Progress 02/01/16 to 01/31/21
Outputs
Target Audience:Our work is of interest to farmers that favor organic farming and promote arthropod diversity and pest resistance management. Farmers may use pheromone emitting trap plants as an alternative to traditional trap crops and the application of traditional pesticides. Outcomes of our project are also of interest to biotechnology companies for sustainable, low-cost production of insect pheromones with biocatalysts instead of chemical synthesis. The pheromones are used by small-hold farmers, especially in developing countries, for mating disruption and pest population control, which leads to reduced plant damage, lower insecticide applications, and increased yields. Changes/Problems:The corona pandemic caused a slowdown in research productivity in the final year of the project. Therefore, manuscript submission and research for milestones under Objective 3 will continue in 2021 beyond the expiration date of the grant. What opportunities for training and professional development has the project provided?The project has provided scientific training and mentoring for Ph.D. student Jason Lancaster and Master's students Bryan Lehner and Sophie LeBlanc at Virginia Tech. Jason Lancaster successfully completed his dissertation in February 2018 and is currently a staff fellow at the U.S. Food and Drug Administration. MS student Lehner successfully completed his thesis in February 2019 and is currently a research technician at the University of Connecticut-Stamford. MS student Sophie LeBlanc is scheduled to complete her thesis in August 2021. The P.D. and the graduate students attended several local, national, or international meetings and conferences. Those include the ESA national and Eastern branch meetings, the annual meetings of the Phytochemical Society of North America and the International Society of Chemical Ecology, the biennial international meeting "Terpnet", and the Gordon Research Conference on Plant Volatiles. How have the results been disseminated to communities of interest?Throughout the funding period, the P.D. and the graduate students have presented their results to the research community in the form of posters or oral presentations at local, national, or international meetings and conferences. Those include the ESA national and Eastern branch meetings, the annual meetings of the Phytochemical Society of North America and the International Society of Chemical Ecology, the biennial international meeting "Terpnet", and the Gordon Research Conference on Plant Volatiles. The P.D. also gave invited seminars on the conducted research at several universities. The P.D. was a co-organizer of the 2019 Eastern Branch Meeting Symposium of the Entomological Society of America "Plant-Insect Chemical Ecology: Multi-Species Interactions and Emerging Applications in Agriculture", Blacksburg, VA. The P.D. provided consulting services to the company Provivi, which uses biotechnology for pheromone production and global application in crop pest control. Media and News: Research conducted in this project was featured by several media outlets: The Academic Times, News article: "Stink bugs could be stopped by engineered pheromones", spring 2021 - https://academictimes.com/stink-bugs-could-be-stopped-by-engineered-pheromones/ Virginia Tech Faculty Inventor Spotlight - https://vt.edu/link/license/faculty-inventors.html The Virginian Pilot, News article about research on insect pheromones and their engineering in the Tholl lab - http://pilotonline.com/business/field-notes/field-notes-scientists-finding-ways-to-out-stink-the-stink/article_acf58886-fcdb-5f85-8aa0-e10df0457033.html, spring 2016 Virginia Farm Bureau, News article about research on insect pheromones and their engineering in the Tholl lab, spring 2016 Virginia Tech News, News article about insect pheromone research and engineering in the Tholl lab - http://vtnews.vt.edu/articles/2016/03/fralin-stinkbugs.html, spring 2016 WDBJ7 news channel feature "Scented plant could be answer to saving crops from stink bugs, Virginia Tech scientists say", spring 2016 What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The problem that this project has addressed: Alternative pest management strategies like trap cropping have become a viable option in farming systems that favor organic farming and promote arthropod diversity and pest resistance management. Pheromones applied in baits can enhance the efficacy of trap plants; however, pheromones are typically produced at high cost by chemical synthesis, which has been a barrier for their low cost application. This project has explored alternative strategies to use plants as sustainable factories of pheromones and generate pheromone emitting trap plants for an augmented attraction of pest insects. Audience to be impacted by the project: Our work is of interest to farmers that favor organic farming and promote arthropod diversity and pest resistance management. Farmers may use pheromone emitting trap plants as an alternative to the application of traditional pesticides. Outcomes of our project are also of interest to biotechnology companies for sustainable production of insect pheromones by fermentation instead of chemical synthesis. Achievements by this project to address the problem: Specific achievements of the different Objectives: Objective 1: Milestone: Identification and functional characterization of the first enzyme (terpene synthase MhTPS) in murgantiol pheromone biosynthesis. This milestone has been completed and the outcomes were published in PNAS (see Lancaster et al., 2018). Milestone: Identification and functional characterization of MhTPS homologues in brown marmorated stink bug (Hyalomorpha halys) and Southern green stink bug (Nezara viridula). This milestone has been completed with one publication in the Journal of Chemical Ecology (see Lancaster et al, 2018) and another manuscript to be submitted (see Products). Milestone: Identification and functional characterization of the gene candidates responsible for the second and/or third enzymatic step of the murgantiol biosynthetic pathway. We were able to identify gene candidates for this milestone; however, characterization of the function of these genes will require more time beyond the funding period. Objective 2: Milestone: Determination of the effect of host plant cues on murgantiol pheromone biosynthesis and release. We have not pursued this objective further because work by the current Masters student Sophie LeBlanc was entirely focused on Objective 3. Objective 3: Milestone: Transient expression of pheromone biosynthetic genes in Nicotiana benthamiana. We have successfully expressed genes involved in the pheromone biosynthesis of stink bugs (harlequin bug, southern green stink bug) and the crucifer flea beetle. While we could not yet engineer the entire pheromone biosynthetic pathways from stink bugs, we were able to establish tobacco plants that temporarily release a major pheromone component of the crucifer flea beetle. The amounts of this pheromone compound emitted by a tobacco plant per day exceed daily emissions by a single flea beetle. Experiments to assess the attraction of the crucifer flea beetle to pheromone emitting and non-emitting tobacco plants are in progress and will continue beyond the funding period. Milestone: Stable transformation and expression of pheromone biosynthetic genes in Arabidopsis thaliana coupled with bioassays. Experiments to establish Arabidopsis plants that stably emit the flea beetle pheromone are still ongoing and continue beyond the funding period. It is possible that new strategies using different transformation constructs have to be considered if no pheromone emission is observed. In the case pheromone-emitting plants are generated, the plants will be tested for flea beetle attraction. Milestone: Stable transformation and expression of pheromone biosynthetic genes in Brassica napus. Stable transformation of B. napus will not be pursued under the current grant and will require renewed funding support as previously indicated in the proposal. Overall achievements and impact: This project has uncovered how agricultural insect pests, especially invasive stink bugs, synthesize pheromones that facilitate aggregation and mating of the pest on crops. The project has made first steps in translating this knowledge to the development of novel and unconventional pest management practices. We have identified individual steps in stink bug pheromone biosynthesis and used metabolic engineering to transfer these pathway steps into plants. To date, our work has successfully applied a transient engineering strategy in tobacco as a model. While work on generating pheromone emitting plants for the attraction of stink bugs remains ongoing, we were able to establish plants that transiently release the pheromone component of the crucifer flea beetle, a major pest of cabbage crops. These findings have laid the foundation for ongoing and future work to generate plants with stable production and emission of pheromones and test them for pest attraction in the field. The genetic tools that have been generated by this project are of immediate interest for the commercial production of pheromones via biofermentation using microbial (e.g. yeast) fermentation systems. The project has resulted in two international patent applications for the commercial use of genes in stink bug pheromone biosynthesis. We have discussed the technology with different companies throughout the duration of the funding period. Our project has been at the forefront of new technology for "green chemistry"-alternatives to conventional synthetic pheromone production and developments of sustainable pest management. The work has been recognized by different media outlets including local TV stations. While we could not accomplish all original goals during this funding period, we plan to continue the work by fully establishing stable production of stink bug and other insect pheromones in plants and possibly microbial production systems.
Publications
- Type:
Journal Articles
Status:
Other
Year Published:
2021
Citation:
Lancaster., J., Khrimian, A., Larose, H., Luck, K., Rebholz, Z., Shewade, L., K�llner, T., Ghosh, S.K.B., Weber, D.C., Gundersen-Rindal, D.E., OMaille, P., and Tholl, D. Formation of a sesquiterpene precursor of the aggregation pheromone in the brown marmorated stink bug; to be submitted to Insect Biochemistry and Molecular Biology
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
LeBlanc, S. and Tholl, D. Toward developing pheromone emitting trap crops: metabolic engineering of an aggregation pheromone of the crucifer flea beetle, Phyllotreta cruciferae, in Nicotiana benthamiana, ASPB Plant Biology 20 Worldwide Summit (virtual poster).
|
Progress 02/01/19 to 01/31/20
Outputs
Target Audience:Entomologists, graduate students, industry and scientists at ESA and other meetings. Changes/Problems:Since we were, so far, unable to identify P450 epoxidase genes involved in the biosynthesis of murgantiol or alpha-bisabolene epoxide pheromones in the harlequin bug and the southern green stink bug, respectively, we changed the focus of Objective 3 to transforming plants with genes from the aggregation pheromone biosynthetic pathway of the striped flea beetle, Phyllotreta striolata. P. striolata and P. cruciferae are pests of crucifer crops including cabbage and oilseed rape. Our transformation strategy relies on two genes to generate plants that produce the sesquiterpene himachaladiene, which is a major attractant of P. cruciferae. Behavioral assays will be performed with this species and himachaladiene emitting, transiently and stably transformed plants. What opportunities for training and professional development has the project provided?The project has provided scientific training and mentoring for Master's students Bryan Lehner and Sophie LeBlanc at Virginia Tech. MS student Lehner successfully completed his thesis in February 2019. How have the results been disseminated to communities of interest?The P.D. and student Sophie LeBlanc have presented their results to the research community in the form of posters or oral presentations at local, national, or international meetings and conferences. Those include the 2019 ESA national and Eastern branch meetings, the 2019 annual meetings of the Phytochemical Society of North America and the International Society of Chemical Ecology and the biennial international meeting "Terpnet". The P.D. gave a seminar at West Virginia University. What do you plan to do during the next reporting period to accomplish the goals?Objective 3 Milestone: Transient expression of pheromone biosynthetic genes in Nicotiana benthamiana Co-expression of the P. striolata terpene synthase and isoprenyl diphosphate synthase (PsTPS, PsIDS3) has been performed by MS student Sophie LeBlanc. The plants will be tested in behavioral assays with the crucifer flea beetle Phyllotreta cruciferae (year 5, no-cost extension). Milestone: Stable transformation and expression of pheromone biosynthetic genes in Arabidopsis thaliana coupled with bioassays. Stable transformation of the P. striolata terpene synthase and isoprenyl diphosphate synthase (PsTPS, PsIDS3) will be performed in year 5 by MS student Sophie LeBlanc. Pheromone emitting transformants will be tested in behavioral assays for attraction of P. cruciferae. Milestone: Stable transformation and expression of pheromone biosynthetic genes in Brassica napus. This work will be performed beyond the currently designated funding period.
Impacts
What was accomplished under these goals?
Objective 1 Milestone: Identification and functional characterization of the first enzyme (terpene synthase MhTPS) in murgantiol pheromone biosynthesis Completed - see report from year 3 Milestone: Identification and functional characterization of MhTPS homologues in brown marmorated stink bug (Hyalomorpha halys) and Southern green stink bug (Nezara viridula) Nearly completed - see report from year 3. A manuscript on the characterization of HhTPS is in preparation pending the outcome of RNAi experiments. Milestone: Identification and functional characterization of the gene candidates responsible for the second and/or third enzymatic step of the murgantiol biosynthetic pathway Comparative analysis of transcriptomes from M. histrionica and N. viridula related to sex, developmental stage and tissue specificity (with support by Michael Sparks, research associate with Dawn Gundersen-Rindal) resulted in a larger collection of P450s with a possible role in pheromone biosynthesis. Characterization of these P450 candidates has not led to the identification of an epoxidase. The work will not be continued at this stage since primary focus has been placed on Objective 3. Objective 2 Milestone: Determination of the effect of host plant cues on murgantiol pheromone biosynthesis and release. We have not pursued this objective further at this time because work by the current Masters student Sophie LeBlanc is entirely focused on Objective 3. Objective 3 Milestone: Transient expression of pheromone biosynthetic genes in Nicotiana benthamiana Completed; We have changed the focus of this objective by performing expression with a TPS from the flea beetle P. striolata (a crucifer pest on cole crops) together with its precursor enzyme (PsIDS3) (Beran et al., 2016; PNAS). The advantage of this strategy is that it should lead to the formation of the aggregation pheromone himachaldiene without requirement for further oxidation. Transient co-expression of the PsTPS and PsID3 genes in N. benthamiana has been successful. Inoculated plants were emitting himachaladiene together with other sesquiterpene products of the PsTPS enzyme. Attraction of the crucifer flea beetle to himachaladiene emitting and non-emitting plants will be tested in year 5 (no-cost extension) using olfactometer assays. Milestone: Stable transformation and expression of pheromone biosynthetic genes in Arabidopsis thaliana coupled with bioassays. In progress; Stable transformations with the PsIDS3 and PsTPS genes will be performed by using different vector constructs throughout year 5 (no-cost extension). In the case himachaladiene-emitting plants are generated, the plants will be tested for the attraction of the crucifer flea beetle P. cruciferae, which responds to himachaladiene as a single compound (Don Weber, unplublished results). Milestone: Stable transformation and expression of pheromone biosynthetic genes in Brassica napus Stable transformation of B. napus will most likely not be pursued under the current grant and will require renewed funding support. Notable findings/impacts and/or unexpected results: A major focus in year 4 has been to establish the formation of a sesquiterpene aggregation pheromone (himachaladiene) from the striped flea beetle Phyllotreta striolata. The primary rationale for changing the transformation approach to produce this pheromone has been that himachaladiene does not require an additional oxidation step and can be tested directly for the attraction of the crucifer beetle Phyllotreta cruciferae. Himachaladiene is sufficient as a single compound to elicit attraction of P. cruciferae. We have been successful with establishing N. benthamiana plants that transiently emit himachaladiene in addition to other sesquiterpene products of the PsTPS enzyme.
Publications
- Type:
Book Chapters
Status:
Awaiting Publication
Year Published:
2019
Citation:
Tholl, D. Biosynthesis of terpene pheromones in Hemiptera/stink bugs. In: Insect Pheromone Biochemistry and Molecular Biology, 2nd edition (Blomquist, G., Vogt, R., eds), Elsevier/Academic Press
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Lancaster, J, Khrimian, A., Young, S., Lehner, B, Wallingford, A., Ghosh, S.K.B., Sparks, M.E., Tittiger, C, Weber, D.C., Gundersen-Rindal, D.E., Kuhar, T.P., and Tholl, D. Aggregation pheromone biosynthesis: New genetic tools for pest management? Symposium Plant-Insect Chemical Ecology: Multi-Species Interactions and Emerging Applications in Agriculture , 2019 Entomological Society of America Eastern Branch Meeting, March 2019, Virginia Tech (oral presentation, no abstract).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Lancaster, J., Lehner, B., Tittiger, C., Khrimian, A., Weber, D.C., and Tholl, D. Do it yourself: De novo biosynthesis of terpene pheromones in stink bugs and beyond Annual Meeting of the International Society of Chemical Ecology, June 2019, Atlanta, GA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
LeBlanc, S. and Tholl, D. Developing a specialized trap crop for the control of striped flea beetle , 58th Annual Meeting of the Phytochemical Society of North America, July 2019, Johnson City, TN.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Rebholz, Z., Lancaster, J., Lehner, B., OMaille, P., and Tholl, D. Exploring the evolution of terpene synthase function in insects Terpnet, August 2019, Halle, Germany
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Tholl, D. Understanding pentatomid pheromone biochemistry for developing new pest management strategies Symposium Advances in Hemipteran Biology and Control , November 2019, Entomological Society of America Annual Meeting, St. Louis, MO (oral presentation, no abstract).
- Type:
Theses/Dissertations
Status:
Awaiting Publication
Year Published:
2020
Citation:
Lehner, B. Aggregation Pheromone Biosynthesis and Engineering in Plants for
Stinkbug Pest Management
|
Progress 02/01/18 to 01/31/19
Outputs
Target Audience:
Nothing Reported
Changes/Problems:No major changes other than those reported in year 2. What opportunities for training and professional development has the project provided?The project has provided scientific training and mentoring for Ph.D. student Jason Lancaster and Master's student Bryan Lehner at Virginia Tech. Ph.D. student Lancaster successfully completed his dissertation in February 2018 and currently holds a position at the Federal Food and Drug Administration. MS student Lehner successfully completed his thesis in February 2019. How have the results been disseminated to communities of interest?The P.D. and students Jason Lancaster and Bryan Lehner have presented their results to the research community in the form of posters or oral presentations at local, national, or international meetings and conferences. Those include the Virginia Tech 2018 Biological Sciences Research Day, the Virginia Tech 2018 Translational Plant Sciences Mini-Symposium, the 2018 Gordon Research Conference on Plant Volatiles (Barga, Italy), the 2018 ESA, ESC, and ESBC Joint Annual Meeting, and the Stewarts of the Future Conference, Communicating with Plants at NC State. The P.D. gave seminars at Purdue University, University of North Texas, and Michigan State University. Media and News: The publication by Lancaster et al. (2018) in PNAS received attention from different media and news outlets including NPR and USDA radio: VT News/Fralin: https://vtnews.vt.edu/articles/2018/08/81618-Fralin-Tholl-stinkbug.html Radio IQ: http://www.wvtf.org/post/new-way-control-stink-bugs (NPR Radio) USDA radio: https://www.usda.gov/media/radio/weekly-features/2018-09-04/using-stink-bug-pheromones-your-advantage WSLS10 News: https://www.wsls.com/news/virginia/reseachers-at-virginia-tech-find-new-ways-to-fight-stink-bugs IFT (Institute of Food Technologists) Next Newsletter: http://www.ift.org/IFTNEXT/011519.aspx What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Milestone: Identification and functional characterization of the gene candidates responsible for the second and/or third enzymatic step of the murgantiol biosynthetic pathway (and terpene pheromone biosynthetic pathways in other stink bugs) Screening of possible P450 candidates from M. histrionica and epoxidases from Nezara viridula will continue in year 4 with involvement of a new graduate student. Objective 2: Milestone: Determination of the effect of host plant cues on murgantiol pheromone biosynthesis and release. This study will be taken on by the P.D. with support by an undergraduate student and performed throughout year 4. Objective 3: Milestone: Transient expression of pheromone biosynthetic genes in Nicotiana benthamiana Co-expression of the M. histrionica and N. viridula terpene synthases (MhTPS, NvTPS) with any promising P450 candidates selected under Objective 1 will be performed in year 4 by a new graduate student. Milestone: Stable transformation and expression of pheromone biosynthetic genes in Arabidopsis thaliana coupled with bioassays. Stable transformation of terpene synthases (MhTPS, NvTPS) will be continued in year 4 by a new graduate student. In addition, an alternative IDS (FPPS)/TPS gene pair from the flea beetle P. striolata will be tested for expression in N. benthamiana and Arabidopsis. This approach has the advantage to produce an aggregation pheromone without including an oxidative step. Milestone: Stable transformation and expression of pheromone biosynthetic genes in Brassica napus. It is possible that this work begins partly for terpene synthases in year 4 but is more likely to be continued in a no-cost extension period and beyond.
Impacts
What was accomplished under these goals?
Objective 1: Milestone: Identification and functional characterization of the first enzyme (terpene synthase MhTPS) in murgantiol pheromone biosynthesis Completed; the function of MhTPS in murgantiol biosynthesis could be confirmed in vivo by an RNAi approach via microinjection; a manuscript has been published in PNAS. Milestone: Identification and functional characterization of MhTPS homologues in brown marmorated stink bug (Hyalomorpha halys) and Southern green stink bug (Nezara viridula) Completed; we have identified a terpene synthase in N. viridula (NvTPS) that specifically produces (Z)-α-bisabolene as the precursor of (Z)-α-bisabolene epoxide sex pheromone isomers. In addition, a MhTPS homologue has been identified in H. halys. As MhTPS, recombinant HhTPS produces sesquipiperitol as the presumed precursor of the H. halys aggregation pheromone. A manuscript on the identification and characterization of NvTPS has been published in the Journal of Chemical Ecology; a manuscript on the characterization of HhTPS is in preparation. Milestone: Identification and functional characterization of the gene candidates responsible for the second and/or third enzymatic step of the murgantiol biosynthetic pathway In progress; so far no enzyme responsible for the enzymatic steps downstream of the terpene synthase catalyzed reactions could be identified in M. histrionica and N. viridula. Comparative analysis of transcriptomes from M. histrionica and N. viridula related to sex, developmental stage and tissue specificity (with support by Michael Sparks, research associate with Dawn Gundersen-Rindal) resulted in a larger collection of P450s with a possible role in pheromone biosynthesis. Characterization of these P450 candidates will continue in year 4 by performing a larger scale characterization. Objective 2: Milestone: Determination of the effect of host plant cues on murgantiol pheromone biosynthesis and release. Delayed; Master's student Lehner focused his work on the identification and characterization of P450 genes (Objective 1); Anna Wallingford did not work on this objective because of her departure from the USDA for a new position. This objective is planned to be pursued in year 4 by the P.D. with support by an undergraduate student. Objective 3: Milestone: Transient expression of pheromone biosynthetic genes in Nicotiana benthamiana In progress; Transient expression of MhTPS1 and NvTPS1 as codon optimized synthetic transgenes in N. benthamiana was successful. The pheromone precursors sesquipiperitol and (Z)-α-bisabolene were emitted from inoculated N. benthamiana leaves. Transient expression of an alternative TPS from the flea beetle P. striolata together with its precursor enzyme is currently examined. This strategy might directly lead to the formation of the aggregation pheromone himachaldiene without requirement for further oxidation. These studies and co-expression with any promising P450 candidates will continue in year 4. Milestone: Stable transformation and expression of pheromone biosynthetic genes in Arabidopsis thaliana coupled with bioassays. In progress; Stable expression of TPS genes in A. thaliana has been tested but will require further optimization in year 4. If identification of P450s turns out to be challenging, we will focus our studies on the aggregation pheromone himachaladiene from P. striolata. Milestone: Stable transformation and expression of pheromone biosynthetic genes in Brassica napus Not yet started; may begin for terpene synthases depending on the results obtained in A. thaliana in year 4. Transformation and expression of P450 gene candidates will depend on success in identifying such genes and might be carried out in a non-cost extension period and beyond. Expression of the terpene synthase and its precursor enzyme from P. striolata will be considered as an alternative approach. Notable findings/impacts and/or unexpected results: Major notable findings and unexpected results for year 3 are the same as in year 2. Additional findings: Transient expression experiments with MhTPS1 and NvTPS in N. benthamiana have been successful by generating sesquipiperitol and (Z)-alpha-bisabolene emitting plants.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Lancaster, J., Lehner, B., Khrimian, A., Muchlinski, A., Luck, K., K�llner, T.G., Weber, D.C., Gundersen-Rindal, D.E., and Tholl, D. (2018) An IDS-type sesquiterpene synthase produces the pheromone precursor (Z)-alpha-bisabolene in Nezara viridula. Journal of Chemical Ecology, doi: 10.1007/s10886-018-1019-0. [Epub ahead of print]
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Lancaster, J., Khrimian, A., Young, S., Lehner, B., Luck, K., Wallingford, A., Ghosh, S.K.B., Zerbe, P., Muchlinski, A., Marek, P.E., Sparks, M.E., Tokuhisa, J.G., Tittiger, C., K�llner, T.G., Weber, D.C., Gundersen-Rindal, D.E., Kuhar, T.P., and Tholl, D. (2018) De novo formation of an aggregation pheromone precursor by an isoprenyl diphosphate synthase-related terpene synthase in the harlequin bug. Proceedings of the National Academy of Sciences USA, 115, E8634-E8641.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2019
Citation:
Beran, F., K�llner, T. G., Gershenzon, J., Tholl, D. Chemical convergence between plants and insects: Biosynthetic origins and functions of common secondary metabolites. Invited Tansley Review , New Phytologist.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Lancaster, J., Khrimian, A., Weber, D.C., Sparks, M.E., Gundersen-Rindal, D.E., Kuhar, T., Tholl, D. Aggregation pheromone biosynthesis in the brown marmorated stink bug Halyomorpha halys: Genetic tools for alternative pest management? 2018 ESA, ESC, and ESBC Joint Annual Meeting, November 11-14, Vancouver, Canada.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Tholl, D. "Volatile Molecules": Chemical Communication of Plants and Pests, Stewarts of the Future Conference, Communicating with Plants, Plants and Sensors, December 2-4, 2018, NC State University, NC.
- Type:
Theses/Dissertations
Status:
Awaiting Publication
Year Published:
2018
Citation:
Lancaster, J. Identification and Functional Characterization of Sesquiterpene Pheromone Biosynthetic Genes in Stink Bugs (Pentatomidae)
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Progress 02/01/17 to 01/31/18
Outputs
Target Audience:The project has provided scientific training for two Ph.D. students, Jason Lancaster and Bryan Lehner in the Tholl lab at Virginia Tech. Furthermore, the project has provided training for a technical assistant (Sharon Young) at the University of Nevada, Reno. Changes/Problems:Objective 1: Identification of P450s involved in the murgantiol biosynthetic pathway may take longer than expected. We may apply an additional screening tool for targeting P450 candidate genes by conducting RNAi via microinjection and headspace analysis of mature M. histrionica males. Objective 3: We plan to include the expression of a TPS from the striped flea beetle Phyllotreta striolata, which makes the sesquiterpene aggregation pheromone himachaladiene (Beran et al., 2016). Since no P450s are involved in the biosynthesis of this pheromone, we expect that transgenic plants emitting the pheromone can be more easily established and used for behavioral assays. What opportunities for training and professional development has the project provided?The project has provided scientific training and mentoring for two Ph.D. students (Jason Lancaster, Bryan Lehner) at Virginia Tech. Ph.D. student Bryan Lehner has worked closely with Co-PD Tittiger (University of Nevada, Reno) on recombinant enzyme expression experiments and assays for the P450 enzymes from M. histrionica. Furthermore, the project has provided training for a technical assistant at the University of Nevada, Reno (Sharon Young). How have the results been disseminated to communities of interest?The P.D. and Ph.D. students Jason Lancaster and Bryan Lehner have presented their results to the research community in the form of posters or oral presentations at local, national, or international meetings and conferences as stated under Products. Research results are further diseminated by manuscripts as listed under Products. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Milestone: Identification and functional characterization of the gene candidates responsible for the second and/or third enzymatic step of the murgantiol biosynthetic pathway Ph. D. student Bryan Lehner, Co-PD Dr. Claus Tittiger: Characterization of possible P450 candidates from M. histrionica and epoxidases from Nezara viridula will continue in year 3. The screening strategy for P450 candidates might be further revised by performing RNAi experiments at Virginia Tech. Objective 2: Milestone: Determination of the effect of host plant cues on murgantiol pheromone biosynthesis and release. Started together with Drs. Don Weber and Anna Wallingford at USDA-ARS, Beltsville; will continue in year 3. Objective 3: Milestone: Transient expression of pheromone biosynthetic genes in Nicotiana benthamiana Ph.D. student Bryan Lehner: Expression with NvTPS will be tested. Co-expression with of any promising P450 candidates that are selected in Objective 1 will be performed in year 3. Milestone: Stable transformation and expression of pheromone biosynthetic genes in Arabidopsis thaliana coupled with bioassays. Ph. D. student Bryan Lehner: Stable transformation has been initiated for terpene synthases (MhTPS, NvTPS) and will be continued in year 3.
Impacts
What was accomplished under these goals?
Objective 1: Milestone 1: Identification and functional characterization of the first enzyme (terpene synthase MhTPS) in murgantiol pheromone biosynthesis Completed; the function of MhTPS in murgantiol biosynthesis could be confirmed in vivo by an RNAi approach via microinjection; a manuscript is in review (PNAS) with the PD, Ph.D. students Jason Lancaster and Bryan Lehner and all co-PDs as authors/co-authors. Milestone 2: Identification and functional characterization of MhTPS homologues in brown marmorated stink bug (Hyalomorpha halys) and Southern green stink bug (Nezara viridula) Completed (Ph.D. students Jason Lancaster, Bryan Lehner); we have identified a terpene synthase in N. viridula that specifically produces (Z)-α-bisabolene as the precursor of (Z)-α-bisabolene epoxide sex pheromone isomers. In addition, a MhTPS homologue has been identified in H. halys. As MhTPS, recombinant HhTPS produces sesquipiperitol as the presumed precursor of the H. halys aggregation pheromone. Manuscripts for the identification and characterization of both enzymes are in preparation. Milestone 3: Identification and functional characterization of the gene candidates responsible for the second and/or third enzymatic step of the murgantiol biosynthetic pathway In progress (Ph.D. student Bryan Lehner); we have mined M. histrionica sex-specific and tissue (cuticle) specific transcriptomes for cytochrome P450 gene candidates with differential expression between males and females. Corresponding RT-qPCR analyses have been performed for nearly all P450s. Three P450 candidates of highest priority in the CYP3 and CYP4 clans were functionally characterized by Dr. Claus Tittiger (UNR) but did not reveal activity with sesquipiperitol as substrate. Objective 2: Milestone: Determination of the effect of host plant cues on murgantiol pheromone biosynthesis and release. In progress (with Co-PD Drs. Don Weber and Anna Wallingford); we determine whether exposure of male HB to host plant volatiles is sufficient or if direct contact and feeding of bugs on the host plant is required to induce the production and release of murgantiol. Objective 3: Milestone: Transient expression of pheromone biosynthetic genes in Nicotiana benthamiana In progress (Ph.D. student Bryan Lehner); we have established N. benthamiana expression protocols by performing positive control expressions of plant-derived terpene synthases in optimized vector systems. Expression of MhTPS1 as a codon optimized synthetic transgene has been successful. The product sesquipiperitol could be detected in leaf tissue as well as in the headspace of N. benthamiana leaves. Expression with NvTPS1 is currently tested. Notable findings/impacts and/or unexpected results We have characterized MhTPS as the first terpene synthase to be identified in pentatomids. MhTPS functions as a sesquiterpene cyclase producing sesquipiperitol, a bisabolene-type sesquiterpene alcohol, as the presumed precursor of the M. histrionica aggregation pheromone murgantiol. The MhTPS gene is highly expressed in mature males and its expression is restricted to the subcuticular tissue of the male sternites. MhTPS activity has been confirmed in crude protein extracts and in vivo by an RNAi approach applying dsRNA injection. We have identified a closely related homologue of MhTPS in the brown marmorated stink bug Halyomorpha halys. HhTPS produces sesquipiperitol as the presumed precursor of the H. halys aggregation pheromone. The enzymatic activity has been confirmed in crude protein extracts of mature males. In contrast to MhTPS, HhTPS appears to be expressed mostly in the fat body. We have also identified an isoprenyl diphosphate synthase in H. halys that makes (E,E)-FPP as the substrate for HhTPS. We have identified NvTPS1 as a unique (Z)-alpha-bisabolene synthase from the Southern green stink bug (N. viridula). The enzymatic activity has been confirmed in crude protein extracts. (Z)-alpha-Bisabolene is the direct precursor to the (Z)-alpha-bisabolene epoxide isomers of N. viridula male sex pheromone. The finding of this gene allows us to perform plant transformation studies and possibly the assembly of the N. viridula pheromone biosynthetic pathway in addition to the outlined expression studies for the murgantiol biosynthetic pathway. Overall, NvTPS1 represents an important tool for the metabolic engineering of (Z)-alpha-bisabolene in plants and microbes since no such isomer-specific terpene synthase has been identified to date from other organisms. MhTPS1, HhTPS, and NvTPS1 enzymes have likely evolved from isoprenyl diphosphate synthases of the terpene biosynthetic pathway. They represent a "new" family of terpene synthases in insects that can be distinguished from plant and microbial terpene synthases. First transient expression experiments with MhTPS1 in N. benthamiana have been successful by generating sesquipiperitol emitting plants. Unexpected result: Screening and functional characterization of cytochrome P450 gene candidates of the murgantiol biosynthetic pathway has resulted in the identification of a gene in the CYP4G family, which functions as an oxidative decarbonylase and may be involved in cuticular hydrocarbon biosynthesis. The finding has initiated further characterization of P450s of the CYP4G family in M. histrionica as part of a proposal to develop cuticular hydrocarbon biosynthesis as a novel target for pest management submitted with Dr. Claus Tittiger (UNR) as PD to the NIFA AFRI Foundational Program "Pests and Beneficial Insects in Agricultural Production Systems".
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2018
Citation:
Lancaster, J., Khrimian, A., K�llner, T.G., Lehner, B., Young, S., Wallingford, A., DiMeglio,T., Tittiger, C., Weber, D.C., Gundersen-Rindal, D.E., Kuhar, T., and Tholl, D. Terpene synthases in stink bugs: De novo formation of an aggregation pheromone precursor in the harlequin bug Murgantia histrionica
- Type:
Journal Articles
Status:
Submitted
Year Published:
2018
Citation:
Tholl, D., Beran, F., Gershenzon, G., and Koellner, T. Tansley Review: Secondary metabolite biosynthetic pathways shared between plants and animals: convergent mechanisms and functions.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Lancaster, J., Lehner, B., Tittiger, C., Young, S., Khrimian, A., Weber, D.C., Sparks, M.E., Gundersen-Rindal, D.E., Kuhar, T., and Tholl, D. Elucidating the biosynthesis of terpene pheromones in stink bugs for the development of alternative pest management strategies. Entomology 2017, Denver, CO, November 2017 (poster).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Tholl, D., Lancaster, J., Lehner, B., Tittiger, C., Young, S., Khrimian, A., Weber, D.C., Sparks, M.E., Gundersen-Rindahl, D.E., Kuhar, T., Luck, K., K�llner, T., and Gershenzon, J. Using different tools: The biosynthesis of terpene pheromones in insects. 13th International Meeting on Biosynthesis, Function, and Synthetic Biology of Isoprenoids (TERPNET), Dalian, China, July 2017 (plenary talk by D. Tholl).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Lehner, B., Lancaster, J., Young, S., Tittiger, C., and Tholl, D. (2017) Deciphering the de novo biosynthesis of insect pheromones, Terpnet 2017, 13th International Meeting on Biosynthesis, Function, and Synthetic Biology of Isoprenoids (TERPNET), Dalian, China, July 2017 (poster).
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Progress 02/01/16 to 01/31/17
Outputs
Target Audience:The project has provided scientific training for two Ph.D. students, Jason Lancaster and Bryan Lehner in the Tholl lab at Virginia Tech. Furthermore, the project has provided training for a junior computational molecular biologist (Josh Rhoades) and a postdoctoral researcher (Saikat Gosh) at the USDA Agricultural Research Service Invasive Insect Biocontrol & Behavior Lab and a technical assistant (Sharon Young) at the University of Nevada, Reno. An outreach activity has been perfomed at the 2016 Virginia Tech Science Festival and research performed under this grant has been featured in several news releases in the local press and media (see Accomplishments). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has provided scientific training and mentoring for two Ph.D. students (Jason Lancaster, Bryan Lehner) at Virginia Tech. Ph.D. student Bryan Lehner works closely with Co-PD Tittiger (University of Nevada, Reno) on recombinant enzyme expression experiments and assays for the TPS1 enzymes from HB and SGSB. Furthermore, the project has provided training for a junior computational molecular biologist at the USDA Agricultural Research Service Invasive Insect Biocontrol & Behavior Lab and a technical assistant at the University of Nevada, Reno (Sharon Young). Ph.D. students Jason Lancaster and Bryan Lehner have presented their research in the form of posters or oral presentations at local, national, or international meetings and conferences as stated under Products. How have the results been disseminated to communities of interest?Ph.D. students Jason Lancaster and Bryan Lehner together wuth MS student Tony DiMeglio (Co-PD Thomas Kuhar, Virginia Tech) showcased their research on HB pheromones to the public at the 2016 Virginia Tech Science Festival. DiMeglio, A.G, Lancaster, J.G, Lehner, B.G, Tholl, D., & Kuhar, T. P. (2016). Bug talk, Poster, Virginia Tech Science Festival. Blacksburg, VA. Research performed under this grant also lead to several news releases in the local press and media: The Virginian Pilot, News article about research on insect pheromones and their engineering in the Tholl lab - http://pilotonline.com/business/field-notes/field-notes-scientists-finding-ways-to-out-stink-the-stink/article_acf58886-fcdb-5f85-8aa0-e10df0457033.html Virginia Farm Bureau, News article about research on insect pheromones and their engineering in the Tholl lab, 04/01/2016 Virginia Tech News, News article about insect pheromone research and engineering in the Tholl lab - http://vtnews.vt.edu/articles/2016/03/fralin-stinkbugs.html WDBJ7 news channel feature - http://www.wdbj7.com/content/news/Scented-plant-could-be-answer-to-saving-crops-from-stink-bugs-Virginia-Tech-scientists-say-376575821.html What do you plan to do during the next reporting period to accomplish the goals? For Objective 1, we are in the process of verifying cytochrome P450 epoxidase gene candidates by quantitative RT-PCR and have begun characterizing these candidates by epression in insect Sf9 cells and funcyional terpene synthase assays (Co-PD Tittiger with technicina Sharon Young, Ph.D. student Bryan Lehner). These efforts will continue throughout year 2 of the funding period. For Objective 2, we will determine whether exposure of male HB to host plant volatiles is sufficient or if direct contact and feeding of bugs on the host plant is required to induce the production and release of murgantiol. These experiments will be performed at the USDA Agricultural Research Service Invasive Insect Biocontrol & Behavior Lab (Beltsville, MD) with Co-PD Weber. For Objective 3, we will begin transient transformation of MhTPS1 and NvTPS1 in Nicotiana benthamiana in the summer 2017 using vector systems that have been optimized for this transformation system (Ph.D. student Bryan Lehner). We will also stably transform these genes into Arabidopsis thaliana and optimize the expression protocol if necessary (Ph.D. student Bryan Lehner).
Impacts
What was accomplished under these goals?
We have made substantial progress under Objective 1 according to our time plan in year 1: To elucidate the first step in the murgantiol biosynthetic pathway, we have identified two prenyltransferase like genes (farnesyl diphosphate synthase 1 and 2 - FPPS1 and 2) in the transcriptome of male harlequin bug (HB). Heterologous protein expression demonstrated that FPPS1 functions as a terpene synthase (now called MhTPS1), which produces the sesquiterpene alcohol, sesquipiperitol, as the presumed precursor of murgantiol (PD Tholl and Ph.D. student Jason Lancaster). MhTPS1 activity has been confirmed in protein extracts of the epidermal tissue from the cuticle of male HB (Ph.D. student Bryan Lehner). An HB colony required for the molecular and biochemical experiments has been maintained by Co-PD Thomas Kuhar (Virginia Tech). Sesquipiperitol was identified by a combination of mass-spectral comparisons to related compounds, chemical reactions to known compounds, and synthesis (Co-PD Khrimian). Determination of the absolute configuration of MhTPS1-produced sesquipiperitol (Co-PD Khrimian) strongly indicated that the MhTPS1 enzymatic product is the precursor of murgantiol. MhTPS1 is predominantly expressed in the epidermal tissue of the abdominal sternites of mature male HB, which supports a sex- and tissue-specific formation of murgantiol (Ph.D. student Jason Lancaster). A manuscript reporting these findings is in preparation to be published in 2017. Another manuscript on the HB transcriptome (Sparks et al.) is currently in revision. We have identified a gene with homology to MhTPS1 in the Southern green stink bug (SGSB, Nezara viridula), which encodes a cis-alpha-bisabolene synthase. cis-alpha-bisabolene is the precursor of bisabolene epoxide pheromones in SGSB (Ph.D. students Jason Lancaster, Bryan Lehner). To identify the gene(s) responsible for the conversion of sesquipiperitol to murgantiol, we have mined transcriptome databases from the epidermal cuticle tissue and whole bugs of mature male and female HB and from whole bugs of mature and immature male HBs. Sex and developmental comparative gene expression analyses have resulted in the identification of several cytochrome P450 candidates with a possible role in catalyzing the epoxidation step leading to the formation of murgantiol (Ph.D. student Bryan Lehner).
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2017
Citation:
Sparks, M.E., Rhoades, J.H., Nelson, D.R., Kuhar, D., Lancaster, J., Lehner, B., Tholl, D., Weber, D.C., and Gundersen-Rindal, D.E.
A Transcriptome Survey Spanning Life Stages and Sexes of the Harlequin Bug, Murgantia histrionica
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Lancaster, J.G, Khrimian, A., Gundersen-Rindal, D.E., Weber, D.C., and Tholl, D.
Discovering sesquiterpenoid pheromone biosynthetic pathways in stink bugs (Pentatomidae). Published abstract, XXV International Congress of Entomology, Orlando, FL.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Lancaster, J.G, Khrimian, A., Gundersen-Rindal, D.E., Kuhar, T.P., and Tholl, D.
Sesquiterpenoid pheromone biosynthetic pathways in stink bugs (Pentatomidae). Published abstract, Entomology Section. In Virginia Journal of Science Vol. 67. Virginia Academy of Science.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Lehner, B.G, Lancaster, J.G, and Tholl, D. (2016). Elucidating an insect terpenoid pheromone pathway to engineer trap crops for pest management. Published abstract, 55th Annual Meeting of the Phytochemical Society of North America. University of California, Davis.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2016
Citation:
Lancaster, J.G, Khrimian, A., Gundersen-Rindahl, D.E., Weber, D.C., Kuhar, T., and Tholl, D.
Elucidating the sesquiterpenoid pheromone biosynthetic pathway in stink bugs (Pentatomidae) to provide tools for alternative pest management strategies. Poster presented at the 2016 Gordon Research Conference on Plant Volatiles, Ventura, CA.
- Type:
Other
Status:
Published
Year Published:
2016
Citation:
DiMeglio, A.G, Lancaster, J.G, Lehner, B.G, Tholl, D., and Kuhar, T. P. (2016). Bug talk, Poster, Virginia Tech Science Festival. Blacksburg, VA.
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