Abstract
Perception of biotic and abiotic stresses often leads to stomatal closure in plants1,2. Rapid influx of calcium ions (Ca2+) across the plasma membrane has an important role in this response, but the identity of the Ca2+ channels involved has remained elusive3,4. Here we report that the Arabidopsis thaliana Ca2+-permeable channel OSCA1.3 controls stomatal closure during immune signalling. OSCA1.3 is rapidly phosphorylated upon perception of pathogen-associated molecular patterns (PAMPs). Biochemical and quantitative phosphoproteomics analyses reveal that the immune receptor-associated cytosolic kinase BIK1 interacts with and phosphorylates the N-terminal cytosolic loop of OSCA1.3 within minutes of treatment with the peptidic PAMP flg22, which is derived from bacterial flagellin. Genetic and electrophysiological data reveal that OSCA1.3 is permeable to Ca2+, and that BIK1-mediated phosphorylation on its N terminus increases this channel activity. Notably, OSCA1.3 and its phosphorylation by BIK1 are critical for stomatal closure during immune signalling, and OSCA1.3 does not regulate stomatal closure upon perception of abscisic acid—a plant hormone associated with abiotic stresses. This study thus identifies a plant Ca2+ channel and its activation mechanisms underlying stomatal closure during immune signalling, and suggests specificity in Ca2+ influx mechanisms in response to different stresses.
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Data availability
Blot source images are presented in Supplementary Fig. 1. Identifiers for publicly available Arabidopsis lines are provided in Methods. Raw data and a detailed description of the analysis presented in Fig. 4a have been deposited on GitHub: https://github.com/TeamMacLean/peak_analysis. All SRM assay information and raw data have been deposited to the Panorama Skyline server and can be accessed via https://panoramaweb.org/Vzao3P.url. Source data are provided with this paper.
Code availability
All codes used for the wavelet analysis are available at https://github.com/TeamMacLean/peak_analysis.
Change history
17 November 2020
A Correction to this paper has been published: https://doi.org/10.1038/s41586-020-2954-9
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Acknowledgements
We thank H. Krutinová and S. Vanneste for assistance in early stages of this project; J. P. Kukkonen for assistance for setting up the HEK293T cell assays; J. Sun for assistance with Ca2+ measurements in guard cells; B. Brandt for help with structural modelling of OSCA1.3; P. He and E. Peiter for providing published materials; J. –M. Zhou for early strategic discussions on this project and for providing published materials; M. Smoker, J. Taylor and J. Lopez from the TSL Plant Transformation support group for plant transformation; the John Innes Centre Horticultural Services for plant care; and all past and current members of the Zipfel group for technical help and fruitful discussions. This work was supported by the European Research Council under the Grant Agreements No. 309858 and 773153 (grants ‘PHOSPHinnATE’ and ‘IMMUNO-PEPTALK’ to C.Z.), The Gatsby Charitable Foundation (to C.Z.), the University of Zürich (to C.Z.), and the Swiss National Science Foundation (grant 31003A_182625 to C.Z.). The Biotechnology and Biological Research Council supported C.Z. and G.E.D.O. with BB/P012574/1. S.J., J.D., T.A.D. and J. Gronnier were supported by post-doctoral fellowships from the European Molecular Biology Organization (EMBO-LTF no. 225-2015; EMBO-LTF no. 683-2018; EMBO-LTF no. 100-2017 and EMBO-LTF no. 438-2018, respectively). Y.K. was supported by JSPS KAKENHI Grant Numbers JP16H06186 and JP16KT0037. Work in the J.F. laboratory was supported by the National Institutes of Health (NIH R01 GM131043), the National Science Foundation (MCB1616437/2016 and MCB1930165/2019) and the University of Maryland. Work in the M.W. laboratory was supported by the Academy of Finland (grant numbers 275632, 283139 and 312498). R.H. and M.R.G.R. were supported by the German Research Foundation (DFG, HE 1640/34-1; HE 1640/40-1; RO2381/6-1 and RO2381/8-1).
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Authors and Affiliations
Contributions
C.Z. designed and supervised the project, and obtained funding. K.T. and S.J. conceived, designed and performed the majority of the plant and biochemical experiments. E.M. and J.F. provided the patch-clamp data in COS-7 cells; J. George performed some of the genetic and phenotypic characterization of the osca1.3/1.7 mutant. P.D. and F.L.H.M. performed the SRM assays. N.L., M.C. and G.E.D.O. provided the yeast complementation assays. K.H. and M.W. provided the HEK cell data. T.A.D. and J.D. performed aequorin and YC3.6 measurements in leaf discs. P.K. and J. Gronnier generated expression constructs for OSCA1.7. L.S. assisted with the genetic characterization of the mutants. Y.K. provided initial data on the BIK1–OSCA1.3 interaction. C.A.B. provided OSCA1.3 localization data. S.S., S.H., M.R.G.R. and R.H. assisted with initial electrophysiological characterization, conducted ion flux measurements and carried out gas-exchange recordings. D.M. analysed the guard-cell Ca2+ measurements. K.T. and C.Z. wrote the manuscript. All authors commented and agreed on the manuscript before submission.
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Extended data figures and tables
Extended Data Fig. 1 Predicted topology of OSCA1.3 with possible BIK1 phosphorylation sites and multiple alignment of loop 1 from Clade 1 OSCA proteins.
a, Topology was visualized using Protter (www.wlab.ethz.ch/protter) version 1.0 on the basis of information from ref. 26. Blue numbers indicate transmembrane regions. Possible BIK1 phosphorylation sites are highlighted in red. b, Protein sequence alignment of OSCA1.1 to OSCA1.8 showing amino acids 30 to 95. Clustal Omega alignments were visualized with Jalview 2.10.5. Possible BIK1 phosphorylation motifs (SxxL/I) are highlighted in red. Blue colour denotes % identity. c, Structural model for OSCA1.3. Arrows indicate the position of S54 located in the cytosolic loop.
Extended Data Fig. 2 OSCA1.3 localizes to the plasma membrane.
Confocal microscopy of osca1.3 cotyledons expressing OSCA1.3-GFP under the control of the OSCA1.3 promoter. Right Panel: Plasmolysis with 2 M NaCl underlines plasma membrane localization. Green: GFP; magenta: chlorophyll autofluorescence. The experiment was performed once.
Extended Data Fig. 3 PBL1 also phosphorylates OSCA1.3.
Differences in PBL1-mediated incorporation of radioactive phosphate in OSCA1.3 and its mutation variants. In vitro kinase assay performed with the corresponding recombinant proteins. For blot source data, see Supplementary Fig. 1. The experiment was performed twice with similar results.
Extended Data Fig. 4 OSCA1.3 promotes calcium influx in HEK cells.
HEK293T cells loaded with the calcium indicator Fura-2 and transfected with OSCA1.3-myc show an increase in fluorescence intensity ratio at 340/380 nm excitation compared to non-transfected cells after addition of sorbitol and calcium to the culture medium, indicating an increase in calcium influx. Data show mean ± s.d. (n = 4 technical replicates). Similar results were obtained in 3 independent biological repeats.
Extended Data Fig. 5 OSCA1.3 and OSCA1.7 are BIK1-activated calcium-permeable channels.
a, Typical currents (left panel) and corresponding I/V curves (right panel) recorded in OSCA1.3 plus BIK1 expressing COS-7 cells increase with increasing calcium concentrations as indicated on the figure legend (n = 3 cells, mean ± s.e.m.). Currents were normalized with current intensities recorded at -100 mV in the standard bath solution (5 mM calcium), and consequently expressed in normalized arbitrary units for easier comparison of reverse potential changes. Note the inward currents increase and the reverse potentials shift to positive values when extracellular calcium concentration increases, indicating a calcium permeation of the channel. See methods for solutions composition. b, Typical traces (left panel) and corresponding statistical analysis (right panel) of currents recorded in whole-cell configuration in COS-7 cells co-transfected with pCI-OSCA1.7 plus pCI-BIK1 (n = 17 cells, mean ± s.e.m.) or plus pCI-BIK1(KD) (n = 9 cells, mean ± s.e.m.) as indicated on the figure legend. OSCA1.7 is a BIK1-activated channel. I/V curves recorded on cells. c, BIK1 kinase activity activates currents in cells expressing both OSCA1.3 and OSCA1.7. Typical currents (left panel) and corresponding I/V curves (right panel) recorded in cells co-transfected with both pCI-OSCA1.3 and pCI-OSCA1.7 plus pCI-BIK1 (n = 10 cells, mean ± s.e.m.) or plus pCI-BIK1(KD) (n = 9 cells, mean ± s.e.m.) as indicated on the figure legend. Note that current intensities are not higher than current intensities recorded in cells expressing either OSCA1.3+BIK1 (Fig. 3b, c) or OSCA1.7+BIK1 (a), giving no indication on functional heteromerization of OSCA1.3 and OSCA1.7. Whole-cell patch clamp protocols used in b and c were identical to the one used in Fig. 3b, c.
Extended Data Fig. 6 T-DNA insertion lines used in this study and transcript levels.
a, Gene structure of OSCA1.3 and OSCA1.7 showing exons (black boxes) and introns (lines) as well as location of T-DNA insertions. Line osca1.3/1.7 was obtained by crossing osca1.3 and osca1.7. Arrows denote location of primers used for genotyping. b, Transcript levels of OSCA1.3 and OSCA1.7 in Col-0, osca1.3, osca1.7 and osca1.3/1.7 as determined by quantitative real-time PCR with reverse transcription. Values are mean +/− s.d. (n = 6, representing 2 independent experiments with 3 biological repeats each). c, Transcript levels of OSCA1.3 in Col-0, osca1.3/1.7 and osca1.3/1.7 complemented with OSCA1.3(WT) or OSCA1.3(S54A), respectively. Expression levels for three independent T1 plants corresponding to Fig. 4f are shown separately, with two technical replicates (leaves). This experiment was repeated three times. Shown are quantitative real-time RT–PCR data relative to U-box (At5g15400). Primers used in b and c are listed in Supplementary Table 2.
Extended Data Fig. 7 Expression pattern of OSCA genes from Clade 1.
Tissue-specific expression patterns were obtained from Genevestigator (www.genevestigator.com). OSCA1.3 shows high expression levels in guard cells and guard cell protoplasts.
Extended Data Fig. 8 Flg22-induced calcium influx measured in leaf discs is comparable between wild-type and osca1.3/1.7 plants.
a, Calcium influx in leaf discs taken of Col-0 and osca1.3/1.7 plants expressing the calcium reporter aequorin. flg22 was added at time point 10 min. Error bars represent mean ± s.d. (n = 12 leaf discs from 6 independent plants). The experiment was performed twice with similar results. b, Average values of FRET ratio changes in leaf discs of Col-0 and osca1.3/1.7 expressing the ratiometric calcium reporter YC3.6 obtained in plate reader-based assays. Error bars show s.e.m., n = 90 leaf discs (Col-0) and 47 leaf discs (osca1.3/1.7), with 6 leaf discs taken per individual plant. The experiment was performed twice with similar results.
Extended Data Fig. 9 Flg22-induced calcium fluxes in osca1.3/1.7 guard cells are reduced compared to wild-type guard cells.
a, Typical flg22-induced spiking patterns and their distribution in Col-0 and osca1.3/1.7 guard cells. Legends show ratio changes of the Yellow Cameleon 3.6 calcium reporter observed over time (flg22 added at time point 10 min, indicated by an arrow). The pattern of every cell (n = 64 for wild-type and n = 61 for osca1.3/1.7) was assigned to one of the categories based on visual assessment. b, Left panel, net calcium fluxes of a representative Col-0 and osca1.3/1.7 guard cell, respectively, measured using Scanning Ion Selective Electrodes (SISE). Right panel, integrated calcium fluxes over 7 min after addition of flg22 are reduced in osca1.3/1.7 compared to Col-0 (n = 29 cells for Col-0, n = 23 cells for osca1.3/1.7; error bars represent mean ± s.e.m. bootstrapped Welch two sample t-test, two-sided P = 0.0464.). c, Left panel, flg22-induced calcium fluxes are blocked by lanthanum. Representative calcium fluxes measured using Scanning Ion Selective Electrodes (SISE) of Col-0 guard cells with or without lanthanum pre-treatment (1 mM lanthanum applied 10 min before flg22 treatment). One micromolar flg22 was added at time point 0 to epidermal strips. Right panel, integrated calcium fluxes over 8 min after addition of flg22 are significantly blocked by lanthanum in Col-0 (n = 8 cells without lanthanum and n = 5 cells with lanthanum; error bars represent mean ± s.e.m.; bootstrapped Welch two sample t-test, two-sided P = 0.0026).
Extended Data Fig. 10 AtPep1-induced decrease in stomatal conductance is impaired in osca1.3/1.7.
Leaf transpiration was recorded in excised intact leaves. AtPep1 was added to the solution at the petioles to a concentration of 3 μM, water was used as control. Data show mean ± s.e.m. (Col-0 mock, Col-0 AtPep1, osca1.3/1.7 AtPep1: n = 8; osca1.3/1.7 mock: n = 11 leaves).
Supplementary information
Supplementary Information
This file contains Supplementary Figure 1: Source data for images for gels and blots. Original source images for all data obtained by SDS-PAGE, western blots, autoradiography scans and Coomassie Blue stained blots and gels; Supplementary Table 1: Specific transitions used for selected reaction monitoring (SRM) with OSCA1.3 and control peptide; Supplementary Table 2: Primers used in this study.
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Thor, K., Jiang, S., Michard, E. et al. The calcium-permeable channel OSCA1.3 regulates plant stomatal immunity. Nature 585, 569–573 (2020). https://doi.org/10.1038/s41586-020-2702-1
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DOI: https://doi.org/10.1038/s41586-020-2702-1
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