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Visualization of RMRs (Receptor Membrane RING-H2) Dimerization in Nicotiana benthamiana Leaves Using a Bimolecular Fluorescence Complementation (BiFC) Assay

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Book cover Plant Vacuolar Trafficking

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1789))

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Abstract

The bimolecular fluorescent complementation (BiFC) is a fluorescent complementation method largely used to investigate protein–protein interaction in living cells. This method is based on the ability of two nonfluorescent fragments to assemble forming a native fluorescent reporter with the same spectral properties of the native reporter. Such fragments are fused to putative protein partners that in case of interaction will bring the two halves in close proximity, allowing for the reconstitution of an active fluorescent reporter. The BiFC has been used to investigate protein–protein interaction in a number of different organisms, including plants. In plant cells, many essential pathways of protein trafficking and subcellular localization necessitate the intervention of several protein units organized in multisubunit complexes. It is well known that vacuolar sorting of many secretory soluble proteins require the intervention of specific transmembrane cargo receptors able to interact forming dimers. In this chapter we describe a BiFC method for the efficient visualization of RMR (Receptor Membrane RING-H2) type 2 dimerization in agro-infiltrated Nicotiana benthamiana leaves. Furthermore, this relatively simple method represents an optimal strategy to test protein–protein interaction using any other putative protein partners of interest in plant cells.

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Acknowledgments

Alessandro Occhialini thanks prof. Jean-Marc Neuhaus (University of Neuchâtel, Switzerland) for helpful discussion and support.

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Authors and Affiliations

  1. Department of Food Science, University of Tennessee, Food Safety and Processing Building, 2600 River Dr., Knoxville, Tennessee, TN, 37996, USA

    Alessandro Occhialini

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  1. Alessandro Occhialini
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Correspondence to Alessandro Occhialini .

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Editors and Affiliations

  1. Faculty of Sciences, University of Porto, Porto, Portugal

    Cláudia Pereira

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Occhialini, A. (2018). Visualization of RMRs (Receptor Membrane RING-H2) Dimerization in Nicotiana benthamiana Leaves Using a Bimolecular Fluorescence Complementation (BiFC) Assay. In: Pereira, C. (eds) Plant Vacuolar Trafficking. Methods in Molecular Biology, vol 1789. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7856-4_14

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  • DOI: https://doi.org/10.1007/978-1-4939-7856-4_14

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7855-7

  • Online ISBN: 978-1-4939-7856-4

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