This work was supported by the National Natural Science Foundation of China (Grant No. 32000197 to X.H.), the Special Financial Grant from the China Postdoctoral Science Foundation (Grant No. 2019T120467 to X.H.)

1. Preparation of plant material
<ol>
	<li>Cucumis sativus (Cucumber) is employed for experimental analysis. Put the seeds in water, incubate at 50 &#176;C for 20 min, and then place the seeds on filter paper on a Petri plate for 12-16 h.</li>
	<li>Afterward, transfer the seeds to pots containing soil (purchased commercially) and grow in a climatic chamber at 23 &#176;C temperature and 16 h light and 8 h dark photoperiod.</li>
	<li>Maintain the plants in the climate chamber for 3-4 weeks until the plants reach 3 or 4 leaf stages for successful agroinfiltration.</li>
</ol>
2. Making AirID construct
<ol>
	<li>Use APRR2 as a target protein and construct APRR2-AirID under the Cauliflower mosaic virus 35S promoter (p35S: APRR2-AirID). Introduce it into the Gateway-compatible binary vector pEarleyGate1006&#160;(provided by Dr. Kathrin Schrick, Kansas State University), and synthesize the PL enzyme directly according to the sequence provided in Supplementary File 1.</li>
</ol>
3. Preparation of competent cells
<ol>
	<li>Preparation of DH5&#945; competent cells
	<ol>
		<li>Inoculate the 2 mL of LB (10 g/L of tryptone, 5 g/L of yeast extract, and 10 g/L of NaCl; pH = 7.0.) with E.Coli DH5&#945; cells (directly from the frozen stock without thawing) and grow overnight (O/N) at 37 &#176;C.</li>
		<li>Freshly inoculate 0.1%-0.5% inoculum from the O/N culture into 100 mL of LB Medium and grow the cells until OD600 reaches between 0.4-0.6, and then Incubate the culture at 4 &#176;C for 30 min.</li>
		<li>Take the culture into two 50 mL centrifuge tubes and spin at 2500 x g&#160;for 5 min at 4 &#176;C.</li>
		<li>Discard the supernatant, resuspend the cells in 10 mL of 0.1 M ice cold CaCl2,&#160;and set on ice for 15 min. Pellet the cells with a 2500 x&#160;g spin for 5 min at 4 &#176;C.</li>
		<li>Resuspend the cells in 1 mL of ice-cold 0.1 M CaCl2 + 20% glycerol, aliquot 100 &#181;L into each 1.5 mL tube, and store them immediately at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Preparation of GV3101 competent cells
	<ol>
		<li>Inoculate 2 mL of LB (containing 50 &#181;g/mL gentamycin and 25 &#181;g/mL rifampicin) with a single GV3101 colony. Incubate the culture O/N at 28 &#176;C while shaking at 250 rpm.</li>
		<li>Inoculate 200 mL of LB with 1 mL of a saturated overnight culture. Shake the culture at 250 rpm while incubating it at 28 &#176;C until the OD600 is equal to 0.5.</li>
		<li>Transfer the culture to four pre-chilled sterile 50 mL centrifuge tubes and set on ice for 30 min.</li>
		<li>Pellet the cells at 2500 x g&#160;for 10 min at 4 &#176;C. Discard the supernatant and place the pellets on ice.</li>
		<li>Resuspend the cells in 10 mL of 0.1 M ice cold CaCl2 solution. Pool the cells together into one pre-chilled 50 mL Oakridge tube.</li>
		<li>Pellet the cells at 1,000 x g for 5 min at 4 &#176;C. Discard the supernatant and resuspend the cells in 10 mL of ice cold CaCl2 solution. Set on ice for 30 min.</li>
		<li>Pellet the cells at 1,000 x g&#160;for 5 min at 4 &#176;C. Discard the supernatant and resuspend the cells in 2 mL of 0.1 M ice cold CaCl2 solution.</li>
		<li>Dispense the cells into a 50 &#181;L aliquot in pre-chilled sterile polypropylene tubes. Store the cells at -80 &#176;C.</li>
	</ol>
	</li>
</ol>
4. Agroinfiltration
<ol>
	<li>First, transfer the plasmid to agrobacterium
	<ol>
		<li>Transfer 2.5 &#181;L of the plasmid generated from step 2.1 to the competent cells of the agrobacterium tumefaciens GV3101 strain and incubate on ice for 30 min.</li>
		<li>Transfer the tube containing the plasmid and the competent cells to liquid nitrogen for 3 min.</li>
		<li>Heat shock at 37 &#176;C for 5 min in an incubator, and then put the tube on ice for 2 min.</li>
		<li>Add 1,000 &#181;Lof LB medium (10 g/L of tryptone, 5 g/L of yeast extract, and 10 g/L of NaCl; pH = 7.0) to the agrobacteriumand incubate at 30 &#176;C and 118 rpm for 1 h.</li>
		<li>Centrifuge at 3,000 x g for 2 min at 4 &#176;C.</li>
		<li>Discard the upper 800 &#181;L of the solution and mix the remaining solution. Then, plate it on LB (as mentioned in step 4.1.4 added with agar and 50 &#181;g/mL Kanamycin for plasmid and 50 &#181;g/mL gentamycin and 25 &#181;g/mL rifampicin for GV3010 competent cells). Incubate the plates at 30 &#176;C for 48 h. 
		NOTE: It is better to pick some colonies and perform PCR to confirm the gene of interest7.</li>
	</ol>
	</li>
	<li>Pick up some bacterial colonies from the plates and put them in LB media plus appropriate antibiotics (see step 4.1.6). Incubate at 30 &#176;C and 218 rpm for 36-48 h.</li>
	<li>Centrifuge the cells at 3,000 x g for 2 minat 4 &#176;C. Resuspend the cells to OD600&#160;= 1.0 in Agroinfiltration buffer (10 mM MgCl2, 10 mM MES, pH = 5.6, 250 &#181;M acetosyringone).</li>
	<li>Infiltrate the inoculum (generated from step 4.3) in the (abaxial) epidermis using a 1 mL needleless syringe. 
	NOTE: It is better to infiltrate the entire leaf and choose to replicate. For 4-5 plants, use one construct. For each leaf, 1.5 mL of resuspended agrobacteria is sufficient.</li>
	<li>Maintain the plants for 36 h in the climate chamber with a 16 h light (about 75 &#181;mol&#183;m-2&#183;s-1)and 8 h dark photoperiod at 23 &#176;C.</li>
	<li>After 36 h of construct infiltration, infiltrate 1 mL of 5 &#181;M Biotin (in 10 mM MgCl2 solution) into the already infiltered leaves of the construct.</li>
	<li>Maintain the treated plants for an additional 4-12 h before harvesting the leaf tissue. 
	NOTE: According to the previous study, the target protein peaks at 36 h, so choose 36 hpi biotin infiltrations4,6. The incubation duration for biotin depends on the target study, but according to the current experiment, an 8 h incubation period is more suitable for labeling proteins.</li>
</ol>
5. Collection of samples
NOTE: All materials for sample collection should be sterile to avoid keratin contamination, and all the protocol steps should be performed in a contamination-free environment.
<ol>
	<li>Cut the infiltrated leaves and quickly transfer them to liquid nitrogen to avoid protein degradation.</li>
	<li>Perform the pre-detection of protein through immunoblot analyses6; this is highly recommended before Co-immunoprecipitation (Co-IP).</li>
</ol>
6. Total protein extraction from leaf
<ol>
	<li>Grind the leaves using a pestle and mortar, quickly add 2 mL of 1x PBS-BSA PH 7.4 and grind slowly.</li>
	<li>Take a 15 mL conical tube, place a quick filtration material filter on top of the conical tube, transfer the sample mix to the tube through the quick filtration material filter, and keep it on ice.</li>
	<li>Transfer the samples to a 2 mL tube and add 1% protein inhibitor cocktail.</li>
	<li>Mix the contents by turning upward and downward 7-8 times and centrifuge at 1,000 x g for 2 min at 4 &#176;C.</li>
	<li>Transfer the upper solvent of samples to new 2 mL tubes, add 10% &#946;-D-maltoside (&#946;-D.M), and place on ice for 5 min. Centrifuge at 20,000 x g for 10 min at 4 &#176;C.</li>
</ol>
7. Equilibrate the desalting column
<ol>
	<li>Centrifuge the column at 1,000 x g for 1 min at 4 &#176;C. 
	NOTE: Mark one side of the column and ensure that the marked side faces outward during all centrifugation processes.</li>
	<li>Remove the top and bottom cover of the column and centrifuge at 1,000 x g for 2 min at 4 &#176;C.</li>
	<li>Discard the liquid solution from the collection tube of the column and add 5 mL of 1x PBS buffer for washing.</li>
	<li>Centrifuge the column at 1,000 x g for 2 min at 4 &#176;C.</li>
	<li>Repeat the steps 7.3 and 7.4 at least five times.</li>
</ol>
8. Magnetic beads washing
<ol>
	<li>Take a 1.5 mL tube and add 50 &#181;L of streptavidin-C1-conjugated magnetic beads.</li>
	<li>Add 1 mL of 1x PBS-BSA for washing, mix thoroughly and place on the magnet stand for 3 min. Discard the supernatant.</li>
	<li>Repeat step 8.2 at least three times.</li>
	<li>After each washing, place the tube on the magnetic rack to adsorb the beads toward one side of the tube to remove the washing buffer.</li>
</ol>
9. Enrichment of biotinylated proteins
<ol>
	<li>Add 2 mL of the samples to the column and centrifuge at 1,000 x g for 8 min at 4 &#176;C.</li>
	<li>Add 1 mL of the desalted protein extract to 50 &#181;L of streptavidin-C1-conjugated magnetic beads to equilibrate them.</li>
	<li>Place the tube on the rotator at normal speed so the solution mixes thoroughly and incubates at room temperature (RT) for 30 min.</li>
	<li>Place the beads on the magnetic rack for 3 min at RT, or until they gather on one side of the tube, to gently remove the supernatant.</li>
	<li>Add 1 mL of wash buffer I (2% SDS in water), rotate at RT for 2 min on a rotator and repeat step 9.4.</li>
	<li>Place the tube on the rotator at RT for 2 min after adding 1 mL of wash buffer II (50 mM HEPES: pH = 7.5, 500 mM NaCl, 1 mM EDTA, 0.1% deoxycholic acid [w/v], and 1% Triton X-100). Repeat step 9.4.</li>
	<li>Add 1 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]), and rotate using a shaker for 2 min at RT. Then, repeat step 9.4.</li>
	<li>To remove the detergent, add 1.7 mL of 50 mM Tris-HCl (pH = 7.5) and repeat step 9.4. Repeat this step one more time.</li>
	<li>Wash the beads six times for 2 min each in 1 mL of 50 mM ammonium bicarbonate buffer at RT and repeat step 9.4.</li>
	<li>Add 50 &#181;L of the protein extract containing 50 mM Tris-HCl (pH 8.0), 12% (w/v) sucrose (Suc), 2% (w/v) lithium lauryl sulfate, and 1.5% (w/v) dithiothreitol to the beads, heat shock in the incubator at 100 &#176;C for 5 min and follow step 9.4. Store the supernatant at -80 &#176;C for LC-MS/MS analysis.</li>
</ol>

<ol>
	<li>Han, 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+identification+of+novel+protein-protein+interactions+in+liver+that+affect+glucagon+receptor+activity.">The identification of novel protein-protein interactions in liver that affect glucagon receptor activity.</a> PloS One. 10 (6), 0129226 (2015).</li><li>Zhao, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+and+identification+of+host+proteins+interacting+with+Theileria+annulata+cysteine+proteinase+(TaCP)+by+yeast-two-hybrid+system.">Screening and identification of host proteins interacting with Theileria annulata cysteine proteinase (TaCP) by yeast-two-hybrid system.</a> Parasites &amp; Vectors. 10 (1), 536 (2017).</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=A+highly+efficient+agrobacterium-mediated+method+for+transient+gene+expression+and+functional+studies+in+multiple+plant+species.">A highly efficient agrobacterium-mediated method for transient gene expression and functional studies in multiple plant species.</a> Plant Communications. 1 (5), 100028 (2020).</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+for+in+planta+identification+of+protein-protein+interaction+networks.">TurboID-based proximity labeling for in planta identification of protein-protein interaction networks.</a> Journal of Visualized Experiments. (159), e60728 (2020).</li><li>Kido, K., 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=AirID,+a+novel+proximity+biotinylation+enzyme,+for+analysis+of+protein-protein+interactions.">AirID, a novel proximity biotinylation enzyme, for analysis of protein-protein interactions.</a> eLife. 9, 54983 (2020).</li><li>Hu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpressing+7-hydroxymethyl+chlorophyll+a+reductase+alleviates+non-programmed+cell+death+during+dark-induced+senescence+in+intact+Arabidopsis+plants.">Overexpressing 7-hydroxymethyl chlorophyll a reductase alleviates non-programmed cell death during dark-induced senescence in intact Arabidopsis plants.</a> Biomolecules. 11 (8), 1143 (2021).</li><li>Ammiraju, 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+Oryza+bacterial+artificial+chromosome+library+resource:+construction+and+analysis+of+12+deep-coverage+large-insert+BAC+libraries+that+represent+the+10+genome+types+of+the+genus+Oryza.">The Oryza bacterial artificial chromosome library resource: construction and analysis of 12 deep-coverage large-insert BAC libraries that represent the 10 genome types of the genus Oryza.</a> Genome Research. 16 (1), 140-147 (2006).</li><li>Liu, 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=Albino+Leaf+2+is+involved+in+the+splicing+of+chloroplast+group+I+and+II+introns+in+rice.">Albino Leaf 2 is involved in the splicing of chloroplast group I and II introns in rice.</a> Journal of Experimental Botany. 67 (18), 5339-5347 (2016).</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>Nakano, S., Niwa, M., Asano, Y., Ito, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Following+the+evolutionary+track+of+a+highly+specific+l-Arginine+oxidase+by+reconstruction+and+biochemical+analysis+of+ancestral+and+native+enzymes.">Following the evolutionary track of a highly specific l-Arginine oxidase by reconstruction and biochemical analysis of ancestral and native enzymes.</a> Applied and Environmental Microbiology. 85 (12), 00459 (2019).</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>Odeh, H. M., Coyaud, E., Raught, B., Matunis, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+SUMO-specific+isopeptidase+SENP2+is+targeted+to+intracellular+membranes+via+a+predicted+N-terminal+amphipathic+&#945;-helix.">The SUMO-specific isopeptidase SENP2 is targeted to intracellular membranes via a predicted N-terminal amphipathic &#945;-helix.</a> Molecular Biology of the Cell. 29 (15), 1878-1890 (2018).</li><li>Motani, K., Kosako, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+stimulator+of+interferon+genes+(STING)+induces+ADAM17-mediated+shedding+of+the+immune+semaphorin+SEMA4D.">Activation of stimulator of interferon genes (STING) induces ADAM17-mediated shedding of the immune semaphorin SEMA4D.</a> Journal of Biological Chemistry. 293 (20), 7717-7726 (2018).</li></ol>

The authors declared no conflicts of interest.

In the current experiment, AirID was used for proximity labeling, which Kido et al. developed through an algorithm of ancestral enzyme reconstruction using a large genome dataset and five conventional BirA enzymes5. Random mutations were used in traditional evolutionary protein engineering to enhance activity9,10 as random mutations cannot produce dynamic sequence changes. Compared to other PL enzymes, AirID has several advantages. Previou...

Various cellular proteins work under the biologically regulatory system, and protein-protein interactions (PPIs) are a part of this system and the basis of many cellular processes. Besides PPIs, the function of natural proteins is post-translationally promoted via various modifications such as the formation of complex, ubiquitination, and phosphorylation. Therefore, studying PPIs is significant to understanding the possible function of target proteins. PPIs have been carried out using various technologies such as mass spectrometry analysis after immunoprecipitation (IP-MS analysis)1, yeast two-hybrid system (Y2H)2, also cell-free based arrays3. These methods explored various vital findings in the field of research. However, these methods have some drawbacks; for example, Y2H is a time-consuming, expensive strategy that necessitates building the target species&#39; Y2H library.
Additionally, the Y2H technique uses yeast, a heterologous single-cell eukaryotic organism, which could not accurately reflect the cellular state of higher eukaryotic cells. The IP-MS is unsuitable for high hydrophobicity proteins and shows low efficiency in capturing weak PPIs. Various essential proteins in plants such as nucleotide-binding domain and leucine-rich repeat-containing (NLR) proteins and receptor-like kinases (RLKs) are expressed at a low level and mostly interact with other proteins transiently; therefore, using these methods is insufficient for understanding the mechanisms underlying the regulation of these proteins3.
A new technique called proximity biotinylation (PB) helps researchers identify PPIs. PB depends on PL enzymes, which attach to the protein of interest (POI), and when partner protein comes near POI, the PL attaches a chemical biotin tag to the partner protein. Further, the tagged protein can be identified and can quickly know which partner protein attaches to the target protein5. Previous studies proved that BioID and TurboID are successful tools for PPIs, especially in plants, but they have certain limitations4. BioID needs a high level of biotin for labeling partner proteins, which takes more than 16 h. Compared to BioID, the TurboID is more beneficial as it labels protein in 10 min and can label the partner protein at room temperature (RT). It is also toxic to cells in certain conditions and tags those proteins that do not show interaction with the protein of interest.
To overcome these issues, AirID, developed by Kido et al., is more efficient than the rest of the labeling enzymes, although the sequence similarity is 82% between BioID and AirID5. To check the efficiency of AirID, we conducted an experiment by using a POI with known associates. This experiment confirmed that AirID could undoubtedly label associated proteins in plant cells. AirID is a valuable enzyme for analyzing PPIs in vitro and in cells. It creates less toxicity and is less erroneous in time taken processes than TurboID to tag non-partners, leading to killing the cell. It demonstrates that AirID is more competitive than other labeling enzymes for proximity biotinylation. It is more accurate, has more potential in time-taking processes, and less toxic in vitro and in living cells. The current protocol describes the identification of interacting proteins of APRR2 using AirID as a PL enzyme; furthermore, the method can be applied to other proteins to investigate PPIs in plant species.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetosyringone</td>
			<td>Beijing solaribo science and technology Co.Ltd</td>
			<td>S1519</td>
			<td></td>
		</tr>
		<tr>
			<td>Acryl/Bis 30% solution</td>
			<td>Sangon Biotech (Shanghai) Co.Ltd</td>
			<td>1510KA4528</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>BioFroxx GmbH</td>
			<td>D64683</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>tsingke (Shanghai) Co.Ltd</td>
			<td>TSJ001</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium bicarbonate</td>
			<td>Sangon Biotech (Shanghai) Co.Ltd</td>
			<td>G313BA0018</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin</td>
			<td>BBI life Sciences</td>
			<td>G908BA0012</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>BBI life Sciences</td>
			<td>E209BA0008</td>
			<td></td>
		</tr>
		<tr>
			<td>Competent cells GV3101</td>
			<td>Made in the current experiment</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Desalting column</td>
			<td>Thermo scientific</td>
			<td>WC321753</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxycholic acid</td>
			<td>Sangon Biotech (Shanghai) Co.Ltd</td>
			<td>G818BA0029</td>
			<td></td>
		</tr>
		<tr>
			<td>DH5&#945; competent cells</td>
			<td>Made in the current experiment</td>
			<td></td>
			<td>E.coli DH5&#945;</td>
		</tr>
		<tr>
			<td>&#946;-D-maltoside</td>
			<td>Beijing Scolario Science and Tech Co.Ltd</td>
			<td>S818</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sangon Biotech (Shanghai) Co.Ltd</td>
			<td>E104BA0029</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sangon Biotech (Shanghai) Co.Ltd</td>
			<td>161BA0031</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Beijing solaribo science and technology Co.Ltd</td>
			<td>H8090</td>
			<td></td>
		</tr>
		<tr>
			<td>LiCl</td>
			<td>Sangon Biotech (Shanghai) Co.Ltd</td>
			<td>H209BA0003</td>
			<td></td>
		</tr>
		<tr>
			<td>MES</td>
			<td>Beijing solaribo science and technology Co.Ltd</td>
			<td>M8019</td>
			<td></td>
		</tr>
		<tr>
			<td>MiraCloth</td>
			<td>EMD Milipore Corp/MERCK kgAa Darmstadt, Germenay</td>
			<td>3429963</td>
			<td>Quick filtration material filter</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Beijing solaribo science and technology Co.Ltd</td>
			<td>20200819</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sangon Biotech (Shanghai) Co.Ltd</td>
			<td>H324BA0003</td>
			<td></td>
		</tr>
		<tr>
			<td>NP40</td>
			<td>Sangon Biotech (Shanghai) Co.Ltd</td>
			<td>N8030</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein inhibitor cocktail</td>
			<td>Beijing Scolario Science and Tech Co.Ltd</td>
			<td>S3450</td>
			<td></td>
		</tr>
		<tr>
			<td>PVDF</td>
			<td>BIO-RAD</td>
			<td>5820172</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Beijing Scolario Science and Tech Co.Ltd</td>
			<td>S1015</td>
			<td></td>
		</tr>
		<tr>
			<td>Silwet</td>
			<td>Sangon Biotech (Shanghai) Co.Ltd</td>
			<td>S9430</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin-C1-conjugated magnetic beads</td>
			<td>Enriching Biotechnology</td>
			<td>7E511E1</td>
			<td>Magnetic beads</td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Servicebio</td>
			<td>G2056</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sangon Biotech (Shanghai) Co.Ltd</td>
			<td>GB03BA007</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Sangon Biotech (Shanghai) Co.Ltd</td>
			<td>F828BA0020</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Thermo scientific</td>
			<td>LP0042</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

According to previous research, the cucumber gene APRR2 is the candidate gene that controls white immature fruit color8. Here, a protocol was developed using AirID as a proximity labeling enzyme to find the interacting partner protein of APRR2 in cucumber. The construct was transferred to the cucumber leaves, and after 36 h post infiltration, biotin was transferred. After 48 h the samples were taken for western blot analysis to confirm the successful transformation. The proteins ...

Watch this Scientific Journal Video about AirID-Based Proximity Labeling for Protein-Protein Interaction in Plants at JoVE.com

AirID-Based Proximity Labeling for Protein-Protein Interaction 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 ...

airid-based-proximity-labeling-for-protein-protein-interaction

Research

JoVE Journal

Biology

1.5K Views.  Yangzhou University. 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.

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

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 ...

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 ...

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. ...

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 ...

Study of Protein-protein Interactions in Autophagy Research

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. The majority of cellular activities require physical interactions between proteins. To analyze and map these interactions, various experimental techniques as well as bioinformatics tools were developed. Autophagy is a cellular recycling mechanism that allows the cells to cope with different stressors, including nutrient deprivation, chemicals, and hypoxia. In order to better understand autophagy-related signaling events and to discover novel factors that regulate protein complexes in autophagy, we performed protein-protein interaction screens. Validation of these screening results requires the use of immunofluorescence and immunoprecipitation techniques. In this system, specific autophagy-related protein-protein interactions that we discovered were tested in Neuro2A (N2A) and HEK293T cell lines. Details of the technical procedures used are explained in this visualized experiment paper.

Presented here are two antibody-based protein-protein interaction research techniques: immunofluorescence and immunoprecipitation. These techniques are suitable for studying physical interactions between proteins for the discovery of novel components of cellular signaling pathways and for understanding protein dynamics.

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. ...

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

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

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

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

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