This work was supported by grants from the National Transgenic Science and Technology Program (2019ZX08010-003 to Y.Z.), the National Natural Science Foundation of China (31872637 to Y.Z.), and the Fundamental Research Funds for the Central Universities (2019TC028 to Y.Z.), and NSF-IOS-1354434, NSF-IOS-1339185, and NIH-GM132582-01 to S.P.D.K.

NOTE: An overview of the method is shown in Figure 1.
1. Plant material preparation
<ol>
	<li>Grow N. benthamiana seeds in wet soil at a high density and maintain them in a climate chamber with a 16 h light (about 75 &#956;mol/m2s) and 8 h dark photoperiod at 23&#8211;25 &#176;C.</li>
	<li>About 1 week later, carefully transfer each young seedling to 4&#39; x 4&#39; pots and keep the seedlings in the same chamber.</li>
	<li>Maint...

<ol>
	<li>Bergg&#229;rd, T., Linse, S., James, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+the+detection+and+analysis+of+protein-protein+interactions.">Methods for the detection and analysis of protein-protein interactions.</a> Proteomics. 7 (16), 2833-2842 (2007).</li><li>Kim, D. I., Roux, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filling+the+void:+proximity-based+labeling+of+proteins+in+living+cells.">Filling the void: proximity-based labeling of proteins in living cells.</a> Trends in Cell Biology. 26 (11), 804-817 (2016).</li><li>Li, P., Li, J., Wang, L., Di, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proximity+labeling+of+interacting+proteins:+Application+of+BioID+as+a+discovery+tool.">Proximity labeling of interacting proteins: Application of BioID as a discovery tool.</a> Proteomics. 17 (20), (2017).</li><li>Roux, K. J., Kim, D. I., Raida, M., Burke, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+promiscuous+biotin+ligase+fusion+protein+identifies+proximal+and+interacting+proteins+in+mammalian+cells.">A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells.</a> Journal of Cell Biology. 196 (6), 801-810 (2012).</li><li>Kim, D. I., 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=Probing+nuclear+pore+complex+architecture+with+proximity-dependent+biotinylation.">Probing nuclear pore complex architecture with proximity-dependent biotinylation.</a> Proceedings of the National Academy of Sciences. 111 (24), 2453-2461 (2014).</li><li>Lin, Q., 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+of+proximal+and+interacting+proteins+in+rice+protoplasts+by+proximity-dependent+biotinylation.">Screening of proximal and interacting proteins in rice protoplasts by proximity-dependent biotinylation.</a> Frontiers in Plant Science. 8 (749), (2017).</li><li>Khan, M., Youn, J. Y., Gingras, A. C., Subramaniam, R., Desveaux, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+planta+proximity+dependent+biotin+identification+(BioID).">In planta proximity dependent biotin identification (BioID).</a> Scientific Reports. 8 (1), 9212 (2018).</li><li>Conlan, B., Stoll, T., Gorman, J. J., Saur, I., Rathjen, 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=Development+of+a+rapid+in+planta+BioID+system+as+a+probe+for+plasma+membrane-associated+immunity+proteins.">Development of a rapid in planta BioID system as a probe for plasma membrane-associated immunity proteins.</a> Frontiers in Plant Science. 9 (1882), (2018).</li><li>Macharia, M. W., Tan, W. Y. Z., Das, P. P., Naqvi, N. I., Wong, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proximity-dependent+biotinylation+screening+identifies+NbHYPK+as+a+novel+interacting+partner+of+ATG8+in+plants.">Proximity-dependent biotinylation screening identifies NbHYPK as a novel interacting partner of ATG8 in plants.</a> BMC Plant Biology. 19 (1), 326 (2019).</li><li>Branon, T. C., 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+proximity+labeling+in+living+cells+and+organisms+with+TurboID.">Efficient proximity labeling in living cells and organisms with TurboID.</a> Nature Biotechnology. 36 (9), 880-887 (2018).</li><li>Zhang, 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=TurboID-based+proximity+labeling+reveals+that+UBR7+is+a+regulator+of+N+NLR+immune+receptor-mediated+immunity.">TurboID-based proximity labeling reveals that UBR7 is a regulator of N NLR immune receptor-mediated immunity.</a> Nature Communications. 10 (1), 3252 (2019).</li><li>Arora, 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=Establishment+of+proximity-dependent+biotinylation+approaches+in+different+plant+model+systems.">Establishment of proximity-dependent biotinylation approaches in different plant model systems.</a> bioRxiv. , (2019).</li><li>Kim, T. W., 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=Application+of+TurboID-mediated+proximity+labeling+for+mapping+a+GSK3+kinase+signaling+network+in+Arabidopsis.">Application of TurboID-mediated proximity labeling for mapping a GSK3 kinase signaling network in Arabidopsis.</a> bioRxiv. , (2019).</li><li>Mair, A., Xu, S. L., Branon, T. C., Ting, A. Y., Bergmann, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proximity+labeling+of+protein+complexes+and+cell-type-specific+organellar+proteomes+in+Arabidopsis+enabled+by+TurboID.">Proximity labeling of protein complexes and cell-type-specific organellar proteomes in Arabidopsis enabled by TurboID.</a> eLife. 8, 47864 (2019).</li><li>Yuan, C., 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=A+high+throughput+Barley+stripe+mosaic+virus+vector+for+virus+induced+gene+silencing+in+monocots+and+dicots.">A high throughput Barley stripe mosaic virus vector for virus induced gene silencing in monocots and dicots.</a> PLoS ONE. 6 (10), 26468 (2011).</li><li>McCormac, A. C., Elliott, M. C., Chen, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+the+production+of+highly+competent+cells+of+Agrobacterium+for+transformation+via+electroporation.">A simple method for the production of highly competent cells of Agrobacterium for transformation via electroporation.</a> Molecular Biotechnology. 9 (2), 155-159 (1998).</li><li>Gingras, A. C., Abe, K. T., Raught, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+to+know+the+neighborhood:+using+proximity-dependent+biotinylation+to+characterize+protein+complexes+and+map+organelles.">Getting to know the neighborhood: using proximity-dependent biotinylation to characterize protein complexes and map organelles.</a> Current Opinion in Chemical Biology. 48, 44-54 (2019).</li><li>Firat-Karalar, E. N., Rauniyar, N., Yates, J. R., Stearns, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proximity+Interactions+among+centrosome+components+identify+regulators+of+centriole+duplication.">Proximity Interactions among centrosome components identify regulators of centriole duplication.</a> Current Biology. 24 (6), 664-670 (2014).</li><li>Shen, 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=Organelle+pH+in+the+Arabidopsis+endomembrane+system.">Organelle pH in the Arabidopsis endomembrane system.</a> Molecular Plant. 6 (5), 1419-1437 (2013).</li><li>Bally, 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=The+rise+and+rise+of+Nicotiana+benthamiana:+a+plant+for+all+reasons.">The rise and rise of Nicotiana benthamiana: a plant for all reasons.</a> Annual Review of Phytopathology. 56 (1), 405-426 (2018).</li></ol>

The authors have nothing to disclose.

The TurboID biotin ligase is generated by yeast display-based directed evolution of the BioID10. It has many advantages over other PL enzymes. TurboID allows the application of PL to other model systems, including flies and worms, whose optimal growth temperature is around 25 &#176;C10. Although the PL approach has been widely used in animal systems, its application in plants is limited. The protocol described here provides a step-by-step procedure for establishing the Turb...

PPIs are the basis of various cellular processes. Traditional methods for identifying PPIs include yeast-two-hybrid (Y2H) screening and immunoprecipitation coupled with mass spectrometry (IP-MS)1. However, both suffer from some disadvantages. For example, Y2H screening requires the availability of Y2H library of the target plant or animal species. Construction of these libraries is labor-intensive and expensive. Furthermore, the Y2H approach is performed in the heterologous single-cell eukaryotic organism yeast, which may not represent the cellular status of higher eukaryotic cells.
In contrast, IP-MS shows low efficiency in capturing transient or weak PPIs, and it is also unsuitable for those proteins with low abundance or high hydrophobicity. Many important proteins involved in the plant signaling pathways such as receptor-like kinases (RLKs) or the NLR family of immune receptors are expressed at low levels and often interact with other proteins transiently. Therefore, it greatly restricts the understanding of mechanisms underlying the regulation of these proteins.
Recently, proximity labeling (PL) methods based on engineered ascorbate peroxidase (APEX) and a mutant Escherichia coli biotin ligase BirAR118G (known as BioID) have been developed and utilized for the study of PPIs2,3,4. The principle of PL is that a target protein of interest is fused with an enzyme, which catalyzes the formation of labile biotinyl-AMP (bio-AMP). These free bio-AMP are released by PL enzymes and diffuse to the vicinity of the target protein, allowing the biotinylation of proximal proteins at the primary amines within an estimated radius of 10 nm5.
This approach has significant advantages over the traditional Y2H and IP-MS approaches, such as the ability to capture transient or weak PPIs. Furthermore, PL allows the labeling of proximal proteins of the target protein in their native cellular environments. Different PL enzymes have unique disadvantages when applying them to different systems. For example, although APEX offers higher tagging kinetics compared to BioID and is successfully applied in mammalian systems, the requirement of toxic hydrogen peroxide (H2O2) in this approach makes it unsuitable for PL studies in plants.
In contrast, BioID-based PL avoids use of the toxic H2O2, but the rate of labeling is slow (requiring 18&#8211;24 h to complete biotinylation), thus making the capture of transient PPIs less efficient. Moreover, the higher incubation temperature (37 &#176;C) required for efficient PL by BioID introduces external stress to some organisms, such as plants4. Therefore, limited deployment of BioID-based PL in plants (i.e., rice protoplasts, Arabidopsis, and N. benthamiana) has been reported6,7,8,9. The recently described TurboID enzyme overcomes the deficiencies of APEX and BioID-based PL. TurboID showed high activity that enables the accomplishment of PL within 10 min at RT10. TurboID-based PL has been successfully applied in mammalian cells, flies, and worms10. Recently, we and other research groups independently optimized and extended the use of TurboID-based PL for studying PPIs in different plant systems, including N. benthamiana and Arabidopsis plants and tomato hairy roots11,12,13,14. Comparative analyses indicated that TurboID performs better for PL in plants compared to BioID11,14. It has also demonstrated the robustness of TurboID-based PL in planta by identifying a number of novel interactions with an NLR immune receptor11, a protein whose interaction partners are usually difficult to obtain using traditional methods.
This protocol illustrates the TurboID-based PL in planta by describing the identification of interaction proteins of the N-terminal TIR domain of the NLR immune receptor in N. benthamiana plants. The method can be extended to any proteins of interest in N. benthamiana. More importantly, it provides an important reference for investigating PPIs in other plant species such as Arabidopsis, tomato, and others.

<table>
	<tbody>
		<tr>
			<td>721 Spectrophotometer</td>
			<td>Metash, made in China</td>
			<td>Q/SXFZ6</td>
			<td>For OD600 measurement</td>
		</tr>
		<tr>
			<td>Ammonium bicarbonate</td>
			<td>Sigma</td>
			<td>A6141-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin</td>
			<td>Sigma</td>
			<td>B4639-1G</td>
			<td>50 mM Stock</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>Centrifuge 5702</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>Centrifuge 5417R</td>
			<td></td>
		</tr>
		<tr>
			<td>cOmplete Protease Inhibitor Cocktail</td>
			<td>Roche</td>
			<td>11697489001</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxycholic acid</td>
			<td>Sigma</td>
			<td>D2510-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>DL-Dithiothreitol (DTT)</td>
			<td>VWR Life Science</td>
			<td>0281-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads MyOne Streptavidin C1</td>
			<td>Invitrogen</td>
			<td>65001</td>
			<td>For affinity purification</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>E6758-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>ELISA plate</td>
			<td>Corning</td>
			<td>Costar 3590</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid (HCl)</td>
			<td>Fisher Scientific</td>
			<td>A144S-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Immobilon-P PVDF membrane</td>
			<td>Millipore</td>
			<td>IPVH00010</td>
			<td>For Western blot analysis</td>
		</tr>
		<tr>
			<td>Lithium chloride solution(LiCl), 8M</td>
			<td>Sigma</td>
			<td>L7026-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Low speed refrigerated centrifuge</td>
			<td>Zonkia, made in China</td>
			<td>KDC-2046</td>
			<td>For desalting</td>
		</tr>
		<tr>
			<td>Magnesium Chloride, Hexahydrate (MgCl2&#183;6H2O)</td>
			<td>Sigma</td>
			<td>M9272-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic rack</td>
			<td>Invitrogen</td>
			<td>123.21D</td>
			<td>For bead adsorption</td>
		</tr>
		<tr>
			<td>Multiskan FC Microplate Photometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>N07710</td>
			<td>For OD595 measurement</td>
		</tr>
		<tr>
			<td>NP-40 (IGEPAL CA-630)</td>
			<td>Sigma</td>
			<td>I8896-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-HA</td>
			<td>Roche</td>
			<td>11867423001</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotational mixer</td>
			<td>Kylin-Bell Lab Instrument</td>
			<td>WH-986</td>
			<td>For IP</td>
		</tr>
		<tr>
			<td>Shock incubator</td>
			<td>Labotery, made in China</td>
			<td>ZQPZ-228</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Fisher Scientific</td>
			<td>S271-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium deoxycholate</td>
			<td>Sigma</td>
			<td>D2510-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate(SDS)</td>
			<td>Sigma</td>
			<td>L4390-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin-HRP</td>
			<td>Abcam</td>
			<td>ab7403</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher Scientific</td>
			<td>BP151-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma base</td>
			<td>Sigma</td>
			<td>T1503-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>Scientific Industries</td>
			<td>G-560E</td>
			<td></td>
		</tr>
		<tr>
			<td>Water-jacket Incubator</td>
			<td>Blue pard, made in China</td>
			<td>GHP-9080</td>
			<td>For Agrobacterium incubation</td>
		</tr>
		<tr>
			<td>Zeba Spin Desalting Column</td>
			<td>Thermo Fisher Scientific</td>
			<td>89893</td>
			<td>For removal of biotin</td>
		</tr>
	</tbody>
</table>

turboid-based proximity labeling for in planta identification of protein-protein interaction networks

Proximity labeling (PL) techniques using engineered ascorbate peroxidase (APEX) or Escherichia coli biotin ligase BirA (known as BioID) have been successfully used for identification of protein-protein interactions (PPIs) in mammalian cells. However, requirements of toxic hydrogen peroxide (H2O2) in APEX-based PL, longer incubation time with biotin (16&#8211;24 h), and higher incubation temperature (37 &#176;C) in BioID-based PL severely limit their applications in plants. The recently described TurboID-based PL addresses many limitations of BioID and APEX. TurboID allows rapid proximity labeling of proteins in just 10&#8201;min under room temperature (RT) conditions. Although the utility of TurboID has been demonstrated in animal models, we recently showed that TurboID-based PL performs better in plants compared to BioID for labeling of proteins that are proximal to a protein of interest. Provided here is a step-by-step protocol for the identification of protein interaction partners using the N-terminal Toll/interleukin-1 receptor (TIR) domain of the nucleotide-binding leucine-rich repeat (NLR) protein family as a model. The method describes vector construction, agroinfiltration of protein expression constructs, biotin treatment, protein extraction and desalting, quantification, and enrichment of the biotinylated proteins by affinity purification. The protocol described here can be easily adapted to study other proteins of interest in Nicotiana and other plant species.

The representative data, which illustrate the expected results based on the described protocol, are adapted from Zhang et al11. Figure 1 summarizes the procedures for performing TurboID-based PL in N. benthamiana. Figure 2 shows the protein expression and biotinylation in the infiltrated N. benthamiana leaves. Figure 3 shows that the biotinylated proteins in the infiltrated leaves were effic...

Watch this Scientific Journal Video about TurboID-Based Proximity Labeling for In Planta Identification of Protein-Protein Interaction Networks at JoVE.com

TurboID-Based Proximity Labeling for In Planta Identification of Protein-Protein Interaction Networks

Described here is a proximity labeling method for identification of interaction partners of the TIR domain of the NLR immune receptor in Nicotiana benthamiana leaf tissue. Also provided is a detailed protocol for the identification of interactions between other proteins of interest using this technique in Nicotiana and other plant species.

Proximity labeling (PL) techniques using engineered ascorbate peroxidase (APEX) or Escherichia coli biotin ligase BirA (known as BioID) have been ...

NLR immune receptors play a crucial role in mediating plant defense against various pathogens. We describe a TurboID-based proximity labeling method for the identification of interaction partners of Toll/interleukin-1 receptor domain containing NLRs such as immune receptors in Nicotiana benthamiana plants. This protocol provides an important reference for investigating protein-protein interactions in other plant species and it can be extended to any protein of interest in Nicotiana benthamiana.The TurboID-based proximity labeling approach has a distinct advantage over the traditional approaches because it can be used to capture transient or weak protein-protein interactions in vivo. Start by growing N.benthamiana seeds in wet soil at a high density. Maintain them in a climate chamber with an 18-hour light and eight-hour dark photo period at 23 to 25 degrees Celsius.Keep the plants in the chamber for about four weeks until they grow to a leaf stage of four to eight for subsequent agroinfiltration. Construct TurboID fusions and transform the plasmids into Agrobacterium tumefaciens competent cells as described in the text protocol. Use a one milliliter needleless syringe to infiltrate the inoculum of the abaxial epidermis of the fully mature N.benthamiana leaves.At 36 hours post-filtration, infiltrate 200 micromolar biotin into the leaves and maintain the plant for an additional three to 12 hours before harvesting the leaf tissue. To collect the leaf sample, cut the infiltrated leaves at the base of the petiole, remove the leaf vein, and flash freeze the tissue in liquid nitrogen. Grind the leaf tissue with a pestle and mortar and store the powder in 15 or 50 milliliter Falcon tubes at minus 80 degrees Celsius.To extract the total protein, transfer about 0.35 grams of leaf powder to a two milliliter tube. Be sure to maintain the tissue at low temperature in the liquid nitrogen during this process. Add 700 microliters of RIPA lysis buffer to the powder.Vortex the tube for 10 minutes and leave the sample on ice for 30 minutes, mixing the contents every four to five minutes by inverting the tubes. Centrifuge the tubes at 20, 000 times g at four degrees Celsius for 10 minutes. Then collect the supernatant.Remove the free biotin by running the sample through a desalting column. Remove the sealer at the bottom of the column and place it in a 50 milliliter tube. Then loosen the cap and centrifuge the column at 1, 000 times g and four degrees Celsius for two minutes.Place the desalting column in a fresh 50 milliliter tube and equilibrate the column three times with five milliliters of RIPA lysis buffer. Centrifuge it at 1, 000 times g and four degrees Celsius for two minutes and discard the flow-through each time. Add 1.5 milliliters of protein extract to the top of the resin in the equilibrated column.And when the extract enters the resin, add another 100 microliters of RIPA lysis buffer. Centrifuge the column for two minutes and leave the desalted samples on ice until further use. To quantify the desalted protein extracts, measure the OD595 using a Bio-Rad spectrophotometer.Draw the standard curve based on the value of the gradient BSA solution and calculate the concentration of the desalted protein samples. Equilibrate the streptavidin C1 conjugated magnetic beads with one milliliter of RIPA lysis buffer for one minute at room temperature. Use the magnetic rack to absorb the beads for three minutes and gently aspirate the solution.Then repeat the wash one more time. Transfer the protein extract to the equilibrated magnetic beads and incubate the tube at four degrees Celsius overnight on a rotator to affinity purify the proteins. On the next day, capture the beads with a magnetic rack and aspirate the supernatant.Next, wash the beads with 1.7 milliliters of wash buffer one by adding it to the tube and incubate it on the rotor for eight minutes. Remove the supernatant as previously described and repeat the wash with 1.7 milliliters of wash buffer two and wash buffer three. Add 1.7 milliliters of 50 millimolar Tris HCL to remove the detergent.Then place the tube on the magnetic rack and remove the supernatant. Repeat the wash with one milliliter of 50 millimolar Tris HCL and transfer the beads to a new 1.5 milliliter tube. Finally, wash the beads six times with 50 millimolar ammonium bicarbonate buffer for five minutes per wash.Use 100 microliters of beads for immunoblot analysis to confirm the enrichment of the biotinylated proteins and flash freeze the rest of the sample to be stored at minus 80 degrees Celsius. Western blots were used to analyze protein expression and biotinylation in the agroinfiltrated N.benthamiana leaves. The biotinylated proteins in the infiltrated leaves were efficiently enriched with streptavidin C1 conjugated magnetic beads for subsequent mass spectrometry analysis.Different proteins with varied sizes were captured and Western blot analysis of the enriched proteins showed smeared bands. When attempting this procedure, please remember to remove the sealer at the bottom of desalting column and loosen the caps before each centrifugation. This protocol is easily adaptable to other proteins of interest in Nicotiana benthamiana and can also be used to extend the TurboID PL in other plant species.

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JoVE Journal

Immunology and Infection

23.2K Views.  China Agricultural University. NLR immune receptors play a crucial role in mediating plant defense against various pathogens. We describe a TurboID-based proximity labeling method for the identification of interaction partners of Toll/interleukin-1 receptor domain containing NLRs such as immune receptors in Nicotiana benthamiana plants. This protocol provides an important reference for investigating protein-protein interactions in other plant species and it can be extended to any protein of interest in Nicotiana benthamian...