The calcium-permeable channel OSCA1.3 regulates plant stomatal immunity

Abstract

Perception of biotic and abiotic stresses often leads to stomatal closure in plants^1,2. 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 elusive^3,4. 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.

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.. 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).

Fig. 1 |. OSCA1.3 associates with BIK1.

a, Co-immunoprecipitation of BIK1–haemagglutinin (HA) and OSCA1.3–GFP transiently expressed in Nicotiana benthamiana leaves treated with or without 1 μM flg22 for 10 min. GFP-LTI6b served as negative control. b, Co-immunoprecipitation of BIK1–HA and OSCA1.3–GFP from A. thaliana lines stably expressing BIK1–HA and OSCA1.3–GFP or GFP–LTI6b, respectively. Immunoprecipitation was performed with GFP agarose beads. Western blots were probed with antibodies against GFP and haemagglutinin. CBB, Coomassie brilliant blue. Uncropped blots are presented in Supplementary Fig. 1. Both experiments were performed three times with similar results.

Fig. 2 |. OSCA1.3 is phosphorylated by BIK1 and S54 is a major phosphorylation site.

a, In vitro GST pull down with recombinant GST–BIK1 and MBP–OSCA1.3 (amino acids 30–95). MBP was used as control. GST pull down was performed with glutathione resin and western blots probed with GST and MBP antibodies. The experiment was repeated three times with similar results. b, In vitro radioactive kinase assay performed with the corresponding recombinant proteins. The experiment was performed three times with similar results. c SRM relative quantification of tryptic phosphorylated peptide SSPLHS[+80] GALVSK at 0 and 5 min after flg22 treatment. Values are individual points and mean ± s.e. (n = 6, representing three biological repeats with two technical repeats each). ***P < 0.0001 (ordinary one-way ANOVA with multiple comparisons; NS, not significant). Uncropped blots are presented in Supplementary Fig. 1.

Fig. 3 |. OSCA1.3 is a BIK1-activated calcium-permeable channel.

a, OSCA1.3 complements growth of the calcium-uptake-deficient yeast mutant cch1/mid1. Filter discs containing 10 μg of the mating pheromone α factor were placed on nascent lawns of wild-type (WT) or cch1/mid1 yeast, or cch1/mid1 yeast complemented with AtOSCA1.3. DsRed served as control. Photographs were taken after 48 h. OSCA1.3, pYES-DEST52-OSCA1.3, DsRed, pYES-DEST52-DsRed. The experiment was repeated three times with similar results. b, Typical currents recorded in whole-cell configuration of COS-7 cells expressing OSCA1.3 or OSCA1.3(S54A) with or without the kinase BIK1 or the mutant BIK1(KD) (BIK1(K105A/K106A)). Voltage pulses were applied from −100 to +60 mV (1.5 s long, 20 mV steps). c, Current–voltage (I/V) curves of currents shown in b (n > 3 cells, mean ± s.e.m.). Solutions had two only main charge carriers, Na⁺ and Ca²⁺, with equilibrium potentials of −66.6 mV (Na⁺) and higher than +60 mV (Ca²⁺), respectively. OSCA1.3-mediated currents crossed the x-axis between −10 mV and −20 mV, compatible with the activity of a non-selective cationic channel permeable to Ca²⁺. Currents recorded at −100 mV in cells expressing OSCA1.3 plus BIK1 were significantly higher than in cells expressing OSCA1.3 alone (one-sided ANOVA, P = 0.004).

Fig. 4 |. OSCA1.3 and OSCA1.7 are required for stomatal immunity.

a, Box and scatter plot showing summed area under the curve (AUC) for wavelet-reconstructed profiles of the first 5 min of flg22-induced calcium spiking in Col-0 YC3.6 and osca1.3/1.7 YC3.6 guard cells. Each point represents the summed AUC for a single cell. Marker shapes represent individual independent experimental repeats and the box plot represents the distribution of all points for Col-0 or osca1.3/1.7. *P = 0.0024 (n = 4 biological replicates of independently grown batches of plants with three technical replicates and up to six cells assayed; linear mixed-effect model plus ANOVA, one-sided F-distribution). Maxima and minima of scatter are 121 to 72.4, respectively, for Col-0 and 113 to 69, respectively, for osca1.3/1.7. In box plots, centre lines show means of 91.6 for Col-0 and 86.7 for osca1.3/1.7; hinges are positioned at the 25th and 75th percentiles, and whiskers extend from the hinges to the largest or smallest value no more than 1.5× the inter-quartile range from the hinge. b, Stomatal aperture of wild-type, osca1.3, osca1.7 and osca1.3/1.7 plants treated with either 5 μM flg22 or water. Individual data points are shown with mean ± s.d. for n > 346 stomata from three experiments. ***P < 0.0001 (ordinary one-way ANOVA with multiple comparisons). c, Stomatal aperture of wild-type and osca1.3/1.7 plants treated with water, 5 μM AtPep1 or 10 μM ABA. Individual data points are shown with mean ± s.d. for n > 410 stomata from three experiments. ***P < 0.0001 (ordinary one-way ANOVA with multiple comparisons). d, Leaf transpiration recorded in excised intact leaves of wild-type and osca1.3/1.7 plants. Stimuli were added to the solution at the petioles at concentrations of 10 μM flg22 and 10 μM ABA, with 0.01% ethanol as control. Data are mean ± s.e.m. for n = 4 (Col-0 mock, osca1.3/1.7 flg22 and Col-0 ABA) or n = 5 (osca1.3/1.7 mock, Col-0 flg22 and osca1.3/1.7 ABA) leaves. The experiment was performed twice with similar results. e, Numbers of P. syringae pv. tomato (Pto) DC3000 COR⁻ bacteria determined 3 days after spray inoculation in Col-0, osca1.3/1.7 and bak1–5 plants. Individual data points are shown with mean ± s.d. for n = 22 to 24 plants from three experiments. *P = 0.012 (ordinary one-way ANOVA with multiple comparisons). f, Stomatal aperture of wild-type, osca1.3/1.7 plants and osca1.3/1.7 plants complemented with pOSCA1.3:OSCA1.3(WT) or pOSCA1.3:OSCA1.3(S54A), treated with 5 μM flg22 or water. Individual data points are shown with mean ± s.d. for n > 108 stomata counted over three independent T1 plants. ***P < 0.0001 (ordinary one-way ANOVA with multiple comparisons). The experiment was repeated three times with similar results.