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 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. Maintain the plants in the chamber for about 4 weeks until they grow to a leaf stage of 4–8 for subsequent agroinfiltration¹⁵....

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