UV‐LASER adjuvant–surfactant‐facilitated delivery of mobile dsRNA to tomato plant vasculature and evidence of biological activity by gene knockdown in the potato psyllid

Abstract

BackgroundDouble‐stranded RNA (dsRNA) biopesticides are of interest for abatement of insect vectors of pathogenic bacteria such as ‘Candidatus Liberibacter’, which infects both its psyllid and plant hosts. Silencing of genes essential for psyllids, or for Liberibacter invasion, is anticipated to lead to mortality or impeded bacterial multiplication. Foliar delivery is preferred for biopesticide application; however, the cuticle impedes dsRNA penetration into vasculature. Here, conditions were established for wounding tomato leaves using UV‐LASER to promote dsRNA penetration into leaves and vasculature.ResultsUV‐LASER treatment with application of select adjuvants‐surfactants resulted in vascular delivery of 100‐, 300‐, and 600‐base pair (bp) dsRNAs that in general were correlated with size. The 100‐bp dsRNA required no pre‐treatment, while 300‐bp and 600‐bp dsRNAs entered the vasculature after UV‐LASER treatment only, and UV‐LASER‐adjuvant/surfactant treatment, respectively. Of six adjuvant/surfactants evaluated, the plant‐derived oil combined with an anionic organosilicon compound performed most optimally. Localization of dsRNAs in the tomato vasculature was documented by fluorometry and fluorescence confocal microscopy. The biological activity of in planta‐delivered dsRNA (200‐250bp) was determined by feeding third instar psyllids on tomato leaves post‐UV‐LASER‐adjuvant/surfactant treatment, with or without psyllid cdc42‐ and gelsolin dsRNAs. Gene knockdown was quantified by quantitative, real‐time RT‐PCR amplification. At 10‐days post‐IAP, knockdown of cdc42 and gelsolin expression was 61% and 56%, respectively, indicating the dsRNAs delivered to the tomato vasculature were mobile and biologically active.ConclusionResults indicated that UV‐LASER‐adjuvant/surfactant treatments facilitated the delivery of mobile, biologically‐active dsRNA molecules to the plant vasculature.This article is protected by copyright. All rights reserved.

Optimal dsRNA Concentration for RNA Interference in Asian Citrus Psyllid

“…Consequently, transgene expression or other means of deployment of dsRNA in planta will likely be necessary to achieve an optimal concentration of continuously available dsRNA that persists in the vascular tissue, in particular, the phloem where psyllids feed. Alternatively, stabilizing dsRNA for foliar or soil applications with time-release technology and/or combined with translaminar adjuvants and surfactants [39] may offer alternative solutions.…”

“…The template for dsRNA synthesis was a 200-250 bp fragment of the coding region for each gene. The optimal dsRNA size of <300 bp was selected based on a previous study [39] that evaluated different lengths of dsRNA on biological activity for the potato psyllid Ca. L. solanacearum (CLso) study system, a fast-track surrogate study system for ACP-CLas [39].…”

“…The optimal dsRNA size of <300 bp was selected based on a previous study [39] that evaluated different lengths of dsRNA on biological activity for the potato psyllid Ca. L. solanacearum (CLso) study system, a fast-track surrogate study system for ACP-CLas [39]. The off-target potential for each gene target was analyzed bioinformatically by considering all possible 21-mers that corresponded to siRNAs expected to result from dsRNA processing by dicer.…”