Using Tripartite Split-sfGFP for the Study of Membrane Protein–Protein Interactions

The study of protein–protein interaction (PPI) is critical for understanding cellular processes within biological systems. The conventional biomolecular fluorescence complementation (BiFC) or bipartite split-fluorescent protein (FP) is a noninvasive fluorescent-based technique that enables direct visualization of PPI in living cells once the two nonfluorescent fragments are brought into close vicinity. However, BiFC can potentially lead to a high background noise arising from an inherent feature of the irreversible self-assembly of the nonfluorescent fragments. Recently, the newly developed tripartite split-sfGFP method was demonstrated to detect membrane PPIs in plant cells without spurious background signals even when fusion proteins are highly expressed and accessible to the compartments of interaction. Here we describe a protocol for using the ß-Estradiol-inducible tripartite split-sfGFP assay for side-by-side analyses of in vivo PPI along with in situ subcellular localization of fusion proteins in agroinfiltrated Nicotiana benthamiana leaves.