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Activation of TIR signalling boosts pattern-triggered immunity

Abstract

Plant immune responses are mainly activated by two types of receptor. Pattern recognition receptors localized on the plasma membrane perceive extracellular microbial features, and nucleotide-binding leucine-rich repeat receptors (NLRs) recognize intracellular effector proteins from pathogens1. NLRs possessing amino-terminal Toll/interleukin-1 receptor (TIR) domains activate defence responses via the NADase activity of the TIR domain2,3. Here we report that activation of TIR signalling has a key role in pattern-triggered immunity (PTI) mediated by pattern recognition receptors. TIR signalling mutants exhibit attenuated PTI responses and decreased resistance against pathogens. Consistently, PTI is compromised in plants with reduced NLR levels. Treatment with the PTI elicitor flg22 or nlp20 rapidly induces many genes encoding TIR-domain-containing proteins, which is likely to be responsible for activating TIR signalling during PTI. Overall, our study reveals that activation of TIR signalling is an important mechanism for boosting plant defence during PTI.

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Fig. 1: Overexpression of SNIPER1 leads to attenuated flg22- and nlp20-induced immunity.
Fig. 2: Contributions of TIR signalling components to flg22- and nlp20-induced immunity.
Fig. 3: PCRK1/2 and PBL19/20 are required for nlp20-induced immunity.

Data availability

All data are included within the article or the Supplementary Information. Statistical analyses are provided in Supplementary Table 5. The RNA-seq data used for analysing nlp20-induced gene expression were from the National Center for Biotechnology Information (GSE133053). Full versions of all gels and blots are provided in Supplementary Fig. 1Source data are provided with this paper.

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Acknowledgements

We thank J. Parker, J. Dangl, J.-M. Zhou, K. Yoshioka and H. Cui for sharing mutant seeds and pathogen strains; L. Tian for help with the high-performance liquid chromatography analysis and biochemical assays; and T. Nuernberger and J. Parker for insightful discussions. This study was financially supported by grants to X.L. and Yuelin Zhang from the Natural Sciences and Engineering Research Council (NSERC) Discovery programme of Canada, NSERC-CREATE-PRoTECT, and the Canadian Foundation for Innovation, a grant to Yanjun Zhang from the National Natural Science Foundation of China (31828008), scholarships to H.T., S.C., W.H. and Z.W. from the Chinese Scholarship Council, and scholarships to K.A. from the Alexander Graham Bell Canada Graduate Scholarship Doctoral Program, and the University of British Columbia Four-Year Fellowship programme.

Author information

Authors and Affiliations

Authors

Contributions

H.T. and S.C. together carried out the genetic analysis of the SNIPER1-overexpression lines, the TIR signalling mutants and the RLCK mutants. H.T. carried out the overexpression analysis of the TIR genes. Z.W. carried out the analyses on protein–protein interactions, quantification of expression levels of the three TIR genes, and test of the effect of SNIPER1 overexpression on the accumulation of PTI signalling components. K.A. generated eds1-24 and performed bioinformatic analyses. W.H. and Yanjun Zhang assisted with SA analysis. F.X. made the ZZ–TEV–FLAG-tagged EDS1 and PAD4 constructs. H.Y. and T.S. generated the combined RLCK mutants. Yuelin Zhang, H.T., Z.W., S.W. and X.L. wrote the manuscript with contributions from all authors.

Corresponding authors

Correspondence to Xin Li or Yuelin Zhang.

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Competing interests

The authors declare no competing interests.

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Peer review information Nature thanks Gitta Coaker and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data figures and tables

Extended Data Fig. 1 Overexpression of SNIPER1 leads to reduced expression of SARD1 and FMO1, lower SA accumulation, increased Pto DC3000 hrcC growth, but has no effect on MAPK activation and ROS production during PTI.

(a, b) Relative expression levels of SARD1 (a) and FMO1 (b) in WT and OX-SNIPER1 lines (L4 and L5) treated with 10 mM MgCl2 (mock) or Pto DC3000 hrcC for 12 h. Bars represent mean ± s.d. (n = 3 biologically independent samples). (c, d) Free SA (c) and SAG (d) levels in the indicated genotypes treated with 10 mM MgCl2 (mock) or Pto DC3000 hrcC (OD600 = 0.05) for 12 h. Bars represent mean ± s.d. (n = 3 biologically independent samples). (e) Growth of Pto DC3000 hrcC in the indicated genotypes. Bars represent mean ± s.d. (n = 4 plants). (f, g, i, j) flg22 and nlp20-induced MAPK activation in the indicated genotypes. Seedlings were treated with 0.1 μM flg22 (f, g) or 0.1 μM nlp20 (i, j). MAPK activation was analyzed by immunoblotting with the anti-pERK antibody. Equal loading is confirmed by Ponceau staining of Rubisco. The signals of phosphorylated MPK6 and MPK3 in samples treated with flg22 (g) or nlp20 (j) detected by western blots were normalized to the loading control. The intensity of MPK6 or MPK3 bands after elicitors treatment in WT plants was set as 1. Bars represent mean ± s.d. (n = 4 different experiments for (g), n = 3 different experiments for (j)). (h, k) ROS production in the indicated genotypes after treatment with 0.1 μM flg22 (h) or 1 μM nlp20 (k) measured as luminescence. The values show the peak of luminescence units through all the time points. Bars represent mean ± s.d. (n = 8 leaf disks from different plants). (l, m) SAG levels in the indicated genotypes treated with H2O, 1 µM nlp20 (l) or 1 µM flg22 (m). Samples were collected for SAG measurement 24 h after 1µM nlp20, or 9 h after 1 µM flg22 treatment. Bars represent mean ± s.d. (n = 3 biologically independent samples). All data were analyzed by one-way ANOVA with Tukey’s test. Exact P values are provided in Supplementary Table 5. The experiments were repeated at least three times with similar results.

Source data

Extended Data Fig. 2 Overexpression of SNIPER1 does not affect the accumulation of FLS2, BAK1, SOBIR1, RLP23, BIK1, PBL19 or PCRK2, as opposed to SNC1.

(a, b) FLS2 and BAK1 protein levels in WT and OX-SNIPER1 lines #4 and #5. Total proteins were extracted from four-week-old soil-grown Arabidopsis plants and analyzed by western blot using anti-FLS2 (a) or anti-BAK1 (b) antibodies. Equal loadings are shown by Ponceau S staining of a non-specific band. The relative bands intensity (n = 3 biologically independent samples) are shown below (normalized to loading control, the protein levels in Col-0 was arbitrarily set at 1). (c-h) Immunoblot analysis of protein levels of SOBIR1-3FLAG, RLP23-ZZ-TEV-FLAG, BIK1-3FLAG, PBL19-3FLAG, PCRK2-3FLAG and SNC1-3FLAG in N. benthamiana leaves co-expressed with HA-SNIPER1. GFP-HATurboID was used as a negative control. Equal loading is shown by Ponceau S staining of a non-specific band. Numbers underneath (n = 3 biologically independent samples) indicate the relative intensity of bands of SOBIR1-3FLAG (c), RLP23-ZZ-TEV-FLAG (d), BIK1-3FLAG (e), PBL19-3FLAG (f), PCRK2-3FLAG (g) and SNC1-3FLAG (h).

Extended Data Fig. 3 Analysis of interactions between RPP4NB/SOBIR1/RLP23/BIK1/PCRK2/PBL19 and SNIPER1H129Y by TurboID-based proximity labeling method.

3 × FLAG-tagged NB domain of RPP4 (RECOGNITION OF PERONOSPORA PARASITICA 4) (RPP4NB) or SOBIR1 (a), BIK1/RLP23 (b), and PCRK2/PBL19 (c) were transiently expressed in N. benthamiana together with SNIPER1H129Y-HATurboID or GFP-HATurboID. SNIPER1H129Y is a dominant-negative mutant of SNIPER1 that does not affect binding to its substrate but lost its E3 ligase activity, which is used to stabilize the protein interactions with the substrates. The 3 × FLAG-tagged RPP4NB was used as a positive control in the experiment. The 3 × FLAG-tagged proteins were immunoprecipitated with anti-FLAG beads and detected using an anti-FLAG antibody. The biotinylated proteins were detected using HRP-Streptavidin. The experiment was repeated twice with similar results.

Extended Data Fig. 4 Expression of SARD1/FMO1, SA accumulation, expression of FLS2/BAK1/BIK1/RLP23/SOBIR1 and growth of Pto DC3000 hrcC in TIR signaling mutants.

(a, b) Relative expression levels of SARD1 (a) and FMO1 (b) in WT, eds1-24, pad4-1, adr1 triple, sag101-1 and nrg1 triple mutant plants after treatment with 10 mM MgCl2 (mock) or Pto DC3000 hrcC (OD600 = 0.05) for 12 h. Bars represent mean ± s.d. (n = 3 biologically independent samples). (c, d) SA (c) and SAG (d) induction in TIR signaling mutants. Plants of the indicated genotypes were treated with Pto DC3000 hrcC (OD600 = 0.05). Samples were collected for SA and SAG measurements 12 h after inoculation of Pto DC3000 hrcC. Bars represent mean ± s.d. (n = 3 biologically independent samples). (e) Growth of Pto DC3000 hrcC in plants of the indicated genotypes. Bars represent mean ± s.d. (n = 4 plants). (f, g) Relative expression levels of SARD1 (f) and FMO1 (g) in the indicated genotypes upon flg22 treatment. Bars represent mean ± s.d. (n = 3 biologically independent samples). (h, i) Relative expression levels of SARD1 (h) and FMO1 (i) in the indicated genotypes upon nlp20 treatment. Bars represent mean ± s.d. (n = 3 biologically independent samples). (j) Expression levels of FLS2, BAK1 and BIK1 in WT and eds1-24 treated with H2O or flg22. Bars represent mean ± s.d. (n = 3 biologically independent samples). (k) Expression levels of RLP23, SOBIR1, BAK1 and BIK1 in WT and eds1-24 treated with H2O or nlp20. Bars represent mean ± s.d. (n = 3 biologically independent samples). (l, m) SAG induction in TIR signaling mutants. Plants of the indicated genotypes were treated with 1 µM flg22 (l) or 1 µM nlp20 (m). Samples were collected for SAG measurements 24 h after treatment with 1 µM nlp20, or 9 h after treatment with 1 µM flg22. Bars represent mean ± s.d. (n = 3 biologically independent samples). For gene expression analysis in (f-k), total RNA was isolated from Arabidopsis seedlings 4 h after spraying with 1 μM elicitor (flg22 or nlp20) or H2O. The expression of each gene in the mock or H2O-treated WT plants was set as 1. Data in (a-i and l-m) were analyzed by one-way ANOVA with Tukey’s test. Exact P values are provided in Supplementary Table 5. Data in (j, k) were analyzed using two-tailed Student’s t-test. Exact P values are provided in Supplementary Table 5. All experiments were repeated three times with similar results.

Source data

Extended Data Fig. 5 flg22 and nlp20-induced MAPK activation and ROS production in WT and TIR signaling mutants.

(a) flg22-induced MAPK activation in WT, eds1-24 and adr1 triple mutants. 12-day-old seedlings were treated with 0.1 μM flg22. MAPK activation was analyzed by immunoblotting with the anti-pERK antibody. Equal loading was confirmed by Ponceau staining of Rubisco. The experiment was repeated twice with similar results. (b) ROS production in the indicated genotypes after treatment with 0.1 μM flg22 measured as luminescence. The values show the peak of luminescence units through all the time points. Bars represent mean ± s.d. (n = 7 leaf disks from different plants). Data were analyzed by one-way ANOVA with Tukey’s test. Exact P values are provided in Supplementary Table 5. The experiment was repeated twice with similar results. (c, d) nlp20-induced MAPK activation in the indicated genotypes treated with 0.1 μM nlp20. MAPK activation was analyzed by immunoblotting with the anti-pERK antibody. Equal loading was confirmed by Ponceau staining of Rubisco. The phosphorylated MPK6 and MPK3 10 min after treatment with nlp20 were quantified in (d). Bars represent mean ± s.d. (n = 4 different experiments). The band intensity of MPK6 or MPK3 after elicitors treatment in WT plants was set as 1. Data were analyzed by one-way ANOVA with Tukey’s test. Exact P values are provided in Supplementary Table 5. (e) ROS production in the indicated genotypes after treatment with 1μM nlp20 measured as luminescence. The values show the peak of luminescence units through all the time points. Bars represent mean ± s.d. (n = 9 leaf disks from different plants). Data were analyzed by one-way ANOVA with Tukey’s test. Exact P values are provided in Supplementary Table 5. The experiment was repeated three times with similar results.

Source data

Extended Data Fig. 6 Growth of Hpa Noco2 on the distal leaves of TIR signaling mutants.

Three-week-old soil-grown plants of the indicated genotypes were pretreated with H2O or 1 μM nlp20 and sprayed with Hpa Noco2 spores (50,000 spores/ml) 24 h later. Disease ratings showing the relative growth of Hpa Noco2 are as described in the Methods. The experiment was repeated three times with similar results.

Source data

Extended Data Fig. 7 nlp20-induced defense responses in wild-type and ndr1-1 mutant plants.

(a) MAPK activation in WT and ndr1-1 plants treated with 0.1μM nlp20. MAPK activation was analyzed by immunoblotting using the anti-pERK antibody. Equal loading was confirmed by Ponceau staining of Rubisco. Quantification of the phosphorylated MPK6 and MPK3 was shown on the right. Bars represent mean ± s.d. (n = 4 different experiments). The band intensity of MPK6 or MPK3 after nlp20 treatment in WT plants was set as 1. Data were analyzed by two-tailed student’s t-test. Exact P values are provided in Supplementary Table 5. (b) ROS production in WT and ndr1-1 treated with 1μM nlp20 measured as luminescence. The values show the peak of luminescence units through all the time points. Bars represent mean ± s.d. (n = 9 leaf disks from different plants). Data were analyzed by two-tailed student’s t-test. Exact P values are provided in Supplementary Table 5. The experiment was repeated three times with similar results. (c) Levels of free SA in WT and ndr1-1 treated with H2O or 1 μM nlp20 for 24 h. Bars represent mean ± s.d. (n = 3 biologically independent samples). Data were analyzed by one-way ANOVA with Tukey’s test. Exact P values are provided in Supplementary Table 5. The experiment was repeated three times with similar results. (d) Growth of Hpa Noco2 on the local leaves of the indicated plants. Three-week-old soil-grown plants were pretreated with H2O or 1 μM nlp20 and sprayed with Hpa Noco2 spores (50,000 spores/ml) 24 h later. Disease ratings showing the relative growth of Hpa Noco2 are as described in the Methods. The experiment was repeated twice with similar results.

Source data

Extended Data Fig. 8 Induction of SA accumulation and the pSARD1::Luc reporter gene expression by overexpression of three TIR genes and up-regulation of the TIR genes by nlp20 and flg22 or in cngc20-4 and cpr22 mutants.

(a, b) Levels of free SA (a) and SAG (b) in N. benthamiana leaves after infiltration of Agrobacterium (OD600 = 0.4) carrying EV (empty vector), the TIR genes At4g11170, At3g04220 or At2g32140. Samples were collected 24h and 36 h post infiltration, before cell death was visible. Bars represent mean ± s.d. (n = 3 biologically independent samples). (c) Induction of the pSARD1::Luc reporter gene by overexpression of At4g11170, At3g04220 or At2g32140 in Arabidopsis protoplasts. Bars represent mean ± s.d. (n = 3 biologically independent samples) of the firefly luciferase activities in Arabidopsis protoplasts co-transformed with the indicated constructs. Empty vector (EV) control was set to 1. (d, e) Induction of the indicated TIR genes by nlp20 (d) or flg22 (e). 10-day-old plate-grown WT plants were transplanted to H2O 1 day before for recovery and then pretreated with H2O or 100 μM GdCl3 for 1 h. Samples were collected 1 h after treatment with 1 μM nlp20 or flg22. Bars represent mean ± s.d. (n = 3 biologically independent samples). (f, g) Induction of the indicated TIR genes by nlp20 (f) or flg22 (g) in WT and the rbohd mutant. Samples were collected 1 h after treatment with 0.1 μM nlp20 or flg22. Bars represent mean ± s.d. (n = 3 biologically independent samples). (h, i) Induction of the indicated TIR genes by nlp20 (h) or flg22 (i) in WT and eds1-24. 10-day-old WT and eds1-24 seedlings were transplanted to H2O 1 day before for recovery and then supplied with 1 μM nlp20 or flg22. Samples were collected 1 h after supplying with nlp20 or flg22. Bars represent mean ± s.d. (n = 3 biologically independent samples). (j, k) Expression levels of the indicated TIR genes in cngc20-4 (j) and cpr22 (k) mutant plants. Total RNA was isolated from 12-d-old plate-grown seedlings. Bars represent mean ± s.d. (n = 3 biologically independent samples). For (d–i), the expression of each gene in the H2O-treated WT plants was set as 1. Data in (c) were analyzed by one-way ANOVA with Tukey’s test. Exact P values are provided in Supplementary Table 5. Data in (a, b, d–k) were analyzed using two-tailed Student’s t-test. Exact P values are provided in Supplementary Table 5. The experiments in (a, b, d–k) were repeated twice with similar results. The experiment in (c) was repeated three times with similar results.

Source data

Extended Data Fig. 9 flg22 or nlp20-induced immune responses in pcrk1/2 pbl19/20 quadruple mutant plants.

(a) Growth of Hpa Noco2 on the local leaves of WT, pcrk1/2, pcrk1/2 pbl19, and pcrk1/2 pbl19/20 quadruple mutant lines (#33 and #47) after 1 µM nlp20 treatment. Disease ratings showing the relative growth of Hpa Noco2 are as described in the Methods. The experiment was repeated three times with similar results. (b) Growth of Pto DC3000 in the leaves of four-week-old WT and pcrk1/2 pbl19/20 quadruple mutant lines (#33 and #47) pre-treated with H2O or 1 μM nlp20. The treated leaves were infiltrated with Pto DC3000 (OD600=0.001) 24 h post treatment with nlp20. Samples were taken 3 days after Pto DC3000 inoculation. Bars represent mean ± s.d. (n = 8 plants). The reduction of bacterial titer after nlp20 treatment in each genotype was regarded as nlp20-induced protection, which was compared among the WT and the mutants. The experiment was repeated three times with similar results. (c) Levels of SAG in four-week-old soil-grown plants of the indicated genotypes treated with H2O or 1 μM nlp20 for 24 h. Bars represent mean ± s.d. (n = 3 biologically independent samples). The experiment was repeated three times with similar results. (d, e) nlp20-induced MAPK activation in the indicated genotypes. 12-day-old seedlings were treated with 0.1 μM nlp20. MAPK activation was analyzed by immunoblotting with the anti-pERK antibody (d). Equal loading is confirmed by Ponceau staining of Rubisco. Quantifications of the phosphorylated MPK6 and MPK3 were shown in (e). Bars represent mean ± s.d. (n = 4 different experiments). (f) nlp20-induced ROS production in the indicated genotypes. Leaf strips were treated with 1 μM nlp20 and production of ROS was measured as luminescence. The values show the peak of luminescence units through all the time points. Bars represent mean ± s.d. (n = 8 leaf disks from different plants). The experiment was repeated three times with similar results. (g, h) Induction of SARD1 (g) and FMO1 (h) in the indicated genotypes by nlp20. Total RNA extracted from 12-day-old plate-grown plants treated with 1 μM nlp20 for 4 h. The expression of each gene in H2O-treated WT was set as 1. Bars represent mean ± s.d. (n = 3 biologically independent samples). The experiment was repeated twice with similar results. (i, j) flg22-induced MAPK activation in the indicated genotypes. 12-day-old seedlings were treated with 0.1 μM flg22. MAPK activation was analyzed by immunoblotting with the anti-pERK antibody. Equal loading is confirmed by Ponceau staining of Rubisco. Quantifications of the phosphorylated MPK6 and MPK3 10 min after treatment with flg22 were shown in (j). Bars represent mean ± s.d. (n = 4 different experiments). (k) ROS production in the indicated genotypes after treatment with 0.1 μM flg22 measured as luminescence. The values show the peak of luminescence units through all the time points. Bars represent mean ± s.d. (n = 8 leaf disks from different plants). The experiment was repeated three times with similar results. (l, m) Relative expression levels of SARD1 (l) and FMO1 (m) in the indicated genotypes. Total RNA was isolated from 12-day-old plate-grown seedlings 4 h after spraying with H2O or 1 μM flg22. The expression of each gene in the H2O -treated WT plants was set as 1. Bars represent mean ± s.d. (n = 3 biologically independent samples). Experiments were repeated twice with similar results. (n) Free SA and SAG levels in four-week-old plants of the indicated genotypes after treatment with H2O or 1 μM flg22 for 9 h. Bars represent mean ± s.d. (n = 3 biologically independent samples). The experiment was repeated three times with similar results. (o) Growth of Pto DC3000 in the leaves of four-week-old plants of the indicated genotypes after treatment with H2O or 1 μM flg22. Bars represent mean ± s.d. (n = 6 plants). The reduction of bacterial titer after flg22 treatment in each genotype was regarded as flg22-induced protection, which was compared among different genotypes. The experiment was repeated three times with similar results. The data in (b, c, e–h, j–o) were analyzed by one-way ANOVA with Tukey’s test. Exact P values are provided in Supplementary Table 5.

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Extended Data Fig. 10 Analysis of the interactions between SOBIR1 and PBL19/EDS1/PAD4/ADR1 by TurboID-based proximity labeling mathod.

Agrobacterium carrying the indicated constructs were infiltrated into N. benthamiana leaves for protein expression. Immunoprecipitation of PBL19-3FLAG (a), ADR1-3FLAG (b), EDS1-ZZ-TEV-FLAG (c), and PAD4-ZZ-TEV-FLAG (d) was carried out with anti-FLAG beads. The FLAG-tagged proteins were detected by western blot using an anti-FLAG antibody. The biotiny lated proteins were detected by western blot using HRP-Streptavidin. The experiments were repeated at least twice with similar results.

Supplementary information

Supplementary Fig. 1

This file contains the uncropped blots.

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Supplementary Tables

This file contains Supplementary Tables 1–4.

Supplementary Table 5

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Tian, H., Wu, Z., Chen, S. et al. Activation of TIR signalling boosts pattern-triggered immunity. Nature 598, 500–503 (2021). https://doi.org/10.1038/s41586-021-03987-1

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