A High-Sensitivity, Microtiter-Based Plate Assay for Plant Pattern-Triggered Immunity.

Abstract

The first step in the plant immune response to pathogen challenge involves the perception of conserved epitopes, called microbe-associated molecular patterns (MAMPs), by cell-surface pattern recognition receptors (PRRs). Given the key roles that MAMPs and PRRs play in plant innate immunity, great effort has been expended to identify these molecules. Current methods for assaying these immune responses are often limited in their resolution and throughput, and consequently, there is a need for medium- to high-throughput methodologies. Here, we describe the development of a 96-well microtiter plate-based assay for plant pattern-triggered immunity that measures the activity of plant peroxidase (POX) enzymes produced in response to treatment with bacterial MAMPs. The system has been optimized to minimize both the amount of plant tissue and MAMPs required and displays up to three orders of magnitude greater sensitivity than the traditional luminol-based reactive oxygen species assay when measuring the plant response to treatment with the bacterial MAMP flg22, reaching detection limits in the picomolar range. This high sensitivity opens the possibility of evaluating the immune-eliciting effects of weaker elicitors. The throughput and material requirements of the assay make it ideal for screens involving quantitative measurement of the plant innate immune response to MAMPs.