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Visualization of Transiently Expressed mRNA in Plants Using MS2

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RNA Tagging

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2166))

Abstract

RNA transport and localization are evolutionarily conserved processes that allow protein translation to occur at specific subcellular sites and thereby having fundamental roles in the determination of cell fates, embryonic patterning, asymmetric cell division, and cell polarity. In addition to localizing RNA molecules to specific subcellular sites, plants have the ability to exchange RNA molecules between cells through plasmodesmata (PD). Plant RNA viruses hijack the mechanisms of intracellular and intercellular RNA transport to establish localized replication centers within infected cells and then to disseminate their infectious genomes between cells and throughout the plant organism with the help of their movement proteins (MP). In this chapter, we describe the transient expression of the tobacco mosaic virus movement protein (TMV-MP) and the application of the MS2 system for the in vivo labeling of the MP-encoding mRNA. The MS2 method is based on the binding of the bacteriophage coat protein (CP) to its origin of assembly (OAS) in the phage RNA. Thus, to label a specific mRNA in vivo, a tandem repetition of a 19-nucleotide-long stem-loop (SL) sequence derived from the MS2 OAS sequence (MSL) is transcriptionally fused to the RNA under investigation. The RNA is detected by the co-expression of fluorescent protein-tagged MS2 CP (MCP), which binds to each of the MSL elements. In providing a detailed protocol for the in vivo visualization of TMV-MP mRNA tagged with the MS2 system in Nicotiana benthamiana epidermal cells, we describe (1) the specific DNA constructs, (2) Agrobacterium tumefaciens-mediated transfection for their transient expression in plants, and (3) imaging conditions required to obtain high-quality mRNA imaging data.

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Acknowledgments

This work was supported by the Agence National de la Recherche (grant ANR-08-BLAN-244 to MH), the Consejo Nacional de Investigaciones Cientificas y Técnicas (CONICET, grant PIP-0847 to EP), and by a binational CNRS-CONICET grant (PICS-2014 to MH and EP).

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Authors and Affiliations

  1. Instituto de Biotecnología y Biología Molecular, CCT—La Plata CONICET, Fac. Cs. Exactas, U.N.L.P, La Plata, Argentina

    Eduardo José Peña

  2. Institut de Biologie Moléculaire des Plantes du CNRS, Université de Strasbourg, Strasbourg, France

    Manfred Heinlein

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  1. Eduardo José Peña
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  2. Manfred Heinlein
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Correspondence to Manfred Heinlein .

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Editors and Affiliations

  1. Institut de Biologie Moléculaire des Plantes (IBMP-CNRS), Université de Strasbourg, Strasbourg, France

    Manfred Heinlein

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Peña, E.J., Heinlein, M. (2020). Visualization of Transiently Expressed mRNA in Plants Using MS2. In: Heinlein, M. (eds) RNA Tagging. Methods in Molecular Biology, vol 2166. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0712-1_6

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  • DOI: https://doi.org/10.1007/978-1-0716-0712-1_6

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0711-4

  • Online ISBN: 978-1-0716-0712-1

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