Name: luciferase complementation imaging assay in nicotiana benthamiana leaves for transiently determining protein-protein interaction dynamics
Uploaded: 2017-11-20T15:00:00
Description: Watch this Scientific Journal Video about Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics at JoVE.com

This work was supported by the Natural Science Foundation of Jiangsu Province (BK20140919), the National Natural Science Foundation of China (31470375), the Priority Academic Program Development of Jiangsu Higher Education Institutions and Qing Lan Project.

1. Preparation of Plants (8 weeks)
<ol>
	<li>Put 50 Nicotiana benthamiana seeds in one 1.5 mL microcentrifuge tube containing 1 mL of 5% NaClO and 0.1% Triton X-100 (V/V) solution and leave for 5 min to sterilize the seeds.</li>
	<li>Rinse the seeds with sterile water 5 times and suspend seeds in 200 &#181;L of sterile water.</li>
	<li>Carefully place individual seeds onto the surface of MS-agar medium (1 L: 1x Murashige &#38; Skoog salts, 10 g sucrose, pH 5.8 (KOH), 1% agar) with fine tips and store medium pl...

<ol>
	<li>Thines, B., 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=JAZ+repressor+proteins+are+targets+of+the+SCF(COI1)+complex+during+jasmonate+signalling.">JAZ repressor proteins are targets of the SCF(COI1) complex during jasmonate signalling.</a> Nature. 448, 661-665 (2007).</li><li>Chini, A., 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+JAZ+family+of+repressors+is+the+missing+link+in+jasmonate+signalling.">The JAZ family of repressors is the missing link in jasmonate signalling.</a> Nature. 448, 666-671 (2007).</li><li>Jones, A. 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=Border+control--a+membrane-linked+interactome+of+Arabidopsis.">Border control--a membrane-linked interactome of Arabidopsis.</a> Science. 344, 711-716 (2014).</li><li>Arabidopsis Interactome Mapping Consortium. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+network+evolution+in+an+Arabidopsis+interactome+map.">Evidence for network evolution in an Arabidopsis interactome map.</a> Science. 333, 601-607 (2011).</li><li>Yazaki, 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=Mapping+transcription+factor+interactome+networks+using+HaloTag+protein+arrays.">Mapping transcription factor interactome networks using HaloTag protein arrays.</a> Proc Natl Acad Sci U S A. 113, 4238-4247 (2016).</li><li>Kenworthy, A. K. <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+using+fluorescence+resonance+energy+transfer+microscopy.">Imaging protein-protein interactions using fluorescence resonance energy transfer microscopy.</a> Methods. 24, 289-296 (2001).</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 J. 40, 428-438 (2004).</li><li>Chen, H., 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=Firefly+luciferase+complementation+imaging+assay+for+protein-protein+interactions+in+plants.">Firefly luciferase complementation imaging assay for protein-protein interactions in plants.</a> Plant Physiol. 146, 368-376 (2008).</li><li>Layton, C. J., Hellinga, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+protein-protein+interactions+by+thermal+stability+shift+analysis.">Quantitation of protein-protein interactions by thermal stability shift analysis.</a> Protein Sci. 20, 1439-1450 (2011).</li><li>Schuck, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliable+determination+of+binding+affinity+and+kinetics+using+surface+plasmon+resonance+biosensors.">Reliable determination of binding affinity and kinetics using surface plasmon resonance biosensors.</a> Curr Opin Biotechnol. 8, 498-502 (1997).</li><li>Huang, X., Ouyang, X., Deng, X. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+repression+of+photomorphogenesis:+role+switching+of+COP/DET/FUS+in+light+signaling.">Beyond repression of photomorphogenesis: role switching of COP/DET/FUS in light signaling.</a> Current opinion in plant biology. 21, 96-103 (2014).</li><li>Liu, B., Zuo, Z., Liu, H., Liu, X., Lin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+cryptochrome+1+interacts+with+SPA1+to+suppress+COP1+activity+in+response+to+blue+light.">Arabidopsis cryptochrome 1 interacts with SPA1 to suppress COP1 activity in response to blue light.</a> Genes Dev. 25, 1029-1034 (2011).</li><li>Lian, H. L., 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=Blue-light-dependent+interaction+of+cryptochrome+1+with+SPA1+defines+a+dynamic+signaling+mechanism.">Blue-light-dependent interaction of cryptochrome 1 with SPA1 defines a dynamic signaling mechanism.</a> Genes Dev. 25, 1023-1028 (2011).</li><li>Zuo, Z., Liu, H., Liu, B., Liu, X., Lin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue+light-dependent+interaction+of+CRY2+with+SPA1+regulates+COP1+activity+and+floral+initiation+in+Arabidopsis.">Blue light-dependent interaction of CRY2 with SPA1 regulates COP1 activity and floral initiation in Arabidopsis.</a> Curr Biol. 21, 841-847 (2011).</li></ol>

The protocol described here is simple and reproducible for studying in vivo protein-protein interactions, and particularly suitable for detecting protein interaction dynamics under exogenous treatment. The key step in this assay is N. benthamiana infiltration. To ensure infiltration success, plants must be very healthy. Another critical factor is when to check luciferase activity after infiltration. There is no correct answer for this question. Researchers are encouraged to monitor luciferase activity a...

In order to coordinate growth with its environment, plants have evolved elegant signaling pathways to sense, transduce, and respond to signaling cues. Like the runners in a relay race, proteins are necessary players in plant signal transduction. It has been widely recognized that protein-protein interaction (PPI) plays a major role for cellular communication. Protein phosphorylation, acetylation, and degradation are all dependent on physical interaction between a target protein and its modifying enzymes. For example, Jasmonate ZIM-domain protein (JAZ) family proteins interact with transcription factor MYC2 and suppress its transcriptional activity1,2. Given the importance of PPI, large-scale protein interactomes in plants have been explored recently3,4,5. These interactome results further reveal the complex regulatory network that coordinates diverse cellular functions.
There are some established approaches for monitoring PPI. The yeast two hybrid (Y2H) assay is the most widely used method for PPI detection. Y2H is easy to perform, and suitable for a quick test to examine protein interactions. Y2H is also widely used for screening unknown interaction partners for the specific protein-of-interest. However, because Y2H is entirely carried out in yeast, it cannot reflect the real scenario in plant cells and brings high false positive rates. Another similar strategy is the pull-down assay. In general, two proteins are expressed and purified from Escherichia coli cells and then mixed and immobilized for detecting protein interactions. Although this method is not time-consuming, it cannot obtain in vivo interaction results either. For in vivo interaction purposes, co-immunoprecipitation (co-IP) is the most popular assay, which requires high quality antibody to immunoprecipitate the protein-of-interest and cannot exclude the possibility of indirect PPIs. Moreover, due to the complicated experimental procedures in co-IP, the results usually vary with individual expertise.
Reporter based reconstitution assays greatly advance the detection of in vivo PPI. These methods include fluorescence resonance energy transfer (FRET)6, bimolecular fluorescence complementation (BiFC)7, and the firefly luciferase complementation imaging (LCI) assay8. Although these three approaches are better than co-IP to reflect the direct in vivo PPI, FRET, and BiFC need specific microscopes to detect fluorescence signals and cannot easily quantify the interaction intensity. In contrast, LCI takes advantage of firefly luciferase, which will glow after reacting with its substrate luciferin. More importantly, PPI intensity can be determined from the value of luciferase activity. Thus, LCI not only indicates whether two proteins interact or not, but also provides information on the interaction dynamics upon treatment. Although there are some established methods for monitoring interaction dynamics (based on surface plasmon resonance or thermo-stability shifts)9,10, these approaches require delicate instruments or specific labeling. In contrast, LCI is easier to perform and detect.
The principle for LCI is that the amino-terminal and carboxyl-terminal halves of luciferase (named N-Luc or C-Luc, respectively) were fused with protein A and B, and these two fusion proteins were simultaneously expressed in plant cells. If protein A interacts with protein B, then the two halves of luciferase will be reconstituted to be an active luciferase enzyme. Luciferase activity can be detected with a luminometer or CCD-camera. It is not necessary to obtain stable transformants for LCI; transient expression is enough to get a high-quality interaction result.
RING-type E3 ubiquitin ligase constitutive photomorphogenic 1 (COP1) interacts with a myriad of transcription factors and promotes their degradation through the 26S proteasome11. Some of these COP1-targeted transcription factors are positive regulators for photomorphogenesis. In darkness, COP1 interacts with suppressor of phytochrome A-105 1 (SPA1), which enhances COP1 E3 activity8. After perception of light, photoreceptors will abrogate the COP1-SPA1 interaction to inhibit COP1 activity and then stabilize COP1 substrates12,13,14. This example illustrates the biological significance of studying PPI and determining PPI dynamics.

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		<col style="width:114pt" width="152" />
	</colgroup>
	<tbody>
		<tr height="28" style="height:21.0pt">
			<td class="xl16" height="28" style="width:293pt;height:21.0pt" width="390">Transformation solution</td>
			<td class="xl16" style="width:140pt" width="186"></td>
			<td class="xl16" style="width:140pt" width="187"></td>
			<td style="width:114pt" width="152"></td>
		</tr>
		<tr height="28" style="height:21.0pt">
			<td class="xl17" height="28" style="width:293pt;height:21.0pt" width="390">10 mM Morpholineethanesulfonic acid</td>
			<td class="xl17" style="width:140pt" width="186">VETEC</td>
			<td class="xl17" style="width:140pt" width="187">V900336&#160;</td>
			<td></td>
		</tr>
		<tr height="28" style="height:21.0pt">
			<td class="xl17" height="28" style="width:293pt;height:21.0pt" width="390">27.8 mM Glucose</td>
			<td class="xl17" style="width:140pt" width="186">VETEC</td>
			<td class="xl17" style="width:140pt" width="187">V900392</td>
			<td></td>
		</tr>
		<tr height="32" style="height:24.0pt">
			<td class="xl18" height="32" style="width:293pt;height:24.0pt" width="390">10 mM MgCl2&#215;6H2O</td>
			<td class="xl17" style="width:140pt" width="186">VETEC</td>
			<td class="xl17" style="width:140pt" width="187">V900020&#160;</td>
			<td></td>
		</tr>
		<tr height="28" style="height:21.0pt">
			<td class="xl17" height="28" style="width:293pt;height:21.0pt" width="390">150 &#956;M Acetosyringone</td>
			<td class="xl17" style="width:140pt" width="186">ALDRICH</td>
			<td class="xl17" style="width:140pt" width="187">D134406&#160;</td>
			<td></td>
		</tr>
		<tr height="28" style="height:21.0pt">
			<td class="xl17" height="28" style="width:293pt;height:21.0pt" width="390">pH 5.7</td>
			<td class="xl17" style="width:140pt" width="186"></td>
			<td class="xl17" style="width:140pt" width="187"></td>
			<td></td>
		</tr>
		<tr height="28" style="height:21.0pt">
			<td class="xl16" height="28" style="width:293pt;height:21.0pt" width="390">Luciferin working buffer</td>
			<td class="xl17" style="width:140pt" width="186"></td>
			<td class="xl17" style="width:140pt" width="187"></td>
			<td></td>
		</tr>
		<tr height="56" style="height:42.0pt">
			<td class="xl17" height="56" style="width:293pt;height:42.0pt" width="390">5 mM Luciferin potassium salt</td>
			<td class="xl17" style="width:140pt" width="186">GOLD BIOTECHNOLOGY</td>
			<td class="xl17" style="width:140pt" width="187">LUCK-100</td>
			<td></td>
		</tr>
		<tr height="28" style="height:21.0pt">
			<td class="xl17" height="28" style="width:293pt;height:21.0pt" width="390">0.025% Triton X-100</td>
			<td class="xl17" style="width:140pt" width="186">VETEC</td>
			<td class="xl17" style="width:140pt" width="187">V900502&#160;</td>
			<td></td>
		</tr>
		<tr height="32" style="height:24.0pt">
			<td class="xl17" height="32" style="width:293pt;height:24.0pt" width="390">H2O to 10 ml</td>
			<td class="xl21"></td>
			<td class="xl21"></td>
			<td></td>
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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.

Three major steps can be singled out in this luciferase complementation protocol for studying protein-protein interactions in vivo, including plant growth, tobacco infiltration, and the luciferase assay. The most crucial step in this protocol is infiltrating liquid A. tumefaciens into N. benthamiana leaves (Figure 1).
Here is an example of the usefulness of this technique ...

Watch this Scientific Journal Video about Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics at JoVE.com

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.

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 ...

The overall goal of this procedure is to quantitatively detect protein-protein interactions in a transient expression system. This measure can help answer key questions in quan-signal transaction fields such as how to quantitatively monitor our protein protein directions after a chemical or environmental treatment. The main advantage of this technic is that it uses a luminometer or a CCD camera to detect the luciferous activity, which represents the protein protein direction intensity so the results are more accurate.To begin this procedure, add one milliliter of a solution containing five percent sodium hypochlorite and zero point one percent triton x one hundred to a one point five milliliter microcentrifuge tube containing seeds. Let the solution rest for five minutes to sterilize the seeds. Then, wash the seeds with sterile water five times.Suspend the washed seeds in two hundred microliters of steril water. Using fine tips, carefully place individual seeds onto the surface of plated MS agar medium. Store the plates at four degrees Celsius for three days to synchronize germination.After this, keep medium plates under normal growth conditions for ten days. Transfer the germinating seedlings into soil, making sure not to damage the roots. Cover the tray with a transparent plastic cover overnight to maintain humidity.Grow plants in a controlled growth room for seven weeks, when they'll be ready for agrobacterium tumefaciens infiltration. To begin, deliver one hundred and fifty nanograms of plasmids into the a-tumefaciens competent cell strain GV3101. Place the tubes containing the a-tumefaciens culture in a shaker, and incubate for four hours.Using a glass rod, transfer the culture from the tubes to LB mediums. Culture a-tumefaciens and LB agar medium at 28 degrees celsius for four days. Next, prepare to inoculate a single a-tumefaciens colony from each plate into its own tube containing three milliliters of LB liquid medium.Shake at 280 RBM at 28 degrees Celsius for 24 hours to propagate the culture. Then, transfer 75 microliters of the bacterial culture into 15 milliliters of fresh LB liquid medium containing 15 micrograms per milliliter of kanamycin and 50 micrograms per milliliter of rifampicin. Culture the bacteria at 220 RPM at 30 degrees Celsius for approximately 8 hours until OD600 is between 0.5 to 1.After this, transfer the culture to 15 milliliters centrifuge tubes. Centrifuge at 4000 times gravity and four degrees Celsius for 15 minutes. Discard the supernatant and resuspend the pallet in 15 milliliters of transformation solution.Centrifuge at 4000 times gravity and 4 degrees Celsius for 15 minutes. Then, repeat the suspension and centrifugation step once more. Discard the supernatant and resuspend the pallet in 5 milliliters of transformation solution.Let the resuspended pallet rest for two hours at room temperature in dark conditions. Then, either add transformation solution into the OD600 is 0.5 or combine two samples with equal volume for testing paired protein interactions. Using a one milliliter needle of syringe, infiltrate suspended cells into the fourth to seventh leaves.After this, immediately cover the plants with a black plastic cover for 12 hours. Firstly, plants should be very healthy to ensure success. Secondly, make sure not to damage any leaves during the infiltration process.Keep the tray in darkness for 12 hours. Then grow the plants under normal conditions for 2 to 4 days before detecting the luciferase activity. To begin the imaging method, detach an infiltrated leaf.Place the entire leaflet on a plate of MS agar medium. Using a 50 milliliter spray bottle, spray luciferon working buffer onto the leafs adaxial side. Then, keep in the dark for 5 minutes to quench the chlorophyll autofluorescence.Use a lower light cooled CCD imagine apparatus cooled to negative 30 degrees Celsius to capture images with a light filled exposure time of 50 milliseconds, and luminescence detection time of 1 minute. After this, cut out leaf fragments from the infiltration area of the N-benthamiana leaves. Immerse the fragments in 100 microliters of deionized water in the wells of 96-well white plate.Add ten microliters of luciferon working buffer. Let the plate rest 5 minutes. Then, perform any desired specific treatment to study protein interaction dynamics, and measure the luminescence directly with a commercial luminometer.Shown here is the demonstration of luciferous activity to represent of COP 1 SPA 1 interactions. The luciferous activity, which is detected by luminescence, is imaged by CCD camera. COP 1 SPA 1 interactions are seen to result in copper mutation of functional luciferase.The luciferase activity under Jasmine 8 treatment over time is then determined. COP 1 SPA 1 interactions represented by luciferase activity are seen to gradually decline in darkness. Treatment with Jasmine 8 further reduces these interactions.Once mastered, this technic can be done in 1 week, after the plants are ready if it's performed properly. While attempting this procedure, it is important to remember to maintain healthy plants. Infiltrate leaves carefully and include controls properly.After it's development, this technic paves the way for researchers in the field of signal transactions to explore protein protein interaction dynamics in a rapid way. After watching this video, you should have a good understanding of how to grow tobacco plants, infiltrate agrobacterias into tobacco leaves, and to detect luciferous activities.

luciferase-complementation-imaging-assay-nicotiana-benthamiana-leaves

Research

JoVE Journal

Biology

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development2,3,4. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host5.
Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript2.
Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS6,7. Inoculation of Nicotiana benthamiana and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase (PDS) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana can be used to silence their respective orthologs in tomato and vice versa8.

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA ...

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 ...

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, ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another 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 ...

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1 to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most commonly used agent for agroinfiltration is Agrobacterium tumefaciens, a pathogen of many dicot plant species. This implies that agroinfiltration can be applied to many plant species. Here, we present our protocols and expected results when applying these methods to the potato (Solanum tuberosum), its related wild tuber-bearing Solanum species (Solanum section Petota) and the model plant Nicotiana benthamiana. In addition to functional analysis of single genes, such as resistance (R) or avirulence (Avr) genes, the agroinfiltration assay is very suitable for recapitulating the R-AVR interactions associated with specific host pathogen interactions by simply delivering R and Avr transgenes into the same cell. However, some plant genotypes can raise nonspecific defense responses to Agrobacterium, as we observed for example for several potato genotypes. Compared to agroinfiltration, detection of AVR activity with PVX agroinfection is more sensitive, more high-throughput in functional screens and less sensitive to nonspecific defense responses to Agrobacterium. However, nonspecific defense to PVX can occur and there is a risk to miss responses due to virus-induced extreme resistance. Despite such limitations, in our experience, agroinfiltration and PVX agroinfection are both suitable and complementary assays that can be used simultaneously to confirm each other&#39;s results.

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are routine functional assays for transient ectopic expression of genes in plants. These methods are efficient assays in effectoromics strategies (rapid resistance and avirulence gene discovery) and crucial to modern research in molecular plant pathology. They meet the demand for robust high-throughput functional analysis in plants.

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most ...

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.

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 ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells

Proteins interact with each other and these interactions determine in a large part their functions. Protein interaction partners can be identified at high-throughput in vivo using a yeast fitness assay based on the dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA).

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often ...