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Quantitative analysis of dynamic protein–protein interactions in planta by a floated‐leaf luciferase complementation imaging (FLuCI) assay using binary Gateway vectors
Author(s) -
Gehl Christian,
Kaufholdt David,
Hamisch Domenica,
Bikker Rolf,
Kudla Jörg,
Mendel Ralf R.,
Hänsch Robert
Publication year - 2011
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1111/j.1365-313x.2011.04607.x
Subject(s) - luciferase , complementation , gateway (web page) , binary number , protein fragment complementation assay , computational biology , microbiology and biotechnology , biology , computer science , genetics , mathematics , gene , mutant , transfection , world wide web , arithmetic
Summary Dynamic protein–protein interactions are essential in all cellular and developmental processes. Protein‐fragment 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 Gateway‐compatible 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.