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At least 19 recordsLinked to original sources
PubMed1997

Proteinase-mediated insect resistance to Bacillus thuringiensis toxins.

Two Bacillus thuringiensis (Bt)-resistant strains of the Indianmeal moth, Plodia interpunctella, lack a major gut proteinase that activates Bt protoxins. The absence of this enzyme is genetically linked to larval survival on Bt-treated diets. When considered with previous data supporting the existence of receptor-mediated insect resistance to Bt, these results provide evidence that insect adaptation to these toxins occurs through multiple physiological mechanisms, which complicate efforts to prevent or manage resistance to Bt toxins in insect control programs.

Animals↗
PubMed1985

Nematoda: susceptibility of the egg to Bacillus thuringiensis toxins.

Crystalline toxins from Bacillus thuringiensis israelensis and B.t. kurstaki were lethal in vitro to eggs of the nematode Trichostrongylus colubriformis. The LD50 values for the two toxins were 0.38 ng and 37.5 micrograms total protein/ml, respectively. After 1 week at ambient temperature, the LD50 of B.t. kurstaki decreased to less than 4 micrograms/ml. Toxin from B.t. israelensis had no effect within 48 hr on survival of adult nematodes or on their feeding in vitro. Third-stage larvae of T. colubriformis were also unaffected by B.t. israelensis toxin. Exposure of third-stage larvae of Nippostrongylus brasiliensis to 1.1 micrograms total protein/ml of B.t. israelensis for 4 hr had no effect on their infectivity in mice, based on recovery of helminths at 7 days after infection. Similar exposure of 5-day-old N. brasiliensis and subsequent transfer into the intestine of mice gave recoveries that were similar to the untreated control. Thirty strains of B. thuringiensis caused mortality in nematode eggs, but over a 77,000-fold range of activity was found, based on the LD50 values. Toxin from B.t. israelensis was lethal to eggs of six zooparasitic and one free-living species of nematode, but the LD50 values varied 28-fold. Addition of B.t. israelensis to feces that contained eggs of T. colubriformis reduced subsequent recovery of larvae, with an LD50 of 260 ng/g of feces.

Animals↗
PubMedDate not supplied

Mechanism of action of Bacillus thuringiensis toxins.

The selectivity of Bacillus thuringiensis toxins is determined both by the toxin structure and by factors inherent to the insect. These toxins contain distinct domains that appear to be functionally important in toxin binding to protein receptors in the midgut of susceptible insects, and the subsequent formation of a pore in the insect midgut epithelium. In this article features necessary for the insecticidal activity of these toxins are discussed. These include toxin structure, toxin processing in the insect midgut, the identification of toxin receptors in susceptible insects, and toxin pore formation in midgut cells. In addition a number of B. thuringiensis toxins act synergistically to exert their full insecticidal activity. This synergistic action is critical not only for expressing the insecticidal activity of these toxins, but could also play a role in delaying the onset of insect resistance.

Animals↗
PubMed2006

Mode of action of mosquitocidal Bacillus thuringiensis toxins.

Cry toxins from Bacillus thuringiensis (Bt) are used for insect control. Their primary action is to lyse midgut epithelial cells. In lepidopteran insects, Cry1A monomeric toxins interact with a first receptor and this interaction triggers toxin oligomerization. The oligomeric structure interacts then with a second GPI-anchored receptor that induces insertion into membrane microdomains and larvae death. In the case of mosquitocidal Bt strains, two different toxins participate, Cry and Cyt. These toxins have a synergistic effect and Cyt1Aa overcomes Cry toxin-resistance. We will summarize recent findings on the identification of Cry receptors in mosquitoes and the mechanism of synergism: Cyt1Aa synergizes or suppresses resistance to Cry toxins by functioning as a Cry membrane-bound receptor.

Animals↗
PubMed2001

Unfolding affects insect cell permeabilization by Bacillus thuringiensis Cry1C toxin.

Bacillus thuringiensis Cry toxins are efficient, environment-friendly biological insecticides. Their molecular mode of action on target insect cells remains largely unknown. The aim of this study was to investigate the relation between the conformational state of the Cry1C toxin and its ionophoric activity on live Sf9 cells of Spodoptera frugiperda, a target insect for this protein. Potassium ion movement induced by Cry1C across the cell membrane was measured with a fluorescent assay developed previously and the conformation of the toxin was studied using tryptophan spectroscopy. Following treatment with 4 M guanidinium hydrochloride, which resulted in the unfolding of its N-terminal half, the toxin retained its full capacity to permeabilize the cells while the fully unfolded toxin did not induce potassium leakage. Therefore, permeabilization of Sf9 cells by Cry1C requires the integrity of the C-terminal half of the toxin and may depend on an initial unfolding step provided by the acidic environment of the cells.

Animals↗
PubMed2006

Genetic variability of Spodoptera frugiperda Smith (Lepidoptera: Noctuidae) populations from Latin America is associated with variations in susceptibility to Bacillus thuringiensis cry toxins.

Bacillus thuringiensis strains isolated from Latin American soil samples that showed toxicity against three Spodoptera frugiperda populations from different geographical areas (Mexico, Colombia, and Brazil) were characterized on the basis of their insecticidal activity, crystal morphology, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of parasporal crystals, plasmid profiles, and cry gene content. We found that the different S. frugiperda populations display different susceptibilities to the selected B. thuringiensis strains and also to pure preparations of Cry1B, Cry1C, and Cry1D toxins. Binding assays performed with pure toxin demonstrated that the differences in the toxin binding capacities of these insect populations correlated with the observed differences in susceptibility to the three Cry toxins analyzed. Finally, the genetic variability of the three insect populations was analyzed by random amplification of polymorphic DNA-PCR, which showed significant genetic diversity among the three S. frugiperda populations analyzed. The data presented here show that the genetic variability of S. frugiperda populations should be carefully considered in the development of insect pest control strategies, including the deployment of genetically modified maize in different geographical regions.

Animals↗
PubMed2001

Carbohydrate analyses of Manduca sexta aminopeptidase N, co-purifying neutral lipids and their functional interactions with Bacillus thuringiensis Cry1Ac toxin.

Bacillus thuringiensis Cry1Ac insecticidal toxin binds specifically to 120kDa aminopeptidase N (APN) (EC 3.4.11.2) in the epithelial brush border membrane of Manduca sexta midguts. The isolated 120-kDa APN is a member of a functional Cry1 toxin receptor complex (FEBS Lett. 412 (1997) 270). The 120-kDa form is glycosyl-phosphatidylinositol (GPI) anchored and converted to a 115-kDa form upon membrane solubilization. The 115-kDa APN also binds Cry1A toxins and Cry1Ac binding is inhibited by N-acetylgalactosamine (GalNAc). Here we determined the monosaccharide composition of APN. APN is 4.2mol% carbohydrate and contains GalNAc, a residue involved in Cry1Ac interaction. APN remained associated with non-covalently bound lipids through anion-exchange column purification. Most associated lipids were separated from APN by hydrophobic interaction chromatography yielding a lipid aggregate. Chemical analyses of the lipid aggregate separated from APN revealed neutral lipids consisting mostly of diacylglycerol and free fatty acids. The fatty acids were long, unsaturated chains ranging from C:14 to C:22. To test the effect of APN-associated lipids on Cry1Ac function, the lipid aggregate and 115-kDa APN were reconstituted into phosphatidylcholine (PC) vesicles. The lipid aggregate increased the amount of Cry1Ac binding, but binding due to the lipid aggregate was not saturable. In contrast the lipid aggregate promoted Cry1Ac-induced release of 86Rb(+) at the lowest Cry1Ac concentration (50nM) tested. The predominant neutral lipid component extracted from the lipid aggregate promoted Cry1Ac-induced 86Rb(+) release from membrane vesicles in the presence of APN.

Animals↗
PubMed1998

Phage display of a biologically active Bacillus thuringiensis toxin.

Activated forms of Bacillus thuringiensis insecticidal toxins have consistently been found to form insoluble and inactive precipitates when they are expressed in Escherichia coli. Genetic engineering of these proteins to improve their effectiveness as biological pesticides would be greatly facilitated by the ability to express them in E. coli, since the molecular biology tools available for Bacillus are limited. To this end, we show that activated B. thuringiensis toxin (Cry1Ac) can be expressed in E. coli as a translational fusion with the minor phage coat protein of filamentous phage. Phage particles displaying this fusion protein were viable, infectious, and as lethal as pure toxin on a molar basis when the phage particles were fed to insects susceptible to native Cry1Ac. Enzyme-linked immunosorbent assay and Western blot analysis showed the fusion protein to be antigenically equivalent to native toxin, and micropanning with anti-Cry1Ac antibody was positive for the toxin-expressing phage. Phage display of B. thuringiensis toxins has many advantages over previous expression systems for these proteins and should make it possible to construct large libraries of toxin variants for screening or biopanning.

Amino Acid Sequence↗
PubMed2002

Transgenic Drosophila reveals a functional in vivo receptor for the Bacillus thuringiensis toxin Cry1Ac1.

The bacterium Bacillus thuringiensis synthesizes toxins (delta-endotoxins) that are highly specific for insects. Once ingested, the activated form of the toxin binds to a specific receptor(s) located on the midgut epithelial cells, inserts into the membrane causing the formation of leakage pores and eventual death of the susceptible insect larvae. Manduca sexta larvae are highly susceptible to Cry1Ac1, a toxin that is believed to bind M. sexta Aminopeptidase N, a glycoprotein located on the apical membrane. However, the binding data obtained to date only support the interaction of Cry1Ac1 with APN in vitro. To explore the in vivo role of APN, we have utilized the GAL4 enhancer trap technique to drive the expression of M. sexta APN in both midgut and mesodermal tissues of Cry1Ac1 insensitive Drosophila larvae. Transgenic Drosophila fed the toxin were now killed, demonstrating that APN can function as a receptor for Cry1Ac1 in vivo.

Animals↗
PubMed2001

Chronic exposure of the European corn borer (Lepidoptera: Crambidae) to Cry1Ab Bacillus thuringiensis toxin.

Transgenic corn expressing the insecticidal toxin from Bacillus thuringiensis Berliner is gaining support as an effective control technology for use against lepidopteran pests, particularly European corn borer, Ostrinia nubilalis Hübner (Lepidoptera: Crambidae). However, there is concern that widespread adoption of transgenic plants will rapidly lead to B. thuringiensis toxin resistance. Thus, long-term selection of O. nubilalis populations with the Cry1Ab B. thuringiensis toxin has been undertaken in several laboratories in the United States and in Europe. We present results from two independent selection experiments performed in laboratories at the University of Nebraska and at the Institut National de la Recherche Agronomique in France. Although the protocols and methods used by the two laboratories were different, the results were comparable. The highest level of resistance occurred at generation 7 (14-fold), generation 9 (13-fold), and generation 9 (32-fold) for three different strains. For each strain, the level of resistance fluctuated from generation to generation, although there were consistently significant decreases in toxin susceptibility across generations for all selected strains. These results suggest that low levels of resistance are common among widely distributed O. nubilalis populations.

Animals↗
PubMed2006

Characterization of chimeric Bacillus thuringiensis Vip3 toxins.

Bacillus thuringiensis vegetative insecticidal proteins (Vip) are potential alternatives for B. thuringiensis endotoxins that are currently utilized in commercial transgenic insect-resistant crops. Screening a large number of B. thuringiensis isolates resulted in the cloning of vip3Ac1. Vip3Ac1 showed high insecticidal activity against the fall armyworm Spodoptera frugiperda and the cotton bollworm Helicoverpa zea but very low activity against the silkworm Bombyx mori. The host specificity of this Vip3 toxin was altered by sequence swapping with a previously identified toxin, Vip3Aa1. While both Vip3Aa1 and Vip3Ac1 showed no detectable toxicity against the European corn borer Ostrinia nubilalis, the chimeric protein Vip3AcAa, consisting of the N-terminal region of Vip3Ac1 and the C-terminal region of Vip3Aa1, became insecticidal to the European corn borer. In addition, the chimeric Vip3AcAa had increased toxicity to the fall armyworm. Furthermore, both Vip3Ac1 and Vip3AcAa are highly insecticidal to a strain of cabbage looper (Trichoplusia ni) that is highly resistant to the B. thuringiensis endotoxin Cry1Ac, thus experimentally showing for the first time the lack of cross-resistance between B. thuringiensis Cry1A proteins and Vip3A toxins. The results in this study demonstrated that vip3Ac1 and its chimeric vip3 genes can be excellent candidates for engineering a new generation of transgenic plants for insect pest control.

Animals↗
PubMed2005

Baseline susceptibility of western corn rootworm (Coleoptera: Crysomelidae) to Cry3Bb1 Bacillus thuringiensis toxin.

Susceptibility to Cry3Bb1 toxin from Bacillus thuringiensis (Bt) was determined for western corn rootworm, Diabrotica virgifera virgifera LeConte, neonates from both laboratory and field populations collected from across the Corn Belt. Rootworm larvae were exposed to artificial diet treated with increasing Cry3Bb1 concentrations, and mortality and growth inhibition were evaluated after 4-7 d. The range of variation in Bt susceptibility indicated by growth inhibition was similar to that indicated by mortality. Although interpopulation variation in susceptibility was observed, the magnitude of the differences was comparable with the variability observed between generations of the same population. In general, the toxin was not highly toxic to larvae and estimated LC50 and EC50 values were several times higher than those reported for lepidopteran-specific Cry toxins by using similar bioassay techniques. These results suggest that the observed susceptibility differences reflect natural variation in Bt susceptibility among rootworm populations and provide a baseline for estimating potential shifts in susceptibility that might result from selection and exposure to Cry3Bb1-expressing corn hybrids.

Animals↗
PubMed2001

Cross-resistance and stability of resistance to Bacillus thuringiensis toxin Cry1C in diamondback moth.

We tested toxins of Bacillus thuringiensis against larvae from susceptible, Cry1C-resistant, and Cry1A-resistant strains of diamondback moth (Plutella xylostella). The Cry1C-resistant strain, which was derived from a field population that had evolved resistance to B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai, was selected repeatedly with Cry1C in the laboratory. The Cry1C-resistant strain had strong cross-resistance to Cry1Ab, Cry1Ac, and Cry1F, low to moderate cross-resistance to Cry1Aa and Cry9Ca, and no cross-resistance to Cry1Bb, Cry1Ja, and Cry2A. Resistance to Cry1C declined when selection was relaxed. Together with previously reported data, the new data on the cross-resistance of a Cry1C-resistant strain reported here suggest that resistance to Cry1A and Cry1C toxins confers little or no cross-resistance to Cry1Bb, Cry2Aa, or Cry9Ca. Therefore, these toxins might be useful in rotations or combinations with Cry1A and Cry1C toxins. Cry9Ca was much more potent than Cry1Bb or Cry2Aa and thus might be especially useful against diamondback moth.

Animals↗
PubMed2002

Changes in protease activity and Cry3Aa toxin binding in the Colorado potato beetle: implications for insect resistance to Bacillus thuringiensis toxins.

Widespread commercial use of Bacillus thuringiensis Cry toxins to control pest insects has increased the likelihood for development of insect resistance to this entomopathogen. In this study, we investigated protease activity profiles and toxin-binding capacities in the midgut of a strain of Colorado potato beetle (CPB) that has developed resistance to the Cry3Aa toxin of B. thuringiensis subsp. tenebrionis. Histological examination revealed that the structural integrity of the midgut tissue in the toxin-resistant (R) insect was retained whereas the same tissue was devastated by toxin action in the susceptible (S) strain. Function-based activity profiling using zymographic gels showed specific proteolytic bands present in midgut extracts and brush border membrane vesicles (BBMV) of the R strain not apparent in the S strain. Aminopeptidase activity associated with insect midgut was higher in the R strain than in the S strain. Enzymatic processing of toxin did not differ in either strain and, apparently, is not a factor in resistance. BBMV from the R strain bound approximately 60% less toxin than BBMV from the S strain, whereas the kinetics of toxin saturation of BBMV was 30 times less in the R strain than in the S strain. However, homologous competition inhibition binding of (125)I-Cry3Aa to BBMV did not reveal any differences in binding affinity (K(d) approximately 0.1 microM) between the S and R strains. The results indicate that resistance by the CPB to the Cry3Aa toxin correlates with specific alterations in protease activity in the midgut as well as with decreased toxin binding. We believe that these features reflect adaptive responses that render the insect refractory to toxin action, making this insect an ideal model to study host innate responses and adaptive changes brought on by bacterial toxin interaction.

Aminopeptidases↗
PubMedDate not supplied

Exploration of receptor binding of Bacillus thuringiensis toxins.

Wild type and mutant toxins of Bacillus thuringiensis delta-endotoxins were examined for their binding to midgut brush border membrane vesicles (BBMV). CryIAa, CryIAb, and CryIAc were examined for their binding to Gypsy moth (Lymantria dispar) BBMV. The binding of CryIAa and CryIAc was directly correlated with their toxicity, while CryIAb was observed to have lower binding than expected from its toxicity. The latter observation confirms the observation of Wolfersberger (1990). The "rule" of reciprocity of binding and toxicity is apparently obeyed by CryIAa and CryIAc, but broken by CryIAb on L. dispar. Alanine substitutions were made in several positions of the putative loops of CryIAa to test the hypothesis that the loops are intimately involved in binding to the receptor. The mutant toxins showed minor shifts in heterologous binding to Bombyx mori BBMV, but not enough to conclude that the residues chosen play critical roles in receptor binding.

Amino Acid Sequence↗
PubMed2002

Control of resistant pink bollworm (Pectinophora gossypiella) by transgenic cotton that produces Bacillus thuringiensis toxin Cry2Ab.

Crops genetically engineered to produce Bacillus thuringiensis toxins for insect control can reduce use of conventional insecticides, but insect resistance could limit the success of this technology. The first generation of transgenic cotton with B. thuringiensis produces a single toxin, Cry1Ac, that is highly effective against susceptible larvae of pink bollworm (Pectinophora gossypiella), a major cotton pest. To counter potential problems with resistance, second-generation transgenic cotton that produces B. thuringiensis toxin Cry2Ab alone or in combination with Cry1Ac has been developed. In greenhouse bioassays, a pink bollworm strain selected in the laboratory for resistance to Cry1Ac survived equally well on transgenic cotton with Cry1Ac and on cotton without Cry1Ac. In contrast, Cry1Ac-resistant pink bollworm had little or no survival on second-generation transgenic cotton with Cry2Ab alone or with Cry1Ac plus Cry2Ab. Artificial diet bioassays showed that resistance to Cry1Ac did not confer strong cross-resistance to Cry2Aa. Strains with >90% larval survival on diet with 10 microg of Cry1Ac per ml showed 0% survival on diet with 3.2 or 10 microg of Cry2Aa per ml. However, the average survival of larvae fed a diet with 1 microg of Cry2Aa per ml was higher for Cry1Ac-resistant strains (2 to 10%) than for susceptible strains (0%). If plants with Cry1Ac plus Cry2Ab are deployed while genes that confer resistance to each of these toxins are rare, and if the inheritance of resistance to both toxins is recessive, the efficacy of transgenic cotton might be greatly extended.

Animal Feed↗
PubMed2004

Contamination of refuges by Bacillus thuringiensis toxin genes from transgenic maize.

Transgenic crops producing insecticidal toxins from Bacillus thuringiensis (Bt) are widely used to control pests, but their benefits will be lost if pests evolve resistance. The mandated high-dose/refuge strategy for delaying pest resistance requires planting refuges of toxin-free crops near Bt crops to promote survival of susceptible pests. We report that pollen-mediated gene flow up to 31 m from Bt maize caused low to moderate Bt toxin levels in kernels of non-Bt maize refuge plants. Immunoassays of non-Bt maize sampled from the field showed that the mean concentration of Bt toxin Cry1Ab in kernels and the percentage of kernels with Cry1Ab decreased with distance from Bt maize. The highest Bt toxin concentration in pooled kernels of non-Bt maize plants was 45% of the mean concentration in kernels from adjacent Bt maize plants. Most previous work on gene flow from transgenic crops has emphasized potential effects of transgene movement on wild relatives of crops, landraces, and organic plantings, whereas implications for pest resistance have been largely ignored. Variable Bt toxin production in seeds of refuge plants undermines the high-dose/refuge strategy and could accelerate pest resistance to Bt crops. Thus, guidelines should be revised to reduce gene flow between Bt crops and refuge plants.

Animals↗
PubMed2001

Mannose phosphate isomerase isoenzymes in Plutella xylostella support common genetic bases of resistance to Bacillus thuringiensis toxins in Llpidopteran species.

A strong correlation between two mannose phosphate isomerase (MPI) isoenzymes and resistance to Cry1A toxins from Bacillus thuringiensis has been found in a Plutella xylostella population. MPI linkage to Cry1A resistance had previously been reported for a Heliothis virescens population. The fact that the two populations share similar biochemical, genetic, and cross-resistance profiles of resistance suggests the occurrence of homologous resistance loci in both species.

Animals↗