Nbeds1 mutant Figure S8. RipP1-induced hypersensitive response (HR) is restored by NbNRG1 (Nicotiana benthamiana N Requirement Gene 1) complementation in the Nbnrg1 mutant Figure S9. Bacterial population of GMI1000 and its RipP1 deletion mutant ΔripP1 in wild-type Nicotiana benthamiana, Nbnrc2/3/4, Nbeds1, and Nbnrg1 leaves in Figure 1T Figure S10. XopJ4-triggered hypersensitive response (HR) is compromised in the Nbeds1 and Nbnrg1 mutants Table S1. Primers used in this study Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.