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

Plant J. 2011 Aug;67(3):542-53. doi: 10.1111/j.1365-313X.2011.04607.x. Epub 2011 Jun 10.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 14-3-3 Proteins / metabolism
  • Arabidopsis Proteins / metabolism
  • Genetic Vectors
  • Immunoblotting / methods
  • Luciferases / genetics
  • Luciferases / metabolism
  • Luminescent Measurements / methods*
  • Microscopy, Confocal / methods*
  • Plant Leaves / genetics
  • Plant Leaves / metabolism
  • Plant Proteins / metabolism*
  • Protein Binding
  • Protein Interaction Mapping / methods*
  • Protein Multimerization
  • Recombinant Fusion Proteins / metabolism
  • Rhizobium
  • Sulfurtransferases / metabolism
  • Tobacco / genetics
  • Tobacco / metabolism

Substances

  • 14-3-3 Proteins
  • Arabidopsis Proteins
  • Cnx protein, Arabidopsis
  • Plant Proteins
  • Recombinant Fusion Proteins
  • Luciferases
  • Sulfurtransferases
  • molybdopterin synthase