title: In vivo phosphorylation of WRKY transcription factor by MAPK.
abstract: 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.
authors.affiliation: RIKEN Center for Sustainable Resource Science (CSRS), Tsurumi, Yokohama, Japan.
authors.forename: Nobuaki
authors.initials: N
authors.lastname: Ishihama
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Adachi
authors.forename: Miki
authors.initials: M
authors.lastname: Yoshioka
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Yoshioka
mesh_terms: D060054:Agrobacterium / Q000235:genetics; D000906:Antibodies / Q000276:immunology / Q000302:isolation & purification; D015151:Immunoblotting; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010748:Phosphopeptides / Q000276:immunology / Q000378:metabolism; D010766:Phosphorylation; D018515:Plant Leaves / Q000201:enzymology; D010940:Plant Proteins / Q000378:metabolism*; D014026:Nicotiana / Q000201:enzymology* / Q000235:genetics; D014157:Transcription Factors / Q000378:metabolism*; D014170:Transformation, Genetic
chemical_list: D000906:Antibodies; D010748:Phosphopeptides; D010940:Plant Proteins; D014157:Transcription Factors; D020928:Mitogen-Activated Protein Kinases