Fig. 2: Transgenic verification of SBRR1 function in ShB resistance and requirement of kinase activity for SBRR1 function.

a, ShB disease severity of SBRR1 knockout lines (sbrr1-ko1/2/3, T2 generation) 14 days after R. solani inoculation in a greenhouse. b, ShB resistance of SBRR1 overexpression lines (SBRR1-OE1/2/3, T₃ generation). c, Immunoblotting assay using a pSer/Thr antibody to detect autophosphorylation of His-SBRR1-ICD (ICD, intracellular domain) and His-SBRR1-ICD^K553E. Ponceau S stains His-SBRR1-ICD and His-SBRR1-ICD^K553E proteins. P.S., Ponceau S staining. d, Complementation by WT (Com, T₃ generation) and kinase-dead (KiD, T₃ generation) constructs for the susceptible phenotype of the sbrr1 mutant (n = 18). e, Comparison of phosphorylation levels of SBRR1 protein in SBRR1-OE1 with or without R. solani infection. Total protein was fractionated in SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) containing Phos-tag or normal SDS-PAGE. ACTIN was used as a loading reference. f, LC-MS/MS (liquid chromatography-tandem mass spectrometry)-detected intensity ratios of phosphorylated (P-peptide) to un-phosphorylated peptide (UP-peptide) at S678/T682/T683/Y733 sites of SBRR1 protein in SBRR1-OE1 line with or without R. solani infection (n = 3). g, Immunoblot assay using a pSer/Thr antibody to detect autophosphorylation of His-SBRR1-ICD^TT682/683AA. P.S., Ponceau S staining. h, ShB resistance of SBRR1^TT682/683AA-OE lines (T2 generation) at 7 days after inoculation. Data in a, b, d, f and h are presented as means ± s.d. Statistical significances in a and b were determined by a two-sided Student’s t-test. Different lowercase letters in d and h indicate significant differences (P < 0.05) based on one-way ANOVA with Duncan’s multiple range test. Phosphatase in c, e and g indicates calf intestinal alkaline phosphatase. Scale bar (a, b and d), 5 cm. The in vitro and in vivo phosphorylation assays were done independently three times.

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