Subcellular Localization Experiments and FRET-FLIM Measurements in Plants

Abstract

Determining the localization of proteins within living cells may be very essential for understanding their biological function. Usually for analysis of subcellular localization, a construct encoding the translational fusion of a cDNA of interest with a fluorescent protein (FP) is engineered, transiently expressed in plant cells and examined with confocal microscopy.In co-localization and interaction studies, two plasmids, each encoding one of the potential interacting/binding partners tagged with an appropriate pair of fluorescence proteins (for instance CFP/YFP) are co-expressed in plant cells. If proteins co-localize in certain cellular compartments it does not necessarily mean that they bind/interact to each other, therefore an additional technique should be applied for in vivo verification of putative interaction, e.g. Fluorescence Lifetime Imaging (FLIM) to detect Fluorescence Resonance Energy Transfer (FRET).The protocol describes in detail the method that has been used to verify interaction between the bacterial effector HopQ1 and a 14-3-3a host protein and additionally to check the necessity of the central serine in the canonical 14-3-3 binding site within HopQ1 (Giska et al., 2013) for this association.