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.
ArabidopsisSignal transduction MAMP PRR MAPK cascades Protoplast transient expression system Anti-phospho-ERK1/2 antibody Myelin basic protein (MBP) Kinase assay
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The authors thank the former and current members of the Sheen Laboratory for their efforts to develop and improve kinase assays and the Arabidopsis mesophyll cell transient expression system. This work was supported by the Gordon and Betty Moore Foundation fellowship to H.S.C through Life Science Research Foundation, and the National Science Foundation grant IOS-0618292 and the National Institute of Health grant R01 GM070567 to J.S.
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