Progress 02/01/10 to 01/31/14
Outputs
Target Audience: Chemical ecology research community, forest management experts, biochemists Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This project provided training for graduate and undergraduate students in molecular biology, functional genomics, cell culture, recombinant protein production, enzyme assays, metabolite characterization, and standard biochemical and molecular lab techniques. These skills are in high demand by employers in molecular life science industries (e.g. biotechnology). How have the results been disseminated to communities of interest? The results have been reported in peer-reviewed scientific publications and presented at national and international conferences. What do you plan to do during the next reporting period to accomplish the goals? There are two remaining publications (both in revision) that are currently in preparation and will be submitted to peer-reviewed scientific journals within the next five months.
Impacts
What was accomplished under these goals?
Pine bark beetles are the most devastating insect pests of western forests in North America, causing significant economic costs due to ecological disturbances, fire suppression costs, and contributions to global climate perturbations. All bark beetles rely on pheromones (chemical signals) to reproduce. Understanding how the beetles make these chemicals is an important first step to understand how they are evolving along with their pine hosts, and to develop potential management strategies that will target the beetles without harming other organisms. The metabolic steps mountain pine beetles use to make their pheromones are virtually unknown. Our major gol is to understand the biochemical tools bark beetles use to make pheromones. The resulting handful of genes represent targets for future management strategies and are also windows into the evolutionary history of the beetles. Understanding how they adapt and have adapted will help predict their potential responses in the future. The three main Objectives of this proposal were: 1. Confirm the metabolic pathway and site of pheromone component biosynthesis 2. Characterize a potential pheromone-biosynthetic enzyme (CYP6CR1) and identify new ones 3. Characterize GPPS/FPPS (another pheromone-biosynthetic enzyme) We leveraged genomic research data to make preliminary identification of five new pheromone-biosynthetic enzymes and confirmed their functions. We also discovered that mountain pine beetles make pheromones in a completely different organ compared to other bark beetles. As a result, we and others investigating insect/plant interactions have a new, better understanding of the flexibility bark beetles can bring to their metabolism. Our research is pioneering work on the unique metabolism of these devastating forest pests. Our discoveries have implications for other pests as well.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Keeling, C.I., C.C. Chiu, T. Aw, M. Li, H. Henderson, C. Tittiger, H.-B. Weng, G.J. Blomquist and J. Bohlman (2013). Frontalin pheromone biosynthesis in the mountain pine beetle, Dendroctonus ponderosae Hopkins: role of isoprenyl diphosphate synthases. Proc. Natl. Acad. Sci. U.S.A. 110(47):18838-43. doi: 10.1073/pnas.1316498110.
- Type:
Journal Articles
Status:
Other
Year Published:
2014
Citation:
Song, M., P. Delaplain, T.T. Nguyen, X. Liu, L. Wickenberg, C. Jeffrey, G.J. Blomquist, and C. Tittiger (2014). exo-Brevicomin biosynthetic pathway enzymes from the mountain pine beetle, Dendroctonus ponderosae. In revision.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Song M, Gorzalski A, Nguyen TT, Liu X, Jeffrey C, Blomquist GJ, Tittiger C. (2014). exo-Brevicomin biosynthesis in the mountain pine beetle, Dendroctonus ponderosae.
J Chem Ecol. 40(2):181-9. doi: 10.1007/s10886-014-0381-9.
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Progress 02/01/12 to 01/31/13
Outputs
OUTPUTS: Objective 1: (confirm metabolic path and site of exo-brevicomin biosynthesis) This objective was completed during the previous reporting period. Objective 2: A preliminary assay using isolated male fat body tissues to determine if exo-brevocomin biosynthesis involves a 9-carbon intermediate yielded inconclusive results. Efforts were initiated to produce a labeled substrate (collaboration with UNR Dept. Chemistry. Objective 3: Experimental activity on this Objective is complete; work began for preparation of a manuscript reporting data . Other: Experiments to determine monoterpene substrate profiles of recombinant CYP9T3 were completed. Fatty acyl-CoA reductase enzymes (FARs) that act upstream of hydrocarbon biosynthesis were identified and functionally assayed. Pheromone-biosynthetic CYP6DH1 was determined to be possibly iso-genic with a detoxification enzyme, CYP6DH2. Efforts were initiated to compare their gene structures in a local mountain pine beetle population. Progress from this work was reported at the 2012 USDA-NIFA Principle Investigators Meeting in Oak Ridge, TN, in October, 2012. PARTICIPANTS: Reese Taylor - undergraduate student (FAR isolation and assay, gene structure studies) Minmin Song - graduate student (CYP6CR1, ZnoDH cloning and assay; substrate profiling) Marina MacLean - graduate student (CPR and FAR characterization) Leah Plaugher - undergraduate/graduate student student (ZnoDH cloning and assay) Karan Sharma - undergraduate student (CPR cloning and assay) Magda Nowinski - undergraduate student (FAR cloning) Aaron Kanow - (FAR cloning and functional assay) Nicole Abbott - graduate student (frontalin biosynthesis feeding experiments) Collaborators: Chris Jeffrey (University of Nevada Reno, Chemistry Department) Christopher I. Keeling (Michael Smith Laboratories, University of British Columbia) Joerg Bohlmann (Michael Smith Laboratories, University of British Columbia) Students gained experience in molecular biology and biochemical techniques, including PCR, qRT-PCR, cloning and sequencing, cell culture, recombinant protein production, enzyme assays, GC/MS analyses, enzyme kinetic analyses, bioinformatics, etc., depending on their roles in the project. Graduate students gained teaching and project management experience by training and mentoring undergrads. All participants gained experience in oral and written presentations. TARGET AUDIENCES: Chemical ecology community, life-science researchers. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Objective 2: Preliminary experiments emphasized the requirement for a labeled substrate (3-nonene) to complete this Objective. Objective 3: With molecular and biochemical data completed earlier in the year, we began to prepare a manuscript for publication. Anticipated submission date ~05/13. Other: There appears to be no practical biochemical difference in using housefly or bark beetle P450 reductase for our in vitro reactions. We can thus continue to use the housefly enzyme with high confidence. We determined that the FAR encoded by Drosophila melanogaster CG18031 accepts long chain (C24) fatty acids as substrate and produces an alcohol. CYP6DH1 and CYP6DH2, despite significantly different expression patterns and activities, appear to be allelic. Molecular sequencing across intronic DNA is necessary to confirm this hypothesis. It appears that all individuals carry CYP6DH2, but approximately half carry CYP6DH1. These data have important implications for mountain pine beetle population structures and the evolutionary pressures they face.
Publications
- Song, M., A.C. Kim, A.L. gorzalski, M. MacLean, S. Young, M.D. Ginzel, G.J. Blomquist, and C. Tittiger. 2013. Functional characterization of myrcene hydroxylases from two geographically distinct Ips pini populations. Insect Biochem. Molec. Biol. 43(4):336-343.
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Progress 02/01/11 to 01/31/12
Outputs
OUTPUTS: Objective 1: (confirm metabolic path and site of exo-brevicomin biosynthesis) This objective was completed during the previous reporting period. Objective 2: A microplate-based assay was developed to measure the amount of active (correctly folded) recombinant CYP6CR1 produced by our Sf9 expression system. Penta-deuterium-labeled substrates (e.g. (6Z)-nonen-2-one) were prepared and used in functional assays to test the hypothesis that CYP6CR1 converts the methyl-ketone to the keto-epoxide (penultimate step in the pathway). Objective 3: Feeding experiments with deuterium-labeled precursors were performed to trace the incorporation of mevalonate pathway intermediates into frontalin. Other: Further experiments on a novel oxidoreductase (ZnoDH) identified that enzyme as functioning upstream of ZnoDH. In vivo assays were performed to test the ability of fat body tissues to interconvert (6Z)-nonen-2-ol and (6Z)-nonen-2-one. The monoterpene substrate profiles of recombinant CYP6DH1, CYP6DH2, and CYP9T3 were explored by functional assays. The utility of bark beetle cytochrome P450 reductase (CPR) as a redox partner for our enzymes (as opposed to the housefly enzyme we have been using) was investigated. Fatty acyl-CoA reductase enzymes (FARs) that act upstream of hydrocarbon biosynthesis were identified and functionally assayed. Progress from this work was reported at the 2011 International Society for Chemical Ecology annual meeting (Vancouver, Canada) and the Entomological Society of America annual meeting (Reno, NV). PARTICIPANTS: Minmin Song - graduate student (CYP6CR1, ZnoDH cloning and assay; substrate profiling) Marina MacLean - graduate student (CPR and FAR characterization) Leah Plaugher - undergraduate/graduate student student (ZnoDH cloning and assay) Karan Sharma - undergraduate student (CPR cloning and assay) Magda Nowinski - undergraduate student (FAR cloning) Aaron Kanow - (FAR cloning and functional assay) Nicole Abbott - graduate student (frontalin biosynthesis feeding experiments) Tidiane Aw - graduate student (FPPG, GGPPS expression profiling) Collaborators: Chris Jeffrey (University of Nevada Reno, Chemistry Department) Christopher I. Keeling (Michael Smith Laboratories, University of British Columbia) Joerg Bohlmann (Michael Smith Laboratories, University of British Columbia) Students gained experience in molecular biology and biochemical techniques, including PCR, qRT-PCR, cloning and sequencing, cell culture, recombinant protein production, enzyme assays, GC/MS analyses, enzyme kinetic analyses, bioinformatics, etc., depending on their roles in the project. Graduate students gained teaching and project management experience by training and mentoring undergrads. All participants gained experience in oral and written presentations. TARGET AUDIENCES: Chemical ecology community, life-science researchers. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Objective 2: CYP6CR1 was confirmed as the epoxide-forming P450 active in the penultimate step of the exo-brevicomin-biosynthetic pathway. The epoxide is biologically stable, indicating that another enzyme catalyzes the cyclization of the epoxide to exo-brevicomin (final step). Objective 3: The feeding experiments were inconclusive, so we are unable to determine what precursor(s) are converted to sulcatone or another frontalin precursor. Intensive sequence confirmations were done to confirm the identities of putative prenyl-transferases. Of three candidates, two encode true enzymes (FPPS/GPPS, and GGPPS) and the third is a pseudogene. qRT-PCR experiments documenting their regulation were completed. Both genes show expression patterns consistent with frontalin production. These data suggest that the length of the terpene precursor to sulcatone/frontalin is not important. Other: Fat bodies readily reduce(6Z)-nonen-2-one to the secondary alcohol. This is a highly significant result because it suggests that early steps in exo-brevicomin biosynthesis involve oxidative decarbonylation of a C10-aldehyde precursor to a C9 hydrocarbon, which is then hydroxylated by a P450 to the secondary alcohol. It is exciting to contemplate the P450 involved, as well as the potential specificity of a short-chain specific fatty acyl-CoA reductase acting upstream of the oxidative decarbonylase. These are two new targets for gene isolation experiments. CYP6DH1 is produced in a pattern consistent with trans-verbenol biosynthesis, while the parologous CYP6DH2 has an expression pattern consistent with resin detoxification. These enzymes have complementary substrate preferences and produce highly specific hydroxylation products depending on the chirality of their substrates (+ or - alpha-pinene). These biochemical data support the hypothesis that pheromone-biosynthetic P450s evolved from enzymes originally used to detoxify host tree resin components. Kinetic assays of bark beetle P450s with bark beetle and hosuefly CPR are in progress. Four different FAR cDNAs have been isolated and are currently being assayed for substrate profiles.
Publications
- Aw. Tidiane. 2011. Functional genomics of mountain pine beetle (Dendroctonus ponderosae) related to pheromone production. Ph.D. Dissertation. Department of Biochemistry and Molecular Biology, University of Nevada, Reno.
- Song, M. (2012) MPB pheromone component biosynthesis, P450s, and monoterpene metabolism in bark beetles. Ph.D. Dissertation. Department of Biochemistry and Molecular Biology, University of Nevada, Reno.
- C. Tittiger, M. Song, G.J. Blomquist (2011). cDNA, encoded enzyme, and product produced therefrom, including use for pheromone production. U.S. Provisional Patent, filed July 22, 2011. UNR11-018.
- Figueroa-Teran, R., W.H. Welch, G.J. Blomquist and C. Tittiger. 2012. Ipsdienol dehydrogenase (IDOLDH), a novel oxidoreductase important for Ips pini pheromone production. Insect Biochem. Molec. Biol. 42(2): 81-90.
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Progress 02/01/10 to 01/31/11
Outputs
OUTPUTS: Objective 1 (confirm metabolic path and site of exo-brevicomin biosynthesis) Two putative oxidoreductases that appeared coordinately regulated with our hypothesized exo-brevicomin biosynthetic P450 (CYP6CR1) were further characterized at the molecular level. The most promising, DPG22F04, was subcloned into a baculoviral expression vector for functional assays. A polyclonal antibody was also developed for DPG22F04. Tissue cultures were performed to confirm the site of exo-brevicomin biosynthesis in males. The contribution of a P450 reaction to exo-Brevicomin production was investigated using tissue cultures in the presence of the non-specific P450 inhibitor, piperonyl butoxide (PPO). The point at which carbon enters the biosynthetic pathway was also examined by incubating male fat bodies with tri-deuterated fatty acid precursors. Objective 2 (Express and functionally characterize CYP6CR1). The CYP6CR1 ORF was placed into the BaculoDirect expression vector and stocks were prepared for functional assays. Conditions necessary to produce active recombinant CYP6CR1 were determined. Functional assays were performed with the predicted substrate (6(Z)-nonen-2-one; unlabeled). A collaboration was established with Dr. Chris Jeffrey (UNR Chemistry) to synthesize labeled precursor. A polyclonal antibody was developed for CYP6CR1 and partially characterized. A microplate-based assay method to determine P450 activity is being developed to increase throughput. Objective 3 (Characterize IPP synthases involved in frontalin biosynthesis). A collaboration was established with Dr. Christopher I. Keeling and Dr. Joerg Bohlmann (University of British Columbia, Michael Smith Laboratories) to investigate these proteins. Experiments were completed to finalize the expression profiles of putative GPPS and GGPPS genes. Deuterium-labeled frontalin precursor (6-methylhepten-2-one) was delivered orally to male beetles to investigate incorporation into frontalin. Progress from this work was reported in a poster at the 2010 USDA NIFA grant awardees' meeting in San Diego, CA, Dec. 2010. PARTICIPANTS: Minmin Song - graduate student (CYP6CR1, DPG022F04 cloning and assay;) Patrick Delaplain - undergraduate student (DPG022F04 cloning and assay) Showell Busby - undergraduate student (DPG022F04 assay) Nicole Abbott - graduate student (frontalin biosynthesis feeding experiments) Tidiane Aw - graduate student (FPPG, GGPPS expression profiling) Collaborators: Chris Jeffrey (University of Nevada Reno, Chemistry Department) Christopher I. Keeling (Michael Smith Laboratories, University of British Columbia) Joerg Bohlmann (Michael Smith Laboratories, University of British Columbia) Students gained experience in molecular biology and biochemical techniques, including PCR, qRT-PCR, cloning and sequencing, cell culture, recombinant protein production, enzyme assays, GC/MS analyses, enzyme kinetic analyses, bioinformatics, etc., depending on their roles in the project. Graduate students gained teaching and project management experience by training and mentoring undergrads. All participants gained experience in oral and written presentations. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Objective 1: Of two potentially pheromone-biosynthetic oxidoreductases, one had an expression pattern distributed throughout the male body, particularly in the head and fat body. The other (DPG202F04), was localized predominantly to the fat body, and was not expressed in females at all. These data support our hypothesis that DPG22F04 may catalyzed a reaction in exo-brevicomin biosynthesis, possibly by providing an alpha-oxidation function required to chain-shorten a C10 fatty acyl precursor to a C9 substrate. The polyclonal antibody was shown to have high affinity, specificity, and avidity for DPG022F04. A western blot using this antibody confirmed that DPG022F04 localizes to the male fat body. Fat bodies incubated with exo-brevicomin precursor in the presence of PPO produced less exo-brevicomin than controls. This experiment confirms the role of a P450 in exo-brevicomin production. Further incubations with deuterium-labeled decanoic acid revealed no incorporation into exo-brevicomin, although the label was converted to nonen-2-one.. Thus, unsaturated fatty acids are likely produced by a desaturase with delta-9 or higher activity, but not delta-7 activity. A very exciting conclusion is that alpha-oxidation is confirmed by these data as leading to precursor (6(Z)-nonen-2-one) production. Objective 2: CO-difference spectra confirm that functional recombinant CYP6CR1 can reliably be produced using or baculoviral/Sf9 cell expression system. However, preliminary assays with unlabeled substrate did not show exo-brevicomin production. Definitive assays await synthesis of a labeled substrate by our collaborator (C. Jeffrey). The anti-CYP6CR1antibody has been partially purified; further characterization is in progress. Objective 3: Expression profiles for GGPPS and FPPS were completed. An agreement was made to share our expression data with functional assays of recombinant enzymes performed by our collaborators (C. Keeling and J. Bohlmann) . Feeding experiments with deuterium-labeled (6-methylhepten-2-one) confirmed that we can use live beetles to investigate frontalin biosynthesis. Future experiments will replicate this pilot study with deuterium-labeled isoprenyl diphosphates.
Publications
- Blomquist, G.J., R. Figueroa Teran, T. Aw, M. Song, A. Gorzalski, N. Abbot, E. Chang and C. Tittiger, 2010. Pheromone production in bark beetles. Insect Biochem. Molec. Biol. 40(10): 699-712.
- M. Song, A. Gorzalski, S. Young, P. Delaplain, G.J. Blomquist and C. Tittiger. The role of CYP6CR1 in mountain pine beetle pheromone production. 10th International Symposium on P450 Biodiversity and Biotechnology. Woods Hole, MA. Oct. 2010.
- Aw. Tidiane. 2011. Functional genomics of mountain pine beetle (Dendroctonus ponderosae) related to pheromone production. Ph.D. Dissertation. Department of Biochemistry and Molecular Biology, University of Nevada, Reno.
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