Efficient detection of long dsRNA in vitro and in vivo using the dsRNA binding domain from FHV B2 protein
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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 in vitro applications, although recent developments have allowed the visualization of dsRNA in vivo. Here, we report the sensitive and rapid detection of long dsRNA both in vitro and in vivo using the dsRNA binding domain of the B2 protein from Flock house virus. In vitro, we adapted the system for the detection of dsRNA either enzymatically by northwestern blotting or by direct fluorescence labeling on fixed samples. In vivo, we produced stable transgenic Nicotiana benthamiana 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 N. benthamiana, 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.
Monsion , B , Incarbone , M , Hleibieh , K , Poignavent , V , Ghannam , A , Dunoyer , P , Daeffler , L , Tilsner , J & Ritzenthaler , C 2018 , ' Efficient detection of long dsRNA in vitro and in vivo using the dsRNA binding domain from FHV B2 protein ' Frontiers in Plant Science , vol 9 , 70 . DOI: 10.3389/fpls.2018.00070
Frontiers in Plant Science
© 2018 Monsion, Incarbone, Hleibieh, Poignavent, Ghannam, Dunoyer, Daeffler, Tilsner and Ritzenthaler. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
BM benefited from an IdEx postdoctoral fellowship from the Université de Strasbourg. Financial support to MI was provided in part by the INTERREG V Upper Rhine program Vitifutur, Transcending borders with every project. Work in the JT laboratory is funded by the U.K. Biotechnology and Biological Sciences Research Council (BB/M007200/1).
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