title: Primary and secondary siRNAs in geminivirus-induced gene silencing.
abstract: 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.
authors.affiliation: Institute of Botany, University of Basel, Basel, Switzerland.
authors.forename: Michael
authors.initials: M
authors.lastname: Aregger
authors.forename: Basanta K
authors.initials: BK
authors.lastname: Borah
authors.forename: Jonathan
authors.initials: J
authors.lastname: Seguin
authors.forename: Rajendran
authors.initials: R
authors.lastname: Rajeswaran
authors.forename: Ekaterina G
authors.initials: EG
authors.lastname: Gubaeva
authors.forename: Anna S
authors.initials: AS
authors.lastname: Zvereva
authors.forename: David
authors.initials: D
authors.lastname: Windels
authors.forename: Franck
authors.initials: F
authors.lastname: Vazquez
authors.forename: Todd
authors.initials: T
authors.lastname: Blevins
authors.forename: Laurent
authors.initials: L
authors.lastname: Farinelli
authors.forename: Mikhail M
authors.initials: MM
authors.lastname: Pooggin
mesh_terms: D020413:3' Untranslated Regions; D020121:5' Untranslated Regions / Q000235:genetics; D017360:Arabidopsis / Q000378:metabolism / Q000821:virology*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism; D017791:Geminiviridae / Q000235:genetics*; D018506:Gene Expression Regulation, Plant; D049452:Green Fluorescent Proteins / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing; D010935:Plant Diseases / Q000235:genetics / Q000821:virology; D034622:RNA Interference; D012319:RNA Polymerase II / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000235:genetics* / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics* / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism
chemical_list: D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D029681:Arabidopsis Proteins; D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering; D012367:RNA, Viral; D049452:Green Fluorescent Proteins; D012319:RNA Polymerase II; C519655:RDR2 protein, Arabidopsis; C514575:RDR6 protein, Arabidopsis; D012324:RNA-Dependent RNA Polymerase