Progress 11/05/06 to 11/04/11
Outputs OUTPUTS: Plant metabolism is highly adaptive and changes rapidly after the sensing of extracellular stimuli. One good example is the rapid induction of phytoalexins, small secondary metabolites with antimicrobial activity in response to pathogen invasion, which is an integral part of plant defense. Biosynthetic pathways of a number of phytoalexins have been elucidated, and it is known that the induction of phytoalexin is associated with the activation of biosynthetic genes. However, the signaling pathway(s), downstream transcription factor(s), and how these signaling event(s) regulate transcription factor(s) are largely unknown. 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 wrky33 mutant. Using a phospho-protein mobility shift assay, we demonstrate that WRKY33 is phosphorylated by MPK3/MPK6 in vivo in response to B. 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. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts In animals and yeast, stress-responsive MAPKs regulate gene expression by direct phosphorylation of transcription factors. It has been speculated that plant stress/pathogen-responsive MAPKs have similar mechanism of action, although the direct evidence is scarce. WRKY transcription factors are unique to plants, and Arabidopsis has more than 70 members. Detailed characterization of WRKY33, a newly identified MPK3/MPK6 substrate, and its target genes will further the understanding of MAPK signaling in plants as well as the functional diversity and signaling specificity of MPK3 and MPK6. The tools generated in this project will have broad applications and will be shared with other groups. This project served as an excellent training environment for students/post-docs by using an integrative approach encompassing both experimental and computational biology to understand the roles of a MAPK cascade and its downstream transcription factor in reprogramming plant metabolism. Training of students and post-docs who can use a combinatory approach to study complex biological processes is critical to the advance of post-genome biology. In addition to scientific methodology, students/post-docs will also receive training in ethics and career development. Students (both graduate and undergraduate) and post-docs from under-represented groups will be actively recruited through institutional programs that reach out to minorities. Post-docs involved in the project will follow a career development plan supported by the research training in the lab, student mentoring experiences, and professional development training. Understanding the signal transduction pathways and the identification of important regulators of plant secondary metabolism could positively impact food/feed/biofuel production and/or quality, which is important to sustain the increasing world population.
Publications
- Mao, G., Meng, X., Liu, Y., Zheng, Z., Chen, Z., and Zhang, S. (2011) Phosphorylation of a WRKY transcription factor by two pathogen-responsive MAPKs drives phytoalexin biosynthesis in Arabidopsis. Plant Cell, 23: 1639-1653. (Highlighted in the Plant Cell In Brief)
- Ren, D., Liu, Y., Yang, K.-Y., Han, L., Mao, G., Glazebrook, J., and Zhang, S. (2008) Fungal-responsive mitogen-activated protein kinase cascade in signaling phytoalexin biosynthesis in Arabidopsis. Proceedings of the National Academy Sciences USA. 105: 5638-5643
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Progress 01/01/09 to 12/31/09
Outputs OUTPUTS: Induction of anti-microbial phytoalexins is an integral part of plant defense response. Camalexin, the major phytoalexin in Arabidopsis, is essential for fungal resistance. We recently demonstrated MPK3 and MPK6, two Arabidopsis pathogen-responsive mitogen-activated protein kinases (MAPKs) play essential role in signaling the Botrytis cinerea-induced camalexin biosynthesis (Ren et al., 2008). Genetic analysis placed the MPK3/MPK6 cascade upstream of PHYTOALEXIN DEFICIENT 2 (PAD2) and PAD3, but independent or downstream of PAD1 and PAD4. Camalexin induction after MPK3/MPK6 activation was preceded by rapid and coordinated up-regulation of multiple genes encoding enzymes in the tryptophan (Trp) biosynthetic pathway, in the conversion of Trp to indole-3-acetaldoxime (IAOx, a branch point between primary and secondary metabolism), and in the camalexin biosynthetic pathway downstream of IAOx. In search of the transcription factors downstream of MPK3/MPK6 in regulating defense-related gene expression, we found that a WRKY transcription factor is required for MPK3/MPK6-induced camalexin biosynthesis. In wrky33 mutant background, both the gain-of-function MPK3/MPK6- and pathogen-induced camalexin production are compromised, which is associated with the loss of activation of camalexin biosynthetic genes. WRKY33 is a pathogen-inducible transcription factor, whose expression is regulated by MPK3/MPK6 cascade. In addition, WRKY33 protein is phosphorylated by MPK3/MPK6 in vivo in response to B. cinerea infection or the activation on MPK3/MPK6 in the gain-of-function transgenic plants. Mutation of MPK3/MPK6-phosphorylation sites in WRKY33 compromises the camalexin induction in response to B. cinerea attack and in conditional gain-of- function MPK3/MPK6 system. We further demonstrated that WRKY33 is phosphorylated by MPK3/MPK6 in vivo in response to B. cinerea. These results provide direct evidences that WRKY33 is a substrate of MPK3/MPK6 and is involved in regulating camalexin biosynthesis in Arabidopsis. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts Activation of biosynthetic genes is known to be associated with the induction of phytoalexins in plants challenged by pathogens. However, the transcription factor(s) and regulatory pathways are largely unknown. Built on the finding of a specific MAPK cascade in signaling the pathogen-induced biosynthesis of camalexin, the major phytoalexin in Arabidopsis, we identified a WRKY transcription factor downstream of MPK3/MPK6 cascade. WRKY33 is regulated at two levels, transcriptional activation and post-translational phosphorylation by MPK3/MPK6. Understanding of the regulation of phytoalexin biosynthesis in plants may lead to the engineering of crops with enhanced resistance.
Publications
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