Protocol to identify protein-protein interaction networks in Solanum tuberosum using transient TurboID-based proximity labeling

• Published in: STAR protocols 2023-12, Vol.4 (4), p.102577-102577, Article 102577
• Main Authors: Shi, Li, Marti Ferrando, Tatiana, Landeo Villanueva, Sergio, Joosten, Matthieu H.A.J., Vleeshouwers, Vivianne G.A.A., Bachem, Christian W.B.
• Format: Article
• Language: English
• Subjects: Biotin; Molecular Biology; Plant Sciences; Plants; Protein Biochemistry; Protein Interaction Maps; Protocol; Solanum tuberosum - genetics; Transcription Factors
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2023.102577

Protein-protein interactions (PPIs) in crop plants remain largely unexplored. Here, we provide a protocol for identifying PPIs in potato (Solanum tuberosum) using TurboID-mediated proximity labeling. We transiently expressed constructs for a nucleus-located transcription factor and a plasma membrane-localized receptor-like kinase fused to TurboID to identify PPIs in potato leaves. We describe the plasmid construction, plant material, agroinfiltration, biotin treatment, protein isolation, free biotin removal, western blot analysis, and enrichment of biotinylated proteins for mass spectrometry analysis. [Display omitted] •TurboID-enabled biotin-based proximity labeling protocol for Solanum tuberosum•Application of TurboID-PL in nuclear and membrane-bound proteins•A detailed protocol from preparing plants material and transgene design to protein identification•Visualization of biotinylated proteins by western blotting Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Protein-protein interactions (PPIs) in crop plants remain largely unexplored. Here, we provide a protocol for identifying PPIs in potato (Solanum tuberosum) using TurboID-mediated proximity labeling. We transiently expressed constructs for a nucleus-located transcription factor and a plasma membrane-localized receptor-like kinase fused to TurboID to identify PPIs in potato leaves. We describe the plasmid construction, plant material, agroinfiltration, biotin treatment, protein isolation, free biotin removal, western blot analysis, and enrichment of biotinylated proteins for mass spectrometry analysis.