Protein-protein interactions (PPI) are essential for a plethora of biological processes. These interactions can be visualized and quantified with spatial resolution using Förster resonance energy transfer (FRET) measured by fluorescence lifetime imaging microscopy (FLIM) technology. Currently, FRET-FLIM is routinely used in cell biology, and it has become a powerful tool to map protein interactions in native environments. However, implementing this technology in living multicellular organism remains challenging, especially when dealing with developing plant embryos where tissues are confined in multiple cell layers preventing direct imaging. In this chapter, we describe a step-by-step protocol for studying PPI using FRET-FLIM of the two transcription factors SCARECROW and SHORTROOT in Arabidopsis embryos. We provide a detailed description from embryo isolation to data analysis and representation.
Keywords: Embryo; FRET-FLIM; Fluorescence lifetime; Protein interactions; SCR-SHR.