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. Author manuscript; available in PMC: 2012 Apr 17.
Published in final edited form as: Cell Host Microbe. 2010 Feb 18;7(2):164–175. doi: 10.1016/j.chom.2010.01.009

Figure 7. RIN4 Mediates PDF1.2 Induction Down-Stream of MPK4.

Figure 7

(A) RIN4 is required for PDF1.2 induction by bacterially delivered AvrB. Plants of the indicated genotypes were infiltrated with the indicated bacterial strains and RNA was isolated 6 hr later for gene expression analysis. (B) RIN4 is required for JA-induced PDF1.2 expression. Plants of the indicated genotypes were treated with MeJA at the indicated times before RNA isolaton. (C) Overexpression of RIN4 constitutively activates PDF1.2 expression. WT or RIN4 transgenic (RIN4-ox) plants were treated with dexmethosome as described (Kim et al., 2005) for the indicated times before RNA was isolated for PDF1.2 expression analysis. (D) RIN4 interacts with MPK4 in vitro. Recombinant GST-MPK4 protein was incubated with bacterial lysates containing RIN4-His or CK-His (negative control as in Fig. 7A). (E) RIN4 interacts with MPK4 in plants. Protein extract from transgenic plants carrying NP-MPK4-3xFLAG was immunoprecipitated with an agarose-conjugated anti-FLAG antibody, and the presence of RIN4 in the immune complex was determined by immunoblot using anti-RIN4 antibodies. The presence of MPK4-3xFLAG was determined by immunoblot using anti-FLAG antibodies. Arrow head indicates MPK4-3xFLAG, whereas asterisk indicates IgG heavy chain from the anti-FLAG antibody used in immunoprecipitation. (F) MPK4 phosphorylates RIN4 in vitro. MPK4-3xFLAG was stimulated with (+) or without (−) flg22 in protoplasts, immunoprecipitated with anti-FLAG antibody, and the isolated MPK4-3xFLAG protein was incubated with recombinant RIN4 protein in an in vitro kinase assay. RIN4 phosphorylation (p-RIN4) was detected by autoradiography. CBB stain indicates amount of RIN4 protein in the gel.