A Re-elicitation Assay to Correlate flg22-Signaling Competency with Ligand-Induced Endocytic Degradation of the FLS2 Receptor

  • Michelle E. Leslie
  • Antje HeeseEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1209)


In the model plant Arabidopsis, the best studied Pattern-triggered immunity (PTI) system is perception of the bacterial pathogen-associated molecular pattern (PAMP) flagellin, or its active peptide-derivative flg22, by the plasma membrane-localized receptor FLAGELLIN SENSING 2 (FLS2). Flg22 perception initiates an array of immune responses including the fast and transient production of reactive oxygen species (ROS). In addition, FLS2 undergoes ligand-induced endocytosis and subsequent degradation within 60 min of flg22-treatment.

Luminol-based assays are routinely used to measure extracellular ROS production within minutes after flg22 treatment. Many mutants in flg22-response pathways display defects in flg22-induced ROS production. Here, we describe a luminol-based ROS Re-elicitation Assay that can be utilized to quantitatively assess flg22-signaling competency of FLS2 at times during which FLS2 is internalized, trafficked through endosomal compartments, and degraded in response to flg22. This assay may also be employed to correlate FLS2 signaling competency with receptor accumulation in vesicular trafficking mutants that either affect FLS2 endocytosis or replenishment of FLS2 through the secretory pathway. In addition, this assay can be extended to studies of other PAMP (ligand)–receptor pairs.

Key words

Signaling competency Ligand-induced endocytosis Pattern recognition receptor (PRR) FLAGELLIN SENSING2 (FLS2) Pathogen-associated molecular pattern (PAMP) Flagellin flg22 Desensitization Reactive oxygen species (ROS) Luminol-based assay 



This work was supported by grants received from the National Science Foundation (NSF-IOS-1147032) and University of Missouri Research Board to A.H. We thank Daniel Salamango for contributions to assay development and John Smith for critical reading of this chapter.


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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Division of Biochemistry, Interdisciplinary Plant Group (IPG)University of Missouri-ColumbiaColumbiaUSA

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