Intron-assisted, viroid-based production of insecticidal circular double-stranded RNA in .

RNA interference (RNAi) is a natural mechanism for protecting against harmful genetic elements and regulating gene expression, which can be artificially triggered by the delivery of homologous double-stranded RNA (dsRNA). This mechanism can be exploited as a highly specific and environmentally friendly pest control strategy. To this aim, systems for producing large amounts of recombinant dsRNA are necessary. We describe a system to efficiently produce large amounts of circular dsRNA in and demonstrate the efficient insecticidal activity of these molecules against Western corn rootworm (WCR, LeConte), a highly damaging pest of corn crops. In our system, the two strands of the dsRNA are expressed in embedded within the very stable scaffold of (ELVd), a small circular non-coding RNA. Stability in of the corresponding plasmids with long inverted repeats was achieved by using a cDNA coding for a group-I autocatalytic intron from as a spacer. RNA circularization and large-scale accumulation in cells was facilitated by co-expression of eggplant tRNA ligase, the enzyme that ligates ELVd during replication in the host plant. The inserted intron efficiently self-spliced from the RNA product during transcription. Circular RNAs containing a dsRNA moiety homologous to () gene exhibited excellent insecticide activity against WCR larvae. Finally, we show that the viroid scaffold can be separated from the final circular dsRNA product using a second self-splicing intron in a permuted form.