Extended Data Fig. 3: CPKs interact with and phosphorylate SNC1TIR in vitro and in planta.
From: TIR immune signalling is blocked by phosphorylation to maintain plant growth
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.
