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.

Prospects, challenges and current status of RNAi through insect feeding

“…A large‐scale production for potential pest management may not yet be cost efficient. In addition, dsRNA delivery via recombinant microorganisms or viruses holds a potential risk of infection in non‐target organisms, but this approach might be useful to produce and extract dsRNA on a large scale . In conclusion, silencing genes by simply supplementing diets with synthetic dsRNA currently seems to be more suitable for laboratory research rather than field applications.…”

C. elegans electrotaxis behavior is modulated by heat shock response and unfolded protein response signaling pathways

“…S7). Next, worms were fed with two plant pathogens, Agrobacterium tumefaciens and Pectobacterium carotovorum [57][58][59] and a nonpathogenic fungus, Cryptococcus aquaticus. Plant pathogens have been reported to have a detrimental effect on the lifespan of worms 60 .…”

A novel plasmid–Escherichia coli system produces large batch dsRNAs for insect gene silencing

“…The loop structure was generated together with VG‐R, avoiding the trouble of additional PCR amplification of the loop sequence. dsRNA induced in each E. coli system was under the optimal conditions of L4440‐HT115(DE3), which ensures the accuracy of the results.…”

“…When the OD 600nm value reached 0.4, isopropyl β‐ d ‐1‐thiogalactopyranoside (1 m m ) was added and then incubated for 4 h under the same conditions. L4440‐vg was transformed into HT115(DE3) and BL21(DE3) RNase III‐ respectively to express ds vg according to the method described by Ahn et al …”