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. 2012 May;24(5):2225-36.
doi: 10.1105/tpc.112.097253. Epub 2012 May 29.

The MEKK1-MKK1/MKK2-MPK4 kinase cascade negatively regulates immunity mediated by a mitogen-activated protein kinase kinase kinase in Arabidopsis

Qing Kong  1 , Na Qu, Minghui Gao, Zhibin Zhang, Xiaojun Ding, Fan Yang, Yingzhong Li, Oliver X Dong, She Chen, Xin Li, Yuelin Zhang
Affiliations

The MEKK1-MKK1/MKK2-MPK4 kinase cascade negatively regulates immunity mediated by a mitogen-activated protein kinase kinase kinase in Arabidopsis

Qing Kong et al. Plant Cell. 2012 May.

Abstract

In Arabidopsis thaliana, the MEKK1-MKK1/MKK2-MPK4 mitogen-activated protein (MAP) kinase cascade represses cell death and immune responses. In mekk1, mkk1 mkk2, and mpk4 mutants, programmed cell death and defense responses are constitutively activated, but the mechanism by which MEKK1, MKK1/MKK2, and MPK4 negatively regulate cell death and immunity was unknown. From a screen for suppressors of mkk1 mkk2, we found that mutations in suppressor of mkk1 mkk2 1 (summ1) suppress the cell death and defense responses not only in mkk1 mkk2 but also in mekk1 and mpk4. SUMM1 encodes the MAP kinase kinase kinase MEKK2. It interacts with MPK4 and is phosphorylated by MPK4 in vitro. Overexpression of SUMM1 activates cell death and defense responses that are dependent on the nucleotide binding-leucine-rich repeat protein SUMM2. Taken together, our data suggest that the MEKK1-MKK1/MKK2-MPK4 kinase cascade negatively regulates MEKK2 and activation of MEKK2 triggers SUMM2-mediated immune responses.

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Figures

Figure 1.
Figure 1.
Suppression of mkk1 mkk2 Mutant Phenotypes by summ1-1 and summ1-2. (A) Morphology of the wild type (WT), mkk1 mkk2 (mkk1/2), summ1-1 mkk1 mkk2, and summ1-2 mkk1 mkk2. The photograph shows 4-week-old soil-grown plants. (B) and (C) Trypan blue (B) and DAB (C) staining of wild-type, mkk1 mkk2 (mkk1/2), summ1-1 mkk1 mkk2, and summ1-2 mkk1 mkk2 seedlings. (D) and (E) PR1 (D) and PR2 (E) expression in wild-type, mkk1 mkk2 (mkk1/2), summ1-1 mkk1 mkk2, and summ1-2 mkk1 mkk2 seedlings. Values were normalized to the expression of ACTIN1. Error bars represent ±sd of three replicates. (F) Growth of H.a. Noco2 on the wild type, mkk1 mkk2 (mkk1/2), summ1-1 mkk1 mkk2, and summ1-2 mkk1 mkk2. Three-week-old seedlings were sprayed with H.a. Noco2 spores (5 × 104 spores/mL). Infections were scored 7 d after inoculation by counting the number of resuspended conidia spores per gram of leaf samples. Error bars represent ±sd of three replicates. This experiment was repeated three times with similar results.
Figure 2.
Figure 2.
Map-Based Cloning of SUMM1. (A) Map position and the mutation in summ1-1. WT, the wild type. (B) Protein structure of SUMM1/MEKK2. a.a., amino acids. (C) Mutations identified in the summ1 alleles and the consequences of mutations to SUMM1/MEKK2 protein.
Figure 3.
Figure 3.
Suppression of mpk4-3 and mekk1-4 Mutant Phenotypes by summ1 Mutants. (A) Morphology of the wild type (WT), mpk4-3 (mpk4), and summ1-1 mpk4-3 (summ1 mpk4). The photograph shows 4-week-old soil grown plants. (B) and (C) Trypan blue (B) and DAB (C) staining of wild-type, mpk4-3, and summ1-1 mpk4-3 seedlings. (D) and (E) PR1 (D) and PR2 (E) expression in wild-type, mpk4-3, and summ1-1 mpk4-3 seedlings. Values were normalized to the expression of ACTIN1. Error bars represent ±sd of three replicates. (F) Morphology of the wild type, npr1-1 (npr1), mekk1-4 npr1-1 (mekk1 npr1), and summ1-3 mekk1-4 npr1-1 (summ1 mekk1 npr1). The photograph was taken on 4-week-old soil-grown plants. (G) and (H) Trypan blue (G) and DAB (H) staining of wild-type, npr1-1, mekk1-4 npr1-1, and summ1-3 mekk1-4 npr1-1 seedlings. (I) and (J) PR1 (I) and PR2 (J) expression in the wild type, npr1-1, mekk1-4 npr1-1, and summ1-3 mekk1-4 npr1-1. Values were normalized to the expression of ACTIN1. Error bars represent ±sd of three replicates.
Figure 4.
Figure 4.
Analysis of Different Immune Responses in summ1-1. (A) flg22-induced oxidative burst in summ1-1. Leaf slices were treated with 1 μM flg22 before ROS was measured. Error bars represent the sd of 12 independent samples. RLU, relative luminescence units; WT, wild type. (B) flg22-induced MAPKs activation. Two-week-old seedlings grown on half-strength MS medium were treated with 10 μM of flg22. Samples were collected at 0, 10, 20, and 30 min and analyzed by immunoblots using an anti-Erk antibody (Cell Signaling; #4370S). (C) and (D) Real-time RT-PCR analysis of the induction of FRK1 (C) and WRKY29 (D) in the wild type and summ1-1 by flg22. Two-week-old seedlings grown on half-strength MS plates were sprayed with 10 μM of flg22 4 h before samples were taken. Error bars represent ±sd of three replicates. (E) to (G) Growth of P.s.t. DC3000 avrRpt2 (E), P.s.t. DC3000 avrRps4 (F), and P.s.t. DC3000 (G) on the indicated genotypes. Five-week-old plants grown under short-day conditions were infiltrated with P.s.t. DC3000 at a concentration of OD600 = 0.002 and P.s.t. DC3000 avrRpt2 and P.s.t. DC3000 avrRps4 at a concentration of OD600 = 0.001. Samples were taken at 0 h (Day 0), 24 h (Day 1), 48 h (Day 2), and 72 h (Day 3) after inoculation, respectively. Error bars represent ±sd of six replicates. (H) Growth of H.a. Noco2 on the wild type and summ1-1. Four-week-old plants were sprayed with spores of H.a. Noco2 at a concentration of 50,000 spores/mL. Error bars represent sd of three replicates. *P < 0.05, statistical difference from the wild type. All experiments in this figure were independently repeated three times with similar results. [See online article for color version of this figure.]
Figure 5.
Figure 5.
Overexpression of SUMM1 Leads to Activation of Cell Death and Defense Responses. (A) Morphology of the wild type (WT) and SUMM1-FLAG transgenic lines OX#1 and OX#2. The photograph shows soil-grown plants ∼4 weeks after planting. (B) and (C) Trypan blue (B) and DAB (C) staining of the seedlings of the wild type and the SUMM1-FLAG transgenic lines OX#1 and OX#2. (D) Real-time RT-PCR analysis of SUMM1 expression in the wild type and the SUMM1-FLAG transgenic lines OX#1 and OX#2. Error bars represent ±sd of three replicates. (E) and (F) PR1 (E) and PR2 (F) expression in the wild type and the SUMM1-FLAG transgenic lines. Error bars represent ±sd of three replicates. (G) and (H) Free SA (G) and SAG (H) levels in the wild type and the SUMM1-FLAG transgenic lines. This experiment was repeated twice with similar results. Error bars represent ±sd of four replicates. (I) Growth of H.a. Noco2 on the wild type and the SUMM1-FLAG transgenic lines. Inoculation of the pathogen and scoring of the infection were performed as shown in Figure 1F. Error bars represent ±sd of three replicates. This experiment was repeated three times with similar results. fw, fresh weight.
Figure 6.
Figure 6.
MPK4 Interacts with MEKK2 and Phosphorylates the N Terminus of MEKK2. (A) Yeast two-hybrid analysis of the interaction between MPK4 and MEKK2. (B) Yeast two-hybrid analysis of the interaction between MPK4 and the N-terminal (MEKK21-494) and C-terminal (MEKK2495-773) domains of MEKK2. (C) Co-IP of MPK4 with MEKK2-3xFLAG in total proteins extracts from SUMM1-3xFLAG transgenic plants. Total protein extracts were subjected to immunoprecipitation with anti-FLAG Sepharose beads. Crude lysates (left panel, Input) and immunoprecipitated proteins (right panels, Elution) were detected with anti-FLAG, anti-MPK4, anti-MPK3, and anti-MPK6 antibodies, respectively. Wild-type plants without the SUMM1-3xFLAG transgene were used as a negative control. This experiment was repeated three times with similar results. (D) Phosphorylation of the N terminus of SUMM1/MEKK2 by MPK4. MPK4 was immunoprecipitated from the wild type (WT) and mpk4-3. After incubation with [γ-32P]ATP and the immunoprecipitated MPK4 or mpk4-3 mutant protein in protein kinase buffer, the E. coli–expressed N-terminal domain of SUMM1 was separated on 10% SDS-PAGE. The autoradiograph of the gel is shown in the top panel, and immunoblot analysis of MPK4 levels is shown in the bottom panel. This experiment was repeated four times with similar results.
Figure 7.
Figure 7.
SUMM2 Is Required for Defense Responses Activated by SUMM1. (A) Morphology of ∼3-week-old SUMM1-FLAG transgenic lines and SUMM1-FLAG expression levels. Line #2 and #3 are two representative SUMMI1-FLAG transgenic lines from the transformation of wild-type plants. Line #4, #5, and #6 are three representative SUMM1-FLAG transgenic lines from the transformation of summ2-8 plants. Rubisco, ribulose-1,5-bis-phosphate carboxylase/oxygenase; WT, the wild type. (B) and (C) Trypan blue staining (B) and DAB staining (C) of the true leaves of indicated genotypes. (D) and (E) Expression levels of PR1 (D) and PR2 (E) as determined by quantitative PCR. Values were normalized relative to the expression of ACTIN1. Error bars represent sd of three measurements.
Figure 8.
Figure 8.
A Working Model for Repression of MEKK2-Mediated Immunity by the MEKK1-MKK1/2-MPK4 MAPK Cascade. MEKK2 functions as a positive regulator of the NB-LRR R protein SUMM2, and its activity is negatively regulated by the MEKK1-MKK1/2-MPK4 kinase cascade. As shown by the dashed arrow, the mechanism by which MEKK2 activates SUMM2 remains to be determined.
See this image and copyright information in PMC

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