In Vivo Phosphorylation of WRKY Transcription Factor by MAPK
Plants activate signaling networks in response to diverse pathogen-derived signals, facilitating transcriptional reprogramming through mitogen-activated protein kinase (MAPK) cascades. Identification of phosphorylation targets of MAPK and in vivo detection of the phosphorylated substrates are important processes to elucidate the signaling pathway in plant immune responses. We have identified a WRKY transcription factor, which is phosphorylated by defense-related MAPKs, SIPK and WIPK. Recent evidence demonstrated that some group I WRKY transcription factors, which contain a conserved motif in the N-terminal region, are activated by MAPK-dependent phosphorylation. In this chapter, we describe protocols for preparation of anti-phosphopeptide antibodies, detection of activated MAPKs using anti-phospho-MAPK antibody, and activated WRKY using anti-phospho-WRKY antibody, respectively.
Key wordsAnti-phosphopeptide antibody MAPK substrate WRKY transcription factor
We thank David C. Baulcombe for p19 construct, Yuko Ohashi and Ichiro Mitsuhara for pEL2 vector and MEK2 variants, and the Leaf Tobacco Research Center for N. benthamiana seeds. This work was partially supported by a Grant-in-Aid for Scientific Research from the Japan Society of the Promotion of Science.
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