Abstract
DNA-binding proteins are involved in the dynamic regulation of various cellular processes such as recombination, replication, and transcription. For investigating dynamic assembly and disassembly of molecular complexes in living cells, fluorescence microscopy represents a tremendous tool in biology. A fluorescence resonance energy transfer (FRET) approach coupled to fluorescence lifetime imaging microscopy (FLIM) has been used recently to monitor protein–DNA associations in plant cells. With this approach, the donor fluorophore is a GFP-tagged binding partner expressed in plant cells. A Sytox® Orange treatment converts nuclear nucleic acids to FRET acceptors. A decrease of GFP lifetime is due to FRET between donor and acceptor, indicating close association of the GFP binding partner and Sytox® Orange-stained DNA. In this chapter, we present a step-by-step protocol for the transient expression in N. benthamiana of GFP-tagged proteins and the fixation and permeabilization procedures used for the preparation of plant material aimed at detecting protein–nucleic acid interactions by FRET-FLIM measurements.
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Acknowledgments
This research was performed in the Laboratoire de Recherche en Sciences Végétales and in the Laboratory of Plant-Microbe Interactions, part of the French Laboratory of Excellence ‘TULIP’ (ANR-10-LABX-41; ANR-11-IDEX-0002-02). The described protocol was optimized with the assistance of the core facilities of the TRI-Genotoul Imagery Platform of Toulouse (France). E.G. was supported by an ANR Jeunes Chercheuses/Jeunes Chercheurs grant (APHANO-Effect, ANR-12-JSV6-0004-01). L.D. was supported by a grant from the Agence Nationale de la Recherche (ANR) (RADAR, ANR-15-CE20-0016-01).
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Escouboué, M., Camborde, L., Jauneau, A., Gaulin, E., Deslandes, L. (2019). Preparation of Plant Material for Analysis of Protein–Nucleic Acid Interactions by FRET-FLIM. In: Gassmann, W. (eds) Plant Innate Immunity. Methods in Molecular Biology, vol 1991. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9458-8_8
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DOI: https://doi.org/10.1007/978-1-4939-9458-8_8
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