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

Flavia Pampolini,Thais B Rodrigues,Tomokazu Kawashima,Lynne K Rieske,Ramya S Leelesh

doi:10.1007/s10340-020-01230-w

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.

Highlights

The RNA interference (RNAi) pathway is a cellular regulatory mechanism that limits the expression of target genes, resulting in gene silencing
emerald ash borer (EAB) neonate larvae were recovered from F. udhei branches exposed to labeled double-stranded RNA (dsRNA), and confocal imaging of the insect tissue (Fig. 7a) confirms the uptake of labeled dsRNA by larvae that fed on treated ash branches exposed to labeled dsRNA
EAB neonate larvae that emerged from eggs exposed to labeled dsRNA were evaluated, and the labeled dsRNA was present within the larvae (Fig. 7b, c)

Summary

Introduction

The RNA interference (RNAi) pathway is a cellular regulatory mechanism that limits the expression of target genes, resulting in gene silencing. The pathway is triggered by the introduction of double-stranded RNA (dsRNA), which is processed by the enzyme dicer into small interference RNAs (siRNAs). The targeted mRNA is cleaved, preventing the translation of the gene, causing its silencing (Agrawal et al 2003; Meister and Tuschl 2004). The RNAi mechanism involves silencing specific 19–21 base pair sequences (siRNAs) of target genes in a given species or closely related species (Bachman et al 2013). As a pest management strategy, this characteristic makes the mechanism more specific than conventional pesticides, minimizing the risk to nontarget and beneficial organisms (Baum and Roberts 2014; Bachman et al 2016). RNAi is emerging as a next-generation pest control strategy and as a tool to manage various agricultural insect pests and pathogens (Mamta and Rajam 2017)

Methods

Results

Conclusion