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First published online July 11, 2008; 10.1104/pp.108.117358 Plant Physiology 148:51-60 (2008) © 2008 American Society of Plant Biologists Combination of Novel Green Fluorescent Protein Mutant TSapphire and DsRed Variant mOrange to Set Up a Versatile in Planta FRET-FLIM Assay1,[W]Laboratory of Plant Developmental Biology, Service of Plant Biology and Environmental Microbiology/Institute for Biotechnology and Environmental Biology, UMR6191 CEA/CNRS/Mediterranean University Aix-Marseille, CEN, Cadarache, 13108 St. Paul Lez Durance, France
Förster 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 strong evidence of FRET. Traditionally, cyan fluorescent protein has been widely used as a donor fluorophore in FRET experiments. However, given its photolabile nature, low quantum yield, and multiexponential lifetime, cyan fluorescent protein is far from an ideal donor in FRET imaging. Here, we report the application and use of the TSapphire mutant of green fluorescent protein as an efficient donor to mOrange in FLIM-based FRET imaging in intact plant cells. Using time-correlated single photon counting-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-amino acid 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 subcellular 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.
Single- and dual-color fluorescence imaging with intrinsically fluorescent proteins is increasingly being used to study the expression, targeting, colocalization, turnover, and associations of diverse proteins involved in different plant signal transduction pathways (for review, see Fricker et al., 2006  ). Concurrent with the use of fluorescence-based cell biology, Förster resonance energy transfer (FRET) has emerged as a convenient tool to study the dynamics of protein associations in vivo. The technique exploits the biophysical phenomenon of nonradiative energy transfer from a donor fluorophore to an appropriately positioned acceptor at a nanometer scale (1–10 nm; Jares-Erijman and Jovin, 2003). In living cells, FRET occurs when two proteins (or different domains within a single protein) fused to suitable donor and acceptor fluorophores physically interact, thus bringing the donor and the acceptor within the favorable proximity for energy transfer (Immink et al., 2002; Bhat et al., 2006). This results in a decrease in the donor's fluorescence intensity (or quantum yield [QY]) and excited-state lifetime (Gadella et al., 1999). Furthermore, if the acceptor molecule is a fluorophore, then FRET additionally results in an increase in the acceptor's emission intensity (sensitized emission; Shah et al., 2001; Bhat et al., 2006).
However, the exploitation and use of fluorescent marker proteins to study protein trafficking and associations in plants can be problematic because plant cells contain a number of autofluorescent compounds (e.g. lignin, chlorophyll, phenols, etc.) whose emission spectra interfere with that of the most commonly used green or red fluorescent protein fluorophores and/or their spectral variants. For example, lignin fluorescence in roots, vascular tissues, and cell walls of aerial plant parts interferes with imaging at wavelengths between 490 and 620 nm, whereas the chlorophyll autofluorescence in green aerial plant parts is prevalent between 630 and 770 nm (Chapman et al., 2005
CFP and YFP have been widely used as a donor-acceptor pair in in planta FRET measurements (Bhat et al., 2006
Recently, TSapphire (Q69M/C70P/V163A/S175G; excitation/emission 399/511 nm), a variant of the Sapphire (T203I) mutant of wild-type GFP with improved folding properties and better pH sensitivity, was described (Zapata-Hommer and Griesbeck, 2003
However, currently there are no reports demonstrating the application and use of TSapphire and monomeric red-shifted fluorophores as donor-acceptor FRET pairs to probe intermolecular protein-protein interactions in vivo. In this article, we demonstrate in vivo FRET-fluorescence lifetime imaging microscopy (FLIM) between the donor TSapphire and the acceptor mOrange. We show that TSapphire expressed in living plant cells decays with a monoexponential lifetime of 2.93 ± 0.09 ns, which is in agreement with the published lifetime for its parent wild-type GFP (3.2 ns; Striker et al., 1999
Construction of p35S Binary Vectors for Expression of C-Terminally Fluorescently Tagged Proteins in Planta
Binary vectors based on the pam-PAT-MCS backbone (accession no. AY436765) were generated to allow convenient and rapid in planta expression of proteins with TSapphire and mOrange fluorescent tags (see "Materials and Methods" for cloning details). The resulting vectors are high copy number in Escherichia coli and confer ampicillin and carbenicillin resistance to E. coli and Agrobacterium tumefaciens, respectively. The pat gene confers Basta herbicide resistance, allowing rapid selection of transgenic plants on soil (Witte et al., 2004
To examine the expression of TSapphire, mOrange, or TSapphire-mOrange chimeric fluorophores in intact living cells, transient gene expression studies were carried out by particle bombardment (Bhat et al., 2005
Excited-State Lifetime Analysis of TSapphire Expressed in N. benthamiana Leaves
N. benthamiana cells transiently expressing the TSapphire fluorophore were subjected to time-correlated single photon counting (TCSPC)-FLIM analysis to ascertain the excited-state lifetime of the fluorophore. Eighty-megahertz repetitive pulses from a 405-nm diode laser were used to excite the TSapphire and build up the photon decay profile over time (40 cycles of 6 s/cycle) as described in "Materials and Methods." The decay profile was built using a single exponential decay model. Figure 2
shows an overview of the lifetime image and the distribution profile along with fitted fluorescence decay at a single pixel (shown by an arrowhead in A) of a N. benthamiana cell expressing TSapphire. The lifetime image (Fig. 2A) and the overall lifetime distribution profile (Fig. 2B) represent the spatial distribution of the fluorescence lifetime in pseudocolor from red (2.1 ns) to blue (3.1 ns). From the lifetime distribution profile (Fig. 2B), it is quite clear that a majority of the photons decay around 3.0 ns. Figure 2C shows the fluorescence decay functions of a 3 x 3 pixel region around the red arrowhead location indicated in Figure 2A using a single exponential fit. The average lifetime of the TSapphire measured in three independent experiments was found to be 2.93 ± 0.09 ns (see Table I
), which is in agreement with the published lifetime for its parent wild-type GFP (3.16 ± 0.03 ns; Volkmer et al., 2000
FRET-FLIM Analysis in Cells Expressing Chimeric TSapphire-mOrange Fusion Protein To examine the changes in the lifetime of the donor TSapphire in the presence of the acceptor mOrange, N. benthamiana cells expressing TSapphire and mOrange either from separate plasmids or from the chimeric construct harboring TSapphire and mOrange separated by 16-amino acid linker were used for FRET-FLIM as shown in Figure 3 . Figure 3, A and C, shows the pseudocolored lifetime images of the donor TSapphire in representative cells either coexpressing both TSapphire and mOrange from separate plasmids or expressing a chimeric TSapphire-mOrange fusion from a single plasmid. Figure 3, B and D, represent the overall lifetime distribution of the photons depicted in Figure 3, A and C, respectively. As can be seen from Figure 3B, majority of the TSapphire photons show a longer lifetime above 2.9 ns. The averaged mean lifetime of TSapphire in cells coexpressing free TSapphire and free mOrange was found to be 2.87 ± 0.08 ns. Using the FRET-FLIM efficiency formula (see "Materials and Methods"), this amounts to an energy transfer of 2% (see Table I). On the other hand, lifetime analysis in cells expressing TSapphire-mOrange chimeric protein results in a significant shift in the lifetime profile of the donor (Fig. 3D). The mean lifetime of TSapphire in cells expressing chimeric protein was found to be 2.48 ± 0.04 ns amounting to 15% energy transfer from the donor TSapphire to the acceptor mOrange (Table I).
Figure 3E shows that the fitted curve around the 3 x 3 pixel region (red arrowhead) in Figure 3C decays faster than the fitted curve around a similar region (red arrowhead) in Figure 3A. To corroborate the energy transfer from TSapphire to mOrange, we used FRET-acceptor photobleaching (FRET-APB) as an independent and complimentary method to our FRET-FLIM assay (see Supplemental Materials and Methods S1 and Supplemental Fig. S1). Bleaching of the acceptor mOrange in living cells expressing chimeric TSapphire-mOrange fusion protein resulted in dequenching of the donor TSapphire, whereas no such dequenching of TSapphire was observed following bleaching of mOrange in the cells expressing free TSapphire and mOrange (Supplemental Fig. S1). The calculated mean FRET-APB efficiencies were 12.28% for cells expressing chimeric TSapphire-mOrange and 0.52% for cells coexpressing free TSapphire and mOrange (Supplemental Fig. S1, columns M and N).
Having confirmed the intramolecular FRET between TSapphire and mOrange, we next tested whether the TSapphire and mOrange pair could successfully and reliably report on the intermolecular interactions using the FRET-FLIM. For this purpose, we fused the donor and acceptor to some known biochemical and/or genetic interactors. The interactions were tested between (1) Arabidopsis (Arabidopsis thaliana) cytosolic proteins (RAR1 and SGT1b) that are known to be involved in R gene-mediated plant defense against pathogens and have been shown to interact genetically and biochemically (Azevedo et al., 2002 We selected the above protein pairs for testing our TSapphire/mOrange FRET-FLIM system because this presented us with the possibility of not only testing interactions in different subcellular compartments (cytosol, nucleus, and at cellular periphery) but also to assess the nature of these interactions (stable versus dynamic interactions as discussed below). RAR1 and SGT1b proteins fused to TSapphire and/or mOrange, expressed in N. benthamiana leaves colocalized in the cytosol (Supplemental Fig. S2). We found that the two proteins interact with each other in the cytosol regardless of whether SGT1b or RAR1 is fused to the donor TSapphire (Fig. 4 ). However, unlike the chimeric TSapphire-mOrange (Fig. 3C), the lifetime reduction was not observed throughout the cell but was restricted to certain peculiar locations (Fig. 4, C and H). This resulted in a mosaic pattern where, at some locations, TSapphire decayed with longer lifetimes (blue color in Fig. 4, C and H), indicating an absence of acceptor in the vicinity, whereas at other locations there was a dramatic reduction in the lifetime characteristic of resonance energy transfer (warmer colors in Fig. 4, C and H). One likely explanation for this would be that in the chimeric construct, TSapphire and mOrange are linked by a 16-amino acid linker and thus always stay at a fixed distance, resulting in a continuous FRET, whereas, in the case of RAR1 and SGT1b, the interaction and hence FRET are dynamic in nature. Fitted decay curves in Figure 4, E and J, from regions showing the color changes in Figure 4, C and H, respectively, confirmed that the photons in these regions decay much faster than the photons in similar-sized regions from Figure 4, A or F. Despite the nonuniformity of lifetime decay, the overall FRET-FLIM efficiency for the energy transfer from SGT1b-TSapphire to RAR1-mOrange in three independent experiments was found to be 12% (Table I). Similar FRET-FLIM efficiencies were obtained when the donor and acceptor fluorophores were swapped between SGT1b and RAR1 (data not shown).
The coexpression of nuclear-encoded maize transcriptional activator O2 and the coactivator ADA2 fused to TSapphire and mOrange, respectively (Supplemental Fig. S3), resulted in a small but reproducible shift in the lifetime of the donor from 2.95 ± 0.02 ns for O2-TSapphire alone to 2.77 ± 0.03 ns for O2-TSapphire in the presence of ADA2-mOrange (Table I) that amounts to a FRET-FLIM efficiency of around 6%. Figure 5, A to H , shows the lifetime images and the overall lifetime distribution profiles of representative nuclei coexpressing free TSapphire and mOrange (Fig. 5, A and B), O2-TSapphire alone (Fig. 5, C and D), O2-TSapphire and ADA2-mOrange (Fig. 5, E and F), or ADA2-TSapphire and O2-mOrange (Fig. 5, G and H). Like the pattern observed in Figure 4, C and H, the lifetime reduction was observed in a nonuniform way, possibly pointing toward the dynamic nature of these protein-protein interactions. Figure 5I compares the fitted decay curve of a 3 x 3 pixel region (red arrowhead) from Figure 5, A, C, E, and G. Clearly the decay is much faster in situations where TSapphire fused to either O2 or ADA2 is in the presence of the acceptor mOrange fused to ADA2 or O2, respectively.
As a final confirmation of the reliability of the FRET-FLIM based on the TSapphire and mOrange donor-acceptor FRET pair, we tested interaction between the integral membrane protein MLO and the cytosolic calcium sensor protein CaM from barley in our transient single-cell FRET-FLIM assay in N. benthamiana leaves (Supplemental Fig. S4). Genetic and physical interaction between MLO and CaM was recently shown to modulate the entry of powdery mildew fungi into barley leaf epidermal cells (Kim et al., 2002 ; Bhat et al., 2005). In accordance with published data, we found that the two proteins interact with each other (Fig. 6, C–F versus A and B
) and there is a reduction in the lifetime of CaM-TSapphire (from 2.98 ± 0.11 ns to 2.71 ± 0.13 ns) when expressed together with MLO-mOrange that results in a FRET-FLIM efficiency of around 9%. Such a lifetime reduction is absent in control cells coexpressing free TSapphire and MLO-mOrange (Fig. 6, G and H; Table I). As shown in Figure 6I, a sample fitted decay curve from Figure 6, C and E, decays much faster than sample decay curves from Figure 6, A and G.
Among many commonly used fluorescence-based methods including bioluminescence resonance energy transfer (Xu et al., 2007 ) and bimolecular fluorescence complementation (Magliery and Regan, 2006), FRET has emerged as a powerful tool to study in vivo protein dynamics and associations. One of the most direct and robust ways to assess FRET is by recording the excited-state lifetime changes in the donor fluorophore in the absence and presence of an acceptor using FLIM (Gadella et al., 1999; van Munster and Gadella, 2005). FRET measurements using FLIM are superior to other methods (like intensity-based sensitized FRET or APB) because fluorescence lifetimes are independent of local chromophore concentrations and moderate photobleaching (Gadella et al., 1999; Immink et al., 2002; Bhat et al., 2006).
In this article, we demonstrate that the combination of TSapphire and mOrange as a donor-acceptor FRET-FLIM pair successfully reports on the intra- and intermolecular protein interactions. TSapphire as a donor is superior to current favorite CFP in many respects. First, the extinction coefficient (
Whereas any of the red-shifted monomeric proteins, like mStrawberry or mCherry, can be used as acceptors with TSapphire, we chose mOrange because it is the brightest red-shifted monomer (Shaner et al., 2005
To make a standard for in planta FRET-FLIM measurements, we took advantage of Gateway cloning technology (Invitrogen) and recombined TSapphire into a Gateway-compatible entry clone harboring mOrange (see "Materials and Methods"). This resulted in a chimeric construct where TSapphire was separated from mOrange by a 16-amino acid linker. FRET-FLIM analysis on this construct in N. benthamiana resulted in a mean FRET efficiency of 15%. Similar FRET efficiencies (12.28%) were obtained using the FRET-APB method for documenting energy transfer (Supplemental Fig. S1), thus corroborating the results obtained by FRET-FLIM. However, our calculated FRET efficiencies are reduced compared to a chimeric construct between Cerulean C1 (CFP variant) and Venus C1 (YFP variant), which exhibited 38% of FRET efficiency in HEK293 cells (Koushik et al., 2006 This study reports on the interaction between different plant proteins in various subcellular compartments under a single set of conditions and criteria demonstrating, therefore, the versatility of the system proposed here. Furthermore, we have developed a set of Gateway technology-compatible plant-specific vectors that can be used by the interested researchers for studying their protein pairs of interest to facilitate the use of this system. Thus, it is our contention that this study should serve as a reference point for researchers attempting to set up the TSapphire-mOrange donor-acceptor FRET-FLIM in planta.
Plasmids
The backbone of all the vectors used in the study was pam-PAT-MCS (accession no. AY436765). To generate fluorescent protein destination vectors for Gateway cloning technology (Invitrogen), PCR-amplified TSapphire (Zapata-Hommer and Griesbeck, 2003
Nicotiana benthamiana was cultivated in the greenhouse as described (Romeis et al., 2001
Transient intracellular fluorescence was observed by CLSM using a Leica SP2 AOBS inverted confocal microscope (Leica Microsystems) equipped with argon ion (458-, 476-, 488-, 496-, and 514-nm laser lines) and the He-Ne (561-nm laser line) lasers. Additionally, a 405-nm diode laser (BDL 405 SMC; Becker and Hickl) operating either in continuous or pulsing mode was also installed into the Leica SP2 AOBS system. TSapphire was excited with a 405-nm diode laser running in continuous mode, whereas mOrange was excited with a 514-nm (and occasionally 561) argon laser line. TSapphire fluorescence was detected using the Leica AOBS system and a custom 485- to 535-nm band-pass emission filter, whereas mOrange fluorescence was detected using the Leica AOBS system and a custom 545- to 600-nm band-pass emission filter. For spectroscopic analysis, the emission spectra of TSapphire and mOrange were collected in the
FLIM analysis was performed using the TCSPC (with SPC 830 card) system from Becker and Hickl (Becker et al., 2004
The lifetime data acquired using the TCSPC system were imported into SPC image software (version 2.9; Becker and Hickl) and analyzed using the default conditions (scatter, shift, and decay components unfixed). The complete lifetime decays were calculated per pixel as per the
To estimate fluorescence lifetimes, an accurate mathematical representation of the fluorescence decay data is crucial. Several assumptions are made to fit the fluorescence decay curves. The goodness-of-fit parameter
The following materials are available in the online version of this article.
We thank Dr. Oliver Griesbeck (Max Planck Institute of Neurobiology Am Klopferspitz, Martinsried, Germany) and Prof. Roger Tsien (Howard Hughes Medical Institute, University of California, San Diego) for the kind donation of cDNA clones encoding TSapphire and mOrange, respectively. Dr. Wendy A. Peer (Purdue University, West Lafayette, IN) is kindly acknowledged for help with APB experiments. The FRET-APB work performed by Dr. Peer was funded by a U.S. Department of Agriculture grant to Roberto Gaxiola and Angus Murphy at Purdue University. We also wish to thank Dr. Laurent Noël (LIPM-INRA/CNRS, Castanet, France) and Dr. Sidney Shaw (Indiana University, Bloomington, IN) for criticism, suggestions, and helpful comments. Received February 5, 2008; accepted July 5, 2008; published July 11, 2008.
1 This work was supported by the French Atomic Energy Agency. 
2 Present address: Department of Biology, Indiana University, Bloomington, IN 47405. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Riyaz A. Bhat (bhatr{at}indiana.edu).
[W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.108.117358 * Corresponding author; e-mail bhatr{at}indiana.edu.
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ABSTRACT
RESULTS
-spectral mode from 450 to 590 nm for TSapphire; 540 to 660 nm for mOrange; and 600 to 720 nm for chlorophyll using a Leica LCS SP2 CLSM.
2 fit, and lifetime (Tm) of a selected region (red arrowhead) in A.
) and QY of TSapphire (
) was calculated as E = 1 – (
one-half of the