Bimolecular Fluorescence Complementation for Imaging Protein Interactions in Plant Hosts of Microbial Pathogens

Protein–protein interactions mediate many aspects of cellular function. Scientists have developed numerous techniques to investigate these interactions, both in vitro and in vivo. Among these, the peptide complementation assay Bimolecular Fluorescence Complementation (BiFC) allows visualization of the subcellular sites of protein–protein interactions in living cells. BiFC comprises a “split GFP” system: GFP protein (or its derivatives) is split into two fragments, neither of which fluoresces on its own. Interacting proteins linked to these peptide fragments may bring them into proximity, allowing them to refold and restore fluorescence. Although this system was first exploited for use in animal cells, we have developed BiFC for use in plants. Pathogens transfer numerous effector proteins into eukaryotic cells and manipulate host cellular processes through interactions between effector and host proteins. BiFC can therefore facilitate studies of host–bacterial interactions. In this chapter, we describe the numerous BiFC vectors we have constructed, their uses, and their limitations.