Extended Data Fig. 7: Expression of wild-type and mutated TIR proteins and CPKs in planta.
From: TIR immune signalling is blocked by phosphorylation to maintain plant growth
a-c, SNC1TIR-YFP, L6TIR-YFP, RBA1-HA, and CPK3CA-cLUC-Flag proteins were transiently expressed in N. benthamiana leaves and detected by western blot. The anti-GFP antibody was used to detect SNC1TIR-YFP and L6TIR-YFP. The anti-HA antibody was used to detect RBA1-HA. The anti-Flag antibody was used to detect CPK3CA-cLUC-Flag. Cell death phenotypes of wild-type TIRs with or without CPK3CA were shown in Fig. 3b. d,e, SNC1TIR-S26A-Flag, full-length SNC1-Flag and CPK3CA-HA were transiently expressed in N. benthamiana leaves and detected by western blot. The anti-Flag antibody was used to detect SNC1TIR-Flag or full-length SNC1-Flag. The anti-HA antibody was used to detect CPK3CA-HA. Cell death phenotypes of SNC1TIR-S26A and full-length SNC1 with or without CPK3CA were shown in Figs. 3c, d, respectively. f-l, Wild-type and mutated TIR proteins were transiently expressed in N. benthamiana leaves and detected by western blot. Total proteins were extracted from N. benthamiana leaves. Ponceau S staining of RuBisCO was used as a loading control. The anti-GFP antibody was used to detect wild-type and mutated TIR-YFP proteins (f-j,l). The anti-HA antibody was used to detect RBA1-HA (k). SNC1TIR (f-i) related cell death phenotype was shown in Fig. 3e and S6; L6TIR (j) related cell death phenotype was shown in Fig. 3f; RBA1 (k) related cell death phenotype was shown in Fig. 3g; while hSARM1tSAM-TIR (l) related cell death phenotype was shown in Fig. 3h. m, Expression of CPK3CA-Myc and Actin in Col-0, cpk3/4/5/6/11 quintuple mutant, and CPK3pro::CPK3CA transgenic plants in Col-0 background was analyzed by reverse transcription PCR. The corresponding plant growth phenotype was shown in Figs. 3l, m. All experiments were repeated at least three times with similar results.
