Microbial-Based Double-Stranded RNA Production to Develop Cost-Effective RNA Interference Application for Insect Pest Management

Abstract

RNA interference (RNAi) is a convenient tool to identify and characterize biological functions in organisms. Recently, it has become an alternative to chemical insecticides as a biologically based control agent. This promising technology has the potential to avoid many problems associated with conventional chemical insecticides. In order for RNAi application to be practical for field use, a major hurdle is the development of a cost-effective system of double-stranded RNA (dsRNA) production for a large quantity of dsRNA. A handful of research reports has demonstrated microbial-based dsRNA production using L4440 vector and HT115 (DE3) Escherichia coli for application to vertebrate and invertebrate systems. However, the dsRNA yield, production efficiency, and biological purity from this in vitro system is still unclear. Thus, our study detailed biochemical and molecular tools for large-scale dsRNA production using the microbial system and investigated the production efficiency and yield of crude and purified dsRNAs. An unrelated insect gene, green fluorescent protein (GFP), and an insect neuropeptide gene, pyrokinin (PK) identified from Drosophila suzukii, were used to construct the recombinant L4440 to be expressed in the HT115 (DE3) cell. A considerable amount of dsRNA, 19.5 µg/mL of liquid culture, was isolated using ultrasonic disruption followed by phenol extraction. The sonication method was further evaluated to extract crude dsRNA without the additional phenol extraction and nuclease treatments and also to reduce potential bacterial viability. The results suggest that the ultrasonic method saved time and costs to isolate crude dsRNA directly from large volumes of cell culture without E coli contamination. We investigated whether the injection of PK dsRNA into flies resulted in increased adult mortality, but it was not statistically significant at 95% confidence level. In this study, the microbial-based dsRNA production has potential for applied RNAi technology to complement current insect pest management practices.

Keywords: RNAi; bacterial dsRNA production; pest control; spotted wing drosophila.

Figure 1.

Schematic diagram of the bacterial-expressed double-stranded RNA (dsRNA) production system. (A) The target gene fragment is inserted in the multiple-cloning site between two T7 promoter regions in inverted orientation in the expression vector (L4440), which is then transformed into the RNase III-deficient E coli strain HT115 (DE3). IPTG induces RNA transcription mediated by T7 promoter. The dsRNA produced can be purified either by phenol/chloroform/isoamyl alcohol extraction or by spin-column filtration. (B) The target dsRNAs isolated from bacterial culture were analyzed by separation through a 1.2% agarose gel by electrophoresis. White arrows indicate the target dsRNAs. Amp^R indicates ampicillin resistance gene; GFP, green fluorescence gene dsRNA; IPTG, isopropyl β-d-1-thiogalactopyranoside; M, GeneRuler 1 kb DNA Ladder (Thermo Scientific); PK, pyrokinin gene dsRNA.

Figure 2.

Bacterial growth and induction of dsRNA production by IPTG. (A) Various concentrations of IPTG were treated at the mid-exponential phase (arrow). (B) GFP-dsRNA extracted from different IPTG treatments were run by 1.2% agarose gel electrophoresis. M indicates TrackIt 1Kb Plus DNA Ladder (Invitrogen). (C) Digestion of dsRNA by various nucleases. The purified bacterial GFP-dsRNA (Lane 1) was degraded by RNase III (Lane 2), but not by DNase I (Lane 3) or RNase A (Lane 4). dsRNA indicates double-stranded RNA; GFP, green fluorescent protein; IPTG, isopropyl β-d-1-thiogalactopyranoside; M, GeneRuler 1 kb DNA Ladder (Thermo Scientific).

Figure 3.

Effect of pre-treatments on cell lysis and dsRNA isolation by different lysis methods. Sonication or heating was performed before the conventional dsRNA isolation using phenol/chloroform/isoamyl alcohol. The dsRNA crude extracts were diluted 100 times and 10 μL aliquots were run in 1.2% agarose gel electrophoresis. Arrow indicates the target dsRNA (GFP-dsRNA). dsRNA indicates double-stranded RNA; GFP, green fluorescent protein; M, TrackIt 1Kb Plus DNA Ladder (Invitrogen); C, control; S, sonication; H, heating.

Figure 4.

Bacterial cell lysis before and after sonication. An aliquot (100 μL) of 1/100 dilution of cell suspension or supernatant was spread on LB agar plate supplemented with ampicillin and tetracycline. (A) Cell suspension without sonication, (B) cell suspension with sonication, (C) supernatant of the cell suspension without sonication, and (D) supernatant of the cell suspension with sonication.