Establishment of TurboID Proximity Labeling Technology in Plants and Bacteria

doi:10.13560/j.cnki.biotech.bull.1985.2025-0222

Abstract

Abstract:

Objective TurboID-mediated proximity labeling technology is a novel protein-protein interaction research tool based on engineered biotin ligase, offering advantages including rapid labeling kinetics, high spatio-temporal resolution, and minimal cellular toxicity. However, its application remains limited to a few model species during current preliminary implementation stages. In this study, we developed TurboID expression systems in multiple representative species - monocots, dicots and prokaryotes - to systematically evaluate their labeling efficiency and applicability across evolutionary distant organisms. Method Recombinant vectors expressing “epitope tag-TurboID” fusion proteins were constructed for monocots (Oryza sativa), dicots (Arabidopsis thaliana, Solanum lycopersicum, and Nicotiana benthamiana), and bacteria (Escherichia coli), followed by transformation into respective species. Total proteins were extracted after biotin incubation, and fusion protein expression was verified via epitope tag-specific Western blot. Biotinylation of endogenous proteins was detected using streptavidin-HRP-based immunoblotting. Result Using ubiquitin promoters in plants and a T7 promoter in bacteria, we successfully achieved high-level expression of a “tag protein + TurboID” fusion protein in A. thaliana, S. lycopersicum, N. benthamiana, rice (Oryza sativa) callus, and E. coli. Western blot analysis confirmed robust expression of the fusion protein across all five systems. Following biotin treatment, multiple endogenous proteins were efficiently biotinylated in each system, indicating that the constructed TurboID system functioned effectively across monocots, dicots, and prokaryotes, demonstrating broad applicability and strong potential for further expansion. Conclusion A TurboID-based proximity labeling system is successfully established in monocotyledonous and dicotyledonous plants, as well as in bacteria, providing an efficient and reliable platform for analyzing complex protein interaction networks.

Key words: TurboID, proximity labeling, biotin, biotin ligase, protein-protein interaction, Arabidopsis thaliana, Oryza sativa

WANG Fang, SHAO Hui-ru, LYU Lin-long, ZHAO Dian, HU Zhen, LYU Jian-zhen, JIANG Liang. Establishment of TurboID Proximity Labeling Technology in Plants and Bacteria[J]. Biotechnology Bulletin, 2025, 41(9): 44-53.

Figures/Tables 8

Fig. 1 Expression vectors for TurboID proximity labeling in plants and bacteriaA: Expression vector (pDEST17-T7pro::mCherry-6×His-TurboID) for TurboID proximity labeling in Escherichia coli. B: Expression vector (pK7-AtUBIpro::3×HA-TurboID-EGFP) for TurboID proximity labeling in dicotyledonous plants, including Arabidopsis thalian, Solanum lycopersicum, and Nicotiana benthamiana. C: Expression vector (pK7-ZmUBIpro::3×HA-TurboID-EGFP) for TurboID proximity labeling in monocotyledonous plant (Oryza sativa)

Fig. 2 Schematic illustration of the TurboID proximity labeling technology principle

Fig. 3 Establishment of the TurboID proximity labeling method in E. coliA: Schematic diagram of the expression elements of the fusion protein mCherry-TurboID3. B: Photographs of bacterial cultures with and without IPTG induction. C: Western-blot detection of the expression of the fusion protein and biotinylation of proximity labeling in two transgenic E. coli strains (#1; #2). "-" and "+" indicate treatment with ddH2O and 50 μmol/L biotin solution respectively, in the Coomassie Brilliant Blue staining image, they indicate the uninduced and induced states respectively

Fig. 4 Optimization of biotin concentration and labeling time used in proximity labelingA: Treat the induced bacterial culture with biotin solutions of different concentrations for 3 h and then perform Western-blot detection using α-AAL-Biotin and α-His antibodies. B: Treat the induced bacterial culture with a 50 μmol/L biotin solution for different durations and then perform Western-blot detection

Fig. 5 Establishment of the TurboID proximity labeling method in ArabidopsisA: Schematic diagram of the expression elements of the fusion protein TurboID-EGFP. B: Phenotypic comparison of 30-day-old seedlings between wild-type and transgenic Arabidopsis thaliana. C: Western-blot detection of the expression of fusion proteins and biotinylation of proximity labeling in two transgenic Arabidopsis lines (#3; #5). "-" and "+" indicate treatment with ddH2O and 100 μmol/L biotin solution respectively. The same below

Fig. 6 Establishment of the TurboID proximity labeling method in S. lycopersicumA: Comparative analysis of 30-day-old wild-type and transgenic Solanum lycopersicum. B: Western-blot detection of the expression of fusion proteins and biotinylation of proximity labeling in the leaves of two transgenic tomato lines (#1; #2)

Fig.7 Establishment of the TurboID proximity labeling method based on tobacco transient expressionA: Image of tobacco leaf injection. Ruby-expressing Agrobacterium-injected tobacco serves as a visual control for tobacco expression, and CK being the leaf not injected with any Agrobacterium. B: Western-blot detection of the expression of fusion proteins and biotinylation of proximity labeling. From left to right, the three samples are tobacco leaves injected with Agrobacterium but not biotin, leaves not injected with any Agrobacterium or biotin, and tobacco leaves injected with both Agrobacterium and 20 μmol/L biotin

Fig. 8 Establishment of the TurboID proximity labeling method in rice callusesA: Schematic diagram of the expression elements of the fusion protein TurboID-EGFP. B: Image of rice calluses. C: Western-blot detection of the expression of the fusion protein and biotinylation of proximity labeling in two transgenic rice lines (#1; #2)

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