Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein
Monsion, Baptiste | Incarbone, Marco | Hleibieh, Kamal | Poignavent, Vianney | Ghannam, Ahmed | Dunoyer, Patrice | Daeffler, Laurent | Tilsner, Jens | Ritzenthaler, Christophe | Biologie et Génétique des Interactions Plante-Parasite (UMR BGPI) ; Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Institut National de la Recherche Agronomique (INRA)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro) | Institut de Biologie Moléculaire des Plantes (IBMP) ; Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS) | Applied Tumour Virology Programme; Divisions F00 ; German Cancer Research Center - Deutsches Krebsforschungszentrum [Heidelberg] (DKFZ)-Institut National de la Santé et de la Recherche Médicale (INSERM) | Institut de mathématiques de Luminy (IML) ; Université de la Méditerranée - Aix-Marseille 2-Centre National de la Recherche Scientifique (CNRS) | Université de Strasbourg (IdEx postdoctoral fellowship) ; INTERREG V Upper Rhine program Vitifutur, Transcending borders with every project ; U.K. Biotechnology and Biological Sciences Research Council [BB/M007200/1]
This article is part of the research topic: Update on Plant Virus Infection - a Cell Biology Perspective
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Показать больше [+] Меньше [-]Английский. Double-stranded RNA (dsRNA) plays essential functions in many biological processes, including the activation of innate immune responses and RNA interference. dsRNA also represents the genetic entity of some viruses and is a hallmark of infections by positive-sense single-stranded RNA viruses. Methods for detecting dsRNA rely essentially on immunological approaches and their use is often limited to <em>in vitro</em> applications, although recent developments have allowed the visualization of dsRNA <em>in vivo</em>. Here, we report the sensitive and rapid detection of long dsRNA both <em>in vitro</em> and <em>in vivo</em> using the dsRNA binding domain of the B2 protein from Flock house virus. <em>In vitro</em>, we adapted the system for the detection of dsRNA either enzymatically by northwestern blotting or by direct fluorescence labeling on fixed samples. <em>In vivo</em>, we produced stable transgenic <em>Nicotiana benthamiana</em> lines allowing the visualization of dsRNA by fluorescence microscopy. Using these techniques, we were able to discriminate healthy and positive-sense single-stranded RNA virus-infected material in plants and insect cells. In <em>N. benthamiana</em>, our system proved to be very potent for the spatio-temporal visualization of replicative RNA intermediates of a broad range of positive-sense RNA viruses, including high- vs. low-copy number viruses.
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