RNA interference (RNAi)-based technology has been proven as a novel approach for insect pest control. However, whether insects could evolve resistance to RNAi and the underlying mechanism is largely unknown. The target gene mutations were thought to be one of the potential ways to develop the resistance. Here we predicted the effective siRNA candidates that could be derived from dsRNA against the Colorado potato beetle (CPB) β-Actin gene (dsACT). By site-directed mutagenesis, we synthesized the dsRNAs with the defect in generation of effective siRNAs (and thus were supposed to have comparable low RNAi efficacy). We showed that, with mismatches to the target gene, all the dsRNA variants caused similar levels of silencing of target gene, mortality and larval growth retardation of CPB. Our results suggest that when the mismatch rate of dsACT and target β-Actin mRNA is less than 3%, the RNAi efficiency is not impaired in CPB, which might imply the low possibility of RNAi resistance evolving through the sequence mismatches between dsRNA and the target gene.

Transplastomic potato plants expressing double-stranded RNA (dsRNA) targeted against essential genes of the Colorado potato beetle (CPB) can be lethal to larvae by triggering an RNA interference (RNAi) response. High accumulation levels of dsRNAs in plastids are crucial to confer an efficient RNAi response in the insects. However, whether length and sequence of the dsRNA determine the efficacy of RNAi and/or influence the level of dsRNA accumulation in plastids is not known. Here we compared the RNAi efficacy of different lengths of dsRNA targeted against the CPB β – Actin gene ( ACT ) by feeding in vitro -synthesized dsRNAs to larvae. We show that, while the 60 bp dsRNA induced only a relatively low RNAi response in CPB, dsRNAs of 200 bp and longer caused high mortality and similar larval growth retardation. When the dsRNAs were expressed from the plastid (chloroplast) genome of potato plants, we found that their accumulation levels were correlated with length. dsRNA accumulation levels were positively associated with the observed mortality, suppression of larval growth and suppression of target gene expression. Importantly, transplastomic potato plants expressing the 200 bp dsRNA were better protected from CPB than plants expressing the 297 bp dsRNA, the best-performing line in our previous study. Our results suggest that the length of dsRNAs is an important factor that influences their accumulation levels in plastids and thus determines the strength of the insecticidal RNAi effect. Our findings will aid the design of optimized dsRNA expression constructs for plant protection by plastid-mediated RNAi.