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A calmodulin-gated calcium channel links pathogen patterns to plant immunity

Abstract

Pathogen-associated molecular patterns (PAMPs) activate innate immunity in both animals and plants. Although calcium has long been recognized as an essential signal for PAMP-triggered immunity in plants, the mechanism of PAMP-induced calcium signalling remains unknown1,2. Here we report that calcium nutrient status is critical for calcium-dependent PAMP-triggered immunity in plants. When calcium supply is sufficient, two genes that encode cyclic nucleotide-gated channel (CNGC) proteins, CNGC2 and CNGC4, are essential for PAMP-induced calcium signalling in Arabidopsis3,4,5,6,7. In a reconstitution system, we find that the CNGC2 and CNGC4 proteins together—but neither alone—assemble into a functional calcium channel that is blocked by calmodulin in the resting state. Upon pathogen attack, the channel is phosphorylated and activated by the effector kinase BOTRYTIS-INDUCED KINASE1 (BIK1) of the pattern-recognition receptor complex, and this triggers an increase in the concentration of cytosolic calcium8,9,10. The CNGC-mediated calcium entry thus provides a critical link between the pattern-recognition receptor complex and calcium-dependent immunity programs in the PAMP-triggered immunity signalling pathway in plants.

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Fig. 1: CNGC2 and CNGC4 are critical determinants of PAMPs- and ROS-induced calcium signals and PTI responses under sufficient external calcium concentrations.
Fig. 2: CNGC2 and CNGC4, but neither alone, form a calcium channel in Xenopus oocytes.
Fig. 3: The CNGC2–CNGC4 calcium channel is blocked by CaM.
Fig. 4: BIK1 phosphorylates and activates the CaM-gated CNGC2–CNGC4 channel.

Data availability

The data supporting the findings of this study are available within the paper and its Supplementary Information files. Source Data (gels and graphs) for Figs. 14 and Extended Data Figs. 110 are provided with the paper.

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Acknowledgements

We thank C. Zipfel and J.-M. Zhou for critical reading of the manuscript. This research was supported in part by the National Science Foundation (to S.L.), the National Key Research and Development Program of China (2016YFD0300102-3) and National Natural Science Foundation of China grants 31270297 and 31470356 (to L.L.). S.H. was a visiting fellow from Jiangxi Agricultural University, China. We thank J.-M. Zhou and G. A. Berkowitz for providing constructs and plant materials; M. C. Wildermuth for providing the hrcC strain; and E. Y. Isacoff for providing oocytes.

Author information

Authors and Affiliations

Authors

Contributions

W.T., L.L., B.J.S. and S.L. conceived and designed the experiments; C.H. performed molecular cloning and biochemical experiments; C.W. performed molecular cloning, protein interaction, in vivo and in vitro phosphorylation assays; F.Z. performed patch-clamp recording in protoplasts; W.T. and Z.R.—with the help of D.D., S.H., L.Z. and Q.N.—conducted mutant screening, bacterial infections, Ca2+ imaging and oxidative-burst assays and oocyte recording. All experiments were independently reproduced in the laboratory. W.T., C.W. and S.L. wrote the manuscript. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Sheng Luan.

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The authors declare no competing interests.

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Peer review information Nature thanks Thorsten Nuernberger and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Fig. 1 Low [Ca2+]ext levels, while supporting normal growth and development, impair plant immunity.

ad, The duration of the plateau of the increase in [Ca2+]cyt induced by flg22 is reduced in Col-0AEQ plants grown under low [Ca2+]ext. Increase in [Ca2+]cyt in leaf discs is represented as relative luminescence (L/Lmax)9,30,31. The time course of [Ca2+]cyt (a), peak [Ca2+]cyt (b), plateau duration (c) and integrated L/Lmax (d) in Col-0AEQ or rbohDAEQ mutant leaf discs after application of 500 nM flg22 are shown. The arrow indicates the time of flg22 addition. The plateau duration is the time period during which the [Ca2+]cyt is higher than 80% of the peak value. Data are mean ± s.e.m., n = 8 biologically independent leaf discs. eh, The duration and total extent of flg22-induced ROS production are reduced in Col-0 plants grown under low [Ca2+]ext. ROS burst was measured in Col-0 or rbohD mutant leaf discs after treatment with 500 nM flg22. The kinetics of ROS production (e), peak value (f), duration (g) and total photon counts over 32 min (h) are shown. Data are mean ± s.e.m., n = 12 biologically independent leaf discs. i, Col-0 plants exhibit reduced resistance to hrcC bacteria when grown under low [Ca2+]ext. Bacterial populations in plant leaves five days after dip inoculation with hrcC are shown here. Data are represented as mean ± s.d., n = 3 biologically independent samples. j, k, [Ca2+]ext in the range of 1.5 mM to 0.1 mM supports normal growth and development in Arabidopsis, as shown by comparable leaf fresh weight (at 5 weeks old) (j) and flowering time (k) of Col-0 plants grown in hydroponic medium containing 1.5, 0.5 or 0.1 mM [Ca2+]ext. Data are mean ± s.e.m., n = 8 biologically independent samples. P values are from two-sided Student’s t-tests. Experiments were repeated three times with similar results.

Source data

Extended Data Fig. 2 The afc1 and afc2 mutants contain mutations in CNGC2 and CNGC4.

ac, The flg22-triggered [Ca2+]cyt burst is reduced in afc1 and afc2 mutants. a, The [Ca2+]cyt burst was measured as relative luminescence units using a Varioskan Flash Spectral Scanning Multimode Reader as previously described9,30,31. The arrow indicates the time at which 500 nM flg22 was added. WT, wild type. b, Relative [Ca2+]cyt levels are depicted as luminescence counts per second relative to total luminescence counts remaining (L/Lmax)9,30,31. The remaining aequorin was discharged by the addition of 100 μl of 2 M CaCl2 with 20% ethanol per well. c, The bar graph represents the peak [Ca2+]cyt in the wild type and the two mutants after application of 500 nM flg22. d, NaCl (300 mM)-induced increases in [Ca2+]cyt in the afc1 and afc2 mutants are comparable to those of the wild type. For ad, data are shown as mean ± s.e.m., n = 8 biologically independent leaf discs; experiments were repeated 3 times with similar results. P values are from two-sided Student’s t-tests. e, Genetic complementation group analysis indicates that AFC1 encodes CNGC2 and AFC2 encodes CNGC4. Scale bar, 2 cm. Plants of different genotypes were analysed at least twice independently and in all cases they show similar growth phenotypes. f, A schematic of CNGC with six-transmembrane domains and the cytosolic C-terminal region. The mutation in afc1 resulted in a truncated CNGC2 protein and the mutation in afc2 resulted in a G381E substitution in CNGC4.

Source data

Extended Data Fig. 3 cngc2, cngc4 and cngc2 cngc4 mutants exhibit comparable growth phenotypes.

a, Gene structure of CNGC2 and CNGC4 showing the positions of exons (boxes), introns (lines), T-DNA insertion sites (triangle) and point mutations (amino acid position ). b, Morphological phenotype of cngc2-1, cngc2-3, cngc4-1, cngc4-5, cngc2-1 cngc4-1 and cngc2-3 cngc4-5 plants. Scale bar, 2 cm. Photographs were taken when the plants were four weeks old. c, Identification of the point mutations in cngc2-1, cngc4-1 and cngc2-1 cngc4-1 by sequencing. d, RT–PCR analysis for A. thaliana CNGC2 and A. thaliana CNGC4 mRNA in cngc2-3, cngc4-5 and cngc2-3 cngc4-5. ACTIN was used as loading control. For b and d, experiments were repeated three times with similar results.

Extended Data Fig. 4 The increase in [Ca2+]cyt induced by multiple PAMPs or DAMPs and by H2O2 is reduced in soil-grown cngc2AEQ, cngc4AEQ and cngc2 cngc4AEQ mutants.

a, b, The increase in [Ca2+]cyt is reduced in soil-grown cngc2AEQ, cngc4AEQ and cngc2 cngc4AEQ mutants in response to 500 nM flg22. [Ca2+]cyt kinetics (a) and peak [Ca2+]cyt (b) in leaf discs from Col-0AEQ, cngc2AEQ, cngc4AEQ and cngc2 cngc4AEQ plants treated with 500 nM flg22 are shown. n = 7 biologically independent leaf discs. c, d, The increase in [Ca2+]cyt is reduced in soil-grown cngc2AEQ, cngc4AEQ and cngc2 cngc4AEQ mutants in response to 500 nM elf18. [Ca2+]cyt kinetics (c) and peak [Ca2+]cyt (d) in leaf discs from Col-0AEQ, cngc2AEQ, cngc4AEQ and cngc2 cngc4AEQ plants treated with 500 nM elf18 are shown. n = 7 biologically independent leaf discs. e, f, The increase in [Ca2+]cyt is reduced in soil-grown cngc2AEQ, cngc4AEQ and cngc2 cngc4AEQ mutants in response to 100 μg ml−1 chitin. Shown are [Ca2+]cyt kinetics (e) and peak [Ca2+]cyt (f) in leaf discs from Col-0AEQ, cngc2AEQ, cngc4AEQ and cngc2 cngc4AEQ plants treated with 100 μg ml−1 chitin. n = 7 biologically independent leaf discs. g, h, The increase in [Ca2+]cyt is reduced in soil-grown cngc2AEQ, cngc4AEQ and cngc2 cngc4AEQ mutants in response to 500 nM AtPep1. [Ca2+]cyt kinetics (g) and peak [Ca2+]cyt (h) in leaf discs from Col-0AEQ, cngc2AEQ, cngc4AEQ and cngc2 cngc4AEQ plants treated with 500 nM AtPep1 are shown. n = 7 biologically independent leaf discs. i, j, The increase in [Ca2+]cyt is reduced in soil-grown cngc2AEQ, cngc4AEQ and cngc2 cngc4AEQ mutants in response to 2.5 mM H2O2. [Ca2+]cyt kinetics (i) and peak [Ca2+]cyt (j) in leaf discs from Col-0AEQ, cngc2AEQ, cngc4AEQ and cngc2 cngc4AEQ plants treated with 2.5 mM H2O2 are shown. n = 5 biologically independent leaf discs. Data are shown as mean ± s.e.m. P values are from two-sided Student’s t-tests. Experiments were repeated three times with similar results.

Source data

Extended Data Fig. 5 The flg22- and H2O2-activated calcium channel activities are impaired in mesophyll cell protoplasts isolated from cngc2, cngc4 and cngc2 cngc4 mutants.

a, Representative traces from patch-clamp whole-cell recording of Ca2+ channel currents in leaf mesophyll cell protoplasts of Col-0, cngc2, cngc4, cngc2 cngc4 or bik1 with or without the addition of 1 μM flg22 in the bath solution. b, Current density at −150 mV from multiple recordings as in a. For a, b, n = 4 biologically independent protoplasts. c, Representative traces from patch-clamp whole-cell recording of Ca2+ channel currents in leaf mesophyll cell protoplasts of Col-0, cngc2, cngc4, cngc2 cngc4 and bik1 with or without the addition of 5 mM H2O2 in the bath solution. d, Current density at −150 mV from multiple recordings as in c. For c, d, n = 4 biologically independent recordings. Data are represented as mean ± s.e.m. P values are from two-sided Student’s t-tests.

Source data

Extended Data Fig. 6 External calcium level affects the phenotypes of cngc2, cngc4 and cngc2 cngc4.

a, b, Limiting the external calcium supply restores the dwarf phenotype of cngc2, cngc4 and cngc2 cngc4 plants. a, Col-0, cngc2-3, cngc4-5 and cngc2-3 cngc4-5 plants were grown in hydroponic medium containing 1.5, 0.5 or 0.1 mM [Ca2+]ext for five weeks. b, Leaf fresh weight of plants from a. Data are means ± s.e.m., n = 10. P values are from two-sided Student’s t-tests. c, flg22-induced MAPK activation is not affected in the cngc2 cngc4 mutant grown under 1.5 mM [Ca2+]ext. d, flg22-induced MAPK activation is not affected by limited [Ca2+]cyt supply in the growth medium. Col-0 and cngc2-3 cngc4-5 plants were grown in hydroponic medium containing 1.5 mM or 0.1 mM [Ca2+]ext. Five-week-old plant leaves were infiltrated with flg22 or H2O (as mock) and samples were collected at indicated times after infiltration. MAPK phosphorylation was measured using an anti-pERK antibody. Experiments were repeated three times with similar results.

Source data

Extended Data Fig. 7 The CNGC2–CNGC4 channel becomes active when oocytes are perfused with Ca2+ concentrations of 0.3 mM or higher.

a, CNGC2–CNGC4 produced inward currents in oocytes when perfused with the indicated concentrations of extracellular Ca2+. b, Current amplitudes at −140 mV from recordings as in a. Data are represented as mean ± s.d. n = 8 biologically independent oocytes. P values are from two-sided Student’s t-tests. Experiments were repeated three times with similar results.

Source data

Extended Data Fig. 8 The PTI defence is not affected in the cam7-3 mutant.

a, Gene structure of CAM7 showing the positions of exons (boxes), introns (lines) and T-DNA insertion (triangle) site. b, CAM7 gene transcript levels in Col-0AEQ, cam7-3AEQ, CaM-OE1AEQ and CaM-OE2AEQ plants, as indicated by quantitative real-time PCR. ACTIN2 was used as an internal control. Data are mean ± s.d., n = 3 biologically independent samples. Two-sided t-test was used to determine significance. c, d, flg22-induced increase in [Ca2+]cyt in the cam7-3 mutant as compared to that in the wild type. The [Ca2+]cyt time course (c) and peak [Ca2+]cyt (d) after the application of 500 nM flg22 are shown. Data are presented as mean ± s.e.m., n = 6 biologically independent leaf discs. e, f, flg22-induced ROS production in the cam7-3 mutant. The ROS burst was measured in leaf discs after treatment with 500 nM flg22. The kinetics of ROS production (e) and total ROS production (f) are shown, as mean values of total photon counts over 32 min. Data are mean ± s.e.m., n = 8 biologically independent leaf discs. gi, DC3000 and hrcC bacteria grown in the cam7-3 mutant. Disease symptoms (g), the bacterial populations in plant leaves five days after dip inoculation with hrcC (h), and the bacterial populations in leaves three days after dip inoculation with DC3000 (i), are shown here. Photographs were taken five days after inoculation with hrcC or three days after inoculation with DC3000. Data are represented as mean ± s.d., n = 3 biologically independent samples. P values are from two-sided Student’s t-tests. Experiments were repeated three times with similar results.

Source data

Extended Data Fig. 9 BIK1(4D) phosphorylates CNGC4.

a, BIK1(4D) phosphorylates CNGC4-NT but not CNGC2-NT in vitro. CNGC2-NT and CNGC4-NT were fused to the trigger factor (TF) chaperone as a soluble fusion tag. b, FLS2-KD or BAK1-KD (KD, kinase domain) does not phosphorylate CNGC4-CT in vitro. The in vitro phosphorylation reaction was performed using purified proteins and [γ-32P]ATP, resolved by SDS–PAGE, and phosphorylated proteins were detected by autoradiography. The Coomassie Brilliant Blue staining of a and b was used to verify the quality of samples and the loading consistency. c, flg22 induces CNGC4 phosphorylation in Arabidopsis mesophyll protoplasts. Col-0 mesophyll protoplasts expressing CNGC4–Flag were treated with 500 nM flg22 for the indicated time or H2O (0) as control. CNGC4–Flag proteins were purified by Flag-M2 beads. The bead-bounded CNGC4 at 5 min after flg22 treatment were incubated with λ protein phosphatase (PPase) alone, or PPase plus phosphatase inhibitors to dephosphorylate CNGC4. Proteins were eluted from beads by SDS loading buffer with boiling. CNGC4 phosphorylation was detected on a Phos-tag SDS–PAGE gel, indicating that CNGC4 can be phosphorylated in response to flg22 and reversibly dephosphorylated by PPase. BIK1–HA was co-expressed with CNGC4 in protoplasts as a positive control, which presented phosphorylation in response to flg22. Total proteins containing BIK1–HA were processed similarly as described above. Ponceau S staining was used as a loading control for BIK1–HA. d, flg22 does not induce CaM phosphorylation in Arabidopsis protoplasts. Col-0 protoplasts expressing the indicated constructs were treated with either flg22 (+) or H2O control (−) for 10 min. CaM mobility shift was not observed on a Phos-tag SDS–PAGE gel. The values to the sides of the SDS–PAGE gels in ad indicate the molecular mass (kDa) of proteins. p, phosphorylated; np, non-phosphorylated. e, f, The CaM-gated CNGC2–CNGC4 calcium channel is not activated by FLS2 or RIPK in oocytes. e, Representative current traces from oocytes expressing CNGC2 + CNGC4, CNGC2 + CNGC4 + CaM, BIK1 + CNGC2 + CNGC4 + CaM, FLS2 + CNGC2 + CNGC4 + CaM or RIPK + CNGC2 + CNGC4 + CaM, perfused with 5 mM Ca2+. f, Current amplitudes at −140 mV from multiple recordings as in e. Data are presented as mean ± s.d., n = 12 biologically independent oocytes. P values are from two-sided Student’s t-tests. For e and f, experiments were repeated three times using different batches of oocytes and similar results were obtained. For images presented in ad, experiments were repeated three times using different biological materials and one representative image is shown.

Source data

Extended Data Fig. 10 Identification of CNGC4-CT phosphorylation sites.

a, Summary of the identified CNGC4-CT phosphorylation sites by liquid chromatography coupled with tandem mass spectrometry analysis. Lowercase letters indicate phosphorylated amino acids. Spectra of identified sites are presented in Supplementary Fig. 1. Experiments were repeated twice with similar results. b, CNGC4 with all the nine phosphorylation sites mutated (CNGC4-CT(9A/F)) completely eliminated phosphorylation by BIK1(4D) in vitro. Coomassie Brilliant Blue staining was used to verify the quality of samples and the loading consistency. Numbers beside the SDS–PAGE gels indicate the molecular mass (kDa) of proteins. Experiments were repeated three times with similar results. c, The mutations S514A, S544A, T613A, T652A, S653A and S655A do not affect the CNGC2–CNGC4 channel activity and the CaM-gated CNGC2–CNGC4 channel activation by BIK1(4D). CNGC4 variants were co-expressed with CNGC2, CNGC2 and CaM, or CNGC2, CaM and BIK1(4D) in oocytes. Current amplitudes at −140 mV from multiple recordings were shown. Data are presented as mean ± s.d., n = 12 biologically independent oocytes. P values are from two-sided Student’s t-tests. d, e, Proposed model for the role of the CaM-gated CNGC2–CNGC4 channel in the calcium-based PTI signalling pathway. d, Before pathogen exposure, the PRRs are inactive and the CNGC2–CNGC4 Ca2+ channel is gated closed, possibly by apo-calmodulin (Ca2+-free form), to maintain a resting [Ca2+]cyt level (around 100 nM). e, When PAMPs bind to PRRs, BIK1 is phosphorylated and activated by PRRs and in turn phosphorylates RBOHD. Active BIK1 also activates the CaM-gated CNGC2–CNGC4 channel complex directly, triggering Ca2+ influx. Ca2+ binding to EF-hand motifs in RBOHD and CDPKs activates RBOHD and boosts ROS production. The produced ROS may be perceived by a ROS sensor, leading to further activation of the CaM-gated CNGC2–CNGC4 channel, and thereby triggering a sustained increase in [Ca2+]cyt. Thus, the CaM-gated CNGC2–CNGC4-channel-mediated Ca2+ influx and ROS production form a positive feedback loop, propagating the defence signal and leading to optimized immune output.

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Tian, W., Hou, C., Ren, Z. et al. A calmodulin-gated calcium channel links pathogen patterns to plant immunity. Nature 572, 131–135 (2019). https://doi.org/10.1038/s41586-019-1413-y

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