Grantee Research Project Results

Final Report: Identification of Receptors of Bacillus Thuringiensis Toxins in Midguts of the European Corn Borer

EPA Grant Number: R829479C014
Subproject: this is subproject number 014 , established and managed by the Center Director under grant R829479
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: Water Innovation Network for Sustainable Small Systems
Center Director: Reckhow, David A.
Title: Identification of Receptors of Bacillus Thuringiensis Toxins in Midguts of the European Corn Borer
Investigators: Siegfried, Blair , Nickerson, Kenneth W.
Institution: University of Nebraska at Lincoln
EPA Project Officer: Aja, Hayley
Project Period: October 1, 2003 through September 30, 2004
RFA: The Consortium for Plant Biotechnology Research, Inc., Environmental Research and Technology Transfer Program (2001) RFA Text |  Recipients Lists
Research Category: Targeted Research , Hazardous Waste/Remediation

Objective:

The objectives of this research project were to:

1. develop techniques to identify the receptors for Cry1Ab endotoxins in midgut preparations of the European corn borer;
2. determine the cross reactivity among Bacillus thuringiensis (Bt) toxins that have potential for insertion and expression in transgenic plants;
3. and determine the biochemical and physiological nature of Bt receptors.

Summary/Accomplishments (Outputs/Outcomes):

Corn plants expressing the toxin from B. thuringiensis (Berliner) have proven to be effective in controlling lepidopteran pests such as the European corn borer, Ostrinianubilalis (Hübner) (Lepidoptera: Crambidae). A number of Bt toxins are being tested and incorporated into crop genomes, although tests for cross-resistance among different toxins have been limited by a lack of resistant colonies. Four different colonies of O. nubilalis , selected with full-length Cry1Ab incorporated into an artificial diet, developed significant levels of resistance (2- to 10-fold) within 10 generations. Additionally, selection with Cry1Ab resulted in decreased susceptibility to a number of other toxins to which the selected colonies were not previously exposed. Significantly, levels of resistance were highest to Cry1Ac with resistance ratios up to 51-fold. Low levels (< 5-fold) of cross-resistance were detected with Cry1F. In contrast, Cry2Ab and Cry9C susceptibility was unaffected by selection with Cry1Ab. These results indicate that the availability of multiple toxins could improve resistance management strategies, provided cross-resistance among toxins is not a factor.

Susceptibility to the Cry1Ab protoxin and toxin from B. thuringiensis and activity of gut proteinases were assessed in both susceptible and Cry1Ab-selected colonies of the European corn borer. Resistance in two different selected colonies was at least 6- and 15-fold for the Cry1Ab protoxin and 108- and 415-fold for the Cry1Ab toxin. Activities of trypsin-like, chymotrypsin-like, and elastase-like proteinases were variable among the colonies tested and not indicative of a major contribution to Cry1Ab resistance. Activation of the 130-kDa Cry1Ab protoxin occurred rapidly in all colonies with no apparent differences among strains. There also were no apparent changes in activated Cry1Ab processing, indicating that proteolytic degradation was not associated with resistance. These results suggest that mechanisms other than proteolytic activation of protoxin and toxin degradation, such as target site modification, may be involved in the resistance to B. thuringiensis Cry1Ab in these O. nubilalis colonies.

Cry1Ab toxin binding analysis was performed to determine whether resistance in O. nubilalis is related to target site interaction. Brush border membrane vesicles (BBMVs) were prepared using dissected midguts from late instars of three different colonies of O. nubilalis. A 220-kDa protein was identified as a cadherin-like receptor, which bound Cry1Ab toxin and cross-reacted with an anticadherin serum developed from O. nubilalis. Two other smaller bands cross-reacted with the anticadherin serum, suggesting that Cry1Ab may bind to multiple receptors or different forms of the same protein. Additionally, reduced binding of Cry1Ab was observed in the Cry1Ab-selected colonies in ligand blot analyses. Real-time analysis of Cry1Ab binding to gut receptors was accomplished using surface plasmon resonance. Affinity constants for Cry1Ab binding to BBMV from two selected colonies (Europe-R; 60.9 ± 6.34 nM and RSTT-R; 94.0 ± 10.69 nM) compared to the unselected control (Europe-S; 34.5 ± 2.85 nM], were significantly (p < 0.05) reduced. Reduced receptor number did not account for reduced binding, as transcript abundance was similar or greater in the selected strains. Although reduced binding appears to be associated with resistance, the precise mechanisms remain unclear.

Journal Articles on this Report : 4 Displayed | Download in RIS Format

Publications Views

Other subproject views:	All 11 publications	4 publications in selected types	All 4 journal articles
Other center views:	All 212 publications	49 publications in selected types	All 45 journal articles

Publications

Type	Citation	Sub Project	Document Sources
Journal Article	Chaufaux J, Seguin M, Swanson JJ, Bourguet D, Siegfried BD. Chronic exposure of the European corn borer (Lepidoptera: Crambidae) to Cry1Ab Bacillus thuringiensis toxin. Journal of Economic Entomology 2001;94(6):1564-1570.	R829479 (2006) R829479 (Final) R829479C014 (Final)	Abstract from PubMed Abstract: BioOne-Abstract Exit
Journal Article	Flannagan RD, Yu C-G, Mathis JP, Meyer TE, Shi XM, Siqueira HAA, Siegfried BD. Identification, cloning and expression of a Cry1Ab cadherin receptor from European corn borer, Ostrinia nubilalis (Hubner) (Lepidoptera: Crambidae). Insect Biochemistry and Molecular Biology 2005;35(1):33-40.	R829479 (2006) R829479 (Final) R829479C014 (Final)	Abstract from PubMed Full-text: ScienceDirect-PDF Exit Abstract: ScienceDirect-Abstract Exit
Journal Article	Siqueira HAA, Nickerson KW, Moellenbeck D, Siegfried BD. Activity of gut proteinases from Cry1Ab-selected colonies of the European corn borer, Ostrinia nubilalis (Lepidoptera: Crambidae). Pest Management Science 2004;60(12):1189-1196.	R829479 (2006) R829479 (Final) R829479C014 (Final)	Abstract from PubMed Full-text: University of Nebraska-Lincoln - PDF Exit Abstract: Wiley-Abstract Exit
Journal Article	Siqueira HAA, Moellenbeck D, Spencer T, Siegfried BD. Cross-resistance of Cry1Ab-selected Ostrinia nubilalis (Lepidoptera: Crambidae) to Bacillus thuringiensis δ-endotoxins. Journal of Economic Entomology 2004;97(3):1049-1057.	R829479 (2006) R829479 (Final) R829479C014 (Final)	Abstract from PubMed Full-text: University of Nebraska-Lincoln - PDF Exit Abstract: Journal of Economic Entomology-Abstract Exit Other: BioOne-Abstract Exit

Supplemental Keywords:

sustainable industry, sustainable business, waste, agricultural engineering, bioremediation, environmental engineering, geochemistry, new technology, innovative technology, bioaccumulation, biodegradation, bioenergy, bioengineering, biotechnology, phytoremediation, plant biotechnology, sustainable industry/business, environmental chemistry,, Scientific Discipline, TREATMENT/CONTROL, Sustainable Industry/Business, Geochemistry, Genetics, Technology, New/Innovative technologies, Environmental Engineering, Agricultural Engineering, agrobacterium, bioengineering, transgenic plants, plant genes, biotechnology, remediation, plant biotechnology, cloning, endotoxin receptors

Relevant Websites:

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Main Center Abstract and Reports:

R829479    Water Innovation Network for Sustainable Small Systems

Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R829479C001 Plant Genes and Agrobacterium T-DNA Integration
R829479C002 Designing Promoters for Precision Targeting of Gene Expression
R829479C003 aka R829479C011 Biological Effects of Epoxy Fatty Acids
R829479C004 Negative Sense Viral Vectors for Improved Expression of Foreign Genes in Insects and Plants
R829479C005 Development of Novel Plastics From Agricultural Oils
R829479C006 Conversion of Paper Sludge to Ethanol
R829479C007 Enhanced Production of Biodegradable Plastics in Plants
R829479C008 Engineering Design of Stable Immobilized Enzymes for the Hydrolysis and Transesterification of Triglycerides
R829479C009 Discovery and Evaluation of SNP Variation in Resistance-Gene Analogs and Other Candidate Genes in Cotton
R829479C010 Woody Biomass Crops for Bioremediating Hydrocarbons and Metals
R829479C011 Biological Effects of Epoxy Fatty Acids
R829479C012 High Strength Degradable Plastics From Starch and Poly(lactic acid)
R829479C013 Development of Herbicide-Tolerant Energy and Biomass Crops
R829479C014 Identification of Receptors of Bacillus Thuringiensis Toxins in Midguts of the European Corn Borer
R829479C015 Coordinated Expression of Multiple Anti-Pest Proteins
R829479C016 A Novel Fermentation Process for Butyric Acid and Butanol Production from Plant Biomass
R829479C017 Molecular Improvement of an Environmentally Friendly Turfgrass
R829479C018 Woody Biomass Crops for Bioremediating Hydrocarbons and Metals. II.
R829479C019 Transgenic Plants for Bioremediation of Atrazine and Related Herbicides
R829479C020 Root Exudate Biostimulation for Polyaromatic Hydrocarbon Phytoremediation
R829479C021 Phytoremediation of Heavy Metal Contamination by Metallohistins, a New Class of Plant Metal-Binding Proteins
R829479C022 Development of Herbicide-Tolerant Energy and Biomass Crops
R829479C023 A Novel Fermentation Process for Butyric Acid and Butanol Production from Plant Biomass
R829479C024 Development of Vectors for the Stoichiometric Accumulation of Multiple Proteins in Transgenic Crops
R829479C025 Chemical Induction of Disease Resistance in Trees
R829479C026 Development of Herbicide-Tolerant Hardwoods
R829479C027 Environmentally Superior Soybean Genome Development
R829479C028 Development of Efficient Methods for the Genetic Transformation of Willow and Cottonwood for Increased Remediation of Pollutants
R829479C029 Development of Tightly Regulated Ecdysone Receptor-Based Gene Switches for Use in Agriculture
R829479C030 Engineered Plant Virus Proteins for Biotechnology