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Immunocapture of dsRNA-bound proteins provides insight into Tobacco rattle virus replication complexes and reveals Arabidopsis DRB2 to be a wide-spectrum antiviral effector

Plant RNA viruses form organized membrane-bound replication complexes to replicate their genomes. This process requires virus- and host-encoded proteins and leads to the production of double-stranded RNA (dsRNA) replication intermediates. Here, we describe the use of Arabidopsis thaliana expressing...

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Autores principales: Incarbone, Marco, Clavel, Marion, Monsion, Baptiste, Kuhn, Lauriane, Scheer, Hélène, Vantard, Émilie, Poignavent, Vianney, Dunoyer, Patrice, Genschik, Pascal, Ritzenthaler, Christophe
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8566308/
https://www.ncbi.nlm.nih.gov/pubmed/34436604
http://dx.doi.org/10.1093/plcell/koab214
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author Incarbone, Marco
Clavel, Marion
Monsion, Baptiste
Kuhn, Lauriane
Scheer, Hélène
Vantard, Émilie
Poignavent, Vianney
Dunoyer, Patrice
Genschik, Pascal
Ritzenthaler, Christophe
author_facet Incarbone, Marco
Clavel, Marion
Monsion, Baptiste
Kuhn, Lauriane
Scheer, Hélène
Vantard, Émilie
Poignavent, Vianney
Dunoyer, Patrice
Genschik, Pascal
Ritzenthaler, Christophe
author_sort Incarbone, Marco
collection PubMed
description Plant RNA viruses form organized membrane-bound replication complexes to replicate their genomes. This process requires virus- and host-encoded proteins and leads to the production of double-stranded RNA (dsRNA) replication intermediates. Here, we describe the use of Arabidopsis thaliana expressing GFP-tagged dsRNA-binding protein (B2:GFP) to pull down dsRNA and associated proteins in planta upon infection with Tobacco rattle virus (TRV). Mass spectrometry analysis of the dsRNA-B2:GFP-bound proteins from infected plants revealed the presence of viral proteins and numerous host proteins. Among a selection of nine host candidate proteins, eight showed relocalization upon infection, and seven of these colocalized with B2-labeled TRV replication complexes. Infection of A. thaliana T-DNA mutant lines for eight such factors revealed that genetic knockout of dsRNA-BINDING PROTEIN 2 (DRB2) leads to increased TRV accumulation and DRB2 overexpression caused a decrease in the accumulation of four different plant RNA viruses, indicating that DRB2 has a potent and wide-ranging antiviral activity. We propose B2:GFP-mediated pull down of dsRNA to be a versatile method to explore virus replication complex proteomes and to discover key host virus replication factors. Given the universality of dsRNA, development of this tool holds great potential to investigate RNA viruses in other host organisms.
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spelling pubmed-85663082021-11-04 Immunocapture of dsRNA-bound proteins provides insight into Tobacco rattle virus replication complexes and reveals Arabidopsis DRB2 to be a wide-spectrum antiviral effector Incarbone, Marco Clavel, Marion Monsion, Baptiste Kuhn, Lauriane Scheer, Hélène Vantard, Émilie Poignavent, Vianney Dunoyer, Patrice Genschik, Pascal Ritzenthaler, Christophe Plant Cell Breakthrough Report Plant RNA viruses form organized membrane-bound replication complexes to replicate their genomes. This process requires virus- and host-encoded proteins and leads to the production of double-stranded RNA (dsRNA) replication intermediates. Here, we describe the use of Arabidopsis thaliana expressing GFP-tagged dsRNA-binding protein (B2:GFP) to pull down dsRNA and associated proteins in planta upon infection with Tobacco rattle virus (TRV). Mass spectrometry analysis of the dsRNA-B2:GFP-bound proteins from infected plants revealed the presence of viral proteins and numerous host proteins. Among a selection of nine host candidate proteins, eight showed relocalization upon infection, and seven of these colocalized with B2-labeled TRV replication complexes. Infection of A. thaliana T-DNA mutant lines for eight such factors revealed that genetic knockout of dsRNA-BINDING PROTEIN 2 (DRB2) leads to increased TRV accumulation and DRB2 overexpression caused a decrease in the accumulation of four different plant RNA viruses, indicating that DRB2 has a potent and wide-ranging antiviral activity. We propose B2:GFP-mediated pull down of dsRNA to be a versatile method to explore virus replication complex proteomes and to discover key host virus replication factors. Given the universality of dsRNA, development of this tool holds great potential to investigate RNA viruses in other host organisms. Oxford University Press 2021-08-26 /pmc/articles/PMC8566308/ /pubmed/34436604 http://dx.doi.org/10.1093/plcell/koab214 Text en © The Author(s) 2021. Published by Oxford University Press on behalf of American Society of Plant Biologists. https://creativecommons.org/licenses/by/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Breakthrough Report
Incarbone, Marco
Clavel, Marion
Monsion, Baptiste
Kuhn, Lauriane
Scheer, Hélène
Vantard, Émilie
Poignavent, Vianney
Dunoyer, Patrice
Genschik, Pascal
Ritzenthaler, Christophe
Immunocapture of dsRNA-bound proteins provides insight into Tobacco rattle virus replication complexes and reveals Arabidopsis DRB2 to be a wide-spectrum antiviral effector
title Immunocapture of dsRNA-bound proteins provides insight into Tobacco rattle virus replication complexes and reveals Arabidopsis DRB2 to be a wide-spectrum antiviral effector
title_full Immunocapture of dsRNA-bound proteins provides insight into Tobacco rattle virus replication complexes and reveals Arabidopsis DRB2 to be a wide-spectrum antiviral effector
title_fullStr Immunocapture of dsRNA-bound proteins provides insight into Tobacco rattle virus replication complexes and reveals Arabidopsis DRB2 to be a wide-spectrum antiviral effector
title_full_unstemmed Immunocapture of dsRNA-bound proteins provides insight into Tobacco rattle virus replication complexes and reveals Arabidopsis DRB2 to be a wide-spectrum antiviral effector
title_short Immunocapture of dsRNA-bound proteins provides insight into Tobacco rattle virus replication complexes and reveals Arabidopsis DRB2 to be a wide-spectrum antiviral effector
title_sort immunocapture of dsrna-bound proteins provides insight into tobacco rattle virus replication complexes and reveals arabidopsis drb2 to be a wide-spectrum antiviral effector
topic Breakthrough Report
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8566308/
https://www.ncbi.nlm.nih.gov/pubmed/34436604
http://dx.doi.org/10.1093/plcell/koab214
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