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. 2015 May 13;168(3):776–787. doi: 10.1104/pp.15.00533

Binary 2in1 Vectors Improve in Planta (Co)localization and Dynamic Protein Interaction Studies1,[OPEN]

Andreas Hecker 1,2,2, Niklas Wallmeroth 1,2,2, Sébastien Peter 1,2, Michael R Blatt 1,2, Klaus Harter 1,2, Christopher Grefen 1,2,*
PMCID: PMC4741326  PMID: 25971551

The combination of new generation fluorescent proteins with the 2in1-cloning technique improves (co)localization and protein interaction analyses in vivo.

Abstract

Fluorescence-based protein-protein interaction techniques are vital tools for understanding in vivo cellular functions on a mechanistic level. However, only under the condition of highly efficient (co)transformation and accumulation can techniques such as Förster resonance energy transfer (FRET) realize their potential for providing highly accurate and quantitative interaction data. FRET as a fluorescence-based method unifies several advantages, such as measuring in an in vivo environment, real-time context, and the ability to include transient interactions as well as detecting the mere proximity of proteins. Here, we introduce a novel vector set that incorporates the benefit of the recombination-based 2in1 cloning system with the latest state-of-the-art fluorescent proteins for optimal coaccumulation and FRET output studies. We demonstrate its utility across a range of methods. Merging the 2in1 cloning system with new-generation FRET fluorophore pairs allows for enhanced detection, speeds up the preparation of clones, and enables colocalization studies and the identification of meaningful protein-protein interactions in vivo.


Two technological advances allowed the field of modern cell biology to emerge: the development of high-resolution microscopes and the groundbreaking work in the development of fluorescent proteins (FPs) that were originally isolated from jellyfish (for review, see Day and Davidson, 2009; Zimmer, 2009). Genetically encoded FPs can be fused directly to proteins of choice, offering insights into their functional properties by allowing researchers to monitor subcellular localization, chemical environment, interaction, movement, and/or turnover rate.

Research on the FPs themselves has exploded in the past two decades, and a range of technological improvements to and variations of the FPs were generated. These improvements include novel spectral properties from all areas of the color palette and from a diverse range of organisms. They also extend to a multitude of parameters, such as brightness, folding efficiency, chromophore oxidation rate, quantum yield, pH, and photostability (Day and Davidson, 2009). Taken together, these optimizations further the possibilities of available methods or prompt the development of new techniques.

In 1946, the German scientist Theodor Förster laid the theoretical foundation for resonance energy transfer, now known as Förster resonance energy transfer (FRET; Forster, 1946). According to this theory, a donor chromophore in an excited state can transfer the excitation energy to a nearby acceptor chromophore via dipole-dipole coupling without the emission of a photon (for review, see Clegg, 2009; Ishikawa-Ankerhold et al., 2012). FRET depends on the spectral overlap between the emission spectrum of the donor and the absorbance spectrum of the acceptor chromophores. It also depends on the fluorescence quantum yield and the excited state lifetime of the donor in the absence of an acceptor as well as the relative orientation of the two chromophores. Most importantly, FRET efficiency obeys an inverse sixth power law as a function of the distance between the donor and acceptor fluorophores. Thus, FRET is most effective for chromophores separated by distances of less than 10 nm, distances typical for proteins that are closely packed in multiprotein complexes. Accordingly, two interacting proteins in close proximity, which are labeled with an appropriate donor-acceptor FP pair, will generally give rise to a strong FRET interaction, whereas proteins that are located in the same compartment but that do not interact will exhibit little or no FRET activity (Peter et al., 2014). FRET has been used not only as a tool to study protein-protein interactions (PPIs) but also as a sensing principle of small molecule sensors for the determination of a number of cellular biochemical parameters such as calcium concentrations (Miyawaki et al., 1997), changes in membrane voltage (Tsutsui et al., 2008), and hormones (Jones et al., 2013).

Recently, we developed a Gateway-based 2in1 cloning technique that allows the simultaneous recombination of two genes of interest into two independent expression cassettes on the same vector backbone (transfer DNA [T-DNA]). Including an additionally expressed red fluorescent protein from this T-DNA, the system allows the application of a ratiometric bimolecular fluorescence complementation (rBiFC) assay, significantly enhancing the credibility of results compared with classical split-FP-based vector systems (Grefen and Blatt, 2012). This system has since been successfully employed to analyze interactions of a range of different proteins (Karnik et al., 2013b, 2015; Perrella et al., 2013; Hachez et al., 2014; Lipka et al., 2014, Zhang et al., 2015).

Here, we have merged the 2in1 cloning system with state-of-the art FPs for optimizing FRET and improving (co)expression. We demonstrate the technological advantages of using a 2in1 T-DNA over conventional two-plasmid transfection procedures. The new vector sets allow for transient as well as stable expression of FP-tagged proteins in plants. Colocalization of proteins as well as their potential interaction through FRET measurements, such as acceptor photobleaching (AB) and fluorescence lifetime imaging microscopy (FLIM), can reliably and easily be assessed.

RESULTS AND DISCUSSION

The 2in1 System for FRET Applications

The 2in1 system makes use of the λ-phage recombination sites that are commercially exploited in the MultiSite Gateway system of Life Technologies (Grefen and Blatt, 2012). The rationale behind the 2in1 approach was to establish two independent, functional expression cassettes on the same vector backbone while maintaining the ease of recombination-based cloning with two different positive selection markers. This strategy thus allows for simultaneous and directional insertion of two genes into two expression cassettes flanked by individual promoters and tags, which then are expressed from the same vector backbone (Fig. 1A). As constructed, one of the cassettes is a modified version of the classical Gateway cassette. It consists of the chloramphenicol acetyl transferase and the Control of Cell Death B (ccdB) gene flanked by attR1 and attR4 sites (as opposed to attR1 and attR2 in the classical Gateway cassette). The ccdB gene is toxic for most Escherichia coli cloning strains, thereby precluding nonrecombinant clones (Bernard et al., 1994). The second recombination cassette contains the lacZ expression cassette delimited by attR3 and attR2 sites on either side. Both cassettes allow directional and site-specific integration of genes of interest via recombination from the Gateway entry vector constructs pDONR221-P3P2 and pDONR221-P1P4. The vectors designed for this work (Fig. 1, B–D) gave recombination efficiencies comparable to previously published 2in1 vectors of the rBiFC set (Grefen and Blatt, 2012). Vectors and constructs that are needed to perform 2in1 cloning are summarized in Table I.

Figure 1.

Figure 1.

Principle of 2in1 cloning using binary plant vectors. A, Cartoon illustrating the 2in1 concept with recombination reactions between two entry constructs and a FRET 2in1 destination vector carrying the two independent cloning cassettes. Shown is the 2in1 LR reaction for the donor-only FRET-FLIM construct BPC6-mEGFP. Side products are grayed out. B to D, Cartoons depicting all available pFRET 2in1 vectors: pFRETtv-2in1 (B), pFRETgc-2in1 (C), and pFRETvr-2in1 (D). RB, Right border of T-DNA; 35S-Ω, Cauliflower mosaic virus 35S promoter and omega translational enhancer sequence; attRl/attR2/attR3/attR4, attachment/recombination sites; ChloramphenicolR, chloramphenicol acetyltransferase resistance gene; ccdB, gyrase inhibitor gene; lacZ, lacZ expression cassette consisting of the β-galactosidase promoter (pLac) and the codon-optimized (E. coli) α-subunit of β-galactosidase; 3xHA, triple hemagglutinin epitope tag; 2xmyc, double c-myc epitope tag; mTRQ2, monomeric Turquoise2 fluorescent protein; ORI, origin of replication; SpectinomycinR, aminoglycoside-3′-adenylyltransferase expression cassette; LB, left border of T-DNA.

Table I. Constructs used/needed for 2in1 FRET analysis.

Vectora att Sites Insert Marker Functionb Reference
pDONR221-P1P4 attP1, attP4 Gateway cassette Kan, Cm, ccdB Entry vector Life Technologies
pDONR221-P3P2 attP3, attP2 Gateway cassette Kan, Cm, ccdB Entry vector Life Technologies
pDONR221-L1-GentR-L4 attL1, attL4 Gentamycin resistance cassette Kan, Gent Create donor-only clones Grefen and Blatt (2012)
pENTR-L3-TetR-L2 attL3, attL2 Tetracycline resistance cassette Kan, Tet Create acceptor-only clones Life Technologies
pFRETtv-2in1, pFRETgc-2in1,
pFRETvr-2in1 (four each) attR1, attR4, attR3, attR2 Gateway cassette and lacZ cassette Spec, Cm, ccdB, lacZ 2in1 destination vectors (CC, CN, NC, and NN) This study
a

tv, mTRQ2/mVenus; gc, mEGFP/mCherry; vr, mVenus/tagRFP.

b

FP tag orientation according to the cloning cassette; the first letter specifying the position of the donor, the second letter specifying the position of the acceptor FP (e.g. CN = C-terminal donor, N-terminal acceptor FP fusion).

Spectral Properties of the 2in1 FPs

Figure 2 shows the normalized absorbance and emission spectra, including the λ4-weighted spectral overlaps of mTRQ2-mVenus, monomeric enhanced green fluorescent protein (mEGFP)-mCherry, and mVenus-tagRFP pairs selected for the FRET 2in1 vectors. Exemplary pseudocolor images of the coexpression of nucleus-localized Basic Pentacysteine6 (BPC6), a GAGA motif-binding protein (Wanke et al., 2011), with itself were chosen for representation of the wavelength emission peaks of each FP (Fig. 2). Table II lists the crucial FRET-related spectral properties of the state-of-the-art FPs that were chosen. Particular attention is given to properties relevant for FRET-FLIM studies (see below), although the fluorophore pairs are also suitable for intensity-based FRET approaches and (co)localization studies. In terms of quantum yield and brightness, the donor chromophores mTRQ2 (Goedhart et al., 2012), mEGFP (Yang et al., 1996), and mVenus (Nagai et al., 2002) are the gold standards within their spectral ranges (Table II). These fluorophores also benefit from photostability, and their folding efficiency is among the highest of the latest generation of FPs (Kremers and Goedhart, 2009). Each donor can be excited with one of the three major argon laser wavelengths used in confocal microscopy, 458 nm (mTRQ2), 488 nm (mEGFP), and 514 nm (mVenus). Due to their excellent spectral overlap, mVenus, mCherry (Shaner et al., 2004), and tagRFP (Merzlyak et al., 2007) were chosen as FRET acceptors, respectively (Fig. 2). Each pair, namely mTRQ2-mVenus, mEGFP-mCherry, and mVenus-tagRFP, was used in generating four different vectors, thus allowing for the expression of all possible combinations of either N- or C-terminal FP fusions (Fig. 1, B–D). In addition, the chromophores chosen in this vector set are all monomeric, thereby avoiding dimerization or quenching due to fluorophore interaction (Zacharias et al., 2002; Zhang et al., 2002). While the FRET pairs mEGFP-mCherry (Tramier et al., 2006) and mTRQ2-mVenus (Goedhart et al., 2012) were demonstrated before (summarized by Müller et al., 2013), to our knowledge, the pair mVenus-tagRFP has not been used previously.

Figure 2.

Figure 2.

Spectral properties of fluorophore pairs used in this work. Shown are normalized absorbance (dashed lines) and emission spectra (solid lines) including λ4-weighted spectral overlap (light gray surface plot) of mTRQ2-mVenus (A), mEGFP-mCherry (B), and mVenus-tagRFP (C). Exemplary confocal images on the right show colocalization of BPC6 with itself for each FRET couple. Pseudocolors are applied for enhanced visualization. The order of images from top to bottom is as follows: donor fluorophore fusion, acceptor fluorophore fusion, and merge with bright field. Confocal laser scanning microscopy (CLSM) settings were as follows: A, excitation 458 nm/emission 465 to 505 nm (mTRQ2), excitation 514 nm/emission 520 to 560 nm (mVenus); B, excitation 488 nm/emission 495 to 530 nm (mEGFP), excitation 561 nm/emission 580 to 630 nm (mCherry); C, excitation 514 nm/emission 520 to 560 nm (mVenus), excitation 561 nm/emission 580 to 630 nm (tagRFP). Bars = 10 nm.

Table II. Physicochemical properties of FRET fluorophores.

FRET Pair
Donor Excitation λmax Acceptor Emission λmax Donor Quantum Yield Acceptor Absorption Coefficient Förster Distance
Donor Acceptor
nm m−1 cm−1 nm
mTRQ2 mVenus 430 529 0.93 92,200 5.7
mEGFP mCherry 488 611 0.6 72,000 5.2
mVenus tagRFP 516 583 0.57 100,000 5.9

Dual Expression from One T-DNA

A prerequisite for PPI analysis is information on subcellular (co)localization of the proteins of choice. In spite of the reservations associated with heterologous (over)expression of genes depleted of their regulatory sequences (promoter, untranslated regions, and introns), transient or stable coexpression of two genes from a 2in1 vector is a feasible and fast way to determine subcellular (co)localization.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor proteins (SNAREs), which drive the final step in membrane fusion events, are often used as membrane markers due to their specific subcellular localization patterns. The plasma membrane (PM) SNARE Syntaxin of Plants121 (SYP121; Leyman et al., 1999), vesicle- and PM-localized Vesicle-Associated Membrane Protein721 (VAMP721; El Kasmi et al., 2013), and endoplasmic reticulum-localized SNARE VAMP723 (Uemura et al., 2004) were used here to assess the vector potential for (co)localization studies. Figure 3 and Supplemental Figure S1 demonstrate the use of pFRETgc 2in1 vectors to determine and distinguish the subcellular localization of the aforementioned proteins while using different transformation procedures and hosts. Figure 3 depicts the spatial differences in subcellular localization of the SNARE proteins VAMP723 and SYP121 in transient transfection of Nicotiana benthamiana leaves (Fig. 3A), Arabidopsis (Arabidopsis thaliana) leaf protoplasts (Fig. 3B), and stably transformed Arabidopsis Columbia-0 plants (Fig. 3C). In addition, Supplemental Figure S1 shows partial colocalization of SYP121 with VAMP721 in the PM while also highlighting vesicular structures that are solely tagged with GFP-VAMP721 in all three transformation procedures. Line histograms reveal the high spatial resolution providing information on each fusion protein’s subcellular (co)localization or its distinction.

Figure 3.

Figure 3.

Colocalization analysis using different transformation approaches. Confocal images show colocalization analysis of endoplasmic reticulum-localized mEGFP-VAMP723 with PM-localized mCherry-SYP121 (using vector pFRETgc-2in1-NN) in transiently transfected N. benthamiana epidermal leaves (A) or Arabidopsis leaf protoplasts (B) and stably transformed Arabidopsis T1 seedlings (C). Histograms at right show the normalized fluorescence intensity of either mEGFP or mCherry channels along the yellow arrows in the merged images (see also Supplemental Fig. S1). a.u., Arbitrary units. White bars = 10 μm; black bar = 5 μm.

Despite heterologous overexpression, all gene products localize to their previously suggested, native subcellular compartments (Leyman et al., 1999; Uemura et al., 2004; El Kasmi et al., 2013). The vector set was primarily anticipated for transient studies in which the 35S promoter is still a powerful driver of expression despite its inherent silencing issues when used in stable transformants (Chalfun-Junior et al., 2003). Nevertheless, the T1 generation of stably transformed Arabidopsis seedlings was analyzed: out of 74 Basta-resistant seedlings that were screened for fluorescence signals, 10% expressed both mEGFP-VAMP723 and mCherry-SYP121 (Fig. 3C). None of the analyzed transformants expressed only one of the two FPs, confirming an expected coexpression and, thus, cotransformation efficiency of 100%. Taken together, the FRET 2in1 vectors prove their feasibility to easily assess subcellular (co)localization in both transient and stable transformation. In the latter case, the vectors avoid the time-consuming step of sequential insertion either by crossing two individually transformed lines or the simultaneous cotransformation (and T-DNA integration) of the two individual plasmids using floral dip transformation, which has been shown previously to yield only between 8% and 34% (Ghedira et al., 2013) cotransformants. Also, cosegregation excluded, the resulting heterozygous T1 lines will yield only 6.25% double homozygous T2 lines, as compared with 25% using one T-DNA (2in1 insertion).

Transient Transformation Efficiency of 2in1 Vectors Compared with Single Plasmid Cotransformation

For (semi)quantitative FRET studies, especially when they are intensity based, the accumulation of equal amounts of donor and acceptor FP fusion proteins is often ideal. Yet, no expression system can guarantee a 1:1 expression ratio of fusion proteins. Posttranscriptional or posttranslational regulation is likely to alter the stoichiometry even when the identical promoter is used.

Agroinfiltration has become a widely used technique for fast and reliable transient transformation of N. benthamiana leaves (Schöb et al., 1997). Many subcellular localization studies in transient N. benthamiana rely on coexpression of the protein of interest with a subcellular marker. While for colocalization, a 100% cotransformation efficiency is not crucial, for other methods, such as FRET, it is often best to have donor and acceptor fusion proteins in the same cell at nearly equal concentrations.

The issue of cotransformation ratios was raised in the paragraph above regarding stable transformation. Generally, literature values vary between 10% and 60% cotransformation probability for transient transformation of Arabidopsis root explants (Ghedira et al., 2013). In transiently transformed Arabidopsis seedlings using Agrobacterium tumefaciens, Marion et al. (2008) found a cotransformation efficiency of up to 70%. To our knowledge, there are no data on coexpression ratios of transient transformation of N. benthamiana leaves.

We have compared the number of cells that expressed both fusion proteins needed for FRET from either two individual plasmids or from the novel 2in1 vectors (Fig. 4). As the exemplary gene for our studies, we used nucleus-localized BPC6 (Wanke et al., 2011). To avoid the complication of comparing across different vectors, we chose to use the same 2in1 vector not only for the actual transformation of one plasmid with two expression cassettes but also for the two individually transformed plasmids that contain only one fusion protein (donor or acceptor). For vectors incorporating only one of the two fusion proteins in the 2in1 LR reaction, an E. coli resistance marker (dummy) was recombined in the unused of the two expression cassettes, thereby preventing translational read through of that expression cassette’s C-terminal FP tag (Fig. 4). The resulting plasmid contained only one FP fusion protein (donor or acceptor), yet the same plasmid backbone was used for the infiltration.

Figure 4.

Figure 4.

Frequency of cotransformation events of two individual plasmids compared with a 2in1 construct. A, Box-plot diagram with mCherry-mEGFP mean fluorescence intensity ratios including outliers of four independent N. benthamiana infiltration experiments. Red boxes comprise the 25th to 75th percentiles of all values, with the horizontal line in the middle being the median. The yellow background (mCherry:mEGFP relative fluorescence ratio of 0.2–5) spans values that were counted for calculating the cotransformation ratio below. B, Absolute numbers of fluorescent nuclei and cotransformation ratios of each individual experiment. Magenta numbers represent values above an mCherry:mEGFP ratio of 5 (only mCherry fluorescence detected), yellow numbers represent values that were counted as cotransformed cells (ratio of 0.2–5), and green numbers represent the number of times only mEGFP fluorescence was detected (mCherry:mEGFP ratio below 0.2).

We analyzed mean fluorescence intensities of nuclei from 30 different square sections (each roughly 0.084 mm2 in size) of four independently transformed N. benthamiana leaves that expressed either BPC6-mCherry, BPC6-mEGFP, or both (in pFRETgc-2in1-CC; for graphical description of the work flow, see Supplemental Fig. S2). Ratios of mCherry to mEGFP fluorescence were normalized by using the median value of all ratios as denominator for the absolute mCherry values. The box-plot diagram in Figure 4A shows ratios of 25% above or below the median in red boxes along with each value for mEGFP/mCherry signal, represented as black dots. Ratios 5 times higher (greater than 5) or lower (less than 0.2) than unity were designated as thresholds for coexpression. This analysis yielded an average cotransformation ratio for all four experiments of 77.6% (n = 1,221) for the individual and 99.3% (n = 1,099) for the 2in1 plasmid transformations. As the same promoter and backbone were used, the individual ratios also allow a rough estimate of coexpression efficiency. This suggests that, despite all potential posttranscriptional or posttranslational events that will be different for each pair of genes and fusion proteins to be analyzed, the 2in1 system allows for less variation in coexpression rates compared with individual plasmid transformation.

Values slightly below 100% coexpression in 2in1 constructs could have resulted from truncations of the T-DNA left border or from silencing of one of the two expression cassettes in individual cells. Nevertheless, the nearly complete cotransformation of both fusion proteins in the 2in1 system illustrates its advantage for assays that are based on transient expression.

Improving Intensity-Based FRET through Dual Expression

As demonstrated in Figure 4, the high variance of cotransformations makes finding cells with nearly equal expression levels cumbersome and time consuming. Naturally, the fluorescence of both fusion proteins would allow one to choose cells (in transient studies) that express both proteins to similar levels, but the temporal advantage of utilizing a dual expressing T-DNA is obvious.

In order to correlate the result of the cotransformation efficiency in Figure 4 with intensity-based FRET approaches, we performed AB experiments and measured the FRET efficiency of cells transfected with 2in1 constructs versus cells with two individual plasmids (Fig. 5). As a positive control, we again employed the nucleus-localized protein BPC6 fused to mEGFP and mCherry and its capacity to dimerize (Wanke et al., 2011). As a negative control, BPC6-mEGFP was coexpressed with nucleus-localized mCherry. Exactly as before, to avoid the complication of comparing across different vectors, the same 2in1 vector was used for individual and dual expression (see above).

Figure 5.

Figure 5.

AB experiments comparing classical transfection of two individual plasmids with 2in1 transfection. A and B, Exemplar confocal images of an AB experiment, with A depicting a positive interaction couple (BPC6 + BPC6) and B depicting the negative control (BPC6 + nuclear mCherry). Acceptor (mCherry) and donor (mEGFP) fluorescence is monitored prior to (pre bleach) and after (post bleach) bleaching. Bars = 10 μm. C, FRET efficiency was calculated according to: FRETeff = (DpostDpre)/Dpost, with Dpost and Dpre as the mean fluorescence intensity of the donor after and before bleaching. The box plot represents data from 30 independent experiments using either two individual plasmids (gray bars) or a single 2in1 plasmid (white bars). Boxes represent values from the 25th (bottom) to the 75th (top) percentiles, with the median as the horizontal line within each box. Whiskers at the top and bottom of the boxes comprise the upper and lower 25% of all values, with the end points marking the value of the farthest outlier. a.u., Arbitrary units.

For AB FRET experiments (Fig. 5), fluorescent nuclei were chosen that expressed mEGFP and mCherry at apparently equal levels (a routine that was more time consuming in the case of individual plasmids as compared with the 2in1 approach). Mean donor fluorescence intensity was measured in suitable nuclei (n = 30) before and after photobleaching of the mCherry acceptor fusion and used to calculate FRET efficiency (Fig. 5C). While the approach using two individual plasmids yielded FRET signals for the positive control sample that were significant when compared with the negative control, the values showed great variability. Contrary to that, the FRET efficiency values obtained using the 2in1 approach not only showed less variance but also resulted in higher FRET signals in general (FRET efficiency median value of 0.27 using 2in1 versus 0.11 using the conventional approach; Fig. 5C). Taken together, these results clearly show that a dual expressing T-DNA not only saves time in both preparing and performing the experiment but also allows for obtaining less variable, and hence more significant, results.

The Use of 2in1 Vectors for in Planta FRET-FLIM Studies

Quantitative as well as semiquantitative FRET-based interaction studies require for each FP a number of parameters to be optimal, such as quantum yield, oligomerization state, pH dependency, and photostability (Gordon et al., 1998; Kaminski et al., 2014). For the FRET pair, the spectral overlap, the acceptor’s absorption coefficient, and the Förster distance (the distance between donor and acceptor where 50% FRET efficiency is reached) are among the most important parameters (Clegg, 2009; Ishikawa-Ankerhold et al., 2012). Intensity-based FRET techniques, such as donor dequenching through AB, are particularly sensitive to the relative concentrations of donor and acceptor, and their ratio should be kept as close to a constant value as possible (Fig. 5; Harter et al., 2012; Peter et al., 2014). Although this aspect can be tackled using the 2in1 approach, intensity-based FRET approaches are also subject to other difficulties, such as a cross talk caused by donor emission, which leaks into the acceptor channel, a direct excitation of the acceptor by the illumination source, or incomplete photobleaching of all acceptors in the microscopic detection volume (Gordon et al., 1998; Malkusch and Holloschi, 2004; Harter et al., 2012; Kaminski et al., 2014; Peter et al., 2014). These problems are circumvented by measuring FRET using FLIM. This technique takes advantage of the radiative rates of the transition from the excited to the ground state of the donor chromophore, which can be related directly to the distance between donor and acceptor fluorophores (Bücherl et al., 2014; Laptenok et al., 2014). If energy transfer to an acceptor occurs, an additional relaxation channel for the donor to lose its excitation energy opens. Accordingly, the radiative transition competes with this additional nonradiative pathway, causing the donor fluorescence lifetime to be shifted to shorter time values. This reduction in the fluorescence lifetime of the quenched donor is connected to the FRET efficiency and can be quantified, resulting in a unique value for the fusion protein pair (Harter et al., 2012; Peter et al., 2014). Although FRET-FLIM requires careful data analysis, this method is much less error prone than intensity-based FRET methods.

Figure 6 illustrates the use of the pFRETgc-2in1 and pFRETtv-2in1 vectors in measuring FRET-FLIM. The fluorescence lifetimes of donor BPC6-mEGFP with various mCherry acceptor fusions were visualized as bar charts (Fig. 6B) and as heat maps (Fig. 6C) that color code the fluorescence lifetime within the area of interest, here the nucleus. The results show that the donor lifetime of BPC6-mEGFP decreased significantly when coexpressed with BPC6-mCherry and, although slightly weaker, with an mCherry fusion protein encoding the nuclear localization signal-DNA-binding domain fusion of BPC1 (BPC1 NLS-DBD-mCherry). There is no lifetime decrease in the absence of an acceptor fusion or when BPC6-mEGFP is coexpressed with a gene coding for nucleus-targeted mCherry (Fig. 6B). These results indicate a specific homomerization of BPC6 and a heterotypic interaction of BPC6 with the DNA-binding domain of BPC1 inside the nucleus. This interaction has been detected before by yeast two-hybrid analysis (Wanke et al., 2011). The same set of proteins was also used to assess the feasibility of the mTRQ2/mVenus pair in FLIM analysis (Fig. 6, D–F), with results that virtually copy the mEGFP/mCherry study. Striking, though, is the difference in maximum donor lifetime of mTRQ2 (approximately 3.8 ns) as opposed to mEGFP (approximately 2.5 ns). This leads to higher absolute lifetime differences between positive and negative control samples, providing a broader range for quantitative and dynamic association measurements.

Figure 6.

Figure 6.

FLIM analysis comparing two different FRET pairs. A and D, T-DNA cartoons illustrating the vector plasmid used for each FLIM setup in B and E, respectively. FLIM measurements are of transiently transformed N. benthamiana epidermal cells expressing BPC6 as donor in the presence of BPC6, BPC1-NLS-DBD (DNA-binding domain), nuclear localization signal (NLS), or alone from either pFRETcg-2in1-CC (mEGFP-mCherry pair; B) or pFRETtv-2in1-CC (mTRQ2-mVenus pair; D). RB, Right border of T-DNA; LB, left border of T-DNA. Error bars indicate se (n ≥ 21 with one-sided P < 0.01 [B] and n = 10 with one-sided P < 0.01 [E]). C and F, Heat maps of representative nuclei used for FLIM measurements in B and E, respectively. Donor lifetimes (BPC6-mEGFP/BPC6-mTRQ2) are color coded according to the scales at left.

Comparing FLIM with rBiFC

Each PPI technique comes with a set of advantages and disadvantages when compared with alternative approaches. A comparison of FLIM with an alternative PPI technique, therefore, seems appropriate. To eliminate a bias against varying expression, we chose our previously published 2in1-based rBiFC (Grefen and Blatt, 2012) as an alternative method. An interaction between the soluble Qb+c SNARE SNAP33 and its corresponding Qa-SNAREs SYP111, SYP121, and/or SYP122 had been shown previously, mainly by using coimmunoprecipitation analysis (Heese et al., 2001; Kwon et al., 2008; Pajonk et al., 2008). To our knowledge, these interactions have not been demonstrated using either FLIM or rBiFC. As biological negative controls, we made use of the Qa-SNARE SYP21, which localizes to the prevacuolar compartment (Bassham and Raikhel, 1999) and does not cross interact functionally with SYP121 or SYP122 (Tyrrell et al., 2007). Additionally, and to use a control that localizes to the PM, we employed a truncated version of SYP121 that only contains its membrane-spanning C-terminal tail devoid of the known cytoplasmic interaction site (Qa motif; Lipka et al., 2007; Grefen and Blatt, 2008).

A direct comparison of the two methods using membrane-localized proteins revealed some differences. As expected, an interaction between SNAP33 and the three SYP1 family SNAREs was identified by both rBiFC (Fig. 7, B and C) and FLIM (Fig. 7, E and F). Both methods also reported a lack of interaction between the noncognate pair of SYP21 and SNAP33. However, SYP121, when truncated to its transmembrane domain only, yielded a significant rBiFC signal with SNAP33, whereas FLIM analysis showed no difference from the background. We suggest that this seemingly contradictory result highlights the potential for the split fluorophore to stabilize interacting proteins and thereby enhance weak or otherwise transient interactions. Qa-SNAREs are able to assemble in homomultimers as well as heterodimers with Qb+c-SNAREs, and this applies equally to SYP121 and SNAP33 (Karnik et al., 2013b). It is possible, therefore, that a weaker association with the SYP121 transmembrane domain, either with SNAP33 or with a SNAP33-SYP121 complex, could become locked through fluorophore annealing, a situation that would not arise when interactions were probed for protein complex dynamics using the FRET fluorophore pairs (Harter et al., 2012).

Figure 7.

Figure 7.

Comparing rBiFC data with FLIM analysis. A, T-DNA cartoon illustrating the vector plasmid used for rBiFC analysis in B and C. RB, Right border of T-DNA; LB, left border of T-DNA. B, Exemplary confocal images of N. benthamiana cells expressing rBiFC constructs with nYFP-SNAP33 and different cYFP-Qa-SNAREs or a truncated version of SYP121 containing solely its C-terminal transmembrane domain (TMD). Bars = 10 μm. C, Bar chart showing the mean fluorescence intensity ratios of complemented yellow fluorescent protein (YFP) to red fluorescent protein (RFP) signal averaged over 40 different leaf sections including se. a.u., Arbitrary units. D, T-DNA cartoon illustrating the vector plasmid used for FLIM analysis in E and C. E, FLIM measurements of transiently transformed N. benthamiana epidermal cells expressing from pFRETvr-2in1-NN (mVenus-tagRFP pair) SNAP33 with the corresponding Qa-SNAREs used in A to C and alone. Error bars indicate se (n ≥ 21 with one-sided P < 0.05). F, Heat map showing representative membrane areas used for FLIM measurements in E. Donor lifetimes (SNAP33-mVenus) are color coded according to the scale at left.

CONCLUSION

The vector set described here features a number of advantages compared with classical vector sets. Combining the ease of recombination-based cloning with the simultaneous insertion of two genes into two independent expression cassettes speeds up the process of preparing recombinant DNA. The resulting plasmids allow the reliable integration of both transgenes on one T-DNA into plants, transiently or stably, thereby increasing the percentage of cotransformation and enabling nearly balanced coexpression ratios.

In addition to the methodical advantages of cotransformation and coexpression efficiency, the choice of state-of-the-art FPs and their pairing for optimized physicochemical properties greatly enhance the feasibility for colocalization and challenging FRET studies. All three vector sets are equally suited for FRET-based experiments, so researchers can use the pair that fits their in-house microscope’s specifications best. Nevertheless, the experiments performed in this study disclosed certain advantages of different FP pairs with respect to the applied FRET method. The mTRQ2/mVenus vector set benefits from a rather long lifetime of the FP mTRQ. A basal fluorescence lifetime of almost twice the GFP’s makes it an ideal donor in FLIM experiments. The mEGFP/mCherry pair, on the other hand, is convenient in AB setups. Under our imaging conditions and in living plant cells, mCherry was very sensitive to photobleaching, an optimal prerequisite for an acceptor FP in AB experiments. This observation is contradictory to in vitro analysis of the fluorophore; however, it is known that the bleaching properties of mCherry depend on imaging modalities (Shaner et al., 2005). While the FRET pair mVenus/tagRFP does not stand out with such apparent optima, its benefit might be revealed in studying multimeric interactions. This vector set in combinatorial expression with the mTRQ2-mVenus FRET pair should enable triple FRET studies in the future (Haustein et al., 2003; Peter et al., 2014).

MATERIALS AND METHODS

Creation of 2in1 FRET Vectors

Vectors were cloned using a combination of gene synthesis and classical cloning. Two different strategies were followed, one for vectors with both FPs at the C termini (CC) and one for the other tag orientations (CN, NC, and NN), as N-terminal FP fusions also carry a C-terminal epitope tag. A base vector (Supplemental Fig. S3A) was created through gene synthesis and subcloning of the synthesized cassette into the binary vector pBiFC-BB (Grefen and Blatt, 2012) via PmeI/SnaBI. This resulted in the binary vector pBBb that carries a codon-optimized (for Arabidopsis [Arabidopsis thaliana]) Basta expression cassette adjacent to the left border of the T-DNA and with four unique blunt-end restriction enzyme sites (RES; AfeI, PmlI, EcoICRI, and SnaBI) 3′ of the right border.

For the first strategy, a cloning helper vector (pUC35S-Tec; Supplemental Fig. S3B) was gene synthesized that contains the cauliflower mosaic virus 35S promoter and omega enhancer, unique XbaI-RES, 3xHA tag, unique MscI-RES, myc tag, unique NaeI-, SpeI-, PsiI-RES, and 35S terminator. This expression cassette is flanked by StuI-RES. pUC57-Tec was digested using XbaI/NaeI, and, using the 2in1 conversion vectors pUC-R3R2 and pUC-R1R4 (Grefen and Blatt, 2012), recombination cassettes were subcloned via SpeI (compatible to XbaI) and PsiI (blunt end, like NaeI). The resulting vectors pUC35S::R3R2 and pUC35S::R1R4 were used to introduce FPs at the C termini of the expression cassettes (via SpeI/PsiI). Venus and EGFP (Grefen et al., 2010) were mutated at residue Ala-206 to Lys via site-directed mutagenesis (Karnik et al., 2013a), rendering FP variants monomeric (Zacharias et al., 2002). Resulting mEGFP, mVenus, and mTRQ2 were introduced in pUC35S::R3R2, and mCherry, tagRFP (Evrogen), and mVenus were introduced in pUC35S::R1R4. C-terminally tagged R3R2 expression cassettes were excised via StuI and inserted into pBBb via SnaBI. The three resulting vectors are pFRETgc-intA, pFRETvr-intA, and pFRETtv-intA. With the exception of the mCherry expression cassette, which was PCR amplified and flanked by HpaI-RES, the R1R4 cassettes were excised with StuI and ligated into the intermediate vectors via AfeI, to yield pFRETgc-2in1-CC, pFRETvr-2in1-CC, and pFRETtv-2in1-CC.

The second strategy required another helper vector to be gene synthesized: pUC35S-Ctags (Supplemental Fig. S3C) contains the 35S promoter/omega enhancer followed by unique SpeI- and MscI-RES, a 3xHA tag, a unique NaeI-RES, a double myc tag, a unique PsiI-RES, and the 35S termination signal sequence. C-terminal 3xHA-tagged expression cassettes were created through the excision of 2xmyc tags via NaeI/PsiI and religation. The resulting vector was linearized using SpeI/MscI, and the R3R2 cassette of pUC-R3R2 was inserted (SpeI/PsiI). Via XbaI/HpaI, the PCR-amplified FPs mEGFP, mVenus, and mTRQ2 were introduced. Expression cassettes were excised via StuI and ligated into pBBb via SnaBI to create intermediate vectors pFRETgc-intB, pFRETvr-intB, and pFRETtv-intB. A C-terminally 2xmyc-tagged R1R4 cassette was generated through replacing the 3xHA tag in pUC35S-Ctags (SpeI/NaeI) with the R1R4 cassette of pUC-R1R4 (SpeI/PsiI). The FPs mCherry, tagRFP, and mVenus were PCR amplified and introduced via XbaI/HpaI 5′ of the R1R4 expression cassette. The resulting expression cassettes were excised via StuI (or PCR amplified in the case of mCherry) and introduced in intermediate vectors A and B. Also, the expression cassettes with C-terminally fused FPs were introduced into pFRET-2in1-intB vectors to generate another nine vectors: pFRETgc-2in1-CN, -NC, and -NN; pFRETvr-2in1-CN, -NC, and -NN; and pFRETtv-2in1-CN, -NC, and -NN. All vectors were verified via restriction digestion and/or Sanger sequencing. All maps, sequences, and/or DNAs are available upon request.

Gateway Cloning

Oligonucleotides for creating entry clones (Supplemental Table S1) were designed according to the guidelines in the Gateway manual (Life Technologies). PCR programs were based on a two-cycle routine using complementary DNA (cDNA) as a template: the first cycle comprises a five times repeated three-step program of denaturing at 95°C, annealing at 54°C, and synthesis at 72°C (20 s kb−1), followed by the second cycle, a 30 times repetition of a two-step routine, denaturing and synthesis, omitting the annealing step. After gel purification of PCR products, BP recombination reactions were performed as described previously (Grefen and Blatt, 2012). The 2in1 LR reaction was performed according to the supplemental data of Grefen and Blatt (2012). A molar ratio of vector to entry clones of 1:3:3 yielded optimal results as described previously (Grefen and Blatt, 2012). Colonies were selected from plates that contained 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside/isopropyl β-d-1-thiogalactopyranoside in addition to spectinomycin to distinguish partly recombined (blue) from fully recombined (white) clones. Plasmid DNA was verified via restriction digest and Sanger sequencing.

Nicotiana benthamiana Transformation

Agrobacterium tumefaciens strain GV3101 (Koncz and Schell, 1986) was used to transiently transform N. benthamiana with 2in1 FRET constructs through syringe-mediated infiltration (Schöb et al., 1997; Sparkes et al., 2006). Overnight cultures of agrobacteria were used to inoculate fresh Luria-Bertani medium (supplemented with 50 µg µL−1 rifampicin, 20 µg µL−1 gentamycin, and 100 µg µL−1 spectinomycin) to an optical density of 0.01 and grown for another 4 to 6 h up to an optical density at 600 nm of 0.5 to 0.8. Agrobacteria were harvested, washed in sterile water, and resuspended in ice-cold AS medium (10 mm MgCl2, 10 mm MES-KOH, pH 5.6, plus 150 μm acetosyringone; Grefen et al., 2008; Blatt and Grefen, 2014). After an incubation time of 30 to 60 min on ice, leaves of 3- to 4-week-old N. benthamiana plants were infiltrated as described previously (Schöb et al., 1997; Sparkes et al., 2006; Blatt and Grefen, 2014). Leaves were subjected to CLSM analysis 2 d post infiltration.

Arabidopsis Leaf Protoplast Preparation and Transfection

Leaves of 4-week-old Arabidopsis plants (Columbia-0), grown under standard greenhouse conditions, were perforated abaxially and incubated in TEX buffer (Gamborg’s B5 salts, 500 mg L−1 MES, 750 mg L−1 CaCl22H2O, 250 mg L−1 NH4NO3, and 0.4 m 13.7% [w/v] saccharose, pH 5.7), supplied with 1.5% (w/v) cellulase and 0.4% (w/v) macerozyme, overnight in the dark. After incubation, the cells were gently released, collected, and centrifuged at 100g. Intact cells, floating on top of the solution, were washed with W5 buffer (154 mm NaCl, 125 mm CaCl2, 5 mm KCl, and 5 mm Glc, pH 5.7).

Transfection of protoplasts was carried out using the small-scale transfection method described previously (Berendzen et al., 2012) with the following modifications. MMg (0.4 m Mannitol, 15 mm MgCl2, 4 mm MES, pH 6) was replaced by MM medium (0.4 m mannitol and 5 mm MES, pH 6), and the pH of the polyethylene glycol solution was decreased to pH 6. The concentration of cells was adjusted to 6.6 × 10−6 cells mL−1 with MM medium, and 2 × 10−5 cells were used per transfection subsequently. For the transfection, 4 µg of the respective plasmids were used according to Berendzen et al. (2012).

Arabidopsis Stable Transformation and Selection

A. tumefaciens strain GV3101 carrying the respective plasmids was precultured overnight at 28°C in 5 mL of Luria-Bertani medium supplied with antibiotics. A total of 200 mL of Luria-Bertani medium (including antibiotics) was inoculated with 200 µL of preculture and grown overnight again. Cells were pelleted and resuspended in 200 mL of infiltration medium (5% [w/v] Suc, 0.01 Silwett, and 10 mm MgSO4). Inflorescences of 5- to 6-week-old Arabidopsis plants were dipped into the suspension according to the floral dip method (Clough and Bent, 1998). The procedure was repeated after 1 week. T1 seeds were grown on soil and treated 7 d post germination with the herbicide Basta to select for successful transformants.

CLSM, AB, FLIM, and rBiFC Analyses

All CLSM, AB, and FLIM measurements were performed using a Leica TCS SP8 confocal microscope (Leica Microsystems) equipped with a FLIM unit (PicoQuant).

Images for Figures 2, 3, 5, 6, and 7 were acquired using a 63×/1.20 water-immersion objective. The rBiFC analysis was performed using a 40×/0.75 water-immersion objective. For excitation and emission of FPs, the following laser settings were used: mTRQ at excitation 458 nm and emission 465 to 505 nm; mVenus at excitation 514 nm and emission 520 to 560 nm; mEGFP at excitation 488 nm and emission 495 to 530 nm; mCherry at excitation 561 nm and emission 580 to 630 nm; tagRFP at excitation 561 nm and emission 580 to 630 nm.

AB was performed using a bleaching routine with the 561-nm laser (mCherry) line at 100% intensity and 10 frames. Data derive from two different biological replicates and measurements of up to 15 nuclei for each replicate. The mean FRET efficiency as well as the se values were calculated using Microsoft Excel.

FLIM data derive from four different biological replicates and measurements of up to 10 nuclei for each replicate. To excite BPC6-mEGFP for FLIM experiments, a 470-nm pulsed laser (LDH-P-C-470) was used, and the corresponding emission was detected with a FLIM-compatible photomultiplier tube from 495 to 530 nm by time-correlated single-photon counting using a Picoharp 300 module (PicoQuant). Each time-correlated single-photon counting histogram was reconvoluted with a corresponding instrument response function and fitted against a monoexponential decay function to unravel the GFP fluorescence lifetime of each nucleus. The average GFP fluorescence lifetimes as well as the se values were calculated using Microsoft Excel.

The measurements for rBiFC were performed as described previously (Grefen and Blatt, 2012). Data derive from four biological replicates with 20 different leaf sections each. Exemplary images define regions of interest for which mean fluorescence intensity was calculated and ratioed.

Vector Maps and Sequences

Vector sequences and maps can be downloaded at http://www.psrg.org.uk and http://www.zmbp.uni-tuebingen.de/dev-genetics/grefen.html and are available upon request through these Web sites.

Supplemental Data

The following supplemental materials are available.

Supplementary Material

Supplemental Data

Acknowledgments

We thank Volker Lipka and Kenneth Berendzen for providing Venus and mCherry cDNAs; Dorus Gadella for providing the mTRQ2 cDNA and spectral data; Sabine Müller for valuable comments regarding figure preparation; and Lisa Asseck, Dietmar Mehlhorn, and Eva Schwörzer for technical support.

Glossary

FP

fluorescent protein

FRET

Förster resonance energy transfer

PPI

protein-protein interaction

T-DNA

transfer DNA

rBiFC

ratiometric bimolecular fluorescence complementation

AB

acceptor photobleaching

FLIM

fluorescence lifetime imaging microscopy

PM

plasma membrane

cDNA

complementary DNA

CLSM

confocal laser scanning microscopy

Footnotes

1

This work was supported by a Carl Zeiss Fellowship (to A.H.), by the Biotechnology and Biological Sciences Research Council (grant nos. BB/H0024867/1, BB/I024496/1, and BB/K015893/1 to M.R.B.), and by the Deutsche Forschungsgemeinschaft (grant no. SFB1101 to K.H. [project D02] and an Emmy Noether Fellowship to C.G. [grant no. GR 4251/1–1]).

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