Progress 05/15/18 to 05/14/21
Outputs
Target Audience:The outcomes of this project are primarily relevant and of interest to audiences in the research communities of the plant sciences and entomology. The results and knowledge obtained during the entire fundingperiod of this project have been communicated to this audience via oral and poster presentations at several national andinternational scientific conferences. Additionalplanned presentations at scientific conferences unfortunately had to be canceled due to the Covid-19 crisis and related travel bans. In addition, theresults obtained over the course of thisproject have been summarized in severalmanuscripts with two manuscripts already published, one manuscript to be submitted for review now, and one additional manuscript in preparation.In the long run the knowledge gained through this project will also be relevant to audiences beyond the research communities, in particular to growers. Therefore we also presented the approach taken in the project and results of theresearch at a Master Gardener Conference. Moreover, this project and its results are also relevant and of interest to students in the WVU School of Agriculture and Food. Thus aspects of this project were integrated in the curriculum of the PLSC206 'Principles of Plant Science' class. In addition,results of the project were also presented in the AGBI199 'Orientation to Biochemistry' class andin the 'Plant Science Graduate Seminar'. Changes/Problems:The project experienced a major delay due to the Covid-19 related intial complete lockdown of the WVU campus andthe subsequent reduced working hours. Considering these circumstances we decided to first use two of the binary vectors that carry the multicistronic expression constructsfor transient transformation experiments. Under these difficult conditions this approach allowed usto still verify thefunction of multicistronic expression constructs as well as to establish all methods and analytical protocols that arealso required for the characterization of the stable transgenic tomato lines that we are obtaining now. In addition the transient transformation approach allowed us to collect data on the metabolic engineering of the desired sesquiterpenes and their effect on aphid performance which we summarized in another manuscript which now will be submitted to BMC Plant Biology. Anotherproblem caused by the Covid crisis is related to our original planto usea plant transformation facility for creating transgenic tomato lines. Due to the Covid crisis these facilties were closed for an extended period and subsequently did not acceptwork requests. Thus we decided to perform the transformation utilizing the sixvector constructs in the PIs lab. While we are now obtaining the expected transgenic tomato lines this change still resulted in additional work and a further delay. What opportunities for training and professional development has the project provided?Thisproject provided training and professional development opportunities for one PhD graduate student and one undergraduate student: Fumin Wang, the graduate student funded through this project, performed all experiments within this project and received training in entomology, chemical ecology, plant biochemistry and molecular biology under the guidance of the PI and Co-PI. The graduate studenthad the opportunity to present the results of his work as poster at the 2019 ASPB Midwestern Section Meeting (won third place in poster competition), as virtual poster at the2020 Annual Meeting of the Entomological Society of America,and as oral presentation in the departmental graduate seminar.The graduate student also had the opportunity to contribute significantly to the preparation, submission and revision of threemanuscripts summarizing results of this project. Meleana Santivasci, an undergraduate student majoring in horticulture, joined the PI's lab in the spring semester 2019 and continued to work until March 2020. The undergraduate studentassisted in the analysis of some of the tomato accessions utilized in the project. The undergraduate student received training in basic laboratory skills and in the analysis of terpenes from tomato leaves under the guidance of the PI and the graduate student. Unfortunately the undergraduate student was not able to work in the project during the 2020/2021 academic year due to the Covid related restrictions. How have the results been disseminated to communities of interest?Over the course of the project the obtained results have been disseminated to the research community through presentations at scientific conferences: i) The Pi presented results in an oral presentation at the 2018 Annual Conference of the American Society of Horticultural Science ASHS (Session: Weed Control and Pest Management). ii) The graduate student presented results as poster at the 2019 Annual Meeting of the Midwestern Section of the American Society of Plant Biologists (ASPB). iii) The PI presented results in an oral presentation at the 14th International Meeting on the Biosynthesis, Function and Synthetic Biology of Isoprenoids, TERPNET 2019(Germany). iv) The graduate student presented results as virtual poster at the 2020 Annual Meeting of the Entomological Society of America (ESA) -Entomology 2020. The results obtained from our analysis of cultivated and wild tomato accessions, and how their different terpene profiles affect the host plant preference and feeding behavior of aphids have been summarizedin two manuscripts which have been published in peer-reviewedscientific journals: 1) Fumin Wang, Yong-Lak Park, and Michael Gutensohn (2020) Glandular trichome-derived sesquiterpenes of wild tomato accessions (Solanum habrochaites) affect aphid performance and feeding behavior. Phytochemistry180:112532; https://doi.org/10.1016/j.phytochem.2020.112532 2) Fumin Wang, Yong-Lak Park, and Michael Gutensohn (2021) Glandular trichome-derived mono- and sesquiterpenes of tomato have contrasting roles in the interaction with the potato aphid Macrosiphum euphorbiae. Journal of Chemical Ecology47:204-214; https://doi.org/10.1007/s10886-021-01243-4 We have also summarized the results obtained from the transient expression of the multicistronic constructs containing terpene biosynthetic genes in cultivated tomato as well as the characterization of the resulting terpene production and effects on the aphid performance in an additional manuscript which we will now submit for review to the journal BMC Plant Biology: 3)Fumin Wang, Yong-Lak Park, and Michael Gutensohn(2021) Epidermis-specific metabolic engineering of sesquiterpene formation in tomato affects the performance of potato aphid Macrosiphum euphorbiae.BMC Plant Biology (to be submitted) In addition, we have started to work on another manuscript to summarize results obtained from the stable transgenic tomato lines expressingterpene biosynthetic genes under the control of tisssue specific promoters. This manuscript will be finished and then submitted for review and publication as soon as the ongoing data collection and analysis from the characterization of the transgenic tomatolines and the performance of aphids exposed to these lines are completed. As an outreach effort to increase public understanding and awareness of research activities the approach and someresults of this project were included in an oral presentation by the PI at the 2019 Annual Conference of the West Virginia Extension Master Gardener Association. Another outreach effort taken duringthis project was the publication of a news release which is now posted at the webpage of the WVU Division of Plant and Soil Sciences and also includes a link to a respective youtube video. As an additional effort to increase interest and understanding in this ongoing research the PI has also included some aspects of this project into one lecture and one lab section of the "Principles of Plant Science" PLSC206 class. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1:We haveanalyzed a collection of tomato accessions (S. habrochaites & S. lycoperiscum) representing 6 different terpene chemotypes for their effect on performance and behavior of potato aphids. Non-choice assays indicated that two groups of S. habrochaitesaccessions producing santalene/bergamotene and caryophyllene/humulene, respectively, had significant effects on the longevity and reproduction of aphids. When terpenes were extracted from tomato accessions and used in aphid feeding assays on artificial diet, extracts from the santalene/bergamotene and caryophyllene/humulene producing accessions significantly affected aphid feeding behavior and survival. Moreover, when these tomato accessions and respective terpene extracts were used for olfactometer choice assays santalene/bergamotene and caryophyllene/humulene had repellent activity towards aphids. To further verify that theeffects onperformance and behavior of aphids areindeed due to respective terpenes additional control experiments were performed. Pure caryophyllene and humulene were used in feeding and choice assays and confirmed their effects on aphids. Since santalene/bergamotene are not available as pure compounds we instead used a tomato introgression line producing these terpenes in non-choice, feeding and choice assays to confirm their effect on aphids. These results were summarized in a manuscriptwhich has been published in apeer-reviewed journal[Wang, Park &Gutensohn(2020)Phytochemistry180:112532]. Based on our resultsthat S. habrochaitesproducing caryophyllene/humuleneaffectedaphid performance andbehavior weperformed additional experiments since these sesquiterpenes are also present in cultivated tomato. Weutilized tomato trichome mutantsthat are affected in mono- and sesquiterpene production to perform non-choice and olfactometer choice assays. While caryophyllene/humulene contributed to host plant resistance against aphids (confirming our previous results), glandular trichome derived monoterpenes in cultivated tomato appear to be exploitedfor host plant orientation by aphids. Thisprovided baseline knowledge prior to the introduction of new terpene traits into cultivated tomato by metabolic engineering. These results weresummarizedin a manuscriptwhich has been published in apeer-reviewed journal[Wang, Park &Gutensohn(2021)Journal of Chemical Ecology47:204-214]. Objective 2 and 3: The goal of Objective 2 is to determine how the expression of respective terpene biosynthetic genes from S. habrochaites under the control of different tissue-specific promoters changes the terpene profile in resulting engineered cultivated tomato lines. We havedesigned and constructedsixrespective expression constructs inbinary vectorsthat are required for the transient and stable transformation of cultivated tomato. In order to engineer the formation of thetwo groups of terpenes (santalene/bergamotene and caryophyllene/humulene) in three different tissues of cultivated tomato (glandular trichomes, epidermis, phloem companion cells) we have amplified the promoter regions of three genes (tomato SlTPS9, Arabidopsis AtCER5, Arabidopsis AtSUC2) by PCR and have cloned these into the multiple cloning site of the pMCS:GW vector. Constructs consisting of the open reading frames of the respective farnesyl diphosphate synthases (ShzFPPS andAtFPPS2, respectively) and terpene synthases (ShSBS andShTPS12, respectively) involved in the biosynthesis of these terpenes as well as enhanced green fluorescent protein (eGFP) linked in frame by viral 2A sequences were designed and then obtained by gene synthesisfrom a commercial provider. These large multicistronic gene constructs were subsequently recombined into theGateway cloning site of the pMCS:GW vector. All six final vector constructs (two different multicistronic gene constructs each under the control of three tissue specific promoter) cloned into thepMCS:GW vector were thenintroduced into Agrobacterium tumefaciens. At this point the project experienced a major delay due to the Covid-19 related intial complete lockdown of the WVU campus andthe subsequent reduced working hours. Considering these circumstances we decided to first use two of the binary vectors that carry the multicistronic expression constructs (ShzFPPS-ShSBS-eGFP and AtFPPS-ShTPS12-eGFP) under the control of the epidermis specificAtCER5 promoter for transient transformation experiments to verify their function (Objective 2). Transient transformation was achieved by infiltration of tomato leaves with Agrobacterium tumefacienscarrying these vector constructs or the empty pMCS:GW vector as control. Upontransient transformation of tomato leaves transcripts ofprenyl transferases and eGFP,the first and last coding region in both constructs, were detected by RT-PCRsuggestingthat the entire multicistronic constructs were expressed.Our analyses of Agrobacterium infiltrated tomato leaves byfluorescence microscopynot only provided further evidence that the entire multicistronic constructs including eGFP are expressed, but also indicated that their expression under the AtCER5 promoter is indeed restricted to the epidermis. Oursubsequent analysis oftomato leaf extracts by combined gas chromatography-mass spectrometry (GC-MS) demonstrated that leaves expressing the AtFPPS-ShTPS12-eGFP construct accumulated the ShTPS12 products β-caryophyllene/α-humulene, whileleavesexpressing theShzFPPS-ShSBS-eGFPconstruct accumulatedthe ShSBS products (-)-endo-α-bergamotene, (+)-α-santalene, (-)-exo-α-bergamotene, (-)-epi-β-santalene, and (+)-endo-β-bergamotene.To further verifytissue specificity of our metabolic engineering approachAgrobacterium infiltratedtomato leaves were separated into epidermis, trichome, mesophyll and vasculature fractions which wereanalyzed for their terpene content. ShTPS12- and ShSBS-derived sesquiterpenes almost exclusivelyaccumulated in the epidermis of the transiently transformed tomato leaves, thus indicating that expression of the multicistronic constructs under control of the AtCER5 promoter indeed resultedin the epidermis specific production of the engineered sesquiterpenes. To characterize the potential of the sesquiterpene formation engineered into the leaf epidermis to affect the potato aphid (Macrosiphum euphorbiae) (Objective 3), we performed non-choice assays utilizing tomato leaves that transiently expressed the multicostronic constructs. Our analysis demonstrated that the formation of theShTPS12- and ShSBS-derived sesquiterpenes in the epidermis of tomato leaves transiently expressing theAtFPPS-ShTPS12-eGFP andShzFPPS-ShSBS-eGFP constructs significantly reduced the longevity andfecundity (number of offspring) of aphids. We have summarized these results in a manuscriptwhich will now be submitted to the peer-reviewed journalBMC Plant Biology for review[Wang, Park &Gutensohn(2021) Epidermis-specific metabolic engineering of sesquiterpene formation in tomato affects the performance of potato aphidMacrosiphum euphorbiae.BMC Plant Biology (to be submitted)]. We originally had proposed to usea plant transformation facility for creating transgenic tomato lines. However, due to the Covid crisis these facilties were closed for an extended period and subsequently did not acceptwork requests. Thus we decided to perform the transformation utilizing the sixvector constructs in the PIs lab. We are now in the process of isolating stabletransgenic tomato lines for eachvector construct, andare characterizing already available transgenic lines expressing the multicistronic constructs in specifictissues. The characterization of the obtained transgenic lines and their effecton the performance and behavior of aphids is now benefitting from the fact that all methods required have already been established through the analysis of tomato leaves transiently expressing the multicistronic constructs.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Fumin Wang, Yong-Lak Park, and Michael Gutensohn*
"Glandular Trichome-Derived Terpenes of Wild Tomato Accessions Affect Aphid Performance and Feeding Behavior"
Oral Presentation, Session: Weed Control and Pest Management 2
2018 Annual Conference of the American Society of Horticultural Science (ASHS)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Fumin Wang*, Yong-Lak Park, and Michael Gutensohn
"The role of trichome-derived monoterpenes and sesquiterpenes in the interaction of cultivated tomato and potato aphid"
Poster Presentation, Session: Graduate Students
2019 Annual Meeting of the Midwestern Section of the American Society of Plant Biologists (ASPB)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Michael Gutensohn*, Fumin Wang, and Yong-Lak Park
"Glandular trichome-derived mono- and sesquiterpenes of cultivated and wild tomato accessions have different effects on
aphid performance and feeding behavior"
Oral Presentation, Session: Terpenoids in chemical ecology
TERPNET 2019, 14th International Meeting on the Biosynthesis, Function and Synthetic Biology of Isoprenoids
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Michael Gutensohn*
"Why do plants smell? The function of plant volatile compounds in pollinator attraction, pest defense and fruit flavor"
Oral Presentation
2019 Annual Conference of the West Virginia Extension Master Gardener Association
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Fumin Wang, Yong-Lak Park, and Michael Gutensohn* (2020)
"Glandular trichome-derived sesquiterpenes of wild tomato accessions (Solanum habrochaites) affect aphid performance and feeding behavior"
Phytochemistry 180:112532
https://doi.org/10.1016/j.phytochem.2020.112532
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Fumin Wang*, Yong-Lak Park, and Michael Gutensohn
"The contrasting roles of trichome-derived monoterpenes and sesquiterpenes in the interaction of cultivated tomato and potato aphid"
Virtual Poster Presentation
Entomology 2020 - Virtual Annual Meeting of the Entomological Society of America (ESA)
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Fumin Wang, Yong-Lak Park, and Michael Gutensohn* (2021)
"Glandular trichome-derived mono- and sesquiterpenes of tomato have contrasting roles in the interaction with the potato aphid Macrosiphum euphorbiae"
Journal of Chemical Ecology 47:204-214.
https://doi.org/10.1007/s10886-021-01243-4
- Type:
Journal Articles
Status:
Other
Year Published:
2021
Citation:
Fumin Wang, Yong-Lak Park, and Michael Gutensohn* (2021)
"Epidermis-specific metabolic engineering of sesquiterpene formation in tomato affects the performance of potato aphid Macrosiphum euphorbiae"
BMC Plant Biology (to be submitted)
|
Progress 05/15/19 to 05/14/20
Outputs
Target Audience:The outcomes of this project during the current reporting period are primarily relevant and of interest to audiences in the research communities of the plant sciences and entomology. The results and knowledge obtained during the current reporting period of this project have been communicated to this audience via an oral presentationat an international scientific conference. A secondplannedpresentation at a national conference had to be canceled due to the Covid-19 related travel ban. In addition, in the current reporting period results of the project have been summarized in two manuscripts that have been submitted for review and have been accepted for publication with minor revisions. However, this project and its results are also relevant and of interest to students in the WVU School of Agriculture and Food. Thus aspects of this project were integrated in the curriculum of the PLSC206 'Principles of Plant Science'class and results of the project were also presented in the AGBI199 'Orientation to Biochemistry' class. Changes/Problems:Due to the Covid-19 related lock down of the WVU campus and the resulting major delay and set back of the project, we have requested and were recently granted a no-cost extension by one year for this project. What opportunities for training and professional development has the project provided?In the current reporting period the project provided training and professional development opportunities for one PhD graduate student and one undergraduate student: Fumin Wang, the graduate student funded through this project, performed all experiments during the current reporting period and received training in entomology, chemical ecology, plant biochemistry and molecular biology under the guidance of the PI and Co-PI. The graduate student also had the opportunity to contribute significantly to the preparation, submission and current revision of two manuscripts summarizing some of the results of this project. Meleana Santivasci, an undergraduate student majoring in horticulture, assisted in the analysis of some of the tomato lines utilized in the project and received training in basic laboratory skills and in the analysis of terpenes under the guidance of the PI and the graduate student. How have the results been disseminated to communities of interest?In the current reporting period the results of the project have been disseminated to the research community through an oral presentation by the PI at the 14th International Meeting on the Biosynthesis, Function and Synthetic Biology of Isoprenoids, TERPNET 2019, Halle/S. Germany: "Glandular trichome-derived mono- and sesquiterpenes of cultivated and wild tomato accessions have different effects on aphid performance and feeding behavior". The results obtained from our analysis of cultivated and wild tomato accessions, and how their different terpene profiles affect the host plant preference and feeding behavior of aphidshave been summarized during the current reporting period in two manuscripts which have been submitted for review and publication to two scientific journals. Both manuscripts have been reviewed favorably and only minor revisions have been requested by the journal editors prior to publication. The graduate student as well as the PI and CoPI are currently working on the revision of both of these manuscripts: 1) Fumin Wang, Yong-Lak Park and Michael Gutensohn (2020) Glandular trichome-derived sesquiterpenes of wild tomato accessions (Solanum habrochaites) affect aphid performance and feeding behavior. Phytochemistry (under revision) 2) Fumin Wang, Yong-Lak Park and Michael Gutensohn (2020) Glandular trichome-derived mono- and sesquiterpenes of tomato have contrasting roles in the interaction with the potato aphid Macrosiphum euphorbiae. Journal of Chemical Ecology (under revision) What do you plan to do during the next reporting period to accomplish the goals?Considering the recentCovid-19 related campus lock-down we envision the following tentative timetable for the completion of the outstanding portions of the project: With research activities partially resumingon the WVU campus (since earlyJune 2020) we anticipate that we will need approximately six month for the stable transformation of cultivated tomato with the available multicistronic expression constructs, as well as the subsequent genetic and biochemical characterization of the resulting transgenic tomato lines (Objective 2B). Unfortunately, the test of the expression constructs via transient expression in tomato leaves will have to be repeated since the ongoing experiments have been lost due to the Covid-19 related campus lock-down. In general, we will take a stacked approach which will allow us to already characterize one set of transgenic lines while working on the plant transformation with the next expression constructs in parallel. As soon as confirmed transgenic tomato lines will be available, we will utilize these lines for non-choice and choice assays with aphids (Objective 3). We anticipate that we will need additional four to six month for the analysis of the aphid performance on the engineered tomato lines.
Impacts
What was accomplished under these goals?
While overall three specific objectives will be addressed in this proposal, we have achieved significant progress on Objectives 1 and 2 during the current reporting period: Objective 1: In the previous reporting period we had analyzed a collection of different tomato accessions (S. habrochaites & S. lycoperiscum) representing 6 different terpene chemotypes for their effect on performance and behavior of potato aphids. Non-choice assays indicated that two groups of tomato accessions producing santalene/bergamotene and caryophyllene/humulene, respectively, had significant effects on the longevity and reproduction of aphids. When terpenes were extracted from tomato accessions and used in aphid feeding assays on artificial diet, extracts from the santalene/bergamotene and caryophyllene/humulene producing accessions significantly affected aphid feeding behavior and survival. Moreover, when these tomato accessions and respective terpene extracts were used for olfactometer choice assays santalene/bergamotene and caryophyllene/humulene had repellent activity towards aphids. To further verify that the observed effects on the performance and behavior of aphids is indeed due to respective terpenes additional control experiments were performed. Commercially available pure caryophyllene and humulene were used in feeding and choice assays and confirmed their effects on aphids. Since santalene and bergamotene are not available as pure compounds we instead used an available tomato introgression line producing these terpenes in non-choice, feeding and choice assays to confirm their effect on aphids.In the current reporting period these results were summarized in a manuscript (see below) which has been submitted to the journal Phytochemistry and has received favorable reviews (minor revisions). Initiated by some results of this previous analysis we have performed additional experiments in the current reporting period utilizing trichome mutants of cultivated tomato (S. lycoperiscum) that are differently affected in mono- and sesquiterpene production. Non-choice and olfactometer choice assays were performed with these trichome mutants to study effects of these terpenes on performance and choice behavior of aphids. While caryophyllene and humulene contributed to host plant resistance against aphids (confirming our previous results obtained with S. habrochaites), glandular trichome derived monoterpenes in cultivated tomato appear to be exploited as cue for host plant orientation by aphids. These results now provide a good baseline knowledge prior to introduction of new terpene traits into cultivated tomato by metabolic engineering. These results were also summarized in the current reporting period in a manuscript (see below) which has been submitted to the Journal of Chemical Ecology and has received favorable reviews (minor revisions). Objective 2: The goal of this objective is to determine how the expression of respective terpene biosynthetic genes from S. habrochaites under the control of different tissue-specific promoters changes the terpene profile in resulting engineered cultivated tomato lines. In the current reporting period we have continued and completed our work on the design, construction, and cloning of the respective expression constructs and vectors required for the transformation of cultivated tomato that we had already started in the previous reporting period. In order to engineer the formation of these two groups of terpenes in three different tissues of cultivated tomato (glandular trichomes, epidermis, phloem companion cells) we have amplified the promoter regions of three genes (tomato SlTPS9, Arabidopsis AtCER5, Arabidopsis AtSUC2) by PCR and have cloned these into the multiple cloning site of the pMCS:GW vector. Constructs consisting of the open reading frames of the respective farnesyl diphosphate synthases and terpene synthases involved in the biosynthesis of these terpenes as well as enhanced green fluorescent protein linked in frame by viral 2A sequences were designed and then ordered from a commercial provider for gene synthesis. Unfortunately, we had to wait much longer (months instead of weeks) until these synthesized gene constructs were finally delivered to us which resulted in a significant delay of the project. In addition, the subsequent recombination of these large gene constructs under the control of the above promoters into the pMCS:GW vector by Gateway cloning turned out to be by far less efficient than expected which resulted in a further delay. We have completed the cloning of all six final vector constructs (two different gene constructs each under the control of three tissue specific promoter) and have subsequently introduced these vectors into Agrobacterium tumefaciens. We have recently performed a number of transient transformation experiments by Agrobacterium infiltration into tomato leaves with some of these vector constructs to verify their function prior to stable transformation. However, these experiments had to be abandoned due to the Covid-19 related lock-down of the WVU campus before the analysis of transgene expression, terpene formation and GFP localization could be performed and/or completed. Due to the transient nature of these transformation events these experiments are now lost and will have to be repeated. While the engineered tomato lines have not yet been available in the current reporting period, we have used an introgression line producing santalene/bergamotene in glandular trichomes for a first set of analyses. This introgression line was previously obtained by crossing a santalene/bergamotene producing S. habrochaites accession with a cultivated tomato accession and a series of subsequent backcrosses into the cultivated tomato background. We used this introgression line for the same type of non-choice and choice assays (Objective 3) as performed with the respective S. habrochaites accessions. The results indicated that formation of santalene/bergamotene in the background of cultivated tomato indeed has an effect on the performance and behavior of aphids. These results were included in the manuscript (see below) that had been submitted to the journal Phytochemistry and that we are currently revising. Since a respective introgression line is not available for caryophyllene/humulene accessions, in the current reporting period we have also crossed a caryophyllene/humulene producing S. habrochaites accession with a cultivated tomato accession. The F1 plants of this cross were tested for their terpene production and subsequently used for additional backcrosses into the cultivated tomato background.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Michael Gutensohn*, Fumin Wang and Yong-Lak Park
"Glandular trichome-derived mono- and sesquiterpenes of cultivated and wild tomato accessions have different effects on
aphid performance and feeding behavior"
Oral Presentation, Session: Terpenoids in chemical ecology
TERPNET 2019, 14th International Meeting on the Biosynthesis, Function and Synthetic Biology of Isoprenoids
- Type:
Journal Articles
Status:
Accepted
Year Published:
2020
Citation:
Fumin Wang, Yong-Lak Park and Michael Gutensohn
"Glandular trichome-derived sesquiterpenes of wild tomato accessions (Solanum habrochaites) affect aphid performance and feeding behavior"
Phytochemistry (accepted with minor revisions)
- Type:
Journal Articles
Status:
Accepted
Year Published:
2020
Citation:
Fumin Wang, Yong-Lak Park and Michael Gutensohn
"Glandular trichome-derived mono- and sesquiterpenes of tomato have contrasting roles in the interaction with the potato aphid Macrosiphum euphorbiae"
Journal of Chemical Ecology (accepted with minor revisions)
|
Progress 05/15/18 to 05/14/19
Outputs
Target Audience:The outcomes of this project during the current reporting period are primarily relevant and of interest to audiences in the research communities of the plant sciences and entomology. The results and knowledge obtained during the current reporting period of this project have been communicated to this audience via oral and poster presentations at several scientific conferences. However, this project and its results are also relevant and of interest to students in the WVU School of Agriculture and Food. Thus aspects of this project were integrated in the curriculum of the 'Principles of Plant Science' (PLSC206) class and results of the project were presented in the Plant Science Graduate Seminar. In the long run the knowledge gained through this project will also be relevant toaudiences beyond the research communities, in particular to growers. Therefore we also presented the approach taken inthe project and results of the ongoing research at a recent Master Gardener Conference. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?In the current reporting period the project provided training and professional development opportunities for one PhD graduate student and one undergraduate student: Fumin Wang, the graduate student funded through this project, performed all experiments during the current reporting period and received training in entomology, chemical ecology, plant biology and molecular biology under the guidance of the PI and Co-PI. The graduate student also had the opportunity to present the results of his work as poster at the ASPB Midwestern Section Meeting (won third place in poster competition) and as oral presentation in the departmental graduate seminar. Meleana Santivasci, an undergraduate student majoring in horticulture, joined the lab of the PI in the spring semester 2019 and assisted in the analysis of some of the tomato accessions utilized in the project. The undergraduate student received traning in basic laboratory skills and in the analysis of terpenes from tomato leaves under the guidance of the PI and the graduate student. How have the results been disseminated to communities of interest?In the current reporting period the results of the project have been disseminated to the research community through presentations at scientific conferences. The Pi presented results in anoral presentation at the2018 Annual Conference of the American Society of Horticultural Science ASHS (Session: Weed Control and Pest Management 2) and the graduate student presented results as poster at the 2019 Annual Meeting of the Midwestern Section of the American Society of Plant Biologists (ASPB). In addition, an abstract for an oral presentation by the PI at the upcoming14th International Meeting on the Biosynthesis, Function and Synthetic Biology of Isoprenoids (TERPNET 2019) has been accepted. Asan outreach effort to increase public understanding and awareness of research activities the approach and some of the results of this project were included in an oral presentation by the PI at the2019 Annual Conference of the West Virginia Extension Master Gardener Association. Another outreach effort taken in the early phase of this project was the publication of a news release which is nowposted at the webpage of the WVU Division of Plant and Soil Sciences and also includes a link to a respective youtube video. As an additional effort to increase interest and understanding in this ongoing research the PI has also included some aspects of this project into one lecture and one lab section of the "Principles of Plant Science" PLSC206 class. What do you plan to do during the next reporting period to accomplish the goals?In the secondreporting period of this project we are planning to accomplish the following goals as also outlined in the original plan: Objective 1, Assess if wild tomato accessions with different terpene profils affect the host preference and feeding behavior of aphids: We are in the process of summarizing the results obtained during the current reporting period in manuscripts which will be submitted for review and publication in scientific journals. Objective 2,Determine how expression of respective terpene biosynthetic genes under the control of different tissue-specific promoters changes the terpene profile in resulting engineered cultivated tomato lines: The assembled vector constructs will now be used for Agrobacterium mediated leaf disc transformation of the S. lycopersicum line MP-1. Since stable transformation will take some time, we will use all constructs for transient transformation by Agrobacterium infiltration of leaves. This will allow us to verify function of constructs and do first analysis on infiltrated leaves prior to obtaining stable lines.By stable transformation we expect to obtain ~20 lines per construct which will be tested for presence of transgenes by PCR on genomic DNA. To screen for terpene production, ground leaves will be extracted with solvent and extracts analyzed by GC-MS. Based on this initial screen 2-3 lines per construct with sufficient terpene formation will be selected. Expression of all transgenes will be confirmed by quantitative RT-PCR. Emitted volatiles will be collected from leaves by closed-loop stripping. Internal terpene pools will be extracted either by dipping leaves in solventor by extracting ground tissue with solvent. Collected volatiles and solvent extracts will be analyzed by GC-MS to verify qualitative and quantitative composition of terpene profiles. To confirm tissue-specificity transgene expression and terpene production in the targeted tissues will be analyzed (glandular trichomes, epidermis, phloem companion cells). We will verify GFP expression in different lines by fluorescence microscopy. In addition, we will isolate fractions enriched in particular tissues and use these for analysis of gene expression and terpene formation. Objective3,Determine how engineering of repellent and/or toxic terpenes in three tissues (trichomes, epidermis and phloem) of cultivated tomatochanges the overall performance and feeding behavior of aphids: Obtained transgenic tomato lines with engineered terpenes will be used to assess the effects of terpene production in the three target tissues on the performance of aphids. Towards this goal we will perform non-choice assaysand olfactometerchoice tests with the transgenic tomato lines and MP1control, similar to the assays performed previously with S.habrochaites accessions. For each of the different constructs two selected transgenic lines will be utilized. The results of these experiments will verify if terpenes produced in engineered tomato lines relative to control affect the development, reproduction, and feeding behavior of aphids. We will also confirm if aphids are acquiring cell or phloem content by analyzing their ingestion of GFP with fluorescence microscopy.
Impacts
What was accomplished under these goals?
Aphids, a group of small pest insects, pose a serious problem in horticultural crop production including tomato, since damage is caused not only by their direct feeding on plants, but even more so by transmission of viruses for which aphids serve as vectors. This results in reduced crop yield and quality, and often plant death even at low levels of aphid infestation. Current control strategies utilizing synthetic insecticides are increasingly inefficient due to emerging resistances and avoidance behavior of aphids, and are also problematic due to adverse effects on the environment and concerns of consumers. Despite tomota being a major vegetable grown throughout the U.S. no tomato plants with resistance against aphids and viruses have been generated previously. Although aphids are a key pest in both field and greenhouse production, there is no cure for tomato plants infected with viruses transmitted by aphids. Therefore, the development of tomato lines resistant to aphids as a preventative control measure is urgently needed. Terpenes, a highly volatile class of compounds naturally synthesized by plants, are known to efficiently contribute to the defense of plants by repelling or intoxicating pest insects. While in wild plants these defensive traits are constantly under positive selection pressure to increase survival, it appears that they have been at least partially lost in crop plants since breeding has favored other agronomic traits. A number of wild tomato species, such as Solanum habrochaites, were previouslyfound to produce a diverse set of volatile terpenes which are not present in cultivated tomato (Solanum lycoperiscum). Thus wild tomato varieties can be a good source for defensive volatile terpene traits that act repellent and toxic against pests and subsequently could be introduced into cultivated tomato. During the current reporting period of this project we have therefore analysed a collection of wild tomato varieties (Solanum habrochaites) to findvarietiesproducing terpenes that show repellent activity against aphids as well as effects on the feeding behavior, reproduction and survival of aphids. Among the varieties that we have tested we sucessfully identifiedtwo groupsproducing two different setsof terpenes that have significant effects on the overall performance and behavior of aphids. Wefurther verified these results with respective pure terpene compounds. This knowledge gained through ourproject now allows to undertake effortsto integrate the formation of these effective terpenes into the background of cultivated tomato. Towards this goalwe have assembled vectors in the current reporting periodthat subsequently will allow the expression of those genes involved in the biosynthesis of the two identified groups ofeffective terpenes in three different tissues of cultivated tomato. In the current reporting period we have in addition utilized a cultivated tomato line that was previously obtained by a classical breeding approach (via crossing wild and cultivated tomato accessions) and producesone of the two groups of effective terpenes in glandular trichomes (hair like structures at the surface of leaves and stems). We have used this available tomato line to study the effect that formation of theeffective terpenes in the background of cultivated tomato has on the performance and behavior of aphids. The knowledge gained will guide us in the analysis of the new engineered tomato lines that we will obtain in the second partof this project. The following specific results were obtained for the three proposed objectives of this project in the current reporting period: Objective 1, Assess if wild tomato accessions with different terpene profils affect the host preference and feeding behavior of aphids:We have analyzed a collection 20 different tomato accessions (Solanum habrochaites&Solanum lycoperiscum) representing 6 different terpene chemotypes for their effect on performance and behavior of potato aphids. A first set of non-choice assays indicated that two groups of tomato accessions producing santalene/bergamotene and caryophyllene/humulene, respectively, had significant effects on the longevity and reproduction of aphids. When terpenes were extracted from tomato accessions and used in aphid feeding assays on artificial diet,extracts from thesantalene/bergamotene and caryophyllene/humulene producing accessions significantly affected aphid feeding behavior and survival. Moreover, when these tomato accessions and respective terpene extracts were used for olfactometer choice assayssantalene/bergamotene and caryophyllene/humulene had repellent activity towards aphids. To further verify that the observed effects on the performance and behavior of aphids is indeed due to respective terpenes additional control experiments were performed. Commercially available purecaryophyllene and humulene was used in feeding and choice assays and confimed their effects on aphids. Sincesantalene and bergamotene are not available as pure compounds we instead used an available tomatointrogression lineproducing these terpenesin non-choice, feeding and choice assays to confirm their effect on aphids. Objective 2,Determine how expression of respective terpene biosynthetic genes under the control of different tissue-specific promoters changes the terpene profile in resulting engineered cultivated tomato lines: In order to engineer the formation of these two groups of terpenes in three different tissues of cultivated tomato (glandular trichomes, epidermis, phloem companion cells) we have amplified the promoter regions of three genes (tomato SlTPS9, Arabidopsis AtCER5, Arabidopsis AtSUC2) by PCR and have cloned these into the multiple cloning site of the pMCS:GW vector. Constructs consisting of the open reading frames of the respective farnesyl diphosphate synthases and terpene synthases involved in the biosynthesis of these terpenes as well as enhanced green fluorescent protein linked in frame by viral 2A sequences were obtained by gene synthesis and recombined under the control of the above promoters into thepMCS:GW vector by Gateway cloning. Objective3,Determine how engineering of repellent and/or toxic terpenes in three tissues (trichomes, epidermis and phloem) of cultivated tomatochanges the overall performance and feeding behavior of aphids: While the new engineered tomato lines producing effective terpenes in three different tissues have not yet been available in the current reporting period, we have used an introgression line producing santalene/bergamotene in glanular trichomes for a first set of analyses. This introgression line was previously obtained by crossing a santalene/bergamotene producingS.habrochaites accession with a cultivated tomato accession and a series of subsequent backcrosses into the cultivated tomato background. We used thisintrogression line for the same type of non-choice, feeding and choice assays as performed with the respective S.habrochaites accessions. The results indicatedthat formation of santalene/bergamotenein the background of cultivated tomato indeed has an effect on the performance and behavior of aphids.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Fumin Wang, Yong-Lak Park and Michael Gutensohn*
"Glandular Trichome-Derived Terpenes of Wild Tomato Accessions Affect Aphid Performance and Feeding Behavior"
Oral Presentation, Session: Weed Control and Pest Management 2
2018 Annual Conference of the American Society of Horticultural Science (ASHS)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Fumin Wang*, Yong-Lak Park, and Michael Gutensohn
"The role of trichome-derived monoterpenes and sesquiterpenes in the interaction of cultivated tomato and potato aphid"
Poster Presentation, Session: Graduate Students
2019 Annual Meeting of the Midwestern Section of the American Society of Plant Biologists (ASPB)
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2019
Citation:
Michael Gutensohn*, Fumin Wang and Yong-Lak Park
"Glandular trichome-derived mono- and sesquiterpenes of cultivated and wild tomato accessions have different effects on aphid performance and feeding behavior"
Oral Presentation, Session: Terpenoids in chemical ecology
TERPNET 2019, 14th International Meeting on the Biosynthesis, Function and Synthetic Biology of Isoprenoids
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2019
Citation:
Michael Gutensohn*
"Why do plants smell? The function of plant volatile compounds in pollinator attraction, pest defense and fruit flavor"
Oral Presentation
2019 Annual Conference of the West Virginia Extension Master Gardener Association
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