Primary and Secondary siRNAs in Geminivirus-induced Gene Silencing

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Date

2012

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Public Library of Science

Abstract

In plants, RNA silencing-based antiviral defense is mediated by Dicer-like (DCL) proteins producing short interfering (si)RNAs. In $\textit{Arabidopsis}$ infected with the bipartite circular DNA geminivirus $\textit{Cabbage leaf curl virus}$ (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 $\textit{GFP}$ transgene by primary viral siRNAs trigger RDR6-dependent production of secondary siRNAs. Viral siRNAs targeting upstream of the $\textit{GFP}$ stop codon induce secondary siRNAs almost exclusively from sequences downstream of the target site. Conversely, viral siRNAs targeting the $\textit{GFP}$ 3′-untranslated region (UTR) induce secondary siRNAs mostly upstream of the target site. RDR6-dependent siRNA production is not necessary for robust $\textit{GFP}$ silencing, except when viral siRNAs targeted $\textit{GFP}$ 5′-UTR. Furthermore, viral siRNAs targeting the transgene enhancer region cause $\textit{GFP}$ 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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Keywords

dicer, dicer like protein, protein, RNA polymerase, small interfering RNA, unclassified drug, 3' untranslated region, Arabidopsis, article, cabbage leaf curl virus, dot hybridization, Geminivirus, gene sequence, gene silencing, genetic analysis, nonhuman, 3' Untranslated Regions, 5' Untranslated Regions, Arabidopsis, Arabidopsis Proteins, Geminiviridae, Gene Expression Regulation, Plant, Green Fluorescent Proteins, High-Throughput Nucleotide Sequencing, Plant Diseases, RNA Interference, RNA Polymerase II, RNA Replicase, RNA, Double-Stranded, RNA, Small Interfering, RNA, Viral, RNA polymerase, 9014-24-8, protein, 67254-75-5, RDR2 protein, Arabidopsis, 2.7.7.48, reen Fluorescent Proteins, 147336-22-9, RDR6 protein, Arabidopsis, 2.7.7.48, RNA Polymerase II, 2.7.7., NA Replicase, 2.7.7.48

Citation

Aregger, M., Borah, B. K., Seguin, J., Rajeswaran, R., Gubaeva, E. G., Zvereva, A. S., . . . Pooggin, M. M. (2012). Primary and secondary siRNAs in geminivirus-induced gene silencing. PLoS Pathogens, 8(9). http://dx.doi.org/10.1371/journal.ppat.1002941

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© 2012 Aregger et al. 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 credited.

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