Induction of Phytoalexin Biosynthesis: WRKY33 Is a Target of MAPK Signaling

Phytoalexins are a group of chemically diverse broad-spectrum antimicrobial compounds synthesized by plants in response to attack from pathogens. A large number of phytoalexins are products of the phenylpropanoid pathway, while others are synthesized through the mevalonic acid pathway and the Trp pathway. Studies have shown that phytoalexins play a role in plant disease resistance against pathogenic fungi and bacteria through rapid accumulation at the site of infection (reviewed in Dixon, 2001). Camalexin (3-thiazol-2′yl-indole), an indolic secondary metabolite, is a major phytoalexin in Arabidopsis thaliana. Its synthesis is stimulated by a variety of microorganisms, including Pseudomonas syringae, Alternaria brassicicola, and Botrytis cinerea and by some abiotic stresses, such as AgNO3 and amino acid starvation, and it has been shown to inhibit the growth of fungal pathogens. However, the signaling pathway connecting pathogen infection to camalexin biosynthesis is not completely known. One of the earliest signaling events following pathogen infection in Arabidopsis is the induction of a mitogen-activated protein kinase (MAPK) cascade involving MPK3 and MPK6. It was previously found that MPK3/MPK6 signaling activates camalexin biosynthetic genes (Ren et al., 2008). It has also been shown that camalexin induction in Arabidopsis infected with P. syringae is dependent on the transcription factor WRKY33, which binds directly to the promoter of the camalexin biosynthesis gene PAD3 (Qiu et al., 2008). Now, Mao et al. (pages 1639–1653) provide a critical missing link in this signaling cascade, with new results demonstrating that MPK3/MPK6 signaling leads directly to phosphorylation of WRKY33, and this drives camalexin production in Arabidopsis challenged by pathogens. First, the authors confirmed that WRKY33 is a pathogen-inducible transcription factor, and camalexin production is inhibited in wrky33 mutant plants. They then showed that WRKY33 is a substrate of MPK3/MPK6. In vitro MAPK phosphorylation assays with recombinant WRKY33 showed that MPK3 and MPK6 strongly phosphorylated WRKY33, whereas the related MPK10 did not. The WRKY33 protein sequence contains a cluster of five potential MAPK phosphorylation sites (Ser residues) near the N terminus. Mutation of these Ser residues abolished phosphorylation by MPK3/MPK6 and compromised the ability of transgenic WRKY33 to restore camalexin induction in the wrky33 mutant (see Figure). It was further demonstrated that WRKY33 is phosphorylated by MPK3/MPK6 in vivo in response to B. cinerea infection using a phospho-protein mobility shift assay. MPK3/MPK6 phosphorylation sites in WRKY33 are important for its function in vivo. Recombinant loss-of-phosphorylation WRKY33 (WRKY33SA) only partially complements the wrky33 mutant. Camalexin accumulation was determined at indicated number of hours after B. cinerea inoculation (Hr + Bc). FW, fresh weight. (Reprinted from Figure 5 ofMao et al. [2011].) This work establishes a direct link between MPK3/MPK6 and WRKY33, demonstrating that WRKY33 is a target of MPK3/MPK6 signaling and is necessary for the induction of camalexin biosynthesis in Arabidopsis following infection by the necrotrophic fungus B. cinerea.