Name: protein-protein interactions visualized by bimolecular fluorescence complementation in tobacco protoplasts and leaves
Uploaded: 2014-03-09T14:00:00
Description: Watch this Scientific Journal Video about Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves at JoVE.com

We would like to thank J&#252;rgen Soll for helpful discussions and Chris Carrie for critical reading of the manuscript. This project was funded by the DFG and Fonds der chemischen Industrie (grants numbers SFB 1035, project A04 to S.S. and Do 187/22 to R.S.).

1. Transformation of BiFC Constructs in Agrobacteria
<ol>
	<li>Cloning of BiFC constructs
	<ol>
		<li>Amplify the gene of interest from an appropriate template using oligonucleotides containing flanking attB-sites. Perform a PCR using a proofreading polymerase. Adapt the length of the annealing step to the designed primer combination and the length of the elongation step according to the fragment size. Check the PCR product by agarose gel electrophoresis and purify it by using a PCR Clean-up Kit.</li>
		<li>Perform the...

<ol>
	<li>Citovsky, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+localization+of+interacting+proteins+by+bimolecular+fluorescence+complementation+in+planta.">Subcellular localization of interacting proteins by bimolecular fluorescence complementation in planta.</a> J. Mol. Biol. 362, 1120-1131 (2006).</li><li>Schutze, K., Harter, K., Chaban, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bimolecular+fluorescence+complementation+(BiFC)+to+study+protein-protein+interactions+in+living+plant+cells.">Bimolecular fluorescence complementation (BiFC) to study protein-protein interactions in living plant cells.</a> Methods Mol. Biol. 479, 189-202 (2009).</li><li>Weinthal, D., Tzfira, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+protein-protein+interactions+in+plant+cells+by+bimolecular+fluorescence+complementation+assay.">Imaging protein-protein interactions in plant cells by bimolecular fluorescence complementation assay.</a> Trends Plant Sci. 14, 59-63 (2009).</li><li>Ohad, N., Shichrur, K., Yalovsky, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+analysis+of+protein-protein+interactions+in+plants+by+bimolecular+fluorescence+complementation.">The analysis of protein-protein interactions in plants by bimolecular fluorescence complementation.</a> Plant Physiol. 145, 1090-1099 (2007).</li><li>Ohad, N., Yalovsky, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilizing+bimolecular+fluorescence+complementation+(BiFC)+to+assay+protein-protein+interaction+in+plants.">Utilizing bimolecular fluorescence complementation (BiFC) to assay protein-protein interaction in plants.</a> Methods Mol. Biol. 655, 347-358 (2010).</li><li>Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+green+fluorescent+protein.">The green fluorescent protein.</a> Annu. Rev. Biochem. 67, 509-544 (1998).</li><li>Lee, L. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+a+cDNA+library+for+protein-protein+interactions+directly+in+planta.">Screening a cDNA library for protein-protein interactions directly in planta.</a> Plant Cell. 24, 1746-1759 (2012).</li><li>McFarlane, H. E., Shin, J. J., Bird, D. A., Samuels, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+ABCG+transporters,+which+are+required+for+export+of+diverse+cuticular+lipids,+dimerize+in+different+combinations.">Arabidopsis ABCG transporters, which are required for export of diverse cuticular lipids, dimerize in different combinations.</a> Plant Cell. 22, 3066-3075 (2010).</li><li>Dunschede, B., Bals, T., Funke, S., Schunemann, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+studies+between+the+chloroplast+signal+recognition+particle+subunit+cpSRP43+and+the+full-length+translocase+Alb3+reveal+a+membrane-embedded+binding+region+in+Alb3+protein.">Interaction studies between the chloroplast signal recognition particle subunit cpSRP43 and the full-length translocase Alb3 reveal a membrane-embedded binding region in Alb3 protein.</a> J. Biol. Chem. 286, 35187-35195 (2011).</li><li>Gehl, C., Waadt, R., Kudla, J., Mendel, R. R., Hansch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+GATEWAY+vectors+for+high+throughput+analyses+of+protein-protein+interactions+by+bimolecular+fluorescence+complementation.">New GATEWAY vectors for high throughput analyses of protein-protein interactions by bimolecular fluorescence complementation.</a> Mol. Plant. 2, 1051-1058 (2009).</li><li>Qbadou, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+chaperone+Hsp90+delivers+precursor+proteins+to+the+chloroplast+import+receptor+Toc64.">The molecular chaperone Hsp90 delivers precursor proteins to the chloroplast import receptor Toc64.</a> EMBO J. 25, 1836-1847 (2006).</li><li>Schweiger, R., Muller, N. C., Schmitt, M. J., Soll, J., Schwenkert, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AtTPR7+is+a+chaperone+docking+protein+of+the+Sec+translocon+in+Arabidopsis.">AtTPR7 is a chaperone docking protein of the Sec translocon in Arabidopsis.</a> J. Cell Sci. , (2012).</li><li>Schweiger, R., S, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AtTPR7+as+part+of+the+Arabidopsis+Sec+post-translocon.">AtTPR7 as part of the Arabidopsis Sec post-translocon.</a> Plant Signal Behav. 8, (2013).</li><li>Nelson, B. 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Upon planning a BiFC experiment several points should be considered. Although no structural information about the proteins of interest is required, the topology has to be known when working with membrane spanning proteins. The fluorescent proteins have to reside in the same subcellular compartment or face the same side of a membrane to allow interaction. Naturally, when analyzing proteins which require an N-terminal targeting sequence, only a C-terminal tag can be considered. Since it is possible that the tag interferes ...

Studying the formation of protein complexes and their localization in plant cells in vivo is essential to investigate cellular networks, signaling and metabolic processes. BiFC allows visualization of protein-protein interactions in their natural environment directly within the living plant cell1-5.
In the BiFC approach the complementation of two nonfluorescent N- and C-terminal fragments of a fluorescent protein lead to a reconstituted fluorescent protein. Fragments of many different fluorescent proteins have been used to detect protein interactions, e.g. the green fluorescent protein (GFP) the chromophore of which is chemically formed by three distinct residues6. Fluorescent proteins can be halved within a loop or &#223;-strand to result in the two nonfluorescent fragments which can be fused to both proteins of interest. The assay can be used to detect interactions in any subcellular compartment in any aerobically growing organism or cells that can be genetically modified to express the fusion proteins. If the two proteins come into close proximity within the cell, fluorescence is reconstituted and can be monitored by microscopy without the addition of exogenous fluorophores or dyes3.
Tobacco (Nicotiana benthamiana) has proven to be a convenient model organism to visualize the interaction of plant proteins, since proteins can easily be expressed by utilizing agrobacterium-mediated transformation of tobacco leaves with the generated constructs. Agrobacteria use a so-called Ti plasmid (tumor inducing) coding for enzymes that mediate the transduction of the gene of interest into plant cells. BiFC is well applicable for soluble as well as for membrane proteins within all cellular compartments and has been successfully used over the past years to identify interacting proteins in vivo as well as to analyze interaction sites within the proteins7-9. Upon expression of the introduced genes, the interaction of the fluorescent proteins can be visualized directly in leaves, which is suitable for larger cellular structures, such as the endoplasmic reticulum (ER), the plasma membrane or chloroplasts. However, to monitor the localization in more refined structures, for example,&#160;the chloroplast envelope, it is advisable to visualize the fluorescence in protoplasts isolated from transformed tobacco leaves. A set of BiFC vectors containing either a C-terminal or an N-terminal fluorescent tag has to be used for the BiFC approach in plants10. The hereafter described protocol was used to study the interaction of cytosolic heat shock protein 90 (HSP90) with the tetratricopeptide repeat (TPR)&#160;domain containing docking proteins Toc64 and AtTPR7 residing in the chloroplast outer envelope and the endoplasmic reticulum, respectively11-13. For this purpose, HSP90 was fused to the C-terminal part of SCFP (SCFPC). The tag was N-terminally fused to the chaperone to ensure accessibility of the C-terminal MEEVD binding motif of HSP90 to clamp-type TPR domains. In parallel, the N-terminal part of Venus (VenusN) was fused to the cytosolic domains of the TPR domain containing docking proteins Toc64 and AtTPR7, respectively. As a negative control we cloned the soluble C-terminal part of SCFPC solely which resides in the cytosol and is therefore an appropriate control.
The fluorescent tags of the studied proteins have to face the same cellular compartment to allow close proximity and thereby reconstitution of the fluorescent signal. To determine the localization of the reconstituted fluorescent signal a marker protein fused to a different fluorescent tag can be cotransformed to demonstrate the subcellular localization of the interaction. An ER marker protein fused to mCherrry was transformed simultaneously in the case of the ER located AtTPR714. The autofluorescence of chlorophyll served as chloroplast marker in case of Toc64. By this not only the in vivo interaction of Toc64 and AtTPR7, respectively, with the cytosolic HSP90 chaperone can be monitored directly in the tobacco leaves but also the subcellular localization of the interaction can be investigated.
BiFC is well suited as a complementary approach to other methods studying protein-protein interactions. Compared to coimmunoprecipitation or in vitro pull-down experiments, for example, no specific antibodies have to be available for the proteins of interest, and the proteins do not have to be recombinantly expressed in vitro, which can be challenging, especially for membrane proteins. Moreover, also transient interactions can be monitored using BiFC, since the proteins are captured by the interaction of the fused fluorescent tags15.

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		<tr height="21">
			<td height="21" style="height:21px;width:293px;">3&#39;,5&#39;-Dimethoxy-4&#39;-hydroxyacetophenone</td>
			<td style="width:147px;">Sigma-Aldrich</td>
			<td style="width:255px;">D134406</td>
			<td style="width:195px;">Acetosyringone</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Cellulase, Onozuka-R10</td>
			<td style="width:147px;">Serva</td>
			<td style="width:255px;">16419</td>
			<td>from Trichoderma viridae</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Macerozyme R-10&#160;</td>
			<td style="width:147px;">Serva</td>
			<td style="width:255px;">28302</td>
			<td>from Rhizopus sp</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:293px;">GATEWAY, BP Clonase II, Enzyme Kit</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:255px;">11789-(020)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">GATEWAY, LR Clonase II, Enzyme Kit</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:255px;">11791-(020)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">QIAprep Spin Miniprep Kit</td>
			<td style="width:147px;">Qiagen</td>
			<td style="width:255px;">27106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">NucleoSpin Gel and PCR Clean-up Kit</td>
			<td style="width:147px;">Macherey-Nagel</td>
			<td style="width:255px;">740609-250</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:293px;">pDEST-GWVYNE</td>
			<td style="width:147px;"></td>
			<td style="width:255px;">Gehl C. et al, 2009, Molecular Plant</td>
			<td>Gateway-cloning (Invitrogen)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:293px;">pDEST-VYNE(R)GW</td>
			<td style="width:147px;"></td>
			<td style="width:255px;">Gehl C. et al, 2009, Molecular Plant</td>
			<td>Gateway-cloning (Invitrogen)</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:293px;">pDEST-SCYCE(R)GW</td>
			<td style="width:147px;"></td>
			<td style="width:255px;">Gehl C. et al, 2009, Molecular Plant</td>
			<td>Gateway-cloning (Invitrogen)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;"></td>
			<td style="width:147px;"></td>
			<td style="width:255px;"></td>
			<td></td>
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protein-protein interactions visualized by bimolecular fluorescence complementation in tobacco protoplasts and leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

In this example we used the BiFC method to monitor the interaction of the cytosolic molecular chaperone HSP90 with the membrane docking proteins AtTPR7 and Toc64. AtTPR7 is part of the Sec translocon and interacts with cytosolic chaperones, which possibly deliver secretory preproteins for post-translational translocation to the ER membrane. Likewise, Toc64 at the chloroplast outer envelope acts in post-translational import by receiving HSP90 associated chloroplast preproteins. Both proteins comprise a cytosolic exposed T...

Watch this Scientific Journal Video about Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves at JoVE.com

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

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