Extended Data Fig. 9: flg22 or nlp20-induced immune responses in pcrk1/2 pbl19/20 quadruple mutant plants.
From: Activation of TIR signalling boosts pattern-triggered immunity
(a) Growth of Hpa Noco2 on the local leaves of WT, pcrk1/2, pcrk1/2 pbl19, and pcrk1/2 pbl19/20 quadruple mutant lines (#33 and #47) after 1 µM nlp20 treatment. Disease ratings showing the relative growth of Hpa Noco2 are as described in the Methods. The experiment was repeated three times with similar results. (b) Growth of Pto DC3000 in the leaves of four-week-old WT and pcrk1/2 pbl19/20 quadruple mutant lines (#33 and #47) pre-treated with H2O or 1 μM nlp20. The treated leaves were infiltrated with Pto DC3000 (OD600=0.001) 24 h post treatment with nlp20. Samples were taken 3 days after Pto DC3000 inoculation. Bars represent mean ± s.d. (n = 8 plants). The reduction of bacterial titer after nlp20 treatment in each genotype was regarded as nlp20-induced protection, which was compared among the WT and the mutants. The experiment was repeated three times with similar results. (c) Levels of SAG in four-week-old soil-grown plants of the indicated genotypes treated with H2O or 1 μM nlp20 for 24 h. Bars represent mean ± s.d. (n = 3 biologically independent samples). The experiment was repeated three times with similar results. (d, e) nlp20-induced MAPK activation in the indicated genotypes. 12-day-old seedlings were treated with 0.1 μM nlp20. MAPK activation was analyzed by immunoblotting with the anti-pERK antibody (d). Equal loading is confirmed by Ponceau staining of Rubisco. Quantifications of the phosphorylated MPK6 and MPK3 were shown in (e). Bars represent mean ± s.d. (n = 4 different experiments). (f) nlp20-induced ROS production in the indicated genotypes. Leaf strips were treated with 1 μM nlp20 and production of ROS was measured as luminescence. The values show the peak of luminescence units through all the time points. Bars represent mean ± s.d. (n = 8 leaf disks from different plants). The experiment was repeated three times with similar results. (g, h) Induction of SARD1 (g) and FMO1 (h) in the indicated genotypes by nlp20. Total RNA extracted from 12-day-old plate-grown plants treated with 1 μM nlp20 for 4 h. The expression of each gene in H2O-treated WT was set as 1. Bars represent mean ± s.d. (n = 3 biologically independent samples). The experiment was repeated twice with similar results. (i, j) flg22-induced MAPK activation in the indicated genotypes. 12-day-old seedlings were treated with 0.1 μM flg22. MAPK activation was analyzed by immunoblotting with the anti-pERK antibody. Equal loading is confirmed by Ponceau staining of Rubisco. Quantifications of the phosphorylated MPK6 and MPK3 10 min after treatment with flg22 were shown in (j). Bars represent mean ± s.d. (n = 4 different experiments). (k) ROS production in the indicated genotypes after treatment with 0.1 μM flg22 measured as luminescence. The values show the peak of luminescence units through all the time points. Bars represent mean ± s.d. (n = 8 leaf disks from different plants). The experiment was repeated three times with similar results. (l, m) Relative expression levels of SARD1 (l) and FMO1 (m) in the indicated genotypes. Total RNA was isolated from 12-day-old plate-grown seedlings 4 h after spraying with H2O or 1 μM flg22. The expression of each gene in the H2O -treated WT plants was set as 1. Bars represent mean ± s.d. (n = 3 biologically independent samples). Experiments were repeated twice with similar results. (n) Free SA and SAG levels in four-week-old plants of the indicated genotypes after treatment with H2O or 1 μM flg22 for 9 h. Bars represent mean ± s.d. (n = 3 biologically independent samples). The experiment was repeated three times with similar results. (o) Growth of Pto DC3000 in the leaves of four-week-old plants of the indicated genotypes after treatment with H2O or 1 μM flg22. Bars represent mean ± s.d. (n = 6 plants). The reduction of bacterial titer after flg22 treatment in each genotype was regarded as flg22-induced protection, which was compared among different genotypes. The experiment was repeated three times with similar results. The data in (b, c, e–h, j–o) were analyzed by one-way ANOVA with Tukey’s test. Exact P values are provided in Supplementary Table 5.
