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MAPK Assays in Arabidopsis MAMP-PRR Signal Transduction

  • Hoo Sun ChungEmail author
  • Jen Sheen
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1578)

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.

Key words

Arabidopsis Signal transduction MAMP PRR MAPK cascades Protoplast transient expression system Anti-phospho-ERK1/2 antibody Myelin basic protein (MBP) Kinase assay 

Notes

Acknowledgment

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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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Department of Molecular Biology and Center for Computational and Integrative BiologyMassachusetts General HospitalBostonUSA
  2. 2.Department of GeneticsHarvard Medical SchoolBostonUSA

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