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 BP reaction with the obtained fragment and the entry vector using a BP recombinase. Mix 15-150 ng of the attB-PCR product with 150 ng of the entry vector, add 2 &#181;l of the BP recombinase and fill up with TE buffer to a total volume of 8 &#181;l. Incubate the reaction for 1 hr at RT and stop the reaction by adding 2 &#181;l Proteinase K for 10 min at 37 &#176;C.</li>
		<li>Transform the entire reaction into competent E. coli DH5&#945; cells and screen the obtained colonies by colony PCR for correct insertion of the DNA fragment into the vector. DNA sequencing of positive plasmids is performed to verify the absence of mutations.</li>
		<li>Use the obtained entry clone for LR recombination with the appropriate destination vector&#160;using a LR recombinase. Mix 50-150 ng of the entry vector with 150 ng of the destination vector, add 1 &#181;l LR recombinase and fill up with TE buffer to a total volume of 5 &#181;l. Incubate the reaction for 1 hr at RT and stop the reaction by adding 1 &#181;l Proteinase K for 10 min at 37 &#176;C.</li>
		<li>Transform the entire reaction into competent E. coli DH5&#945; cells and screen obtained colonies by colony PCR for correct insertion of the DNA fragment into the vector. DNA sequencing is not necessary at this step.</li>
		<li>Isolate plasmid DNA with a plasmid Mini kit to ensure a high degree of purity.</li>
	</ol>
	</li>
	<li>Preparation of chemically competent Agrobacteria (strain AGL1, Rifampicin and Carbenicillin resistance)
	<ol>
		<li>Streak out agrobacteria from a stock culture and grow for 24 hr at 28 &#176;C.</li>
		<li>Inoculate 5 ml LB medium with a single colony and incubate overnight at 28 &#176;C.</li>
		<li>Inoculate 50 ml LB medium with 2 ml of the overnight culture and grow for approximately&#160;4 hr at 28 &#176;C to an OD600 of 1.0.</li>
		<li>Centrifuge the cells at 3,000 x g for 15 min at 4 &#176;C and resuspend the pellet in 1 ml sterilized ice-cold CaCl2 (10 mM). Keep cells on ice after this step.</li>
		<li>Prepare aliquots (100 &#181;l) of the cells, freeze immediately in liquid nitrogen and store at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Transformation of chemically competent Agrobacteria
	<ol>
		<li>Thaw one aliquot of competent AGL1 cells on ice. Add 1-2 &#181;g of plasmid DNA to the cells. Incubate for 5 min on ice, 5 min in liquid nitrogen and 5 min at 37 &#176;C. Add 600 &#181;l LB medium to the cells and shake at 650 rpm for 4 hr at 28 &#176;C.</li>
		<li>Centrifuge the cells for 1 min at 8,000 x g and discard the supernatant. Resuspend the pellet in 50 &#181;l of the remaining LB medium and plate on LB plates containing the appropriate antibiotics. Seal the plates and incubate for 2 days at 28 &#176;C. Screen colonies for the presence of the plasmid by colony PCR.</li>
		<li>Inoculate one positive colony in 5 ml LB medium containing the appropriate antibiotics and incubate at 28 &#176;C over night. Mix 500 &#181;l of the overnight culture with 500 &#181;l 50% sterilized glycerol and freeze at -80 &#176;C.</li>
		<li>Inoculate the agrobacteria in LB medium containing the appropriate antibiotics directly from the glycerol stock for transient transformation of tobacco leaves.</li>
	</ol>
	</li>
</ol>
2. Transient Transformation of Tobacco Leaves
<ol>
	<li>Agrobacteria growth
	<ol>
		<li>Prepare the following stock solutions: Acetosyringone (150 mM, dissolve in 70% EtOH), store as aliquots at -20 &#176;C. MES/KOH (0.1 M) pH 5.7, store at 4 &#176;C (sterile&#160;filter through a 0.2 &#181;M filter to prevent bacterial growth during longer storage),&#160;MgCl2 (1 M) stock, store at RT.</li>
		<li>Inoculate 10 ml LB medium containing the appropriate antibiotics with 50 &#181;l of the AGL1 glycerol stock culture containing the plasmid of interest in a sterile 50 ml tube and incubate at 28 &#176;C for at least 24 hr shaking at 190 rpm until an OD600 between 1.0-2.0 is reached.</li>
		<li>Centrifuge bacteria at 3,000 x g for 15 min. Resuspend the pellet in freshly made infiltration medium [MgCl2 (10 mM), MES/KOH (10 mM) pH 5.7, Acetosyringone (150 &#181;M)] and adjust the suspension to an OD600 of 1.0.</li>
		<li>Incubate the agrobacteria cells in an overhead shaker for 2 hr in darkness. The cells can then be used for infiltration.</li>
	</ol>
	</li>
	<li>Infiltration of tobacco leaves
	<ol>
		<li>Use three week old tobacco (Nicotiana benthamiana) plants. Choose several older leaves for infiltration.</li>
		<li>Mix equal volumes of the agrobacteria carrying the constructs of interest (3 ml each). Take a 5 ml syringe without a needle for infiltration. Infiltrate the cell suspension carefully into the tobacco leaves by pressing the syringe on the bottom side of the leaves in several places.</li>
		<li>Water the plants and leave them in the darkness for two days.</li>
	</ol>
	</li>
</ol>
3. Protoplast Preparation
Protoplast preparation of tobacco leaves was adapted from Koop et al.16 and slightly modified.
<ol>
	<li>Buffer preparation
	<ol>
		<li>Prepare F-PCN medium: Macro-salts [KNO3 (1012 &#181;g/ml), CaCl2&#8226;2H2O (440 &#181;g/ml), MgSO4&#8226;7H2O (370 &#181;g/ml), KH2PO4 (170 &#181;g/ml), NH4-succinate [(20 mM; prepare a 2 M stock solution (succinate (236 &#181;g/ml) and NH4Cl (106 &#181;g/ml), adjust to pH 5.8 to dissolve)], Micro-salts [EDTA-Fe(III) x Na-salt (40 &#181;g/ml), KJ (0.75 &#181;g/ml), H3BO3 (3 &#181;g/ml), MnSO4&#8226;H2O (10 &#181;g/ml), ZnSO4&#8226;7H2O (2 &#181;g/ml), Na2MoO4&#8226;7H2O (0.25 &#181;g/ml), CuSO4&#8226;5H2O (0.025 &#181;g/ml), CoCl2&#8226;6H2O (0.025 &#181;g/ml)], MES (390 &#181;g/ml), glucose (approximately&#160;80 &#181;g/ml) osmolarity 550 mOsm, pH 5.8 (KOH). Store in aliquots at -20 &#176;C.</li>
		<li>Prepare F-PIN medium: All ingredients as F-PCN but instead of glucose use sucrose (approximately&#160;110 &#181;g/ml), osmolarity 550 mOsm, pH 5.8 (KOH). Store in aliquots at -20 &#176;C.</li>
		<li>Prepare W5 medium: 150 mM NaCl, 125 mM CaCl2, 5 mM KCl, 2 mM MES, osmolarity 550-580 mOsm, pH 5.7 (KOH). Store at 4 &#176;C (sterile&#160;filter through a 0.2 &#181;m filter to prevent bacterial growth during longer storage).</li>
		<li>Prepare fresh enzyme solution for protoplast isolation (0.1 g cellulase, 0.03 g macerozym in 10 ml F-PIN). Incubate the solution at 55 &#176;C for 10 min and cool to RT. Add 100 &#181;l of 10% BSA to 10 ml solution.</li>
	</ol>
	</li>
	<li>Isolation of protoplasts
	<ol>
		<li>Place one infiltrated leaf into a Petri dish and add the fresh enzyme solution. Use a new razorblade to cut the leaf into approximately&#160;0.5 cm2 sized pieces. Transfer the leaf-pieces with the enzyme solution into a vacuum-infiltration flask and vacuum infiltrate for approximately&#160;20 sec until air bubbles emerge from the leaves (release vacuum very carefully).</li>
		<li>Shake the flask for 90 min at 40 rpm in darkness.</li>
		<li>Release protoplasts by shaking for 1 min at 90 rpm. Filter the solution through gauze (100 &#181;M) into a 15 ml centrifugation tube (round bottom).</li>
		<li>Overlay the protoplast solution with 2 ml F-PCN buffer and centrifuge for 10 min at 70 x g (slow acceleration and deceleration) at RT.</li>
		<li>Intact protoplasts accumulate at the interface of enzyme solution and F-PCN. Take a wide orifice 1 ml pipette tip to transfer the intact protoplasts into a fresh centrifuge tube and fill up with W5 buffer. Centrifuge for 2 min at 100 x g (slow acceleration and deceleration) to pellet the protoplasts.</li>
		<li>Remove the supernatant carefully by using a pipette and resuspend pellet in approximately&#160;200 &#181;l W5 buffer, depending on the amount of protoplasts.</li>
		<li>Always use wide orifice tips to prevent rupturing of intact protoplasts.</li>
	</ol>
	</li>
</ol>
4. Laser Scanning Microscopy
<ol>
	<li>Sample preparation
	<ol>
		<li>Paste two small strips of sealant around a microscope slide (2 cm apart). Place 20 &#181;l of the protoplast solution between the strips and carefully place a cover glass on top. The sealant strips make sure that the protoplasts are not squashed by the cover glass.</li>
		<li>For total leaf samples cut a 1 cm piece from the leaf and place it onto a microscope slide with the bottom side of the leaf facing upwards. Add approximately&#160;30 &#181;l H2O, place a cover glass on top and fix it tightly with adhesive tape on both sides.</li>
	</ol>
	</li>
	<li>Confocal imaging and microscope settings
	<ol>
		<li>Imaging is performed with a confocal laser scanning microscope from Leica, Type: TCS SP5. For magnification use an objective lens (HCX PL APO CS) with a magnitude of 63X with glycerol as imaging medium. Set the numerical aperture to 1.3. Use the Leica Application Suite/Advanced Fluorescence software for evaluation (see Supplemental Data S1).</li>
		<li>Set the Argon laser to 30% and the laser power at 488 nm to an intensity of 18% to monitor the reconstituted BiFC signal at 515 nm and set one PMT detector emission bandwidth from 495-550.</li>
		<li>To monitor chlorophyll autofluorescence set a second PMT detector emission bandwidth from 650-705.</li>
		<li>To monitor mCherry signal use the HeNe 561 laser, set the intensity of laser 561 to 18% and the emission bandwidth of a third PMT detector from 587-610.</li>
		<li>Make sure that pictures of all PMT detector channels are taken with the same gain settings (gain should be between 800-900 to exclude background signals).</li>
		<li>Take pictures in a format width/height of 1024 x 1024 pixels with a scan speed of 100 Hz.</li>
		<li>For Z-stackings use a maximum distance of 0.5 &#181;m between each stack.</li>
	</ol>
	</li>
</ol>

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	<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. K., Cai, X., Nebenfuhr, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multicolored+set+of+in+vivo+organelle+markers+for+co-localization+studies+in+Arabidopsis+and+other+plants.">A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants.</a> Plant J. 51, 1126-1136 (2007).</li><li>Kerppola, T. K. <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)+analysis+as+a+probe+of+protein+interactions+in+living+cells.">Bimolecular fluorescence complementation (BiFC) analysis as a probe of protein interactions in living cells.</a> Annu. Rev. Biophys. 37, 465-487 (2008).</li><li>Koop, H. U., 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=Integration+of+foreign+sequences+into+the+tobacco+plastome+via+polyethylene+glycol-mediated+protoplast+transformation.">Integration of foreign sequences into the tobacco plastome via polyethylene glycol-mediated protoplast transformation.</a> Planta. 199, 193-201 (1996).</li></ol>

The authors have nothing to disclose.

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.

<table border="0" cellpadding="0" cellspacing="0" width="888">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<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>
		</tr>
	</tbody>
</table>

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...

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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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Research

JoVE Journal

Biology

20.8K Views.  Ludwig-Maximilians-Universität, München.  Formazione di complessi proteici in vivo può essere visualizzato da bimolecular fluorescenza complementazione. Partner di interazione sono fusi a parti complementari tag fluorescenti e transitoriamente espressi in foglie di tabacco, risultando in un segnale fluorescente ricostituito upon vicinanza delle due proteine. 

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

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay1 allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)2 and the visualization of their interactions mediated by BiFC in onion epidermal cells.

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC)

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of methodological options for addressing molecular interactions in cells have been developed, but most of these methods suffer from being rather indirect and therefore hardly quantitative. On the contrary, a few high-end quantitative approaches were introduced, which however are difficult to extend to high throughput. To combine high throughput capabilities with the possibility to extract quantitative information, we recently developed a new concept for identifying protein-protein interactions (Schwarzenbacher et al., 2008). Here, we describe a detailed protocol for the design and the construction of this system which allows for analyzing interactions between a fluorophore-labeled protein ("prey") and a membrane protein ("bait") in-vivo. Cells are plated on micropatterned surfaces functionalized with antibodies against the bait exoplasmic domain. Bait-prey interactions are assayed via the redistribution of the fluorescent prey. The method is characterized by high sensitivity down to the level of single molecules, the capability to detect weak interactions, and high throughput capability, making it applicable as screening tool.

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

This video shows experiments with subsequent analysis of protein-protein interactions by the use of micro-patterned surfaces. The approach offers the possibility to detect protein interactions in living cells and combines high throughput capabilities with the possibility to extract quantitative information.

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of ...

Cell Electrofusion Visualized with Fluorescence Microscopy

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves application of short high-voltage electric pulses to cells that are in close contact. Application of short, high-voltage electric pulses causes destabilization of cell plasma membranes. Destabilized membranes are more permeable for different molecules and also prone to fusion with any neighboring destabilized membranes. Electrofusion is thus a convenient method to achieve a non-specific fusion of very different cells in vitro. In order to obtain fusion, cell membranes, destabilized by electric field, must be in a close contact to allow merging of their lipid bilayers and consequently their cytoplasm. In this video, we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method. In this method, cells are allowed to attach only slightly to the surface of the well, so that medium can be exchanged and cells still preserve their spherical shape. Fusion visualization is assessed by pre-labeling of the cytoplasm of cells with different fluorescent cell tracker dyes; half of the cells are labeled with orange CMRA and the other half with green CMFDA. Fusion yield is determined as the number of dually fluorescent cells divided with the number of all cells multiplied by two.

In this video we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method using electroporation and the subsequent detection of fused cells visualization with fluorescence microscopy.

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves ...

Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

Bimolecular Fluorescence Complementation

Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as immunoprecipitation do not provide information on the spatial localization of complexes. In contrast, BiFC monitors the interaction and subcellular compartmentalization of protein complexes. In this method, a fluororescent protein is split into amino- and carboxy-terminal non-fluorescent fragments which are then fused to two proteins of interest. Interaction of the proteins results in reconstitution of the fluorophore (Figure 1)1,2. A limitation of BiFC is that once the fragmented fluorophore is reconstituted the complex is irreversible3. This limitation is advantageous in detecting transient or weak interactions, but precludes a kinetic analysis of complex dynamics. An additional caveat is that the reconstituted flourophore requires 30min to mature and fluoresce, again precluding the observation of real time interactions4. BiFC is a specific example of the protein fragment complementation assay (PCA) which employs reporter proteins such as green fluorescent protein variants (BiFC), dihydrofolate reductase, b-lactamase, and luciferase to measure protein:protein interactions5,6. Alternative methods to study protein:protein interactions in cells include fluorescence co-localization and F&ouml;rster resonance energy transfer (FRET)7. For co-localization, two proteins are individually tagged either directly with a fluorophore or by indirect immunofluorescence. However, this approach leads to high background of non-interacting proteins making it difficult to interpret co-localization data. In addition, due to the limits of resolution of confocal microscopy, two proteins may appear co-localized without necessarily interacting. With BiFC, fluorescence is only observed when the two proteins of interest interact. FRET is another excellent method for studying protein:protein interactions, but can be technically challenging. FRET experiments require the donor and acceptor to be of similar brightness and stoichiometry in the cell. In addition, one must account for bleed through of the donor into the acceptor channel and vice versa. Unlike FRET, BiFC has little background fluorescence, little post processing of image data, does not require high overexpression, and can detect weak or transient interactions. Bioluminescence resonance energy transfer (BRET) is a method similar to FRET except the donor is an enzyme (e.g. luciferase) that catalyzes a substrate to become bioluminescent thereby exciting an acceptor. BRET lacks the technical problems of bleed through and high background fluorescence but lacks the ability to provide spatial information due to the lack of substrate localization to specific compartments8. Overall, BiFC is an excellent method for visualizing subcellular localization of protein complexes to gain insight into compartmentalized signaling.

The subcellular localization of proteins is important in determining the spatio-temporal regulation of cell signaling. Here, we describe bimolecular fluorescence complementation (BiFC) as a straightforward method for monitoring the spatial interactions of proteins in the cell.

Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as ...

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein (YFP) into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest. These constructs can then be co-transformed into Nicotiana benthamiana via Agrobacterium mediated transformation, allowing the transit expression of fusion proteins. The reconstitution of YFP signal only occurs when the inquest proteins interact 1-7. To test and validate the protein-protein interactions, BiFC can be used together with yeast two hybrid (Y2H) assay. This may detect indirect interactions which can be overlooked in the Y2H. Gateway technology is a universal platform that enables researchers to shuttle the gene of interest (GOI) into as many expression and functional analysis systems as possible8,9. Both the orientation and reading frame can be maintained without using restriction enzymes or ligation to make expression-ready clones. As a result, one can eliminate all the re-sequencing steps to ensure consistent results throughout the experiments. We have created a series of Gateway compatible BiFC and Y2H vectors which provide researchers with easy-to-use tools to perform both BiFC and Y2H assays10. Here, we demonstrate the ease of using our BiFC system to test protein-protein interactions in N. benthamiana plants.

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System

We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein ...

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells.
BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.

Flow cytometric analysis of Bimolecular Fluorescence Complementation provides a high throughput quantitative method to study protein-protein interaction. This methodology can be applied to mapping protein binding sites and for screening factors that regulate protein-protein interaction.

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis1,2 to determine a high confidence set of protein interactions for downstream validation in vivo.

Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in high-throughput.

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...