Source: UNIVERSITY OF GEORGIA submitted to NRP
FUNCTIONAL INTERACTIONS BETWEEN BACILLUS THURINGIENSIS TOXINS AND INSECTS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0193565
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Sep 1, 2002
Project End Date
Aug 31, 2008
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
UNIVERSITY OF GEORGIA
200 D.W. BROOKS DR
ATHENS,GA 30602-5016
Performing Department
ENTOMOLOGY
Non Technical Summary
Insects become resistant to Bacillus thuringiensis insecticidal proteins. New insecticidal proteins are needed for insect control. This project examines how insects are killed by Bacillus thuringiensis proteins. We will also investigate mechanisms of Bt resistance in insects.
Animal Health Component
30%
Research Effort Categories
Basic
70%
Applied
30%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2111719104025%
2111719113025%
7216010104025%
7216010113025%
Knowledge Area
211 - Insects, Mites, and Other Arthropods Affecting Plants; 721 - Insects and Other Pests Affecting Humans;

Subject Of Investigation
1719 - Cotton, other; 6010 - Individuals;

Field Of Science
1040 - Molecular biology; 1130 - Entomology and acarology;
Goals / Objectives
1)Objective. Characterize interactions between Bt Cry proteins and target molecules in insects. Molecules are identified that bind Bt toxins, yet their function as receptors is uncertain. We propose to determine which known binding molecules function as receptors for differenct Bt toxins. We also propose to localize Bt binding site on receptors. 2)Objective. Investigate mechanisms of resistance to Bt Cry proteins in pest insects. In this project we will develop a greater understanding of mechanisms by which that insects acquire resistance to Bt. Our approach will be to focus on target molecules for Bt in insect midgut. Additionally, we will investigate factors in the midgut of insects that determine susceptibility or resistance. This information will assist in the development of strategies to delay or overcome insect resistance. 3)Objective. Identify molecules in midguts of Lepidoptera and Diptera that determine Cry protein toxicity. A number of important insect species have not been investigated with regards to Bt toxin receptors and specificity. Among the Lepidoptera, we propose to investigate Bt receptors in the cotton pests Helicoverpa zea and Spodoptera species. Mosquitoes that vector disease contribute to the spread of emerging diseases. Using our knowledge of Bt receptors in Lepidoptera we are positioned to investigate Bt receptors in mosquitoes including Aedes, Culex and Anopheles species.
Project Methods
Objective 1. Characterize interactions between Bt Cry proteins and target molecules in insects. (i) Approach. A cultured cell assay was developed using Cry1 toxins and cDNAs encoding known receptors. Candidate receptor proteins are expressed transiently on the cell surface and tested for the ability to mediate toxin binding. This assay system was validated using Cry1 toxins and cloned APNs and cadherins from Lepidoptera. (ii) Approach. Toxin binding peptides will be identified using a mini-receptor assay and cultured cells. (iii) Approach. We propose to over-express Bt Cry protein receptors for toxin binding and structural analyses. Objective 2. Investigate mechanisms of resistance to Bt Cry proteins in pest insects. (i) Approach. Is binding and pore formation altered in Bt-resistant H. virescens strains (other than YHD2) and in H. zea? Cry1Aa and Cry1Ac binding will be measured for resistant strains of H. virescens and H. zea. Light scattering assays to measure membrane permeation (i.e. pore formation) will be conducted when appropriate based on binding data. (ii) Approach. Altered glycosylation as a mechanism of resistance. We determined that Bt-resistant H. virescens YHD2 larvae have altered N-acetylgalactosamine (GalNAc) composition. This discovery will be investigated in more detail by lectin blot analysis. We propose to identify proteins having altered glycosylation. Objective 3. Identify molecules in midguts of Lepidoptera and Diptera that determine Cry protein toxicity. (i) Biochemical approaches. One-dimensional gel ligand blots. The ligand blot technique has become a standard technique for identifying Cry toxin-binding molecules. We will confirm putative binding molecules identified by ligand blotting with non-denaturing; binding assays. 2D gel electrophoresis ligand blots. BBMV prepared from insect larvae have complex protein patterns and 1D SDS-PAGE does not always resolve individual proteins. Proteins will be separated by 2D gel electrophoresis, blotted and probed with labeled Bt toxins. Proteins that bind toxin on blots will be identified using mass spectrometry. Affinity selection and conventional column chromatography. This approach uses Bt toxin coupled to beads to purify toxin-binding proteins from solubilized BBMV. Bound proteins are eluted and identified by N-terminal and internal amino acid sequence analysis. (ii) Molecular Screening Approach: Functional genomics and expression screening. This approach is designed to capture unknown receptors from insect larvae. Midgut cDNA will be expressed on a cell surface. We will identify clones that express a toxin-binding protein with a labeled Bt toxin and fluorescent-assisted cell sorting.

Progress 09/01/02 to 08/31/08

Outputs
OUTPUTS: The results of this research project were disseminated in three international symposia since 2006 and at seminars to faculty in Entomology departments at universities in the United States. Activities conducted included the directing and management of research of seven graduate students, two postdoctoral researchers, a visiting scientist and an Assistant Research scientists. About five undergraduate researchers were mentored in the laboratory in the past two years. Products include: METHODS AND MATERIALS FOR IDENTIFYING NOVEL PESTICIDE AGENTS Adang, M.J., and Luo, K. 2006. U.S. Patent 7,011,975. PEPTIDES FOR INHIBITING INSECTS WITH AN INSECT CADHERIN ECTODOMAIN Adang, M.J., Hua, G., Chen, J., Abdullah, M.A.F. 2008. U. S. Patent 7,396,813. Patents applications: MODIFIED PEPTIDES HAVING TOXIN-ENHANCING EFFECTS. Abdullah, M.A.F. and Adang, M.J. Application published 2/19/2009. PCT Application No. PCT/US2008/072812U.S. Serial No. 60/964,249. INSECT CADHERIN FRAGMENTS. Adang, M.J. and Abdullah, M.A.F. PCT Application published 2/19/2009. PCT Application No. PCT/US2008/072806 IDENTIFICATION OF TOXIN-BINDING PROTEINS INVOLVED IN RESISTANCE TO CRY1 TOXINS, AND RELATED SCREENING METHODS. U.S. Patent Application No. 20050064386. PARTICIPANTS: Individuals who worked on the project. Dr. Gang Hua was an Assistant Research Scientist on the Project. Dr. Juan Luis Jurat Fuentes, was a co-project director before taking an Assistant Professor position at the University of Tennessee. Drs. Youngjin Part and Mohd Amir Abdullah were postdoctoral researchers on the project. Mr. K. Rahman and J. Chen were graduate students who conducted research on the project. The University of Tennessee was a partner organization on the project through the collaboration of Dr. Juan Luis Jurat Fuentes. The USDA laboratory at Stoneville, MS was also a partner through a collaboration with Dr. Perrera. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The tobacco budworm Heliothis virescens, a major pest of cotton in the U.S., is effectively controlled by Bacillus thuringiensis (Bt) Cry proteins produced in transgenic cotton. We investigated proteins in the midgut of tobacco budworm that are involved in susceptibility to Bt Cry proteins and then examine the involvement of these protein in resistance to Bt Cry proteins. Our first objective was to establish if a midgut cadherin functions as a receptor for Cry1Ac and Cry1Fa Cry proteins. We expressed H. virescens cadherin on the surface of Drosophila DS2 cells and tested for function as aCry1A and Cry1F receptor. We demonstrated the role of cadherin as a Cry1A but not Cry1F receptor. These somewhat unexpected results were used as background information in registration of a Bt cotton for commercial planting in the U.S. A stacked combination of Cry1Ac and Cry1Fa are registered and commercially planted. While investigating Bt cadherin interactions, we discovered that fragments of insect cadherins can enhance Bt toxicity to pest insects and, in some cases help overcome resistance. The second part of our project identified midgut alkaline alkaline phosphatase (HvALP) as a receptor for Bt Cry1Ac protein in H. virescens. Through differential proteomics analyses of three Cry1Ac-resistant strains of H. virescens we identified HvALP as a marker for resistance to Cry toxins. In each strain examined, resistance correlated with reduced levels of HvALP. We also detected differential expression of midgut proteases in resistant strains of H. virescens. This information is crucial for the development of methods to detect evolution of resistance in field populations of H. virescens and to design strategies to control resistance episodes.

Publications

  • Abdullah, M.A., Moussa, s., Taylor, M.D., and Adang, M.J. 2009. Manduca sexta (Lepidoptera: Sphingidae) cadherin fragments function as synergists for Cry1A and Cry1C Bacillus thuringiensis toxins against noctuid moths, Helicoverpa zea, Agrotis ipsilon, and Spodoptera exigua. 65. 1097-1103.
  • Park, Y., Abdullah, M.A.F., Taylor, M.D., Rahman, K. and Adang, M.J. 2009. Enhancement of Bacillus thuringiensis Cry3Aa and Cry3Bb toxicities to coleropteran larvae by a toxin-binding fragment of an insect cadherin. Appl. Environ. Microbiol. 75. 3086-3092.
  • Perera, O.P., Willis, J.D., Adang, M.J. and Jurat-Fuentes, J.L. 2009. Cloning and characterization of the Cry1Ac-binding alkaline phosphatase (HvALP) from Heliothis virescens. Insect Biochem. Molec. Biol. 39(4) 294-302.
  • Chen, J., Hua, G.H., Jurat-Fuentes, J.L., Abdullah, M.A., and Adang, M.J. 2007. Synergism of Bacillus thuringiensis toxins by a fragment of a toxin-binding cadherin. Proc. Natl. Acad. Sci. U.S.A. 104(35). 13901-13906.
  • Jurat-Fuentes. J.L. and Adang, M.J. 2007. A proteomic approach to study Cry1Ac binding proteins and their alterations in Heliothis virescens larvae. J. Invertbr. Pathol. 95(3). 187-191.


Progress 01/01/06 to 12/31/06

Outputs
Even though Bacillus thuringiensis (Bt) insecticidal proteins (Bt toxins have been extensively used in insecticidal mixtures and transgenic crops, it is not completly understood how these proteins kill insects. Even less is known about how insects change to become resistant to Bt. Our studies of an important cotton pest, Heliothis virescens, provide key information for the identification and characterization of Cry toxin receptors and their mode of action. Two proteins, a cadherin type protein and an alkaline phosphatase, were identified as putative Cry1 receptors by detection of their alteration in resistant larvae. Down-regulation of receptor proteins is a mechanism of resistance to Bt Cry1Ac toxin. We discovered that a particular Bt toxin receptor, called alkaline phosphatase is reduced in activity in a number of resistant Heliothis virescens strains. These results indicate that testing for reduction of phosphatase activity may be a more sensitive method to differentiate susceptible from resistant larvae. The characterization of the genetic alteration responsible for the lower levels of alkaline phosphatase in resistant larvae is crucial to develop more sensitive and specific DNA-based testing methods.

Impacts
Control of pest insects is an ongoing challenge to crop production. In recent years Bt cotton and Bt corn have reduced chemical pesticide usage and added value to production agriculture. Results from our recent research have fundamental basic and applied impacts. A functional cell-based assay for Bt toxin receptor function was developed. This assay has contributed to a greater understanding of the specificificity between Bt toxins and receptors. Our analyses of toxin binding patterns to the HevCaLP contribute to a rational basis for selecting Bt toxins to be expressed in future versions of Bt cotton. The involvement of alkaline phosphatase in Bt resistance, although poorly understood at a mechanistic level, provides a practical tool that may be useful in monitoring for Bt resistance in field populations of tobacco budworm. Our basic research on Bt receptors led to the discovery of a method and tools for utilizing Bt receptors for enhanced insect control.

Publications

  • Krishnamoorthy, M., Jurat-Fuentes, J.L., McNall, R.J., Andacht, T., and Adang, M.J. 2007. Identification of novel Cry1Ac binding proteins in midgut membranes from Heliothis virescens using proteomic analyses. Insect Biochem. Molec. Biol. Epub ahead of print.
  • Karumbaiah, L., Oppert, B., Juan L. Jurat-Fuentes, J.-L., and Adang, M.J. 2007. Analysis of midgut proteinases from Bacillus thuringiensis- susceptible and -resistant Heliothis virescens (Lepidoptera: Noctuidae). Comp. Physiol. Biochem. Oct 27, epub ahead of print.
  • Jurat-Fuentes, J.L and Adang, M.J. 2006. The Heliothis virescens cadherin protein expressed in Drosophila S2 cells functions as a receptor for Bacillus thuringiensis Cry1A but not Cry1Fa toxins. Biochem. 45: 9688-9695.
  • Jurat-Fuentes, J.L. and Adang, M.J. 2006. Cry toxin mode of action in susceptible and resistant Heliothis virescens larvae. J. Invertbr. Pathol. 92: 166-171.
  • Adang, M.J. and Jurat-Fuentes, J.L. 2006. Bacillus thuringiensis Cry proteins. In J.N. All and M.F. Treacy, eds. Use and Management of Insecticides, Acaricides and Transgenic Crops. Entomological Society of America. Lanham, MD..


Progress 01/01/05 to 12/31/05

Outputs
Insects pests of crops and insect vectors of human disease are effectively controlled by the insecticidal proteins of Bacillus thuringiensis (Bt). Lepidoptera were the primary pests of cotton prior to the extensive planting of Bt cotton. Since that time, the tobacco budworm, Heliothis virescens, has been effectively controlled by Bt cotton. Avoiding or managing insect resistance is critical to the long-term use of Bt cotton. This project investigates proteins in the midgut of lepidopteran larvae that function as Bt toxin receptors. Aminopeptidases, cadherins, alkaline phosphatases are all considered functional Bt toxin receptors in insects. Recently, we established functional assay for Bt toxin receptors using genes expressed in Drosophila S2 cells. These investigations led to our discovery that fragments of a Bt receptor when fed to an insect with a Bt toxin, actually increase the insecticidal potency of the toxin. In H. virescens our research has focused on the Bt receptors and insect resistance. Disruption of the BtR-4 gene encoding the cadherin protein (HevCaLP) is linked to high levels of Cry1Ac resistance in the H.virescens strain YHD2 larvae. We determined that disruption of HevCaLP in YHD2 larvae caused loss of Cry1Aa binding to brush border membranes, but only a slight reduction in Cry1Ab and Cry1Ac binding. This is significant because it resolved discrepancies in published studies of Cry1Ac binding. Our results also led to the search for a second genetic change in larvae that conferred higher levels of Bt resistance and a reduction in Cry1Ab and Cry1Ac binding. An alkaline phosphatase (HvALP) was identified in brush border membrane from H. virescens larvae that functions as a Cry1Ac-receptor. Importantly, reduced amounts of HvALP correlate with increased resistance to Bt and loss of Cry1A toxin binding. The potential use of HvALP alterations as markers for resistance to Bacillus thuringiensis toxins was established.

Impacts
Control of pest insects is an ongoing challenge to crop production. In recent years Bt cotton and Bt corn have reduced chemical pesticide usage and added value to production agriculture. Results from our recent research have fundamental basic and applied impacts. A functional cell-based assay for Bt toxin receptor function was developed. This assay has contributed to a greater understanding of the specificificity between Bt toxins and receptors. Our analyses of toxin binding patterns to the HevCaLP contribute to a rational basis for selecting Bt toxins to be expressed in future versions of Bt cotton. The involvement of alkaline phosphatase in Bt resistance, although poorly understood at a mechanistic level, provides a practical tool that may be useful in monitoring for Bt resistance in field populations of tobacco budworm. Our basic research on Bt receptors led to the discovery of a method and tools for utilizing Bt receptors for enhanced insect control.

Publications

  • Jurat-Fuentes, J.L., Gahan, L.J., Gould, F.L., Heckel, D.G. and Adang, M.J. 2004. The HevCaLP protein mediates binding specificity of the Cry1A class of Bacillus thuringiensis toxins in Heliothis virescens 43(44):14299-305
  • Griffitts J.S., Haslam S.M., Yang T, Garczynski S.F, Mulloy B., Morris H., Cremer P.S., Dell A., Adang M.J. and Aroian R.V. 2005. Glycolipids as receptors for Bacillus thuringiensis crystal toxin. Science 2005; 307: 922-925.
  • Chen, J, Brown, MR, and Hua, G, and Adang, MJ. 2005. Comparison and localization of Bacillus thuringiensis Cry1A delta-endotoxins and their binding proteins in larval midgut of tobacco hornworm. Cell Tissue Research 321; 123-129.
  • Adang MJ, McNall, R, and Fuentes, JLJ. 2005. Identification of toxin-binding protein involved in resistance to Cry1 toxins, and related screening methods. U.S. Patent Application 20050064386.
  • Adang, M.J., Hua, G., Chen, J., Abdullah, M.A.F. 2005. Peptides for inhibiting insects. U.S. Patent Application 20050283857.


Progress 01/01/04 to 12/31/04

Outputs
The overall goal of this project is to identify molecules in insects that are the targets of Bacillus thuringiensis insecticidal Cry proteins. Bt the bacterium is a valuable resource for insect control when used directly as a biopesticide and indirectly as a source of genes encoding pesticidal proteins. Bt cotton and corn (planted on about 60% and 30% of the U.S. farm acreage, respectively) have become essential tools for crop production in the U.S. We investigated cadherin-like proteins located in the midgut of lepidopteran larvae, because they serve as receptor proteins for Cry1 toxins. Our approach relied on a fluorescent-based assay to analyze function of cadherins in insect cells. We cloned a Bt-R1 cDNA from Manduca sexta, expressed the cDNA in Drosophila melanogaster S2 cells and demonstrated receptor function of the expressed protein using Cry1A toxins. We then built truncated versions of BtR1 consisting of cadherin repeat (CR) units and expressed those units on the surface of S2 cells. Using toxin binding and cytotoxicity assays we identified CR12 as the critical Cry1Ab receptor region on Bt-R1. Receptors for Bt proteins in the cotton pest Heliothis virescens change as the insect adapts, i.e. becomes resistant, to Bt. For example, strain YHD2, selected for Bt Cry1Ac resistance in the laboratory, has a disrupted BtR-4 gene encoding the cadherin protein HevCaLP. We analyzed strain YHD2 plus the KCBhyb, and CXC strains for disruption of BtRr-4 and loss of HevCaLP protein. Results showed that a wild-type Bt-R4 allele is necessary for HevCaLP production and Cry1Aa toxin binding supporting the hypothesis that cadherin is an essential receptor for Cry1A toxins. Our results also provided evidence that YHD2 has acquired a separate genetic change after appearance of cadherin disruption, conferring even higher resistance in the resulting strain YHD2-B. Reasoning that H. virescens strain YHD2 has multiple mechanisms for Bt resistance, we searched for additional alterations that may relate to resistance. We identified a 68-kDa glycoprotein as a membrane-bound form of alkaline phosphatase, we called this protein HvALP. Importantly, HvALP has the amino sugar N-acetylgalactosamine that is known to cause Cry1Ac binding. Since larvae of the YHD2 strain of H. virescens have reduced amounts of HvALP in their midguts it is likely that reduction of alkaline phosphatase contributes to Bt resistance in strain YHD2. If correct, this is a novel mechanism of Bt resistance in insects.

Impacts
Control of pest insects is an ongoing challenge to crop production. In recent years Bt cotton and Bt corn have reduced chemical pesticide usage and added value to production agriculture. Results from this project have basic and applied impacts. The cell culture assay for Bt toxins and the identification of a novel toxin binding region on a Bt receptor (the cadherin protein BtR1) contribute towards the understanding of how Bt toins interact with receptors in the insect midgut. Our identification of alkaline phosphatase as associated with Bt resistance in tobacco budworm identifies another mechanism that an insect can deploy to acquire Bt resistance. In both areas of research, characterization of Bt receptors and mechanisms of Bt resistance, our results suggest avenues for improving Bt toxins and delaying the onset of insect resistance to Bt cotton and corn.

Publications

  • Jurat-Fuentes, J.L., Gahan, L.J., Gould, F.L., Heckel, D.G., and Adang M.J. 2004. The HevCaLP protein mediates binding specificity of the Cry1A class of Bacillus thuringiensis toxins in Heliothis virescens. Biochemistry 44: 14299-14305.
  • Jurat-Fuentes, J.L., and Adang, M.J. 2004. Characterization of a Cry1Ac-receptor alkaline phosphatase in susceptible and resistant Heliothis virescens larvae. Eur. J. Biochem. 271: 3127-3135.
  • Hua, G. Jurat-Fuentes, J.L., and Adang, M.J. 2004. Bt-R1a extracellular cadherin repeat 12 mediates Bacillus thuringiensis Cry1Ab binding and cytotoxicity. J. Biol. Chem. 279, 28051-28056.
  • Hua, G., Jurat-Fuentes, and Adang, M.J. 2004. Fluorescent-based assays establish Manduca sexta Bt-R1a cadherin as a receptor for multiple Bacillus thuringiensis Cry1A toxin in Drosophila S2 cells. Insect Biochem. Molec. Biol. 34: 193-202.
  • Adang, M.J. 2004. [M638] Insect aminopeptidase N. In. A., and F. Woessner, eds. Handbook of Proteolytic Enzymes, 2nd Edition. Elsevier Science. Oxford.


Progress 09/01/02 to 12/31/02

Outputs
The binding and pore formation abilities of Cry1A and Cry1Fa Bacillus thuringiensis Cry toxins were analyzed by using brush border membrane vesicles (BBMV) prepared from sensitive (YDK) and resistant (YHD2) Heliothis virescens. Labeled Cry toxins did not bind to BBMV from the resistant strain. In agreement with this reduction in binding, neither Cry1A or CryFa toxins altered the permeability of membrane vesicles from resitant larvae. We discovered that resistant larvae have altered glycoproteins of 63- and 68-kDa in the brush border membrane. This alteration is associated with changes in N-acetylgalactosamine on those proteins. These results are evidence that a dramatic reduction in binding is responsible for Bt resistance and cross-resistance, and that this trait correlates with altered glycosylation.

Impacts
A novel mechanism of resistance to Bacillus thuringiensis was discovered in the tobacco budworm, Heliothis virescens. Knowledge of this mechanism may be used to delay resistance to Bt and design new Bt toxins for insect control

Publications

  • Jurat-Fuentes, J.L., F.L. Gould, and M.J. Adang. 2002. Altered glycosylation of 63- and 68-kilodalton microvillar proteins in Heliothis virescens correlates with reduced Cry1 toxin binding, decreased pore formation, and increased resistance to Bacillus thuringiensis Cry1 toxins. Applied and Environmental Microbiology. 68: 5711-5717.