title: MAPK Assays in Arabidopsis MAMP-PRR Signal Transduction.
abstract: Activation of MAPK (Mitogen-Activated Protein Kinase) cascades after MAMP (Microbe-Associated Molecular Pattern) perception through PRR (Pattern Recognition Receptor) is one of the first conserved responses when plants encounter microbial organisms. Phosphorylation of various cellular factors in the MAMP-PRR pathway by MAPK cascades is critical for broad-spectrum plant innate immunity. Measurement of MAPK activation and identification of MAPK phosphorylation targets in the MAMP-PRR signal transduction pathway are essential to understand how plants reprogram their cellular processes to cope with unfavorable microbial attack. Here, we describe detailed protocols of three assays measuring MAPK activity after MAMP perception: (1) immune-blotting analysis with anti-phospho ERK1/2 antibody; (2) in-gel kinase assay using a general substrate myelin basic protein (MBP); (3) an in vitro kinase assay to evaluate phosphorylation of MAPK substrate candidates during MAMP-PRR signaling based on a protoplast expression system.
authors.affiliation: Department of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General Hospital, 185 Cambridge Street, Boston, MA, 02114, USA. hschung@molbio.mgh.harvard.edu.|Department of Genetics, Harvard Medical School, Boston, MA, 02114, USA. hschung@molbio.mgh.harvard.edu.
authors.forename: Hoo Sun
authors.initials: HS
authors.lastname: Chung
authors.affiliation: Department of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General Hospital, 185 Cambridge Street, Boston, MA, 02114, USA.|Department of Genetics, Harvard Medical School, Boston, MA, 02114, USA.
authors.forename: Jen
authors.initials: J
authors.lastname: Sheen
mesh_terms: D017360:Arabidopsis / Q000254:growth & development / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000378:metabolism; D015153:Blotting, Western; D057075:Enzyme Assays; D020935:MAP Kinase Signaling System; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010766:Phosphorylation; D057865:Plant Immunity
chemical_list: D029681:Arabidopsis Proteins; D020928:Mitogen-Activated Protein Kinases
keywords: Anti-phospho-ERK1/2 antibody; Arabidopsis; Kinase assay; MAMP; MAPK cascades; Myelin basic protein (MBP); PRR; Protoplast transient expression system; Signal transduction