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.
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References
Nakagami H, Pitzschke A, Hirt H (2005) Emerging MAP kinase pathways in plant stress signalling. Trends Plant Sci 7:339–346
Asai S, Ohta K, Yoshioka H (2008) MAPK signaling regulates nitric oxide and NADPH oxidase-dependent oxidative bursts in Nicotiana benthamiana. Plant Cell 20:1390–1406
Yang K-Y, Liu Y, Zhang S (2001) Activation of a mitogen-activated protein kinase pathway is involved in disease resistance in tobacco. Proc Natl Acad Sci U S A 98:741–746
Ishihama N, Yamada R, Yoshioka M, Katou S, Yoshioka H (2011) Phosphorylation of the Nicotiana benthamiana WRKY8 transcription factor by MAPK functions in the defense response. Plant Cell 23:1153–1170
Mao G, Meng X, Liu Y, Zheng Z, Chen Z, Zhang S (2011) Phosphorylation of a WRKY transcription factor by two pathogen-responsive MAPKs drives phytoalexin biosynthesis in Arabidopsis. Plant Cell 23:1639–1653
Andreasson E, Jenkins T, Brodersen P, Thorgrimsen S, Petersen NH, Zhu S, Qiu JL, Micheelsen P, Rocher A, Petersen M, Newman MA, Bjørn NH, Hirt H, Somssich I, Mattsson O, Mundy J (2005) The MAP kinase substrate MKS1 is a regulator of plant defense responses. EMBO J 24:2579–2589
Menke FL, Kang HG, Chen Z, Park JM, Kumar D, Klessig DF (2005) Tobacco transcription factor WRKY1 is phosphorylated by the MAP kinase SIPK and mediates HR-like cell death in tobacco. Mol Plant Microbe Interact 18:1027–1034
Ishihama N, Yoshioka H (2012) Post-translational regulation of WRKY transcription factors in plant immunity. Curr Opin Plant Biol 15:431–437
Bombarely A, Rosli HG, Vrebalov J, Moffett P, Mueller LA, Martin GB (2012) A draft genome sequence of Nicotiana benthamiana to enhance molecular plant–microbe biology research. Mol Plant Microbe Interact 25:1523–1530
Popescu SC, Popescu GV, Bachan S, Zhang Z, Gerstein M, Snyder M, Dinesh-Kumar SP (2009) MAPK target networks in Arabidopsis thaliana revealed using functional protein microarrays. Genes Dev 23:80–92
Mitsuhara I, Ugaki M, Hirochika H, Ohshima M, Murakami T, Gotoh Y, Katayose Y, Nakamura S, Honkura R, Nishimiya S, Ueno K, Mochizuki A, Tanimoto H, Tsugawa H, Otsuki Y, Ohashi Y (1996) Efficient promoter cassettes for enhanced expression of foreign genes in dicotyledonous and monocotyledonous plants. Plant Cell Physiol 37:49–59
Voinnet O, Rivas S, Mestre P, Baulcombe DC (2003) An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J 33:949–956
Cooper HM, Paterson Y (2008) Production of polyclonal antisera. Curr Protoc Mol Biol Chapter 11, Unit 11.12
Acknowledgments
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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Ishihama, N., Adachi, H., Yoshioka, M., Yoshioka, H. (2014). In Vivo Phosphorylation of WRKY Transcription Factor by MAPK. In: Komis, G., Å amaj, J. (eds) Plant MAP Kinases. Methods in Molecular Biology, vol 1171. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0922-3_14
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DOI: https://doi.org/10.1007/978-1-4939-0922-3_14
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