High-throughput imaging of plant immune responses.

Fluorescence confocal microscopy has emerged in the past decade as an important method for studying the cellular changes associated with plant-microbe interactions. One such change is the internalization into endosomes of the cell surface receptor FLAGELLIN SENSING 2 (FLS2) upon activation by its ligand, bacterial flagellin (flg22). Quantification of endosomes containing FLS2 can thus be used as a direct readout of immune response activation at the cellular level. High-throughput imaging of cellular events is routinely applied in chemical screening for pharmaceutical drug discovery, and we have adapted this system for quantification of plant leaf cellular parameters. In this chapter we describe the instrument setup for high-throughput imaging of leaves, protocols for flg22-induced endocytosis, image acquisition for fluorescent-tagged FLS2 receptors and subcellular markers, automated image analysis of cellular parameters, and data outputs of FLS2 endocytosis.