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.

Identification and characterization of a novel geminivirus with a monopartite genome infecting apple trees

“…Interestingly, v-sRNAs of 24 nt were also detected, in line with previous data from v-sRNAs accumulating in plant tissues infected by other geminiviruses, including cabbage leaf curl virus (CaLCuV) (Aregger et al, 2012), tomato yellow leaf curl China virus (TYLCCNV) (Yang et al, 2011) and tomato yellow leaf curl Sardinia virus (TYLCSV) (Miozzi et al, 2013). Regarding AGV sRNAs biogenesis, altogether these data are consistent with the involvement of multiple DCLs, as suggested also for other geminiviruses infecting herbaceous hosts (Aregger et al, 2012;Miozzi et al, 2013;Yang et al, 2011). Supporting this conclusion, apple genes coding for proteins homologous to Arabidopsis DCLs (DCL1-DCL4) have been annotated in GenBank and/or Phytozome databases (http://www.phytozome.net/apple.php).…”

“…Indeed, GC-rich regions were reported to be preferred for DCL activity (Ho et al, 2007), with data supporting that a decrease of viral DNA G+C content from 50 to 42% may negatively affect DCL targeting (Ho et al, 2010). A relatively similar higher accumulation of v-sRNAs in the coding regions and lower accumulation in the AU-rich IR was also reported previously in the case of CaLCuV, TYLCCNV and TYLCSV (Aregger et al, 2012;Miozzi et al, 2013;Yang et al, 2011). Based on the accumulation and the distribution profiles of AGV sRNAs of both polarity strands along the viral genome, dsRNA substrate(s) seem(s) to play major role in their biogenesis, as previously proposed for other geminiviruses (Aregger et al, 2012;Yang et al, 2011).…”

Identification and molecular characterization of a novel monopartite geminivirus associated with mulberry mosaic dwarf disease

“…In line with other geminiviruses (Aregger et al, 2012;Miozzi et al, 2013;Yang et al, 2011), MMDaV-sRNAs of both polarities and with a size distribution profile with prevalent peaks of 21, 22 and 24 nt were identified in the sRNA library from the symptomatic mulberry (Fig. S4a), suggesting that MMDaV-sRNAs are generated by multiple DCLs homologous to DCL1 to -4 of Arabidopsis thaliana.…”

Literature review of baseline information to support the risk assessment of RNAi‐based GM plants

“…It participates in biogenesis of endogenous siRNAs (natsiRNAs) (Brosnan et al, 2007;Borges and Martienssen, 2015). RDR2 is not required for production of viral siRNAs from the Cauliflower mosaic pararetrovirus (Blevins et al, 2011), Cabbage leaf curl geminivirus (Aregger et al, 2012). RDR3 converts PolIV transcripts into dsRNA, which is processed by DCL3 into 24 nt siRNAs loaded onto AGO4 (Pontes et al, 2006;Zhang et al, 2007).…”