Quantitative analysis of dynamic protein–protein interactions in planta by a floated‐leaf luciferase complementation imaging (FLuCI) assay using binary Gateway vectors

Abstract

SUMMARYDynamic protein-protein interactions are essential in all cellular and developmental processes. Proteinfragment complementation assays allow such protein-protein interactions to be investigated in vivo. In contrast to other protein-fragment complementation assays, the split-luciferase (split-LUC) complementation approach facilitates dynamic and quantitative in vivo analysis of protein interactions, as the restoration of luciferase activity upon protein-protein interaction of investigated proteins is reversible. Here, we describe the development of a floated-leaf luciferase complementation imaging (FLuCI) assay that enables rapid and quantitative in vivo analyses of protein interactions in leaf discs floating on a luciferin infiltration solution after transient expression of split-LUC-labelled interacting proteins in Nicotiana benthamiana. We generated a set of eight Gateway-compatible split-LUC destination vectors, enabling fast, and almost fail-safe cloning of candidate proteins to the LUC termini in all possible constellations. We demonstrate their functionality by visualizing the well-established homodimerization of the 14-3-3 regulator proteins. Quantitative interaction analyses of the molybdenum co-factor biosynthesis proteins CNX6 and CNX7 show that the luciferase-based protein-fragment complementation assay allows direct real-time monitoring of absolute values of protein complex assembly. Furthermore, the split-LUC assay is established as valuable tool to investigate the dynamics of protein interactions by monitoring the disassembly of actin filaments in planta. The new Gatewaycompatible split-LUC destination vector system, in combination with the FLuCI assay, provides a useful means to facilitate quantitative analyses of interactions between large numbers of proteins constituting interaction networks in plant cells.

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

“…Ideally, the confirmatory method can also be performed in vivo, in intact plant tissue. Examples of such techniques are FRET-FLIM and splitluciferase assays (Remy and Michnick, 2006;Bayle et al, 2008;Gehl et al, 2011). If the protein-protein interaction results in a subcellular translocation of at least one of the proteins, coexpression and translocation assays can also be applied (Piljić and Schultz, 2008;Schlü cking et al, 2013;Offenborn et al, 2015).…”

Split luciferase complementation assay to detect regulated protein-protein interactions in rice protoplasts in a large-scale format

“…Since then, the assay has been applied to detect interactions between membrane proteins (Kato et al [2009]), bacterial effector proteins and their protein targets (Chen et al [2008]), auxin response factors (Li et al [2011]), 14-3-3 regulator proteins (Gehl et al [2011]), coiled-coil–nucleotide-binding site–leucine-rich repeat (CC-NB-LRR) protein (Inoue et al [2013]), and kinase (Schmidt et al [2013]). We previously showed with the SLC assay that protein-protein interactions in Arabidopsis protoplasts are sensitive to environmental conditions (Kato and Bai [2010]), indicating that the SLC assay would be well-suited for analyzing regulated protein-protein interactions in plant cells.…”

Protein Fragment Bimolecular Fluorescence Complementation Analyses for the In vivo Study of Protein-Protein Interactions and Cellular Protein Complex Localizations

“…The splicing based split luciferase system combines split luciferase with split intein (DnaE), where PPI induces the splicing and ligation of luciferase fragments to recover an irreversible luciferase activity (27). Reversible split luciferase assays have also been developed for luciferases with different emission properties and from different species (28–32) and have been successfully applied to plants (33–35). Split luciferase assays have two drawbacks, namely the need for coelenteracin/luciferin as an externally applied substrate and a low sub-cellular resolution, due to the low intensity of this bioluminescence reporter, which usually requires integrated signal detection from whole tissues, or groups of cells.…”