Name: airid-based proximity labeling for protein-protein interaction in plants
Uploaded: 2022-09-16T14:20:00.000+00:00
Duration: 8 min 36 s
Description: Watch this Scientific Journal Video about AirID-Based Proximity Labeling for Protein-Protein Interaction in Plants at JoVE.com

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>
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(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. 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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. Previously this PL method was only applicable to animal systems. In this study, it was used to investigate protein-protein interactions in plants. The protocol outlines how to set up the AirID-based PL in plants step by step, including preparing leaf samples, removing free biotin, quantifying extracted proteins, and enriching biotinylated proteins.
Using the well-established Agrobacterium-mediated transient expression in the Cucumber, this approach is used to find PPIs of the target protein of interest in Cucumber. The transient expression may result in overexpression of the fusion proteins. The AirID fusion must also be tested to ensure that it does not interfere with the function of the target protein of interest, which is another crucial consideration. However, while PL provides several advantages over standard IP-MS techniques for detecting transitory or weak PPIs, it also has some limitations. First and foremost, discovering a candidate interaction protein does not automatically imply a direct or indirect interaction with the bait protein but instead reflects near proximity to the bait protein9. PPIs can be further verified in vivo using independent assays (e.g., co-immunoprecipitation, bimolecular fluorescence complementation (BiFC), or in vitro GST-pull down assay, which can be carried out to verify PPIs further.
The study found that the proximal biotinylation activity of AirID was significantly lower than that of TurboID. In vitro and in cells, TurboID had the highest proximal biotinylation activity. This enzyme may be utilized within 10 min to biotinylate9,11. However, in cells treated for a prolonged incubation of over 6 h and higher biotin concentrations, highest TurboID activity led to extra biotinylation on unexpected proteins, such as tubulin and GFP (such as 50 mM biotin). In contrast to being used as a proximal biotinylation enzyme for PPIs, TurboID was first reported as a biotin-labeling enzyme4,6,5,7. If TurboID were to evaluate PPIs, it would function best under limiting conditions, such as time duration is short, about 1 h in cells. The AirID, biotinylation of tubulin and GFP were not found under the same conditions as those observed for TurboID biotinylation. AirID-fusion proteins were found to be capable of biotinylation of each well-known interactor in both transients, as demonstrated by the streptavidin-pull down assay and the LC-MS/MS analysis5. In low ATP concentrations (1 mM), the formation of biotinoyl-5-AMP was stronger for AirID than for TurboID. It favors lower concentrations of biotin (with 5 mM biotin or without biotin supplement) than TurboID, which prefers higher concentrations. In addition, an examination of biotinylation sites using LC-MS/MS revealed that AirID biotinylation occurred with no unique sequence preference on a nearby Lys residue, indicating that the biotinylation process was non-preferentia5. Although the sequence similarity between BioID and AirID is 82%, AirID showed high biotinylation activity against interacting proteins. Kido et al., 2020 found that AirID can analyze protein-protein interactions in vitro and in cells. Their findings suggest that biotinylation dependent on AirID may be useful for PPI analysis of chemical compounds. The identification of in vivo partners of target proteins is crucial for understanding biological functions12,13, and it has revealed new PPIs, so in vivo proximity biotinylation using BioID has been used in numerous studies. Even when biotin was supplemented, stable expression of AirID did not result in cell-growth inhibition, indicating that the expression of AirID-fusion proteins would be very low-toxic5. AirID is projected to improve PPI-dependent biotinylation accuracy in combination; in summary, it is concluded that AirID is ideal for PPI analysis in plants.

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><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>CaCl&lt;sub&gt;2&lt;/sub&gt;</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&amp;alpha; competent cells</td><td>Made in the current experiment</td><td></td><td>E.coli DH5&amp;alpha;</td></tr><tr><td>&amp;beta;-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>MgCl&lt;sub&gt;2&lt;/sub&gt;</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 were extracted as mentioned above in the protocol for western blot. The representative data in Figure 1 indicated the protein expression and biotinylation in the infiltrated cucumber leaves, which were the expected findings based on the newly modified technique. The results showed a higher expression level of labeled proteins and extra bands as compared to the control. This confirms that AirID successfully tagged the target proteins of the gene of interest (GOI). Further, the results were also confirmed using anti-flag and anti-mouse antibodies, which successfully showed the target band with anti-flag antibody (Figure 2). After confirmation through western blot analysis, we further performed Co-IP using the protocol mentioned above, and the infiltrating leaves&#39; biotinylated proteins were successfully enriched for mass spectrometry analysis, as shown in Figure 3. After enriching biotinylated proteins with streptavidin C1 conjugated magnetic beads, multiple proteins of different sizes were observed, and western blot analysis revealed smeared bands (Figure 3).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64428/64428fig01.jpg" /> 
Figure 1:&#160;Western blot analysis of proteins after transformation to leaves to confirm the function of AirID using streptavidin-HRP. The figure confirms that biotinylated proteins have four independent replicates for the construct transformed samples. Wild-type leaves were used as a control. For each replicate, 10 &#181;L of the samples were loaded for western blot analysis. Streptavidin-HRP (1:6000 dilution) was used to analyze biotinylated proteins in different samples. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64428/64428fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64428/64428fig02.jpg" /> 
Figure 2: Western blot analysis of proteins after transformation to leaves to detect the Taq sequence. The 3x-Flag tag sequence was fused with the gene of interest to confirm the successful transformation. The taq sequence was identified through western blot analysis in all the transformed samples. Wild-type cucumber leaf samples were used as a control. A total 10 &#181;L of the samples were loaded for western blot analysis, and the results were confirmed using anti-flag as a first antibody at dilution of 1:3000 and anti-mouse as a second antibody at dilution 1:10000. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64428/64428fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64428/64428fig03.jpg" /> 
Figure 3: Immunoblot analysis of protein extracts. After Co-immunoprecipitation (Co-IP), Western blot analysis of biotinylated proteins in the agroinfiltrated leaves of Cucumis sativus (Cucumber) with streptavidin-HRP was performed. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64428/64428fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary File 1. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64428/Supplementary File 1 (2).docx" target="_blank">Please click here to download this File.</a>

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

Nanyang Technological University

Research

JoVE Journal

Biology

1.6K Views.  Yangzhou University. AirID is een waardevol enzym voor eiwit-eiwitinteractie, en het creëert minder toxiciteit en minder fouten in tijdgebonden processen dan andere nabijheidslabelende enzymen. Er wordt een stapsgewijs protocol gepresenteerd voor het uitvoeren van het nabijheidslabeling-experiment in komkommer of Cucumis sativus met AT4G18020 APRR2-AirID-eiwit als model. Ahmad Zada, Imran Khan, Yuting Cheng en Min Zhang uit mijn laboratorium zullen de procedure demonstreren.Begin met het overbrengen van d...

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

mRNA Interactome Capture from Plant Protoplasts

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional gene regulation (PTGR) of messenger RNAs (mRNAs). In the past few years, a number of mRNA-bound proteomes from yeast and mammalian cell lines have been successfully isolated through the use of a novel method called &#34;mRNA interactome capture,&#34; which allows for the identification of mRNA-binding proteins (mRBPs) directly from a physiological environment. The method is composed of in vivo ultraviolet (UV) crosslinking, pull-down and purification of messenger ribonucleoprotein complexes (mRNPs) by oligo(dT) beads, and the subsequent identification of the crosslinked proteins by mass spectrometry (MS). Very recently, by applying the same method, several plant mRNA-bound proteomes have been reported simultaneously from different Arabidopsis tissue sources: etiolated seedlings, leaf tissue, leaf mesophyll protoplasts, and cultured root cells. Here, we present the optimized mRNA interactome capture method for Arabidopsis thaliana leaf mesophyll protoplasts, a cell type that serves as a versatile tool for experiments that include various cellular assays. The conditions for optimal protein yield include the amount of starting tissue and the duration of UV irradiation. In the mRNA-bound proteome obtained from a medium-scale experiment (107 cells), RBPs noted to have RNA-binding capacity were found to be overrepresented, and many novel RBPs were identified. The experiment can be scaled up (109 cells), and the optimized method can be applied to other plant cell types and species to broadly isolate, catalog, and compare mRNA-bound proteomes in plants.

Here, we present an interactome capture protocol applied to Arabidopsis thaliana leaf mesophyll protoplasts. This method critically relies on in vivo UV crosslinking and allows for the isolation and identification of plant mRNA-binding proteins from a physiological environment.

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional ...

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 ...

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein (YFP) into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest. These constructs can then be co-transformed into Nicotiana benthamiana via Agrobacterium mediated transformation, allowing the transit expression of fusion proteins. The reconstitution of YFP signal only occurs when the inquest proteins interact 1-7. To test and validate the protein-protein interactions, BiFC can be used together with yeast two hybrid (Y2H) assay. This may detect indirect interactions which can be overlooked in the Y2H. Gateway technology is a universal platform that enables researchers to shuttle the gene of interest (GOI) into as many expression and functional analysis systems as possible8,9. Both the orientation and reading frame can be maintained without using restriction enzymes or ligation to make expression-ready clones. As a result, one can eliminate all the re-sequencing steps to ensure consistent results throughout the experiments. We have created a series of Gateway compatible BiFC and Y2H vectors which provide researchers with easy-to-use tools to perform both BiFC and Y2H assays10. Here, we demonstrate the ease of using our BiFC system to test protein-protein interactions in N. benthamiana plants.

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System

We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent 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 ...

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.

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 ...

Immunology and Infection

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

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 ...

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

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