In vitro and in vivo application of RNA interference for targeting genes involved in peritrophic matrix synthesis in a lepidopteran system

Abstract

The midgut of most insects is lined with a semipermeable acellular tube, the peritrophic matrix (PM), composed of chitin and proteins. Although various genes encoding PM proteins have been characterized, our understanding of their roles in PM structure and function is very limited. One promising approach for obtaining functional information is RNA interference, which has been used to reduce the levels of specific mRNAs using double-stranded RNAs administered to larvae by either injection or feeding. Although this method is well documented in dipterans and coleopterans, reports of its success in lepidopterans are varied. In the current study, the silencing midgut genes encoding PM proteins (insect intestinal mucin 1, insect intestinal mucin 4, PM protein 1) and the chitin biosynthetic or modifying enzymes (chitin synthase-B and chitin deacetylase 1) in a noctuid lepidopteran, Mamestra configurata, was examined in vitro and in vivo. In vitro studies in primary midgut epithelial cell preparations revealed an acute and rapid silencing (by 24 h) for the gene encoding chitin deacetylase 1 and a slower rate of silencing (by 72 h) for the gene encoding PM protein 1. Genes encoding insect intestinal mucins were slightly silenced by 72 h, whereas no silencing was detected for the gene encoding chitin synthase-B. In vivo experiments focused on chitin deacetylase 1, as the gene was silenced to the greatest extent in vitro. Continuous feeding of neonates and fourth instar larvae with double-stranded RNA resulted in silencing of chitin deacetylase 1 by 24 and 36 h, respectively. Feeding a single dose to neonates also resulted in silencing by 24 h. The current study demonstrates that genes encoding PM proteins can be silenced and outlines conditions for RNA interference by per os feeding in lepidopterans.

Improved insect‐proofing: expressing double‐stranded RNA in chloroplasts

“…It is also possible that RNAi can spread without the need for amplification, if sufficient dsRNA is continuously taken up. Indeed, single high‐dose feeding of dsRNA was less effective than continuous diet‐based application in Coleoptera . There is clear evidence that the RNAi response can be systemic in insects lacking a canonical RdRp .…”

Development of an improved RNA interference vector system for Agrobacterium-mediated plant transformation

“…Total RNA was extracted using TRIzol reagent (Invitrogen) from 2-3 small leaves from 10 T1 plants. The presence of the transgene was confirmed by RT-PCR for pMDC32/McCDA1, pMDC32/McCHS, pMDC32/McIIM4, and pMDC32/McPM1 plants using total leaf RNA and the GUS-specific primers (Fpgus and Rpgus) representing the common spacer element (Table), as previously described (Toprak et al, 2013). (Figure 1, left panel).…”

“…Furthermore, the target of dsRNA, midgut epithelial cells, is accessible through feeding (Turner et al, 2006). Various insect midgut genes, including those encoding structural peritrophic matrix proteins (Toprak et al 2013), digestive enzymes (Rajagopal et al, 2002), chitin deacetylase (Toprak et al, 2013), chitinases (Zhu et al, 2008), and chitin synthase-B (Arakane et al, 2005) have been successfully silenced by RNAi.…”

“…The vector was tested by transforming Arabidopsis plants with 4 different dsRNA constructs specific to genes encoding insect intestinal mucin 1/4 (McIIM1/4) (Shi et al, 2004;Toprak et al, 2010), peritrophic matrix protein 1 (McPM1) (Shi et al, 2004), chitin deacetylase 1 (McCDA1) (Toprak et al, 2008), and chitin synthase-B (McCHS-B) from Mamestra configurata, one of the major pests of Brassica crops in North America. (Toprak et al, 2013). The sense primers (Fpsen and Rpsen) and antisense primers (Fpant and Rpant) used for each amplicon are shown in the Table. In the design of primers, a T7 RNA polymerase promoter was incorporated into the Fpsen and Rpant primers allowing in vitro synthesis of dsRNA from the vectors (Table).…”

Implementing the sterile insect technique with RNA interference – a review

“…Therefore, both non-cell autonomous RNAi and eRNAi occur in a primary cell in direct response to dsRNA, whereas systemic RNAi is initiated in a secondary cell in response to an as yet undefined signal received from a primary cell ( Figure 2). eRNAi can be achieved via the introduction of dsRNAs via food (Asokan et al, 2014;Coleman et al, 2015;Li et al, 2015b;Sandoval-Mojica & Scharf, 2016) or through topical delivery (Toprak et al, 2013;Whyard et al, 2015). For reasons that are as yet not entirely clear, the capacity of insects to express systemic RNAi and eRNAi varies both within and between species (Baum & Roberts, 2014;Li et al, 2015a;Shukla et al, 2016;Sugahara et al, 2017).…”