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* These authors contributed equally
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 effic....
NOTE: An overview of the method is shown in Figure 1.
1. Plant material preparation
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.......
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.......
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.
....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 |
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