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Double-stranded RNA (dsRNA)-degrading enzyme (dsRNase) is one of the negative factors influencing the RNA interference (RNAi) effect in Colorado potato beetle (CPB). We have previously shown that plastid-mediated RNAi (PM-RNAi) can be utilized to control CPB. In this study, we aimed to test whether silencing dsRNase could increase the RNAi effect in CPB via PM-RNAi. We generated transplastomic potato (Solanum tuberosum) plants expressing dsRNA targeting shibire (dsSHI) of CPB. St-dsSHI plants (transplastomic plants expressing dsSHI) showed much less potent RNAi response than transplastomic potato plants expressing dsRNAs targeting CPB Shrub (St-dsSHR) or β-Actin (St-dsACT), two CPB-resistant transplastomic lines generated previously. The three types of transplastomic lines have weak (St-dsSHI), moderate (St-dsSHR) and strong (St-dsACT) resistant levels to CPB. Treatment with in vitro-synthesized dsdsRNase1/2 (a fusion dsRNA targeting dsRNase1 and dsRNase2 of CPB) led to enhanced suppression of dsRNase1 and dsRNase2 in CPB larvae fed with St-dsSHI plants and dsRNase2 in CPB larvae fed with St-dsSHR plants. St-dsSHR or St-dsSHI plants showed significantly reduced CPB larva weight or damaged area by CPB feeding. These results demonstrate that dsRNase-silencing approach could improve the effect of PM-RNAi when the silencing of target genes has weak or moderate strength of the insecticidal effects. The findings provide a new proof-of-concept to enhance crop resistance to insect herbivores to via an “RNAi-of-RNAi” approach. [ABSTRACT FROM AUTHOR]