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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 effic....

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. Maint.......

Representative 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.......

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
721 SpectrophotometerMetash, made in ChinaQ/SXFZ6For OD600 measurement
Ammonium bicarbonateSigmaA6141-500G
BiotinSigmaB4639-1G50 mM Stock
CentrifugeEppendorfCentrifuge 5702
CentrifugeEppendorfCentrifuge 5417R
cOmplete Protease Inhibitor CocktailRoche11697489001
Deoxycholic acidSigmaD2510-100G
DL-Dithiothreitol (DTT)VWR Life Science0281-25G
Dynabeads MyOne Streptavidin C1Invitrogen65001For affinity purification
EDTASigmaE6758-500G
ELISA plateCorningCostar 3590
HEPESSigmaH3375-1KG
Hydrochloric acid (HCl)Fisher ScientificA144S-212
Immobilon-P PVDF membraneMilliporeIPVH00010For Western blot analysis
Lithium chloride solution(LiCl), 8MSigmaL7026-500ML
Low speed refrigerated centrifugeZonkia, made in ChinaKDC-2046For desalting
Magnesium Chloride, Hexahydrate (MgCl2·6H2O)SigmaM9272-500G
Magnetic rackInvitrogen123.21DFor bead adsorption
Multiskan FC Microplate PhotometerThermo Fisher ScientificN07710For OD595 measurement
NP-40 (IGEPAL CA-630)SigmaI8896-100ML
Rat anti-HARoche11867423001
Rotational mixerKylin-Bell Lab InstrumentWH-986For IP
Shock incubatorLabotery, made in ChinaZQPZ-228
Sodium Chloride (NaCl)Fisher ScientificS271-3
Sodium deoxycholateSigmaD2510-100G
Sodium dodecyl sulfate(SDS)SigmaL4390-1KG
Streptavidin-HRPAbcamab7403
Triton X-100Fisher ScientificBP151-100
Trizma baseSigmaT1503-1KG
VortexScientific IndustriesG-560E
Water-jacket IncubatorBlue pard, made in ChinaGHP-9080For Agrobacterium incubation
Zeba Spin Desalting ColumnThermo Fisher Scientific89893For removal of biotin

References

  1. Berggård, T., Linse, S., James, P. Methods for the detection and analysis of protein-protein interactions. Proteomics. 7 (16), 2833-2842 (2007).
  2. Kim, D. I., Roux, K. J. Filling the void: proximity-based lab....

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TurboIDProximity LabelingProtein protein InteractionsNLR Immune ReceptorsNicotiana BenthamianaAgroinfiltrationBiotinRIPA Lysis BufferDesalting Column

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