Bimolecular fluorescence complementation (BiFC) assay is one of the sensitive techniques that allows to investigate direct protein-protein interactions (PPI) in vivo and visualize the subcellular localization of interacting proteins. It is based on splitting of a fluorescent protein into two nonfluorescent parts accordingly fused to two putative interacting partners. If interaction between studied proteins is possible, nonfluorescent parts come to close proximity resulting in reconstitution of the functional fluorescent protein and giving fluorescence under certain wavelength. BiFC analysis implies transient or stable expression of the proteins of interest and can be used as a method to test or validate the direct PPI in various biological pathways, including the regulation of gametogenesis, which is the main focus of this book. In our protocol we give detailed information for beginners about three main steps of BiFC analysis of centromeric protein interactions. These steps include (1) generation of appropriate expression clones with the help of Gateway cloning technology, (2) infiltration of Nicotiana benthamiana plants by Agrobacteria containing generated constructs, and (3) microscopic analysis of plants under fluorescence microscope. Also, we discuss appropriate negative controls that can be used for evaluation as well as recommendable vector systems, possible artifacts and measures to avoid artifactual interactions for BiFC assay.
Keywords: Agroinfiltration; BiFC; Centromeric proteins; Protein–protein interactions; Subcellular localization.
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