Microbial-based double-stranded RNA production to develop cost-effective RNA interference application for insect pest management

Authors: AHN, SEUNG-JOON - Oregon State University; Donahue, Kelly; KOH, YOUNGHO - Hallym University; Martin, Robert; Choi, Man-Yeon

Submitted to: International Journal of Insect Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/5/2019
Publication Date: 4/24/2019
Citation: Ahn, S., Donahue, K.M., Koh, Y., Martin, R.R., Choi, M.Y. 2019. Microbial-based double-stranded RNA production to develop cost-effective RNA interference application for insect pest management. International Journal of Insect Science. 11:1-8. https://doi.org/10.1177/1179543319840323.
DOI: https://doi.org/10.1177/1179543319840323

Interpretive Summary: RNA interference (RNAi) is a post-transcriptional gene silencing mechanism that is initiated by the presence of double-stranded RNA (dsRNA). During the past two decades, research by academic and commercial entities on the application of RNAi technology for insect pest management has led to great advancements. Although RNAi technology is a new approach and promising tool for insect pest management, there are still technical challenges to successfully develop a next generation pesticide. RNAi approach to pest management presents three major challenges: 1) identifying a suitable target gene and/or physiological system; 2) developing suitable RNAi delivery into the target pest; and 3) providing cost-effective dsRNA production. Scientists from USDA-ARS and Oregon State University, Corvallis, OR, developed a microbial-based dsRNA production for providing large quantities of dsRNA. DsRNA yields obtained from crude and purified stages were determined and evaluated with isolation methods used to extract dsRNA in cell culture. These methods of expression and isolation have potential towards the development of biologically-based pest management.

Technical Abstract: RNAi is a convenient tool to identify and characterize biological functions in organisms. 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 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) E. 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 fluorescence 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 per 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, 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 the E coli contamination. Microbial-based dsRNA production has potential for applied RNAi technology to complement current insect pest management practices.