Background

Throughout evolution, plants developed the ability to detect microbe-derived molecules and mount immune responses that seize detrimental interactions (Boutrot and Zipfel, 2017). The microbial-associated molecular patterns (MAMPs) that induce such immune responses are often broadly conserved structural components of microbes, such as chitin from fungi and flagellin from bacteria (Cook et al., 2015). Recognition of MAMPs by extracellular plant receptors leads to MAMP-triggered immunity (MTI), which contributes to halt microbial invaders and maintain plant health (Böhm et al., 2014).

At the molecular level, MAMP perception is followed by an orchestrated set of biochemical events. Firstly, within seconds to few minutes, influx of Ca²⁺ and H⁺ ions occurs, leading to membrane depolarization and extracellular alkalinization (Ranf et al., 2011). Subsequently, production of reactive oxygen species (ROS) is observed, which may locally block pathogen growth and mediate downstream signaling (Bigeard et al., 2015). Phosphorylation of MAP kinases occurs within minutes after MAMP perception and promotes transcriptional activation of defense-related genes, many of which encode antimicrobial proteins or enzymes involved in the synthesis of hormones and secondary metabolites (Bigeard et al., 2015). Lastly, accumulation of antimicrobial compounds and callose deposition are observed, further hampering microbial growth and spread.

Mechanistic understanding of the plant immune system and its activity in plant-microbe interactions has advanced through methods that monitor many of the biochemical events underlying MTI. The most widely used experimental approaches are based on detection of ROS burst, MAP kinase activation, induction of defense-related marker genes and medium alkalinization of cell suspensions (Flury et al., 2013; Bisceglia et al., 2015; Liu et al., 2018). ROS measurement is typically performed by means of luminol-based chemiluminesce assays. MAP kinase activation is detected by western blots using specific antibodies. Transcriptional reprogramming is usually accessed by monitoring the expression levels of one or a few marker genes using real-time PCR. Medium alkalinization assays are based on recording the pH changes in MAMP-treated plant cell suspensions over time using a pH sensor for small volumes. Compared to the other methods, medium alkalinization assays are less laborious and require low cost laboratory equipment. Both model plant species and crops have been studied using medium alkalinization assays (Nühse et al., 2000; Moroz et al., 2017).