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.

The Arabidopsis NUCLEUS- AND PHRAGMOPLAST-LOCALIZED KINASE1-Related Protein Kinases Are Required for Elicitor-Induced Oxidative Burst and Immunity

“…This possibility is supported by the observation that MEKK2 transcripts do not increase in the silenced amiR3 triple mutant, slightly increase in the anp2 anp3 double mutant, and do not further increase in the silenced amiR1 mutants (anp2 anp3 background). On the other hand, increase of MEKK2 expression up to 2.4-fold, as previously shown, is not sufficient to cause activation of the downstream autoimmunity cascade and dwarfism (Kong et al, 2012;Su et al, 2013). These data also suggest that cytokinesis defects of ANP mutants are independent of MEKK2, in agreement with what has been found in mpk4 (Kong et al, 2012;Su et al, 2013).…”

“…This mutant, which lacks the activation of the MEKK2-dependent autoimmunity response, exhibits both a normal morphological phenotype and strongly decreased levels of SA-dependent responses compared with mekk1 (Su et al, 2013). As expected, in the mekk1/mekk2/mekk3 mutant, mekk2 expression was absent (data not shown).…”

“…Thus, in silenced amiR3 mutants, the elevated basal defense gene expression is not accompanied by an increase of MEKK2 transcripts, strongly suggesting that MEKK2 does not mediate this effect. On the other hand, the moderate increase of MEKK2 transcripts in anp2 anp3 and amiR1 mutants, albeit lower than that observed in the mekk1 mutant (4-fold higher than that of the wild type; Su et al, 2013), may still reflect a role of MEKK2 in the increased basal expression of the defense marker genes of these mutants.…”

“…3 The increased basal expression of the defense-related marker genes in anp2 anp3 and in the silenced triple mutants recalls the phenotype of another MAP3K mutant, mekk1, which also shows a reduced growth (SuarezRodriguez et al, 2007). Both phenotypes of mekk1 have been demonstrated to be caused by an increased expression of the MAP3K MEKK2 (At4g08480) that takes place as a guard mechanism for the integrity of the MAPK cascade, leading to the activation of an SA-dependent autoimmune response (Kong et al, 2012;Su et al, 2013). We therefore investigated whether the higher basal expression of defense genes observed in the anp mutants is mediated by the MEKK2 pathway.…”

“…Three immunerelated MAPK modules have been identified. A module comprising the MAP3K MEKK1, the MAP2Ks MKK1 and MKK2, and the MAPK MPK4 (MEKK1/MKK1-MKK2/ MPK4) negatively controls defense responses (Kong et al, 2012;Rasmussen et al, 2012) by negatively regulating the expression of the MAP3K MEKK2, which triggers a salicylate (SA)-dependent autoimmunity response when the cascade is compromised (Berriri et al, 2012;Su et al, 2013). Two other modules, MEKK1-MAPKKKa/MKK4-MKK5/MPK3-MPK6 and MKK9/ MPK3-MPK6 (Xu et al, 2008), mediate the activation of defense responses, including the synthesis of ethylene and camalexin, i.e.…”