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

NOTE: An overview of the method is shown in Figure 1.
1. Plant material preparation
<ol>
	<li>Grow N. benthamiana seeds in wet soil at a high density and maintain them in a climate chamber with a 16 h light (about 75 &#956;mol/m2s) and 8 h dark photoperiod at 23&#8211;25 &#176;C.</li>
	<li>About 1 week later, carefully transfer each young seedling to 4&#39; x 4&#39; pots and keep the seedlings in the same chamber.</li>
	<li>Maintain the plants in the chamber for about 4 weeks until they grow to a leaf stage of 4&#8211;8 for subsequent agroinfiltration15.</li>
</ol>
2. Construction of TurboID fusions
<ol>
	<li>Use standard molecular cloning technique to generate fusion of the target protein with TurboID (PCR TurboID from Addgene plasmid #107177). Here, we used the N-terminal TIR domain of the NLR immune receptor as the target protein of interest and constructed TIR fused to TurboID under the control of Cauliflower mosaic virus 35S promoter (p35S::TIR-TurboID). 
	NOTE: Fusion of the TurboID enzyme to the carboxyl-terminus or amino-terminus of the target protein will depend on the protein of interest. Usually, for a cytoplasmic protein, both termini should be acceptable, as long as the TurboID fusion does not affect the function of the target protein. However, for a membrane localized protein, the protein topology should be characterized in advance prior to determining which terminus is best for TurboID fusion.</li>
	<li>Construct a TurboID-fused citrine under the same promoter to serve as the control for subsequent quantitative proteomic analysis. 
	NOTE: It is important to construct a TurboID control for identifying the proteome proximal to the target protein. The TurboID fusion control should show an expression level similar to that of the TurboID-fused target protein. This can be empirically determined by adjusting the Agrobacterium concentration during agroinfiltration. In addition, it is important that the control protein has a subcellular localization pattern similar to that of the target protein of interest.</li>
</ol>
3. Agroinfiltration
<ol>
	<li>Transformation of the plasmids to Agrobacterium
	<ol>
		<li>Add 0.5 &#956;g of the plasmids generated from steps 2.1 and 2.2 to 50 &#956;L of Agrobacterium tumefaciens strain GV3101 competent cells, prepared as described previously16. 
		NOTE: Other Agrobacterium strains, such as GV2260, can also be used.</li>
		<li>Incubate on ice for 30 min.</li>
		<li>Heat shock in a water bath at 42 &#176;C for 60 s.</li>
		<li>Add 400 &#956;L of LB medium (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl; pH = 7.0) to the Agrobacterium and incubate at 28 &#176;C for 90 min.</li>
		<li>Plate the entire content in the tube on LB agar supplemented with appropriate antibiotics to select the plasmid, as well as for the Agrobacterium (for GV3101: 50 mg/L gentamicin and 50 mg/L rifampicin).</li>
		<li>Incubate plates at 28 &#176;C for 36&#8211;48 h until individual colonies are visible.</li>
	</ol>
	</li>
	<li>Pick and streak several individual colonies onto a fresh LB agar plate with antibiotics (see step 3.1.5) and grow at 28 &#176;C overnight. 
	NOTE: It is more optimal to perform the colony PCR to confirm the presence of the specific binary construct in the Agrobacterium. In our experience, over 95% of the colonies contain the introduced binary constructs.</li>
	<li>Inoculate 3 mL of LB medium plus appropriate antibiotics (see step 3.1.4) with Agrobacterium colony harboring the construct of interest, and incubate by shaking overnight at 28 &#176;C until the OD600 of the Agrobacterium culture reaches 2.0.</li>
	<li>Centrifuge the cells at 3,000 x g and resuspend them to OD600 = 1.0 in agroinfiltration buffer (10 mM MgCl2, 10 mM MES [pH = 5.6], 250 &#956;M acetosyringone). 
	NOTE: Although it is optimal to incubate the inoculum for 2 h at RT prior to agroinfiltration, in our experience with the GV3101 strain, there has not been a large difference between the target protein expression with vs. without incubation.</li>
	<li>Use a 1 mL needleless syringe to infiltrate the inoculum in the (abaxial) epidermis of the fully mature N. benthamiana leaves. 
	NOTE: To prepare a sufficient amount of leaf materials for three biological replicates: an entire leaf is usually infiltrated, three leaves are infiltrated per plant, and three to four plants are used for each construct. For each leaf, 1.5&#8211;2.0 mL of resuspended agrobacteria is sufficient.</li>
	<li>After 36 h post-infiltration (hpi), infiltrate 200 &#956;M biotin (in 10 mM MgCl2 solution) into the leaves pre-infiltrated with TurboID constructs.</li>
	<li>Maintain the plant for additional 3&#8211;12 h before harvesting the leaf tissue as described in section 4. 
	NOTE: The reason for choosing the 36 hpi for biotin infiltration is that the target protein expression peaks at this timepoint according to previous studies11. It is advised to determine the time required for optimal expression of the target protein of interest. The incubation time post-biotin infiltration depends on the experimental design and target protein under study. Usually, 3&#8211;12 h of biotin treatment allows the labeling of most proteins proximal to the target protein by the TurboID fusions.</li>
</ol>
4. Leaf sample collection
NOTE: For subsequent processing of the leaf samples, wear sterile gloves to avoid keratin contamination of the samples. All reagents should also be as keratin-free as possible.
<ol>
	<li>Cut the infiltrated leaves at the base of the petiole, remove the leaf vein, then flash-freeze the leaf tissue in liquid nitrogen.</li>
	<li>Grind the leaf tissue using a pestle and mortar and store the leaf powder in 15 mL or 50 mL falcon tubes at -80 &#176;C for subsequent use. 
	NOTE: Take three to four pieces of leaves for each of the three biological replicates from different plants. The protocol can be paused here. Prior to subsequent steps, it is recommended to assess protein expression and biotinylation of the target protein by immunoblot analyses. Typical western blots are shown in Figure 2.</li>
</ol>
5. Extraction of leaf total protein
<ol>
	<li>Transfer about 0.35 g of leaf powder to a 2 mL tube. Prepare two tubes for each sample. 
	CAUTION: Wear gloves when touching any object cooled by liquid nitrogen.</li>
	<li>Add 700 &#181;L of RIPA lysis buffer (50 mM Tris-HCl [pH = 7.5], 500 mM NaCl, 1 mM EDTA, 1% NP40 [v/v], 0.1% SDS [w/v], 0.5% sodium deoxycholate [w/v], 1 mM DTT, 1 tablet of protease inhibitor cocktail) to 0.35 g of leaf powder.</li>
	<li>Vortex the tubes for 10 min.</li>
	<li>Leave the samples on ice for 30 min.</li>
	<li>Mix the contents every 4&#8211;5 min by turning the tubes upside down several times.</li>
</ol>
6. Removal of free biotin by desalting
NOTE: This section takes about 50 min.
<ol>
	<li>Equilibrate the desalting column.
	<ol>
		<li>Remove the sealer at the bottom of the desalting column and put the column in a 50 mL tube.</li>
		<li>Remove the storage solution by centrifugation at 1000 x g and 4 &#176;C for 2 min and ensure that that the cap is loosened.</li>
		<li>Put the desalting column in a 50 mL tube. Equilibrate the column 3x with 5 mL of RIPA lysis buffer, each time centrifuging at 1000 x g and 4 &#176;C for 2 min and discarding the flowthrough.</li>
		<li>Transfer the desalting column into a new 50 mL tube and store temporarily at 4 &#176;C for subsequent use.</li>
	</ol>
	</li>
	<li>Spin the tubes from step 5.4 at 16,500 x g and 4 &#176;C for 10 min and transfer the supernatant from the two tubes into a new 2 mL tube.</li>
	<li>Add 1,500 &#181;L of protein extract to the top of the resin of the equilibrated desalting column (from step 6.1.4). When the protein extract enters the resin, add another 100 &#181;L of RIPA lysis buffer. 
	NOTE: Although 1,400 &#956;L of RIPA lysis buffer was used for protein extraction from the leaves, the total volume after protein extraction and desalting was invariably increased to some extent relative to the original volume. Therefore, a combination of the samples from each group as described in steps 5.1 and 5.4 can result in at least 1,500 &#181;L of protein extract per sample.</li>
	<li>Centrifuge at 1000 x g and 4 &#176;C for 2 min and leave the desalted samples on ice temporarily.</li>
</ol>
7. Quantification of the desalted protein extracts using a Bradford assay
<ol>
	<li>Prepare 50 &#181;L of each gradient BSA solution: 0 mg/mL, 0.2 mg/mL, 0.4 mg/mL, 0.6 mg/mL, 0.8 mg/mL, and 1 mg/mL.</li>
	<li>Dilute the desalted protein extract by mixing a 5 &#181;L sample with 45 &#181;L of ddH2O.</li>
	<li>Prepare 1x Bradford regent by diluting the 5x Bradford regent (100 mg of Coomassie brilliant blue G250, 47 mL of methanol, 100 mL of 85% phosphoric acid, 53 mL of ddH2O).</li>
	<li>Add 50 &#181;L of the each gradient BSA solution and 50 &#181;L of diluted protein extract to the 2.5 mL of 1x Bradford regent.</li>
	<li>Incubate at RT for 10 min.</li>
	<li>Add 200 &#181;L of solution of each sample to one well of an ELISA plate (three technical replicates per sample).</li>
	<li>Measure the OD595 using a microplate reader.</li>
	<li>Draw the standard curve based on the value of the gradient BSA solution and calculate the concentration of the desalted protein samples. Usually, the total protein concentration obtained from 0.7 g of leaves ranges from 3&#8211;6 mg/mL.</li>
	<li>Prepare 6&#8211;8 mg of desalted protein extract for subsequent affinity purification.</li>
</ol>
8. Enrichment of biotinylated proteins
<ol>
	<li>Take 200 &#181;L of streptavidn-C1-conjugated magnetic beads into a 2 mL tube.</li>
	<li>Equilibrate the streptavidn-C1-conjugated magnetic beads with 1 mL of RIPA lysis buffer for 1 min at RT.</li>
	<li>After each washing, use the magnetic rack to adsorb the beads for 3 min and gently remove the solution by pipetting.</li>
	<li>Repeat steps 8.2 and 8.3.</li>
	<li>Transfer the desalted protein extract to the equilibrated streptavidn-C1-conjugated magnetic beads.</li>
	<li>Incubate the tube at 4 &#176;C for 12 h (or overnight) on a rotator to affinity-purify the biotinylated proteins.</li>
	<li>Capture the beads on a magnetic rack for 4 min at RT until the beads collect at one side of the tube, then gently remove the supernatant by pipette.</li>
	<li>Add 1.7 mL of wash buffer I (2% SDS in water) to the tube and keep it on the rotator at RT for 8 min. Repeat step 8.3.</li>
	<li>Add 1.7 mL of wash buffer II (50 mM HEPES: pH = 7.5, 500 mM NaCl, 1 mM EDTA, 0.1% deoxycholic acid [w/v], 1% Triton X-100) to the tube and keep it on the rotator at RT for 8 min. Repeat step 8.3.</li>
	<li>Add 1.7 mL of wash buffer III (10 mM Tris-HCl: pH = 7.4, 250 mM LiCl, 1 mM EDTA, 0.1% deoxycholic acid [w/v], 1% NP40 [v/v]) to the tube and keep it on the rotator at RT for 8 min. Repeat the step 8.3.</li>
	<li>Add 1.7 mL of 50 mM Tris-HCl (pH = 7.5) to remove the detergent, then repeat step 8.3.</li>
	<li>Transfer the beads to a new 1.5 mL tube, and repeat steps 8.11 and 8.3.</li>
	<li>Wash the beads 6x for 5 min each with 50 mM ammonium bicarbonate buffer at RT.</li>
	<li>Add 1 mL of 50 mM ammonium bicarbonate buffer to the magnetic beads and mix well.</li>
	<li>Remove 100 &#181;L of beads for immunoblot analysis to confirm the enrichment of biotinylated proteins. A typical western blot is shown in Figure 3.</li>
	<li>Flash-freeze the rest of the protein samples and stored at -80 &#176;C or send them immediately for LC-MS/MS analysis on dry ice. 
	NOTE: Typical MS results are displayed in a previous publication (Zhang et al. 2019; Figure 2, Supplementary Data 1, and Supplementary Data 2)11. The whole datasets are available on MassIVE (found at &#60;http://massive.ucsd.edu&#62;) using the identifier: MSV000083018 and MSV000083019.</li>
</ol>

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

The authors have nothing to disclose.

The TurboID biotin ligase is generated by yeast display-based directed evolution of the BioID10. It has many advantages over other PL enzymes. TurboID allows the application of PL to other model systems, including flies and worms, whose optimal growth temperature is around 25 &#176;C10. Although the PL approach has been widely used in animal systems, its application in plants is limited. The protocol described here provides a step-by-step procedure for establishing the TurboID-based PL in N. benthamiana, a model plant that has been widely used in plant-pathogen interaction studies. This protocol outlines leaf sample preparation, removal of free biotin, quantification of the extracted proteins, and enrichment of the biotinylated proteins.
Although free biotin in animal cell culture systems can be largely removed by washing the cells with PBS buffer4, the free biotin in leaf tissue cannot be cleared by simple washing. A recent study indicated that the free biotin can severely impact subsequent enrichment of the biotinylated proteins11. In this protocol, a desalting column is utilized to successfully remove free biotin in the protein extracts, allowing the efficient binding of biotinylated proteins to the streptavidin beads.
Moreover, this protocol serves as an important reference for conducting PL in other plant systems. Recently, three reports have used TurboID-based PL in Arabidopsis12,13,14 and tomato hairy root12, besides the N. benthamiana described here. Similar to animal cell culture systems, the removal of the free biotin was achieved by washing the plant protoplasts prior to protein extraction6. However, lower amounts of free biotin molecules that were not integrated to the protein existed in the interior of the cell, which impacted the efficiency of subsequent enrichment of the biotinylated proteins. Therefore, a desalting procedure is recommended for complete removal of free biotin, thereby increasing the recovery efficiency of biotinylated proteins.
Two types of columns, PD-10 and Zeba, have been used for free biotin removal11,12,13,14. It may be of interest in the future to compare the efficiency of these two columns in removing free biotin in leaf protein extracts. Moreover, this protocol employs a canonical syringe-mediated agroinfiltration method to transiently express the target protein of interest in N. benthamiana leaves. Then, the biotin substrate is reinfiltrated into the leaves for labeling proteins that are proximal to the target protein. Similar operations have also been utilized in other two recently reported studies12,13. As an alternative method, vacuum-mediated infiltration of biotin is applicable for both N. benthamiana and Arabidopsis14. In addition, in plants that are not suitable for agroinfiltration, cell cultures can be transformed for target protein expression, followed by biotin treatment and proximity labeling assay12. These recent studies have underpinned the robustness of TurboID-based PL in studying PPIs and have laid the foundation for future applications of TurboID-based PL in different plant species.
In this protocol, well-established Agrobacterium-mediated transient expression in N. benthamiana is employed to identify PPIs of the target protein of interest. Transient expression may lead to overexpression of the fusion proteins. Therefore, a more optimal alternative is to engineer the TurboID fusion under the control of a native promoter and express it by agroinfiltration or generation of stable transgenic lines. With the development of genome editing technology, it is also possible to knock-in the TurboID fragment directly into the native genomic loci of genes of interest.
Another important factor to be considered is to ensure that the TurboID fusion does not alter the function of the target protein of interest. In a previous study, the function of the NLR immune receptor fused to TurboID was confirmed by testing its ability to induce the defense-mediated cell death in the presence of Tobacco mosaic virus p50 effector11. TurboID is relatively larger (i.e., 35 kDa) than GFP, and its fusion to a target protein can affect the function of a target protein. In such cases, the smaller miniTurboID10 can be used.
It is also important to determine which terminus of the target protein is suitable for fusing with TurboID. A general approach for such functional tests is determining whether the TurboID fusion will complement the mutant plant line of the target gene. Alternatively, analysis of the interaction of the TurboID fusion with a previously known interaction partner of the target protein can be used. In most cases, for cytoplasmic proteins, N-terminal or C-terminal tagging of the TurboID can produce comparable datasets in subsequent mass spectrometry analysis. However, for membrane-localized proteins, it is important to determine the topology before vector construction. Otherwise, fusion of TurboID upstream or downstream of the coding sequence of genes of interest will generate completely different results. Therefore, it is important to determine the facing sides (e.g., cytoplasmic- or lumen-facing) of the N-terminus or C-terminus of the membrane-localized proteins. This ensures that the expected proximal proteome of the target protein can be obtained.
Although PL has several advantages over the traditional IP-MS approaches for detecting transient or weak PPIs, it has its own intrinsic limitations. First, identification of a candidate interaction proteins does not immediately imply a direct or indirect interaction with the bait protein, but it only reflects close proximity2. Therefore, independent in vivo assays (i.e., co-immunoprecipitation, bimolecular fluorescence complementation [BiFC], or in vitro GST-pull down assay) can be carried out to further verify PPIs.
Second, false negatives or false positives may arise from PL assay due to various reasons. For example, false negatives can occur when the protein lacking accessible primary amines. In addition, recent studies of BIN2 interactors in plants showed a partial overlap between data from two experiments13, indicating the existence of false negative results due to inadequate MS coverage. Some interactors of the target protein may also be weakly biotinylated by the TurboID-fused control, resulting in a fold enrichment below the cutoff threshold and eventually the loss of some true interactors13,14. Therefore, sufficient biological replicates with appropriate controls and cutoff values should be set to minimize the number of false negatives11,14.
Moreover, a long biotin treatment period can increase the biotinylation of nonspecific proteins, resulting in false positives10. Therefore, it is important to optimize in vivo labeling time windows to reduce the nonspecific biotinylation, while also not affecting the production of biotinylated proteins for analysis11,12,13,14. In addition, harsh extraction and stringent wash conditions is recommended to reduce the false positives derived from nonspecific binding of proteins to the beads17. Besides the caveats described above, the design of a proper negative control is crucial to distinguish true interactors from false interactors and avoid missing of true interactors11,12,13,14.
TurboID showed greatly improved labeling kinetics compared to that of BioID10,11. However, this can also lead to a higher background during PL analysis. Therefore, appropriate controls must be included to eliminate the background signals. We used the citrine-fused TurboID in this protocol11, and a similar control has been utilized in previous studies18. For the target proteins that localize to a specific organelle membrane, TurboID fusing with a signal peptide that targets the same organelle membrane makes a better control than TurboID fusing with one that is expressed in the cytoplasm14.
Furthermore, if information about the key domain or amino acids in the target protein that determine its interactions with other partners are known, fusing the TurboID with a target protein with a mutation in the key domain or amino acids should be the best control and will maximally reduce the background signals generated by the target protein. In addition, TurboID enzymes require specific temperatures as well as proper pH conditions. For most organelles in the cell such as the ER, nucleus, and mitochondria, TurboID successfully labeled the proximal proteins10. However, some special organelles (i.e., vacuoles in plant cells, whose pH is very low19) may be not suitable for studying PPIs using the PL approach. Moreover, changes in the pH levels or oxidation-reduction states of the subcellular environment during the stress response may affect the labeling efficiency of the TurboID.
In summary, the protocol described here provide the basis for investigating PPIs using TurboID in N. benthamiana, a model plant system that has been widely used in many aspects of plant biology20. With the recently described TurboID-based PL studies in Arabidopsis plants and tomato hairy roots12,13,14, it is expected that this method will become applicable to other plant species. It is anticipated that TurboID-based PL will play an important role in studying PPIs in plant biology research.

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

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

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

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

The representative data, which illustrate the expected results based on the described protocol, are adapted from Zhang et al11. Figure 1 summarizes the procedures for performing TurboID-based PL in N. benthamiana. Figure 2 shows the protein expression and biotinylation in the infiltrated N. benthamiana leaves. Figure 3 shows that the biotinylated proteins in the infiltrated leaves were efficiently enriched for subsequent mass spectrometry analysis. It should be noted that after enrichment of the biotinylated proteins using streptavidn-C1-conjugated magnetic beads, different proteins with varied sizes were captured, and western blot analysis of the enriched proteins showed smeared bands (Figure 3). Similar observations have been made in several recently published studies6,7,13,14.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60728/60728fig1v2.jpg" /> 
Figure 1: Overview of the TurboID-based PL method in N. benthamiana.&#160;Agrobacterium harboring the TurboID-fusion constructs were infiltrated into N. benthamiana leaves. 36 h post-infiltration, 200 &#181;M biotin is infiltrated to the same leaves to initiate biotinylation of the endogenous proteins that are proximal to the TurboID-fused target protein. The infiltrated plants are then incubated at RT for 3&#8211;12 h, followed by leaf harvesting and grinding in liquid nitrogen. Leaf powder is lysed in the RIPA lysis buffer, and a desalting column is employed to remove the free biotin in the protein extract. The biotinylated proteins were then affinity-purified with streptavidin-conjugated beads and identified by mass spectrometry. This figure is adapted from Supplementary Figure 3 from Zhang et al11. <a href="https://www.jove.com/files/ftp_upload/60728/60728fig1v2large.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/60728/60728fig2v2.jpg" /> 
Figure 2: Immunoblot analysis of protein extracts obtained in step 4.2.&#160;(A) Western blot analysis of proteins extracted from the agroinfiltrated leaves with antibody against HA tag. (B) Western blot analysis of biotinylated proteins in the agroinfiltrated leaves with streptavidin-HRP. This figure is adapted from Supplementary Figure 6B of Zhang et al11. <a href="https://www.jove.com/files/ftp_upload/60728/60728fig2v2large.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/60728/60728fig3v2.jpg" /> 
Figure 3: Western blot analysis of beads obtained in step 8.15 to confirm the enrichment of biotinylated proteins.&#160;For each construct, there are three independent replicates (1, 2, and 3). Streptavidin-HRP was used for analysis of biotinylated proteins in different samples. This figure is adapted from Supplementary Figure 7 from Zhang et al11. <a href="https://www.jove.com/files/ftp_upload/60728/60728fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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

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

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

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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

23.6K Views.  China Agricultural University. 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.

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

Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral cytokine production1,2. In order to colonize the host, viruses are obligate to evade host antiviral responses and manipulate signaling pathways. Unraveling the host-virus interaction will shed light on the development of novel therapeutic strategies against viral infection.
Murine &gamma;HV68 is closely related to human oncogenic Kaposi's sarcoma-associated herpesvirus and Epsten-Barr virus3,4. &gamma;HV68 infection in laboratory mice provides a tractable small animal model to examine the entire course of host responses and viral infection in vivo, which are not available for human herpesviruses. In this protocol, we present a panel of methods for phenotypic characterization and molecular dissection of host signaling components in &gamma;HV68 lytic replication both in vivo and ex vivo. The availability of genetically modified mouse strains permits the interrogation of the roles of host signaling pathways during &gamma;HV68 acute infection in vivo. Additionally, mouse embryonic fibroblasts (MEFs) isolated from these deficient mouse strains can be used to further dissect roles of these molecules during &gamma;HV68 lytic replication ex vivo.
Using virological and molecular biology assays, we can pinpoint the molecular mechanism of host-virus interactions and identify host and viral genes essential for viral lytic replication. Finally, a bacterial artificial chromosome (BAC) system facilitates the introduction of mutations into the viral factor(s) that specifically interrupt the host-virus interaction. Recombinant &gamma;HV68 carrying these mutations can be used to recapitulate the phenotypes of &gamma;HV68 lytic replication in MEFs deficient in key host signaling components. This protocol offers an excellent strategy to interrogate host-pathogen interaction at multiple levels of intervention in vivo and ex vivo.
Recently, we have discovered that &gamma;HV68 usurps an innate immune signaling pathway to promote viral lytic replication5. Specifically, &gamma;HV68 de novo infection activates the immune kinase IKK&beta; and activated IKK&beta; phosphorylates the master viral transcription factor, replication and transactivator (RTA), to promote viral transcriptional activation. In doing so, &gamma;HV68 efficiently couples its transcriptional activation to host innate immune activation, thereby facilitating viral transcription and lytic replication. This study provides an excellent example that can be applied to other viruses to interrogate host-virus interaction.

We describe a protocol to identify key roles of host signaling molecules in lytic replication of a model herpesvirus, gamma herpesvirus 68 (&gamma;HV68). Utilizing genetically modified mouse strains and embryonic fibroblasts for &gamma;HV68 lytic replication, the protocol permits both phenotypic characterization and molecular interrogation of virus-host interactions in viral lytic replication.

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral ...

Visualizing Dengue Virus through Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development 
of methods to probe this interaction would be useful. Recent advancements in fluorophores development1-3 and imaging technology4 can be exploited to 
improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually.

The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, 
which contains a single positive strand RNA genome5. The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized 
through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium 
bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer&#x2019;s protocol 
for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block 
productive infection of virus and also induce cell cytotoxicity6. The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. 
Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine2, making them ideal for 
studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue 
virus by virus-specific antibody and its putative receptors in host cells7. This method could have useful applications in virological studies.

Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell.

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

A Method for Labeling Vasculature in Embryonic Mice

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in the thymus proper vasculature formation and patterning is essential for thymocyte entry into the organ and mature T-cell exit to the periphery. The spatial arrangement of blood vessels in the thymus is dependent upon signals from the local microenvironment, namely thymic epithelial cells (TEC). Several recent reports suggest that disruption of these signals results in thymus blood vessel defects 1,2. Previous studies have described techniques used to label the neonatal and adult thymus vasculature 1,2. We demonstrate here a technique for labeling blood vessels in the embryonic thymus. This method combines the use of FITC-dextran or Griffonia (Bandeiraea) Simplicifolia Lectin I (GSL 1 - isolectin B4) facial vein injections and CD31 antibody staining to identify thymus vascular structures and PDGFR-&beta; to label thymic perivascular mesenchyme 3-5. The option of using cryosections or vibratome sections is also provided. This protocol can be used to identify thymus vascular defects, which is critical for defining the roles of TEC-derived molecules in thymus blood vessel formation. As the method labels the entire vasculature, it can also be used to analyze the vascular networks in multiple organs and tissues throughout the embryo including skin and heart 6-10.

This article describes a method for labeling embryonic skin and thymus blood vessels.

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in ...

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Significant efforts were gathered to generate large-scale comprehensive protein-protein interaction network maps. This is instrumental to understand the pathogen-host relationships and was essentially performed by genetic screenings in yeast two-hybrid systems. The recent improvement of protein-protein interaction detection by a Gaussia luciferase-based fragment complementation assay now offers the opportunity to develop integrative comparative interactomic approaches necessary to rigorously compare interaction profiles of proteins from different pathogen strain variants against a common set of cellular factors.
This paper specifically focuses on the utility of combining two orthogonal methods to generate protein-protein interaction datasets: yeast two-hybrid (Y2H) and a new assay, high-throughput Gaussia princeps protein complementation assay (HT-GPCA) performed in mammalian cells.
A large-scale identification of cellular partners of a pathogen protein is performed by mating-based yeast two-hybrid screenings of cDNA libraries using multiple pathogen strain variants. A subset of interacting partners selected on a high-confidence statistical scoring is further validated in mammalian cells for pair-wise interactions with the whole set of pathogen variants proteins using HT-GPCA. This combination of two complementary methods improves the robustness of the interaction dataset, and allows the performance of a stringent comparative interaction analysis. Such comparative interactomics constitute a reliable and powerful strategy to decipher any pathogen-host interplays.

This article focuses on the identification of high-confident interaction datasets between host and pathogen proteins using a combination of two orthogonal methods: yeast two-hybrid followed by a high-throughput interaction assay in mammalian cells called HT-GPCA.

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

Assays for the Identification of Novel Antivirals against Bluetongue Virus

To identify potential antivirals against BTV, we have developed, optimized and validated three assays presented here. The CPE-based assay was the first assay developed to evaluate whether a compound showed any antiviral efficacy and have been used to screen large compound library. Meanwhile, cytotoxicity of antivirals could also be evaluated using the CPE-based assay. The dose-response assay was designed to determine the range of efficacy for the selected antiviral, i.e. 50% inhibitory concentration (IC50) or effective concentration (EC50), as well as its range of cytotoxicity (CC50). The ToA assay was employed for the initial MoA study to determine the underlying mechanism of the novel antivirals during BTV viral lifecycle or the possible effect on host cellular machinery. These assays are vital for the evaluation of antiviral efficacy in cell culture system, and have been used for our recent researches leading to the identification of a number of novel antivirals against BTV.

Three assays, including the cytopathic effect (CPE)-based assay, dose-response assay and Time-of-Addition (ToA) assay have been developed, optimized, validated and utilized to identify novel antivirals against Bluetongue virus (BTV), as well as to determine the possible Mechanism-of-Action (MoA) for newly identified antivirals.

To identify potential antivirals against BTV, we have  developed, optimized and validated three assays presented here. The CPE-based  assay was the first ...

VIGS-Mediated Forward Genetics Screening for Identification of Genes Involved in Nonhost Resistance

Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important for developing durable disease-resistant plants against wide range of pathogens. Virus-induced gene silencing (VIGS)-based forward genetics screening is a useful approach for identification of plant defense genes imparting nonhost resistance. Tobacco rattle virus (TRV)-based VIGS vector is the most efficient VIGS vector to date and has been efficiently used to silence endogenous target genes in Nicotiana benthamiana.

	In this manuscript, we demonstrate a forward genetics screening approach for silencing of individual clones from a cDNA library in N. benthamiana and assessing the response of gene silenced plants for compromised nonhost resistance against nonhost pathogens, Pseudomonas syringae pv. tomato T1, P. syringae pv. glycinea, and X. campestris pv. vesicatoria. These bacterial pathogens are engineered to express GFPuv protein and their green fluorescing colonies can be seen by naked eye under UV light in the nonhost pathogen inoculated plants if the silenced target gene is involved in imparting nonhost resistance. This facilitates reliable and faster identification of gene silenced plants susceptible to nonhost pathogens. Further, promising candidate gene information can be known by sequencing the plant gene insert in TRV vector. Here we demonstrate the high throughput capability of VIGS-mediated forward genetics to identify genes involved in nonhost resistance. Approximately, 100 cDNAs can be individually silenced in about two to three weeks and their relevance in nonhost resistance against several nonhost bacterial pathogens can be studied in a week thereafter. In this manuscript, we enumerate the detailed steps involved in this screening. VIGS-mediated forward genetics screening approach can be extended not only to identifying genes involved in nonhost resistance but also to studying genes imparting several biotic and abiotic stress tolerances in various plant species.

Virus-induced gene silencing is an useful tool for identifying genes involved in nonhost resistance of plants. We demonstrate the use of bacterial pathogens expressing GFPuv in identifying gene silenced plants susceptible to nonhost pathogens. This approach is easy, fast and facilitates large scale screening and similar protocol can be applied to studying various other plant-microbe interactions.


	Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. Whereas Pd transport of small molecules, such as ions and water, is presumed to occur passively, cell-to-cell transport of biological macromolecules, such proteins, most likely occurs via an active mechanism that involves specific targeting signals on the transported molecule. The scarcity of identified plasmodesmata (Pd) localization signals (PLSs) has severely restricted the understanding of protein-sorting pathways involved in plant cell-to-cell macromolecular transport and communication. From a wealth of plant endogenous and viral proteins known to traffic through Pd, only three PLSs have been reported to date, all of them from endogenous plant proteins. Thus, it is important to develop a reliable and systematic experimental strategy to identify a functional PLS sequence, that is both necessary and sufficient for Pd targeting, directly in the living plant cells. Here, we describe one such strategy using as a paradigm the cell-to-cell movement protein (MP) of the Tobacco mosaic virus (TMV). These experiments, that identified and characterized the first plant viral PLS, can be adapted for discovery of PLS sequences in most Pd-targeted proteins.

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plant intercellular connections, the plasmodesmata (Pd), play central roles in plant physiology and plant-virus interactions. Critical to Pd transport are sorting signals that direct proteins to Pd. However, our knowledge about these sequences is still in its infancy. We describe a strategy to identify Pd localization signals in Pd-targeted proteins.

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. ...

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

What differentiates Mycobacterium abscessus from other saprophytic mycobacteria is the ability to resist phagocytosis by human macrophages and the ability to multiply inside such cells. These virulence traits render M. abscessus pathogenic, especially in vulnerable hosts with underlying structural lung disease, such as cystic fibrosis, bronchiectasis or tuberculosis. How patients become infected with M. abscessus remains unclear. Unlike many mycobacteria, M. abscessus is not found in the environment but might reside inside amoebae, environmental phagocytes that represent a potential reservoir for M. abscessus. Indeed, M. abscessus is resistant to amoebal phagocytosis and the intra-amoeba life seems to increase M. abscessus virulence in an experimental model of infection. However, little is known about M. abscessus virulence in itself. To decipher the genes conferring an advantage to M. abscessus intracellular life, a screening of a M. abscessus transposon mutant library was developed. In parallel, a method of RNA extraction from intracellular Mycobacteria after co-culture with amoebae was developed. This method was validated and allowed the sequencing of whole M. abscessus transcriptomes inside the cells; providing, for the first time, a global view on M. abscessus adaptation to intracellular life. Both approaches give us an insight into M. abscessus virulence factors that enable M. abscessus to colonize the airways in humans.

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

Here, we present two protocols to study Phagocyte-Mycobacterium abscessus interactions: the screening of a transposon mutant library for bacterial intracellular deficiency and the determination of bacterial intracellular transcriptome from RNA sequencing. Both approaches provide insight into the genomic advantages and transcriptomic adaptations enhancing intracellular bacteria fitness.

What differentiates Mycobacterium abscessus from other saprophytic mycobacteria is the ability to resist phagocytosis by human macrophages and the ability ...