title: In vivo RNA labeling using MS2.
abstract: The trafficking and asymmetric distribution of cytoplasmic RNA is a fundamental process during development and signaling across phyla. Plants support the intercellular trafficking of RNA molecules such as gene transcripts, small RNAs, and viral RNA genomes by targeting these RNA molecules to plasmodesmata (PD). Intercellular transport of RNA molecules through PD has fundamental implications in the cell-to-cell and systemic signaling during plant development and in the systemic spread of viral disease. Recent advances in time-lapse microscopy allow researchers to approach dynamic biological processes at the molecular level in living cells and tissues. These advances include the ability to label RNA molecules in vivo and thus to monitor their distribution and trafficking. In a broadly used RNA labeling approach, the MS2 method, the RNA of interest is tagged with a specific stem-loop (SL) RNA sequence derived from the origin of assembly region of the bacteriophage MS2 genome that binds to the bacteriophage coat protein (CP) and which, if fused to a fluorescent protein, allows the visualization of the tagged RNA by fluorescence microscopy. Here we describe a protocol for the in vivo visualization of transiently expressed SL-tagged RNA and discuss key aspects to study RNA localization and trafficking to and through plasmodesmata in Nicotiana benthamiana plants.
authors.affiliation: Institut de Biologie Moléculaire des Plantes (IBMP), Centre National de la Recherche Scientifique (CNRS), 12 rue du Général Zimmer, 67084, Strasbourg, France.
authors.forename: Eduardo
authors.initials: E
authors.lastname: Peña
authors.forename: Manfred
authors.initials: M
authors.lastname: Heinlein
authors.forename: Adrian
authors.initials: A
authors.lastname: Sambade
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics / Q000378:metabolism; D001692:Biological Transport; D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D055029:Inverted Repeat Sequences; D017909:Levivirus / Q000235:genetics* / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods*; D018515:Plant Leaves / Q000235:genetics* / Q000378:metabolism / Q000821:virology; D030821:Plants, Genetically Modified; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D031425:Plasmodesmata / Q000235:genetics* / Q000378:metabolism / Q000821:virology; D011485:Protein Binding; D015202:Protein Engineering; D012367:RNA, Viral / Q000032:analysis* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D059008:Time-Lapse Imaging; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism / Q000821:virology; D014027:Tobacco Mosaic Virus / Q000235:genetics* / Q000378:metabolism; D000097573:Red Fluorescent Protein
chemical_list: D036022:Capsid Proteins; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein