Detection and Analyses of Endocytosis of Plant Receptor Kinases.

Genetic dissection has led to a sophisticated understanding of receptor kinases in plant development and responses to abiotic and biotic stresses. Fluorescence confocal microscopy is essential to identify the (sub)cellular locations of resting and signaling receptor kinases that trigger molecular events in plant cells upon ligand perception. In this regard, the internalization of plasma membrane-localized FLAGELLIN SENSING 2 (FLS2) into endosomes induced by its ligand flg22, a peptide derived from bacterial flagellin, is a model system for studying activation status-dependent and endosomal receptor kinase trafficking routes and can be used in screens to identify pathogen effectors that target these trafficking routes for virulence promotion. In this chapter we describe approaches of visualizing fluorescently tagged FLS2, including protocols for flg22-induced endocytosis, instrument parameters, and image analysis. These approaches can be easily adapted for other receptor kinases, using the fast transient expression system in Nicotiana benthamiana for microscopic inspection.