Quantifying MAMP-induced Production of Reactive Oxygen Species in Sorghum and Maize

Background

In plants, pattern recognition receptors (PRRs) at the plasma membrane recognize microbe-associated molecular patterns (MAMPs, also known as pathogen-associated molecular patterns or PAMPs). MAMPs are molecules that are generally highly conserved among large groups of microbes and are not directly associated with pathogenesis (Segonzac and Zipfel, 2011). The most widely studied MAMP is flg22, a 22-amino acid epitope of bacterial flagellin (Zipfel et al., 2004; Sun et al., 2013). Chitin, a component of the fungal cell wall, has also been studied extensively (Newman et al., 2013). MAMP recognition by PRRs leads to a defense response termed the MAMP response or the MAMP-triggered immunity (MTI) response. The MAMP response can include phenomena such as cell wall reinforcement by callose and lignin deposition, changes in ion flux across the plasma membrane, changes in phytohormone concentrations, induction or repression of plant defense-related genes, and production of reactive oxygen species (ROS) and nitric oxide (NO) (Thomma et al., 2011). Several methods have been used to measure the MAMP response, these include measurement of: ROS production, NO production, growth inhibition, gene expression, MAP Kinase phosphorylation, callose deposition and lignification, seedling growth inhibition, and induced disease resistance (Vetter et al., 2012; Valdés-López et al., 2014; Lloyd et al., 2017; Zhang et al., 2017).

Studies have identified genetically-controlled variation in the MAMP response in a number of species including Arabidopsis thaliana (Vetter et al., 2012; Vetter et al., 2016), maize (Zhang et al., 2017), soybean (Valdés-López et al., 2011), tomato (Veluchamy et al., 2014) and sorghum (authors’ unpublished results) and in many cases quantitative trait loci (QTL) associated with variation in these responses have been identified. It is also becoming clear that quantitation of the MAMP response is complex. Relative rankings of lines in a population can vary substantially depending upon the assay (Lloyd et al., 2014; Zhang et al., 2017) and the MAMP used (Veluchamy et al., 2014; Vetter et al., 2016; Lloyd et al., 2017), the environmental conditions (Cheng et al., 2013) as well as the condition and growth stage of the plants (Singh et al., 2014; Zou et al., 2018).

Measurement of ROS production induced by the MAMP flg22 and chitin is probably the most commonly-used method of measuring the MAMP response. Details of the procedure used for measuring MAMP induced ROS production in corn and sorghum seedlings are provided below along with discussion of various considerations and caveats to optimize measurements when using this technique.