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. 2007 Feb;143(2):661-9.
doi: 10.1104/pp.106.091389. Epub 2006 Dec 1.

MEKK1 is required for flg22-induced MPK4 activation in Arabidopsis plants

Maria Cristina Suarez-Rodriguez  1 , Lori Adams-Phillips, Yidong Liu, Huachun Wang, Shih-Heng Su, Peter J Jester, Shuqun Zhang, Andrew F Bent, Patrick J Krysan
Affiliations

MEKK1 is required for flg22-induced MPK4 activation in Arabidopsis plants

Maria Cristina Suarez-Rodriguez et al. Plant Physiol. 2007 Feb.

Abstract

The Arabidopsis (Arabidopsis thaliana) gene MEKK1 encodes a mitogen-activated protein kinase kinase kinase that has been implicated in the activation of the map kinases MPK3 and MPK6 in response to the flagellin elicitor peptide flg22. In this study, analysis of plants carrying T-DNA knockout alleles indicated that MEKK1 is required for flg22-induced activation of MPK4 but not MPK3 or MPK6. Experiments performed using a kinase-impaired version of MEKK1 (K361M) showed that the kinase activity of MEKK1 may not be required for flg22-induced MPK4 activation or for other macroscopic FLS2-mediated responses. MEKK1 may play a structural role in signaling, independent of its protein kinase activity. mekk1 knockout mutants display a severe dwarf phenotype, constitutive callose deposition, and constitutive expression of pathogen response genes. This dwarf phenotype was largely rescued by introduction into mekk1 knockout plants of either the MEKK1 (K361M) construct or a nahG transgene that degrades salicylic acid. When treated with pathogenic bacteria, the K361M plants were slightly more susceptible to an avirulent strain of Pseudomonas syringae and showed a delayed hypersensitive response, suggesting a role for MEKK1 kinase activity in this aspect of plant disease resistance. Our results indicate that MEKK1 acts upstream of MPK4 as a negative regulator of pathogen response pathways, a function that may not require MEKK1's full kinase activity.

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Figures

Figure 1.
Figure 1.
Mutation of mekk1 causes a dwarf phenotype. A, Location of T-DNA insertions. Thick lines are exons. Thin lines are introns and untranslated regions. B, Twelve-day-old homozygous mekk1-1 and mekk1-2 plants grown at 24°C are indicated by arrows. Larger plants are wild-type Col-0. Bar = 1 cm. C, Twelve-day-old plants grown at 24°C on agar plates. Bar = 0.5 cm. D, Environmental scanning electron microscope images of primary leaf epidermis of 10-d-old plants grown at 24°C. Bar = 50 μm. E, RT-PCR of mekk1-1 and mekk1-2 plants. PCR primer locations indicated by arrowheads in A. Actin gene Act2 used as a control. F, Plants grown on soil for 3 weeks at 32°C. Bar = 1 cm.
Figure 2.
Figure 2.
The mekk1 dwarf phenotype is caused by constitutive expression of stress response genes. A, Condition tree comparing the gene expression profile of mekk1-1 plants with various stress-treated Arabidopsis samples from a public database. Dark red indicates highly up-regulated genes, dark blue indicates highly down-regulated genes, and yellow indicates genes with little change in expression. B, RT-PCR of eight genes showing a high level of up-regulation in mekk1-1 plants in the microarray experiment. Three independent biological replicates were tested (Rep 1–Rep 3). 32°, Plants grown for 3 weeks at 32°C. 24°, Plants grown at 32°C for 3 weeks followed by 24 h at 24°C. Fold change indicates the relative expression level of each gene in mekk1-1 compared to wild-type Col-0 grown in the same conditions. C, Eleven-day-old seedlings stained for callose deposition and visualized using UV epifluorescence microscopy. No callose-inducing treatment was given to the seedlings. D, Two-week-old plants grown at 27°C. mekk1-2 + nahG is a homozygous mekk1-2 plant stably transformed with the nahG transgene. White arrows indicate representative primary leaves for comparison. One leaf is missing from the mekk1-2 + nahG plant, because it was harvested for genotyping. Bars = 0.5 cm.
Figure 3.
Figure 3.
Kinase-inactive MEKK1 (K361M) rescues the mekk1 dwarf phenotype. A, Three-week-old plants grown on soil at 24°C. Bars = 1 cm. B, Agarose gel of RT-PCR products generated by 22, 32, or 42 PCR cycles using gene-specific primer pairs for PR-1 or an actin control gene. mekk1 + K361M, mekk1-1 mutant stably transformed with MEKK1 (K361M). mekk1 + MEKK1, mekk1-1 mutant stably transformed with the wild-type MEKK1 gene. [See online article for color version of this figure.]
Figure 4.
Figure 4.
MAPK activation in response to flg22 peptide. Crude protein extracts were prepared from 14-d-old seedlings and subjected to an in-gel kinase assay using myelin basic protein as the embedded substrate. Kinase activity corresponding to MPK3, MPK4, and MPK6 is indicated. Seedlings were harvested at the indicated number of minutes following flg22 treatment. 0, No flg22 treatment. A, mekk1 mutant versus wild type. B, mekk1 + MEKK1, mekk1-1 mutant stably transformed with the wild-type MEKK1 gene. mekk1 + K361M, mekk1-1 mutant stably transformed with MEKK1 (K361M).
Figure 5.
Figure 5.
K361M seedlings are responsive to treatment with flg22 peptide. A, Five-day-old seedlings subsequently treated with and without 10 μm flg22 peptide for 10 d. Error bars indicate se. B, Ten-day-old seedlings treated with 10 μm flg22 peptide for 24 h and stained for callose deposition. Independent replicated experiments gave similar results for both A and B. mekk1 + MEKK1, mekk1-1 mutant stably transformed with the wild-type MEKK1 gene. mekk1 + K361M, mekk1-1 mutant stably transformed with MEKK1 (K361M). fls2, fls2-101/fls2-101 T-DNA mutant.
Figure 6.
Figure 6.
K361M plants have altered responses to Pst DC3000. A, Bacterial leaf populations 3 d after inoculation with Pst DC3000 ± avrRpt2. Four biological replicates were performed and a representative graph is shown (mean ± se of the mean). ANOVA using a generalized linear model was applied to the data from all four replicates, and Tukey's simultaneous test was used to determine the likelihood that bacterial populations were similar in a given plant genotype compared to the wild-type control treated with the same bacterial strain (*, P < 0.05; **, P < 0.01). mekk1 + MEKK1, mekk1-1 mutant stably transformed with the wild-type MEKK1 gene. mekk1 + K361M, mekk1-1 mutant stably transformed with MEKK1 (K361M). B, Leaves were tested for the HR by syringe inoculation with P. syringae pv Glya Race 4 pV288 (avrRpt+). The progression of the HR was monitored over a period of 24 h and was recorded using a scale from 0 = no tissue collapse to 5 = extensive tissue collapse. Data are mean ± se of the mean. Independent replicated experiments gave similar results. mekk1 + MEKK1, mekk1-1 mutant stably transformed with the wild-type MEKK1 gene. mekk1 + K361M, mekk1-1 mutant stably transformed with MEKK1 (K361M).
See this image and copyright information in PMC

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