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Juan Ferre - One of the best experts on this subject based on the ideXlab platform.
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unraveling the composition of insecticidal Crystal Proteins in bacillus thuringiensis a proteomics approach
Applied and Environmental Microbiology, 2020Co-Authors: Javier Caballero, Juan Ferre, Nerea Jimenezmoreno, Irene Orera, Trevor Williams, Ana Beatriz Fernandez, Maite Villanueva, Primitivo Caballero, Carmen AncinazpilicuetaAbstract:ABSTRACT Bacillus thuringiensis (Bt) is the most widely used active ingredient for biological insecticides. The composition of δ-endotoxins (Cry and Cyt Proteins) in the parasporal Crystal determines the toxicity profile of each Bt strain. However, a reliable method for their identification and quantification has not been available, due to the high sequence identity of the genes that encode the δ-endotoxins and the toxins themselves. Here, we have developed an accurate and reproducible mass spectrometry-based method (liquid chromatography-tandem mass spectrometry-multiple reaction monitoring [LC-MS/MS-MRM]) using isotopically labeled proteotypic peptides for each protein in a particular mixture to determine the relative proportion of each δ-endotoxin within the Crystal. To validate the method, artificial mixtures containing Cry1Aa, Cry2Aa, and Cry6Aa were analyzed. Determination of the relative abundance of Proteins (in molarity) with our method was in good agreement with the expected values. This method was then applied to the most common commercial Bt-based products, DiPel DF, XenTari GD, VectoBac 12S, and Novodor, in which between three and six δ-endotoxins were identified and quantified in each product. This novel approach is of great value for the characterization of Bt-based products, not only providing information on host range, but also for monitoring industrial Crystal production and quality control and product registration for Bt-based insecticides. IMPORTANCEBacillus thuringiensis (Bt)-based biological insecticides are used extensively to control insect pests and vectors of human diseases. Bt-based products provide greater specificity and biosafety than broad-spectrum synthetic insecticides. The biological activity of this bacterium resides in spores and Crystals comprising complex mixtures of toxic Proteins. We developed and validated a fast, accurate, and reproducible method for quantitative determination of the Crystal components of Bt-based products. This method will find clear applications in the improvement of various aspects of the industrial production process of Bt. An important aspect of the production of Bt-based insecticides is its quality control. By specifically quantifying the relative proportion of each of the toxins that make up the Crystal, our method represents the most consistent and repeatable evaluation procedure in the quality control of different batches produced in successive fermentations. This method can also contribute to the design of specific culture media and fermentation conditions that optimize Bt Crystal composition across a range of Bt strains that target different pestiferous insects. Quantitative information on Crystal composition should also prove valuable to phytosanitary product registration authorities that oversee the safety and efficacy of crop protection products.
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biochemistry and genetics of insect resistance to bacillus thuringiensis
Annual Review of Entomology, 2002Co-Authors: Juan Ferre, Jeroen Van RieAbstract:Bacillus thuringiensis (Bt) is a valuable source of insecticidal Proteins for use in conventional sprayable formulations and in transgenic crops, and it is the most promising alternative to synthetic insecticides. However, evolution of resistance in insect populations is a serious threat to this technology. So far, only one insect species has evolved significant levels of resistance in the field, but laboratory selection experiments have shown the high potential of other species to evolve resistance against Bt. We have reviewed the current knowledge on the biochemical mechanisms and genetics of resistance to Bt products and insecticidal Crystal Proteins. The understanding of the biochemical and genetic basis of resistance to Bt can help design appropriate management tactics to delay or reduce the evolution of resistance in insect populations.
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Insect resistance to Bacillus thuringiensis insecticidal Crystal Proteins
Entomopathogenic Bacteria: from Laboratory to Field Application, 2000Co-Authors: Jeroen Van Rie, Juan FerreAbstract:Several insect species have developed resistance to insecticidal Crystal Proteins from Bacillus thuringiensis, either through laboratory selection, or under field conditions. In this chapter we review the current knowledge on the biochemical and genetic mechanisms of resistance to B. thuringiensis. This knowledge will be important in the design of appropriate tactics to manage the development of resistance in insect populations.
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Screening for Bacillus thuringiensis Crystal Proteins active against the cabbage looper, Trichoplusia ni.
Journal of invertebrate pathology, 2000Co-Authors: Maria M. Iracheta, Benito Pereyra-alférez, Luis J. Galán-wong, Juan FerreAbstract:Abstract Toxicity tests were performed to find among Cry1 and Cry2 Bacillus thuringiensis Crystal Proteins those with high activity against the cabbage looper. Tests were performed with neonate larvae on surface-contaminated artificial diet. The Crystal Proteins found to be toxic were, from higher to lower toxicity: Cry1Ac, Cry1Ab, Cry1C, Cry2Aa, Cry1J, and Cry1F (LC50 of 1.1–4.1, 3.4–4.4, 12, 34, 87, and 250 ng/cm2, respectively). Cry1B, Cry1D, and Cry1E can be considered nontoxic (LC50 higher than 2500 ng/cm2). Cry1Aa was moderately toxic to nontoxic, depending on the source (LC50 of 420 ng/cm2 from PGS and 8100 ng/cm2 from Ecogen). In vitro binding assays with trypsin-activated 125I-labeled Cry1Aa, Cry1Ab, and Cry1Ac Crystal Proteins and brush border membrane vesicles from midgut larvae showed a direct correlation between toxicity and binding affinity. Heterologous competition experiments indicated that Cry1Aa and Cry1F bind, though only at very high concentrations, to the Cry1Ab/Cry1Ac shared high-affinity binding site.
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integrative model for binding of bacillus thuringiensis toxins in susceptible and resistant larvae of the diamondback moth plutella xylostella
Applied and Environmental Microbiology, 1999Co-Authors: Victoria Ballester, Thomas M. Malvar, Francisco Granero, Bruce E Tabashnik, Juan FerreAbstract:Insecticidal Crystal Proteins from Bacillus thuringiensis in sprays and transgenic crops are extremely useful for environmentally sound pest management, but their long-term efficacy is threatened by evolution of resistance by target pests. The diamondback moth (Plutella xylostella) is the first insect to evolve resistance to B. thuringiensis in open-field populations. The only known mechanism of resistance to B. thuringiensis in the diamondback moth is reduced binding of toxin to midgut binding sites. In the present work we analyzed competitive binding of B. thuringiensis toxins Cry1Aa, Cry1Ab, Cry1Ac, and Cry1F to brush border membrane vesicles from larval midguts in a susceptible strain and in resistant strains from the Philippines, Hawaii, and Pennsylvania. Based on the results, we propose a model for binding of B. thuringiensis Crystal Proteins in susceptible larvae with two binding sites for Cry1Aa, one of which is shared with Cry1Ab, Cry1Ac, and Cry1F. Our results show that the common binding site is altered in each of the three resistant strains. In the strain from the Philippines, the alteration reduced binding of Cry1Ab but did not affect binding of the other Crystal Proteins. In the resistant strains from Hawaii and Pennsylvania, the alteration affected binding of Cry1Aa, Cry1Ab, Cry1Ac, and Cry1F. Previously reported evidence that a single mutation can confer resistance to Cry1Ab, Cry1Ac, and Cry1F corresponds to expectations based on the binding model. However, the following two other observations do not: the mutation in the Philippines strain affected binding of only Cry1Ab, and one mutation was sufficient for resistance to Cry1Aa. The imperfect correspondence between the model and observations suggests that reduced binding is not the only mechanism of resistance in the diamondback moth and that some, but not all, patterns of resistance and cross-resistance can be predicted correctly from the results of competitive binding analyses of susceptible strains.
William P. Donovan - One of the best experts on this subject based on the ideXlab platform.
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binary toxins from bacillus thuringiensis active against the western corn rootworm diabrotica virgifera virgifera leconte
Applied and Environmental Microbiology, 2004Co-Authors: James A Baum, William P. Donovan, Mark J. Rupar, Thomas M. Malvar, Chirei Chu, Gregory R Brown, Joseph E Huesing, Oliver Ilagan, Michael Pleau, Matthew R WaltersAbstract:The western corn rootworm, Diabrotica virgifera virgifera LeConte, is a significant pest of corn in the United States. The development of transgenic corn hybrids resistant to rootworm feeding damage depends on the identification of genes encoding insecticidal Proteins toxic to rootworm larvae. In this study, a bioassay screen was used to identify several isolates of the bacterium Bacillus thuringiensis active against rootworm. These bacterial isolates each produce distinct Crystal Proteins with approximate molecular masses of 13 to 15 kDa and 44 kDa. Insect bioassays demonstrated that both protein classes are required for insecticidal activity against this rootworm species. The genes encoding these Proteins are organized in apparent operons and are associated with other genes encoding Crystal Proteins of unknown function. The antirootworm Proteins produced by B. thuringiensis strains EG5899 and EG9444 closely resemble previously described Crystal Proteins of the Cry34A and Cry35A classes. The antirootworm Proteins produced by strain EG4851, designated Cry34Ba1 and Cry35Ba1, represent a new binary toxin. Genes encoding these Proteins could become an important component of a sustainable resistance management strategy against this insect pest.
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Cloning of the nprA gene for neutral protease A of Bacillus thuringiensis and effect of in vivo deletion of nprA on insecticidal Crystal protein.
Applied and environmental microbiology, 1997Co-Authors: William P. Donovan, Yuping Tan, Annette C. SlaneyAbstract:The nprA gene, encoding Bacillus thuringiensis neutral protease A, was cloned by the use of gene-specific oligonucleotides. The size of neutral protease A deduced from the nprA sequence was 566 amino acids (60,982 Da). The cloned nprA gene was partially deleted in vitro, and the deleted allele, designated nprA3, was used to construct an nprA3 strain (neutral protease A-deficient strain) of B. thuringiensis. Growth and sporulation of the nprA3 strain were similar to those of an isogenic nprA+ strain, although the extracellular proteolytic activity of the nprA3 strain was significantly less than that of the nprA+ strain. The nprA3 strain produced insecticidal Crystal Proteins that were more stable than those of the isogenic nprA+ strain after solubilization in vitro, and sporulated cultures of the nprA3 strain contained higher concentrations of full-length insecticidal Crystal Proteins than did those of its isogenic counterpart. The absence of neutral protease A did not affect the insecticidal activity of a lepidopteran-specific Crystal protein of B. thuringiensis. These results indicate that Crystal protein stability and yield may be improved by deletion of specific proteases from B. thuringiensis.
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Characterization of two genes encoding Bacillus thuringiensis insecticidal Crystal Proteins toxic to Coleoptera species.
Applied and Environmental Microbiology, 1992Co-Authors: William P. Donovan, Mark J. Rupar, Annette C. Slaney, Thomas M. Malvar, M C Gawron-burke, T B JohnsonAbstract:Bacillus thuringiensis EG2838 and EG4961 are highly toxic to Colorado potato beetle larvae, and only strain EG4961 is toxic to southern corn rootworm larvae. To investigate the cause of the different insecticidal activities of EG2838 and EG4961, cryIII-type genes toxic to coleopterans were cloned from each strain. The cryIIIB gene, cloned as part of an 8.0-kb EcoRI fragment of EG2838 DNA, encoded a Crystal protein (CryIIIB) of 74,237 Da. The cryIIIB2 gene, cloned as part of an 8.3-kb PstI-Asp718 fragment of EG4961 DNA, encoded a Crystal protein (CryIIIB2) of 74,393 Da that was 94% identical to CryIIIB. Analysis of the transcriptional start sites showed that cryIIIB and cryIIIB2 were initiated from a conserved region located within 130 nucleotides upstream from the translation start sites of both genes. Although the CryIIIB and CryIIIB2 Proteins were similar in sequence, they displayed distinct insecticidal activities: CryIIIB was one-third as toxic as CryIIIB2 to Colorado potato beetle larvae, and CryIIIB2, but not CryIIIB, was toxic to southern corn rootworm larvae. Genes encoding Crystal Proteins of approximately 32 and 31 kDa were located adjacent to the cryIIIB and cryIIIB2 genes, respectively. The 32- and 31-kDa Crystal Proteins failed to enhance the insecticidal activities of CryIIIB and CryIIIB2. Images
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Two novel strains of Bacillus thuringiensis toxic to coleopterans.
Applied and Environmental Microbiology, 1991Co-Authors: Mark J. Rupar, Jean-françois Charles, Véronique Cosmao Dumanoir, William P. Donovan, Annette C. Slaney, T B Johnson, R G Groat, J W Mattison, H De BarjacAbstract:Two novel strains of Bacillus thuringiensis were isolated from native habitats by the use of genes coding for Proteins toxic to coleopterans (cryIII genes) as hybridization probes. Strain EG2838 (isolated by the use of the cryIIIA probe) contained a cryIIIA-hybridizing plasmid of approximately 100 MDa and synthesized Crystal Proteins of approximately 200 (doublet), 74, 70, 32, and 28 kDa. Strain EG4961 (isolated by the use of a cryIIIA-related probe) contained a cryIIIA-hybridizing plasmid of approximately 95 MDa and synthesized Crystal Proteins of 74, 70, and 30 kDa. Structural relationships among the Crystal Proteins of strains EG2838 and EG4961 were detected; antibodies to the CryIIIA protein toxic to coleopterans reacted with the 74- and 70-kDa Proteins of EG2838 and EG4961, antibodies to the 32-kDa plus 28-kDa Proteins of EG2838 reacted with the 30-kDa protein of EG4961, and antibodies to the 200-kDa Proteins of EG2838 reacted with the 28-kDa protein of EG2838. Experiments with B. thuringiensis flagella antibody reagents demonstrated that EG2838 belongs to H serotype 9 (reference strain B. thuringiensis subsp. tolworthi) and that EG4961 belongs to H serotype 18 (reference strain B. thuringiensis subsp. kumamotoensis). A mixture of spores plus Crystal Proteins of either EG2838 or EG4961 was toxic to the larvae of Colorado potato beetle (Leptinotarsa decemlineata), and significantly, the EG4961 mixture was also toxic to the larvae of southern corn rootworm (Diabrotica undecimpunctata howardi). DNA restriction blot analysis suggested that strains EG2838 and EG4961 each contained a unique gene coding for a protein toxic to coleopterans.
Marnix Peferoen - One of the best experts on this subject based on the ideXlab platform.
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interactions of bacillus thuringiensis Crystal Proteins with the midgut epithelial cells of spodoptera frugiperda lepidoptera noctuidae
Journal of Invertebrate Pathology, 1996Co-Authors: Eduardo Aranda, Marnix Peferoen, Jorge Sanchez, Leopoldo Guereca, Alejandra BravoAbstract:Binding of different Bacillus thuringiensis insecticidal Crystal Proteins (ICPs) to the midgut epithelium of Spodoptera frugiperda larvae was characterized by binding experiments with midgut tissue sections and isolated brush border membrane vesicles. Our results show that ICPs interact with the microvilli of epithelial cells of S. frugiperda in two different ways. The first is typical of highly toxic Proteins (like CryIC and CryID); this interaction is saturable and specific. In contrast, some nontoxic Proteins (like CryIAb) interact nonspecifically with the microvilli, since the binding of this toxin is not affected by the presence of high concentrations of homologous competitor. The CryIC toxin binds to two brush border Proteins of 40 and 44 kDa and the CryIAb toxin binds to a single protein of 150 kDa. Immunological detection of ingested B. thuringiensis ICPs on gut sections of S. frugiperda larvae revealed that CryIC and CryID toxins bound along the epithelial brush border microvilli membrane. Binding of the nontoxic protein CryIAb was also observed in the epithelial brush border membrane of fed larvae, but it was extremely weak, implying that this type of interaction occurs also in vivo although it is not related to toxicity.
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biotinylation of bacillus thuringiensis insecticidal Crystal Proteins
Applied and Environmental Microbiology, 1993Co-Authors: Peter Denolf, Danny Degheele, Marnix Peferoen, Stefan Jansens, S Van Houdt, J Van RieAbstract:Abstract Biotinylation of Bacillus thuringiensis insecticidal Crystal Proteins (ICPs) was evaluated for its potential use in an alternative ICP screening method and in the characterization of ICP receptors. In vivo biological activity of CryIA(b), as inferred from bioassays with Manduca sexta and Ostrinia nubilalis and from histopathological effects on O. nubilalis midgut cells induced by force feeding, was not affected by biotinylation at moderate biotinylation ratios. A competitive radioreceptor assay showed that there was only a minor reduction in binding affinity of biotin-labeled CryIA(b) for M. sexta brush border membrane vesicles. On midgut tissue sections, the binding pattern along the midgut epithelium and the staining intensity of biotinylated ICPs detected with streptavidin-enzyme conjugate were virtually identical to the binding pattern and staining intensity of native CryIA(b) detected with antibodies. The specificity of biotinylated ICP binding to larval midgut tissue was demonstrated by performing homologous competition experiments. The relationship between different ICP receptor types in Plutella xylostella, as inferred from radioligand binding studies, was confirmed by the results of heterologous competition experiments performed with biotinylated and native ICPs.
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two different bacillus thuringiensis delta endotoxin receptors in the midgut brush border membrane of the european corn borer ostrinia nubilalis hubner lepidoptera pyralidae
Applied and Environmental Microbiology, 1993Co-Authors: Peter Denolf, Stefan Jansens, Marnix Peferoen, Danny DegheeleAbstract:Abstract Binding of three Bacillus thuringiensis insecticidal Crystal Proteins (ICPs) to the midgut epithelium of Ostrinia nubilalis larvae was characterized by performing binding experiments with both isolated brush border membrane vesicles and gut tissue sections. Our results demonstrate that two independent ICP receptors are present in the brush border of O. nubilalis gut epithelium. From competition binding experiments performed with 125I-labeled and native ICPs it was concluded that CryIA(b) and CryIA(c) are recognized by the same receptor. An 11-fold-higher binding affinity of CryIA(b) for this receptor correlated with a 10-fold-higher toxicity of this ICP compared with CryIA(c). The CryIB toxin did not compete for the binding site of CryIA(b) and CryIA(c). Immunological detection of ingested B. thuringiensis ICPs on gut sections of O. nubilalis larvae revealed binding only along the epithelial brush border membrane. CryID and CryIE, two ICPs that are not toxic to O. nubilalis, were not bound to the apical microvilli of gut epithelial cells. In vitro binding experiments performed with native and biotinylated ICPs on tissue sections confirmed the correlation between ICP binding and toxicity. Moreover, by performing heterologous competition experiments with biotinylated and native ICPs, it was confirmed that the CryIB receptor is different from the receptor for CryIA(b) and CryIA(c). Retention of activated Crystal Proteins by the peritrophic membrane was not correlated with toxicity. Furthermore, it was demonstrated that CryIA(b), CryIA(c), and CryIB toxins interact in vitro with the epithelial microvilli of Malpighian tubules. In addition, CryIA(c) toxin also adheres to the basement membrane of the midgut epithelium.
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Immunocytochemical analysis of specific binding of Bacillus thuringiensis insecticidal Crystal Proteins to lepidopteran and coleopteran mudgut membranes
Journal of Invertebrate Pathology, 1992Co-Authors: Alejandra Bravo, Koen Hendrickx, Stefan Jansens, Marnix PeferoenAbstract:Abstract The in vitro binding of four insecticidal Crystal Proteins (ICPs) from Bacillus thuringiensis to midgut tissue sections of two lepidopteran (Manduca sexta and Plutella xylostella) and one coleopteran (Leptinotarsa decemlineata) species was immunocytochemically analyzed. Monoclonal antibodies, highly specific against the different ICPs, were used. Light microscopy observations showed that the Crystal Proteins accumulated at the exposed peritrophic membrane of all lepidopteran insects and at the apical microvilli of sensitive lepidopteran insects. No accumulation was observed at the microvilli present in the goblet cell cavity or at the Malpighian tubules. For all lepidopteran insects the binding of the different ICPs to the apical microvilli of the midgut epithelium appears to be highly specific: only the ICPs which were toxic bound to the apical microvilli of midgut tissue sections. As an exception, the nontoxic CryIB bound to the apical microvilli of M. sexta. The binding of ICPs to the peritrophic membrane was not correlated to toxicity. For the first time binding of an ICP to a coleopteran insect larva has been shown. Only the coleopteran-specific toxin CryIIIA bound to the brushborder and to the peritrophic membrane. The binding is preferential to the apical microvilli in the posterior part of the midgut. These results suggest that the mechanism of ICP toxicity in coleopteran insects may be slightly different from that in Lepidopteran and/or Diptera. The present procedure is sensitive and rapid, can be applied to individual insects, and as such may prove very useful for analyzing the binding of different ICPs to small insects.
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immunocytochemical localization of bacillus thuringiensis insecticidal Crystal Proteins in intoxicated insects
Journal of Invertebrate Pathology, 1992Co-Authors: Alejandra Bravo, Stefan Jansens, Marnix PeferoenAbstract:The damage of different insecticidal Crystal Proteins (ICPs) from Bacillus thuringiensis to the midgut of Manduca sexta, Plutella xylostella, and Leptinotarsa decemlineata larvae was determined by light microscopic observations. During the intoxication process, the distribution of the different ICPs in the larval body was monitored with specific antibodies. In lepidopteran and in coleopteran insects, histopathological changes include disruption of the brush border, vacuolization of the cytoplasm, hypertrophy of the epithelial cells, and disintegration of the cell. After ingestion by the insect larvae, the ICPs rapidly accumulate in the peritrophic membrane. However, the binding to the peritrophic membrane does not correlate with toxicity, since the coleopteran-specific toxin (CryIIIA) is also retained in the peritrophic membrane of lepidopteran insects, while the lepidopteran-specific toxin CryIA(b) binds to the peritrophic membrane of the Colorado potato beetle larvae. In contrast, ICPs bind to the microvilli of the midgut epithelial cells of susceptible insects only, confirming a correlation between toxicity and binding to the brush border membrane. In the lepidopteran larvae, the lepidopteran-specific toxic ICPs initially accumulate at the apical microvilli of the epithelial cells in the anterior part of the midgut. In the Colorado potato beetle larvae, CryIIIA is primarily retained by the microvilli of the epithelial cells from the posterior part of the midgut. During the intoxication process, internalization of the ICPs into midgut epithelial cells is not detected, even several hours after toxin ingestion. Apparently, the ICPs are retained within the gut since they were not detected in other organs such as the Malpighian tubules.
Kenneth E Narva - One of the best experts on this subject based on the ideXlab platform.
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characterization of cry34 cry35 binary insecticidal Proteins from diverse bacillus thuringiensis strain collections
Applied and Environmental Microbiology, 2005Co-Authors: Ernest H Schnepf, Kenneth E Narva, Stacey Lynn Lee, Joanna Dojillo, Paula Burmeister, Kristin J Fencil, Lisa M Morera, Linda Nygaard, Jeffrey D WoltAbstract:Bacillus thuringiensis Crystal Proteins of the Cry34 and Cry35 classes function as binary toxins showing activity on the western corn rootworm, Diabrotica virgifera virgifera LeConte. We surveyed 6,499 B. thuringiensis isolates by hybridization for sequences related to cry35A genes, identifying 78 strains. Proteins of the appropriate molecular mass (ca. 44 kDa) for Cry35 were observed in 42 of the strains. Full-length, or nearly full-length, sequences of 34 cry34 genes and 16 cry35 genes were also obtained from cloning, PCR analysis, and DNA sequencing. These included representatives of all known Cry34A, Cry34B, Cry35A, and Cry35B classes, as well as a novel Cry34A/Cry35A-like pair. Bioassay analysis indicated that cry35-hybridizing strains not producing a ca. 14-kDa protein, indicative of Cry34, were not active on corn rootworms, and that the previously identified Cry34A/Cry35A pairs were more active than the Cry34B/Cry35B pairs. The cry35-hybridizing B. thuringiensis strains were found in locales and materials typical for other B. thuringiensis strains. Comparison of the sequences with the geographic origins of the strains showed that identical, or nearly identical, sequences were found in strains from both Australasia and the Americas. Sequence similarity searches revealed that Cry34 Proteins are similar to predicted Proteins in Photorhabdus luminescens and Dictyostelium discoidium, and that Cry35Ab1 contains a segment similar to beta-trefoil domains that may be a binding motif. The binary Cry34/Cry35 B. thuringiensis Crystal Proteins thus appear closely related to each other, are environmentally ubiquitous, and share sequence similarities consistent with activity through membrane disruption in target organisms.
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novel bacillus thuringiensis binary insecticidal Crystal Proteins active on western corn rootworm diabrotica virgifera virgifera leconte
Applied and Environmental Microbiology, 2002Co-Authors: Tracy R Ellis, Brian A Stockhoff, Lisa Stamp, Ernest H Schnepf, George E Schwab, Mark Knuth, Josh Russell, Guy A Cardineau, Kenneth E NarvaAbstract:A new family of insecticidal Crystal Proteins was discovered by screening sporulated Bacillus thuringiensis cultures for oral activity against western corn rootworm (WCR) larvae. B. thuringiensis isolates PS80JJ1, PS149B1, and PS167H2 have WCR insecticidal activity attributable to parasporal inclusion bodies containing Proteins with molecular masses of ca. 14 and 44 kDa. The genes encoding these polypeptides reside in apparent operons, and the 14-kDa protein open reading frame (ORF) precedes the 44-kDa protein ORF. Mutagenesis of either gene in the apparent operons dramatically reduced insecticidal activity of the corresponding recombinant B. thuringiensis strain. Bioassays performed with separately expressed, biochemically purified 14- and 44-kDa polypeptides also demonstrated that both Proteins are required for WCR mortality. Sequence comparisons with other known B. thuringiensis insecticidal Proteins failed to reveal homology with previously described Cry, Cyt, or Vip Proteins. However, there is evidence that the 44-kDa polypeptide and the 41.9- and 51.4-kDa binary dipteran insecticidal Proteins from Bacillus sphaericus are evolutionarily related. The 14- and 44-kDa polypeptides from isolates PS80JJ1, PS149B1, and PS167H2 have been designated Cry34Aa1, Cry34Ab1, and Cry34Ac1, respectively, and the 44-kDa polypeptides from these isolates have been designated Cry35Aa1, Cry35Ab1, and Cry35Ac1, respectively.
Matthew R Walters - One of the best experts on this subject based on the ideXlab platform.
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binary toxins from bacillus thuringiensis active against the western corn rootworm diabrotica virgifera virgifera leconte
Applied and Environmental Microbiology, 2004Co-Authors: James A Baum, William P. Donovan, Mark J. Rupar, Thomas M. Malvar, Chirei Chu, Gregory R Brown, Joseph E Huesing, Oliver Ilagan, Michael Pleau, Matthew R WaltersAbstract:The western corn rootworm, Diabrotica virgifera virgifera LeConte, is a significant pest of corn in the United States. The development of transgenic corn hybrids resistant to rootworm feeding damage depends on the identification of genes encoding insecticidal Proteins toxic to rootworm larvae. In this study, a bioassay screen was used to identify several isolates of the bacterium Bacillus thuringiensis active against rootworm. These bacterial isolates each produce distinct Crystal Proteins with approximate molecular masses of 13 to 15 kDa and 44 kDa. Insect bioassays demonstrated that both protein classes are required for insecticidal activity against this rootworm species. The genes encoding these Proteins are organized in apparent operons and are associated with other genes encoding Crystal Proteins of unknown function. The antirootworm Proteins produced by B. thuringiensis strains EG5899 and EG9444 closely resemble previously described Crystal Proteins of the Cry34A and Cry35A classes. The antirootworm Proteins produced by strain EG4851, designated Cry34Ba1 and Cry35Ba1, represent a new binary toxin. Genes encoding these Proteins could become an important component of a sustainable resistance management strategy against this insect pest.