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Bimolecular Fluorescence Complementation (BiFC) in Host-Virus Interactions.

Fredy Davi Albuquerque Silva1,2, João Paulo Batista Machado3,4, Pedro Augusto Braga Dos Reis5,6

  • 1Department of Biochemistry and Molecular Biology/Bioagro, Universidade Federal de Viçosa, Viçosa, MG, Brazil.

Methods in Molecular Biology (Clifton, N.J.)
|November 21, 2023
PubMed
Summary

Bimolecular fluorescence complementation (BiFC) visualizes protein interactions using fluorescent protein fragments. This method is effective for studying viral and host protein interactions in plants like Nicotiana benthamiana.

Keywords:
AgroinfiltrationBiFCConfocal microscopyViral protein–host protein interactions

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Plant Science

Background:

  • Bimolecular fluorescence complementation (BiFC) is a key technique for studying protein-protein interactions and protein localization within cells.
  • The BiFC assay relies on fusing proteins of interest to complementary, non-fluorescent fragments of a reporter protein, typically a fluorescent protein.

Purpose of the Study:

  • To detail a robust protocol for utilizing BiFC to investigate virus-host protein interactions.
  • To provide a methodological framework for preparing expression cassettes, performing Agrobacterium-mediated transformations, and agroinfiltration in Nicotiana benthamiana.

Main Methods:

  • Proteins of interest are fused to non-fluorescent yellow fluorescent protein (YFP) fragments.
  • Expression cassettes are prepared for transient expression in plant cells.
  • Agrobacterium tumefaciens-mediated agroinfiltration is performed on Nicotiana benthamiana leaves.
  • Confocal microscopy is employed for high-resolution imaging and analysis of protein interactions.

Main Results:

  • Successful reconstitution of fluorescence indicates protein-protein interactions in vivo.
  • The protocol facilitates the visualization of virus-host protein interactions within the plant cellular environment.
  • High-resolution images of protein interactions can be acquired using confocal microscopy.

Conclusions:

  • The described BiFC methodology provides a reliable approach for studying protein-protein interactions, particularly in the context of virus-host interactions.
  • Agroinfiltration of Nicotiana benthamiana offers a practical system for BiFC analysis.
  • This technique enables detailed subcellular localization and interaction studies.