Name: determination of tripartite interaction between two monomers of a mads-box transcription factor and a calcium sensor protein by bifc-fret-flim assay
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Description: Watch this Scientific Journal Video about Determination of Tripartite Interaction between Two Monomers of a MADS-box Transcription Factor and a Calcium Sensor Protein by BiFC-FRET-FLIM Assay at JoVE.c

NB, GG, SB, KC sincerely thank the University Grants Commission (UGC), UGC-BSR, DBT-INSPIRE, and Council for Scientific and Industrial Research (CSIR) for their research fellowships. We thankfully acknowledge the Department of Biotechnology (DBT), Government of India, the Department of Science and Technology (DST-FIST), Government of India for financial support.

1. Cloning of genes in entry and destination vectors (Figure 2)
<ol>
	<li>Amplify the coding sequence (CDS) of the genes of interest (M and C genes in our case) by PCR and clone them in appropriate entry vectors (e.g., pENTR/D-TOPO vector; see Table 1 for vectors used in this experiment).</li>
	<li>Grow the clones on plates containing antibiotics. Validate clones that are selected on the antibiotics by restriction digestion and DN...

<ol>
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M., Sharick, J. T., Gillette, A. A., Skala, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+lifetime+imaging+microscopy:+fundamentals+and+advances+in+instrumentation,+analysis,+and+applications.">Fluorescence lifetime imaging microscopy: fundamentals and advances in instrumentation, analysis, and applications.</a> Journal of Biomedical Optics. 25 (07), 1 (2020).</li><li>Long, 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=Optimizing+FRET-FLIM+labeling+conditions+to+detect+nuclear+protein+interactions+at+native+expression+levels+in+living+Arabidopsis+roots.">Optimizing FRET-FLIM labeling conditions to detect nuclear protein interactions at native expression levels in living Arabidopsis roots.</a> Frontiers in Plant Science. 9, 1-13 (2018).</li><li>Burman, N., Chandran, D., Khurana, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Rapid+and+highly+efficient+method+for+transient+gene+expression+in+rice+plants.">A Rapid and highly efficient method for transient gene expression in rice plants.</a> Frontiers in Plant Science. , 11 (2020).</li><li>Kudla, J., Bock, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lighting+the+way+to+protein-protein+interactions:+Recommendations+on+best+practices+for+bimolecular+fluorescence+complementation+analyses.">Lighting the way to protein-protein interactions: Recommendations on best practices for bimolecular fluorescence complementation analyses.</a> Plant Cell. 28 (5), 1002-1008 (2016).</li><li>Shyu, Y. J., Hu, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+complementation:+an+emerging+tool+for+biological+research.">Fluorescence complementation: an emerging tool for biological research.</a> Trends in Biotechnology. 26 (11), 622-630 (2008).</li><li>Kodama, Y., Hu, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+bimolecular+fluorescence+complementation+assay+with+a+high+signal-to-noise+ratio.">An improved bimolecular fluorescence complementation assay with a high signal-to-noise ratio.</a> BioTechniques. 49 (5), 793-803 (2010).</li><li>Kodama, Y., Hu, C. D. <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):+A+5-year+update+and+future+perspectives.">Bimolecular fluorescence complementation (BiFC): A 5-year update and future perspectives.</a> BioTechniques. 53 (5), 285-298 (2012).</li><li>Piston, D. W., Kremers, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+protein+FRET:+the+good,+the+bad+and+the+ugly.">Fluorescent protein FRET: the good, the bad and the ugly.</a> Trends in Biochemical Sciences. 32 (9), 407-414 (2007).</li><li>Chakrabarty, R., 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=pSITE+vectors+for+stable+integration+or+transient+expression+of+autofluorescent+protein+fusions+in+plants:+Probing+Nicotiana+benthamiana-+Virus+Interactions.">pSITE vectors for stable integration or transient expression of autofluorescent protein fusions in plants: Probing Nicotiana benthamiana- Virus Interactions.</a> Molecular Plant-Microbe Interactions. 20 (7), 740-750 (2007).</li><li>Walter, M., 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=Visualization+of+protein+interactions+in+living+plant+cells+using+bimolecular+fluorescence+complementation.">Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation.</a> Plant Journal. 40 (3), 428-438 (2004).</li><li>Hellens, R., Mullineaux, P., Klee, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+Agrobacterium+binary+Ti+vectors.">A guide to Agrobacterium binary Ti vectors.</a> Trends in Plant Science. 5 (10), 446-451 (2000).</li><li>Van Der Hoorn, R. A. L., Rivas, S., Wulff, B. B. H., Jones, J. D. G., Joosten, M. H. A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+migration+in+gel+filtration+of+the+Cf-4+and+Cf-9+resistance+proteins+is+an+intrinsic+property+of+Cf+proteins+and+not+because+of+their+association+with+high-molecular-weight+proteins.">Rapid migration in gel filtration of the Cf-4 and Cf-9 resistance proteins is an intrinsic property of Cf proteins and not because of their association with high-molecular-weight proteins.</a> Plant Journal. 35 (3), 305-315 (2003).</li><li>Xie, X., 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=Engineering+SARS-CoV-2+using+a+reverse+genetic+system.">Engineering SARS-CoV-2 using a reverse genetic system.</a> Nature Protocols. 16, (2021).</li><li>Xu, J., 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=Optimized+plasmid+construction+strategy+for+Cas9.">Optimized plasmid construction strategy for Cas9.</a> Cellular Physiology and Biochemistry. 48, 131-137 (2018).</li><li>Mattanovich, D., 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=Efficient+transformation+of+Agrobacterium+spp.+by+electroporation.">Efficient transformation of Agrobacterium spp. by electroporation.</a> Nucleic Acids Research. 17 (16), 6747 (1989).</li><li>Rizzo, M. 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J., 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=SP8+FALCON:+a+novel+concept+in+fluorescence+lifetime+imaging+enabling+video-rate+confocal+FLIM.">SP8 FALCON: a novel concept in fluorescence lifetime imaging enabling video-rate confocal FLIM.</a> Nature Methods. 20, 2-4 (2019).</li><li>Postma, M., Goedhart, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plotsofdata-a+web+app+for+visualizing+data+together+with+their+summaries.">Plotsofdata-a web app for visualizing data together with their summaries.</a> PLoS Biology. 17 (3), 1-8 (2019).</li><li>Galperin, E., Verkhusha, V. 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The present protocol demonstrates the use of BiFC-based FRET-FLIM assay to ascertain the formation of a ternary complex between two monomers of a MADS-box protein and a calcium sensor protein. The protocol is adapted from a report by Y. John Shyu et al. where they have developed a BiFC-based FRET method to visualize ternary complex formed between Fos-Jun heterodimers and NFAT or p65 using the sensitized emission method7. Earlier, a three-fluorophore FRET system was developed by Galperin and co-wor...

Protein-protein interactions (PPIs) form the basis of the proper functioning of the eukaryotic cells by regulating various metabolic and developmental processes. Some PPIs are stable, while others are transient in nature. The interactions may be categorized based on the number and type of members in the interaction such as dimeric, trimeric, tetrameric homomeric, and heteromeric1. The identification and characterization of protein interactions may lead to a better understanding of protein functions and regulatory networks.
Transcription factors are proteins that are involved in regulatory functions. They regulate the rate of transcription of their downstream genes by binding to the DNA. Sometimes oligomerization or formation of higher-order complexes by proteins is a prerequisite for carrying out their functions2. Plant MADS-box transcription factors are homeotic genes that regulate various processes such as floral transition, floral organ development, fertilization, seed development, senescence, and vegetative development. They are known to form higher-order complexes which bind to the DNA3,4. Studying PPI networks among transcription factors and their interactors provides insights into the complexity underlying transcriptional regulation.
Transient protein expression in Nicotiana benthamiana has been a popular approach to study protein localization or protein-protein interactions in vivo5. BiFC and FRET are methods for studying protein-protein interactions in vivo using fluorescent reporter systems6. A combination of these two techniques has been shown to reveal the interaction between three proteins7. FRET is measured using acceptor photobleaching, sensitized emission, and Fluorescence Lifetime Imaging (FLIM) technique. FLIM-based FRET assay has emerged as a tool that provides accurate quantification and spatiotemporal specificity to energy transfer measurements between two molecules based on their fluorescence lifetimes8. FLIM measures the time a fluorophore stays in an excited state before emitting a photon and is better than techniques that use intensity measurements alone9,10. Besides heterologous systems such as Nicotiana benthamiana and onion epidermal peels, more recent reports have demonstrated the use of Arabidopsis roots and young rice seedlings, etc., for in vivo analysis of protein-protein interactions under native conditions11,12.
Other than a suitable expression system, the selection of interacting partners for BiFC and FRET assays is also crucial for the success of this experiment. The PPI among partners used in BiFC configuration should be validated using appropriate controls prior to their use as a conjugated partner in the FRET experiment13. BiFC utilizes the structural complementation of N- and C-terminal parts of the fluorescent protein. A common limitation in most if not all fluorescent proteins used in BiFC assays has been the self-assembly between the two derivative nonfluorescent fragments, contributing to false-positive fluorescence and decreases the signal-to-noise (S/N) ratio14. Recent developments, including point mutations or position of splitting the fluorescent protein, have given rise to BiFC pairs with increased intensity, higher specificity, high S/N ratio15,16. These fluorescent proteins can also be used for carrying out BiFC depending on the suitability of the experiment.
Traditionally, CFP and YFP have been used as the donor and acceptor pair in FRET experiments17. However, YFP or m-Citrine were found to be better FRET donors (when used with RFP as the acceptor) because of the high quantum yield (QY) during the native expression of target proteins in the Arabidopsis root system. The selection of promoters (constitutive versus native/endogenous) and fluorophore also play a crucial role in designing a successful BiFC-FRET-FLIM experiment. It is essential to note that the efficiency of FRET donors and suitability of FRET pairs tend to change with the change in the promoter and the biological system being used for expression. The QY of the fluorophore, which relates to its brightness, depends on the pH, temperature, and the biological system in use. We suggest that these criteria be thoroughly considered before choosing the fluorophore pair for the FRET experiment. The biological system, promoters, and the proteins used for this protocol worked well with CFP-YFP fluorophores for the BiFC FRET-FLIM experiment.
In the present study, we incorporate the feature of FLIM to visualize the interaction between three protein molecules using BiFC based FRET. In this technique, two proteins are tagged with split YFP protein and the third protein with CFP. Since we were interested in studying the interaction of a MADS-box protein (M) homodimer with a Calcium sensor protein (C), these proteins were tagged with fluorescent proteins in pSITE-1CA and pSITE-3CA vectors18. Two of the interacting partners, in this assay, were tagged with N- and C-terminal parts of the YFP in pSPYNE-35S and pSPYCE-35S vectors19, and their interaction results in the reconstitution of the functional YFP that acts as a FRET acceptor to the third interacting partner, which is tagged with CFP (acting as FRET donor) (Figure 1). In this particular case, the PPI between two M monomers and between M and C has been validated by performing BiFC in three different systems along with the yeast-two-hybrid system. These vectors were mobilized into Agrobacterium tumifaciens GV3101 strain by electroporation. The GV3101 strain has a disarmed Ti plasmid pMP90 (pTiC58DT-DNA) with gentamicin resistance20. A p19 Agrobacterium strain was added along with all infiltrations to prevent transgene silencing21. We recommend that the three proteins should also be used in opposite conformations to validate the tripartite interactions.
In this technique, we have employed FLIM, where first, the fluorescence lifetime of the donor (unquenched donor lifetime) is measured in the absence of an acceptor. After that, its lifetime is measured in the presence of the acceptor (quenched donor lifetime). This difference in donor fluorescence lifetimes is used to calculate FRET efficiency, which depends on the number of photons exhibiting a reduction in fluorescence lifetime. Mentioned below is a detailed protocol to determine the formation of a ternary complex between any three proteins by transiently expressing the fluorescent-tagged proteins in Nicotiana benthamiana and assaying their interaction by BiFC-FRET-FLIM.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 ml Syringes without needles</td>
			<td>Dispovan</td>
			<td>&#160;-</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetosyringone</td>
			<td>Sigma-Aldrich</td>
			<td>D134406</td>
			<td></td>
		</tr>
		<tr>
			<td>Gateway&#160; LR Clonase II Enzyme mix</td>
			<td>Thermo Fischer Scientific</td>
			<td>11791020</td>
			<td>The vectors used in the study are Gateway based</td>
		</tr>
		<tr>
			<td>Gentamycin Sulphate</td>
			<td>Himedia</td>
			<td>CMS461</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin Sulphate</td>
			<td>Himedia</td>
			<td>MB105</td>
			<td></td>
		</tr>
		<tr>
			<td>MES hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>M2933</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>M2670</td>
			<td></td>
		</tr>
		<tr>
			<td>pENTR/D-TOPO Cloning Kit</td>
			<td>Thermo Fischer Scientific</td>
			<td>K240020</td>
			<td>The vectors used in the study are Gateway based</td>
		</tr>
		<tr>
			<td>Phusion high fidelity Taq DNA polymerase</td>
			<td>Thermo Fischer Scientific</td>
			<td>F530-S</td>
			<td>Any High fidelity Polymerase can work</td>
		</tr>
		<tr>
			<td>Rifampicin</td>
			<td>Himedia</td>
			<td>CMS1889</td>
			<td></td>
		</tr>
		<tr>
			<td>SP8 FALCON Confocal laser scanning microscope</td>
			<td>Leica</td>
			<td>SP8 FALCON</td>
			<td>Any CLSM with FLIM capabilities can be used for this analysis</td>
		</tr>
		<tr>
			<td>Spectinomycin dihydrochloride pentahydrate</td>
			<td>Himedia</td>
			<td>TC034</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

This protocol represents an optimized method to study in vivo tripartite protein-protein interactions in plants. The basic principle of the protocol is to combine two fluorescence-tagged protein-interaction techniques, i.e., BiFC and FRET, to create an assay to measure ternary complex formation between three protein partners. Here, we have used FLIM to measure the fluorescence lifetime of the FRET donor partner in the presence and absence of the FRET acceptor. A reduction in the fluorescence lifetime of the dono...

Watch this Scientific Journal Video about Determination of Tripartite Interaction between Two Monomers of a MADS-box Transcription Factor and a Calcium Sensor Protein by BiFC-FRET-FLIM Assay at JoVE.c

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

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 ...

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