MAPK Assays in Arabidopsis MAMP-PRR Signal Transduction

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


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 



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.


  1. 1.
    Felix G et al (1999) Plants have a sensitive perception system for the most conserved domain of bacterial flagellin. Plant J 18:265–276CrossRefPubMedGoogle Scholar
  2. 2.
    Kunze G et al (2004) The N terminus of bacterial elongation factor Tu elicits innate immunity in Arabidopsis plants. Plant Cell 16:3496–3507CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Tena G et al (2011) Protein kinase signaling networks in plant innate immunity. Curr Opin Plant Biol 14:519–529CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Tena G et al (2001) Plant mitogen-activated protein kinase signaling cascades. Curr Opin Plant Biol 4:392–400CrossRefPubMedGoogle Scholar
  5. 5.
    Asai T et al (2002) MAP kinase signaling cascade in Arabidopsis innate immunity. Nature 415:977–983CrossRefPubMedGoogle Scholar
  6. 6.
    Arthur J et al (2013) Mitogen-activated protein kinases in innate immunity. Nat Rev Immunol 13:679–692CrossRefPubMedGoogle Scholar
  7. 7.
    Cheng Z et al (2014) Pathogen-secreted protease activate a novel plant immune pathway. Nature 521:213–216CrossRefGoogle Scholar
  8. 8.
    Ranf S et al (2015) A lectin S-domain receptor kinase mediates lipopolysaccharide sensing in Arabidopsis thaliana. Nat Immunol 16:426–433CrossRefPubMedGoogle Scholar
  9. 9.
    Chow S et al (2001) Measurement of MAP kinase activation by flow cytometry using phospho-specific antibodies to MEK and ERK: potential for pharmacodynamic monitoring of signal transduction inhibitors. Cytometry 46:72–78CrossRefPubMedGoogle Scholar
  10. 10.
    Atkins C et al (1999) Regulation of myelin basic protein phosphorylation by mitogen-activated protein kinase during increased action potential firing in the hippocampus. J Neurochem 73:1090–1097CrossRefPubMedGoogle Scholar
  11. 11.
    Popescu S et al (2009) MAPK target networks in Arabidopsis thaliana revealed using functional protein microarrays. Genes Dev 23:80–92CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Cocking EC (1960) A method for the isolation of plant protoplasts and vacuoles. Nature 187:927–929CrossRefGoogle Scholar
  13. 13.
    Sheen J (2001) Signal transduction in maize and Arabidopsis mesophyll protoplast. Plant Physiol 127:1466–1475CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Yoo S et al (2007) Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2:1565–1572CrossRefPubMedGoogle Scholar

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

Personalised recommendations