Development of RNAi-based Control Technologies for Use in Plant Health Emergencies

The primary purpose of this agreement is to support and further advance the proof of concept research for developing RN A interference-based methods to control invasive pests in plant health emergencies. I nsecticide treatments of trees are currently used as a crit ical tool in the fight against the Asian longhorned beetle (ALB) erad ication program. However. the broad spectrum toxicity of these chemical insect icides ra ises environmental concerns and prevents their integration with other environmentally sustainable management strategies such as biological control in natural forest ecosystems. I n contrast, a novel class of target-specific RN Ai­ based technology can be an effect ive alternative to the current ly registered insecticides and has the potential to integrate with natural enemies for effective erad ication of ALB and other pest coleopteran insects within forest ecosystems. RN A interference (RNAi) is a posttranscriptional gene-silencing mechanism where double­ stranded RN As (dsRN A) knockdown genes by targeting mRNA in a cel l. RNAi has recent ly become a powerfu l reverse genet ic tool for fi.mct ional characterizat ion of genes in different model and non-model organisms. I n add it ion, technologies are being developed to apply this method in crop improvement, pest control and RNAi therapeut ics. RN Ai efficiency is variable among insect species ranging from highly efficient (e.g. coleopterans) to relatively inefficient (e.g. lepidopterans). Most of the beetles studied d isplay a robust systemic RN Ai response, except for the d ifferences observed due to the use of d ifferent dsRNA del ivery methods. The application of RNAi technology for pest management by expressing dsRNA in plants to control coleopteran pests such as the corn rootwonn and Colorado potato beetle is under commercial development. A bacterial stra in that is deficient in RNase llI has been developed to mass produce dsRNA at a relatively low cost. Previous work by the Pl using the Colorado potato beet le ident ified heat killed bacteria that prod uced dsRNA that effic ient ly knocked out five housekeeping genes. This resulted in larval mo11ality in about 5-7 days as well as a decrease in feed ing and insect growth. Add it ional target sites to enhance feed ing cessat ion and mortal ity to between 24-48 hours after treatment were also ident ified. Nano formulations of dsRNA have been developed for efficient delivery to insects. Recently, the genome sequence (completed by an i5K funding initiat ive) of ALB was examined and found to have core RNAi pathway genes includ ing Dicer 2, Loquacious, Argonaute 2, SilA, SilC, Rsd3 and R2D2. These data suggest that RN Ai function is conserved in this beetle, and therefore, systemic appl ication (e.g., trunk inject ion or soil irrigation) of dsRNAs may be an effective method to del iver dsRNA to ALB larvae and adu lts.