Deletion of a tandem gene family in Arabidopsis: increased MEKK2 abundance triggers autoimmunity when the MEKK1-MKK1/2-MPK4 signaling cascade is disrupted

Abstract

An Arabidopsis thaliana mitogen-activated protein (MAP) kinase cascade composed of MEKK1, MKK1/MKK2, and MPK4 was previously described as a negative regulator of defense response. MEKK1 encodes a MAP kinase kinase kinase and is a member of a tandemly duplicated gene family with MEKK2 and MEKK3. Using T-DNA insertion lines, we isolated a novel deletion mutant disrupting this gene family and found it to be phenotypically wild-type, in contrast with the mekk1 dwarf phenotype. Follow-up genetic analyses indicated that MEKK2 is required for the mekk1, mkk1 mkk2, and mpk4 autoimmune phenotypes. We next analyzed a T-DNA insertion in the MEKK2 promoter region and found that although it does not reduce the basal expression of MEKK2, it does prevent the upregulation of MEKK2 that is observed in mpk4 plants. This mekk2 allele can rescue the mpk4 autoimmune phenotype in a dosage-dependent manner. We also found that expression of constitutively active MPK4 restored MEKK2 abundance to wild-type levels in mekk1 mutant plants. Finally, using mass spectrometry, we showed that MEKK2 protein levels mirror MEKK2 mRNA levels. Taken together, our results indicate that activated MPK4 is responsible for regulating MEKK2 RNA abundance. In turn, the abundance of MEKK2 appears to be under cellular surveillance such that a modest increase can trigger defense response activation.

Figure 1.

MEKK2 and MEKK3 Are Upregulated in mekk1 Dwarf Plants. (A) Wild-type (Col-0) and mekk1 mutant plants grown for 19 d on soil. Bars = 1 cm. (B) and (C) mRNA levels for MEKK2, MEKK3, and PR1 are shown relative to the wild type (Col-0) as determined by qRT-PCR using the PP2A gene as an internal standard. Values are the mean of three independent biological replicates with error bars indicating the se. Asterisks indicate a significant difference from the Col-0 sample as determined by a t test with P < 0.001. [See online article for color version of this figure.]

Figure 2.

Structural Analysis of the mekk1/2/3 Deletion Allele. (A) and (B) Locations of the mekk1 and mekk3 T-DNA insertions in the single mutant lines used to generate the mekk1/2/3 deletion. Thick black lines indicate exons, and arrows indicate direction of transcription. The numbered red lines indicate the locations of the PCR amplicons tested in (E). 1F, 1R, 3F, and 3R are gene-specific PCR primers. LB indicates a T-DNA left border–specific primer. (C) Predicted structure of a hypothetical mekk1 mekk3 double mutant. 2F1, 2R1, 2F2, and 2R2 are MEKK2 gene-specific PCR primers. (D) Predicted structure of the mekk1/2/3 deletion allele. Numbered black lines indicate PCR amplicons that were detected in mekk1/2/3 plants, while gray bars indicate those that were not. The region of the genome predicted to be missing in the mekk1/2/3 allele is indicated in gray. (E) Agarose gel electrophoresis of PCR products. Numbers to the left of each gel refer to the amplicon numbers shown in (A) and (B). “Col-0” is wild-type Columbia, and “no template” is the negative control. The primer pairs used for each PCR amplification are indicated to the right of each gel image.

Figure 3.

MEKK2 Is Responsible for the mekk1 Autoimmune Phenotype. (A) Plants with the indicated genotypes grown for 16 d on soil. mekk1/2/3 + MEKK2 and mekk1/2/3 + MEKK3 indicate lines carrying a genomic rescue construct for the indicated gene. Bars = 1 cm. (B) and (C) mRNA levels for MEKK2, MEKK3, and PR1 are shown relative to the wild type (Col-0) as determined by qRT-PCR using the PP2A gene as an internal standard. Values are the mean of three independent biological replicates with error bars indicating the se. Because the MEKK2 and MEKK3 loci are deleted in the mekk1/2/3 line, no PCR products are produced for those genes when mekk1/2/3 plants are analyzed. For this reason, no values are present on the graph for those samples. Three asterisks indicate a significant difference from the Col-0 sample as determined by a t test with P < 0.001, and one asterisk indicates P < 0.05. (D) Trypan blue staining was used to visualize cell death in the leaves of 2-week-old plants. Bars = 0.5 mm. (E) DAB staining was used to visualize hydrogen peroxide accumulation in the leaves of 2-week-old plants. Bars = 0.5 mm.

Figure 4.

The mekk1/2/3 Deletion Rescues the mkk1 mkk2 Autoimmune Phenotype in a Dosage-Dependent Manner. (A) Plants with the indicated genotypes grown for 17 d on soil. Bars = 1 cm. (B) and (C) mRNA levels for MEKK2 and PR1 are shown relative to the wild type (Col-0) as determined by qRT-PCR using the PP2A gene as an internal standard. Values are the mean of three independent biological replicates with error bars indicating the se. Because the MEKK2 locus is deleted in the mekk1/2/3 line, no PCR products are produced for that gene when mekk1/2/3 plants are analyzed. For this reason, no value is shown on the MEKK2 expression graph for the mekk1/2/3 genotype. An analysis of variance (ANOVA) was performed on the gene expression data, followed by Tukey's post-hoc analysis (α = 0.05). Means not sharing the same letter are significantly different. [See online article for color version of this figure.]

Figure 5.

A T-DNA Insertion in the Promoter Region of MEKK2 Rescues the mpk4 Autoimmune Phenotype in a Dosage-Dependent Manner. (A) Structure of the MEKK2 gene. Black boxes indicate exons, and the white box indicates the 3′-untranslated region. The white triangle indicates the location of the T-DNA insertion allele used in our study. Bar = 100 bp. (B) Plants with the indicated genotypes grown for 17 d on soil. Bars = 1 cm. (C) and (D) mRNA levels for MEKK2 and PR1 are shown relative to the wild type (Col-0) as determined by qRT-PCR using the PP2A gene as an internal standard. Values are the mean of three independent biological replicates with error bars indicating the se. Because the MEKK2 locus is deleted in the mekk1/2/3 line, no PCR products are produced for that gene when mekk1/2/3 plants are analyzed. For this reason, no value is shown on the MEKK2 expression graph for the mekk1/2/3 genotype. ANOVA was performed on the gene expression data followed by Tukey's post-hoc analysis (α = 0.05). Means not sharing the same letter are significantly different. [See online article for color version of this figure.]

Figure 6.

Overexpression of MEKK2 Causes an Autoimmune Phenotype. (A) The wild type (Col-0), three independent transgenic lines expressing MEKK2 from its native promoter (Col-0 + MEKK2), and three independent transgenic lines expressing MEKK2 via the cauliflower mosaic virus 35S promoter (Col-0 + 35S:MEKK2) grown on soil for 17 d. All transgenic lines are primary transformants in the wild-type (Col-0) genetic background. Bars = 1 cm. (B) and (C) mRNA levels for MEKK2 and PR1 are shown relative to the wild type (Col-0) as determined by qRT-PCR using the PP2A gene as an internal standard. Values are the mean of three independent biological replicates with error bars indicating the se. ANOVA was performed on the gene expression data followed by Tukey's post-hoc analysis (α = 0.05). Means not sharing the same letter are significantly different. [See online article for color version of this figure.]

Figure 7.

Genetic Background Potentiates the Effect of MEKK2 Overexpression. (A) The wild type (Col-0) and plants derived from two independent transgenic events carrying the MEKK2 native promoter expression construct grown for 17 d on soil. The independent transgenic events, labeled “event 1” and “event 2,” were originally produced in the wild-type Col-0 background. Each independent event was then transferred into the mekk1/2/3 genetic background by genetic crossing. Bars = 1 cm. (B) and (C) mRNA levels for MEKK2 and PR1 are shown relative to the wild type (Col-0) as determined by qRT-PCR using the PP2A gene as an internal standard. Values are the mean of three independent biological replicates with error bars indicating the se. ANOVA was performed on the gene expression data followed by Tukey's post-hoc analysis (α = 0.05). Means not sharing the same letter are significantly different. [See online article for color version of this figure.]

Figure 8.

MEKK2 Protein Abundance as Determined by SRM Mass Spectrometry. (A) Extracted ion chromatograms (XICs) for MEKK2 peptide EVVDGGTVESK. Three parent-ion to fragment-ion transitions were monitored for this peptide: y7 is represented by the purple trace, y8 by blue, and y9 by green. Only the strongest transition, y8, was used for quantification. The left panel displays XIC for the isotope-labeled peptide standard, while the right trace shows XIC for the endogenous peptide extracted from wild-type plants. The standard and endogenous peptides assayed in the same run have the same retention times and patterns of relative fragment ion intensity. (B) XIC for protein extracted from mekk1/2/3 plants as a negative control. No endogenous MEKK2 peptide was observed. (C) The abundance of the indicated MEKK2 peptides is presented relative to their levels in wild-type Col-0. For each genotype, the abundance of the peptide was first determined relative to the isotope-labeled internal standard peptide. These values were then normalized to the average value obtained for wild-type Col-0. Three independent biological replicates were measured for each genotype, and each biological replicate was analyzed using two to four injection replicates. XICs for the SLDFPNR peptide are presented in Supplemental Figure 6 online. ANOVA was performed separately for each peptide followed by Tukey's post-hoc analysis (α = 0.05). Means not sharing the same letter are significantly different.

Figure 9.

Constitutively Active MPK4 Reduces the Level of MEKK2 Expression in the mekk1 and mekk1 mpk4 Mutant Backgrounds. (A) Plants with the indicated genotypes grown for 17 d on soil. “+ MPK4-CA” indicates plants carrying a transgene expressing a constitutively active variant of MPK4 due to its D198G and E202A mutations. Bars = 1 cm. (B) and (C) RNA levels for MEKK2 and PR1 are shown relative to the wild type (Col-0) as determined by qRT-PCR using the PP2A gene as an internal standard. Values are the mean of three independent biological replicates with error bars indicating the se. Asterisks indicate a significant difference from the Col-0 sample as determined by a t test with P < 0.001. [See online article for color version of this figure.]

Figure 10.

Model for the Role of MEKK2 Regulation in Autoimmunity. MPK4 acts downstream of MEKK1 and MKK1/2 as a negative regulator of MEKK2 RNA abundance. Activated MPK4 is required for the repression of MEKK2 expression. In a wild-type plant, MEKK2 is expressed at a basal level. When the MEKK1-MKK1/2-MPK4 signaling pathway is disrupted, MEKK2 abundance increases, which in turn triggers the autoimmune response.