Progress 07/01/07 to 06/30/11
Outputs OUTPUTS: ACTIVITIES - EXPERIMENTS: We bioassayed ten A. nidulans G protein-coupled receptor (GPCR) mutants and the wild type for their response to pure oxylipins (13S HpODE and 9S HpODE). Oxylipin-responsive, GPCR-mutant strains were treated with four pure oxylipins (9S-HODE, 9S-HpODE, 13S-HODE, and 13S-HpODE) as well as anti-aflatoxigenic extracts of Pacific silver fir needles and their cAMP response was quantified. RACE-PCR was used to describe genuine transcripts and ORFs of each GPCR gene studied. We attempted construction of the following GPCR mutant strains of Aspergillus flavus: 1) gprA::pyrG (gprA deletion mutant); 2) gprD::pyrG (gprD deletion mutant); 3) gpdA::gprA (mutant constitutively expressing gprA); 4)gpdA::gprD (mutant constitutively expressing gprD); 5) nopA::pyrG (nopA deletion mutant) and 6) gpdA::nopA (mutant constitutively expressing nopA). Repeated efforts by ourselves and other colleagues have been unsuccessful, so despite continued efforts we recognize that these mutants may be lethal. We found that Abies amabilis extracts affect sporulation and the aflatoxin biosynthetic pathway by A. nidulans, and the fungus reacts to these extracts in plate bioassays similarly to pure oxylipins. We conducted a survey of A. amabilis in the North Cascades and the Chuckanut Range in WA, and tested samples for bioactivity and for the requirement of GprA and GprD in the response. We chemically fractionated active extracts, and began structural identification in collaboration with Dr. Jim Vyvyan at Western Washington University. ACTIVITIES - MENTORING: A total of fifteen undergraduate students spent substantial time (from six months to several years each) working on this project. PRODUCTS - INSTRUMENTS: This project spurred a successful NSF-MRI proposal, funding the purchase of a multi-well plate reader for use in this project (and also shared with other faculty and used in laboratory courses). PRODUCTS - COLLABORATIONS: This project has fostered collaborations with Dr. Jeffery Young (Biology Dept., WWU), Dr. Jim Vyvyan (Chemistry Dept., WWU), Dr. Perry Fizzano (Computer Science Dept., WWU), Dr. Nancy Keller (UW-Madison), Dr. Mike Kolomiets (TAMU), Dr. Matt Parsek (Univ. of Washington) and Dr. Brad Borlee (Univ. of Washington). PRODUCTS - METHODS: While attempting to generate an inexpensive, safe method for aflatoxin detection, our group has created a methods for loop-mediated amplification of DNA (LAMP) to detect aflatoxin genes. PRODUCTS - STUDENTS GRADUATED: All but one of the fifteen undergraduate students who worked on this project have now graduated and are in graduate programs around the country or at jobs at the University of Washington, the Fred Hutchinson Cancer Research Center, and various biotech companies in Washington State. DISSEMINATION: During the past year I reported our results at: i) the "Plant-Microbe Interactions" graduate course in the College of Forest Resources, University of Washington; ii) NW Division Meeting of the American Society for Microbiology; iii)University of Puget Sound (biology departmental seminar) and iv) Oregon State University (Botany and Plant Pathology departmental seminar). PARTICIPANTS: 2010/07 TO 2011/06, INDIVIDUALS: Marion Brodhagen (PI/PD): 248 paid hours and approximately 180 unpaid hours to repeat and finish bioassays and subsequent cAMP assays, and complile and submit data for submission to PLoS ONE. No work on project after September 2011 due to new job. Michael Miller (158 paid hours) assisted with molecular work. 2009/07 TO 2010/06, INDIVIDUALS: Marion Brodhagen (PI/PD): approximately 1,000 unpaid hours. Directed project and trained students. Lucien Barnes, Graham Bailes, and Andrew Borst (approximately 350, 300, and 150 unpaid hours, respectivly): performed molecular biology tasks related to screening GPCR mutants in A. flavus and A. nidulans. Performed bioassay-guided purification of active fractions from A. amabilis needles. Michael Miller (417 hours paid): performed molecular biology tasks related to screening GPCR mutants in A. flavus and A. nidulans. Zoe Shaw (120 hours paid by WWU) performed general laboratory maintenance and assisted with bioassays. 2008/07/01 TO 2009/06/30, INDIVIDUALS: Marion Brodhagen (PI/PD; 96 paid hours and approximately 1000 unpaid hours between July 1, 2008 and June 30, 2009): Directs project and trains students. Colin Lappala (325 hours paid between July 1, 2008 and June 30, 2009): Found optimal conditions for gprA, gprD, and nopA expression in A. flavus and A. nidulans. Characterized transcripts of these genes. Made constructs of A. nidulans and A. flavus gprA, gprD, and nopA open reading frames for cloning into yeast expression vectors. Graham Bailes & Lucien Barnes (300 hours each; unfunded; use reagents supplied by grant): same as above. Zoe Shaw (180 hours paid by WWU): same as above. 2008/07/01 TO 2010/06,PARTNER ORGANIZATIONS: WWU paid Zoe Shaw's salary as a student hourly. 2007/07/01 TO 2008/06/30, INDIVIDUALS: Marion Brodhagen (PI/PD): Directs project and trains students. Kyle Fowler (400 hours funded by ASM): Found optimal conditions for gprA and gprD expression in A. nidulans and characterized transcripts of these genes. Colin Lappala (530 paid hours) Found optimal conditions for gprA, gprD, and nopA expression in A. flavus and characterized transcripts of these genes. Junwen Law (unfunded): assisted in PCRs for GPCR transcript characterization. Neva Watson and Tori Talkington: (116 and 181 paid hours, respectively): Did laboratory maintenance, optimized oxylipin bioassays and screened native Pacific Northwest plants for oxylipin mimics. 2007/07/01 TO 2008/06/30, PARTNER ORGANIZATIONS: NSF-MRI program: funds to purchase a microplate reader. American Society for Microbiology Undergraduate Research Fellowship program: funded Kyle Fowler's salary, summer 2007 - summer 2008. WWU Foundation Program: funds to Kyle Fowler summer 2007 - summer 2008. COLLABORATIONS OR CONTACTS: Dr. Nancy Keller (University of Wisconsin, Dr. Mikhail Kolomiets (TAMU), Dr. Jim Vyvyan (Dept. of Chemistry, WWU), Dr. Jeffery Young (Dept. of Biology, WWU), Dr. Perry Fizzano (Dept. of Computer Science, WWU)and Drs. Matt Parsek and Brad Borlee (University of Washington). TRAINING AND PROFESSIONAL DEVELOPMENT: Please see Target Audiences/Efforts below. TARGET AUDIENCES: TARGET AUDIENCES include undergraduate researchers who have been impacted positively by the opportunity to participate in this project. These students would otherwise have no exposure to agricultural research during their undergraduate experience. A total of fifteen undergraduate students participated in this project. EFFORTS: The students worked one-on-one with Dr. Brodhagen in the laboratory, met with Dr. Brodhagen weekly, presented data in weekly lab meetings and participated in weekly journal clubs. They presented their work at university-level symposia and one national-level meeting. They became strongly grounded in molecular, chemical, and data analysis skills. They practiced critical thinking and became engaged in the bigger research picture by exploring the literature and discussing current experiments with their labmates. In addition, our results were disseminated in five talks (one given by undergraduate Kyle Fowler at the national ASM meeting) and one invited graduate lecture (see OUTPUTS/DISSEMINATION above). Four undergraduate fellowships were awarded for work on this project. PROJECT MODIFICATIONS: NO-COST EXTENSIONS: Due to the slower nature of research at an undergraduate institution, two no-cost extensions were granted and gratefully accepted for continuation of this work. SETBACKS/HURDLES: We did not achieve our goal of constructing yeast reporter strains bearing Aspergillus GPCRs, which was slated for summer 2010. Instead I finished cAMP assays during that time, while the necessary equipment was available to me at WWU. Then in September 2010, I started a new position as Research Microbiologist at the USDA-ARS Forage Seed and Cereal Research Laboratory in Corvallis, OR. Prior to accepting the position my understanding was that I would be permitted to pursue this work in the capacity of my new position but that did not turn out to be the case. Therefore, the work remained undone and is now outlined in a grant to my collaborators Drs. Keller and Kolomiets from the NSF. It will be completed in their laboratories. I hope to remain an active collaborator though I am not a co-PI for the reasons above. Another major hurdle was construction of GPCR deletion and overexpression mutants in A. flavus. Though constructs were successfully completed, two efforts by me and two more efforts by colleagues in Dr. Keller's lab to transform these into the fungus failed to yield the desired mutants. A third effort is currently underway. UNEXPECTED DISCOVERIES: During the course of the grant we made two unexpected advances. First, we discovered that A. amabilis needles contain a consituent that alters aflatoxin and spore production by Aspergillus spp.. We spent effort in this direction because our preliminary chemical and biological evidence suggests that the active fraction contains an oxylipin-like compound. Preliminary work shows that a gprD mutant is unresponsive to the active fraction. Second, in pursuing a safer, more inexpensive aflatoxin-detecting method, we created a method for using loop-mediated amplification of DNA (LAMP) to detect aflatoxin genes. We received a seed grant to pursue this work and will publish our results soon. However, the idea would never have arisen outside of working on this project.
Impacts CHANGES IN KNOWLEDGE: i) We have characterized transcripts and ORFs of Aspergillus GPCRs and are disseminating some of this knowledge (A. nidulans gprD) in a submitted paper. The remaining information will be published in a future publication. We have demonstrated that GprD perceives plant-generated oxylipins and that oxylipin perception is mediated through a cAMP/protein kinase A pathway. (Though gprA and gprG mutants are not oxylipin-responsive, they do not appear to mediate oxylipin perception via this pathway.) This information was of such value to the community that our data are being included in a forthcoming review article, simultaneous to publication of the research article (which is currently in review). iii) We discovered that A. amabilis needles contain a chemical(s) that, like certain oxylipins, inhibits aflatoxin and spore production in Aspergillus spp. without killing the fungus. Dr. J. Vyvyan and I are submitting a grant to pursue this research further. METHODS: We developed a method for aflatoxin gene detection using LAMP and will publish our results shortly. POSITIVE LIFE CHOICES AMONG YOUTH AND ADULTS: Fifteen undergraduate students participated in this project. All worked for at least six months; most for several years. Three nationally competitive and one university fellowship were awarded for student work on this project and one student gave an invited talk at a national meeting. For each of these students, their research experience played a major role in their decisions about their future careers, and helped them secure their next job or graduate position. At my primarily undergraduate institution, undergraduate research is a scarce and prized opportunity to synthesize theoretical knowledge, practice quantitative and instrumental skills, and hone critical thinking skills. These students were excited to be part of a solution to a real agricultural problem, they remain proud of their work, and all of them have matured into excellent, creative, and independent scientists during the time they spent on this project.
Publications
- Brodhagen, M., Affeldt, K.J., Lappala, C., Fowler, K., and Keller, N.P. 2011. The Aspergillus G Protein-Coupled Receptor GprD Mediates Recognition of Sporogenic Plant and Mammalian Oxylipins. PLoS ONE, In Review.
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Progress 07/01/09 to 06/30/10
Outputs OUTPUTS: EXPERIMENTS: Section 4.2.1 of the original proposal calls for verification and characterization of oxylipin perception by Aspergillus receptors gprA and gprD. I optimized a bioassay for observing the response of A. nidulans to purified oxylipins (13S HpODE and 9S HpODE). I then tested ten G protein-coupled receptor (GPCR) mutants and the wild type for their response to the pure oxylipins using this bioassay. Next, I quantified cyclic AMP after treating tissues of wild type and GPCR-mutant fungal strains with four pure oxylipins (9S-HODE, 9S-HpODE, 13S-HODE, and 13S-HpODE). As described in Section 4.2.2 of the original proposal, we constructed and tested the genotypes and phenotypes of the following mutant strains of Aspergillus flavus: 1) gprA::pyrG (gprA deletion mutant); 2) gprD::pyrG (gprD deletion mutant); 3) gpdA::gprA (mutant constitutively expressing gprA); and 4)gpdA::gprD (mutant constitutively expressing gprD). We also constructed and tested the genotypes and phenotypes of the following mutant strains of A. nidulans: 5) nopA::pyrG (nopA deletion mutant) and 6) gpdA::nopA (mutant constitutively expressing nopA). No successful mutants were derived, so we will re-attempt the mutagenesis using different parental strains. We found that Pacific Silver Fir (Abies amabilis) extracts have a profound effect on sporulation by A. nidulans, and the fungus reacts to these extracts in plate bioassays similarly to pure oxylipins. We conducted a survey of A. amabilis in the North Cascades and the Chuckanut Range in WA, and tested samples for bioactivity. We chemically fractionated active extracts, and began structural identification in collaboration with Dr. Jim Vyvyan at Western Washington University. MENTORING/ENCOURAGING POSITIVE ACTIONS: Five undergraduate students (Lucien Barnes, Graham Bailes, Andrew Borst, Michael Miller, and Zoe Shaw) worked directly or indirectly on this project between July 1, 2009 and June 30, 2010. This work was a venue for the students to become engaged in, and mature as, scientists. DISSEMINATION: This work was presented in January at a departmental seminar at Oregon State University, and in February as a departmental seminar at Texas A&M University. I have been invited to present the results of this work at the upcoming Northwest Branch ASM Annual Meeting in Seattle, WA in October 2010. I have also been invited to give departmental seminars in Spring 2011 at Washington State University and the University of Puget Sound on these results. COLLABORATIONS: Ongoing collaborations among myself, Dr. Nancy Keller (UW-Madison), Dr. Mikhailo Kolomiets (TAMU), and Dr. Ivo Feussner (Georg-August-University, Goettingen) continue to facilitate this work. A new collaboration with Dr. Jim Vyvyan (Western Washington University) resulted from our efforts to identify the active compound(s) in Pacific silver fir. A new collaboration with Dr. Jeffery Young (Western Washington University) has yielded secondary metabolite mutants of Arabidopsis thaliana that are susceptible to Aspergillus seed infection, and we are currently preparing a grant proposal to follow up on our preliminary results. PARTICIPANTS: INDIVIDUALS: Marion Brodhagen (PI/PD): approximately 1,000 unpaid hours paid between July 1, 2009 and June 30, 2010. Directed project, and trained/mentored students, and performed benchwork described below along with students. Lucien Barnes (unpaid undergraduate research credits; used supplies purchased from grant): approximately 350 unpaid hours between July 1, 2009 and June 30, 2010. Performed molecular biology tasks related to screening GPCR mutants in A. flavus and A. nidulans. Collected, extracted, and purified silver fir needles using column chromatography and thin layer chromatography, and performed bioassays to test effects of purified fractions on sporulation and aflatoxin production. Organized and interpreted chemical data. Graham Bailes (unpaid undergraduate research credits; used supplies purchased from grant): approximately 300 unpaid hours between July 1, 2009 and June 30, 2010. Collected, extracted, and purified silver fir needles, and used fractions in bioassays to test effects on sporulation and aflatoxin production. Performed quantitative and visual analysis of bioassays. Andrew Borst (unpaid undergraduate research credits; used supplies purchased from grant): approximately 150 unpaid hours between July 1, 2009 and June 30, 2010. Collected, extracted, and purified silver fir needles, and used fractions in bioassays to test effects on sporulation and aflatoxin production. Michael Miller: 417 hours paid between July 1, 2009 and June 30, 2010. Performed molecular biology tasks related to screening GPCR mutants in A. flavus and A. nidulans. Performed data analysis and graphed bioassay data from tests of pure oxylipins and silver fir extract fractions on WT and GPCR mutants of Aspergillus. Zoe Shaw: (120 hours paid as a student hourly by Western Washington University; used supplies purchased from grant): Performed general laboratory maintenance and assisted with bioassays. COLLABORATIONS OR CONTACTS: Dr. Nancy Keller, University of Wisconsin. Dr. Keller invited me to perform fungal transformations in her laboratory because they are more easily performed there than in my less-equipped laboratory. She also encourages my work and maintains an interest in my career as my former postdoc mentor. Dr. Mikhail Kolomiets. Dr. Kolomiets has offered to donate kernels from lox-deleted maize lines for testing in the GPCR bioassay. Dr. Keller, Dr. Kolomiets and I co-wrote a grant proposal to the NSF to continue this work on characterization of GPCRs in Aspergillus. Dr. Ivo Feussner, Georg-August-University, Goettingen. Dr. Feussner continues to share in his work to purify fungal oxylipins. Dr. Jim Vyvyan, Western Washington University. Dr. Vyvyan is helping in the purification and structural identification of silver fir compounds that inhibit aflatoxin and spore production in Aspergillus. Dr. Jeffery Young, Western Washington University. Dr. Young has provided mutants of Arabidopsis thaliana altered in secondary metabolite production, and we have found some of these mutants to be susceptible to seed infection by Aspergillus. TRAINING AND PROFESSIONAL DEVELOPMENT: Please see Target Audiences/Efforts below. TARGET AUDIENCES: TARGET AUDIENCES include undergraduate researchers who have been impacted positively by the opportunity to participate in this project. These students would otherwise have no exposure to agricultural research during their undergraduate experience. A total of thirteen undergraduate students have so far been involved in the work described in the original proposal. EFFORTS: The students worked one-on-one with Dr. Brodhagen in the laboratory, met with Dr. Brodhagen weekly, presented data in weekly lab meetings and participated in weekly journal clubs. They became strongly grounded in molecular, chemical, and data analysis skills. They practiced critical thinking and became engaged in the bigger research picture by exploring the literature and discussing current experiments with their labmates. For each of them, their research experience has played a large role in helping them to decide what to do next in their lives, and in securing the next job or graduate position. At Western Washington University, a primarily undergraduate university, research opportunities are valued but scarce. The work of these students is a rare but necessary capstone to their coursework in which they synthesize theoretical knowledge, practice quantitative and instrumental skills, and hone critical thinking skills. These students are excited to be a part of a solution to a real agricultural problem, they are proud of their work, and all of them have matured into excellent, creative, and independent scientists in the year or more that they have spent in my laboratory. PROJECT MODIFICATIONS: In May, two changes were requested and approved by NIFA: 1) A budget revision to transfer of funds from the Materials/Supplies, Printing, and Fringe categories of the budget to the Salaries category to cover two months of Dr. Brodhagen's 2010 summer salary and to the Travel budget to cover two local meetings in the summer of 2010. 2) A second no-cost extension of one year, so that the grant expires on June 30, 2011. The new projected timeline (submitted in May) is below. April 2010 - July 2010: Completion of genotype confirmation and phenotype testing of A. flavus GPCR deletion mutants (complete as of September 2010; mutagenesis was unsuccessful must be re-done). July 2010 - September 2010: Construction of Aspergillus GPCR-expressing reporter strains of Saccharomyces cerevisiae strain JKY127-36-1. September 2010 - October 2010: Assay of beta-galactosidase in yeast cells upon exposure to pure oxylipins, crude fungal extracts, and crude maize kernel extracts. October 2010 - June 2011: Confirmatory tests for GPCR/ligand associations via cAMP and G protein activation assays (complete for A. nidulans GPCR mutants as of September 2010). October 2010 - June 2011: Writeup of results in manuscripts for publication. As of September, Dr. Brodhagen took an appointment at the USDA-ARS Forage Seed and Cereal Research Unit in Corvallis, OR. A multiwell plate reader required for some of the planned experiments (above) does not exist at the new location, so July-September was spent performing cAMP assays rather than constructing GPCR-expressing reporter strains. The latter will be done at the new location in the upcoming months.
Impacts NEW KNOWLEDGE: 1. In addition to GprA and GprD, we identified GprG as a third GPCR required for WT-level oxylipin perception in A. nidulans. 2. We demonstrated (by quantifying cAMP levels in response to individual oxylipins) that in addition to 13S-HpODE, wild type A. nidulans (but not gprA, gprD, and gprG mutants) also respond via G protein signaling to 9S-HpODE, as well as to the hydroxides 9S-HODE and 13S-HODE. The hydroxides are more stable and cheaper than the hydroperoxides originally shown to affect spore and mycotoxin production in Aspergillus. 3. Regarding Pacific silver fir, we found that i) extracts of all silver fir, but not of other fir or conifer species, alter spore and mycotoxin production by Aspergillus; ii) extracts active against A. nidulans were also active against A. flavus and A. parasiticus; iii) young needles (fresh buds) have the highest activity; iv) following chemical fractionation of the extracts, aflatoxin production by A. flavus and A. parasiticus was inhibited by certain fractions and enhanced by others. Most compellingly, we showed that v) one bioactive fraction, which affects mycotoxin and spore production by A. flavus, A. parasiticus, and A. nidulans, apparently requires for activity GprA, GprD, and GprG - the same G protein-coupled receptors required in A. nidulans for response to plant oxylipins (9S and 13S-HpODE ). vi) We also saw a change in cAMP levels when we applied this fraction to A. nidulans tissues - suggesting GPCR activation. CHANGE IN ACTIONS: We will include GprG among Aspergillus GPCRs to be tested in future work, and will add to the list of putative oxylipin ligands for these receptors 9S-HpODE, 9S-HODE, and 13S-HODE, as well as Pacific silver fir active fractions. POSITIVE LIFE CHOICES AMONG YOUTH AND ADULTS: A total of thirteen undergraduate students have so far been involved in the work described in the original proposal. Ten of these students have graduated. One works at Western Washington University, one is a technician in the University of Washington's Microbiology Department, two are technicians at the Fred Hutchinson Cancer Research Center, one is enrolled in medical school, one is enrolled in dental school, two are enrolled in the MD/PhD program at SUNY-Syracuse, one is applying to plant pathology graduate programs, and one is applying to bioinformatics graduate programs. For each of them, their research experience played a large role in helping them to decide what to do next in their lives, and in securing the next job or graduate position. At my primarily undergraduate university, undergraduate research is a scarce and prized opportunity to synthesize theoretical knowledge, practice quantitative and instrumental skills, and hone critical thinking skills. These students are excited to be a part of a solution to a real agricultural problem, they are proud of their work, and all of them have matured into excellent, creative, and independent scientists in the year or more that they have spent in my laboratory.
Publications
- No publications reported this period
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Progress 07/01/08 to 06/30/09
Outputs OUTPUTS: EXPERIMENTS: As described in Section 4.2.2 of the original proposal, we constructed and are now confirming the genotypes and phenotypes of the following mutant strains of Aspergillus flavus: 1) delta gprA::pyrG (gprA deletion mutant); 2) delta gprD::pyrG (gprA deletion mutant); 3) gpdA::gprA (mutant constitutively expressing gprA); and 4)gpdA::gprA (mutant constitutively expressing gprD). We also constructed and are now confirming the genotypes and phenotypes of the following mutant strains of A. nidulans: 5) delta nopA::pyrG (nopA deletion mutant) and 6) gpdA::nopA (mutant constitutively expressing nopA). Work is also continuing in the examination of compound(s) in Pacific Silver Fir (Abies amabilis) extracts that we have found to have a profound effect on sporulation by A. nidulans. We conducted a survey of A. amabilis in the North Cascades and the Chuckanut Range and tested samples for bioactivity. MENTORING: Seven undergraduate students worked directly or indirectly on this project between July 1, 2008 and June 30, 2009. Three of these (Lucien Barnes, Graham Bailes, and Zoe Shaw) remain in the laboratory and are continuing to gain research experience. The other four graduated (described immediately below). STUDENTS GRADUATED: One student (Colin Lappala) has now graduated and is working in Dr. Caroline Harwood's laboratory at the University of Washington (Department of Microbiology). A student who worked peripherally on the project (Rachel Blumhagen) also graduated and spent her summer internship with Dr. Joyce Loper at Oregon State University (Department of Botany and Plant Pathology). Rachel won two nationally competitive fellowships based on her research: 1) ASM Undergraduate Research Fellowship from the American Society of Microbiology (30 recipients nationwide); 2) American Phytopathological Society undergraduate research fellowship (2 recipients nationwide). Rachel also won the Biology Department's undergraduate research award. Another student (Leslie Whalen) has applied to dental school, and the fourth (Alyse Douglass) is enrolled in an MD/PhD program at SUNY-Syracuse (Department of Microbiology and Immunology). DISSEMINATION: Kyle Fowler gave an invited talk at the ASM General Meeting in Boston in June 2008 to present his findings. His talk was entitled "G protein-coupled receptors in host oxylipin perception in Aspergillus", and was co-authored with his fellow undergraduate researcher, Colin Lappala. COLLABORATIONS: Ongoing collaborations among myself, Dr. Nancy Keller, Dr. Mikhailo Kolomiets (TAMU), and Dr. Ivo Feussner continue to facilitate this work. I spent three weeks in Dr. Keller's laboratory during July, constructing the mutant strains described above. During that time, Dr. Keller, Dr. Kolomiets and I co-wrote a grant proposal to the NSF to continue this work on characterization of GPCRs in Aspergillus. We specifically proposed to continue to include students from Western Washington University in the research, and fund summer internships for them to continue their work in Dr. Keller's and Dr. Kolomiets' labs where they can experience research at a large, land-grant university with a focus on agricultural research. PARTICIPANTS: INDIVIDUALS: Marion Brodhagen (PI/PD; 96 hours paid between July 1, 2008 and June 30, 2009): Directs project and trains students. Colin Lappala (325 hours paid between July 1, 2008 and June 30, 2009): Found optimal conditions for gprA, gprD, and nopA expression in A. flavus and A. nidulans. Characterized transcripts of these genes. Made constructs of A. nidulans and A. flavus gprA, gprD, and nopA open reading frames for cloning into yeast expression vectors. Graham Bailes & Lucien Barnes (300 hours each; unfunded; use reagents supplied by grant): Perform molecular biology tasks related to screening GPCR mutants in A. flavus and A. nidulans. Collect and extract silver fir needles and use extracts in bioassay to test effects on sporulation. Zoe Shaw (180 hours paid as a student hourly by Western Washington University; uses reagents supplied by grant): Performs general laboratory maintenance. PARTNER ORGANIZATIONS: National Science Foundation Major Research and Instrumentation program. Awarded funds to purchase a microplate reader to be used for cAMP and GTP/GDP exchange assays described in Section 4.3.3 of the original proposal. Western Washington University paid Zoe Shaw's salary as a student hourly during the 2008/09 academic year. COLLABORATIONS OR CONTACTS: Dr. Perry Fizzano, Department of Computer Science, Western Washington University. Dr. Fizzano and I are using bioinformatics approaches to study positive selection in gprA, gprD, and nopA homologs. Dr. Matt Parsek, University of Washington. Dr. Parsek, Dr. Borlee, and I are testing a lox gene from Pseudomonas aeruginosa to query whether it encodes a LOX enzyme whose product(s) crosstalk with Aspergillus oxylipin receptors. Dr. Brad Borlee, University of Washington. See above. Dr. Nancy Keller, University of Wisconsin. Dr. Keller invited me to perform fungal transformations in her laboratory because they are more easily performed there than in my less-equipped laboratory. She also encourages my work and maintains an interest in my career as my former postdoc mentor. Dr. Mikhail Kolomiets. Dr. Kolomiets has offered to donate kernels from lox-deleted maize lines for testing in the GPCR bioassay. Dr. Keller, Dr. Kolomiets and I co-wrote a grant proposal to the NSF to continue this work on characterization of GPCRs in Aspergillus. TRAINING AND PROFESSIONAL DEVELOPMENT: All individual participants listed in the first section above are undergraduate students whose work has doubled as training and professional development as research scientists. The students meet with Dr. Brodhagen weekly, present data in weekly lab meetings and participate in weekly journal clubs. TARGET AUDIENCES: TARGET AUDIENCES include undergraduate researchers who have been impacted positively by the opportunity to participate in this project. These students would otherwise have no exposure to agricultural research during their undergraduate experience. A total of ten undergraduate students have so far been involved in the work described in the original proposal. EFFORTS: The students work one-on-one with Dr. Brodhagen in the laboratory, meet with Dr. Brodhagen weekly, present data in weekly lab meetings and participate in weekly journal clubs. For each of them, their research experience has played a large role in helping them to decide what to do next in their lives, and in securing the next job or graduate position. At Western Washington University, a primarily undergraduate university, research opportunities are valued but scarce. The work of these students is a rare but necessary capstone to their coursework in which they synthesize theoretical knowledge, practice quantitative and instrumental skills, and hone critical thinking skills. These students are excited to be a part of a solution to a real agricultural problem, they are proud of their work, and all of them have matured into excellent, creative, and independent scientists in the year or more that they have spent in my laboratory. PROJECT MODIFICATIONS: MAJOR CHANGES: Dr. Brodhagen is a new investigator whose primary responsibility is teaching at a primarily undergraduate institution. Progress has been somewhat slower than anticipated (e.g. as outlined in Section 4.6 of the original proposal). This is primarily due to the lack of any graduate students, technicians, or postdoctoral scientists to assist with the research and help oversee undergraduate students' research and training. A no-cost extension of one year was requested and granted. Completion of the project goals are anticipated by June of 2010. The addition of nopA as an additional candidate to test as a putative oxylipin receptor reflects the only other major change to the experimental plan.
Impacts NEW KNOWLEDGE: Previously, the transcripts for gprA and gprD in A. nidulans and A. flavus were characterized. Among our other observations, of note is that the A. nidulans gprA transcript appears to include two separate start sites, which contain distinct putative open reading frames and may encode two separate and functionally different proteins. Also, third potential oxylipin-binding GPCR (NopA) in Aspergillus was identified as the result of this research. We have mutated the gene in A. nidulans and will include this GPCR in our yeast reporter strains. We also found that a compound(s) in Pacific Silver Fir extracts has a profound effect on sporulation by A. nidulans. We intend to test the extract's effect on genes whose expression is known to be controlled by oxylipins, and we will also test the extract (and/or purified compounds, if available) in GPCR assays once the yeast reporter strains described in Section 4.3.1 of the original proposal are constructed. POSITIVE LIFE CHOICES AMONG YOUTH AND ADULTS: A total of ten undergraduate students have so far been involved in the work described in the original proposal. Seven of these students have graduated four are working at the Washington Native Plant Society, the University of Washington's Microbiology Department, the Fred Hutchinson Cancer Research Center, and as an ambulance driver for Cascade Ambulance. Two more are enrolled in the MD/PhD program at SUNY-Syracuse. One is applying to dental school. For each of them, their research experience played a large role in helping them to decide what to do next in their lives, and in securing the next job or graduate position. At my primarily undergraduate university, research opportunities are valued but scarce. The work of these students is a rare but necessary capstone to their coursework in which they synthesize theoretical knowledge, practice quantitative and instrumental skills, and hone critical thinking skills. These students are excited to be a part of a solution to a real agricultural problem, they are proud of their work, and all of them have matured into excellent, creative, and independent scientists in the year or more that they have spent in my laboratory. CHANGE IN CONDITIONS: This funding continues to permit me - a new researcher at a primarily undergraduate institution - to set up my laboratory and my research program. The College of Science is proud of our work and visitors are frequently taken through our lab on tours. The connections and momentum I've gained through this work have also helped me to successfully apply as a co-PI for two grants in a separate area (the use of biodegradable plastic mulches in high tunnels). Thank you for giving a young assistant professor a much-needed first chance! Last year, this research project was also part of the impetus for a successful proposal to the NSF-MRI program that funded the purchase a new instrument (a microplate reader) to be used for cAMP and GTP/GDP exchange assays described in Section 4.3.3 of the original proposal. The microplate reader has already been used in a number of undergraduate laboratory courses and has been a boon to our biology department.
Publications
- PUBLICATIONS: Gao, X., Brodhagen, M., Isakeit, T., Horowitz Brown, S., Goebel, C., Betran, J., Feussner, I., Keller, N.P., and Kolomiets, M. 2009. Inactivation of the lipoxygenase ZmLOX3 increases susceptibility of maize to Aspergillus spp. Molecular Plant-Microbe Interactions 22(2): 222-231.
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Progress 07/01/07 to 06/30/08
Outputs OUTPUTS: ACTIVITIES: As described in Section 4.3.1 of the original proposal, we used RT-PCR and Northern analysis to find culture conditions that permitted expression of gprA and gprD in A. flavus. RNA was extracted from A. nidulans and A. flavus in parallel, and cDNA derived. Next, 5' and 3' RACE experiments were performed on gprA and gprD genes from both A. flavus and A. nidulans. Transcripts were characterized for each gene, and introns and alternative start sites were identified. NEW KNOWLEDGE: The transcripts for gprA and gprD in A. nidulans and A. flavus are characterized. Among our other observations, of note is that the A. nidulans gprA transcript appears to include two separate start sites, which contain distinct putative open reading frames and may encode two separate and functionally different proteins. Also, third potential oxylipin-binding GPCR (NopA) in Aspergillus was identified as the result of this research, and a deletion vector has been constructed for use in knocking out this potential oxylipin receptor in A. nidulans. We will collaborate with Dr. Nancy Keller (UW-Madison) on this addition to our project. TEACHING AND MENTORING: These completed experiments were performed by myself and three undergraduate students: Kyle Fowler, Junwen Law, and Colin Lappala. For his work, Kyle Fowler received the competitive American Society for Microbiology (ASM) Undergraduate Research Fellowship from the American Society of Microbiology to pursue research in my laboratory from summer 2007 - summer 2008. Mr. Fowler also received an internal award from the WWU Foundation Program in Support of Undergraduate Research and Scholarship Excellence. All three students have graduated. Two other undergraduate students worked on this project: Neva Watson and Tori Talkington performed organic extracts of native Pacific Northwest plants for use in bioassays to reveal potential oxylipin mimics. Their work revealed several potential leads to be tested in GPCR assays once these are constructed. Two undergraduate students have just begun work in the laboratory this month and will continue to work on the project: Graham Bailes and Lucien Barnes. This research project was part of the impetus for a successful proposal to the NSF-MRI program that funded the purchase a new instrument (a microplate reader) to be used for cAMP and GTP/GDP exchange assays described in Section 4.3.3 of the original proposal. The microplate reader will be used in a number of undergraduate laboratory courses and has been a boon to our biology department. DISSEMINATION: Kyle Fowler gave an invited talk at the ASM General Meeting in Boston in June 2008 to present his findings. His talk was entitled "G protein-coupled receptors in host oxylipin perception in Aspergillus", and was co-authored with his fellow undergraduate researcher, Colin Lappala. COLLABORATIONS: Ongoing collaborations among myself, Dr. Nancy Keller, Dr. Mikhailo Kolomiets (TAMU), and Dr. Ivo Feussner continue to facilitate this work. PARTICIPANTS: INDIVIDUALS: Marion Brodhagen (PI/PD): Directs project and trains students. Kyle Fowler (funded by ASM): Found optimal conditions for gprA and gprD expression in A. nidulans and characterized transcripts of these genes. Was invited to speak at the national ASM 108th General meeting. Colin Lappala (530 hours) Found optimal conditions for gprA, gprD, and nopA expression in A. flavus and characterized transcripts of these genes. Junwen Law (unfunded; used reagents supplied by grant): Assisted in PCRs for GPCR transcript characterization. Neva Watson (116 hours): Performed general laboratory maintenance (e.g. reagent and media making and dishwashing), optimized oxylipin bioassays and screened native Pacific Northwest plants for oxylipin mimics. Tori Talkington (181 hours): Performed general laboratory maintenance (e.g. reagent and media making and dishwashing), optimized oxylipin bioassays and screened native Pacific Northwest plants for oxylipin mimics. Graham Bailes (unfunded; uses reagents supplied by grant): Recently joined group. Lucien Barnes (unfunded; uses reagents supplied by grant): ): Recently joined group. PARTNER ORGANIZATIONS: National Science Foundation Major Research and Instrumentation program. Awarded funds to purchase a microplate reader to be used for cAMP and GTP/GDP exchange assays described in Section 4.3.3 of the original proposal. American Society for Microbiology Undergraduate Research Fellowship program. Awarded funds to Kyle Fowler for his work from summer 2007 - summer 2008. WWU Foundation Program in Support of Undergraduate Research and Scholarship Excellence. Awarded funds to Kyle Fowler for his work from summer 2007 - summer 2008. COLLABORATIONS OR CONTACTS: Dr. Perry Fizzano, Department of Computer Science, Western Washington University. Dr. Fizzano and I are using bioinformatics approaches to study positive selection in gprA, gprD, and nopA homologs. Dr. Nancy Keller, University of Wisconsin. Dr. Keller and I collaborate in that she has offered to allow myself or my students visit her laboratory to complete facets of the project that cannot easily be completed here, such as fungal transformations. She also encourages my work and maintains an interest in my career as my former postdoc mentor. Dr. Mikhail Kolomiets. Dr. Kolomiets has offered to donate kernels from lox-deleted maize lines for testing in the GPCR bioassay. Dr. Matt Parsek, University of Washington. Dr. Parsek, Dr. Borlee, and I are testing a lox gene from Pseudomonas aeruginosa to query whether it encodes a LOX enzyme whose product(s) crosstalk with Aspergillus oxylipin receptors. Dr. Brad Borlee, University of Washington. See above. TRAINING AND PROFESSIONAL DEVELOPMENT: All individual participants listed in the first section above are undergraduate students whose work has doubled as training and professional development as research scientists. The students meet with Dr. Brodhagen once a week, present data in weekly lab meetings and partipate in weekly journal clubs. They are encouraged to become more and more independent in their experimental work as they progress in their training. TARGET AUDIENCES: The individuals listed as participants above are all undergraduate students who received extensive training in molecular biology, microbiology, mycology, bioinformatics and sequence analysis, and general laboratory, recordkeeping, critical thinking, and presentation skills during their tenure in my laboratory. PROJECT MODIFICATIONS: Dr. Brodhagen is a new investigator whose primary responsibility is teaching at a primarily undergraduate institution. Getting both her courses and laboratory up and running and training all students without the help of a postdoc, graduate student, or technician has slowed work on this project. Progress has been somewhat slower than anticipated (e.g. as outlined in Section 4.6 of the original proposal). However, no significant changes to the plan are expected aside from the addition of nopA as an additional candidate to test as a putative oxylipin receptor.
Impacts NEW KNOWLEDGE: The transcripts for gprA and gprD in A. nidulans and A. flavus are characterized. Among our other observations, of note is that the A. nidulans gprA transcript appears to include two separate start sites, which contain distinct putative open reading frames and may encode two separate and functionally different proteins. Also, third potential oxylipin-binding GPCR (NopA) in Aspergillus was identified as the result of this research, and a deletion vector has been constructed for use in knocking out this potential oxylipin receptor in A. nidulans. We will collaborate with Dr. Nancy Keller (UW-Madison) on this addition to our project. POSITIVE LIFE CHOICES among youth and adults: A total of seven undergraduate students were or are directly involved in the work described in the original proposal. Four of these students have graduated; one of the four plans to go on to study agricultural science. Another is employed as a research assistant at the Fred Hutchinson Cancer Research Center. Two others are seeking employment in the Seattle area. I cannot stress enough how this project has helped these students gain confidence and a sense of personal contribution. At my primarily undergraduate university, research opportunities are valued but scarce. The work of these students is a rare but necessary capstone to their coursework in which they synthesize their theoretical knowledge, quantitative and instrumental skills, and critical thinking skills. These students are excited to be a part of a solution to a real agricultural problem, they are proud of their work, and all of them have matured into excellent, creative, and independent scientists in the year or more that they have spent in my laboratory. CHANGE IN CONDITIONS: First, this funding permitted me - a new researcher at a primarily undergraduate institution - to transform my laboratory to a cavernous empty space filled with empty shelves to a functional, busy, productive laboratory filled with students. The College of Science is proud of our work and visitors are frequently taken through our lab on tours. My students have been featured in a promotional video produced by the university. The research momentum gained by this funding has spurred me to successfully apply for an internal grant in a related area: development of a low-cost, low-technology method for aflatoxin screening. Thank you for giving a young assistant professor a much-needed first chance! This research project was also part of the impetus for a successful proposal to the NSF-MRI program that funded the purchase a new instrument (a microplate reader) to be used for cAMP and GTP/GDP exchange assays described in Section 4.3.3 of the original proposal. The microplate reader will be used in a number of undergraduate laboratory courses and has been a boon to our biology department.
Publications
- Fowler, K., Lappala, C., Keller, N.P., and M. Brodhagen. G protein-coupled receptors in host oxylipin perception in Aspergillus. 108th General Meeting of the American Society for Microbiology, Boston, MA, 2008. (Invited student talk)
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