Studying Protein-Protein Interactions at Plasmodesmata by Measuring Förster Resonance Energy Transfer.

Plasmodesmata (PD) provide interconnectivity between plant cells to enable the intercellular transport and communication that is requisite to multicellularity. Being at the interface of the apoplast, plasma membrane (PM), endoplasmic reticulum (ER), and symplast, PD are uniquely positioned to integrate exogenously and endogenously derived signals with plant developmental and physiological responses. The distinct membrane curvature and composition of PD allow them to function as microdomains to facilitate dynamic protein-protein interactions. Förster resonance energy transfer (FRET) combined with fluorescence lifetime imaging microscopy (FLIM) and fluorescence anisotropic decay measurements provides valuable tools to analyze these interactions in vivo and in planta. Here we describe a detailed methodology to perform FRET-FLIM and fluorescence anisotropy measurements to analyze protein-protein interactions at PD in a transient expression system using Nicotiana benthamiana; however this can be adapted to other plant species and subcellular compartments.