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TurboID-Based Proximity Labeling: A Method to Decipher Protein–Protein Interactions in Plants

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Plant-Virus Interactions

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2724))

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Abstract

Proteins form complex networks through interaction to drive biological processes. Thus, dissecting protein–protein interactions (PPIs) is essential for interpreting cellular processes. To overcome the drawbacks of traditional approaches for analyzing PPIs, enzyme-catalyzed proximity labeling (PL) techniques based on peroxidases or biotin ligases have been developed and successfully utilized in mammalian systems. However, the use of toxic H2O2 in peroxidase-based PL, the requirement of long incubation time (16–24 h), and higher incubation temperature (37 °C) with biotin in BioID-based PL significantly restricted their applications in plants. TurboID-based PL, a recently developed approach, circumvents the limitations of these methods by providing rapid PL of proteins under room temperature. We recently optimized the use of TurboID-based PL in plants and demonstrated that it performs better than BioID in labeling endogenous proteins. Here, we describe a step-by-step protocol for TurboID-based PL in studying PPIs in planta, including Agrobacterium-based transient expression of proteins, biotin treatment, protein extraction, removal of free biotin, quantification, and enrichment of the biotinylated proteins by affinity purification. We describe the PL using plant viral immune receptor N, which belongs to the nucleotide-binding leucine-rich repeat (NLR) class of immune receptors, as a model. The method described could be easily adapted to study PPI networks of other proteins in Nicotiana benthamiana and provides valuable information for future application of TurboID-based PL in other plant species.

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Acknowledgments

This work was supported by grants from NIH-GM132582, NSF-MCB-EAGER-2028283 and NSF-IOS-2139987 to SPD-K.

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Authors and Affiliations

  1. Department of Plant Biology and The Genome Center, College of Biological Sciences, University of California, Davis, Davis, CA, USA

    Yuanyuan Li & Savithramma P. Dinesh-Kumar

  2. State Key Laboratory of Plant Environmental Resilience and Ministry of Agriculture Key Laboratory of Soil Microbiology, College of Biological Sciences, China Agricultural University, Beijing, China

    Yongliang Zhang

Authors
  1. Yuanyuan Li
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  2. Yongliang Zhang
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  3. Savithramma P. Dinesh-Kumar
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Corresponding author

Correspondence to Savithramma P. Dinesh-Kumar .

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Editors and Affiliations

  1. Departamento de Bioquimica e Biologia Molecular, Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil

    Elizabeth P.B. Fontes

  2. Department of Agricultural Sciences, University of Helsinki, Helsinki, Finland

    Kristiina Mäkinen

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© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Li, Y., Zhang, Y., Dinesh-Kumar, S.P. (2024). TurboID-Based Proximity Labeling: A Method to Decipher Protein–Protein Interactions in Plants. In: Fontes, E.P., Mäkinen, K. (eds) Plant-Virus Interactions. Methods in Molecular Biology, vol 2724. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3485-1_19

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  • DOI: https://doi.org/10.1007/978-1-0716-3485-1_19

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3484-4

  • Online ISBN: 978-1-0716-3485-1

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