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Figure 1 Real-time PCR quantitation of PAD3, CYP71A13, NUDT6 and PPIase mRNAs. (A) Expression in Col-0 and wrky33 before or 24 h after treatment with BTH. (B) Expression in Col-0 and wrky33 before or after treatment with flg22, Pst DC3000 or Pst DC3000 (avrRpm1) for 30 min, 1, 2 and 4 h. The samples were tested in triplicate and normalized to ACTIN2. Means s.d. are shown. d.p.i.: days post-infection. h.p.i.: hours post-infection.
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 | Figure 2 WRKY33 binding to and activation from the PAD3 promoter. (A, B) Enrichment of P1 amplicons from chromatin immunoprecipitates with anti-WRKY33 antibodies from regions of the PAD3 promoter over background (measured with primers to a promoter region of the ROC5 control gene) in Col-0 and wrky33 treated with flg22 for 1.5 h (A) and after treatment with Pst DC3000 (avrRpm1) for 4 h (B). The samples were tested in triplicate and normalized to ROC5. Meanss.d. are shown. (C) Transient expression assays using a 2.4-kb fragment of the PAD3 promoter fused to the GUS reporter gene (PAD3:GUS) inoculated with Pst DC3000 (avrRpm1). Bombardments of detached leaves of Col-0 and wrky33 mutant were done with PAD3:GUS in combination with an empty vector or an effector of the CaMV 35S promoter driving WRKY33 or WRKY22 full-length cDNA. A 35S:LUC construct was used as a control for the efficiency of transient transformation. GUS activity in each sample was expressed relative to LUC activity to normalize data for variation in transformation efficiency.
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Figure 3 Levels of PAD3 mRNA and camalexin in Col-0 and wrky33 plants after infection with pathogens. (A) Camalexin levels after inoculation with Pst DC3000 or Pst DC3000 (avrRpm1). (B) Real-time PCR detection of PAD3 mRNA after inoculation with A. brassicicola. The samples were tested in triplicate and normalized to ACTIN2. (C) Camalexin levels after inoculation with A. brassicicola. Meanss.d. are shown. d.p.i.: days post-infection. h.p.i.: hours post-infection.
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 | Figure 4 Complexes between WRKY33, MKS1 and MPK4. (A) Co-immunoprecipitation of WRKY33 and MKS1 from nuclear extracts from: Bottom, Col-0 and wrky33 mutant immunoprecipitated with anti-WRKY33 and stained (WB) with anti-MKS1; Top, Ler and mks1 mutant immunoprecipitated with anti-MKS1 and stained with anti-WRKY33. One-tenth of inputs was stained with the same antibodies as a loading control. IgH: Ig heavy chain. (B) Top, co-immunoprecipitation of WRKY33 and MPK4 before and after treatment of Pst DC3000 (avrRpm1). Nuclear extracts of transgenic plants expressing MPK4-HA in the mpk4 background were immunoprecipitated with anti-WRKY33 and stained with anti-HA. Wild-type Ler plants were used as a negative control. One-tenth of input before and after the immunoprecipitation was stained with anti-HA as a control. Infected samples were harvested after 4 h. Bottom, co-immunoprecipitation performed as above for WRKY33 and MPK4 in Ler, mks1 and wrky33.
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Figure 5 Effects of infection on MPK4 activity and MKS1 protein, and of mks1 mutation on PAD3 mRNA levels. (A) Kinase activity of HA-tagged MPK4 immunoprecipitated from total protein extracts was assayed using 32P-labelled ATP and myelin basic protein (MBP) as substrate. h.p.i., hours post-infection. (B) Plants were treated with Pst DC3000 (avrRpm1) for 4 h. Total nuclear extracts in the presence of 10  M MG-132 were resolved on 12% SDS–PAGE, blotted and stained with anti-MKS1. Nonspecific band was used as a loading control. (C) Co-immunoprecipitation of MKS1 and WRKY33 4 h after P. syringae treatments. Nuclear extracts from untreated Ler and plants treated with Pst DC3000 (avrRpm1) were immunoprecipitated with anti-WRKY33 and stained with anti-MKS1. (D) Real-time PCR quantitation of PAD3 mRNA in Ler and mks1 mutant before or after treatment with Pst DC3000 (avrRpm1) for 4 and 21 h. (E) Camalexin levels after inoculation with Pst DC3000 or Pst DC3000 (avrRpm1).
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 | Figure 6 WRKY33 functions downstream of MPK4. (A) Expression of PAD3 in Ler, mpk4 and mpk4–nahG. (B) Effect of wrky33 mutation on constitutive PAD3 expression in mpk4. Samples were tested in triplicate and normalized to ACTIN2. Meanss.d. are shown. (C) Partial rescue of the mpk4 dwarf phenotype by wrky33 mutation.
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