A Naturally Occurring Plant Cysteine Protease Possesses Remarkable Toxicity against Insect Pests and Synergizes Bacillus thuringiensis Toxin

When caterpillars feed on maize (Zea maize L.) lines with native resistance to several Lepidopteran pests, a defensive cysteine protease, Mir1-CP, rapidly accumulates at the wound site. Mir1-CP has been shown to inhibit caterpillar growth in vivo by attacking and permeabilizing the insect's peritrophic matrix (PM), a structure that surrounds the food bolus, assists in digestion and protects the midgut from microbes and toxins. PM permeabilization weakens the caterpillar defenses by facilitating the movement of other insecticidal proteins in the diet to the midgut microvilli and thereby enhancing their toxicity. To directly determine the toxicity of Mir1-CP, the purified recombinant enzyme was directly tested against four economically significant Lepidopteran pests in bioassays. Mir1-CP LC50 values were 1.8, 3.6, 0.6, and 8.0 ppm for corn earworm, tobacco budworm, fall armyworm and southwestern corn borer, respectively. These values were the same order of magnitude as those determined for the Bacillus thuringiensis toxin Bt-CryIIA. In addition to being directly toxic to the larvae, 60 ppb Mir1-CP synergized sublethal concentrations of Bt-CryIIA in all four species. Permeabilization of the PM by Mir1-CP probably provides ready access to Bt-binding sites on the midgut microvilli and increases its activity. Consequently, Mir1-CP could be used for controlling caterpillar pests in maize using non-transgenic approaches and potentially could be used in other crops either singly or in combination with Bt-toxins

Identification of a Novel Aminopeptidase P-Like Gene (OnAPP) Possibly Involved in Bt Toxicity and Resistance in a Major Corn Pest (Ostrinia nubilalis)

Studies to understand the Bacillus thuringiensis (Bt) resistance mechanism in European corn borer (ECB, Ostrinia nubilalis) suggest that resistance may be due to changes in the midgut-specific Bt toxin receptor. In this study, we identified 10 aminopeptidase-like genes, which have previously been identified as putative Bt toxin receptors in other insects and examined their expression in relation to Cry1Ab toxicity and resistance. Expression analysis for the 10 aminopeptidase-like genes revealed that most of these genes were expressed predominantly in the larval midgut, but there was no difference in the expression of these genes in Cry1Ab resistant and susceptible strains. This suggested that altered expression of these genes was unlikely to be responsible for resistance in these ECB strains. However, we found that there were changes in two amino acid residues of the aminopeptidase-P like gene (OnAPP) involving Glu305 to Lys305 and Arg307 to Leu307 in the two Cry1Ab-resistant strains as compared with three Cry1Ab-susceptible strains. The mature OnAPP contains 682 amino acid residues and has a putative signal peptide at the N-terminus, a predicted glycosylphosphatidyl-inositol (GPI)-anchor signal at the C-terminal, three predicted N-glycosylation sites at residues N178, N278 and N417, and an O-glycosylation site at residue T653. We used a feeding based-RNA interference assay to examine the role of the OnAPP gene in Cry1Ab toxicity and resistance. Bioassays of Cry1Ab in larvae fed diet containing OnAPP dsRNA resulted in a 38% reduction in the transcript level of OnAPP and a 25% reduction in the susceptibility to Cry1Ab as compared with larvae fed GFP dsRNA or water. These results strongly suggest that the OnAPP gene could be involved in binding the Cry1Ab toxin in the ECB larval midgut and that mutations in this gene may be associated with Bt resistance in these two ECB strains

Recherche de la toxicité de diverses souches de Bacillus thuringiensis Berliner vis-à-vis de trois espèces de limaces

La toxicité de plusieurs préparations à base de Bacillus thuringiensis a été montrée vis-à-vis de la limace grise Deroceras reticulatum (Müller) et de l'escargot aquatique Biomphalaria alexandrina (Ehrenberg) par de précédents auteurs, laissant envisager une méthode de lutte microbiologique contre les gastéropodes. C'est pourquoi la toxicité de certains de ces produits commerciaux et de diverses souches non formulées ayant des activités insecticides différentes a été éprouvée sur trois espèces de limaces : D reticulatum, Arion distinctus Mabille et Limax valentianus Férussac et comparée à celle de produits molluscicides de référence. Parmi les produits testés, deux étaient à base de β-exotoxine (Bitoxibacillin 5 et le surnageant H1). Cependant, aucune préparation à base de Bacillus ne s'est montrée toxique vis-à-vis des trois espèces de limaces. Seuls les produits molluscicides ont confirmé leur efficacité contre les limaces.Research on the toxicity of various Bacillus thuringiensis Berliner strains to three species of slugs. Toxicity of several Bacillus thuringiensis preparations had been shown against the grey field slug Deroceras reticulatum (Müller) and the aquatic snail Biomphalaria alexandrina (Ehrenberg) by previous authors, letting view a method of microbiological control of gastropods. Therefore, the toxicities of some of these products and of several unformulated strains, having different insecticide activities, were studied on three species of slugs: D reticulatum, Arion distinctus Mabille and Limax valentianus Férussac, and compared to molluscicide products of reference. Amongst the products tested, two contained the β-exotoxin (Bitoxibacillin 5 and the surnatant H1). However, no strain of Bacillus showed to be toxic for the three slug species. Only the molluscicide products confirmed their efficiency against the slugs

Recherche de la toxicité de diverses souches de Bacillus thuringiensis Berliner vis-à-vis de trois espèces de limaces

La toxicité de plusieurs préparations à base de Bacillus thuringiensis a été montrée vis-à-vis de la limace grise Deroceras reticulatum (Müller) et de l'escargot aquatique Biomphalaria alexandrina (Ehrenberg) par de précédents auteurs, laissant envisager une méthode de lutte microbiologique contre les gastéropodes. C'est pourquoi la toxicité de certains de ces produits commerciaux et de diverses souches non formulées ayant des activités insecticides différentes a été éprouvée sur trois espèces de limaces : D reticulatum, Arion distinctus Mabille et Limax valentianus Férussac et comparée à celle de produits molluscicides de référence. Parmi les produits testés, deux étaient à base de β-exotoxine (Bitoxibacillin 5 et le surnageant H1). Cependant, aucune préparation à base de Bacillus ne s'est montrée toxique vis-à-vis des trois espèces de limaces. Seuls les produits molluscicides ont confirmé leur efficacité contre les limaces.Research on the toxicity of various Bacillus thuringiensis Berliner strains to three species of slugs. Toxicity of several Bacillus thuringiensis preparations had been shown against the grey field slug Deroceras reticulatum (Müller) and the aquatic snail Biomphalaria alexandrina (Ehrenberg) by previous authors, letting view a method of microbiological control of gastropods. Therefore, the toxicities of some of these products and of several unformulated strains, having different insecticide activities, were studied on three species of slugs: D reticulatum, Arion distinctus Mabille and Limax valentianus Férussac, and compared to molluscicide products of reference. Amongst the products tested, two contained the β-exotoxin (Bitoxibacillin 5 and the surnatant H1). However, no strain of Bacillus showed to be toxic for the three slug species. Only the molluscicide products confirmed their efficiency against the slugs