Silencing of Midgut Aminopeptidase N of Spodoptera litura by Double-stranded RNA Establishes Its Role asBacillus thuringiensis Toxin Receptor

Abstract

Insecticidal crystal proteins of Bacillus thuringiensis bind to receptors in the midgut of susceptible insects leading to pore formation and death of the insect. The identity of the receptor is not clearly established. Recently a direct interaction between a cloned and heterologously expressed aminopeptidase (slapn) from Spodoptera litura and the Cry1C protein was demonstrated by immunofluorescence and in vitro ligand blot interaction. Here we show that administration of slapn doublestranded RNA to S. litura larvae reduces its expression. As a consequence of the reduced expression, a corresponding decrease in the sensitivity of these larvae to Cry1C toxin was observed. The gene silencing was retained during the insect's moulting and development and transmitted to the subsequent generation albeit with a reduced effect. These results directly implicate larval midgut aminopeptidase N as receptor for Bacillus thuringiensis insecticidal proteins. The bacterium Bacillus thuringiensis (Bt)1 produces insecticidal crystal proteins, which upon ingestion by susceptible larvae get activated in the midgut, interact with specific receptor and form pores in the epithelium, resulting in the death of the larvae (1). Understanding the mechanism of action of Bt toxin and development of resistance in insects is fundamental in sustaining the use of Cry proteins in integrated pest management. One of the mechanisms of resistance development is an alteration in the binding ability and/or a decrease in the population of receptor molecules, which bind Bt toxin in the insect midgut (2). There have been intense efforts to characterize the nature of this receptor. As a result of several independent experiments employing ligand blot analysis and fluorescent labeling of insecticidal proteins, cadherin and aminopeptidase N (APN) have emerged as main putative receptor molecules (Ref. 3 and references there in). While the role of a receptor molecule in mediating the effect of Cry toxin is acknowledged, the identity of this receptor is still being worked out.Aminopeptidase N from Manduca sexta was the first molecule to be tentatively identified as a Cry toxin-binding protein (4, 5), and APN is the most extensively studied putative receptor, having been identified and isolated subsequently from other lepidopteran insect pests. Independently, a 210-kDa cadherin-like protein from M. sexta was shown to interact with Cry1Ab toxin (6) and later its presence and toxin interaction was also demonstrated from another insect, Bombyx mori (7). Relative abundance of APN in the posterior midgut (8) and lower binding constants of Cry toxin toward cadherin as compared with APN (9) raised apprehension about the role of APN as a receptor for Bt toxin in the insect midgut. Moreover,

In vivo and in vitro knockdown of FREP2 gene expression in the snail Biomphalaria glabrata using RNA interference

“…The efficiency of RNAi varies, depending on the species, tissue, or genes targeted. For example, knockdown of Toll, Dif and Drs genes in adult Drosophila were 26, 47 and 62%, respectively [15], and the knockdown of aminopeptidase (slapn) in Spodoptera litura larvae was up to 95% based on the intensity of RT-PCR bands [14].…”

“…Most in vivo RNAi studies have been performed with invertebrates, mainly arthropods, with the aim of directly assessing phenotype changes. These invertebrates include Caenorhabditis elegans [9,10], digenetic trematodes [11,12], silkworms [13], moth Spodoptera liturs [14], Drosophila [15][16][17], mosquitoes [18,19], honeybee [20], and ticks [21]. In vitro RNAi assays have also been widely applied to cell lines from humans and model organisms such as C. elegans [10], Drosophila [22,23], mosquitoes [24], and mice [25,26].…”

Diet-delivered RNAi in Helicoverpa armigera – Progresses and challenges

“…Nevertheless, efficient downregulation of aminopeptidase-N was reported by microinjection of dsRNA into the haemocoel of Spodoptera litura, but feeding of dsRNA was unsuccessful in generating an RNAi response (Ahmad et al, 2003;Rajagopal et al, 2002). Likewise, microinjection of 15 µg dsRNA achieved 75% silencing of nitrophorin 2 in Rhodnius prolixus, as compared to 42% knockdown when fed 13 µg dsRNA (Araujo et al, 2006).…”

Sequence variation in the cadherin gene of Ostrinia nubilalis: a tool for field monitoring

“…Cadherin was implicated as a resistance factor by phenotypic association in P. gossypiella (Morin et al, 2003) and transposon knockout in H. virescens (Gahan et al, 2001). For Spodoptera litura, transcript knockdown by RNA interference (RNAi) showed the role of aminopeptidase in Bt resistance (Rajagopal et al, 2002). Inheritance of resistance in an O. nubilalis strain selected for survival on Dipels Bt formulations was autosomal and incompletely dominant (Huang et al, 1999).…”