Progress 04/01/08 to 03/31/13
Outputs
Target Audience: The major target audience of this project is researchers in academia and industry in the areas of Bt-resistance and agricultural biotechnology. Audience of the project also includes insect IPM professionals and growers. The target audiences were reached by publication of our findings in scientific journals, presentation of the findings in professional meetings and seminars, communication with colleagues in the field, and presentation and face-to-face meetings through department out-reach activities. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This project provided valuable opportunities for learning and career development to all participants of the project. This project provided unique training opportunities to a postdoc, students and technical staff. The postdoctoral associate, Dr. Kasorn Tiewsiri, a key participant of this project, received extensive training in insect midgut biochemistry and proteomics by participating in this project. Upon completion of her postdoctoral research with this project, she secured a position as the manager of a genetics lab in a major healthcare company. This project provided Ms. Xiaozhao Song, a PhD student, an excellent opportunity for her graduate training. Song had previous training in fishery and ecology for her BS and MS. This project provided her with the training opportunity in insect biochemistry and molecular biology and a research opportunity for her PhD thesis research. A graduate student, Mr. Xiaoning Nan, learned gene cloning and molecular analysis of gene expression in this project. His participation in this project contributed to his PhD thesis research. This project greatly helped the PI to train and advise researchers and students, and allowed him to keep abreast of the new proteomics and next generation sequencing technologies for research in the field. How have the results been disseminated to communities of interest? The results from this project have been disseminated to communities of interest through our publications in scientific journals and presentations in professional meetings. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
This project aimed to identify the genes/proteins associated with the resistance to Bt toxin Cry1Ac in the cabbage looper, Trichoplusia ni, that evolved Bt-resistance in commercial greenhouses. The three major goals proposed in this project were successfully accomplished. Goal 1. To identify the midgut brush border membrane proteins which are differentially present between the susceptible and Bt Cry1Ac-resistant T. ni larvae using multiple approaches to answer the question “what candidate Cry1Ac binding proteins in the midgut are altered to confer the greenhouse-evolved resistance in T. ni?” – Using biochemical, proteomic and genetic approaches, we identified that the Cry1Ac-resistance in T. ni was associated with down-regulation of aminopeptidase N1 (APN1) and up-regulation of aminopeptidase N6 (APN6) at the protein level in the midgut brush border membrane. The major Cry toxin receptor, cadherin, in the larval midgut does not have alteration in either protein sequence or expression level in the resistant T. ni. (Tiewsiri and Wang, 2011. PNAS, 108: 14037-14042; Zhang et al., 2012. PLoS One, 7:e35991; Zhang et al., 2013. Insect Biochem. Mol. Biol., in press) Goal 2. To identify the gene(s) for the candidate Cry1Ac binding protein(s) and their mutations in the resistant T. ni to answer the question “what mutations of the binding protein genes confer the greenhouse-evolved resistance in T. ni?” – Using genetic and molecular approaches, we identified that the resistance in T. ni is associated with the down-regulation of the APN1 gene. However, the APN1 gene is not genetically linked with the resistance and the down-regulation of APN1 expression is conferred by a trans-regulatory mechanism. The resistance gene was mapped to the ABC transporter, ABCC2, gene locus in T. ni. The cadherin gene in T. ni was identified to be highly variable, but the resistance to Cry1Ac in T. ni populations selected in greenhouses is not conferred by cadherin gene mutations. (Tiewsiri and Wang, 2011. PNAS, 108: 14037-14042; Baxter et al., 2011. Genetics, 189: 675-679; Zhang et al., 2012. PLoS One, 7:e35991; Zhang et al., 2013. Insect Biochem. Mol. Biol. In press) Goal 3. To determine the differential expression of the candidate Cry1Ac binding protein genes in the resistant T. ni to answer the question “is resistance in T. ni conferred by differential expression of the Cry1Ac binding protein genes?” – Using genetic, biochemical and molecular approaches, we identified that the Cry1Ac resistance in greenhouse-selected T. ni populations is associated with differential expression of the APN1 and APN6 genes. The down-regulation of APN1 was directly correlated with the resistance to Cry1Ac. The expression of other putative midgut receptors for Cry1Ac is not altered in Cry1Ac-resistant T. ni. (Tiewsiri and Wang, 2011. PNAS, 108: 14037-14042; Zhang et al., 2012. PLoS One, 7:e35991) The findings from this project represent the first identification of a molecular mechanism of insect resistance to Bt toxins evolved in an agricultural system, and a novel Bt resistance mechanism associated with alteration of an APN gene expression conferred by a trans-regulatory genetic mechanism. The results from this project provided us with a new insight into the molecular genetic mechanisms for Bt resistance in insect pests and new fundamental knowledge important to the field of Bt resistance management of insect pests in agriculture.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Zhang, X, Tiewsiri, K., Kain, W., Huang, L. and Wang, P. (2012) Resistance of Trichoplusia ni to Bacillus thuringiensis Toxin Cry1Ac is independent of alteration of the cadherin-like receptor for Cry toxins. PLoS ONE 7: e35991. doi:10.1371/journal.pone.0035991.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Tian, J. C., Wang, X. P., Long, L. P., Romeis, J., Naranio, S. E. Hellmich, R. L., Wang, P., Earle, E. D. and Shelton, A. M. (2013) Bt Crops producing Cry1Ac, Cry2Ab and Cry1F do not harm the green lacewing, Chrysoperla rufilabris. PLoS One 8 (3): e60125.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2013
Citation:
Zhang, X., Kain, W. and Wang, P. (2013) Sequence variation and differential splicing of the midgut cadherin gene in Trichoplusia ni. Insect Biochemistry and Molecular Biology (in press)
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Progress 04/01/11 to 03/31/12
Outputs
OUTPUTS: Cases of insect resistance to Bt toxins have been reported in agricultural settings in several species, but the molecular genetic mechanism for field-evolved resistance has yet to be understood. In this project, we took genetic, biochemical, molecular and proteomic approaches to identify the genes/proteins involved in the resistance to Bt toxin Cry1Ac in the cabbage looper, Trichoplusia ni, that evolved Bt-resistance in commercial greenhouses. Genetic analysis of the genes coding for candidate Bt toxin receptors and their allelic association with the Cry1Ac-resistance were conducted and the results indicated that the Bt resistance in the greenhouse population of T. ni involves a novel molecular genetic mechanism. With the near-isogenic Bt-resistant and susceptible T. ni strains generated in this project, midgut proteins were globally examined by biochemical and proteomic analysis to identify midgut proteins that bind to Bt Cry1A toxins and are differentially expressed in association with the resistance. We identified that the resistance to the Bt toxin Cry1Ac evolved in greenhouse populations of T. ni is associated with differential alteration of two midgut aminopeptidases N, APN1 and APN6, conferred by a trans-regulatory mechanism. Biochemical, proteomic and molecular analyses determined that in the Cry1Ac resistant T. ni, the expression of APN1 gene was significantly down-regulated and, in contrast, the expression of APN6 gene was significantly up-regulated. The Cry1Ac resistance was correlated with down-regulation of APN1 but not with the up-regulation of APN6. The up-regulation of APN6 concurrent with down-regulation of APN1 may play a role in compensating for the loss of APN1 to minimize the fitness costs of the resistance. In addition to identification of reduced expression of APN1 as the molecular basis of Bt-resistance selected in an agricultural setting, the results demonstrated the importance of APN1 in the mode of action of Bt toxin Cry1Ac. The findings from this project represent the first identification of a molecular mechanism of insect resistance to Bt toxins evolved in an agricultural system. To further understand the trans-acting gene controlling the resistance, we took a genetic linkage mapping approach to identify genes with genetic linkage to the resistance and have mapped the Cry1Ac-resistance gene to the ABCC2 gene locus. PARTICIPANTS: Ping Wang, PD of this project; Wendy Kain, Research Specialist; Xiaozhao Song, Graduate Student TARGET AUDIENCES: Researchers in academia and industry, insect IPM professionals and growers PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Bacillus thuringiensis (Bt) has continued to be the most successfully used microbial insecticide and its genes coding for insecticidal toxins are the primary transgenes utilized in current insect-resistant transgenic crops. Development of resistance to Bt toxins in insect populations is the foremost threat to the sustainable application of Bt biopesticides and Bt crops. Resistance of insect pest populations to both Bt formulations and Bt-crops has occurred in agricultural systems. In this project, we identified the molecular mechanism of high level Bt resistance evolved in greenhouse populations of T. ni, which is the first identification of a molecular mechanism of Bt-resistance selected in agriculture, and genetically mapped the resistance gene locus. Our finding of the molecular basis of the Bt-resistance evolved in T. ni populations in agriculture provided us with a new insight into the molecular genetic mechanisms for Bt resistance in insect pests and new fundamental knowledge important to the field of Bt resistance management of insect pests in agriculture.
Publications
- Baxter, S. W., Badenes-Perez, F. R., Morrison, A., Vogel, H., Crickmore, N., Kain, W., Wang, P., Heckel, D. G. and Jiggins, C. D. (2011) Parallel evolution of Bacillus thuringiensis toxin resistance in Lepidoptera. Genetics, 189: 675-679.
- Tiewsiri, K. and Wang, P. (2011) Differential alteration of two aminopeptidases N associated with resistance to Bacillus thuringiensis toxin Cry1Ac in cabbage looper. Proc. Natl. Acad. Sci. USA 108: 14037-14042.
- Li, Y., Romeis, J., Wang, P., Peng, Y. and Shelton, A. M. (2011) A Comprehensive assessment of the effects of Bt cotton on Coleomegilla maculata demonstrates no detrimental effects by Cry1Ac and Cry2Ab. PLoS ONE 6, e22185. doi:10.1371/journal.pone.0022185
- Terenius, O. et al. (2011) RNA interference in Lepidoptera: An overview of successful and unsuccessful studies and implications for experimental design. Journal of Insect Physiology 57: 231-245.
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Progress 04/01/10 to 03/31/11
Outputs
OUTPUTS: To date, cases of resistance to Bt toxins have been reported in agricultural settings in six insect species, but the molecular mechanism for any of these cases of resistance has not been identified. In this project, we took genetic, biochemical, molecular and proteomic approaches to identify the genes/proteins involved in the resistance to Bt toxin Cry1Ac in populations of the cabbage looper, Trichoplusia ni, that evolved Bt-resistance in commercial greenhouses. Genetic analysis of insect midgut genes coding for candidate Bt toxin receptors and quantitative analysis of the expression of these candidate genes at both transcript and protein levels were conducted and the results indicated that the Bt resistance in the greenhouse population of T. ni involves a novel genetic mechanism. With the near-isogenic Bt-resistant and susceptible T. ni strains generated in the project, midgut proteins were globally studied by biochemical and proteomic analysis, aiming to identify midgut proteins that bind to Bt Cry1A toxins, and that are differentially expressed and associated with the resistance. We identified that the resistance to the Bt toxin Cry1Ac in T. ni is associated with differential alteration of two midgut aminopeptidases N, APN1 and APN6, conferred by a trans-regulatory mechanism. Biochemical, proteomic and molecular analyses determined that in the Cry1Ac resistant T. ni, APN1 was significantly down-regulated and, in contrast, APN6 was significantly up-regulated. The Cry1Ac resistance was correlated with down-regulation of APN1 but not with the up-regulation of APN6. The up-regulation of APN6 concurrent with down-regulation of APN1 may play a role in compensating for the loss of APN1 to minimize the fitness costs of the resistance. In addition to identification of reduced expression of APN1 as the molecular basis of Bt-resistance selected in an agricultural setting, the results demonstrated the importance of APN1 in the mode of action of Bt toxin Cry1Ac. The findings from this project represent the first identification of a molecular mechanism of insect resistance to Bt toxins evolved in an agricultural system. PARTICIPANTS: Ping Wang, PD of this project Kasorn Tiewsiri, Postdoctoral Associate Wendy Kain, Research Specialist Xiaozhao Song, Graduate Student TARGET AUDIENCES: Researchers in academia and industry, insect IPM professionals and growers. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Bacillus thuringiensis (Bt) has been the most successfully used microbial insecticide and its genes coding for insecticidal toxins are the primary transgenes utilized in current insect-resistant transgenic crops. Evolution of insect resistance to Bt toxins is the foremost threat to the long-term future of Bt biopesticides and Bt crops. Resistance of insect pest populations to Bt formulations and Bt-crops has occurred in agricultural systems. In this reporting period of the project, we identified the molecular mechanism of high level Bt resistance evolved in greenhouse populations of T. ni, which is the first identification of a molecular mechanism of Bt-resistance selected in agriculture. Our finding of the novel molecular basis of the Bt-resistance evolved in T. ni populations in agriculture provided us with a new insight into the diverse genetic mechanisms for Bt resistance in insects and new fundamental knowledge important to the field of Bt resistance management of insect pests in agriculture.
Publications
- No publications reported this period
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Progress 04/01/09 to 03/31/10
Outputs
OUTPUTS: To date, Bt resistance has been documented in populations of five Lepidoptera species in agricultural systems. The common type of high level resistance to Bt Cry1A toxins identified in lepidopterans is "Mode 1" type resistance. "Mode 1" type resistance is conferred by reduced binding of Cry1A toxins to the host midgut binding sites, which is often associated with mutations of the midgut cadherin gene in host insects. However, the "Mode 1" type resistance in greenhouse-evolved resistant populations of Trichoplusia ni and field-evolved resistant populations of Plutella xylostella is conferred by a novel, but unknown, molecular genetic mechanism. To understand the molecular mechanism(s) of the "Mode 1" type resistance in insect populations that developed resistance in agricultural systems, in this project multiple approaches, including genetic, biochemical, molecular and proteomic approaches, were used to identify genes/proteins involved in the resistance mechanism in T. ni. Genetic linkage analysis of multiple cadherin gene alleles and quantitative analysis of cadherin gene expression and protein levels were conducted and the results further indicated that the Bt resistance in the greenhouse population of T. ni involves a novel genetic mechanism. With the near-isogenic Bt-resistant and susceptible T. ni strains, generated in the project, midgut proteins were comparatively studied by biochemical and proteomic analysis, aiming to identify midgut proteins that bind to Bt Cry1A toxins and are differentially present between the susceptible and resistant larvae. Genetic linkage analysis of sets of midgut genes was conducted to investigate their potential roles in the resistance evolved in greenhouse populations of T. ni. In parallel to the analysis of midgut proteins, the T. ni genes coding for candidate Bt toxin binding proteins and their expression were analyzed to investigate their linkage with the resistance. The mechanism of Bt Cry1Ac resistance in the greenhouse evolved population of T. ni is being studied at gene, mRNA and protein levels. PARTICIPANTS: Ping Wang, PD of this project; Kasorn Tiewsiri, Postdoctoral Associate; Xiaoning Nan, Visiting Scientist; Wendy Kain, Research Specialist; Xiaozhao Song, Graduate Student. TARGET AUDIENCES: Researchers in academia and industry, insect IPM professionals and growers PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Resistance of insect pest populations to Bt formulations and Bt-crops has occurred in agricultural systems. The development of Bt-resistance in insect populations threatens the sustainable application of Bt-based biological control technologies. In this reporting period of the project, we have further confirmed that the high level Bt resistance evolved in greenhouse populations of T. ni involves a novel mechanism distinctly different from the currently known genetic mechanisms of Bt resistance in insects. Results from this project to identify the molecular mechanism of the greenhouse-evolved Bt resistance in T. ni will not only reveal the mechanism for the Bt resistance evolved in a greenhouse population of T. ni, but will also facilitate understanding the development of Bt resistance in other insects in agricultural systems and will provide fundamental knowledge to the field of Bt resistance management of insect pests in agriculture.
Publications
- No publications reported this period
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Progress 04/01/08 to 03/31/09
Outputs
OUTPUTS: The most common type of high level insect resistance to Bt Cry1A toxins is "Mode 1" type resistance, which results from reduced binding of Cry1A toxins to the host midgut binding sites. The reduced binding of Cry toxins to the insect midgut is often associated with mutations of the midgut cadherin gene in insects. However, the "Mode 1" type resistance in greenhouse-evolved resistant populations of Trichoplusia ni and field-evolved resistant populations of Plutella xylostella is conferred by a novel, but unknown, molecular genetic mechanism. To understand the molecular mechanism(s) of the "Mode 1" type resistance in insect populations that developed resistance in agricultural systems, multiple approaches, including genetic, biochemical, molecular and proteomic approaches, are being used in this project to identify genes/proteins involved in the resistance mechanism in T. ni. For identification of genes/proteins that are differentially present between the susceptible and resistant T. ni strains, the resistant backcross strain of T. ni (GLEN-Cry1Ac-BCS) was further backcrossed with a susceptible laboratory strain four more times to introgress the resistance gene into the genetic background of the susceptible strain. Successful introgression was confirmed by allelic analysis of a nuclear gene. Genetic linkage analysis of multiple cadherin gene alleles and quantitative analysis of cadherin gene expression were conducted and the results further indicated that the greenhouse selected resistance in T. ni involves a novel genetic mechanism. Our previous studies have shown that the Cry1Ac resistance in T. ni is due to lack of Cry1A toxin binding to the larval midgut brush border membranes. Therefore, in this project the effort of cloning genes coding for putative Bt binding proteins was continued. Two additional genes coding for putative midgut binding proteins for Bt toxins were cloned, leading to completion of gene cloning for putative major Cry1A binding proteins, including the membrane bound alkaline phosphatase, 6 aminopeptidases N and the midgut cadherin. To examine the midgut proteins that bind to Cry1A toxins, midgut brush border membrane proteins were analyzed by affinity chromatography and proteins that bind to activated-Cry1Ac toxin were detected, which provides the basis for biochemical identification of midgut proteins involved in the resistance. More extensive proteomic analyses of midgut proteins are being conducted in search for the proteins involved in the resistance. Results from this project have been presented in scientific meetings. PARTICIPANTS: Ping Wang, PD of this project; Kasorn Tiewsiri, Postdoctoral Associate; Xiaoning Nan, Visiting Scientist; Wendy Kain, Research Specialist TARGET AUDIENCES: Researchers in academia and industry and insect IPM professionals PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Development of Bt-resistance in insect populations in agricultural systems threatens the sustainable application of Bt-crops and other Bt-based biological control technologies. In this reporting period of the project, we have further confirmed that the greenhouse evolved high level Bt resistance in T. ni involves a novel mechanism. Results from this project to identify the molecular mechanism of the greenhouse-evolved Bt resistance in T. ni will not only reveal the mechanism for the Bt resistance in T. ni, but will also facilitate understanding the development of Bt resistance in other insects in agricultural systems and provide important knowledge to the field of Bt resistance management of insect pests in agriculture.
Publications
- No publications reported this period
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