Downregulation of a Chitin Deacetylase-Like Protein in Response to Baculovirus Infection and Its Application for Improving Baculovirus Infectivity
ABSTRACT Several expressed sequence tags (ESTs) with homology to chitin deacetylase-like protein (CDA) were selected from a group of Helicoverpa armigera genes whose expression changed after infection with H. armigera single nucleopolyhedrovirus (HearNPV). Some of these ESTs coded for a midgut protein containing a chitin deacetylase domain (CDAD). The expressed protein, HaCDA5a, did not show chitin deacetylase activity, but it showed a strong affinity for binding to chitin. Sequence analysis showed the lack of any chitin binding domain, described for all currently known peritrophic membrane (PM) proteins. HaCDA5a has previously been detected in the H. armigera PM. Such localization, togethe…
Binding Site Alteration Is Responsible for Field-Isolated Resistance to Bacillus thuringiensis Cry2A Insecticidal Proteins in Two Helicoverpa Species
Background Evolution of resistance by target pests is the main threat to the long-term efficacy of crops expressing Bacillus thuringiensis (Bt) insecticidal proteins. Cry2 proteins play a pivotal role in current Bt spray formulations and transgenic crops and they complement Cry1A proteins because of their different mode of action. Their presence is critical in the control of those lepidopteran species, such as Helicoverpa spp., which are not highly susceptible to Cry1A proteins. In Australia, a transgenic variety of cotton expressing Cry1Ac and Cry2Ab (Bollgard II) comprises at least 80% of the total cotton area. Prior to the widespread adoption of Bollgard II, the frequency of alleles conf…
Bacillus thuringiensis Cry1Ac Toxin-Binding and Pore-Forming Activity in Brush Border Membrane Vesicles Prepared from Anterior and Posterior Midgut Regions of Lepidopteran Larvae
ABSTRACT It is generally accepted that Bacillus thuringiensis Cry toxins insert into the apical membrane of the larval midgut after binding to specific receptors, and there is evidence that the distribution of binding molecules along the midgut is not uniform. By use of the voltage-sensitive dye DiSC 3 (5) and 125 I-labeled Cry1Ac, we have measured the effect of Cry1Ac in terms of permeabilization capacity and of binding parameters on brush border membrane vesicles (BBMV) prepared from the anterior and the posterior regions of the larval midgut from two insect species, Manduca sexta and Helicoverpa armigera . The permeabilizing activity was significantly higher with BBMV from the posterior …
Susceptibility of Spodoptera frugiperda and S. exigua to Bacillus thuringiensis Vip3Aa insecticidal protein
The Vip3Aa protein is an insecticidal protein secreted by Bacillus thuringiensis during the vegetative stage of growth. The activity of this protein has been tested after different steps/protocols of purification using Spodoptera frugiperda as a control insect. The results showed that the Vip3Aa protoxin was stable and retained full toxicity after being subjected to common biochemical steps used in protein purification. Bioassays with the protoxin in S. frugiperda and S. exigua showed pronounced differences in LC(50) values when mortality was measured at 7 vs. 10d. At 7d most live larvae were arrested in their development. LC(50) values of "functional mortality" (dead larvae plus larvae rem…
Proteolytic processing of Bacillus thuringiensis Vip3A proteins by two Spodoptera species
Abstract Vip3 proteins have been described to be secreted by Bacillus thuringiensis during the vegetative growth phase and to display a broad insecticidal spectrum against lepidopteran larvae. Vip3Aa protoxin has been reported to be significantly more toxic to Spodoptera frugiperda than to Spodoptera exigua and differences in the midgut processing have been proposed to be responsible. In contrast, we have found that Vip3Ae is essentially equally toxic against these two species. Proteolysis experiments were performed to study the stability of Vip3A proteins to peptidase digestion and to see whether the differences found could explain differences in toxicity against these two Spodoptera speci…
Midgut microbiota and host immunocompetence underlie Bacillus thuringiensis killing mechanism
Bacillus thuringiensis is a widely used bacterial entomopathogen producing insecticidal toxins, some of which are expressed in insect-resistant transgenic crops. Surprisingly, the killing mechanism of B. thuringiensis remains controversial. In particular, the importance of the septicemia induced by the host midgut microbiota is still debated as a result of the lack of experimental evidence obtained without drastic manipulation of the midgut and its content. Here this key issue is addressed by RNAi-mediated silencing of an immune gene in a lepidopteran host Spodoptera littoralis, leaving the midgut microbiota unaltered. The resulting cellular immunosuppression was characterized by a reduced …
Toxicity and mode of action of Bacillus thuringiensis Cry proteins in the Mediterranean corn borer, Sesamia nonagrioides (Lefebvre)
ABSTRACT Sesamia nonagrioides is one of the most damaging pests of corn in Spain and other Mediterranean countries. Bt corn expressing the Bacillus thuringiensis Cry1Ab toxin is being grown on about 58,000 ha in Spain. Here we studied the mode of action of this Cry protein on S. nonagrioides (binding to specific receptors, stability of binding, and pore formation) and the modes of action of other Cry proteins that were found to be active in this work (Cry1Ac, Cry1Ca, and Cry1Fa). Binding assays were performed with 125 I- or biotin-labeled toxins and larval brush border membrane vesicles (BBMV). Competition experiments indicated that these toxins bind specifically and that Cry1Aa, Cry1Ab, an…
Constitutive Activation of the Midgut Response to Bacillus thuringiensis in Bt-Resistant Spodoptera exigua
Bacillus thuringiensis is the most effective microbial control agent for controlling numerous species from different insect orders. The main threat for the long term use of B. thuringiensis in pest control is the ability of insects to develop resistance. Thus, the identification of insect genes involved in conferring resistance is of paramount importance. A colony of Spodoptera exigua (Lepidoptera: Noctuidae) was selected for 15 years in the laboratory for resistance to Xentari (TM), a B. thuringiensis-based insecticide, reaching a final resistance level of greater than 1,000-fold. Around 600 midgut ESTs were analyzed by DNA-macroarray in order to find differences in midgut gene expression …
Association of Cry1Ac toxin resistance in Helicoverpa zea (Boddie) with increased alkaline phosphatase levels in the midgut lumen.
ABSTRACT Resistance to Bacillus thuringiensis Cry1Ac toxin was characterized in a population of Helicoverpa zea larvae previously shown not to have an alteration in toxin binding as the primary resistance mechanism to this toxin. Cry1Ac-selected larvae (AR1) were resistant to protoxins and toxins of Cry1Ab, Cry1Ac, and the corresponding modified proteins lacking helix α-1 (Cry1AbMod and Cry1AcMod). When comparing brush border membrane vesicles (BBMVs) prepared from susceptible (LC) and AR1 larval midguts, there were only negligible differences in overall Cry1Ac toxin binding, though AR1 had 18% reversible binding, in contrast to LC, in which all binding was irreversible. However, no differe…