Name: airid-based proximity labeling for protein-protein interaction in plants
Uploaded: 2022-09-16T14:20:00
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...

<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. 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>

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 ...

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