Confocal microscopy provides visual evidence and confirms the feasibility of dsRNA delivery to emerald ash borer through plant tissues

Abstract

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.

Emerald Ash Borer Specific Gene Silencing Has No Effect on Non-target Organisms

“…Safety protocols dictate that exposures used for toxicity studies with non-target arthropods must exceed the maximum amount of dsRNA expected to be available in the environment (EFSA European Food Safety Authority, 2014). Previous studies demonstrated that adult EAB experiences significant gene silencing and mortality when exposed to ash leaves treated with dsRNA solution at 70 ng/µl (Pampolini et al, 2020). In the present study, our model NTOs were exposed to EAB dsRNAs in diets at concentrations ∼140 times higher than that inducing mortality in EAB delivered topically on plant foliage.…”

“…Implementation of crop protection strategies applying RNAi technology are under development (Poelchau et al, 2016;Vogel et al, 2019). Potential applications of environmental or dietary RNAi for pest control include applications of dsRNA in foliar sprays (San Miguel and Scott, 2016); systemic uptake through plant material (Hunter et al, 2012;Dalakouras et al, 2018;Pampolini et al, 2020), oral administration of dsRNA in baits (Zhou et al, 2008), embedded in nanoparticles (Zhang et al, 2010;Yan et al, 2020), expressed in genetically engineered microorganisms (Zhu et al, 2011;Joga et al, 2016), or expressed in transgenic plants (Baum et al, 2007;Mao et al, 2007;Ghag, 2017;Zhang et al, 2017).…”

“…In addition to its application for agricultural purposes, RNAi technology has been investigated in forest pests including the southern pine beetle, Dendroctonus frontalis (Coleoptera: Curculionidae) (Kyre et al, 2019), the congeneric mountain pine beetle, D. ponderosae (Kyre et al, 2020), the wood-boring Asian longhorned beetle, Anoplophora glabripennis (Coleoptera: Cerambycidae) (Rodrigues et al, 2017a), and the emerald ash borer (EAB), Agrilus planipennis Fairmaire (Coleoptera: Buprestidae) (Rodrigues et al, 2017b(Rodrigues et al, , 2018Pampolini et al, 2020).…”

“…throughout its invaded range (Cappaert et al, 2005;Poland and McCullough, 2006), devastating urban forests, posing a threat to forest ecosystems, reducing biodiversity, and causing unprecedented economic losses (Aukema et al, 2011). Fortunately, previous studies evaluating the susceptibility of EAB to RNAi (Zhao et al, 2015) and the effectiveness of this technology against this pest (Rodrigues et al, 2017b(Rodrigues et al, , 2018Pampolini et al, 2020) shows promise, and suggests that RNAi could be a viable option when implemented as a component of an integrated management strategy for EAB.…”

Oral Ingestion of Bacterially Expressed dsRNA Can Silence Genes and Cause Mortality in a Highly Invasive, Tree-Killing Pest, the Emerald Ash Borer

“…Recombinant bacteria with EAB-specific dsRNA could be sprayed on foliage to be ingested by feeding beetles or on ash stems to be ingested by newly hatched neonates. Naked dsRNAs applied topically or through root drenching can be assimilated into and moved through ash plant tissues [ 27 ], suggesting crude extract of bacterially-expressed dsRNA may also be able to be translocated through the plant via soil drench, trunk sprays or injection [ 28 ]. Recombinant bacteria producing EAB-specific dsRNAs also make the genetic transformation of ash trees with dsRNA-expressing constructs a more plausible goal [ 29 ].…”