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. 2018 Jun 5;30(7):1543–1561. doi: 10.1105/tpc.17.00981

Figure 7.

Figure 7.

Phosphorylation of MAPKKK5 Ser-682/692 Enhances MPK3/6 Activation and Immunity.

(A) and (B) Phosphorylation of Ser-682/692 in MAPKKK5 is required for MAPK activation induced by chitin (A) and flg22 (B). Seedlings of mapkkk3-2 mapkkk5-2 mutant T2 transgenic lines complemented with EV, wild-type MAPKKK5 (WT), MAPKKK5S682/S692A (2A), or MAPKKK5S682/692E (2E) were sprayed with chitin (A) or flg22 (B), and total protein was subjected to immunoblot analysis with anti-pERK and anti-HA antibodies. Numbers indicate arbitrary densitometry units of phosphorylated MPK3/6 normalized to EV. All assays were performed at least three times, and a representative photograph is shown.

(C) Phospho-mimetic mutations at Ser682/692 prolong chitin-triggered MPK3/6 activation. Seedlings of mapkkk3-2 mapkkk5-2 transgenic lines complemented with MAPKKK5S682/692E (2E) and Col-0 were treated with chitin for the indicated times, and total protein was subjected to immunoblotting with anti-pERK antibodies. Numbers indicate arbitrary densitometry units of phosphorylated MPK3/6 normalized to Rubisco. All assays were performed at least three times, and a representative photograph is shown.

(D) Phospho-mimetic mutations at Ser-599/682/692 do not further enhance chitin-triggered MPK3/6 activation compared with phospho-mimetic mutations at Ser-682/692. Protoplasts of the mapkkk3-2 mapkkk5-2 double mutant expressing EV, the wild type, phospho-dead (S599D), phospho-mimicking Ser682/692E (2E), and phospho-mimicking S599D, S682/692E (S599D,2E) forms of MAPKKK5 were treated with chitin. Total protein was subjected to immunoblot analysis with anti-pERK antibody. Numbers indicate relative protein band density of phosphorylated MPKs normalized to Rubisco. All assays were performed at least three times, and a representative photograph is shown.

(E) and (F) Phosphorylation of Ser-682/692 in MAPKKK5 is required for pattern-induced FRK1 expression. Ten-day-old seedlings were sprayed with chitin (E) and flg22 (F), and the samples were harvested at 3 h after treatment. ACTIN1 was used as the internal standard. Data are presented as mean ± sd. Different letters indicate significant difference at P < 0.05 (n = 3, one-way ANOVA, Tukey post-test, three independent experiments).

(G) Phosphorylation of Ser-682/692 in MAPKKK5 is required for immunity to P. syringae DC3000 and B. cinerea. Plants were infiltrated with Pto DC3000, and bacterial population size in the leaf and diseased lesions was determined 3 d after inoculation. Data are presented as mean ± sd. Different letters indicate significant difference at P < 0.05 (n ≥ 8, one-way ANOVA, Tukey post-test, three independent experiments).

(H) Phosphorylation of Ser-682/692 in MAPKKK5 is required for immunity to B. cinerea. Plants were inoculated with B. cinerea, and disease lesions were recorded 3 d after inoculation with B. cinerea. Lesion size represents mean ± sd. Different letters indicate significant difference at P < 0.05 (n ≥ 14, one-way ANOVA, Tukey post-test, three independent experiments).