FRET-FLIM for visualizing and quantifying protein interactions in live plant cells

Proteins are the workhorses that control most biological processes in living cells. Although proteins can accomplish their functions independently, the vast majority of functions require proteins to interact with other proteins or biomacromolecules. Protein interactions can be investigated through biochemical assays such as co-immunoprecipitation (co-IP) or Western blot analysis, but such assays lack spatial information. Here we describe a well-developed imaging method, Förster resonance energy transfer (FRET) analyzed by fluorescence lifetime imaging microscopy (FLIM), that can be used to visualize protein interactions with both spatial and temporal resolution in live cells. We demonstrate its use in plant developmental research by visualizing in vivo dimerization of AUXIN RESPONSE FACTOR (ARF) proteins, mediating auxin responses.