Double-stranded RNA (dsRNA)-mediated gene silencing, or RNA interference (RNAi), is an emerging biotechnology that has been a breakthrough tool for crop protection. Exogenous dsRNA triggers the RNAi pathway, silences genes, disrupts protein function, and can cause insect mortality. However, effective delivery of the dsRNA is problematic, particularly in systems with long-lived, endophagous insects such as the emerald ash borer (EAB), Agrilus planipennis, a tree-killing nonnative invader that attacks ash, Fraxinus spp. Larvae feed on cambial tissue causing rapid tree death. EAB is susceptible to RNAi, but we lack a practical means of delivery. Here we evaluated delivery of dsRNA to green, F. pennsylvanica, and tropical ash, F. uhdei, through root and/or petiole absorption, and also demonstrated dsRNA absorption through the EAB egg chorion. We labeled exogenous dsRNA using a fluorescing label and then used confocal microscopy and RT-qPCR to evaluate its distribution in plant and insect tissues. Labeled dsRNAs are detectable in root, stem, and leaf tissues 48-h postapplication. In excised ash branches, labeled dsRNA is detectable in the inner bark and in recovered EAB neonates 8-day postapplication. Eggs and larvae emerging from treated eggs also presented fluorescing dsRNA under confocal imaging. Adult EAB-fed tropical ash leaves treated with in vitro synthesized EAB-specific dsSHI through petiole absorption experience a significant knockdown of the shi gene and a significant mortality. Our findings provide a proof of concept that delivery of dsRNAs through topical or systemic application methods is a feasible means of suppressing EAB, providing hope for future tree protection.

The sequence complementarity of the RNA interference (RNAi) pathway allows for targeted suppression of genes essential for insect survival, and enables development of pest management strategies specific to a given species while reducing the likelihood of adversely impacting non-target organisms (NTOs). The feasibility of manipulating the RNAi pathway to cause mortality in the highly invasive emerald ash borer (EAB) has been demonstrated. Here the spectrum of activity of three double stranded RNAs (dsRNAs) targeting the genes hsp, shi, and sn-rnp in EAB was evaluated in model insects representing five functional guilds including herbivore, predator, detritivore, pollinator, parasitoid; the last represented by the classical biological control agents currently deployed for EAB management in North America. All NTOs were exposed to EAB-specific dsRNAs in diet bioassays that measured potential lethal effects. Gene expression and in silico analysis were also assessed on NTOs for which gene sequences were publicly available. Bioassays demonstrated no lethal effects on our model insects, suggesting a narrow spectrum of activity for the three EAB-specific dsRNAs evaluated. The gene expression and in silico analyses suggest potential sublethal effects on our model pollinator; however we found no effects on insect survival. Overall, our results suggest no adverse effects of the RNAi strategy targeting EAB genes on the survival of the selected non-target organisms we evaluated. The results from this study provide guidance for future RNAi risk analyses that will allow this technology to move forward to a deployment stage.

All North American ash (Fraxinus spp.) species are threatened by the emerald ash borer (EAB; Agrilus planipennis), an exotic beetle which has already destroyed millions of ash trees in the U.S. and Canada. Although both chemical insecticides and biological control can be effective, and host resistance appears possible, the speed of the invasion has defied traditional management approaches. One potential, innovative approach to managing this destructive insect is to develop a host tree-induced gene silencing strategy using RNA interference (RNAi) constructs targeting EAB-specific genes. An important requirement for applying RNAi technology is a reliable transformation/regeneration system for the host tree species. We developed an Agrobacterium-mediated gene transfer system for white ash (F. americana) and green ash (F. pennsylvanica) using the embryogenic cultures of these species as target material. Embryogenic suspension cultures of multiple genotypes of both species were plated and inoculated with A. tumefaciens carrying the pFHI-GUSi expression vector, which carries the nptII selectable marker and intron-GUS reporter genes, followed by selection on a semi-solid medium containing geneticin. Putative transgenic events showed expression of the GUS gene at all tested developmental stages from callus to plantlets, and transgene presence in the leaves of regenerated plants was confirmed using PCR. The overall average transformation efficiency achieved was 14.5 transgenic events per gram of tissue. Transgenic somatic seedlings of two white ash and three green ash genotypes were produced and acclimated to greenhouse conditions.