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 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.
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 18–24 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 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.
NOTE: An overview of the method is shown in Figure 1.
1. Plant material preparation
2. Construction of TurboID fusions
3. Agroinfiltration
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.
5. Extraction of leaf total protein
6. Removal of free biotin by desalting
NOTE: This section takes about 50 min.
7. Quantification of the desalted protein extracts using a Bradford assay
8. Enrichment of biotinylated proteins
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.
Figure 1: Overview of the TurboID-based PL method in N. benthamiana. Agrobacterium harboring the TurboID-fusion constructs were infiltrated into N. benthamiana leaves. 36 h post-infiltration, 200 µM 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 3–12 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. Please click here to view a larger version of this figure.
Figure 2: Immunoblot analysis of protein extracts obtained in step 4.2. (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. Please click here to view a larger version of this figure.
Figure 3: Western blot analysis of beads obtained in step 8.15 to confirm the enrichment of biotinylated proteins. 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. Please click here to view a larger version of this figure.
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 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.
The authors have nothing to disclose.
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.
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 |