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>Maintain the plants in the chamber for about 4 weeks until they grow to a leaf stage of 4&#8211;8 for subsequent agroinfiltration15.</li>
</ol>
2. Construction of TurboID fusions
<ol>
	<li>Use standard molecular cloning technique to generate fusion of the target protein with TurboID (PCR TurboID from Addgene plasmid #107177). Here, we used the N-terminal TIR domain of the NLR immune receptor as the target protein of interest and constructed TIR fused to TurboID under the control of Cauliflower mosaic virus 35S promoter (p35S::TIR-TurboID). 
	NOTE: Fusion of the TurboID enzyme to the carboxyl-terminus or amino-terminus of the target protein will depend on the protein of interest. Usually, for a cytoplasmic protein, both termini should be acceptable, as long as the TurboID fusion does not affect the function of the target protein. However, for a membrane localized protein, the protein topology should be characterized in advance prior to determining which terminus is best for TurboID fusion.</li>
	<li>Construct a TurboID-fused citrine under the same promoter to serve as the control for subsequent quantitative proteomic analysis. 
	NOTE: It is important to construct a TurboID control for identifying the proteome proximal to the target protein. The TurboID fusion control should show an expression level similar to that of the TurboID-fused target protein. This can be empirically determined by adjusting the Agrobacterium concentration during agroinfiltration. In addition, it is important that the control protein has a subcellular localization pattern similar to that of the target protein of interest.</li>
</ol>
3. Agroinfiltration
<ol>
	<li>Transformation of the plasmids to Agrobacterium
	<ol>
		<li>Add 0.5 &#956;g of the plasmids generated from steps 2.1 and 2.2 to 50 &#956;L of Agrobacterium tumefaciens strain GV3101 competent cells, prepared as described previously16. 
		NOTE: Other Agrobacterium strains, such as GV2260, can also be used.</li>
		<li>Incubate on ice for 30 min.</li>
		<li>Heat shock in a water bath at 42 &#176;C for 60 s.</li>
		<li>Add 400 &#956;L of LB medium (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl; pH = 7.0) to the Agrobacterium and incubate at 28 &#176;C for 90 min.</li>
		<li>Plate the entire content in the tube on LB agar supplemented with appropriate antibiotics to select the plasmid, as well as for the Agrobacterium (for GV3101: 50 mg/L gentamicin and 50 mg/L rifampicin).</li>
		<li>Incubate plates at 28 &#176;C for 36&#8211;48 h until individual colonies are visible.</li>
	</ol>
	</li>
	<li>Pick and streak several individual colonies onto a fresh LB agar plate with antibiotics (see step 3.1.5) and grow at 28 &#176;C overnight. 
	NOTE: It is more optimal to perform the colony PCR to confirm the presence of the specific binary construct in the Agrobacterium. In our experience, over 95% of the colonies contain the introduced binary constructs.</li>
	<li>Inoculate 3 mL of LB medium plus appropriate antibiotics (see step 3.1.4) with Agrobacterium colony harboring the construct of interest, and incubate by shaking overnight at 28 &#176;C until the OD600 of the Agrobacterium culture reaches 2.0.</li>
	<li>Centrifuge the cells at 3,000 x g and resuspend them to OD600 = 1.0 in agroinfiltration buffer (10 mM MgCl2, 10 mM MES [pH = 5.6], 250 &#956;M acetosyringone). 
	NOTE: Although it is optimal to incubate the inoculum for 2 h at RT prior to agroinfiltration, in our experience with the GV3101 strain, there has not been a large difference between the target protein expression with vs. without incubation.</li>
	<li>Use a 1 mL needleless syringe to infiltrate the inoculum in the (abaxial) epidermis of the fully mature N. benthamiana leaves. 
	NOTE: To prepare a sufficient amount of leaf materials for three biological replicates: an entire leaf is usually infiltrated, three leaves are infiltrated per plant, and three to four plants are used for each construct. For each leaf, 1.5&#8211;2.0 mL of resuspended agrobacteria is sufficient.</li>
	<li>After 36 h post-infiltration (hpi), infiltrate 200 &#956;M biotin (in 10 mM MgCl2 solution) into the leaves pre-infiltrated with TurboID constructs.</li>
	<li>Maintain the plant for additional 3&#8211;12 h before harvesting the leaf tissue as described in section 4. 
	NOTE: The reason for choosing the 36 hpi for biotin infiltration is that the target protein expression peaks at this timepoint according to previous studies11. It is advised to determine the time required for optimal expression of the target protein of interest. The incubation time post-biotin infiltration depends on the experimental design and target protein under study. Usually, 3&#8211;12 h of biotin treatment allows the labeling of most proteins proximal to the target protein by the TurboID fusions.</li>
</ol>
4. Leaf sample collection
NOTE: For subsequent processing of the leaf samples, wear sterile gloves to avoid keratin contamination of the samples. All reagents should also be as keratin-free as possible.
<ol>
	<li>Cut the infiltrated leaves at the base of the petiole, remove the leaf vein, then flash-freeze the leaf tissue in liquid nitrogen.</li>
	<li>Grind the leaf tissue using a pestle and mortar and store the leaf powder in 15 mL or 50 mL falcon tubes at -80 &#176;C for subsequent use. 
	NOTE: Take three to four pieces of leaves for each of the three biological replicates from different plants. The protocol can be paused here. Prior to subsequent steps, it is recommended to assess protein expression and biotinylation of the target protein by immunoblot analyses. Typical western blots are shown in Figure 2.</li>
</ol>
5. Extraction of leaf total protein
<ol>
	<li>Transfer about 0.35 g of leaf powder to a 2 mL tube. Prepare two tubes for each sample. 
	CAUTION: Wear gloves when touching any object cooled by liquid nitrogen.</li>
	<li>Add 700 &#181;L of RIPA lysis buffer (50 mM Tris-HCl [pH = 7.5], 500 mM NaCl, 1 mM EDTA, 1% NP40 [v/v], 0.1% SDS [w/v], 0.5% sodium deoxycholate [w/v], 1 mM DTT, 1 tablet of protease inhibitor cocktail) to 0.35 g of leaf powder.</li>
	<li>Vortex the tubes for 10 min.</li>
	<li>Leave the samples on ice for 30 min.</li>
	<li>Mix the contents every 4&#8211;5 min by turning the tubes upside down several times.</li>
</ol>
6. Removal of free biotin by desalting
NOTE: This section takes about 50 min.
<ol>
	<li>Equilibrate the desalting column.
	<ol>
		<li>Remove the sealer at the bottom of the desalting column and put the column in a 50 mL tube.</li>
		<li>Remove the storage solution by centrifugation at 1000 x g and 4 &#176;C for 2 min and ensure that that the cap is loosened.</li>
		<li>Put the desalting column in a 50 mL tube. Equilibrate the column 3x with 5 mL of RIPA lysis buffer, each time centrifuging at 1000 x g and 4 &#176;C for 2 min and discarding the flowthrough.</li>
		<li>Transfer the desalting column into a new 50 mL tube and store temporarily at 4 &#176;C for subsequent use.</li>
	</ol>
	</li>
	<li>Spin the tubes from step 5.4 at 16,500 x g and 4 &#176;C for 10 min and transfer the supernatant from the two tubes into a new 2 mL tube.</li>
	<li>Add 1,500 &#181;L of protein extract to the top of the resin of the equilibrated desalting column (from step 6.1.4). When the protein extract enters the resin, add another 100 &#181;L of RIPA lysis buffer. 
	NOTE: Although 1,400 &#956;L of RIPA lysis buffer was used for protein extraction from the leaves, the total volume after protein extraction and desalting was invariably increased to some extent relative to the original volume. Therefore, a combination of the samples from each group as described in steps 5.1 and 5.4 can result in at least 1,500 &#181;L of protein extract per sample.</li>
	<li>Centrifuge at 1000 x g and 4 &#176;C for 2 min and leave the desalted samples on ice temporarily.</li>
</ol>
7. Quantification of the desalted protein extracts using a Bradford assay
<ol>
	<li>Prepare 50 &#181;L of each gradient BSA solution: 0 mg/mL, 0.2 mg/mL, 0.4 mg/mL, 0.6 mg/mL, 0.8 mg/mL, and 1 mg/mL.</li>
	<li>Dilute the desalted protein extract by mixing a 5 &#181;L sample with 45 &#181;L of ddH2O.</li>
	<li>Prepare 1x Bradford regent by diluting the 5x Bradford regent (100 mg of Coomassie brilliant blue G250, 47 mL of methanol, 100 mL of 85% phosphoric acid, 53 mL of ddH2O).</li>
	<li>Add 50 &#181;L of the each gradient BSA solution and 50 &#181;L of diluted protein extract to the 2.5 mL of 1x Bradford regent.</li>
	<li>Incubate at RT for 10 min.</li>
	<li>Add 200 &#181;L of solution of each sample to one well of an ELISA plate (three technical replicates per sample).</li>
	<li>Measure the OD595 using a microplate reader.</li>
	<li>Draw the standard curve based on the value of the gradient BSA solution and calculate the concentration of the desalted protein samples. Usually, the total protein concentration obtained from 0.7 g of leaves ranges from 3&#8211;6 mg/mL.</li>
	<li>Prepare 6&#8211;8 mg of desalted protein extract for subsequent affinity purification.</li>
</ol>
8. Enrichment of biotinylated proteins
<ol>
	<li>Take 200 &#181;L of streptavidn-C1-conjugated magnetic beads into a 2 mL tube.</li>
	<li>Equilibrate the streptavidn-C1-conjugated magnetic beads with 1 mL of RIPA lysis buffer for 1 min at RT.</li>
	<li>After each washing, use the magnetic rack to adsorb the beads for 3 min and gently remove the solution by pipetting.</li>
	<li>Repeat steps 8.2 and 8.3.</li>
	<li>Transfer the desalted protein extract to the equilibrated streptavidn-C1-conjugated magnetic beads.</li>
	<li>Incubate the tube at 4 &#176;C for 12 h (or overnight) on a rotator to affinity-purify the biotinylated proteins.</li>
	<li>Capture the beads on a magnetic rack for 4 min at RT until the beads collect at one side of the tube, then gently remove the supernatant by pipette.</li>
	<li>Add 1.7 mL of wash buffer I (2% SDS in water) to the tube and keep it on the rotator at RT for 8 min. Repeat step 8.3.</li>
	<li>Add 1.7 mL of wash buffer II (50 mM HEPES: pH = 7.5, 500 mM NaCl, 1 mM EDTA, 0.1% deoxycholic acid [w/v], 1% Triton X-100) to the tube and keep it on the rotator at RT for 8 min. Repeat step 8.3.</li>
	<li>Add 1.7 mL of wash buffer III (10 mM Tris-HCl: pH = 7.4, 250 mM LiCl, 1 mM EDTA, 0.1% deoxycholic acid [w/v], 1% NP40 [v/v]) to the tube and keep it on the rotator at RT for 8 min. Repeat the step 8.3.</li>
	<li>Add 1.7 mL of 50 mM Tris-HCl (pH = 7.5) to remove the detergent, then repeat step 8.3.</li>
	<li>Transfer the beads to a new 1.5 mL tube, and repeat steps 8.11 and 8.3.</li>
	<li>Wash the beads 6x for 5 min each with 50 mM ammonium bicarbonate buffer at RT.</li>
	<li>Add 1 mL of 50 mM ammonium bicarbonate buffer to the magnetic beads and mix well.</li>
	<li>Remove 100 &#181;L of beads for immunoblot analysis to confirm the enrichment of biotinylated proteins. A typical western blot is shown in Figure 3.</li>
	<li>Flash-freeze the rest of the protein samples and stored at -80 &#176;C or send them immediately for LC-MS/MS analysis on dry ice. 
	NOTE: Typical MS results are displayed in a previous publication (Zhang et al. 2019; Figure 2, Supplementary Data 1, and Supplementary Data 2)11. The whole datasets are available on MassIVE (found at &#60;http://massive.ucsd.edu&#62;) using the identifier: MSV000083018 and MSV000083019.</li>
</ol>

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<li>Khan, M., Youn, J. Y., Gingras, A. C., Subramaniam, R., Desveaux, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+planta+proximity+dependent+biotin+identification+%28BioID%29%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">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/pubmed?term=((Development+of+a+rapid+in+planta+BioID+system+as+a+probe+for+plasma+membrane-associated+immunity+proteins%5BTitle%5D)+AND+%22Frontiers+in+Plant+Science%22%5BJournal%5D)">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>
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<li>Branon, T. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+proximity+labeling+in+living+cells+and+organisms+with+TurboID%5BTitle%5D)+AND+%22Nature+Biotechnology%22%5BJournal%5D)">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/pubmed?term=((TurboID-based+proximity+labeling+reveals+that+UBR7+is+a+regulator+of+N+NLR+immune+receptor-mediated+immunity%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">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/pubmed?term=((Establishment+of+proximity-dependent+biotinylation+approaches+in+different+plant+model+systems%5BTitle%5D)+AND+%22bioRxiv%22%5BJournal%5D)">Establishment of proximity-dependent biotinylation approaches in different plant model systems.</a> bioRxiv. , (2019).</li>
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</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 OD&lt;sub&gt;600&lt;/sub&gt; 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 (MgCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;6H&lt;sub&gt;2&lt;/sub&gt;O)</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 OD&lt;sub&gt;595&lt;/sub&gt; 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 &lt;em&gt;Agrobacterium&lt;/em&gt; 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 ...

turboid-based-proximity-labeling-for-planta-identification-protein

Research

JoVE Journal

Immunology and Infection

24.0K Views.  China Agricultural University. NLR immuunreceptoren spelen een cruciale rol bij het bemiddelen van plantenverdediging tegen verschillende ziekteverwekkers. We beschrijven een TurboID-gebaseerde nabijheidslabelmethode voor de identificatie van interactiepartners van Toll/interleukine-1 receptordomein met NLR's zoals immuunreceptoren in Nicotiana benthamiana planten. Dit protocol biedt een belangrijke referentie voor het onderzoeken van eiwit-eiwit interacties in andere plantensoorten en het kan worden uitgebreid tot elk eiw...

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

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation is a popular technique to detect protein-protein interactions. Subsequent Western blot analysis is the most common method to visualize co-immunoprecipitated proteins. Recently, the proximity ligation assay has become a powerful tool to visualize protein-protein interactions in situ and provides the possibility to quantify protein-protein interactions by this method. Similar to conventional immunocytochemistry, the proximity ligation assay technique is also based on the accessibility of primary antibodies to the antigens, but in contrast, proximity ligation assay detects protein-protein interactions with a unique technique involving rolling-circle PCR, while conventional immunocytochemistry only shows co-localization of proteins.
Nuclear factor 90 (NF90) and RNA-binding motif protein 3 (RBM3) have been previously demonstrated as interacting partners. They are predominantly localized in the nucleus, but also migrate into the cytoplasm and regulate signaling pathways in the cytoplasmic compartment. Here, we compared NF90-RBM3 interaction in both the nucleus and the cytoplasm by co-immunoprecipitation and proximity ligation assay. In addition, we discussed the advantages and limitations of these two techniques in visualizing protein-protein interactions in respect to spatial distribution and the properties of protein-protein interactions.

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Protein-protein interactions can occur in both the nucleus and the cytoplasm of a cell. To investigate these interactions, traditional co-immunoprecipitation and modern proximity ligation assay are applied. In this study, we compare these two methods to visualize the distribution of NF90-RBM3 interactions in the nucleus and the cytoplasm.

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation ...

Biochemistry

Split-BioID &#8212; Proteomic Analysis of Context-specific Protein Complexes in Their Native Cellular Environment

To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced that allow the proximity-dependent labeling of proteins in living cells. One such enzyme, BirA* (used in the BioID approach), mediates the biotinylation of proteins within a range of approximately 10 nm. Hence, when fused to a protein of interest and expressed in cells, it allows the labeling of proximal proteins in their native environment. As opposed to AP that relies on the purification of assembled protein complexes, BioID detects proteins that have been marked within cells no matter whether they are still interacting with the protein of interest when they are isolated. Since it biotinylates proximal proteins, one can moreover capitalize on the exceptional affinity of streptavidin for biotin to very efficiently isolate them. While BioID performs better than AP for identifying transient or weak interactions, both AP- and BioID-mass spectrometry approaches provide an overview of all possible interactions a given protein may have. However, they do not provide information on the context of each identified PPI. Indeed, most proteins are typically part of several complexes, corresponding to distinct maturation steps or different functional units. To address this common limitation of both methods, we have engineered a protein-fragments complementation assay based on the BirA* enzyme. In this assay, two inactive fragments of BirA* can reassemble into an active enzyme when brought in close proximity by two interacting proteins to which they are fused. The resulting split-BioID assay thus allows the labeling of proteins that assemble around a pair of interacting proteins. Provided these two only interact in a given context, split-BioID then allows the analysis of specific context-dependent functional units in their native cellular environment. Here, we provide a step-by-step protocol to test and apply split-BioID to a pair of interacting proteins.

We provide a step-by-step protocol for split-BioID, a protein fragments-complementation assay based on the proximity-labeling technique BioID. Activated on the interaction of two given proteins, it allows the proteomics analysis of context-dependent protein complexes in their native cellular environment. The method is simple, cost-effective and only requires standard laboratory equipment.

To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced ...

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in understanding how such pathways are organized and how they function. There are many methods to detect protein-protein interactions in cells, but relatively few can be used to detect interactions between endogenous proteins. One such method, the proximity ligation assay (PLA), has several advantages to recommend its use. Compared to other common methods of protein-protein interaction analysis, PLA has relatively high sensitivity and specificity, can be performed with minimal cell manipulation, and, in the protocol described herein, requires only two target-specific antibodies derived from different species (e.g., from mouse and rabbit) and one specialized reagent: a set of secondary antibodies that are covalently linked to specific oligonucleotides that, when brought in close proximity of one another, create an amplifiable platform for in situ PCR or rolling circle amplification. In this presentation, we show how to apply the PLA technique to visualize changes in MST1 and MST2 proximity in fixed cells. The technique described in this manuscript is particularly applicable for the analysis of cell signaling studies.

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Here, we show how to use a Proximity Ligation Assay (PLA) to visualize MST1/MST2 heterodimerization in fixed cells with high sensitivity.

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in ...

Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This publication presents a complete interaction proteomics workflow designed for identifying low abundance protein-protein interactions from the nucleus that could also be applied to other subcellular compartments. This workflow includes subcellular fractionation, immunoprecipitation, sample preparation, offline cleanup, single-shot label-free mass spectrometry, and downstream computational analysis and data visualization. Our protocol is optimized for detecting compartmentalized, low abundance interactions that are difficult to identify from whole cell lysates (e.g., transcription factor interactions in the nucleus) by immunoprecipitation of endogenous proteins from fractionated subcellular compartments. The sample preparation pipeline outlined here provides detailed instructions for the preparation of HeLa cell nuclear extract, immunoaffinity purification of endogenous bait protein, and quantitative mass spectrometry analysis. We also discuss methodological considerations for performing large-scale immunoprecipitation in mass spectrometry-based interaction profiling experiments and provide guidelines for evaluating data quality to distinguish true positive protein interactions from nonspecific interactions. This approach is demonstrated here by investigating the nuclear interactome of the CMGC kinase, DYRK1A, a low abundance protein kinase with poorly defined interactions within the nucleus.

Described is a proteomics workflow for identifying protein interaction partners from a nuclear subcellular fraction using immunoaffinity enrichment of a given protein of interest and label-free mass spectrometry. The workflow includes subcellular fractionation, immunoprecipitation, filter aided sample preparation, offline cleanup, mass spectrometry, and a downstream bioinformatics pipeline.

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This ...

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

Biology

Characterization of Neuronal Lysosome Interactome with Proximity Labeling Proteomics

Lysosomes frequently communicate with a variety of biomolecules to achieve the degradation and other diverse cellular functions. Lysosomes are critical to human brain function, as neurons are postmitotic and rely heavily on the autophagy-lysosome pathway to maintain cellular homeostasis. Despite advancements in the understanding of various lysosomal functions, capturing the highly dynamic communications between lysosomes and other cellular components is technically challenging, particularly in a high-throughput fashion. Here, a detailed protocol is provided for the recently published endogenous (knock-in) lysosome proximity labeling proteomic method in human induced pluripotent stem cell (hiPSC)-derived neurons. 
Both lysosomal membrane proteins and proteins surrounding lysosomes within a 10-20 nm radius can be confidently identified and accurately quantified in live human neurons. Each step of the protocol is described in detail, i.e., hiPSC-neuron culture, proximity labeling, neuron harvest, fluorescence microscopy, biotinylated protein enrichment, protein digestion, LC-MS analysis, and data analysis. In summary, this unique endogenous lysosomal proximity labeling proteomics method provides a high-throughput and robust analytical tool to study the highly dynamic lysosomal activities in live human neurons.

A neuronal lysosome proximity labeling proteomics protocol is described here to characterize the dynamic lysosomal microenvironment in human induced pluripotent stem cell-derived neurons. Lysosomal membrane proteins and proteins that interact with lysosomes (stably or transiently) can be accurately quantified in this method with excellent intracellular spatial resolution in live human neurons.

Lysosomes frequently communicate with a variety of biomolecules to achieve the degradation and other diverse cellular functions. Lysosomes are critical to ...

Neuroscience

Inducible LAP-tagged Stable Cell Lines for Investigating Protein Function, Spatiotemporal Localization and Protein Interaction Networks

Multi-protein complexes, rather than single proteins acting in isolation, often govern molecular pathways regulating cellular homeostasis. Based on this principle, the purification of critical proteins required for the functioning of these pathways along with their native interacting partners has not only allowed the mapping of the protein constituents of these pathways, but has also provided a deeper understanding of how these proteins coordinate to regulate these pathways. Within this context, understanding a protein's spatiotemporal localization and its protein-protein interaction network can aid in defining its role within a pathway, as well as how its misregulation may lead to disease pathogenesis. To address this need, several approaches for protein purification such as tandem affinity purification (TAP) and localization and affinity purification (LAP) have been designed and used successfully. Nevertheless, in order to apply these approaches to pathway-scale proteomic analyses, these strategies must be supplemented with modern technological developments in cloning and mammalian stable cell line generation. Here, we describe a method for generating LAP-tagged human inducible stable cell lines for investigating protein subcellular localization and protein-protein interaction networks. This approach has been successfully applied to the dissection of multiple cellular pathways including cell division and is compatible with high-throughput proteomic analyses.

We describe a method for generating localization and affinity purification (LAP)-tagged inducible stable cell lines for investigating protein function, spatiotemporal subcellular localization and protein-protein interaction networks.

Multi-protein complexes, rather than single proteins acting in isolation, often govern molecular pathways regulating cellular homeostasis. Based on this ...

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the JDP family could form transient heterocomplexes in eukaryotes to fine-tune substrate selection for the 70 kDa heat shock protein (Hsp70) chaperone-based protein disaggregases. The JDP complexes target acute/chronic stress induced aggregated proteins and presumably help assemble the disaggregases by recruiting multiple Hsp70s to the surface of protein aggregates. The extent of the protein quality control (PQC) network formed by these physically interacting JDPs remains largely uncharacterized in vivo. Here, we describe a microscopy-based in situ protein interaction assay named the proximity ligation assay (PLA), which is able to robustly capture&#160;these transiently formed chaperone complexes in distinct cellular compartments of eukaryotic cells. Our work expands the employment of PLA from human cells to yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli), thus rendering an important tool to monitor the dynamics of transiently formed protein assemblies in both prokaryotic and eukaryotic cells.

Cognate J-domain proteins cooperate with the Hsp70 chaperone to assist in a myriad of biological processes ranging from protein folding to degradation. Here, we describe an in situ proximity ligation assay, which allows the monitoring of these transiently formed chaperone machineries in bacterial, yeast and human cells.

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the ...

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 Networks

An organism can have thousands of different proteins, and these proteins must cooperate to ensure the health of an organism. Proteins bind to other proteins and form complexes to carry out their functions. Many proteins interact with multiple other proteins creating a complex network of protein interactions.
These interactions can be represented through maps depicting protein-protein interaction networks, represented as nodes and edges. Nodes are circles that are representative of a protein, and edges are lines that connect two interacting proteins. These networks provide a way to visualize the complexity of the protein-protein interactions in a system. These maps can include both stable interactions, like the ones formed in protein complexes, as well as transient interactions. The protein interactions occurring in a cell, an organism, or a specific biological context can be collectively called an &#8216;interactome&#8217;.
Protein networks can be studied using various biochemical and computational methods. One of the first steps in studying protein interactions is to isolate a protein of interest along with the other associated proteins. This can be carried out by tagging the protein of interest with an affinity tag, such as a histidine tag. This tag can then be used to separate the protein along with the other proteins using affinity chromatography. Isolated proteins are then digested with a protease, such as trypsin,&#160; and then analyzed using liquid chromatography-mass spectrometry (LC-MS). The peptide mass can then be compared to a database with known protein sequences to determined its identity.
Computationally, protein-protein interactions can be analyzed using databases as well as prediction tools. There are various databases, such as IntAct managed by EMBL-EBI, that consist of experimentally validated and predicted protein interactions. Other tools like STRING by the Swiss Institute of Bioinformatics can be used to predict these interaction networks.
The study of protein networks can lead to scientific discoveries, such as determining the function of an unknown protein. Examining the changes in these networks can help elucidate the differences between healthy and diseased cells. This information can also be used for crucial applications such as designing drugs for the treatment of diseases.&#160; Analysis of protein networks can identify highly connected nodes that may be crucial for cellular survival, which can be targeted in cancer and diseases where cell death is desired but would be unsuitable for most diseases. On the other hand, less connected nodes that only interact with a few specific pathways may be targeted if a specific cell function is affected, and designing drugs that interact with these less connected nodes may lead to fewer side effects.

Protein Networks and PredictionTools: IntAct and STRING

An organism can have thousands of different proteins, and these proteins must cooperate to ensure the health of an organism. Proteins bind to other ...

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

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