Extended Data Fig. 6: NPR4-point-mutant expression and their differential phenotypic effects.
From: Structural basis of salicylic acid perception by Arabidopsis NPR proteins
Related to Fig. 4. a, Western blot analysis of transgenic npr3 npr4 (npr3/4) seedlings expressing similar amounts of the NPR4–GFP variants 24 h after treatment with 0.1 mM SA. An antibody against GFP (anti-GFP) was used. Asterisk denotes a nonspecific band. Experiments were repeated two times with similar results. b, Western blot depicting cell-free protein-degradation assays comparing the rate of endogenous NPR1 degradation in protein extracts from a; quantifications of the data are shown in Fig. 4a. Arrows, endogenous NPR1; MG115/132, proteasome inhibitors. The ratios listed below each sample indicate NPR1 levels compared to 0 min for the degradation assay or 30 min for samples containing MG115/132. An antibody against NPR1 (anti-NPR1) was used. Experiments were repeated three times with similar results. c, d, In planta protein-degradation assays comparing the rate of endogenous NPR1 degradation in seedlings pretreated with 0.1 mM SA for 24 h. NPR1 was detected using an anti-NPR1 antibody (c) and the relative band intensities were quantified (d). n = 3 independent biological samples. Error bars indicate s.d. (centre values). e, Western blot analysis of transgenic npr3 npr4 seedlings expressing NPR4–GFP or NPR4(F426L)–GFP after a 24-h treatment with 0.1 mM SA. L1, L2, L6 and L7, independent transgenic lines; TPE, total protein extract; CBB, Coomassie brilliant blue. An antibody against GFP (anti-GFP) was used. Asterisks denotes a non-specific band. f, Fold change of PR1 expression in seedlings from e 24 h after 0.1 mM SA treatments. The data are normalized to UBQ5 expression, error bars indicate s.d. (n = 3). Statistical significance was determined by one-way ANOVA on log-transformed data, followed by Tukey’s multiple comparison correction; letters indicate statistical significance, P < 0.05. g, Western blot analysis of mature leaves from transgenic npr3 npr4 plants expressing NPR4–GFP, NPR4(F426L)–GFP or NPR4(F426L/T459G)–GFP after a 6-h treatment with 0.5 mM SA spray. L8 and L11 denote independent transgenic lines. An antibody against GFP (anti-GFP) was used. Asterisk denotes a non-specific band. h, Fold change of PR1 expression in leaves from g 6 h after mock or 0.5 mM SA spray. The data are normalized to UBQ5 expression. n = 5 biologically independent samples. Error bars indicate s.d. (centre values). Statistical significance was determined by one-way ANOVA on log-transformed data, followed by Tukey’s multiple comparison correction; letters indicate statistical significance, P < 0.05. i, j, SA protection against P. syringae pv. maculicola ES4326 infection. Images of the development of disease symptoms (i) and bacterial growth in infected leaves (j) were recorded 3 d after inoculation at OD600 nm = 0.001. Light grey bars, mock; dark grey bars, 0.1 mM SA. Colony-forming units (cfu) were determined for three experiments and combined using linear mixed effect model (lme4) with experiment as random effects. n = 3 experiments each with 8 biological repeats per genotype and treatment. Error bars indicate s.d. (centre value). Statistical significance was determined by two-way ANOVA on log-transformed data. NS P = 0.6, *P = 0.03; **P = 0.008; ***P = 0.0004. Experiments in i were repeated three times with similar results. k, Relative band intensities were quantified after in planta protein degradation assays comparing the rate of endogenous NPR1 degradation in seedlings pretreated with 1 mM SA for 24 h as in c, d. n = 5 biologically independent samples. Error bars indicate s.e.m. (centre values).
