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TIR immune signalling is blocked by phosphorylation to maintain plant growth

Nature Plants volume 11, pages 1193–1204 (2025)Cite this article

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Abstract

Toll/interleukin-1 receptor (TIR) ___domain proteins are immune signalling components and function as NAD+-cleaving enzymes to activate defence responses. In plants, TIR activation triggers cell death and severely represses growth, especially under osmotic stress, while in animals, it promotes axon degeneration. However, the mechanisms regulating TIR suppression remain unclear. Here we show that TIR NADase activity requires a conserved serine residue spatially close to the catalytic glutamate. The osmotic-stress-activated plant Ca2+-dependent protein kinases (CPKs), the mammalian Ca2+/calmodulin-dependent protein kinase II delta (CAMK2D) and TANK-binding kinase 1 (TBK1) phosphorylate TIR domains at this conserved serine, which blocks TIR NADase activities and functions, thereby maintaining growth in plants and suppressing SARM1 TIR signalling in animals. Our findings define a fundamental molecular mechanism by which phosphorylation at a conserved serine residue inhibits TIR signalling in plants and animals and sustains plant growth.

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Fig. 1: CPK3 phosphorylates a conserved serine in the TIR ___domain and represses TIR signalling to restore plant growth.
Fig. 2: Phosphorylation of TIR represses its activity.
Fig. 3: CPK-mediated phosphorylation of TIR domains represses TIR functions to maintain plant growth.
Fig. 4: TBK1 phosphorylates SARM1TIR and represses SARM1TIR signalling.

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Data availability

All data supporting the findings of this study are available in the main text or the supplementary files. The biological materials are available from the corresponding author upon reasonable request. The mass spectrometric data on in vitro and in planta CPK3-mediated phosphorylation of SNC1TIR have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers PXD060014 and PXD060129, respectively. The genes mentioned in this study can be found in the TAIR database (https://www.arabidopsis.org/) or the NCBI database (https://www.ncbi.nlm.nih.gov/) under the following identifiers: AT5G61900 (BON1), AT4G16890 (SNC1), AT1G47370 (RBA1), U27081 (L6), NM_015077 (SARM1), AT4G23650 (CPK3), AT4G09570 (CPK4), AT4G35310 (CPK5), AT2G17290 (CPK6), AT1G35670 (CPK11), AT5G66210 (CPK28), AT2G43790 (MPK6), AT1G51660 (MKK4), AT4G33950 (SnRK2.6), Q13557 (CAMK2D), Q9UQM7 (CAMK2A), Q9UHD2 (TBK1) and O43318 (TAK1). Source data are provided with this paper.

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Acknowledgements

We thank J. L. Dangl and M. T. Nishimura for careful reading and discussion of the manuscript. We thank the members of the Zhao Lab for helpful discussions. We thank X. Xu and S. Wang of the Core Facility Center, CEMPS, for their help in mass spectrometry. We are grateful to M. Li (Zhongshan Hospital, Fudan University) for her contributions to the construction of NRK1- or TBK1/NRK1-HEK293T stable cell lines and the related transfection experiments. This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences grant nos. XDB063020102 (to Y.Z.) and XDB27040214 (to L.W.), the Project of Stable Support for Youth Teams in Basic Research Field of the Chinese Academy of Sciences (no. YSBR-119 to Y.Z.), and the Science and Technology Commission of Shanghai Municipality grant no. 23ZR1470700 (to B.Y.). Y.Z. was supported by the Key Laboratory of Plant Carbon Capture and the Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences. L.W. was supported by the State Key Laboratory of Plant Trait Design at the Institute of Plant Physiology and Ecology/Center for Excellence in Molecular Plant Sciences.

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Author notes

  1. These authors contributed equally: Jun Li, Sisi Chen, Bo Yu.

Authors and Affiliations

  1. Key Laboratory of Plant Carbon Capture, Shanghai Center for Plant Stress Biology, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China

    Jun Li, Bo Yu, Qingzhong Li, Ruijia Liu & Yang Zhao

  2. State Key Laboratory of Plant Trait Design, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China

    Sisi Chen, Zaiqing Wang & Li Wan

  3. University of Chinese Academy of Sciences, Beijing, China

    Sisi Chen, Ruijia Liu & Yang Zhao

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  1. Jun Li
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Contributions

Y.Z. and L.W. conceived and designed the research. J.L., S.C., B.Y., Q.L., R.L. and Z.W. performed the experiments. J.L., S.C., B.Y., L.W. and Y.Z. analysed the results. Y.Z., J.L. and L.W. wrote the manuscript.

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Correspondence to Li Wan or Yang Zhao.

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Patent applications related to this work have been submitted by Y.Z., L.W., J.L., S.C. and B.Y. The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Dominant-negative SNC1 mutations suppress NLR signaling in bon1 mutants.

a, Gene structure of SNC1. The mutations of SNC1 in the insensitive to osmotic stress (ios) mutants are indicated. The ios mutants suppress bon1-7 based on plant growth under osmotic stress. The ios26 (snc1-17, a single amino acid change V518I) and ios102 (snc1-18, a single amino acid change G491S) mutations were distributed on the NB-ARC ___domain, likely affecting SNC1 oligomerization. The ios112 (snc1-20, a nonsense mutation resulting in a premature termination at codon 734), and ios113 (snc1-21, a single amino acid change L647F) mutations were distributed on the LRR ___domain, likely affecting SNC1 autoinhibition. The ios22 (snc1-16, a single amino acid change G1343D) was distributed on the C-terminal jelly-roll/Ig-like ___domain (C-JID). Interestingly, the ios109 (snc1-19, a single amino acid change S609N) mutation was distributed in the linker between the two domains (NL linker), adjacent to the autoimmune mutation of the gain-of-function snc1-1 (a single amino acid change E561K). b,c,e,f, Shoot growth of Col-0, bon1, ios22 (snc1-16) and ios26 (snc1-17) mutants, and bon1 transformed with OE-SNC1G1343D (corresponding to snc1-16 mutation) or CPK3pro::CPK3CA 15 days post-transfer to 1/2 MS medium (e,f) or 1/2 MS medium containing 100 mM mannitol (b,c). d, Morphology of 6-week-old soil-grown bon1 and ios26 (snc1-17), and OE-SNC1V518I (corresponding to snc1-17 mutation) transgenic plants in bon1-1 background. g-i, Free SA levels (g) and PR1 and PR2 expression (h,i) in AEQsig6 (wild type), bon1-7 and ios22, and bon1-7 transformed with OE-SNC1G1343D 5 days post-transfer to 1/2 MS medium containing 0 or 100 mM mannitol. j,k, Protein abundance of SNC1G1343D (j, snc1-16) and SNC1V518I (k, snc1-17) in OE-SNC1G1343D and OE-SNC1V518I transgenic plants in bon1 background was detected by anti-Myc antibody. Actin was used as a loading control. l, Shoot growth of Col-0 (wild-type) and snc1-1 mutant 15 days post-transfer to 1/2 MS medium containing 0 or 100 mM mannitol. Scale bars, 0.5 cm (b,c,e,f), and 2 cm (d). Error bars represent mean ± SD (n = 3 biological replicates) (g-i). Statistical analysis was performed using a two-way ANOVA with Tukey’s test; NS, not significant, P > 0.05, ****P < 0.0001.

Source data

Extended Data Fig. 2 Constitutively active CPK3 suppresses NLR signaling in bon1 mutants.

a, CPKs were co-immunoprecipitated with SNC1 in OE-SNC1-Myc transgenic plants treated with 600 mM mannitol. b, Venn diagram represents the overlap of CPKs detected as candidate SNC1 interacting proteins (a) and candidate osmotic stress-responsive proteins from the in vivo phosphoproteomics data from (https://www.pnas.org/doi/10.1073/pnas.1308974110). c, Expression of CPK3, 4, 5, 6, and 11 in Arabidopsis according to the Arabidopsis electronic fluorescent pictograph (eFP) browser. Expression is colored with increasing saturation from light blue to dark blue. d-f, Activation of CPK3 by osmotic stress in seedlings. CPK3 was immunoprecipitated from 7-day-old CPK3pro::CPK3-4Myc transgenic seedlings treated with 0 or 0.6 M mannitol for 30 min, and used for phosphorylation assay with [γ-³²P]ATP (d) or the ATP analog ATPγS (e). Autoradiography exhibits CPK3 phosphorylation (d). The thiophosphate ester groups were detected by anti-thiophosphate ester antibody (e), and relative band intensity was quantified (f). Total CPK3 was detected by anti-Myc antibody. g-i, Levels of free SA (g) and expression of PR1 (h) and PR2 (i) in Col-0, bon1-1, and bon1-1 transformed with CPK3pro::CPK3CA 5 days post-transfer to 1/2 MS medium containing 0 or 100 mM mannitol. j, The structure of CPK3 (top) and Sanger sequencing (bottom) show wild-type CPK3 and mutations on CPK3 in CPK3pro::CPK3CA transgenic plants in a bon1-1 mutant background, with the kinase-dead mutations (K107M and ΔK107). k, In planta kinase activity of CPK3CA-HA and its kinase-dead mutant (K107M) immunoprecipitated from N. benthamiana leaves 48 h post-agroinfiltration. Anti-thiophosphate ester antibody detected thiophosphate ester groups (top). Total CPK3CA and CPK3CA-K107M proteins were detected by an anti-HA antibody. l, Expression of wild-type and mutated CPK3CA-Myc and Actin in Col-0, bon1-1, and CPK3pro::CPK3CA, CPK3pro::CPK3CA-K107M, and CPK3pro::CPK3CA-ΔK107 in bon1-1 background. m, Protein abundance of SNC1-Myc, CPK3CA-Myc, and Tubulin in Col-0 and OE-SNC1-4Myc transgenic plants with or without CPK3pro::CPK3CA in Col-0 background. Error bars represent mean ± SD (n = 3 biological replicates) (g-i). Statistical analysis was performed using a two-way ANOVA with Tukey’s test; ***P = 0.0002, ****P < 0.0001. All experiments except (a, once; b and c, previously published data) were repeated at least three times with similar results.

Source data

Extended Data Fig. 3 CPKs interact with and phosphorylate SNC1TIR in vitro and in planta.

a, CPK3-SNC1TIR interaction in a co-immunoprecipitation assay using N. benthamiana eds1 mutant leaves expressing SNC1TIR-Myc, CPK3-YFP, and YFP-YFP. Total proteins were immunoprecipitated with anti-GFP agarose and detected with anti-Myc antibody. *CPK3-YFP; **YFP-YFP; ***SNC1TIR-Myc. b, CPK3-SNC1TIR interaction in split-LUC assays using N. benthamiana eds1 mutant leaves expressing CPK3-nLUC and cLUC-SNC1TIR. c, CPK3CA-SNC1TIR or CPK3CA-K107M-SNC1TIR interaction in N. benthamiana leaves in a co-immunoprecipitation assay. Total proteins were immunoprecipitated with anti-HA beads and detected with an anti-Flag antibody. d,e, Mass spectrometric analysis of GST-SNC1TIR phosphorylation at Ser18 (d) and Ser26 (e) by GST-CPK3. f, Phosphorylation of GST-SNC1TIR by GST-CPK3. Anti-thiophosphate ester antibody detected thiophosphate ester groups (top), anti-GST was used as loading control (bottom). g, Ser26 phosphorylation in SNC1TIR by GST-CPK3, without or with λ-protein phosphatase (λ-PPase). Anti-phospho-Ser26-SNC1TIR antibody (anti-pS26) detected phosphorylation (top), anti-GST was used as loading control (bottom). h-j, In planta Ser26 phosphorylation in SNC1TIR-Flag (h) or full-length SNC1-Flag (i) by CPK3CA-HA. SNC1TIR-Flag or SNC1-Flag was immunoprecipitated from N. benthamiana leaves co-expressing CPK3CA-HA. Anti-pS26 detected phosphorylation (top), and relative band intensity was quantified (j). Anti-Flag and anti-HA antibodies were used as loading controls. Error bars represent mean ± SD (n = 3). Two-sided Student’s t-test, ****P < 0.0001. k, In planta Ser26 phosphorylation in SNC1TIR by CPK3CA was detected by data-independent acquisition (DIA)-based mass spectrometry using immunoprecipitated SNC1TIR from N. benthamiana leaves co-expressing CPK3CA-HA. l, Phosphorylation of SNC1TIR by CPK3-4Myc immunoprecipitated from transgenic seedlings treated with 0.6 M mannitol for 30 min. Anti-thiophosphate ester antibody detected the phosphorylation (top), anti-SNC1 antibody was used as loading control. m-o, Phosphorylation of SNC1TIR by GST-CPK4/6/11 (m), GST-CPK5 (n) and GST-CPK28 (o). Autoradiography (top) and Coomassie staining (bottom) exhibit phosphorylation and loading. p, Phosphorylation of RBA1 and RBA1-S19A by GST-CPK3. Autoradiography exhibits phosphorylation (top). Anti-Flag antibody was used as a loading control (bottom). All experiments except (d,e,k, once) were repeated at least three times with similar results.

Source data

Extended Data Fig. 4 MPK6 and SnRK2.6 cannot phosphorylate the TIR ___domain of SNC1.

a,b, Phosphorylation of wild-type SNC1TIR by CPK3, MPK6 or SnRK2.6. GST-CPK3 was used as a positive control. Autoradiography (top) and Coomassie staining (bottom) exhibit phosphorylation and the loading of recombinant MBP-MAPK6, MKK4DD, GST-CPK3, MBP-SnRK2.6 and GST-SNC1TIR (a). The anti-thiophosphate ester antibody was used to detect the phosphorylation of SNC1TIR, CPK3, MAPK6 and SnRK2.6 (b, top panel). The anti-MBP and anti-GST antibodies were used to detect the loading of recombinant GST-SNC1TIR, MBP-MAPK6, MBP-MKK4DD, and MBP-SnRK2.6 (b, middle panel and bottom panel). All experiments were repeated at least three times independently with similar results.

Extended Data Fig. 5 Phosphorylation of TIR represses its NADase activity.

a, Cartoon structures of SNC1TIR (PDB: 5TEC) and hSARM1TIR (PDB: 8D0J) showing catalytic glutamate (orange) and conserved serine (red) as sticks. The distances are shown for the carboxylic acid group of the catalytic glutamate with the hydroxyl group of the conserved serine. b-g, Purified wild-type RBA1 protein, but not the catalytically dead RBA1-E86A mutant, exhibited strong NADase activity. LC-MS/MS results showing that RBA1 could degrade NAD+ (b,c) and produce Nam (d,e) and 2′cADPR (f,g). LC-MS/MS chromatograms (b,d,f) and areas under curves (c,e,g) indicating that NADase activity of RBA1 depends on the catalytic glutamate. The purified RBA1-S19A and RBA1-S19D proteins were also used for in vitro NADase assays (Fig. 2c–f). h,i, LC-MS/MS analysis of the generation of Nam by wild-type RBA1, RBA1-S19A, and RBA1-S19D in vitro. Error bars represent mean ± SEM (n = 3). Two-tailed Student’s t-test (c,e,g), or one-way ANOVA with Tukey’s test (i), NSP = 0.0752, *P = 0.0372, **P = 0.0022 (g) and 0.0074 (i), ***P = 0.0009. RBA1-S19D could not produce Nam, degrade NAD+ (Fig. 2c, d) or produce 2′cADPR (Fig. 2e, f). j, Wild-type and mutated RBA1-Flag proteins purified from insect cells were detected by anti-Flag antibody. The purified RBA1 proteins were used for in vitro NADase assays (Fig. 2c–f, and b–i). k, Wild-type and mutated RBA1-YFP proteins were expressed in N. benthamiana eds1-2 mutant and detected by anti-GFP antibody. The resulting accumulation of 2′cADPR was shown in Fig. 2g. l, RBA1-Flag and RBA1-S19A-Flag were stably expressed in HEK293T cells, with transient expression of CPK3CA-Myc. RBA1-Flag, RBA1-S19A-Flag and CPK3CA-Myc were detected by anti-Flag and anti-Myc antibodies. Actin was detected with anti-actin antibody. The resulting degradation of NAD+ was shown in Fig. 2h. All experiments except (a) were repeated at least three times with similar results.

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Extended Data Fig. 6 Phosphomimetic mutations of TIR repress TIR-mediated cell death.

Cell death phenotypes of wild-type and mutated TIRs upon transient expression in N. benthamiana leaves. The cell-death phenotype was visualized under visible light (left panel) or UV (right panel) and imaged 3 days after agroinfiltration inoculation. Different degrees of cell death were defined as the cell death score (0, blue; 1, coral pink; 2, orange). Each color on stacked bars shows the proportions (in numbers) of the corresponding degrees of cell death. a, Expression of SNC1TIR-YFP, non-phosphorylatable SNC1TIR-S18A-YFP, and phospho-mimic SNC1TIR-S18D-YFP. Plant leaves were infiltrated with Agrobacterium tumefaciens bacteria with OD600 at 0.2 (left) or 0.4 (right). b, Expression of SNC1TIR-YFP, non-phosphorylatable SNC1TIR-S18A/S26A (AA)-YFP, phospho-mimic SNC1TIR- S18D/S26D (DD)-YFP, and catalytically dead SNC1TIR-E93A-YFP. c, Expression of AbTIR-YFP, non-phosphorylatable AbTIR-S140A-YFP, phospho-mimic AbTIR-S140D-YFP, and catalytically dead AbTIR-E208A-YFP. All experiments were repeated at least three times with similar results.

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Extended Data Fig. 7 Expression of wild-type and mutated TIR proteins and CPKs in planta.

a-c, SNC1TIR-YFP, L6TIR-YFP, RBA1-HA, and CPK3CA-cLUC-Flag proteins were transiently expressed in N. benthamiana leaves and detected by western blot. The anti-GFP antibody was used to detect SNC1TIR-YFP and L6TIR-YFP. The anti-HA antibody was used to detect RBA1-HA. The anti-Flag antibody was used to detect CPK3CA-cLUC-Flag. Cell death phenotypes of wild-type TIRs with or without CPK3CA were shown in Fig. 3b. d,e, SNC1TIR-S26A-Flag, full-length SNC1-Flag and CPK3CA-HA were transiently expressed in N. benthamiana leaves and detected by western blot. The anti-Flag antibody was used to detect SNC1TIR-Flag or full-length SNC1-Flag. The anti-HA antibody was used to detect CPK3CA-HA. Cell death phenotypes of SNC1TIR-S26A and full-length SNC1 with or without CPK3CA were shown in Figs. 3c, d, respectively. f-l, Wild-type and mutated TIR proteins were transiently expressed in N. benthamiana leaves and detected by western blot. Total proteins were extracted from N. benthamiana leaves. Ponceau S staining of RuBisCO was used as a loading control. The anti-GFP antibody was used to detect wild-type and mutated TIR-YFP proteins (f-j,l). The anti-HA antibody was used to detect RBA1-HA (k). SNC1TIR (f-i) related cell death phenotype was shown in Fig. 3e and S6; L6TIR (j) related cell death phenotype was shown in Fig. 3f; RBA1 (k) related cell death phenotype was shown in Fig. 3g; while hSARM1tSAM-TIR (l) related cell death phenotype was shown in Fig. 3h. m, Expression of CPK3CA-Myc and Actin in Col-0, cpk3/4/5/6/11 quintuple mutant, and CPK3pro::CPK3CA transgenic plants in Col-0 background was analyzed by reverse transcription PCR. The corresponding plant growth phenotype was shown in Figs. 3l, m. All experiments were repeated at least three times with similar results.

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Extended Data Fig. 8 Phosphomimetic mutation of SNC1TIR represses its immune function and maintains plant growth.

a-c, Levels of relative expression of PR1 (a), PR2 (b) and free SA (c) in Col-0 and OE-SNC1-Myc transgenic plants expressing wild-type or S18D/S26D (DD)-mutated SNC1 in Col-0 background five days after transfer to the 1/2 MS medium containing 0 or 100 mM mannitol. d-f, Pst DC3000 triggered plant immunity in Col-0 and OE-SNC1-4Myc transgenic plants expressing wild-type or S18D/S26D (DD)-mutated SNC1 in Col-0 background. Plant leaves were infiltrated with Pst DC3000 bacteria with OD600 at 0.002. Disease symptoms (d), bacterial populations (e), and levels of free SA (f) were monitored 3 days post-infection. Error bars represent mean ± SD (n = 3 experiments) for (a-c), ± SD (n = 4 experiments) in (e), and ± SEM (n = 3 experiments) in (f). Letters denote significant differences (two-way ANOVAs, Tukey’s test; a,b). Asterisks denote significant differences: two-way ANOVA with Tukey’s test (c,f) and one-way ANOVA with Tukey’s test (e), NS P > 0.05, *P < 0.05, ****P < 0.0001. g, Protein abundance of SNC1 and SNC1S18D/S26D (DD) in OE-SNC1-4Myc transgenic plants expressing wild-type or S18D/S26D (DD)-mutated SNC1 in Col-0 background. The anti-Myc antibody was used to detect SNC1 and SNC1S18D/S26D (DD). All experiments were repeated at least three times with similar results.

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Extended Data Fig. 9 The cpk3/4/5/6/11 quintuple mutant exhibited shoot growth defects and elevated levels of defense marker gene expression under hyperosmotic stress.

a,b, Shoot growth of Col-0, cpk3-2 mutant (a), and cpk3/4/5/6/11 quintuple mutant (b), 15 days post-transfer to 1/2 MS medium containing 0 mM or 100 mM mannitol. Scale bars, 0.5 cm. c,d, Levels of relative expression of PR1 (c) and PR2 (d) in Col-0 and cpk3/4/5/6/11 quintuple mutant five days after transfer to the 1/2 MS medium containing 0 or 100 mM mannitol. All experiments were repeated at least three times with similar results. Error bars represent means ± SD (n = 3). Asterisks denote significant differences (two-way ANOVA with Tukey’s test, ****P < 0.0001).

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Extended Data Fig. 10 Regulation of SARM1TIR by TBK1.

a, Expression of hTBK1-Flag and Actin in N. benthamiana leaves transiently expressing hSARM1TIR-GFP with or without hTBK1-Flag was analyzed by reverse transcription PCR. The anti-GFP antibody was used to detect hSARM1TIR-GFP proteins. b, Human SARM1TIR-GFP-Flag and hSARM1TIR-S567A-GFP-Flag proteins were transiently expressed in the NRK1-HEK293T or TBK1/NRK1-HEK293T stable cell lines and detected by western blot. The anti-Flag antibody was used to detect wild-type and mutated hSARM1TIR-GFP-Flag. The anti-Myc antibody was used to detect TBK1-Myc. The anti-actin was used as a loading control. All experiments were repeated at least three times with similar results.

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Supplementary information

Supplementary Information

Supplementary methods.

Reporting Summary

Supplementary Data 1

IP–MS data for SNC1–Myc under osmotic stress.

Supplementary Data 2

Primers used in this study.

Source data

Source Data Fig. 1

Unprocessed western blots for Fig. 1.

Source Data Figs. 1–4 and Extended Data Figs. 1–3, 5, 6, 8 and 9

Statistical source data for Figs. 1–4 and Extended Data Figs. 1–3, 5, 6, 8 and 9.

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Unprocessed western blots for Extended Data Fig. 1.

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Unprocessed western blots and gels for Extended Data Fig. 2.

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Unprocessed western blots for Extended Data Fig. 3.

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Unprocessed western blots for Extended Data Fig. 5.

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Unprocessed western blots and gels for Extended Data Fig. 7.

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Unprocessed western blots for Extended Data Fig. 8.

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Unprocessed western blots and gels for Extended Data Fig. 10.

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Li, J., Chen, S., Yu, B. et al. TIR immune signalling is blocked by phosphorylation to maintain plant growth. Nat. Plants 11, 1193–1204 (2025). https://doi.org/10.1038/s41477-025-02012-x

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