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. 2020 Jul 16;11(7):449.
doi: 10.3390/insects11070449.

Low Mismatch Rate between Double-Stranded RNA and Target mRNA Does Not Affect RNA Interference Efficiency in Colorado Potato Beetle

Wanwan He  1 , Wenbo Xu  1 , Kaiyun Fu  2 , Wenchao Guo  3 , Jiang Zhang  1
Affiliations

Low Mismatch Rate between Double-Stranded RNA and Target mRNA Does Not Affect RNA Interference Efficiency in Colorado Potato Beetle

Wanwan He et al. Insects. .

Abstract

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.

Keywords: Colorado potato beetle; RNA interference; double-stranded RNA; resistance development; sequence mutation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Introduction and in vitro synthesis of dsACTs with point mutations. (A) The relative location of effective siRNA candidates at ACT200a which were predicted by indicated bioinformatic toolboxes (DNAMAN, siDirect and siRNA Wizard). (B) Alignment of original ACT200a sequence with mutated ACT200a variants. The high consensus base pairs are represented as blue. The mutated base pairs are indicated as red. (C) Summary of the number and location of mutations in (B). (D) Analysis of the synthesized dsACTs by electrophoresis. M, 1 kb DNA ladder.
Figure 2
Figure 2
RNAi effects on Colorado potato beetle (CPB) larvae fed with different in vitro synthesized dsRNA variants. (A) Kaplan-Meier survival curves of third-instar CPB larvae fed with potato leaves that had been painted with identical amounts (4 ng/cm2) of indicated dsACTs. The log-rank test was used to assess the significance of differences between two survival curves. *** p < 0.001; NS, not significant. (B) Mean weight of surviving CPB larvae at the indicated days of feeding. Data are means ± SE (n = 30). (C) Relative expression levels of β-Actin in the CPB larvae in (A) at day 3. Gene expression levels were set as one in CPB larvae fed with H2O-painted control leaves. Data are means ± SE (n = 3). The letters above each bar in (B,C) indicate the significance of differences as determined by one-way ANOVA in SPSS (Bonferroni’s test, p < 0.05).
Figure 3
Figure 3
Leaves painted by H2O and dsACT200a and its mutations were fed to third-instar CPB after 3 days.
See this image and copyright information in PMC

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