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>

<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. 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><tbody><tr><td>3&amp;#039;,5&amp;#039;-Dimethoxy-4&amp;#039;-hydroxyacetophenone</td><td>Sigma-Aldrich</td><td>D134406</td><td>Acetosyringone</td></tr><tr><td>Cellulase, Onozuka-R10</td><td>Serva</td><td>16419</td><td>from &lt;em&gt;Trichoderma viridae&lt;/em&gt;</td></tr><tr><td>Macerozyme R-10&amp;nbsp;</td><td>Serva</td><td>28302</td><td>from &lt;em&gt;Rhizopus sp.&lt;/em&gt;</td></tr><tr><td>GATEWAY, BP Clonase II, Enzyme Kit</td><td>Invitrogen</td><td>11789-(020)</td><td></td></tr><tr><td>GATEWAY, LR Clonase II, Enzyme Kit</td><td>Invitrogen</td><td>11791-(020)</td><td></td></tr><tr><td>QIAprep Spin Miniprep Kit</td><td>Qiagen</td><td>27106</td><td></td></tr><tr><td>NucleoSpin Gel and PCR Clean-up Kit</td><td>Macherey-Nagel</td><td>740609-250</td><td></td></tr><tr><td>pDEST-&lt;sup&gt;GW&lt;/sup&gt;VYNE</td><td>Invitrogen</td><td></td><td>Gateway-cloning</td></tr><tr><td>pDEST-VYNE(R)&lt;sup&gt;GW&lt;/sup&gt;</td><td>Invitrogen</td><td></td><td>Gateway-cloning</td></tr><tr><td>pDEST-SCYCE(R)&lt;sup&gt;GW&lt;/sup&gt;</td><td>Invitrogen</td><td></td><td>Gateway-cloning</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...

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 ...

protein-protein-interactions-visualized-bimolecular-fluorescence

Research

JoVE Journal

Biology

20.8K Views.  Ludwig-Maximilians-Universität, München. 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.

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

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately using complex regulatory strategies including differential promoters and/or signal sequences. Metabolic engineers and synthetic biologists interested in targeting enzymes to a particular organelle are faced with a challenge: For a protein that is to be localized to more than one organelle, the engineer must clone the same gene multiple times. This work presents a solution to this strategy: harnessing alternative splicing of mRNA. This technology takes advantage of established chloroplast and peroxisome targeting sequences and combines them into a single mRNA that is alternatively spliced. Some splice variants are sent to the chloroplast, some to the peroxisome, and some to the cytosol. Here the system is designed for multiple-organelle targeting with alternative splicing. In this work, GFP was expected to be expressed in the chloroplast, cytosol, and peroxisome by a series of rationally designed 5&#8217; mRNA tags. These tags have the potential to reduce the amount of cloning required when heterologous genes need to be expressed in multiple subcellular organelles. The constructs were designed in previous work11, and were cloned using Gibson assembly, a ligation independent cloning method that does not require restriction enzymes. The resultant plasmids were introduced into Nicotiana benthamiana epidermal leaf cells with a modified Gene Gun protocol. Finally, transformed leaves were observed with confocal microscopy.

This work describes a novel method for selectively targeting subcellular organelles in plants, assayed using the BioRad Gene Gun.

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately ...

Environment

Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in an Efficient Way in Plants

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent proteins and generation of fluorescent fusion proteins have been wildly used as an effective tool to directly visualize the protein localization and dynamics in cells. It is especially useful to compare them with well-known organelle markers after co-expression with the protein of interest. Nevertheless, classical approaches for protein co-expression in plants usually involve multiple independent expression plasmids, and therefore have drawbacks that include low co-expression efficiency, expression-level variation, and high time expenditure in genetic crossing and screening. In this study, we describe a robust and novel method for co-expression of multiple chimeric fluorescent proteins in plants. It overcomes the limitations of the conventional methods by using a single expression vector that is composed of multiple semi-independent expressing cassettes. Each protein expression cassette contains its own functional protein expression elements, and therefore it can be flexibly adjusted to meet diverse expression demand. Also, it is easy and convenient to perform the assembly and manipulation of DNA fragments in the expression plasmid by using an optimized one-step reaction without additional digestion and ligation steps. Furthermore, it is fully compatible with current fluorescent protein derived bio-imaging technologies and applications, such as FRET and BiFC. As a validation of the method, we employed this new system to co-express fluorescently fused vacuolar sorting receptor and secretory carrier membrane proteins. The results show that their perspective subcellular localizations are the same as in previous studies by both transient expression and genetic transformation in plants.

We have developed a novel method for co-expressing multiple chimeric fluorescent fusion proteins in plants to overcome the difficulties of conventional methods. It takes advantage of using a single expression plasmid that contains multiple functionally independent protein expressing cassettes to achieve protein co-expression.

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent ...

Chemistry

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 ...

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 ...

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level. There are several methods, both in vitro and in vivo, to evaluate protein binding, and at least two methods that complement the shortcomings of each other should be conducted to obtain reliable insights. 
For an in vivo assay, the bimolecular fluorescence complementation (BiFC) assay represents the most popular and least invasive approach that enables to detect protein-protein interaction within living cells, as well as identify the intracellular localization of the interacting proteins 1,2. In this assay, non-fluorescent N- and C-terminal halves of GFP or its variants are fused to tested proteins, and when the two fusion proteins are brought together due to the tested proteins&#x2019; interactions, the fluorescent signal is reconstituted3-6. Because its signal is readily detectable by epifluorescence or confocal microscopy, BiFC has emerged as a powerful tool of choice among cell biologists for studying about protein-protein interactions in living cells 3. This assay, however, can sometimes produce false positive results. For example, the fluorescent signal can be reconstituted by two GFP fragments arranged as far as 7 nm from each other due to close packing in a small subcellular compartment, rather that due to specific interactions7.
Due to these limitations, the results obtained from live cell imaging technologies should be confirmed by an independent approach based on a different principle for detecting protein interactions. Co-immunoprecipitation (Co-IP) or glutathione transferase (GST) pull-down assays represent such alternative methods that are commonly used to analyze protein-protein interactions in vitro. However, iIn these assays, however, the tested proteins must be readily soluble in the buffer that supportsused for the binding reaction. Therefore, specific interactions involving an insoluble protein cannot be assessed by these techniques.
 Here, we illustrate the protocol for the protein membrane overlay binding assay, which circumvents this difficulty. In this technique, interaction between soluble and insoluble proteins can be reliably tested because one of the proteins is immobilized on a membrane matrix. This method, in combination with in vivo experiments, such as BiFC, provides a reliable approach to investigate and characterize interactions faithfully between soluble and insoluble proteins. In this article, binding between Tobacco mosaic virus (TMV) movement protein (MP), which exerts multiple functions during viral cell-to-cell transport8-14, and a recently identified plant cellular interactor, tobacco ankyrin repeat-containing protein (ANK) 15, is demonstrated using this technique.

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Testing protein-protein interaction is indispensable for dissection of protein functionality. Here, we introduce an in vitro protein-protein binding assay to probe a membrane-immobilized protein with a soluble protein. This assay provides a reliable method to test interaction between an insoluble protein and a protein in solution.

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level.  There are several ...

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 ...

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel protein-protein interaction pairs and monitoring protein interaction dynamics are of particular interest for revealing how plants respond to environmental factors and/or developmental signals. A plethora of approaches have been developed to examine protein-protein interactions, either in vitro or in vivo. Among them, the recently established luciferase complementation imaging (LCI) assay is the simplest and fastest method for demonstrating in vivo protein-protein interactions. In this assay, protein A or protein B is fused with the amino-terminal or carboxyl-terminal half of luciferase, respectively. When protein A interacts with protein B, the two halves of luciferase will be reconstituted to form a functional and active luciferase enzyme. Luciferase activity can be recorded with a luminometer or CCD-camera. Compared with other approaches, the LCI assay shows protein-protein interactions both qualitatively and quantitatively. Agrobacterium infiltration in Nicotiana benthamiana leaves is a widely used system for transient protein expression. With the combination of LCI and transient expression, these approaches show that the physical interaction between COP1 and SPA1 was gradually reduced after jasmonate treatment.

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

This manuscript describes an easy and rapid experimental procedure for determining protein-protein interactions based on the measurement of luciferase activity.

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel ...

Determination of Tripartite Interaction between Two Monomers of a MADS-box Transcription Factor and a Calcium Sensor Protein by BiFC-FRET-FLIM Assay

Protein-protein interactions are an integral part of all biological processes in the cells as they play a crucial role in regulating, maintaining, and amending cellular functions. These interactions are involved in a wide range of phenomena such as signal transduction, pathogen response, cell-cell interactions, metabolic and developmental processes. In the case of transcription factors, these interactions may lead to oligomerization of subunits, sequestering in specific subcellular contexts such as the nucleus, cytoplasm, etc., which, in turn, might have a more profound effect on the expression of the downstream genes. Here, we demonstrate a methodology to visualize in vivo tripartite interaction using Bimolecular Fluorescence Complementation (BiFC) based F&#246;rster Resonance Energy Transfer (FRET) involving Fluorescence Lifetime Imaging (FLIM). Two of the proteins selected for this demonstration interact as BiFC partners, and their reconstituted fluorescence activity is used to assay FRET-FLIM with the third partner. Four to five-week-old growth-chamber-grown Nicotiana benthamiana plants have been used as the model plant system for this demonstration.

Here we present, a method to visualize ternary complex formation between three protein partners using fluorescent-tagged proteins by BiFC based FRET-FLIM assay. This method is valuable for studying protein-protein interaction complexes in vivo.

Protein-protein interactions are an integral part of all biological processes in the cells as they play a crucial role in regulating, maintaining, and ...

AirID-Based Proximity Labeling for Protein-Protein Interaction in Plants

In mammalian cells and plants, proximity labeling (PL) approaches using modified ascorbate peroxidase (APEX) or the Escherichia coli biotin ligase BirA (known as BioID) have proven successful in identifying protein-protein interactions (PPIs). APEX, BioID, and TurboID, a revised version of BioID have some restrictions in addition to being valuable technologies. The recently developed AirID, a novel version of BioID for proximity identification in protein-protein interactions, overcame these restrictions. Previously, AirID has been used in animal models, while the current study demonstrates the use of AirID in plants, and the results confirmed that AirID performs better in plant systems as compared to other PL enzymes such as BioID and TurboID for protein labeling that are proximal to the target proteins. Here is a step-by-step protocol for identifying protein interaction partners using AT4G18020 (APRR2) protein as a model. The methods describe the construction of vector, the transformation of construct through agroinfiltration, biotin transformation, extraction of proteins, and enrichment of biotin-labeled proteins through affinity purification technique. The results conclude that AirID is a novel and ideal enzyme for analyzing PPIs in plants. The method can be applied to study other proteins in plants.

Here, we present a step-by-step protocol for performing the proximity labeling (PL) experiment in cucumber (Cucumis sativus L.) using AT4G18020 (APRR2)-AirID protein as a model. The method describes the construction of a vector, the transformation of a construct through agroinfiltration, biotin infiltration, protein extraction, and purification of biotin-labeled proteins through affinity purification technique.

In mammalian cells and plants, proximity labeling (PL) approaches using modified ascorbate peroxidase (APEX) or the Escherichia coli biotin ligase BirA ...

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

Summary

Explore More Videos

Chapters in this video

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in an Efficient Way in Plants

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

Bimolecular Fluorescence Complementation

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

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

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

Determination of Tripartite Interaction between Two Monomers of a MADS-box Transcription Factor and a Calcium Sensor Protein by BiFC-FRET-FLIM Assay

AirID-Based Proximity Labeling for Protein-Protein Interaction in Plants