Bimolecular Fluorescence Complementation (BiFC) in Host–Virus Interactions

Bimolecular fluorescence complementation (BiFC) is an assay widely used for studying protein–protein interactions and determining the subcellular localization of proteins. This technique involves fusing the proteins of interest to separate structural domains of a fluorescent protein, followed by transient expression in cells. The interaction between the proteins of interest in vivo allows the reconstitution of the fluorescence that can be visualized by fluorescence microscopy. BiFC has been particularly useful in investigating the interactions between viral and host proteins. Here, we describe the steps involved in preparing expression cassettes that allow the expression of proteins of interest fused to nonfluorescent fragments of yellow fluorescent protein (YFP), Agrobacterium transformations, and agroinfiltration of Nicotiana benthamiana leaves to facilitate virus protein–host protein interactions. Finally, high-resolution images can be obtained by analyzing the leaves under a confocal microscope.