Detection of protein–protein interactions in plants using bimolecular fluorescence complementation

Abstract

SummaryProtein function is often mediated via formation of stable or transient complexes. Here we report the determination of protein-protein interactions in plants using bimolecular fluorescence complementation (BiFC). The yellow fluorescent protein (YFP) was split into two non-overlapping N-terminal (YN) and C-terminal (YC) fragments. Each fragment was cloned in-frame to a gene of interest, enabling expression of fusion proteins. To demonstrate the feasibility of BiFC in plants, two pairs of interacting proteins were utilized: (i) the a and b subunits of the Arabidopsis protein farnesyltransferase (PFT), and (ii) the polycomb proteins, FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) and MEDEA (MEA). Members of each protein pair were transiently co-expressed in leaf epidermal cells of Nicotiana benthamiana or Arabidopsis. Reconstitution of a fluorescing YFP chromophore occurred only when the inquest proteins interacted. No fluorescence was detected following co-expression of free non-fused YN and YC or non-interacting protein pairs. Yellow fluorescence was detected in the cytoplasm of cells that expressed PFT a and b subunits, or in nuclei and cytoplasm of cells that expressed FIE and MEA. In vivo measurements of fluorescence spectra emitted from reconstituted YFPs were identical to that of a non-split YFP, confirming reconstitution of the chromophore. Expression of the inquest proteins was verified by immunoblot analysis using monoclonal antibodies directed against tags within the hybrid proteins. In addition, protein interactions were confirmed by immunoprecipitations. These results demonstrate that plant BiFC is a simple, reliable and relatively fast method for determining protein-protein interactions in plants.

Endosperm: an integrator of seed growth and development

“…The rbr1 mutant phenotype is likely to be compound. RBR1 can interact with components of the FIS-complex [21], but could also act upstream of the FIS Pc-G complex and together with MSI1 to directly control cell cycle progression in the female gametophyte. In analogy to its function in root stem cells, RBR1 might also be involved in specifying gamete identity [22].…”

Lighting the Way to Protein-Protein Interactions: Recommendations on Best Practices for Bimolecular Fluorescence Complementation Analyses

“…To this end, an FP is separated into nonfluorescent N-terminal and C-terminal fragments that are translationally fused with the two proteins of interest (i.e., a pair of potentially interacting proteins). Upon interaction of the fusion proteins in living cells, the N-and C-terminal FP fragments are brought into close proximity resulting in reassembly of a functional fluorophore ( Figure 1A; Hu et al, 2002;Walter et al, 2004;Bracha-Drori et al, 2004). Consequently, BiFC analyses not only allow the detection of protein-protein interactions but also provide information about the subcellular localization of the observed protein complex.…”

“…This (O) and (P) Possible orientations of the protein fusions in BiFC assays. It is important to note that the orientation can have a strong impact on the propensity of spontaneous FP reconstitution (i.e., the formation of false positive interactions; Bracha-Drori et al, 2004;Horstman et al, 2014). Hence, it is essential that, for the negative controls, exactly the same orientations are used as for the positive interaction.…”

“…Inappropriate controls for BiFC experiments that, unfortunately, are frequently seen in the literature include, for example, combination of one of the interaction partners with an empty vector (expressing the unfused FP fragment) or the expression of only one of the fusion proteins (Horstman et al, 2014; Figures 1G to 1N). In addition, the orientation of the protein fusions ( Figures 1O and 1P) is known to influence the propensity of spontaneous FP reconstitution in BiFC assays (Bracha-Drori et al, 2004;Horstman et al, 2014). Therefore, for the negative controls to be conclusive, exactly the same orientations must be used as for the positive interaction.…”

Advances in Fluorescent Protein-Based Imaging for the Analysis of Plant Endomembranes

“…Increasingly popular methods for analyzing proteinprotein interactions in plants are the fluorescence complementation protocols such as the split-YFP (or bimolecular fluorescence complementation [BiFC]) and split-luciferase systems (Bracha-Drori et al, 2004;Walter et al, 2004;Fujikawa and Kato, 2007).…”