Reciprocal phosphorylation between SOAK1 and SOBIR1 fine-tunes receptor-like protein (RLP)-mediated plant immunity

Abstract

SUPPRESSOR OF BIR1-1 (SOBIR1) is a receptor-like kinase (RLK) that acts as a coreceptor for multiple receptor-like proteins (RLPs) to mediate pathogen-associated molecular pattern)-triggered immunity. However, the regulation of SOBIR1 homeostasis and activity remains largely unknown. Our study reveals that SOBIR1-ASSOCIATED PROTEIN KINASE 1 (SOAK1), a member of the receptor-like cytoplasmic kinase (RLCK)-V subfamily with a transmembrane domain, negatively regulates multiple RLP-mediated immune responses. SOAK1 constitutively interacts with SOBIR1 and modulates SOBIR1-dependent immune signaling. SOAK1 directly phosphorylates SOBIR1 at serine-406, substantially impairing its ability to transphosphorylate itself and BAK1. The conservation of serine-406 residue among various flowering plants suggests that phosphorylation at this site plays a critical role in regulating plant immunity. Conversely, SOBIR1 also phosphorylates SOAK1 primarily at serine-73, inhibiting SOAK1's kinase activity and derepressing SOBIR1 activity. This study elucidates a regulatory mechanism for SOBIR1 activity and highlights an uncharacterized role of RLCK-V subfamily members in plant immunity.

Fig. 1.. SOAK1 associates with SOBIR1.

(A) Identification of representative proteins associating with SOBIR1 by protein immunoprecipitation and mass spectrum analysis. To identify SOBIR1-interacting proteins, SOBIR1 and its potential interacting proteins were immunoprecipitated by α-HA agarose beads from the SOBIR1-HA overexpression plants and analyzed by mass spectrometry. (B) Schematic diagram of the SOAK1 protein structure. The green box represents the predicted transmembrane domain, the blue box represents the predicted kinase domain, and the red line indicates the conserved ATP-binding site. The number represents the position of amino acids (aa). (C) Subcellular localization of SOAK1 in Arabidopsis transgenic plants overexpressing SOAK1. Four-week-old Arabidopsis mature leaves were visualized using a confocal microscope. Scale bars, 10 μm. (D) SOAK1 associates with SOBIR1 in Arabidopsis protoplast. Protoplasts from Col-0 coexpressing SOBIR1-HA and SOAK1-FLAG were treated with or without 1 μM pg13 for 10 min. Coexpressing SOBIR1-HA and LTI6B-FLAG or RLCK-V (AT5G18500)-FLAG as controls. Total proteins were immunoprecipitated with α-FLAG agarose beads and analyzed by immunoblotting with α-HA or α-FLAG. The input proteins were shown on the right two panels. (E) SOAK1 associates with SOBIR1 in Arabidopsis transgenic plants. Four-week-old transgenic plants carrying 35S::SOAK1-GFP/Col-0 or Col-0 were treated with or without 1 μM pg13 for 10 min. (F) SOAK1 associates with SOBIR1 in BiFC assay. SOAK1-nYFP (SOAK1 fused with nYFP), SOBIR1-cYFP (SOBIR1 fused with cYFP), RLCK-V-nYFP (AT5G18500 fused with nYFP), or LTI6B-cYFP (LTI6B fused with cYFP) as negative controls. Different combinations were coexpressed in N. benthamiana leaves, and YFP signals were observed using a confocal microscopy. Scale bars, 10 μm. (G) SOBIR1 interacts with the SOAK1 in a pull-down assay. GST-FLS2^KD and GST-SOAK1^KD immobilized on glutathione Sepharose beads were incubated with HIS-SOBIR1^KD proteins. Eluted proteins were subjected to immunoblotting with α-GST and α-HIS antibodies, respectively. Input proteins were shown by Coomassie brilliant blue (CBB) staining.

Fig. 2.. SOAK1 negatively regulates pg13-mediated immune responses.

(A) The soak1 mutants showed enhanced pg13-induced MAPK activation. Ten-day-old seedlings were treated with 1 μM pg13 at different time points. The MAPK activation was analyzed by immunoblotting with α-pERK antibody, and the protein loading is shown by Ponceau S staining for Rubisco (RBC). (B and C) Increased expression of PAD3 and CYP71A13 in soak1 mutants. Col-0, soak1-1, and soak1-2 seedlings were treated with ddH₂O (Mock) or 1 μM pg13 for 3 hours. Relative transcript levels of defense-related gene PAD3 (B) and CYP71A13 (C) were normalized to that of ACTIN1. The data are shown as means ± SD (n = 4). (D) The indicated four-week-old plants were preinfiltrated with ddH₂O (Mock) or 1 μM pg13 for 24 hours before inoculation with 8 μl of B. cinerea spores, and lesion areas were photographed 40 hours after inoculation. Scale bars, 1 cm. (E) Lesion areas were measured by ImageJ. The data are shown as means ± SD (n = 6). (F) The sobir1 soak1 exhibited similar MAPK activation to sobir1. Ten-day-old seedlings of the indicated plants were treated with 1 μM pg13 for 0 and 15 min. (G and H) Mutation of SOBIR1 suppresses the enhanced pg13-triggered resistance to B. cinerea in soak1. (G) The indicated four-week-old plants were preinfiltrated with ddH₂O (Mock) or 1 μM pg13 for 24 hours before inoculation with 8 μl of B. cinerea spores, and lesion areas were photographed 40 hours after inoculation. Scale bars, 1 cm. (H) Lesion areas were measured by ImageJ. The data are shown as means ± SD (n = 6). Different letters denote statistically significant differences according to two-way ANOVA followed by the Tukey’s test (P < 0.05). All above experiments were repeated at least three times with similar results.

Fig. 3.. The kinase activity of SOAK1 is indispensable for its immune function.

(A) SOAK1 exhibits autophosphorylation activity in vitro. The MBP-SOAK1^CD-HIS and MBP-SOAK1^CD-KM-HIS purified from E. coli were subjected to in vitro kinase assay. After separation by SDS-PAGE, the phosphorylated proteins were detected by autoradiography (Autorad; top panel). Inputs were assessed by Coomassie brilliant blue staining (bottom panel). (B and C) pg13-triggered MAPK activation in SOAK1-complemented plants and SOAK1^KM-complemented plants. Ten-day-old seedlings of Col-0, soak1, (B) SOAK1-complemented plants, and (C) SOAK1^KM-complemented plants were treated with 1 μM pg13 for 0, 5, and 15 min. The MAPK activation was analyzed by immunoblotting with α-pERK antibody (top panel). SOAK1 or SOAK1^KM was detected by immunoblotting with α-FLAG antibody (middle panel), and the protein loading is shown by Ponceau S staining for Rubisco (RBC) (bottom panel). (D) B. cinerea resistance of soak1 and SOAK1^KM-complemented plants. Four-week-old Col-0, soak1-1, and SOAK1^KM-complemented plants were preinfiltrated with ddH₂O (Mock) or 1 μM pg13 for 24 hours before inoculation with 8 μl of B. cinerea spores, and lesion areas were photographed 40 hours after inoculation. Scale bars, 1 cm. (E) Lesion areas were measured by ImageJ, and error bars indicate the SD from six biological replicates. Different letters indicate significant difference according to two-way ANOVA followed by the Tukey’s test (P < 0.05). All above experiments were repeated at least twice with similar results.

Fig. 4.. SOAK1 directly phosphorylates SOBIR1 at Ser⁴⁰⁶ and suppresses SOBIR1 transphosphorylation activity.

(A) SOAK1^CD phosphorylates SOBIR1^KD-KM in vitro. Purified GST- SOBIR1^KD-KM was incubated with MBP-SOAK1^CD-HIS or MBP-SOAK1^CD-KM-HIS in a kinase reaction buffer. After separation by SDS-PAGE, the phosphorylated proteins were detected by autoradiography (Autorad; top panel). Inputs were assessed by Coomassie brilliant blue staining (bottom panel). (B) The Ser⁴⁰⁶ residue of SOBIR1^KD is phosphorylated by SOAK1^CD in LC-MS/MS analysis. m/z, mass/charge ratio. (C) The Ser⁴⁰⁶ residue of SOBIR1 is the major SOAK1 phosphorylation site in vitro. The GST-SOBIR^KD-KM, GST-SOBIR1^KD-KM-S406A, GST-SOBIR1^KD-KM-S524A, and GST-SOBIR1^KD-KM-S592A were incubated with MBP-SOAK1^CD-HIS in a kinase reaction buffer, respectively. The phosphorylated proteins were detected by autoradiography. (D) pg13 triggers phosphorylation of SOBIR1 at Ser⁴⁰⁶ by SOAK1. Ten-day-old transgenic line pSOAK1::SOAK1-FLAG/soak1 was treated with ddH₂O or 1 μM pg13 for 5 min. Total protein was immunoprecipitated with α-FLAG agarose beads. The immunoprecipitates were inoculated with purified GST-SOBIR1^KD-KM or SOBIR1^KD-KM-S406A in a kinase reaction buffer, respectively. Phosphorylation was analyzed by autoradiography. (E) Compromised transphosphorylation of SOBIR1 by SOBIR1^S406D in vitro. The GST-SOBIR^KD-KM was incubated with HIS-SOBIR^KD, HIS-SOBIR^KD-KM, or HIS-SOBIR^KD-S406D in a kinase reaction buffer, respectively. Phosphorylation was analyzed by autoradiography. (F) Compromised transphosphorylation of BAK1 by SOBIR1^S406D in vitro. The GST-BAK1^CD-KM was incubated with HIS-SOBIR^KD or HIS-SOBIR^KD-S406D in a kinase reaction buffer, respectively. Phosphorylation was analyzed by autoradiography. (G) The SOBIR1 immunoprecipitants obtained from soak1 exhibited stronger phosphorylation than that from wild-type plants. The SOBIR1-HA was expressed in Col-0 and soak1 protoplast, respectively, and immunoprecipitated by α-HA agarose beads. The immunoprecipitants was incubated with GST-BAK1^KD-KM in a kinase reaction buffer. (H) Compromised transphosphorylation of PBL14, PBL11, and PBL31 by SOBIR1^S406D in vitro. The GST-PBL14^KM, GST-PBL11^KM, and GST-PBL31^KM were incubated with HIS-SOBIR1^KD or HIS-SOBIR^KD-S406D in a kinase reaction buffer, respectively. Phosphorylation was analyzed by autoradiography. The experiments, except (B), were repeated at least three times with similar results.

Fig. 5.. The Ser⁴⁰⁶ of SOBIR1 is indispensable for its immune function.

(A) SOBIR1^S406D-complemented plants failed to restore pg13-induced MAPK activation. Ten-day-old seedlings were treated with 1 μM pg13 for 0 and 15 min. The MAPK activation was analyzed by immunoblotting with α-pERK antibody, and the protein loading is shown by Ponceau S staining for Rubisco (RBC). (B and C) The SOBIR1^S406D-complemented plants failed to prime pg13-induced (B) PAD3 and (C) CYP71A13 expression in the sobir1-13 mutant. (D) The SOBIR1^S406D-complemented plants failed to prime pg13-triggered resistance to B. cinerea in sobir1-13 mutants. Scale bars, 1 cm. (E) Lesion areas were measured by ImageJ. Error bars indicate the SD from eight biological replicates, and the asterisk indicates statistically significant differences from Mock according to Student’s t test (**P < 0.01). (F) The SOBIR1/soak1sobir1 plants showed enhanced MAPK activation, but SOBIR1^S406D/soak1sobir1 plants showed compromised MAPK activation. Ten-day-old seedlings were treated with 1 μM pg13 for 0 and 15 min. The MAPK activation was analyzed by immunoblotting with α-pERK antibody, and the protein loading is shown by Ponceau S staining for Rubisco (RBC). (G and H) The expression of PAD3 (G) and CYP71A13 (H) was increased in the SOBIR1/soak1sobir1 plants but decreased in SOBIR1^S406D/soak1sobir1 plants. Ten-day-old seedlings were treated with ddH₂O (Mock) or 1 μM pg13 for 3 hours. Relative transcript levels of defense-related gene PAD3 and CYP71A13 were normalized to that of ACTIN1. Error bars indicate the SD from three biological replicates. The asterisk indicates statistically significant differences from the wild type within the same time point according to one-way ANOVA followed by the Dunnett’s test (**P < 0.01). n.s., not significant. All above experiments were repeated at least three times with similar results.

Fig. 6.. The conserved SOBIR1 S406D mutation suppresses its function among different species.

(A) The Ser⁴⁰⁶ of SOBIR1 is a conserved site across plant species. Multiple sequence alignment by DNAMAN9 (top panel) and WebLogo analyses (bottom panel) of SOBIR1 residues surrounding AtSOBIR1 Ser⁴⁰⁶ from different plant species. At, A. thaliana; As, Arabidopsis suecica; Al, Arabidopsis lyrata; Ca, Cardamine amara; Me, Microthlaspi erraticum; Cr, Capsella rubella; Cs, Camelina sativa; Bc, Brassica carinata; Ev, Eruca vesicaria subsp. sativa; Aa, Arabis alpina; Es, Eutrema salsugineum; Bn, Brassica napus; Bcr, Brassica cretica; Br, Brassica rapa; Rs, Raphanus sativus; Sa, Sinapis alba; Hi, Hirschfeldia incana; Th, Tarenaya hassleriana; Asa, Acer saccharum; Xs, Xanthoceras sorbifolium; An, Acer negundo; Ds, Dipteronia sinensis; St, Solanum tuberosum; Nb, N. benthamiana; Os, O. sativa; Zm, Zea mays. The preserved serine residue is pointed toward by a black triangle. Protein sequence data can be found in the NCBI (National Center for Biotechnology Information) database following accession numbers from table S2. (B) The expression of NbSOBIR1 or OsSOBIR1, but not NbSOBIR1^S400D or OsSOBIR1^S252D, can substantially restore the INF1-induced cell death response in the Nbsobir1 mutant. Cell death was visualized and photographed under UV light 48 hours after infiltration. Scale bars, 1 cm. The experiments in (B) were repeated twice with similar results.

Fig. 7.. SOBIR1 predominantly phosphorylates SOAK1 at Ser⁷³, counteracting the negative role of SOAK1 in plant immunity.

(A) SOBIR1 phosphorylates SOAK1 in vitro. Purified HIS-SOBIR1^KD was incubated with GST-SOAK1^CD-KM in a kinase reaction buffer. After separation by SDS-PAGE, the phosphorylated proteins were detected by autoradiography (Autorad; top panel). The protein inputs were assessed by Coomassie brilliant blue staining (bottom panel). (B) The Ser⁷³ residue of SOAK^CD is phosphorylated by SOBIR1^KD in LC-MS/MS analysis. (C) The Ser⁷³ residue of SOAK1 is the major SOBIR1 phosphorylation site. Phosphorylation of the MBP-SOAK1^CD-KM-HIS, MBP-SOAK1^CD-KM-S73A-HIS by HIS-SOBIR^KD was detected by autoradiography (Autorad; top panel). The protein inputs were assessed by Coomassie brilliant blue staining (bottom panel). (D) SOAK1^CD-S73D impairs the capacity of SOAK1 to transphosphorylate SOBIR1. The MBP-SOAK1^CD-S73D-HIS was incubated with GST-SOBIR1^KD-KM in a kinase reaction buffer. After separation by SDS-PAGE, the phosphorylated proteins were detected by autoradiography (Autorad; top panel). The protein inputs were assessed by Coomassie brilliant blue staining (bottom panel). (E) pg13-triggered MAPK activation in SOAK1^S73A-complemented and SOAK1^S73D-complemented plants. Ten-day-old seedlings of Col-0, soak1, SOAK1^S73A-complemented plants, and SOAK1^S73D-complemented plants were treated with 1 μM pg13 for 0 and 15 min. The MAPK activation was analyzed by immunoblotting with α-pERK antibody (top panel), and the protein loading is shown by Ponceau S staining for Rubisco (RBC) (bottom panel). (F and G) pg13-induced the expression of (F) PAD3 and (G) CYP71A13 in SOAK1^S73A-complemented and SOAK1^S73D-complemented plants. Ten-day-old seedlings of Col-0, soak1, SOAK1^S73A-complemented plants, and SOAK1^S73D-complemented plants were treated with ddH₂O (Mock) or 1 μM pg13 for 3 hours. Error bars indicate the SD from four biological replicates. The asterisk indicates statistically significant differences from Col-0 according to one-way ANOVA followed by the Dunnett’s test (**P < 0.01). The experiments, except (B), were repeated at least three times with similar results.