Progress 10/01/08 to 09/30/13
Outputs
Target Audience: Our research targets scientists in the US and abroad interested in the mode of action of Cry toxins from Bacillus thuringiensis (Bt) and the resistance mechanisms that insects evolve against these toxins. In addition, our research activities also provide outputs relevant to the characterization of the insect gut epithelial response to damage, which is of interest to scientists focused on insect pathophysiology. Outputs from our project also target companies and farmers developing or using insecticidal technologies based on Bt toxins, and regulatory agencies responsible for risk assessment for current and novel Bt transgenic technologies. Changes/Problems: There were some problems related to the use of the Indian meal moth as a model for our mode of action and resistance objectives (goals one and two). Although we originally proposed to use this insect due to availability of Bt-resistant colonies, we then received access to Bt-resistant strains of Heliothis virescens and Spodoptera frugiperda that we maintained in our laboratory. Consequently, we decided to focus our research goals using these two species as models. It is highly plausible that findings obtained in these model species may be extended to the Indian meal moth and other lepidopteran pests. In addition, limited access to Bt-resistant Helicoverpa zea restricted the work that we were able to perform on this insect species. What opportunities for training and professional development has the project provided? In addition to the project principal and co-principal investigators, three students directly participated in the research activities as part of their PhD program. All participant students were trained in the techniques necessary to achieve the project goals. An undergraduate student also assisted in maintenance of insect colonies, purification to bacterial toxins, performance of insect bioassays and data collection. Moreover, a postdoctoral researcher also participated in completion of objectives two and three of the project. All student (graduate and undergraduate) participating in the project were able to also obtain relevant experience in presentation of research to peers during oral and poster presentations during national and international scientific meetings. All these students were recognized through travel grants to assist to scientific meetings or presentation awards. How have the results been disseminated to communities of interest? Results obtained from completion of the project objectives have been disseminated through oral and poster presentations at scientific meetings and in peer reviewed publications. Attendees at these scientific meeting sessions included academic students and principal investigators as well as scientific representatives from government and industry groups. What do you plan to do during the next reporting period to accomplish the goals? This is the final report for this project. Goals have been accomplished.
Impacts
What was accomplished under these goals?
Completion of the first proposed objective has identified proteins critical for susceptibility to Bt toxins present in pesticides and transgenic crops. This identification allows for the use of these proteins to screen for novel insecticidal proteins and to develop improved insecticidal proteins with enhanced interactions with the receptors. Information gathered from completion of our second objective has identified mechanism of resistance to Bt pesticides and transgenic Bt crops. This information allows for the development of new technologies to detect the identified resistance mechanisms and alleles involved. Monitoring for resistance is considered a critical component for effective insect resistance management programs aimed at maintaining utility of Bt pesticides and transgenic crops for the future. Information from completion of our third objective has provided the first clues supporting similarities in the Cry mode of action between lepidopteran and coleopteran insects. Identification of functional Cry3Aa receptors in coleopteran pests will allow for the screening of novel toxins recognizing this critical receptor and the engineering of novel toxins with enhanced interactions with Cry toxins. In this regard, our data demonstrating that a peptide designed from the identified Cry3A receptor enhances activity of Cry toxins opens new avenues of research to develop new products to increase effectivity and range of activity of Bt products.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2011
Citation:
Castagnola, A., S. Eda, and J. L. Jurat-Fuentes (2011) Monitoring stem cell proliferation and differentiation in primary midgut cell cultures from Heliothis virescens larvae using flow cytometry. Differentiation 81(3): 192-198.
- Type:
Journal Articles
Status:
Published
Year Published:
2009
Citation:
Morris, K., Lorenzen, M.D., Hiromasa, Y., Tomich, J., Oppert, C., Elpidina, E.N.,Vinokurov, K., Jurat-Fuentes, J.L., Fabrick, J., and B. Oppert (2009) Tribolium castaneum larval gut transcriptome and proteome: a resource for the study of the coleopteran gut J. Proteome Res. 8(8):3889-3898
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Information obtained from the performance of the project has been disseminated through presentations at the Annual meeting of the Entomological Society of America (Knoxville, TN) and the Society for Invertebrate Pathology (Buenos Aires, Argentina), and the 2nd International Insect Midgut Biology Symposium (Guangzhou, China). Audiences in these meetings included students (undergraduate and graduate), research staff and laboratory directors from national and international research groups, as well as researchers from biopesticide and biotechnology companies. Dissemination of this information has allowed for a more thorough characterization of the response to Cry intoxication in insects and has helped to identify potential molecular targets responsible for this response. In addition, our research has presented novel models of resistance mechanisms against Cry toxins in highly relevant insect pests. This information will assist in designing improved Bt-based products and assessing current resistance management practices for improvement. PARTICIPANTS: Dr. Juan Luis Jurat-Fuentes; Principal Investigator for the Bt mode of action in Lepidoptera objectives, Associate Professor, Department of Entomology and Plant Pathology, University of Tennessee, Knoxville, TN 37996. Dr. Jurat-Fuentes oversees the activities of graduate students assigned to this project. Additional responsibilities of Dr. Jurat-Fuentes include assistance with performance of experiments for the Bt mode of action in Coleoptera, progress report preparation, funding administration, and manuscript preparation and submission. Dr. Omaththage P. Perera; Co-Principal Investigator, Research Entomologist, USDA-ARS Southeastern Insect Management Research Unit, Stoneville, MS 38776. Dr. Perera directs the microarray analysis of resistance and co-directs with Dr. Jurat-Fuentes the characterization of Cry1Ac mode of action in H. virescens. Dr. Brenda Oppert; Co-Principal Investigator, Research Molecular Biologist, USDA-ARS Grain Production and Marketing Research Center, Manhattan, KS 66502. Dr. Oppert directs the characterization of Bt intoxication in Coleoptera. Dr. Jeff Fabrick; Co-principal Investigator, Research Entomologist, USDA-ARS Arid-Land Agricultural Research center, Maricopa, AZ 85238. Dr. Fabrick designs and performs research related to the characterization of Bt mode of action in Coleoptera. Dr. Cris Oppert; Assistant Research Professor, Department of Entomology and Plant Pathology, University of Tennessee, Knoxville, TN, 37996. Dr. Oppert has assisted in both objectives focused on Bt mode of action in Lepidoptera and Coleoptera. TARGET AUDIENCES: Our research targets scientists in the US and abroad interested in the mode of action of Cry toxins from Bacillus thuringiensis (Bt) and the resistance mechanisms that insects evolve against these toxins. In addition, our research activities also provide outputs relevant to the characterization of the insect gut epithelial response to damage, which is of interest to scientists focused on insect pathophysiology. Outputs from our project also target companies and farmers developing or using insecticidal technologies based on Bt toxins, and regulatory agencies responsible for risk assessment for current and novel Bt transgenic technologies. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Through collaboration with Dr. Alejandra Bravo (Universidad Nacional Autonoma de Mexico, Mexico), we have functionally identified proteins with differential expression in response to Cry toxin in mosquito larvae. These functional analyses have identified candidate proteins that allow insects to recover from sublethal Cry intoxication, which should guide future efforts to increase potency and potentially expand range of activity for Bt insecticidal products. In collaboration with Dr. Juan Ferre (Universitat de Valencia, Spain), we have described a novel mechanism of resistance against Cry1Ac toxin in a highly relevant insect pest, Helicoverpa zea. This novel mechanism is in agreement with our previous findings on resistant insects and highlights the importance of alkaline phosphatase in Cry intoxication and resistance against Cry toxins. Our research on the response to Cry3Aa intoxication in the beetle Tenebrio molitor has identified proteins and genes that are involved in recovery from sublethal intoxication as well as toxicity. Our data represent the most detailed characterization to date of Cry intoxication in Coleoptera, one of the most agriculturally important insect groups. Data from these studies will help identify steps in the mode of action of Cry toxins that may be improved through Cry toxin engineering and contribute to the assessment of current resistance management practices and monitoring methods for prevention and detection of field resistance episodes.
Publications
- Caccia, S., Moar, W. J., Chandrashekhar, J., Oppert, C., Anilkumar, K. J., Jurat-Fuentes, J. L., and J. Ferre (2012) "Resistance to Cry1Ac toxin in Helicoverpa zea (Boddie) is associated with increased alkaline phosphatasde levels in the midgut lumen" Appl. Environ. Microbiol. In Press.
- Cancino-Rodezno, A., Lozano, L., Oppert, C., Castro, J. L., Lanz-Mendoza, H., Encarnacion, S., Evans, A. E., Gill, S. S., Soberon, M., Jurat-Fuentes, J. L., and A. Bravo (2012) "Comparative proteomic analysis of Aedes aegypti larval midgut after intoxication with Cry11Aa toxin from Bacillus thuringiensis" PLoS One 7(5): e37034.
- Oppert, B., Dowd, S. E., Bouffard, P., Li, L., Conesa, A., Lorenzen, M., Touytges, M., Marshall, J., Huestis, D. L., Fabrick, J., Oppert, C., and J. L. Jurat-Fuentes (2012) "Transcriptome profiling of the intoxication response of Tenebrio molitor larvae to Bacillus thuringiensis Cry3Aa protoxin" PLoS One 7(4): e34624.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: The main output for this time period from our project has been the use of combined proteomic and genomic approaches to characterize resistance mechanisms to diverse Cry toxins in Heliothis virescens, and the development of an enhancer of Bt toxin activity. We expect that this characterization of resistance mechanisms and fitness of resistant insects will greatly contribute the optimization of strategies and mandates aimed at delaying the onset of resistance to Bt crops. Data from this research have been disseminated through publication and presentations at national and international scientific meetings. Information on the characterization of resistance in strains of H. virescens was presented at invited presentations in US universities and meetings of the Entomological Society of America, and in contributing presentations at the annual meeting of the Society for Invertebrate Pathology. Data from testing of a fragment of a Tenebrio molitor Cry3Aa receptor cadherin as an enhancer of Cry toxicity was shared at invited presentations at US universities and was used to prepare a patent application protecting this technology. Audiences in these meetings included students (undergraduate and graduate), research staff and laboratory directors from national and international research groups, as well as researchers from biopesticide and biotechnology companies. Dissemination of this information has allowed for increased recognition of the role of non-binding related mechanisms in resistance to diverse Cry toxins and has greatly increased our knowledge resistance to Bt toxins. This information will assist in designing improved Bt-based products and developing effective resistance management practices. In this regard, one of the major outputs from our work has been the identification of a potential sensitive marker for resistance to Cry toxins that is independent of the resistance mechanism or Cry toxin-insect combinations considered. This discovery represents a crucial step towards development of sensitive resistance monitoring approaches that could be applied to a wide array of currently commercialized transgenic Bt crops. PARTICIPANTS: Dr. Juan Luis Jurat-Fuentes; Principal Investigator for the Bt mode of action in Lepidoptera objectives, Assistant Professor, Department of Entomology and Plant Pathology, University of Tennessee, Knoxville, TN 37996. Dr. Jurat-Fuentes oversees the activities of a Postdoctoral Associate (Dr. Oppert) and graduate students assigned to this project. Additional responsibilities of Dr. Jurat-Fuentes include performance of experiments for the Bt mode of action in Coleoptera objective, progress report preparation, funding administration, and manuscript preparation and submission. Dr. Omaththage P. Perera; Co-Principal Investigator, Research Entomologist, USDA-ARS Southeastern Insect Management Research Unit, Stoneville, MS 38776. Dr. Perera directs the microarray analysis of resistance and co-directs with Dr. Jurat-Fuentes the characterization of Cry1Ac mode of action in H. virescens. Dr. Brenda Oppert; Co-Principal Investigator, Research Molecular Biologist, USDA-ARS Grain Production and Marketing Research Center, Manhattan, KS 66502. Dr. Oppert directs the characterization of Bt intoxication in the Coleoptera objective. Dr. Jeff Fabrick; Co-principal Investigator, Research Entomologist, USDA-ARS Arid-Land Agricultural Research center, Maricopa, AZ 85238. Dr. Fabrick designs and performs research related to the characterization of Bt mode of action in Coleoptera. Dr. Cris Oppert; Postdoctoral Research Associate, Department of Entomology and Plant Pathology, University of Tennessee, Knoxville, TN, 37996. Dr. Oppert is responsible for the proteomic 2D-DIGE differential analysis of resistance and testing of HevCaLP as Cry2Ab receptor. Dr. Oppert also designs and performs experiments for the Bt mode of action in Coleoptera objective. Anais S. Castagnola; Graduate Research Assistant; Department of Entomology and Plant Pathology, University of Tennessee, Knoxville, TN, 37996. Ms. Castagnola is the student assigned to the characterization of resistance to diverse Cry toxins in H. virescens. She designs and performs experiments related to this part of the project. TARGET AUDIENCES: Our research targets scientists in the US and abroad interested in the mode of action of Cry toxins from Bacillus thuringiensis (Bt) and the resistance mechanisms that insects evolve against these toxins. Data from our project also targets companies and farmers developing or using insecticidal technologies based on Bt toxins, and regulatory agencies responsible for risk assessment for current and novel Bt transgenic technologies. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Through collaboration with Dr. Yulin Gao (Chinese Academy of Agricultural Sciences) we have documented that a peptide derived from the Cry3Aa receptor cadherin TmCad1 enhances Cry3Aa toxicity against chrysomelid larvae that are devastating pests of vegetables. These data demonstrate the potential use of this peptide to develop novel and efficient Cry-based methods against difficult to control pests. Using a genomic approach (microarrays and pyrosequencing), we have performed a study to identify altered gene expression in T. molitor larvae after exposure to Cry3Aa toxins. Data from this research is identifying genes participating in the intoxication and healing processes, which can be targeted for the development of novel insecticidal strategies. In addition, information derived from this study represents the most thorough characterization of the Cry intoxication process in a coleopteran model, and is expected to guide the design of Bt-based technologies for the control of coleopteran pests. We have combined proteomic and genomic (microarray and transcriptome profiling) analyses to identify genes and proteins that are differentially expressed when H. virescens larvae are exposed to diverse Cry toxins. From this work we have characterized the response to Cry intoxication in larvae and how this process can be modified to result in resistance to diverse Cry toxins in selected H. virescens strains. We have identified a number of proteins and genes involved in digestion, tissue regeneration, and signaling that change their expression upon intoxication with diverse Cry toxins, and will be evaluated for their role in the Cry intoxication process. Data from these studies will help evaluate current resistance management practices and design methods to overcome field resistance episodes.
Publications
- Gouffon, C., Van Vliet, A., Van Rie, J., Jansens, S., and J. L. Jurat-Fuentes (2011) "Binding sites for Cry2Ae toxin from Bacillus thuringiensis on heliothine brush border membrane vesicles are not shared with Cry1A, Cry1F or Vip3A toxins" Appl. Environ. Microbiol. 77(10): 3182-3188.
- Jurat-Fuentes, J. L., Karumbaiah, L., Jakka, S. R. K., Ning, C., Liu, C., Wu, K., Jackson, J., Gould, F., Blanco, C., Portilla, M., Perera, O. P., and M. J. Adang (2011) "Reduced levels of membrane-bound alkaline phosphatase are common to lepidopteran strains resistant to Cry toxins from Bacillus thuringiensis" PLOS One 6(3): e17606.
- Gao, Y., Jurat-Fuentes, J.L., Oppert, B., Fabrick, J.A., Liu, C., Gao, J., and Z. Lei (2011) "Increased toxicity of Bacillus thuringiensis Cry3Aa against Crioceris quatuordecimpunctata, Phaedon brassicae and Colaphellus bowringi by a Tenebrio molitor cadherin fragment" Pest. Manag. Sci. 67(9): 1076-1081.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Experimental data from this research project have been disseminated through presentations at national and international scientific meetings. The identification of a fragment of a Tenebrio molitor Cry3Aa receptor cadherin as an enhancer of Cry toxicity against coleopteran and lepidopteran pests has been presented in invited oral presentations to the Department of Biochemistry and Molecular Biology at the University of Tennessee (Knoxville, TN), the Institute of Plant Protection of the Chinese Academy of Sciences (Beijing, China), the 54th Livestock Insect Workers Conference (Knoxville, TN), and at the Tennessee Technology Development Corporation Innovation Showcase (Nashville, TN). Results from our proteomic and genomic characterization of the Cry intoxication process were included in two contributing oral presentations and a poster display at 43rd Annual Meeting of the Society for Invertebrate Pathology (Trabzon, Turkey). Data on resistance to Cry toxins expressed in Bt transgenic corn in field-collected strains of Spodoptera frugiperda were presented at the 59th Annual Meeting of the Southeastern branch of the Entomological Society of America (Cancun, Mexico). Audiences in these meeting included students, research staff and laboratory directors from national and international research groups, as well as representatives and scientists from chemical and biological pesticide companies. PARTICIPANTS: Dr. Juan Luis Jurat-Fuentes; Principal Investigator for the Bt mode of action in Lepidoptera objectives, Assistant Professor, Department of Entomology and Plant Pathology, University of Tennessee, Knoxville, TN 37996. Dr. Jurat-Fuentes oversees the activities of a Postdoctoral Associate (Dr. Oppert) and graduate students assigned to this project. Additional responsibilities of Dr. Jurat-Fuentes include performance of experiments for the Bt mode of action in Coleoptera objective, progress report preparation, funding administration, and manuscript preparation and submission. Dr. Omaththage P. Perera; Co-Principal Investigator, Research Entomologist, USDA-ARS Southeastern Insect Management Research Unit, Stoneville, MS 38776. Dr. Perera directs the microarray analysis of resistance and co-directs with Dr. Jurat-Fuentes the characterization of Cry1Ac mode of action in H. virescens. Dr. Carlos Blanco; Co-Principal Investigator, Research Entomologist, USDA-ARS Southeastern Insect Management Research Unit, Stoneville, MS 38776. Dr. Blanco developed the Bt corn-resistant strains of S. frugiperda used in this project. He also performed initial characterization of resistance and cross-resistance in these insects. Dr. Brenda Oppert; Co-Principal Investigator, Research Molecular Biologist, USDA-ARS Grain Production and Marketing Research Center, Manhattan, KS 66502. Dr. Oppert directs the characterization of Bt intoxication in the Coleoptera objective. Dr. Jeff Fabrick; Co-principal Investigator, Research Entomologist, USDA-ARS Arid-Land Agricultural Research center, Maricopa, AZ 85238. Dr. Fabrick designs and performs research related to the characterization of Bt mode of action in Coleoptera. Dr. Cris Oppert; Postdoctoral Research Associate, Department of Entomology and Plant Pathology, University of Tennessee, Knoxville, TN, 37996. Dr. Oppert is responsible for the proteomic 2D-DIGE differential analysis of resistance, evaluation of Bt resistance fitness costs, and testing of HevCaLP as Cry2Ab receptor. Dr. Oppert also designs and performs experiments for the Bt mode of action in Coleoptera objective. Anais S. Castagnola; Graduate Research Assistant; Department of Entomology and Plant Pathology, University of Tennessee, Knoxville, TN, 37996. Ms. Castagnola is the student assigned to the characterization of the gut healing response as a potential mechanism of resistance. She designs and performs experiments related to this part of the project, and also assists with larval intoxication assays and insect dissections for microarrays. TARGET AUDIENCES: Our research targets scientists in the US and abroad interested in the mode of action of Cry toxins from Bacillus thuringiensis (Bt) and the resistance mechanisms that insects evolve against these toxins. Data from our project also targets companies and farmers developing or using insecticidal technologies based on Bt toxins, and regulatory agencies responsible for risk assessment for current and novel Bt transgenic technologies. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We have documented that a peptide derived from the Cry3Aa receptor cadherin, previously identified as TmCad1, enhances toxicity when mixed with Cry toxin preparations against both coleopteran and lepidopteran pests. These data have been generated for diverse species that are not currently controlled efficiently with Cry toxins, and demonstrate the potential use of this peptide to develop novel and efficient Cry-based methods to control difficult pests. We have combined microarray and proteomic analyses to identify genes and proteins that are differentially expressed when Heliothis virescens larvae are exposed to Cry toxins. In microarray assays, diverse gene families involved in digestion, tissue regeneration, and signaling that change their expression upon Cry intoxication have been identified as targets for further analysis of their role in the Cry intoxication process. Proteomics analysis of gut fluids after exposure to Cry toxins has identified proteins that are differentially expressed by gut cells after exposure to Cry toxins, which will allow us to further characterize the gut epithelium response to Cry intoxication. As part of our research aimed at characterization of resistance to Cry toxins, we have maintained field-isolated colonies of S. frugiperda that are resistant to Bt corn expressing Cry1Fa. Characterization of resistance mechanisms and fitness of resistant insects will greatly contribute to our overall goal of optimization of strategies and mandates aimed at delaying the onset of resistance to Bt crops. We have also used Bt corn-resistant larvae to develop a potential biomarker for resistance to Bt (alkaline phosphatase). Through collaboration with Dr. Kongming Wu (Institute of Plant Protection, Beijing, China) we characterized alkaline phosphatase as putative Cry1Ac toxin receptor that is down-regulated in Cry1Ac-resistant insects. Identification of a sensitive resistance biomarker is crucial to develop and implement methodologies to monitor for resistance to Bt transgenic crops. Using a 1DGeLC/MS/MS proteomic approach, we have identified proteins that are secreted by H. virescens gut cells after exposure to Cry1Ac toxin. Using the same method, we have compared secretomes from susceptible and Cry-resistant strains of H. virescens, to identify alterations in the gut regenerative process correlating with enhanced healing capacity in resistant insects. Both these studies have identified proteins with a key role in gut regeneration that can be targeted in novel pesticidal technologies to inhibit gut healing after intoxication with entomopathogens.
Publications
- Blanco, C., Portilla, M., Jurat-Fuentes, J. L., Sanchez, J. F., Viteri, D., Vega-Aquino,P., Teran-Vargas, A. P., Azuara-Dominguez, A., Lopez, J. D., Arias, R., Zhu, Y.-C., Lugo-Barrera, D., and R. Jackson (2010) "Susceptibility of isofamilies of Spodoptera frugiperda (Lepidoptera: Noctuidae) to Cry1Ac and Cry1Fa proteins of Bacillus thuringiensis" Southwest. Entomol. 35(3): 409-415.
- Ning, C., Wu, K., Liu, C., Gao, Y., Jurat-Fuentes, J. L., and X. Gao (2010) "Characterization of a Cry1Ac toxin-binding alkaline phosphatase in the midgut from Helicoverpa armigera (Hubner) larvae". J. Insect Physiol. 56(6):666-672.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: Experimental data from this research project have been disseminated through presentations at national and international scientific meetings. The identification of a Tenebrio molitor cadherin as a Cry3Aa receptor is the first report of a functional Cry receptor in a coleopteran insect. This information was presented in a contributing oral presentation at the 41st Annual Meeting of the Society for Invertebrate Pathology (August 2008, Warwick, United Kingdom). Our proteomic analysis of the Cry intoxication process was presented as an invited oral presentation at the 56th Annual Meeting of the Entomological Society of America (November 2008, Reno, NV). Data on gut regeneration as a putative mechanisms of resistance to diverse Cry toxins (Cry1Ac and Cry2A) in laboratory-selected H. virescens strains were presented in invited talks at the 83rd Annual Meeting of the Southeastern branch of the Entomological Society of America (March 2009, Montgomery, AL), the 12th European Meeting of the International Organisation for Biological and Integrated Control of Noxious Animals and Plants (IOBC) West Paleartic Region Section (WPRS) in Pamplona, Spain (June 22-25, 2009), the 42nd Annual International Meeting of the Society for Invertebrate Pathology (August 16-20, 2009, Park City, Utah, USA), and the Department of Entomology at Clemson University (November 2008, Clemson, SC). Preliminary data from the validation of our Heliothis virescens microarray through comparison of Cry1Ac-treated and untreated larvae were presented in contributing oral presentations at the 56th National Annual Meeting of the Entomological Society of America (November 16-19, 2008, Reno, NV, USA), the 12th European Meeting of the IOBC/WPRS in Pamplona, Spain (June 22-25, 2009), the 41st Annual International Meeting of the Society for Invertebrate Pathology (August 3-7, 2008, Warwick, UK). Audiences in these meeting include students, research staff and laboratory directors from national and international research groups, as well as representatives and scientists from chemical and biological pesticide companies. PARTICIPANTS: Dr. Juan Luis Jurat-Fuentes; Principal Investigator for the Bt mode of action in Lepidoptera objectives, Assistant Professor, Department of Entomology and Plant Pathology, University of Tennessee, Knoxville, TN 37996. Dr. Jurat-Fuentes oversees the activities of a Postdoctoral Associate (Dr. Oppert) and graduate students assigned to this project. Additional responsibilities of Dr. Jurat-Fuentes include performance of experiments for the Bt mode of action in Coleoptera objective, progress report preparation, funding administration, and manuscript preparation and submission. Dr. Omaththage P. Perera; Co-Principal Investigator, Research Entomologist, USDA-ARS Southeastern Insect Management Research Unit, Stoneville, MS 38776. Dr. Perera directs the microarray analysis of resistance and co-directs with Dr. Jurat-Fuentes the characterization of Cry1Ac mode of action in H. virescens. Dr. Carlos Blanco; Co-Principal Investigator, Research Entomologist, USDA-ARS Southeastern Insect Management Research Unit, Stoneville, MS 38776. Dr. Blanco designs and performs research related to the mechanisms of resistance to Cry1Ac in H. virescens, more specifically the effect of diet ingredients in susceptibility to Cry1Ac toxin in bioassays. Dr. Brenda Oppert; Co-Principal Investigator, Research Molecular Biologist, USDA-ARS Grain Production and Marketing Research Center, Manhattan, KS 66502. Dr. Oppert directs the characterization of Bt intoxication in Coleoptera objective. Dr. Jeff Fabrick; Co-principal Investigator, Research Entomologist, USDA-ARS Arid-Land Agricultural Research center, Maricopa, AZ 85238. Dr. Fabrick designs and performs research related to the characterization of Bt mode of action in Coleoptera. Dr. Cris Oppert; Postdoctoral Research Associate, Department of Entomology and Plant Pathology, University of Tennessee, Knoxville, TN, 37996. Dr. Oppert is responsible for the proteomic 2D-DIGE differential analysis of resistance, evaluation of Bt resistance fitness costs, and testing of HevCaLP as Cry2Ab receptor. Dr. Oppert also designs and performs experiments for the Bt mode of action in Coleoptera objective. Anais S. Castagnola; Graduate Research Assistant; Department of Entomology and Plant Pathology, University of Tennessee, Knoxville, TN, 37996. Ms. Castagnola is the student assigned to the characterization of the gut healing response as a potential mechanism of resistance. She designs and performs experiments related to this part of the project, and also assists with larval intoxication assays and insect dissections for microarrays. TARGET AUDIENCES: Our research targets scientists in the US and abroad interested in the mode of action of Cry toxins from Bacillus thuringiensis (Bt) and the resistance mechanisms insects evolve against these toxins. Data from our project also targets companies and farmers developing or using insecticidal technologies based on Bt toxins, and regulatory agencies responsible for risk assessment for current and novel Bt transgenic technologies. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We have purified Bacillus thuringiensis toxins (Cry1Ac, Cry2Ab, and Cry3Aa) to use for all our research objectives. Our proteomic analysis of Cry3Aa intoxication in Tenebrio molitor larvae identified a midgut cadherin protein that we named TmCad1 as interacting with this toxin. To demonstrate the role of TmCad1 in Cry3Aa intoxication, we cloned the tmcad1 full length cDNA and expressed a fragment of TmCad1 that includes predicted Cry3A toxin binding regions in heterologous systems. The purified TmCad1 peptide bound Cry3Aa and increased formation of Cry3Aa oligomers, which have been reported to be crucial for toxicity. In bioassays, this TmCad1 peptide increased Cry toxicity or delayed time-to-kill against coleopteran, lepidopteran, and dipteran pests, which may be related to increased oligomer formation. To test the functional role of TmCad1 as Cry3Aa receptor we down-regulated its expression in larvae and demonstrated that lower TmCad1 expression levels results in lack of susceptibility to Cry3Aa. To characterize Cry1Ac intoxication in lepidopteran pests, we used a Heliothis virescens microarray containing 14,584 ESTs (assembled from 39,760 nucleotide sequences from the H. virescens gut transcriptome) and RNA from midguts of susceptible H. virescens larvae treated with Cry1Ac toxin, and compared the gene expression to larvae from resistant H virescens strains. A number of genes, including reported Cry1Ac receptors such as aminopeptidase and alkaline phosphatase, displayed dramatic changes in their levels of expression. During preparation of the H. virescens microarray we identified and cloned five isoforms of alkaline phosphatase, a previously predicted Cry1Ac receptor. We expressed one of these proteins (HvmALP1) in Escherichia coli to test the importance of glycosylation for HvmALP1-Cry1Ac interactions. We have subcloned and expressed glycosylated HvmALP1 in S2 insect cell cultures and are testing interactions with Cry1Ac and the role of these interactions on toxicity. As part of our research on the Cry1Ac intoxication process, we discovered the effect of diet on the susceptibility of H. virescens larvae to this toxin. This information is crucial to develop and implement methodologies to monitor for resistance to Cry1Ac-producing transgenic crops. Through quantification of midgut stem cell proliferation as a direct indicator of midgut regeneration after Cry intoxication, we have been able to identify enhanced regeneration in the CXC and KCB strains (both resistant to Cry1Ac and Cry2A toxins) of H. virescens as a potential mechanism of resistance to diverse Cry toxins. Midgut stem cell primary cultures from resistant strains showed increased proliferation when treated with factors secreted by mature midgut cells treated with Cry1Ac or Cry3Aa toxin.
Publications
- Blanco, C. A., Gould, F., Vega-Aquino, P., Jurat-Fuentes, J. L., Perera, O. P., and C. A. Abel (2009). Response of Heliothis virescens (Lepidoptera: Noctuidae) strains to Bacillus thuringiensis Cry1Ac incorporated into different insect artificial diets. J. Econ. Entomol., 102(4):1599-1606.
- Fabrick J., Oppert C., Lorenzen M. D., Morris K., Oppert B., and J. L. Jurat-Fuentes (2009). A novel Tenebrio molitor cadherin is a functional receptor for Bacillus thuringiensis Cry3Aa toxin. J. Biol. Chem., 284(27):18401-18410.
- Perera, O. P., Willis, J. D., Adang, M. J., and J. L. Jurat-Fuentes (2009). Cloning and characterization of the Cry1Ac-binding alkaline phosphatase (HvALP) from Heliothis virescens. Insect Biochem. Molec. Biol. 39(4): 294-302.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: None to report at this time. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
None to report at this time.
Publications
- No publications reported this period
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