Expression of dsRNA

The L4440 plasmid, comprising two T7 promoters in inverted orientation flanking the multiple cloning site, was applied for target dsRNA inducible expression. Double restriction enzyme digestion was used to cut pMD-18T-genes and L4440 plasmid, respectively. The restriction enzyme digestion sites were checked by using Primer Premier 5.0 to ensure their presence exclusively in pMD-18T and L4440 plasmid, other than in that of the target genes (Table S2). The target fragments were excised from pMD-18T-genes and ligated to L4440 plasmid by T4 DNA Ligase (TaKaRa, China). The recombinant vectors (L4440-lola, -topi, -per, -aly, -rac, -rho, -upd, -magu) were transformed to Escherichia coli HT115 competent cells which lack RNase III (Competent Cell Preparation Kit, TaKaRa, China). Single colonies of HT115 were cultured in Luria Broth (LB) media at 37 °C with shaking at 220 rpm overnight. The culture was diluted 100-fold in 2 L LB with 100 μg/ml ampicillin cultured at 37 °C and 0.6 optical density 600. Synthesis of T7 polymerase was induced with 0.4 mM IPTG and the bacteria were incubated with shaking for an additional 4 h at 37 °C. Bacteria solutions of HT115 were centrifuged at 4,000 g for 5 min and re-suspended in 4 ml distilled water to condense the concentration to 500×.