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,

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Several investigators have independently identified membrane-associated aminopeptidases in the midgut of insect larvae as the initial interacting ligand to the insecticidal crystal proteins of Bacillus thuringiensis. Though several isoenzymes of aminopeptidases have been identified from the midgut of an insect and their corresponding cDNA cloned, only one of the isoform has been expressed heterologously and studied for its binding to Cry toxins. Here we report the cloning and expression of two aminopeptidases N from Helicoverpa armigera (American cotton bollworm) (HaAPNs). The full-length cDNA of H. armigera APN1 (haapn1) is 3205 bp in size and encodes a 1000-amino-acid protein, while H. armigera APN2 (haapn2) is 3116 bp in size and corresponds to a 1012-amino-acid protein. Structurally these proteins show sequence similarity to other insect aminopeptidases and possess characteristic aminopeptidase motifs. Both the genes have been expressed in Trichoplusia ni (cabbage looper) cells using a baculovirus expression vector. The expressed aminopeptidases are membrane-associated, catalytically active and glycosylated. Ligand-blot analysis of both these aminopeptidases with bioactive Cry1Aa, Cry1Ab and Cry1Ac proteins displayed differential interaction. All the three toxins bound to HaAPN1, whereas only Cry1Ac interacted with HaAPN2. This is the first report demonstrating differential Cry-toxin-binding abilities of two different aminopeptidases from a susceptible insect.

The sap-sucking insects (order Hemiptera), including aphids, planthoppers, whiteflies and stink bugs, present one of the greatest challenges for pest management in global agriculture. Insect neurotoxins offer an alternative to chemical insecticides for controlling these pests, but require delivery into the insect hemocoel. Here we use the coat protein of a luteovirus, an aphid-vectored plant virus, to deliver a spider-derived, insect-specific toxin that acts within the hemocoel. The luteovirid coat protein is sufficient for delivery of fused proteins into the hemocoel of pea aphids, Acyrthosiphon pisum, without virion assembly. We show that when four aphid pest species-A. pisum, Rhopalosiphum padi, Aphis glycines and Myzus persicae-feed on a recombinant coat protein-toxin fusion, either in an experimental membrane sachet or in transgenic Arabidopsis plants, they experience significant mortality. Aphids fed on these fusion proteins showed signs of neurotoxin-induced paralysis. Luteovirid coat protein-insect neurotoxin fusions represent a promising strategy for transgenic control of aphids and potentially other hemipteran pests.

Development of ways to block virus transmission by aphids could lead to novel and broad-spectrum means of controlling plant viruses. Viruses in the Luteoviridae enhanced are obligately transmitted by aphids in a persistent manner that requires virion accumulation in the aphid hemocoel. To enter the hemocoel, the virion must bind and traverse the aphid gut epithelium. By screening a phage display library, we identified a 12-residue gut binding peptide (GBP3.1) that binds to the midgut and hindgut of the pea aphid Acyrthosiphon pisum. Binding was confirmed by labeling the aphid gut with a GBP3.1-green fluorescent protein fusion. GBP3.1 reduced uptake of Pea enation mosaic virus (Luteoviridae) from the pea aphid gut into the hemocoel. GBP3.1 also bound to the gut epithelia of the green peach aphid and the soybean aphid. These results suggest a novel strategy for inhibiting plant virus transmission by at least three major aphid pest species.