title: Quantitative analysis of dynamic protein-protein interactions in planta by a floated-leaf luciferase complementation imaging (FLuCI) assay using binary Gateway vectors.
abstract: 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.
authors.affiliation: Institut für Pflanzenbiologie, Technische Universität Braunschweig, Humboldtstraße 1, D-38106 Braunschweig, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Gehl
authors.forename: David
authors.initials: D
authors.lastname: Kaufholdt
authors.forename: Domenica
authors.initials: D
authors.lastname: Hamisch
authors.forename: Rolf
authors.initials: R
authors.lastname: Bikker
authors.forename: Jörg
authors.initials: J
authors.lastname: Kudla
authors.forename: Ralf R
authors.initials: RR
authors.lastname: Mendel
authors.forename: Robert
authors.initials: R
authors.lastname: Hänsch
mesh_terms: D048948:14-3-3 Proteins / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000378:metabolism; D005822:Genetic Vectors; D015151:Immunoblotting / Q000379:methods; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008163:Luminescent Measurements / Q000379:methods*; D018613:Microscopy, Confocal / Q000379:methods*; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012231:Rhizobium; D013466:Sulfurtransferases / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism
chemical_list: D048948:14-3-3 Proteins; D029681:Arabidopsis Proteins; C092535:Cnx protein, Arabidopsis; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D008156:Luciferases; D013466:Sulfurtransferases; C085585:molybdopterin synthase