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.
This is a preview of subscription content, log in to check access.
Springer Nature is developing a new tool to find and evaluate Protocols. Learn more
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.
Monaghan J, Zipfel C (2012) Plant pattern recognition receptor complexes at the plasma membrane. Curr Opin Plant Biol 15:349–357PubMedCrossRefGoogle Scholar
Nicaise V, Roux M, Zipfel C (2009) Recent advances in PAMP-triggered immunity against bacteria: pattern recognition receptors watch over and raise alarm. Plant Physiol 150:1638–1647PubMedCentralPubMedCrossRefGoogle Scholar
Beck M, Zhou J, Faulkner C et al (2012) Spatio-temporal cellular dynamics of the Arabidopsis flagellin receptor reveal activation status-dependent endosomal sorting. Plant Cell 24:4205–4219PubMedCentralPubMedCrossRefGoogle Scholar
Smith JM, Salamango DJ, Leslie ME et al (2014) Sensitivity to flg22 is modulated by ligand-induced degradation and de novo synthesis of the endogenous flagellin-receptor FLS2. Plant Physiol 164:440–454PubMedCentralPubMedCrossRefGoogle Scholar
Mueller K, Chinchilla D, Albert M et al (2012) Contamination risks in work with synthetic peptides: flg22 as an example of a pirate in commercial peptide preparations. Plant Cell 24:3193–3197PubMedCentralPubMedCrossRefGoogle Scholar
Felix G, Duran JD, Volko S et al (1999) Plants have a sensitive perception system for the most conserved domain of bacterial flagellin. Plant J 18:265–276PubMedCrossRefGoogle Scholar
Heese A, Hann DR, Gimenez-Ibanez S et al (2007) The receptor-like kinase SERK3/BAK1 is a central regulator of innate immunity in plants. Proc Natl Acad Sci U S A 104:12217–12222PubMedCentralPubMedCrossRefGoogle Scholar