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Published on July 11, 2008; 10.1104/pp.108.117358

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Received February 5, 2008
Accepted July 5, 2008

Combination of Novel GFP Mutant TSapphire and DsRed Variant mOrange to Set Up a Versatile in planta FLIM-FRET Assay

Vincent Bayle , Laurent Nussaume , and Riyaz A. Bhat *

Laboratory of Plant Developmental Biology (LBDP); SBVME/Institute for Biotechnology and Environmental Biology (iBEB),; UMR6191 CEA-CNRS, Mediterranean University Aix-Marseille; CEN, Cadarache, 13108, St Paul Lez Durance, France

* Corresponding author; email: bhatr{at}indiana.edu.

Forster resonance energy transfer (FRET) measurements based on fluorescence lifetime imaging microscopy (FLIM) are increasingly being used to assess molecular conformations and associations in living systems. Reduction in the excited state lifetime of the donor fluorophore in the presence of an appropriately positioned acceptor is taken as a strong evidence of FRET. Traditionally CFP has been widely used as a donor fluorophore in FRET experiments. However given its photolabile nature, low quantum yield and multiexponential lifetime, CFP is far from an ideal donor in FRET imaging. Here we report the application and use of TSapphire mutant of GFP as an efficient donor to mOrange in FLIM based-FRET imaging in intact plant cells. Using time correlated single photon counting (TSCPC)-FLIM we show that TSapphire expressed in living plant cells decays with lifetime of 2.93 ± 0.09 ns. Chimerically linked TSapphire and mOrange (with 16 aa linker in-between) exhibit substantial energy transfer based on the reduction in the lifetime of TSapphire in the presence of the acceptor mOrange. Experiments performed with various genetically and/or biochemically-known interacting plant proteins demonstrate the versatility of the FRET-FLIM system presented here in different sub-cellular compartments tested (cytosol, nucleus and at plasma-membrane). The better spectral overlap with red monomers, higher photostability and monoexponential lifetime of TSapphire makes it an ideal FRET-FLIM donor to study protein-protein interactions in diverse eukaryotic systems overcoming in particular many technical challenges encountered (like autofluorescence of cell walls and fluorescence of pigments associated with photosynthetic apparatus) while studying plant protein dynamics and interactions.






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