The Plant Journal (2004) 40, 428-438 doi: 10.1111/J.1365-313X.2004.02219.X TECHNICAL ADVANCE Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation Michael Walter1, Christina Chaban2, Katia Schütze2, Oliver Bat ist ic1, Katrin Weckermann3, Christian Näke2, Dragica Blazevic1, Christopher Grefen2, Karin Schumacher3, Claudia (Decking3, Klaus Harter2'* and Jörg Kudla1 * Institut für Botanik und Botanischer Garten, Molekulare Entwicklungsbiologie der Pflanzen, Universität Münster, Schlossplatz 4, 48149 Münster, Germany, 2Botanisches Institut, Universität zu Köln, Gyrhofstr. 15, 50931 Köln, Germany, and 3ZMBP, Pflanzenphysiologie, Universität Tübingen, Auf der Morgenstelle 1, 72076 Tübingen, Germany Received 24 June 2004; revised 6 August 2004; accepted 12 August 2004. "For correspondence (fax +49 251 83 23311; e-mail jkudla@uni-muenster.de: fax +49 221 470 7765; e-mail klaus.harter@uni-koeln.de). Dynamic networks of protein-protein interactions regulate numerous cellular processes and determine the ability to respond appropriately to environmental stimuli. However, the investigation of protein complex formation in living plant cells by methods such as fluorescence resonance energy transfer has remained experimentally difficult, time consuming and requires sophisticated technical equipment. Here, we report the implementation of a bimolecular fluorescence complementation (BiFC) technique for visualization of protein-protein interactions in plant cells. This approach relies on the formation of a fluorescent complex by two non-fluorescent fragments of the yellow fluorescent protein brought together by association of interacting proteins fused to these fragments (Hu et al., 2002). To enable BiFC analyses in plant cells, we generated different complementary sets of expression vectors, which enable protein interaction studies in transiently or stably transformed cells. These vectors were used to investigate and visualize homodimerization of the basic leucine zipper (bZIP) transcription factor bZIP63 and the zinc finger protein lesion simulating disease 1 (LSD1) from Arabidopsis as well as the dimer formation of the tobacco 14-3-3 protein T14-3c. The interaction analyses of these model proteins established the feasibility of BiFC analyses for efficient visualization of structurally distinct proteins in different cellular compartments. Our investigations revealed a remarkable signal fluorescence intensity of interacting protein complexes as well as a high reproducibility and technical simplicity of the method in different plant systems. Consequently, the BiFC approach should significantly facilitate the visualization of the subcellular sites of protein interactions under conditions that closely reflect the normal physiological environment. Keywords: bimolecular fluorescence complementation, protein-protein interaction, intracellular localization, bZIP transcription factor, 14-3-3 proteins, LSD1. Summary Introduction The regulation and execution of biological processes requires specific interactions of numerous proteins. Tightly regulated protein interaction networks mediate cellular responses to environmental cues and direct the implementation of developmental programs. The selectivity of protein-protein interactions and their appropriate temporal and spatial regulation determine the developmental potential of the cell and its response to endogenous and exogenous signals. On the molecular level differential protein-protein interactions are thought to determine the operation of complex regulatory circuits and signal transduction systems. The complete sequencing of an increasing number of eukaryotic genomes has provided a wealth of information about the number and complexity of protein functions 428 © 2004 Blackwell Publishing Ltd Protein interaction in plant cells 429 required to build up an organism. However, the regulation and interplays of these proteins remain to become explored in order to appreciate the molecular mechanisms of their action. Several methods have been developed to identify, examine and visualize protein interactions and protein complexes in living cells. Among them, the yeast two-hybrid system has significantly advanced the speed and extent of protein interaction studies. However, this system bears intrinsic limitations as for example systematic'false-positive' and 'false-negative' interactions and, moreover, usually combines protein pairs in a heterologous environment (Field and Song, 1989; Stephens and Banting, 2000). The most widely used approach for the visualization of protein interactions in living cells is fluorescence resonance energy transfer (FRET) between spectral variants of the green fluorescence protein (GFP) fused to the associating proteins (Chen efa/., 2003; Periasamy, 2000). However, to enable observation and quantification of small alterations in fluorescence emission, the GFP fluorophores have to join in close spatial proximity and the fusion proteins generally have to be expressed in high levels. Furthermore, verification, whether changes in fluorescence emission are caused by energy transfer, requires complicated irreversible photobleaching or fluorescence lifetime imaging techniques (Chen et al., 2003; Periasamy, 2000). However, the instrumental equipment necessary for these techniques is not widely available and FRET requires intensive methodical training. For these reasons reports about FRET-based protein-protein interaction investigations in living cells have remained rare especially in plant science (Immink efa/., 2002; Mas efa/., 2000; Shah ef al., 2002; Vermeer ef al., 2004). Alternatively, protein interactions can also be investigated in vivo if the protein complex formation can be visualized by the restoration of a detectable activity. In this regard, the principle of intragenic complementation of the lacZ locus from Escherichia co//was adapted to detect protein interactions (Rossi efa/., 1997; Ullmann efa/., 1967). In this experimental system the detection of protein-protein interactions by restoration of p-galactosidase activity was enabled by using p-galactosidase fragments, which could associate only when fused to interacting proteins. Similarly, fragments of the dihydrofolate reductase have been used in protein interaction studies based on complementation of protein function (Pelletier efa/., 1998). However, these techniques require the application of extrinsic fluorophores to visualize the complex formation. An alternative experimental approach for the visualization of protein interactions is based on the formation of a fluorescent complex by fragments of the enhanced yellow fluorescent protein (YFP) when brought together by the interaction of two associating partners fused to these fragments. Recently, Kerppola and colleagues (2002) reported a proof-of-concept for such an approach for the investigation of protein interactions in living mammalian cells and designa- ted this technique as bimolecular fluorescence complementation (BiFC). The unique characteristic of the BiFC approach is that the bright intrinsic fluorescence of the bimolecular complex allows direct visualization of the complex formation in living mammalian cells. Moreover, by analyzing the interactions between members of the basic leucine zipper (bZIP) and Rel transcription factor families, the BiFC approach provided direct evidence of the intracellular locations where the protein association occurs (Hu ef al., 2002). The application of the BiFC approach has recently been extended to the investigation of the interaction pattern and intracellular localization of G-protein complexes in mammalian cells and Dictyostelium discoideum (Hynes ef al., 2004) and to the visualization of 1-aminocyclopropane-1-carboxylase synthase heterodimer formation in E. coli (Tsuchisaka and Theologis, 2004). Furthermore, by introducing a large number of different GFP variants the technique was extended to multicolor BiFC, which allows the direct visualization of multiple protein interactions within the same cell (Grinberg ef al., 2004; Hu and Kerppola, 2003). In this report we describe the generation of several sets of plant-compatible BiFC vectors. We used these vectors for investigating the interaction of plant nuclear and cytoplasmic proteins in different plant systems. Our study attests the general applicability of the BiFC technique and that this assay represents an efficient and convenient tool to investigate protein-protein interactions in living plant cells. Results Generation of plant-compatible BiFC transformation vectors To develop the BiFC technology for the visualization of protein-protein interactions in living plant cells, we constructed four pairs of vectors (Figure 1; for further details see Experimental procedures and Supplementary material). These vectors have been designated pSPYNE and pSPYCE (for split YFP N-terminal/C-terminal fragment expression) respectively. Each vector pair enables the expression of proteins of interest fused either to the N-terminal 155 amino acids (YFPN) or to the C-terminal 86 amino acids of YFP (YFPC; Hu ef al., 2002). Moreover the plasmids contain either a c-myc (pSPYNE) or HA (pSPYCE) affinity tag for detection of fusion protein expression in cell extracts (Figure 1). The binary pSPYNE-KAN and pSPYCE-BAR vectors enable the expression of YFP fragment-fused genomic DNA or of YFP-fragment constructs driven by any promoter of interest (Figure 1). Strong and constitutive expression of fusion proteins in plant cells is ensured by the binary pSPYNE-35S and pSPYCE-35S plasmids which contain the 35S promoter of the cauliflower mosaic virus. For selection of transgenic plants pSPYNE-KAN and pSPYNE-35S carry a nos promoter-driven kanamycin resistance gene (nptll), whereas pSPYCE-BAR and pSPYCE-35S harbor the bar gene conferring © Blackwell Publishing Ltd, The Plant Journal, (2004), 40, 428-438 430 Michael Walter et al. (a) pSPYNE-Kan pSPYCE-Bar c-myc MCS MCS [ HA [ YFP' (aa 1-155) NosT yfp (aa 156-239) NosT (b) pSPYNE-35S/pUC-SPYNE c-myc 35S MCS I YFP' (aa 1-155) NosT pSPYCE-35S/pUC-SPYCE HA ^ 35S | MCsJ yfp (aa 156-239) NosT (C) pUC-SPYNE0 ss ^35S pUC-SPYCEG __ 35S rpulOI ACC65I ^mal ^3tul AscI |SpeI pamHI plal |SalI |XhoI | |KpnI | ^Smal CTCTAGAGTTAACCGGGCTCAGGCCTGGCGCGCCACTAGTGGATCCATCGATAGTACTGTCGACCTCGAGGGTACCGCTCCCGGGATG SRVNRAQAWRAT SGS IDS TVDLEGTAPGM attR1 cdB c-myc attR2 YFP (aa 1-155) NosT attR 1 - &cdB HA attR2 YFP" (aa 156-239) NosT Figure 1. Schematic representation of plant-compatible BiFC vectors. (a) pSPYNE-Kan and pSPYCE-Bar. (b) pSPYNE-35S/pUC-SPYNE and pSPYCE-35S/pUC-SPYCE. (c) pUC-SPYNEG and pUC-SPYCEG. Details of the plasmid construction and vector back bones are given in Experimental procedures and in Figures SI and S2. c-myc, c-myc affinity tag; HA, hemagglutinin affinity tag; MCS, multi-cloning site; 35S, 35S promoter of the cauliflower mosaic virus; NosT, terminator of the A/osgene; YFPN, N-terminal fragment of YFP reaching from amino acid (aa) 1 to 155; YFPC, C-terminal fragment of YFP reaching from amino acid 156 to 239; attR1-CmR-ccdB-attR2, Gateway conversion cassette. insensitivity to the herbicide glufosinate (Figure 1). In addition, we generated two additional sets of vectors based on pUC19 that are specially designed for transient plant cell transformation approaches. pUC-SPYNE and pUC-SPYCE contain the entire expression cassette of pSPYNE-35S or pSPYCE-35S, respectively, and harbor a more variable multi-cloning site (MCS) (Figure 1). Furthermore, in a second set the entire MCSs of pUC-SPYNE and pUC-SPYCE were replaced by the Gateway conversion cassette providing the attR1 and attR2 recombination sites for use with the Gateway cloning system (pUC-SPYNEG, pUC-SPYCE0, Figure 1). BiFC analysis of Arabidopsis nuclear bZIP63 To address the feasibility of BiFC for visualization of protein-protein interaction in living plant cells we first choose a member of the Arabidopsis bZIP factor family (bZIP63, AGI: At5g28770) as a model protein. bZIP63 belongs to subfamily C of Arabidopsis bZIP factors (Jakoby et al., 2002). This transcription factor binds to promoter elements containing the CACGTG or GACGTC sequence in vitro and is localized to the nucleus of plant cells (Nake, 2001). Moreover, bZIP transcription factors are known to form homodimers and heterodimers via the C-terminal leucine zipper domain (Siberil et al., 2001). To first investigate the interaction potential of bZIP63 by an independent experimental approach, we performed an interaction analysis in the yeast two-hybrid system. As shown in Figure 2, bZIP63 formed homodimers in vivo as demonstrated by the growth of transformants on interaction selective medium and induction of p-galactosidase reporter activity above background level. To corroborate that the © Blackwell Publishing Ltd, The Plant Journal, (2004), 40, 428-438 Protein interaction in plant cells 431 I II O 0 5 1 0 1,5 2-0 2.5 3,0 3.5 4,0 CSM - L, W CSNI - L, W, A gal actos Was« acti vi ty (un its) Figure 2. Homodimerization of bZIP63 and bZIP63pp in yeast. The indicated Gal4 DNA-binding domain (BD) and activation domain (AD) constructs were transformed into yeast strain PJ69-4A. Transformants were assayed for the activity of protein-protein interaction reporting genes either by growth in decreasing densities (narrowing triangle) on selective medium (I: CSM-L,W,A) or determination of p-galactosidase activity (II). CSM-L,W(I) depicts a dilution series on non-selective control plates. bZIP63pp represents a mutated version of bZIP63 in which Leu188 and Leu195 have been mutated to Pro. homodimer formation is mediated by the leucine zipper we introduced two point mutations in the bZIP63 sequence which changed Leu188 and Leu195 into Pro. Leu188 and Leu195 are the first two hydrophobic amino acids in the C-terminal zipper-forming amphipathic a-helix of the bZIP63 monomers. Therefore, conversion of these positions into prolines is likely to interfere with the dimerization potential of the protein (Siberil et ai, 2001). The combination of wild type bZIP63 with the mutated version (bZIP63pp) reduced expression of the reporter genes (Figure 2). It is noteworthy that in yeast mutation of Leu188 and Leu195 to proline does not completely abolish homodimerization of bZIP63 (Figure 2). In summary, these data indicate that the leucine zipper domain is predominantly responsible for bZIP homodimer formation in yeast. We next attempted the direct visualization of homodimerization in living plant cells. To this end, we transiently transfected Arabidopsis eel I culture protoplasts with various pUC-SPYNE/pUC-SPYCE constructs of bZIP63 and, in addition to microscopic analysis, quantified the fluorescence intensity. Whereas cells transfected with single plasmids and any combination with empty vectors produced no or only background fluorescence, a strong signal was observed when bZIP63-YFPN was co-expressed with bZIP63-YFPc (Figure 3a,b). Significantly weaker fluorescence signals were observed when combinations of bZIP63pp with wild type bZIP63were transfected, thereby reflecting reduction in homodimerization by these mutations (Figure 3a,b). Generally the number of BiFC signal-emitting protoplasts was about the half compared with cells expressing full-length GFP fusion proteins. This difference corresponds to the reduced efficiency when more than one construct is used for transfection. To test the functionality of the binary BiFC vectors in planta, the wild type bZIP63 and bZIP63pp cDNAs were cloned into pSPYNE-35Sand pSPYCE-35S, respectively. The constructs were delivered into leaf cells of tobacco (Nicoti-ana benthamiana) by Agrobacterium infiltration (Voinnet et ai, 2003; Witte et ai, 2004). Similar to the situation in Arabidopsis protoplasts strong YFP fluorescence was observed when wild type combinations of bZIP63 were expressed (Figure 4a, panels I, II). Pairwise expression of bZIP63 and bZIP63pp, bZIP63pp alone or in combination with the YFP fragments induced no or only weak fluorescence signals (Figure 4a, panels I, II and data not shown). Using HA- and c-myc-tag-specific antibodies the expression of all fusion proteins in tobacco cells was demonstrated (Figure 4a, panel III). Notably, we observed that the transformation efficiency of /4grobacfe/7um-infiltrated tobacco cells strongly depends on the constructs used. For instance, whereas 80% of epidermal cells which have been infiltrated with bZIP63-YFPN and bZIP63-YFPc-carrying Agrobacteria © Blackwell Publishing Ltd, The Plant Journal, (2004), 40, 428-438 432 Michael Walter et al. bzi p&app-yfpn*zi pea pp-yfp0 t*-% Trk'%. -5, bZIP63-VFPNJbZl P63pp-Y FPC - v • f-■ i * As t)ZIP63PP-GFF Figure 3. BiFC visualization of bZIP63 dimeriza-tion in transiently transfected Arabidopsis thali-ana cell culture protoplasts. (a) Epifluorescence (I) and bright field images (II) images of Arabidopsis cell culture protoplasts co-transfected with constructs encoding the indicated fusion proteins. (b) Quantification of fluorescence intensities in transiently transfected Arabidopsis cell culture protoplasts. Fluorescence intensity (arbitrary units) was determined using the Metamorph software. The mean and standard deviation of three independent measurements are shown. (c) bZIP63-GFP and bZIP63pp-GFP are localized to the nucleus. Epifluorescence (I) and bright field (II) images of protoplasts transfected with constructs expressing the indicated fusion proteins. Scale bars, 20 (jm. t>ZIPe3-GFP vfp%zip63-yfpc showed BiFC-induced fluorescence, LSD1 homodimer formation (see Figure 7) was observed in only 20% of the cells. In transfected Arabidopsis protoplasts and infiltrated tobacco leaves the homodimerization-induced YFP fluorescence appeared exclusively inside the nucleus which is in agreement with the observation that bZIP63-GFP and bZIP63pp-GFP are nuclear proteins (Figures 3c and 4b). BiFC analysis of proteins in the cytoplasm of plant cells To further extend the applicability of BiFC beyond bZIP transcription factors we analyzed a 14-3-3 protein (isoform T14-3c from N. tabacum; GenBank: NTU91724) and the zinc finger protein LSD1 from Arabidopsis thaliana (Dietrich et al., 1997) by BiFC analyses in Arabidopsis protoplasts and /4grobacfe/7um-infiltrated tobacco leaves respectively. 14-3-3 proteins form a conserved family of eukaryotic polypeptides that were the first signaling molecules identified as discrete phosphoserine/phosphothreonine-binding modules. They associate to homodimers or heterodimers with a saddle-shaped structure, with each monomer forming an extended groove that allows binding of the phosphoryl-ated sequence motif (Rittinger et al., 1999; Wurtele et al., 2003). Dimerization of 14-3-3 proteins occurs via their N-terminal region. Accordingly, yeast two-hybrid analyses revealed that an N-terminally truncated version of T14-3c is © Blackwell Publishing Ltd, The Plant Journal, (2004), 40, 428-438 (a) I II III bz i pea- YFpt>c/i>zi pea- yfp" bZI P S3PP-YFP bc/bZ IP63-YFP" bZIPB3-YFPbc/YFPN (B) bZ IP63 u G fp bZI P63pp: :G FP Figure 4. BiFC visualization of bZIP63 dimerization in Agrobacterium-infiltrated tobacco {Nicotiana benthamiana) leaves. (a) Epifluorescence (I) and bright field (II) images of epidermal leaf cells infiltrated with a mixture of Agrobacterium suspensions harboring constructs encoding the indicated fusion proteins. In addition, the second panel from above shows a confocal image of BiFC-induced bZIP63 dimerization. For technical details of infiltration see Experimental procedures. The expression of the proteins (III) is demonstrated by immunodetection with anti-HA (a-HA) antibodies for YFPC fusions and anti-c-myc (a-c-myc) for YFPN fusions. *, degradation product. (b) bZIP63-GFP and bZIP63pp-GFP are both localized to the nucleus of plant cells. Epifluorescence images of Agrofcacfer/um-infiltrated tobacco (A/, benthamiana) epidermal cells are shown. Scale bars, 50 (jm. no longer able to homodimerize (Jaspert and Oecking, 2002). For BiFC studies the cDNAs encoding wild type T14-3c (T14) and the mutant version (THAN) were cloned into pUC-SPYNE and pUC-SPYCE or pSPYNE-35S and pSPYCE-35S, respectively, and transformed into either Arabidopsis protoplasts or tobacco leaf cells. Upon co-expression of T14- Figure 5. The tobacco 14-3-3 protein T14-3c interacts in Arabidopsis cell culture protoplasts. (a) Bright field (I) and epifluorescence (II) images of Arabidopsis cell culture protoplasts co-transfected with constructs encoding the indicated fusion proteins. (b) T14-3c-GFP is localized to the cytoplasm and nucleus. Bright field (I) and epifluorescence (II) images of protoplasts transfected with a construct expressing T14-3c-GFP are depicted. (c) Demonstration of protein expression by immunodetection with anti-HA (a-HA) antibodies for YFPC fusions and anti-c-myc (a-c-myc) for YFPN fusions. Extracts from protoplasts co-transfected with the constructs indicated in (a) are shown (lanes 1-4). Scale bars, 20 (jm. YFPN and T14-YFPC, strong YFP fluorescence was detected throughout the cytoplasm and the nucleus in both systems indicating that homodimerization occurs in both compartments (Figures 5a and 6a). In transformed epidermal cells of tobacco the cytoplasmic fluorescence typically appears as a thin area between the cell wall and the turgescent vacuole. Plasmolysis of the cells by treatment with 500 itim mannitol demonstrated the localization of the BiFC signal in the cytoplasm (data not shown). Importantly, the localization of homodimer formation corresponds to the subcellular distribution of T14-3c, when expressed as GFP fusion in protoplasts or tobacco leaf cells under the control of the 35S promoter (Figures 5b and 6b). In contrast, expression of neither the N-terminal truncated form THAN fused to the YFP fragments nor any combination of wild type with the truncated version resulted in a YFP signal (Figures 5a and 6a). Western blot analysis confirmed that the expression of © Blackwell Publishing Ltd, The Plant Journal, (2004), 40, 428-438 434 Michael Walter et al. (a) T14-YFPN/ T14AN-YFPN/ (b) T14- YFPC T14A N-YFPC T14-3C-G FP 1 ,—' 2 Vf ' '» ■ ■"■ ; ■, * ■ ■ , i i ..■■<.-' , - * ■ /\ "JA (c) kDa 1 2 48 - " Ij^^^m, acfer/um-infiltrated tobacco leaf discs were extracted under denaturing conditions using boiling SDS-sample buffer supplemented with 4 m urea (Harter et al., 1993). Protein assay, SDS-PAGE, Western blottransfer and immunodetection using HA- (Roche, Basel, Switzerland) and c-myc-specific (Sigma, St. Louis, MO, USA) antibodies has been described elsewhere (Harter era/., 1993). Acknowledgements We thank D. Baulcombe and T. Romeisfor the Agrobacterium strain C58C1, and the p19 construct and C. Frankhauser for the pCF203 plasmid. We are also very thankful to G. Freymark who introduced to us the Agrobacterium infiltration technique, to G. Fiene, C. Brancato, D. Kreuder, L. Rößner and C. Spitzer for excellent technical assistance and to D. Wanke for help in image processing. This work was supported by grants of the Deutsche Forschungsgemeinschaft to CO. (SFB 446), K.H. (SFB 635, HA 2146/3-2, 5-1) and to J.K. (931/4-1, 4-2). Note added in proof The usefulness of BiFC for in planta-protein interaction studies is also presented in the paper by Bracha-Drori et al. (2004), in this issue. Supplementary material The following material is available from http://www. blackwellpublishing.com/products/journals/suppmat/TPJ/TPJ2219/ TPJ2219sm.htm. Figure S1. (a) Schematic depiction of the series of pGPTVII vectors (kan, bar, hyg). Unique restriction sites are given in bold letter, others in italics. (b) Multiple cloning site of the pGPTVII vector series. Unique restriction sites (above the nucleotide sequence) are illustrated in normal letters. Restriction sites marked with * are not unique in pGPTVII.Bar. Figure S2. (a) Schematic depiction of the series of pGPTVII.GFP vectors (kan, bar, hyg). Unique restriction sites are given in bold letters, others in italics. 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