Nucleases as a barrier to gene silencing in the cotton boll weevil, Anthonomus grandis.

Rayssa Almeida Garcia,Muhammad Faheem,Clidia Eduarda Moreira-Pinto,Danila Cabral do Nascimento,Maria Cristina Mattar Silva,Maria Fatima Grossi-de-Sa,François-Xavier Gillet,Angelina Maria Moreschi Basso,Leonardo Lima Pepino Macedo,Juan Luis Jurat-Fuentes

doi:10.1371/journal.pone.0189600

Rayssa Almeida Garcia, Muhammad Faheem + Show 8 more

Open Access

https://doi.org/10.1371/journal.pone.0189600

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Abstract

RNA interference (RNAi) approaches have been applied as a biotechnological tool for controlling plant insect pests via selective gene down regulation. However, the inefficiency of RNAi mechanism in insects is associated with several barriers, including dsRNA delivery and uptake by the cell, dsRNA interaction with the cellular membrane receptor and dsRNA exposure to insect gut nucleases during feeding. The cotton boll weevil (Anthonomus grandis) is a coleopteran in which RNAi-mediated gene silencing does not function efficiently through dsRNA feeding, and the factors involved in the mechanism remain unknown. Herein, we identified three nucleases in the cotton boll weevil transcriptome denoted AgraNuc1, AgraNuc2, and AgraNuc3, and the influences of these nucleases on the gene silencing of A. grandis chitin synthase II (AgraChSII) were evaluated through oral dsRNA feeding trials. A phylogenetic analysis showed that all three nucleases share high similarity with the DNA/RNA non-specific endonuclease family of other insects. These nucleases were found to be mainly expressed in the posterior midgut region of the insect. Two days after nuclease RNAi-mediated gene silencing, dsRNA degradation by the gut juice was substantially reduced. Notably, after nucleases gene silencing, the orally delivered dsRNA against the AgraChSII gene resulted in improved gene silencing efficiency when compared to the control (non-silenced nucleases). The data presented here demonstrates that A. grandis midgut nucleases are effectively one of the main barriers to dsRNA delivery and emphasize the need to develop novel RNAi delivery strategies focusing on protecting the dsRNA from gut nucleases and enhancing its oral delivery and uptake to crop insect pests.

Highlights

The availability of different insect’s transcriptomes allows the evaluation and identification of genes that can be potentially used for insect control using different biotechnological approaches [1,2,3]
The main advantage of RNA interference technology in controlling crop insect pests is the high specificity for the target gene
The Double-stranded RNA (dsRNA) concentration and length are critical for the effectiveness of the RNA interference (RNAi) response because these features affect the cellular uptake of dsRNA [28, 76,77,78]

Summary

Introduction

The availability of different insect’s transcriptomes allows the evaluation and identification of genes that can be potentially used for insect control using different biotechnological approaches [1,2,3]. Adult females lay eggs inside cotton buds and squares, where the larvae break out and feed on the plant tissue [9, 10]. This CBW’s endophytic habit substantially affects cotton buds flowering and thereby, the productivity, making its control difficult by using chemical pesticides. CBW takes approximately three weeks until reaching adulthood and each female can lay up to 300 eggs in cotton buds and squares, being able to generate 7 to 10 generations per year [3]. Adult CBW can live about 20 to 40 days, and its life cycle can last approximately 50 days [14]

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