Progress 01/15/09 to 01/14/12
Outputs
OUTPUTS: Bacillus thuringiensis(Bt) Cry toxins are used in the control of insect pests. They are pore-forming toxins with a complex mechanism that involves the sequential interaction with receptors. They are produced as protoxins, which are activated by midgut proteases. Activated toxin binds to cadherin receptor, inducing an extra cleavage including helix α-1, facilitating the formation of a pre-pore oligomer. The toxin oligomer binds to secondary receptors such as aminopeptidase and inserts into lipid rafts forming pores and causing larval death. The primary threat to efficacy of Bt-toxins is the evolution of insect resistance. Engineered Cry1AMod toxins, devoid of helix alpha-1, could be used for the control of resistance in lepidopterans by bypassing the altered cadherin receptor, killing resistant insects affected in this receptor. We analyzed the mechanism of action of Cry1AbMod. We found that alkaline pH and the presence of membrane lipids facilitates the oligomerization of Cry1AbMod. In addition, tryptophan fluorescence emission spectra, ELISA binding to pure aminopeptidase receptor, calcein release assay and analysis of ionic-conductance in planar lipid bilayers, indicated that the secondary steps in mode of action that take place after interaction with cadherin receptor such as oligomerization, receptor binding and pore formation are similar in the Cry1AbMod and in the wild type Cry1Ab. Finally, the membrane-associated structure of Cry1AbMod oligomer was analyzed by electron crystallography showing that it forms a complex with a trimeric organization. Transgenic crops that produce Bacillus thuringiensis (Bt) toxins are grown widely for pest control, but insect adaptation can reduce their efficacy. Genetically modified Bt toxins Cry1AbMod and Cry1AcMod were designed to counter insect resistance to native Bt toxins Cry1Ab and Cry1Ac. Previous results suggested that the modified toxins would be effective if and only if resistance was linked with mutations in genes encoding toxin-binding cadherin proteins. We found evidence from five major crop pests refuting this hypothesis. Relative to native toxins, the potency of modified toxins was >350-fold higher against resistant strains of Plutella xylostella and Ostrinia nubilalis in which resistance was not linked with cadherin mutations. Conversely, the modified toxins provided little or no advantage against some resistant strains of three other pests with altered cadherin. Independent of the presence of cadherin mutations, the relative potency of the modified toxins was generally higher against the most resistant strains. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Target audiences include farmers, regulators, policy makers, environmentalists, the pest control industry, and scientists. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
With Bt toxins increasingly important for pest control and the threat of resistance also mounting, the modified toxins could help to protect the nation's food supply and promote sustainable, environmentally friendly agriculture. The project was featured in a PBS Documentary: "The Botany of Desire" (broadcast nationally October 2009) and in an Inside Science Television (ISTV) program produced in 2011 by the American Institute of Physics: "Smarter Pest Control for the Cotton Crop" that was broadcast in many US cities.
Publications
- Tabashnik, B. E., J. B. J. Van Rensburg and Y. Carriere. 2009. Field-evolved insect resistance to Bt crops: definition, theory, and data. Journal of Economic Entomology 102: 2011-2025.
- Franklin, M. T., C. L. Nieman, A. F. Janmaat, M. Soberon, A. Bravo, B. E. Tabashnik and J. H. Myers. 2009. Modified Bacillus thuringiensis toxins and a hybrid B. thuringiensis strain counter greenhouse-selected resistance in Trichoplusia ni. Applied and Environmental Microbiology 75: 5739-5741.
- Tabashnik, B. E, F. Huang, M. N. Ghimire, B. R. Leonard, B. D. Siegfried, M. Rangasamy, Y. Yang, Y. Wu, L. J. Gahan, D. G. Heckel, A. Bravo and M. Soberon. 2011. Efficacy of genetically modified Bt toxins against insects with different mechanisms of resistance. Nature Biotechnology 29: 1128-1131.
- Porta, H., Jimenez, G., Cordoba, E., Leon, P., Soberon, M., and A. Bravo 2011.Tobacco plants expressing the Cry1AbMod toxin suppressed the insect tolerance to Cry1Ab toxin of Manduca sexta cadherin-silenced larvae. Insect Biochem. Mol. Biol. 41: 513-519.
- Porta, H., Munoz-Minutti, C., Soberon, M. and A. Bravo. 2011. Induction of Manduca sexta larvae caspases expression in midgut cells by Bacillus thuringiensis Cry1Ab toxin. Psyche 2011, Article ID 938249 doi:10.1155/2011/938249.
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Progress 01/15/10 to 01/14/11
Outputs
OUTPUTS: Bacillus thuringiensis Cry toxins are used in the control of insect pests. They are pore-forming toxins with a complex mechanism that involves the sequential interaction with receptors. They are produced as protoxins, which are activated by midgut proteases. Activated toxin binds to cadherin receptor, inducing an extra cleavage including helix α-1, facilitating the formation of a pre-pore oligomer. The toxin oligomer binds to secondary receptors such as aminopeptidase and inserts into lipid rafts forming pores and causing larval death. The primary threat to efficacy of Bt-toxins is the evolution of insect resistance. Engineered Cry1AMod toxins, devoid of helix alpha-1, could be used for the control of resistance in lepidopterans by bypassing the altered cadherin receptor, killing resistant insects affected in this receptor. We analyzed the mechanism of action of Cry1AbMod. We found that alkaline pH and the presence of membrane lipids facilitates the oligomerization of Cry1AbMod. In addition, tryptophan fluorescence emission spectra, ELISA binding to pure aminopeptidase receptor, calcein release assay and analysis of ionic-conductance in planar lipid bilayers, indicated that the secondary steps in mode of action that take place after interaction with cadherin receptor such as oligomerization, receptor binding and pore formation are similar in the Cry1AbMod and in the wild type Cry1Ab. Finally, the membrane-associated structure of Cry1AbMod oligomer was analyzed by electron crystallography showing that it forms a complex with a trimeric organization. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Target audiences include farmers, regulators, policy makers, environmentalists, the pest control industry, and scientists. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
With Bt toxins increasingly important for pest control and the threat of resistance also mounting, the modified toxins could help to protect the nation's food supply and promote sustainable, environmentally friendly agriculture. The project was featured in a PBS Documentary: "The Botany of Desire" (broadcast nationally October 2009).
Publications
- Munoz-Garay, C. L. Portugal, L. Pardo-Lopez, N. Jimenez-Juarez, I. Arenas, I. Gomez, R. Sanchez-Lopez, R. Arroyo, A. Holzenburg, C. G. Savva, M. Soberon and A. Bravo. 2009. Characterization of the mechanism of action of the genetically modified Cry1AbMod toxin that is active against Cry1Ab-resistant insects. Biochimica et Biophysica Acta 1788: 2229-2237.
- Tabashnik, B. E., J. B. J. Van Rensburg and Y. Carriere. 2009. Field-evolved insect resistance to Bt crops: definition, theory, and data. Journal of Economic Entomology 102: 2011-2025.
- Carriere, Y., D. W. Crowder and B. E. Tabashnik. 2010. Evolutionary ecology of insect adaptation to Bt crops. Evolutionary Applications 3: 561-573.
- Fernandez-Luna, M. T., B. E. Tabashnik, H. Lanz-Mendoza, A. Bravo, M. Soberon, J. Miranda-Rios. 2010. Single concentration tests show synergism among Bacillus thuringiensis subsp. israelensis toxins against the malaria vector mosquito Anopheles albimanus. J. Invert. Pathol. 104: 231-233.
- Munoz-Garay, C., M. Soberon, and A. Bravo. 2010. Mode of action of Bacillus thuringiensis-genetically modified Cry1AbMod and Cry1AcMod Toxins: Role of alkaline pH in toxin oligomerization. Southwest. Entomol. 35: 383-386.
- Tabashnik, B. E. 2010. Communal benefits of transgenic corn. Science 330: 189-190.
- Tabashnik, B. E., M. S. Sisterson, P. C. Ellsworth, T. J. Dennehy, L. Antilla, L. Liesner, M. Whitlow, R. T Staten, J. A. Fabrick, G. C. Unnithan, A. J. Yelich, C. Ellers-Kirk, V. S. Harpold, X. Li and Y. Carriere. 2010. Suppressing resistance to Bt cotton with sterile insect releases. Nature Biotechnology 28: 1304-1307.
- Wang, J., E. D. Miller, G. S. Simmons, T. A. Miller, B. E. Tabashnik and Y. Park. 2010. PiggyBac-like elements in the pink bollworm, Pectinophora gossypiella. Insect Mol. Biol. 19: 177-184.
- Wang, J., G. S. Simmons, T. A. Miller, B. E. Tabashnik and Y. Park. 2011. Global variation in the piggyBac-like element of the pink bollworm, Pectinophora gossypiella. Journal of Asia-Pacific Entomology 14: 131-135.
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Progress 01/15/09 to 01/14/10
Outputs
OUTPUTS: Insecticidal proteins from Bacillus thuringiensis are used widely for environmentally friendly insect pest control in sprays and transgenic plants. However, evolution of resistance by insect pests threatens the continued success of B. thuringiensis toxins. Resistance to B. thuringiensis toxins has been documented in pest populations attacking crops in fields or greenhouses for five species of Lepidoptera. As part of this project, we focused on understanding and countering resistance to B. thuringiensis toxins that evolved in commercial greenhouse populations of a major crop pest, the cabbage looper, Trichoplusia ni. We suspected that two approaches might be useful for countering greenhouse-selected resistance of T. ni. First, we hypothesized that toxicity to resistant T. ni would be greater for genetically modified toxins Cry1AbMod and Cry1AcMod than for the native toxins Cry1Ab and Cry1Ac, which are in Dipel. Cry1AbMod and Cry1AcMod are genetically engineered variants of Cry1Ab and Cry1Ac, respectively, in which an amino-terminal region including helix α-1 has been removed. Cry1AbMod and Cry1AcMod were toxic to Pectinophora gossypiella that were highly resistant to Cry1Ab and Cry1Ac. Because greenhouse-selected resistance of T. ni and laboratory-selected resistance of P. gossypiella both fit the mode 1 pattern of resistance, we hypothesized that the modified toxins would kill resistant T. ni, even though linkage with cadherin has not been reported for T. ni resistance. Second, we hypothesized that toxicity to resistant T. ni would be greater for B. thuringiensis subsp. aizawai than B. thuringiensis subsp. kurstaki. Formulations of B. thuringiensis subsp. aizawai contain substantial amounts of Cry1C and Cry1D toxins, which are not in B. thuringiensis subsp. kurstaki. Given that greenhouse-selected resistance in T. ni evolved in response to sprays of B. thuringiensis subsp. kurstaki and did not include cross-resistance to Cry1C and Cry1D, we hypothesized that the Cry1C and Cry1D in B. thuringiensis subsp. aizawai would be effective against the resistant strains. We tested these hypotheses by using bioassays to compare responses of susceptible and resistant strains of T. ni. PARTICIPANTS: Co-pIs on this project are Drs. Mario Soberon and Alejandra Bravo at the Universidad Nacional Autonoma de Mexico. Collaborating scientists also include Michelle Franklin and Judith Myers at the University of British Columbia. TARGET AUDIENCES: Target audiences include farmers, regulators, policy makers, environmentalists, the pest control industry, and scientists. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
In laboratory bioassays with artificial diet, we evaluated susceptibility of greenhouse-selected, resistant strains and susceptible strains of T. ni to single toxins and formulations of B. thuringiensis. We compared the efficacy of native toxins Cry1Ab and Cry1Ac versus genetically modified toxins Cry1AbMod and Cry1AcMod that lack helix α-1 and are thought to skip binding to the cadherin receptor protein that occurs with Cry1Ab and Cry1Ac. We also compared the efficacy of DiPel versus Agree, a Bacillus thuringiensis subsp. aizawai product that contains the toxins Cry1C and Cry1D, which are not present in DiPel. Based on the concentration killing 50% (LC50) of a resistant strain divided by the LC50 of a susceptible strain, resistance was 590-fold for Cry1Ab and 1400-fold for Cry1Ac, but only 5.5-fold for Cry1AbMod and 9.6-fold for Cry1AcMod. Thus, resistance to the modified toxins was more than 100-fold lower than resistance to the native toxins. Resistance to Agree was 4- to 37-fold less than resistance to DiPel. Against resistant strains, the modified toxins were more potent than the native toxins and Agree was more potent than DiPel, but the reverse was true against susceptible strains. The results suggest that the modified toxins and B. thuringiensis subsp. aizawai may be useful for countering greenhouse-selected resistance of T. ni to B. thuringiensis subsp. kurstaki.
Publications
- Franklin, M. T., C. L. Nieman, A. F. Janmaat, M. Soberon, A. Bravo, B. E. Tabashnik and J. H. Myers. 2009. Modified Bacillus thuringiensis toxins and a hybrid B. thuringiensis strain counter greenhouse-selected resistance in Trichoplusia ni. Appl. Environ. Microbiol. 75: 5739-5741.
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