Arabidopsis MAPKKK δ-1 is required for full immunity against bacterial and fungal infection

Abstract

The genome of Arabidopsis encodes more than 60 mitogen-activated protein kinase kinase (MAPKK) kinases (MAPKKKs); however, the functions of most MAPKKKs and their downstream MAPKKs are largely unknown. Here, MAPKKK δ-1 (MKD1), a novel Raf-like MAPKKK, was isolated from Arabidopsis as a subunit of a complex including the transcription factor AtNFXL1, which is involved in the trichothecene phytotoxin response and in disease resistance against the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (PstDC3000). A MKD1-dependent cascade positively regulates disease resistance against PstDC3000 and the trichothecene mycotoxin-producing fungal pathogen Fusarium sporotrichioides. MKD1 expression was induced by trichothecenes derived from Fusarium species. MKD1 directly interacted with MKK1 and MKK5 in vivo, and phosphorylated MKK1 and MKK5 in vitro. Correspondingly, mkk1 mutants and MKK5RNAi transgenic plants showed enhanced susceptibility to F. sporotrichioides. MKD1 was required for full activation of two MAPKs (MPK3 and MPK6) by the T-2 toxin and flg22. Finally, quantitative phosphoproteomics suggested that an MKD1-dependent cascade controlled phosphorylation of a disease resistance protein, SUMO, and a mycotoxin-detoxifying enzyme. Our findings suggest that the MKD1-MKK1/MKK5-MPK3/MPK6-dependent signaling cascade is involved in the full immune responses against both bacterial and fungal infection.

Keywords: Fusarium; Disease resistance; MAPK cascade; MAPKKK; Raf kinase; immune response; protein phosphorylation; proteomics.

Figure 1.

Protein–protein interaction between MKDI and AtNFXL1. (A) SDS-PAGE of the AtNFXL1 protein-containing complex purified from T-2 toxin-treated WT and atnfxll mutant plants using an anti-AtNFXL1C antibody column. Designations on the left side indicate identified subunits specifically observed in WT. Asterisks indicate non-specific proteins. (B) Western blot analysis of purified AtNFXL1-containing complex using anti-AtNFXL1C antibody. (C) Schematic diagram of full length and partial AtNFXL1 using yeast two-hybrid analysis. (D) The interaction between AtNFXL1 and MKDI was investigated by yeast two-hybrid analysis. The concentrations of 3-amino-1,2,4-triazole (3-AT) are shown on the left. P53+T and LamC+T represent the positive and negative controls, respectively. Similar results were obtained in three independent experiments. (E) The binding of AtNFXL1 protein to the MKD1 protein was examined by pull-down assays. His epitope-tagged AtNFXL1DNDZn protein was applied to a Ni Sepharose High Performance column. Biotin–MKD1 was detected by Transcend™ Non -Radioactive Translation Detection Systems.

Figure 2.

Induction of MKD1 mRNA by trichothecenes. (A) Expression levels of MKD1 after T-2 toxin treatment were analysed by RT-qPCR. Circles and squares show the data from T-2 toxin- and mock-treated samples, respectively. Data points represent the mean ±SD (n=3). *P<0.05, based on Student’s t-test. (B) Expression levels of MKD1 6 h after DAS, DON, or flg22 treatment were analysed by RT-qPCR. Data points represent means ±SD (n=5). *P<0.05, **P<0.01, based on Student’s t-test. Similar results were obtained in three independent experiments. (C) Representative photos of GUS staining in PMKD1:GUS plants grown on MS agar medium with or without trichothecene (T-2 toxin, DAS, or DON). Scale bars: 1 mm. Similar results were obtained for more than 10 transgenic plants with each trichothecene treatment. (This figure is available in color at JXB online.)

Figure 3.

MKD1 is involved in disease resistance against bacterial and fungal phytopathogens. (A) Schematic structure of the MKD1 protein. (B) Position of the T-DNA insertion in the mkd1 mutant. Boxes show exons. Triangle indicates the insertion position of the T-DNA. (C) RNA gel blot analysis of MKD1 mRNA in WT and the mkd1 mutant. (D) Representative images of WT, the mkd1 mutant, and the complementation line (mkd1;gMKD1 transgenic plants) 2 d after inoculation with F. sporotrichioides conidia. Scale bars: 1 cm. T-2 toxin/tissue indicates the concentration of T-2 toxin in leaves. ND: not detected. Similar results were obtained in three independent experiments. (E) Trypan blue staining of F. sporotrichioides-inoculated leaves after 2 d. Scale bars: 100 µm. (F) Relative values for the classification of disease symptoms in F. sporotrichioides-inoculated leaves (n=17–30). The bars show disease severity. White (class 1): normal, light gray (class 2): leaf turned black; dark gray (class 3): partial hyphae; black (class 4): expanded aerial hyphae. *P<0.05, based on Man–Whitney U-test. (G) Representative photos of WT and mkd1 mutant; the complementation lines were grown on MS agar medium with or without 0.5 µM T-2 toxin for 2 weeks. Similar results were obtained in three independent experiments. Scale bars: 1 cm.

Figure 4.

Enhanced susceptibility of mkd1 mutant plants to the virulent pathogen PstDC3000. (A) Colony forming units (CFU) are shown as means ±SD (n=6). A significant difference between WT and the mkd1 mutant was observed in the number of CFU/g fresh weight (P<0.05, ANOVA). (B) Representative photos of WT and mkd1 mutant; the complementation lines were inoculated with the PstDC3000. Similar results were obtained in two independent experiments. (This figure is available in color at JXB online.)

Figure 5.

Downstream MKKs of the MKD1-dependent signaling cascade. (A) Protein–protein interactions between MKD1 and MKKs were examined by yeast two-hybrid analysis. The interactions were evaluated by β-galactosidase activity units per number of cells and incubation time. Black and white bars represent the values observed for the full-length MKD1 and for the kinase domain of MKD1, respectively. Results shown are means ±SD (n=3). *P<0.05, **P<0.01, based on Student’s t-test. Similar results were obtained in three independent experiments. (B) In vivo interactions of MKD1 with MKK1, MKK2, and MKK5 were examined by BiFC analysis. Images show the YFP signal in the root tip. Scale bars: 10 µm. Similar results were obtained in two independent experiment. (C) Phosphorylation of MKK1, MKK2, and MKK5 by constitutively active MKD1 (ΔMKD1) investigated by in vitro kinase assays. −, without ΔMKD1. (D) Phosphorylation sites on MKK1 and MKK5 targeted by MKD1. Phosphorylation sites targeted by MKD1 are shown above; autophosphorylation sites are shown below. S, serine; T, threonine. (This figure is available in color at JXB online.)

Figure 6.

Resistance of mkk1, mkk2, and MKK5RNAi transgenic plants against F. sporotrichioides. (A) Suppression of MKK5 mRNA in MKK5RNAi transgenic plants grown on MS medium containing 0.5 µM T-2 toxin. Amounts of MKK5 mRNA were normalized against ACTIN2, 8. MKK5 mRNA levels in MKK5RNAi transgenic plants are represented as fold changes of the WT level (n=5).**P<0.01, based on Student’s t-test. Similar results were obtained in two independent experiment. (B) Representative images of WT, mkk1, mkk2, and MKK5RNAi leaves 2 d after inoculation with F. sporotrichioides. Similar results were obtained in three independent experiments. Scale bars: 1 cm. (C) Relative values for the classification of disease symptoms (n=17–30). Bars describe data as explained in Fig. 3F.*P<0.05, based on Man–Whitney U-test. (This figure is available in color at JXB online.)

Figure 7.

Downstream MAPKs of the MKD1-dependent signaling cascade in response to T-2 toxin. (A) MAP kinase activities were investigated in mock- or T-2 toxin-treated WT and mkd1 mutant plants by in-gel kinase assays. Similar results were obtained in two independent experiments. (B) Immunoprecipitation kinase assay carried out with T-2 toxin-treated WT and mkd1 mutant plants using an anti-MPK6 antibody. Similar results were obtained in two independent experiments. (C) p44 and p47 MAPK correspond to MPK3 and MPK6, respectively. MAP kinase activities were examined in WT, mpk3, and mpk6 mutant plants after 3h of T-2 toxin treatment. (D) Activation of MPK3 and MPK6 by T-2 toxin was suppressed in the mkd1 mutant. These MAPK activities were investigated in WT and mkd1 mutant plants after 3 h of T-2 toxin treatment by in-gel kinase assays (n=3). Then, corresponding bands were quantified. *P<0.05, **P<0.01, based on Student’s t-test.

Figure 8.

Downstream MAPKs of the MKD1-dependent signaling cascade in response to flg22. (A) MAP kinase activities were investigated in mock- or flg22-treated WT and mkd1 mutant plants by in-gel kinase assays. Similar results were obtained in two independent experiments. (B) Immunoprecipitation kinase assay carried out with flg22-treated WT and mkd1 mutant plants using an anti-MPK3, -MPK4, and -MPK6 antibody. Similar results were obtained in two independent experiments.