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Primary and Secondary siRNAs in Geminivirus-induced Gene Silencing

In plants, RNA silencing-based antiviral defense is mediated by Dicer-like (DCL) proteins producing short interfering (si)RNAs. In Arabidopsis infected with the bipartite circular DNA geminivirus Cabbage leaf curl virus (CaLCuV), four distinct DCLs produce 21, 22 and 24 nt viral siRNAs. Using deep s...

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Autores principales: Aregger, Michael, Borah, Basanta K., Seguin, Jonathan, Rajeswaran, Rajendran, Gubaeva, Ekaterina G., Zvereva, Anna S., Windels, David, Vazquez, Franck, Blevins, Todd, Farinelli, Laurent, Pooggin, Mikhail M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3460622/
https://www.ncbi.nlm.nih.gov/pubmed/23028332
http://dx.doi.org/10.1371/journal.ppat.1002941
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author Aregger, Michael
Borah, Basanta K.
Seguin, Jonathan
Rajeswaran, Rajendran
Gubaeva, Ekaterina G.
Zvereva, Anna S.
Windels, David
Vazquez, Franck
Blevins, Todd
Farinelli, Laurent
Pooggin, Mikhail M.
author_facet Aregger, Michael
Borah, Basanta K.
Seguin, Jonathan
Rajeswaran, Rajendran
Gubaeva, Ekaterina G.
Zvereva, Anna S.
Windels, David
Vazquez, Franck
Blevins, Todd
Farinelli, Laurent
Pooggin, Mikhail M.
author_sort Aregger, Michael
collection PubMed
description In plants, RNA silencing-based antiviral defense is mediated by Dicer-like (DCL) proteins producing short interfering (si)RNAs. In Arabidopsis infected with the bipartite circular DNA geminivirus Cabbage leaf curl virus (CaLCuV), four distinct DCLs produce 21, 22 and 24 nt viral siRNAs. Using deep sequencing and blot hybridization, we found that viral siRNAs of each size-class densely cover the entire viral genome sequences in both polarities, but highly abundant siRNAs correspond primarily to the leftward and rightward transcription units. Double-stranded RNA precursors of viral siRNAs can potentially be generated by host RDR-dependent RNA polymerase (RDR). However, genetic evidence revealed that CaLCuV siRNA biogenesis does not require RDR1, RDR2, or RDR6. By contrast, CaLCuV derivatives engineered to target 30 nt sequences of a GFP transgene by primary viral siRNAs trigger RDR6-dependent production of secondary siRNAs. Viral siRNAs targeting upstream of the GFP stop codon induce secondary siRNAs almost exclusively from sequences downstream of the target site. Conversely, viral siRNAs targeting the GFP 3′-untranslated region (UTR) induce secondary siRNAs mostly upstream of the target site. RDR6-dependent siRNA production is not necessary for robust GFP silencing, except when viral siRNAs targeted GFP 5′-UTR. Furthermore, viral siRNAs targeting the transgene enhancer region cause GFP silencing without secondary siRNA production. We conclude that the majority of viral siRNAs accumulating during geminiviral infection are RDR1/2/6-independent primary siRNAs. Double-stranded RNA precursors of these siRNAs are likely generated by bidirectional readthrough transcription of circular viral DNA by RNA polymerase II. Unlike transgenic mRNA, geminiviral mRNAs appear to be poor templates for RDR-dependent production of secondary siRNAs.
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spelling pubmed-34606222012-10-01 Primary and Secondary siRNAs in Geminivirus-induced Gene Silencing Aregger, Michael Borah, Basanta K. Seguin, Jonathan Rajeswaran, Rajendran Gubaeva, Ekaterina G. Zvereva, Anna S. Windels, David Vazquez, Franck Blevins, Todd Farinelli, Laurent Pooggin, Mikhail M. PLoS Pathog Research Article In plants, RNA silencing-based antiviral defense is mediated by Dicer-like (DCL) proteins producing short interfering (si)RNAs. In Arabidopsis infected with the bipartite circular DNA geminivirus Cabbage leaf curl virus (CaLCuV), four distinct DCLs produce 21, 22 and 24 nt viral siRNAs. Using deep sequencing and blot hybridization, we found that viral siRNAs of each size-class densely cover the entire viral genome sequences in both polarities, but highly abundant siRNAs correspond primarily to the leftward and rightward transcription units. Double-stranded RNA precursors of viral siRNAs can potentially be generated by host RDR-dependent RNA polymerase (RDR). However, genetic evidence revealed that CaLCuV siRNA biogenesis does not require RDR1, RDR2, or RDR6. By contrast, CaLCuV derivatives engineered to target 30 nt sequences of a GFP transgene by primary viral siRNAs trigger RDR6-dependent production of secondary siRNAs. Viral siRNAs targeting upstream of the GFP stop codon induce secondary siRNAs almost exclusively from sequences downstream of the target site. Conversely, viral siRNAs targeting the GFP 3′-untranslated region (UTR) induce secondary siRNAs mostly upstream of the target site. RDR6-dependent siRNA production is not necessary for robust GFP silencing, except when viral siRNAs targeted GFP 5′-UTR. Furthermore, viral siRNAs targeting the transgene enhancer region cause GFP silencing without secondary siRNA production. We conclude that the majority of viral siRNAs accumulating during geminiviral infection are RDR1/2/6-independent primary siRNAs. Double-stranded RNA precursors of these siRNAs are likely generated by bidirectional readthrough transcription of circular viral DNA by RNA polymerase II. Unlike transgenic mRNA, geminiviral mRNAs appear to be poor templates for RDR-dependent production of secondary siRNAs. Public Library of Science 2012-09-27 /pmc/articles/PMC3460622/ /pubmed/23028332 http://dx.doi.org/10.1371/journal.ppat.1002941 Text en © 2012 Aregger et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Aregger, Michael
Borah, Basanta K.
Seguin, Jonathan
Rajeswaran, Rajendran
Gubaeva, Ekaterina G.
Zvereva, Anna S.
Windels, David
Vazquez, Franck
Blevins, Todd
Farinelli, Laurent
Pooggin, Mikhail M.
Primary and Secondary siRNAs in Geminivirus-induced Gene Silencing
title Primary and Secondary siRNAs in Geminivirus-induced Gene Silencing
title_full Primary and Secondary siRNAs in Geminivirus-induced Gene Silencing
title_fullStr Primary and Secondary siRNAs in Geminivirus-induced Gene Silencing
title_full_unstemmed Primary and Secondary siRNAs in Geminivirus-induced Gene Silencing
title_short Primary and Secondary siRNAs in Geminivirus-induced Gene Silencing
title_sort primary and secondary sirnas in geminivirus-induced gene silencing
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3460622/
https://www.ncbi.nlm.nih.gov/pubmed/23028332
http://dx.doi.org/10.1371/journal.ppat.1002941
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