Ishihama Nobuaki, Adachi Hiroaki, Yoshioka Miki, Yoshioka Hirofumi
RIKEN Center for Sustainable Resource Science (CSRS), Tsurumi, Yokohama, Japan.
Methods Mol Biol. 2014;1171:171-81. doi: 10.1007/978-1-4939-0922-3_14.
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.
植物会响应多种病原体衍生信号激活信号网络，通过丝裂原活化蛋白激酶（MAPK）级联反应促进转录重编程。鉴定MAPK的磷酸化靶点以及体内检测磷酸化底物是阐明植物免疫反应信号通路的重要过程。我们已鉴定出一种WRKY转录因子，它被防御相关的MAPK、SIPK和WIPK磷酸化。最近的证据表明，一些I组WRKY转录因子在N端区域含有保守基序，它们通过MAPK依赖性磷酸化被激活。在本章中，我们分别描述了抗磷酸肽抗体的制备方案、使用抗磷酸化MAPK抗体检测活化的MAPK以及使用抗磷酸化WRKY抗体检测活化的WRKY的方案。
