Phosphorylation of a WRKY Transcription Factor by Two Pathogen-Responsive MAPKs Drives Phytoalexin Biosynthesis in Arabidopsis    

Abstract

Plant sensing of invading pathogens triggers massive metabolic reprogramming, including the induction of secondary antimicrobial compounds known as phytoalexins. We recently reported that MPK3 and MPK6, two pathogen-responsive mitogen-activated protein kinases, play essential roles in the induction of camalexin, the major phytoalexin in Arabidopsis thaliana. In search of the transcription factors downstream of MPK3/MPK6, we found that WRKY33 is required for MPK3/MPK6-induced camalexin biosynthesis. In wrky33 mutants, both gain-of-function MPK3/MPK6- and pathogen-induced camalexin production are compromised, which is associated with the loss of camalexin biosynthetic gene activation. WRKY33 is a pathogen-inducible transcription factor, whose expression is regulated by the MPK3/MPK6 cascade. Chromatin immunoprecipitation assays reveal that WRKY33 binds to its own promoter in vivo, suggesting a potential positive feedback regulatory loop. Furthermore, WRKY33 is a substrate of MPK3/MPK6. Mutation of MPK3/MPK6 phosphorylation sites in WRKY33 compromises its ability to complement the camalexin induction in the wrky33 mutant. Using a phospho-protein mobility shift assay, we demonstrate that WRKY33 is phosphorylated by MPK3/MPK6 in vivo in response to Botrytis cinerea infection. Based on these data, we conclude that WRKY33 functions downstream of MPK3/MPK6 in reprogramming the expression of camalexin biosynthetic genes, which drives the metabolic flow to camalexin production in Arabidopsis challenged by pathogens.

The Arabidopsis Mediator Complex Subunit16 Is a Key Component of Basal Resistance against the Necrotrophic Fungal Pathogen Sclerotinia sclerotiorum

“…The MED16pro: MED16-33FLAG transgene was then crossed into the previously characterized 35S:43Myc-WRKY33 transgenic line (Mao et al, 2011). We chose this transgenic line because the fusion protein has been demonstrated to be functional and can be easily detected using an anti-Myc antibody (Mao et al, 2011). Additionally, overexpression of WRKY33 has been shown to elevate basal levels of PDF1.2 (Zheng et al, 2006), which would allow us to detect the interaction between MED16 and WRKY33 in the absence of any treatment.…”

“…These bands were not present in med16-1 and wild-type plants, confirming the specificity of the anti-FLAG immunoblot analysis. The MED16pro: MED16-33FLAG transgene was then crossed into the previously characterized 35S:43Myc-WRKY33 transgenic line (Mao et al, 2011). We chose this transgenic line because the fusion protein has been demonstrated to be functional and can be easily detected using an anti-Myc antibody (Mao et al, 2011).…”

MED18 interaction with distinct transcription factors regulates multiple plant functions

“…The DNAbinding activity of WRKY33 is controlled by SIB1 (SIGMA FACTOR-BINDING PROTEIN1) and SIB2 (ref. 17), and its transcription activity is enhanced by MPK3/MPK6 phosphorylation 18 . Modification of histone tails is another layer of transcriptional control that was found to affect plant immune responses [19][20][21] .…”

Soybean Homologs of MPK4 Negatively Regulate Defense Responses and Positively Regulate Growth and Development

“…Among the 20 Arabidopsis MPKs, MPK3, MPK4, and MPK6 are implicated in defense responses and have been most extensively studied (Innes, 2001;Pitzschke et al, 2009b). While MPK3 and MPK6 act as positive regulators of defense responses (Asai et al, 2002;Menke et al, 2005;Takahashi et al, 2007;Ren et al, 2008;Mao et al, 2011), MPK4 negatively regulates defense responses (Petersen et al, 2000;Brodersen et al, 2006;Gao et al, 2008). Besides its role in defense, MPK4 also plays a role in cytokinesis (Kosetsu et al, 2010;Beck et al, 2011;Zeng et al, 2011).…”