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

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.

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 al.

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 al.

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 al.