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

Summary

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.

Abstract

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 (16–24 h), and higher incubation temperature (37 °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 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.

Introduction

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 BirA^R118G (known as BioID) have been developed and utilized for the study of PPIs^2,3,4. 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 18–24 h to complete biotinylation), thus making the capture of transient PPIs less efficient. Moreover, the higher incubation temperature (37 °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 reported^6,7,8,9. 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 roots^11,12,13,14. Comparative analyses indicated that TurboID performs better for PL in plants compared to BioID^11,14. 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.

Protocol

NOTE: An overview of the method is shown in Figure 1.

1. Plant material preparation

1. 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 μmol/m²s) and 8 h dark photoperiod at 23–25 °C.
2. About 1 week later, carefully transfer each young seedling to 4' x 4' pots and keep the seedlings in the same chamber.
3. Maintain the plants in the chamber for about 4 weeks until they grow to a leaf stage of 4–8 for subsequent agroinfiltration¹⁵.

2. Construction of TurboID fusions

1. 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.
2. 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.

3. Agroinfiltration

1. Transformation of the plasmids to Agrobacterium
   1. Add 0.5 μg of the plasmids generated from steps 2.1 and 2.2 to 50 μL of Agrobacterium tumefaciens strain GV3101 competent cells, prepared as described previously¹⁶.
      NOTE: Other Agrobacterium strains, such as GV2260, can also be used.
   2. Incubate on ice for 30 min.
   3. Heat shock in a water bath at 42 °C for 60 s.
   4. Add 400 μL 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 °C for 90 min.
   5. 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).
   6. Incubate plates at 28 °C for 36–48 h until individual colonies are visible.
2. Pick and streak several individual colonies onto a fresh LB agar plate with antibiotics (see step 3.1.5) and grow at 28 °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.
3. 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 °C until the OD₆₀₀ of the Agrobacterium culture reaches 2.0.
4. 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 μM 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.
5. 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, 1.5–2.0 mL of resuspended agrobacteria is sufficient.
6. After 36 h post-infiltration (hpi), infiltrate 200 μM biotin (in 10 mM MgCl₂ solution) into the leaves pre-infiltrated with TurboID constructs.
7. Maintain the plant for additional 3–12 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, 3–12 h of biotin treatment allows the labeling of most proteins proximal to the target protein by the TurboID fusions.

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.

1. Cut the infiltrated leaves at the base of the petiole, remove the leaf vein, then flash-freeze the leaf tissue in liquid nitrogen.
2. Grind the leaf tissue using a pestle and mortar and store the leaf powder in 15 mL or 50 mL falcon tubes at -80 °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.

5. Extraction of leaf total protein

1. 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.
2. Add 700 µL 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.
3. Vortex the tubes for 10 min.
4. Leave the samples on ice for 30 min.
5. Mix the contents every 4–5 min by turning the tubes upside down several times.

6. Removal of free biotin by desalting

NOTE: This section takes about 50 min.

1. Equilibrate the desalting column.
   1. Remove the sealer at the bottom of the desalting column and put the column in a 50 mL tube.
   2. Remove the storage solution by centrifugation at 1000 x g and 4 °C for 2 min and ensure that that the cap is loosened.
   3. 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 °C for 2 min and discarding the flowthrough.
   4. Transfer the desalting column into a new 50 mL tube and store temporarily at 4 °C for subsequent use.
2. Spin the tubes from step 5.4 at 16,500 x g and 4 °C for 10 min and transfer the supernatant from the two tubes into a new 2 mL tube.
3. Add 1,500 µL 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 µL of RIPA lysis buffer.
   NOTE: Although 1,400 μL 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 µL of protein extract per sample.
4. Centrifuge at 1000 x g and 4 °C for 2 min and leave the desalted samples on ice temporarily.

7. Quantification of the desalted protein extracts using a Bradford assay

1. Prepare 50 µL 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.
2. Dilute the desalted protein extract by mixing a 5 µL sample with 45 µL of ddH₂O.
3. 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).
4. Add 50 µL of the each gradient BSA solution and 50 µL of diluted protein extract to the 2.5 mL of 1x Bradford regent.
5. Incubate at RT for 10 min.
6. Add 200 µL of solution of each sample to one well of an ELISA plate (three technical replicates per sample).
7. Measure the OD₅₉₅ using a microplate reader.
8. 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 3–6 mg/mL.
9. Prepare 6–8 mg of desalted protein extract for subsequent affinity purification.

8. Enrichment of biotinylated proteins

1. Take 200 µL of streptavidn-C1-conjugated magnetic beads into a 2 mL tube.
2. Equilibrate the streptavidn-C1-conjugated magnetic beads with 1 mL of RIPA lysis buffer for 1 min at RT.
3. After each washing, use the magnetic rack to adsorb the beads for 3 min and gently remove the solution by pipetting.
4. Repeat steps 8.2 and 8.3.
5. Transfer the desalted protein extract to the equilibrated streptavidn-C1-conjugated magnetic beads.
6. Incubate the tube at 4 °C for 12 h (or overnight) on a rotator to affinity-purify the biotinylated proteins.
7. 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.
8. 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.
9. 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.
10. 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.
11. Add 1.7 mL of 50 mM Tris-HCl (pH = 7.5) to remove the detergent, then repeat step 8.3.
12. Transfer the beads to a new 1.5 mL tube, and repeat steps 8.11 and 8.3.
13. Wash the beads 6x for 5 min each with 50 mM ammonium bicarbonate buffer at RT.
14. Add 1 mL of 50 mM ammonium bicarbonate buffer to the magnetic beads and mix well.
15. Remove 100 µL of beads for immunoblot analysis to confirm the enrichment of biotinylated proteins. A typical western blot is shown in Figure 3.
16. Flash-freeze the rest of the protein samples and stored at -80 °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 <http://massive.ucsd.edu>) using the identifier: MSV000083018 and MSV000083019.

Results

The representative data, which illustrate the expected results based on the described protocol, are adapted from Zhang et al¹¹. Figure 1 summarizes the procedures for performing TurboID-based PL in N. benthamiana. Figure 2 shows the protein expression and biotinylation in the infiltrated N. benthamiana leaves. Figure 3 shows that the biotinylated proteins in the infiltrated leaves were effic...

Discussion

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 °C¹⁰. Although the PL approach has been widely used in animal systems, its application in plants is limited. The protocol described here provides a step-by-step procedure for establishing the Turb...

Materials

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