Source: UNIVERSITY OF KENTUCKY submitted to NRP
ELUCIDATING AND MANIPULATING ALKALOID BIOSYNTHESIS PATHWAYS IN THE PLANT-SYMBIOTIC EPICHLOE AND NEOTYPHODIUM SPECIES OF FUNGI
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0231524
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Oct 1, 2012
Project End Date
Sep 30, 2017
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
UNIVERSITY OF KENTUCKY
500 S LIMESTONE 109 KINKEAD HALL
LEXINGTON,KY 40526-0001
Performing Department
Plant Pathology
Non Technical Summary
Tall fescue is the most widely planted pasture and forage grass in the U.S.A., and its exceptional stress tolerance is largely attributable to the fact that it typically harbors a seed-transmissible fungal symbiont (endophyte). In Kentucky, tall fescue is extremely important for pastured cattle and other livestock. In 2008, Kentucky beef production, valued at $574 million, and milk production, at $238 million, constituted 16.8% of Kentucky agricultural production in 2008. Total livestock production was 60.1% of Kentucky agricultural production in 2008. Kentucky sits at the heart of the tall fescue belt, which extends from eastern Kansas and Oklahoma to the Atlantic seaboard. The tall fescue endophyte increases resistance of this pasture plant to insects and nematodes, enhances its drought tolerance, and provides various growth improvements. Unfortunately, these benefits are often offset by livestock toxicity caused by certain substances, called ergot alkaloids, which are produced by the endophyte. On the other hand, two other kinds of substances made by endophytes - lolines and peramine - are major components of the endophyte arsenal against insects, helping to protect the tall fescue plants and improve their persistence and productivity in the field. In this study the genes for production of ergot alkaloids and lolines will be identified and characterized, and the endophyte will be modified to eliminate its production of ergot alkaloids. Outputs/Impacts: This work will generate endophytes that do not make ergot alkaloids, but produce lolines and peramine. These modified endophytes can be tested for anti-insect activity and other characteristics beneficial to pastures and livestock. Such endophytes, may ultimately be suited for use in tall fescue cultivars, and the results and knowledge gained will enhance capabilities to use endophytes in forage grasses in order to improve sustainability of pastured livestock production.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3144020100033%
3144020104034%
3144020110233%
Knowledge Area
314 - Toxic Chemicals, Poisonous Plants, Naturally Occurring Toxins, and Other Hazards Affecting Animals;

Subject Of Investigation
4020 - Fungi;

Field Of Science
1040 - Molecular biology; 1102 - Mycology; 1000 - Biochemistry and biophysics;
Goals / Objectives
Tall fescue is the most widely planted pasture and forage grass in the U.S.A., and its exceptional stress tolerance is largely attributable to the fact that it typically harbors a seed-transmissible fungal symbiont (endophyte), Neotyphodium coenophialum. In Kentucky, tall fescue is extremely important for pastured cattle and other livestock. In 2008, Kentucky beef production, valued at $574 million, and milk production, at $238 million, constituted 16.8% of Kentucky agricultural production in 2008. Total livestock production was 60.1% of Kentucky agricultural production in 2008. Kentucky sits at the heart of the tall fescue belt, which extends from eastern Kansas and Oklahoma to the Atlantic seaboard. Agronomic traits that are enhanced by the tall fescue endophyte include increased resistance to insects and nematodes, enhanced drought tolerance, and various growth improvements. Three classes of alkaloids - lolines, peramine and ergot alkaloids - are major components of the endophyte arsenal against insects. Unfortunately, agronomic benefits of the endophyte are often offset by toxicity of the ergot alkaloids to livestock. This study will focus on two alkaloid classes: ergot alkaloids, which are neurotoxic to mammalian grazers, and loline alkaloids, which are more specifically active against insects. Genes and enzymatic steps in the biosynthetic pathways will be identified and characterized, and N. coenophialum will be modified to eliminate its production of ergot alkaloids. Objectives are: 1. Identify genes and enzymes for key steps in loline alkaloid biosynthesis by epichloid fungi (endophytes) symbiotic with grasses. 2. Alter and eliminate ergot alkaloid biosynthesis by the tall fescue endophyte, Neotyphodium coenophialum. Outputs: Products: This work will generate endophytes with different alkaloid profiles that can be tested for anti-insect activity, effects on mammals, and other agronomic characteristics, and may ultimately be suited for use in tall fescue cultivars. Results and knowledge gained will enhance capabilities to deploy endophytes in forage grasses in order to improve sustainability of pastured livestock production.
Project Methods
Objective 1: Identify genes and enzymes for key steps in loline alkaloid biosynthesis (LOL). The overall strategy to test each step in the proposed biosynthetic pathway will be to mutate (knock out) genes for loline alkaloid biosynthesis, or to identify natural knock-out mutants, test for accumulation of precursors in the mutants, test for chemical complementation of the mutants, and test for the ability of yeast expressing LOL genes to synthesize intermediates. A comprehensive screening of natural endophytes in native grasses will be conducted to identify those with loline alkaloid gene mutations and differences in profiles of loline alkaloids and their precursors. When necessary, gene knockouts will be generated by standard marker-exchange mutagenesis, replacing each loline gene with an antibiotic resistance gene. The mutants will be introduced into their host grasses, which will then be analyzed for lolines and postulated biosynthetic precursors. Chemical complementation tests will be conducted by purchasing or synthesizing postulated enzyme products, feeding them to plants with the mutant endophytes and checking for loline alkaloid production. Sets of LOL genes will be expressed in yeast cells, which will be fed appropriate precursors or intermediates, then extracted and analyzed for the predicted products. Objective 2. Alter and eliminate ergot alkaloid biosynthesis by a tall fescue endophyte. The overall strategy is to eliminate from the genome of N. coenophialum isolate e19 (from tall fescue 'Kentucky 31') the genes for ergot alkaloid biosynthesis, and then to generate tall fescue lines with this modified endophyte. This fungus has two copies of the ergot alkaloid synthesis (EAS) genes, which genome-sequencing results have indicated are located in clusters near the ends of chromosomes. The strategy will be to transform the fungus by introducing a synthetic DNA plasmid designed to replace the chromosome end with an antibiotic resistance marker, together with a telomeric repeat structure that is required to stabilize the chromosome end. Transformants will be screened by standard molecular techniques for those that have lost the targeted EAS genes. The process will be conducted twice in series in order to eliminate both EAS clusters. After each EAS cluster is successfully removed, the modified fungus will be introduced into tall fescue plants to test for alkaloid production.

Progress 10/01/12 to 09/30/17

Outputs
Target Audience:Agricultural and biological researchers were targeted through peer-reviewed publications, and through scientific meetings. Livestock and forage producers were targeted through local meetings. Also consulted were representatives of AgResearch U.S.A. and Grasslanz Technology Ltd., which are companies that specialize in development and marketing of forage grass seeds. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One postdoctoral scholar and one graduate student were involved in the project. Both of whom presented their work at three international meetings. The graduate student was an author on six articles and chapters, two as first author, and the postdoctoral scholar was an author on 18 articles and chapters, eight as first author. The postdoctoral scholar also was a co-inventor for a patent application. An undergraduate research intern majoring in education, and four local high school students also participated in this research. How have the results been disseminated to communities of interest?Results have been disseminated primarily through research publications listed in this report, as well as presentations by the P.I. at two local meetings with stakeholders that included livestock producers and forage producers. The PI presented relevant seminars at universities and research institutes in Japan, New Zealand and Taiwan, Indiana University, the University of Massachusetts, and the North Carolina Biotech Consortium in North Carolina, as well as international meetings in Australia, China, France, Taiwan, Massachusetts, New Hampshire, Texas and Utah. The PI also presented at three NC1183 USDA multistate project meetings. The postdoctoral scholar gave oral presentations at international meetings in France, Spain, and California, and the graduate student gave an oral presentation at the meeting in Texas and poster presentations in China, California and Massachusetts. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Epichloë coenophiala (formerly Neotyphodium coenophialum), the fungal endophyte of the forage grass, tall fescue (Schedonorus arundinaceus = Lolium arundinaceum), provides the grass protection from stresses such as drought, from invertebrates such as insects and nematodes, and from small and large grazing animals. Protection from invertebrates and small mammals is agriculturally beneficial, but toxicity to livestock is a problem. The endophyte produces chemicals called alkaloids as part of the plant defense. Among these, all (and especially lolines) are active against invertebrates, but complex ergot alkaloids also affect mammals. A complex ergot alkaloid, ergovaline, is particularly toxic to livestock. It is crucial to understand the importance of different alkaloids, and to identify endophytes or genetically alter them for the most beneficial alkaloid profiles. Individual genes and groups of genes were eliminated, and in some cases, replaced in order to test their roles in production of lolines or ergot alkaloids. Several different endophytes were used for these experiments because their natural variation in alkaloid gene contents helped facilitate this strategy to clearly determine what genes were important for which steps in the alkaloid biosynthetic pathways. Metabolic and enzymatic studies further elucidated details of the pathway and mechanisms of one of the key enzymes involved in loline alkaloid biosynthesis. Finally, a strain lacking ergovaline was generated by elimination of one of two clusters of ergot alkaloid biosynthesis genes, and that strain and the novel method to produce it are the subject of a patent application. Objective 1. A total of 11 genes apparently encoding biosynthetic enzymes have been associated with loline alkaloid production by Epichloë species. The genes designated lolM and lolN were added to the previously characterized lolP gene as those that direct the chemical diversification of lolines. Also, the non-heme iron 2-oxoglutarate-dependent dioxygenase enzyme encoded by lolO was demonstrated to catalyze ether bridge formation. Upon analysis of natural lolO mutants, the intermediate, 1-acetamidopyrrolizidine (AcAP), was discovered as a novel metabolite that the LolO enzyme converts into the first loline alkaloid, N-acetylnorloline (NANL). AcAP was also found to be the pathway end product in some endophytes of native grasses such as Canadian wild rye (Elymus canadensis) and long-awned wood grass (Brachyelytrum erectum). Similarly, NANL is the end product in some other native grasses such as foul managrass (Glyceria striata) and some lines of Canadian wild rye. Thus, the pathway was elucidated whereby LolO catalyzes ether-bridge formation on AcAP to give the NANL, then LolN catalyzes conversion of NANL to norloline, which is then N-methylated by the enzyme encoded by lolM to give loline and N-methylloline (NML). A plant enzyme converts loline to N-acetylloline (NAL), and the fungal LolP cytochrome P450 monooxygenase converts NML to N-formylloline (NFL). NAL and NFL are the most abundant alkaloids in tall fescue with common strains of E. coenophiala, and are well characterized protectants against invertebrates. In a collaboration with structural biochemists at Pennsylvania State University and a chemist at the University of Kentucky, details of the multistep mechanism of ether bridge formation by the LolO enzyme were elucidated. Isotopologues of AcAP and L-aspartate were synthesized with positional deuterium labels and used, respectively, for in vitro and in vivo studies. The results indicated that LolO catalyzes both oxidation steps at unactivated carbons, C2 and C7 of AcAP, and that LolO is solely required for insertion of the ether bridge that is the most unusual chemical feature of loline alkaloids. Characterization of the LolO catalytic mechanism should open avenues to production of other molecules useful as pharmaceuticals or for other purposes. Objective 2. The genome of an E. coenophiala from tall fescue cultivar Kentucky 31 was sequenced. Two homeologous clusters of genes were identified and designated EAS1 and EAS2, of which EAS1 had 11 ergot alkaloid biosynthesis genes, and EAS2 had copies of all 11 genes but with one inactivated by natural mutation. Taking advantage of the location of EAS1 near a chromosome end, a novel method was developed to efficiently eliminate all of the EAS1 genes, and the resulting eas1 deletion mutant (exgenic strain) was introduced into tall fescue plants. Plants with the exgenic strain lacked the ergovaline toxin as expected, surprisingly also lacked most other intermediate ergot alkaloids, and accumulated only chanoclavine and a related spur product from this pathway. When the wild-type lpsB gene from the EAS cluster of a related species was introduced into the exgenic strain it restored ergovaline production. Genome sequence results indicated that no exogenous genes were present in the exgenic strain, and the only exogenous DNA was a 45-bp oligonucleotide introduced by the procedure adjacent to the telomere sequence repeats that protect the end of the chromosome from which the EAS1 gene cluster had been removed. Plants and seeds with the exgenic strain are to be increased in preparation for tests of their effects on animals.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Saikkonen, K., C.A. Young, M. Helander, and C.L. Schardl. 2016. Endophytic Epichlo� species and their grass hosts: from evolution to applications. Plant Molecular Biology 90:665-675. doi: 10.1007/s11103-015-0399-6.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Florea, S., D.G. Panaccione, and C.L. Schardl. 2017. Ergot alkaloids of the family Clavicipitaceae. Phytopathology 107:504-518. doi: 10.1094/phyto-12-16-0435-rvw.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Schmid, J., R. Day, N.X. Zhang, P.Y. Dupont, M.P. Cox, C.L. Schardl, N. Minards, M. Truglio, N. Moore, D.R. Harris, and Y.F. Zhou. 2017. Host tissue environment directs activities of an Epichlo� endophyte, while it induces systemic hormone and defense responses in its native perennial ryegrass host. Molecular Plant-Microbe Interactions 30:138-149. doi: 10.1094/mpmi-10-16-0215-r
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Pan, J., M. Bhardwaj, J.R. Faulkner, P. Nagabhyru, N.D. Charlton, R.M. Higashi, A.-F. Miller, C.A. Young, R.B. Grossman, and C.L. Schardl. 2014. Ether bridge formation in loline alkaloid biosynthesis. Phytochemistry 98:60-68. doi: 10.1016/j.phytochem.2013.11.015.
  • Type: Book Chapters Status: Published Year Published: 2014 Citation: Schardl, C.L., L. Chen, and C.A. Young. 2014. Fungal endophytes of grasses and morning glories, and their bioprotective alkaloids. Pages 125-145 IN: A. Osbourn, R.J. Goss and G.T. Carter, ed. Natural products: discourse, diversity, and design Vol.Published online: doi: 10.1002/9781118794623.ch7 John Wiley & Sons, Inc., Hoboken, New Jersey.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Young, C.A., C.L. Schardl, D.G. Panaccione, S. Florea, J.E. Takach, N.D. Charlton, N. Moore, J.S. Webb, and J. Jaromczyk. 2015. Genetics, genomics and evolution of ergot alkaloid diversity. Toxins (Basel) 7:1273-1302. doi: 10.3390/toxins7041273.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Schardl, C.L., C.A. Young, N. Moore, N. Krom, P.-Y. Dupont, J. Pan, S. Florea, J.S. Webb, J. Jaromczyk, J.W. Jaromczyk, M.P. Cox, and M.L. Farman. 2014. Genomes of plant-associated Clavicipitaceae. Advances in Botanical Research 70:291-327. doi: 10.1016/B978-0-12-397940-7.00010-0.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Chen, L., X.Z. Li, C.J. Li, G.A. Swoboda, C.A. Young, K. Sugawara, A. Leuchtmann, and C.L. Schardl. 2015. Two distinct Epichlo� species symbiotic with Achnatherum inebrians, drunken horse grass. Mycologia 107:863-873. doi: 10.3852/15-019.
  • Type: Book Chapters Status: Published Year Published: 2015 Citation: Florea, S., C.L. Schardl, and W. Hollin. 2015. Detection and isolation of Epichlo� species, fungal endophytes of grasses. Current Protocols in Microbiology 38:19A.11.11-19A.11.24. doi: 10.1002/9780471729259.mc19a01s38.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Florea, S., T.D. Phillips, D.G. Panaccione, M.L. Farman, and C.L. Schardl. 2016. Chromosome-end knockoff strategy to reshape alkaloid profiles of a fungal endophyte. G3: Genes - Genomes - Genetics 6:2601-2610. doi: 10.1534/g3.116.029686.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Mulinti, P., S. Florea, C.L. Schardl, and D.G. Panaccione. 2016. Modulation of ergot alkaloids in a grassendophyte symbiosis by alteration of mRNA concentrations of an ergot alkaloid synthesis gene. Journal of Agricultural and Food Chemistry 64:4982-4989. doi: 10.1021/acs.jafc.6b01604.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Schardl, C.L., S. Florea, J. Pan, P. Nagabhyru, S. Bec, and P.J. Calie. 2013. The epichloae: alkaloid diversity and roles in symbiosis with grasses. Current Opinion in Plant Biology 16:480-488. doi: 10.1016/j.pbi.2013.06.012.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Schardl, C.L., C.A. Young, U. Hesse, S.G. Amyotte, K. Andreeva, P.J. Calie, D.J. Fleetwood, D.C. Haws, N. Moore, B. Oeser, D.G. Panaccione, K.K. Schweri, C.R. Voisey, M.L. Farman, J.W. Jaromczyk, B.A. Roe, D.M. OSullivan, B. Scott, P. Tudzynski, Z. An, E.G. Arnaoudova, C.T. Bullock, N.D. Charlton, L. Chen, M. Cox, R.D. Dinkins, S. Florea, A.E. Glenn, A. Gordon, U. G�ldener, D.R. Harris, W. Hollin, J. Jaromczyk, R.D. Johnson, A.K. Khan, E. Leistner, A. Leuchtmann, C. Li, J. Liu, J. Liu, M. Liu, W. Mace, C. Machado, P. Nagabhyru, J. Pan, J. Schmid, K. Sugawara, U. Steiner, J. Takach, E. Tanaka, J.S. Webb, E.V. Wilson, J.L. Wiseman, R. Yoshida, and Z. Zeng. 2013. Plant-symbiotic fungi as chemical engineers: multi-genome analysis of the Clavicipitaceae reveals dynamics of alkaloid loci. PLoS Genetics 9:e1003323. doi: 10.1371/journal.pgen.1003323.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Schardl, C.L., C.A. Young, J. Pan, S. Florea, J.E. Takach, D.G. Panaccione, M.L. Farman, J.S. Webb, J. Jaromczyk, N.D. Charlton, P. Nagabhyru, L. Chen, C. Shi, and A. Leuchtmann. 2013. Currencies of mutualisms: sources of alkaloid genes in vertically transmitted epichloae. Toxins (Basel) 5:1064-1088. doi: 10.3390/toxins5061064.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Pan, J., M. Bhardwaj, P. Nagabhyru, R.B. Grossman, and C.L. Schardl. 2014. Enzymes from fungal and plant origin required for chemical diversification of insecticidal loline alkaloids in grass-Epichlo� symbiota. PLoS ONE 9:e115590. doi: 10.1371/journal.pone.0115590.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Leuchtmann, A., C.W. Bacon, C.L. Schardl, J.F. White, and M. Tadych. 2014. Nomenclatural realignment of Neotyphodium species with genus Epichlo�. Mycologia 106:202-215. doi: 10.3852/106.2.202.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Schardl, C.L. 2015. Introduction to the Toxins special issue on ergot alkaloids. Toxins (Basel) 7:4232-4237. doi: 10.3390/toxins7104232.
  • Type: Theses/Dissertations Status: Published Year Published: 2014 Citation: Pan, J. 2014. Ether bridge formation and chemical diversification in loline alkaloid biosynthesis. Ph.D. University of Kentucky, Lexington, Kentucky. 139 pp.


Progress 10/01/15 to 09/30/16

Outputs
Target Audience:We targeted fellow agricultural and biological researchers through peer-reviewed publications, and through scientific meetings. We also consulted with representatives of AgResearch U.S.A. and Grasslanz Technology Ltd., which are companies that specialize in development and marketing of forage grass seeds. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?An undergraduate research intern was involved in loline alkaloid analyses. A postdoctoral scholar mentored four local high school students in grass endophyte research techniques. How have the results been disseminated to communities of interest?Results were published in in G3: Genes - Genomes - Genetics and Journal of Agricultural and Food Chemistry, and a review was published in Plant Molecular Biology. Presentations were made at the European Conference on Fungal Genetics, the Mycological Society of America annual meeting, and the Phytochemical Society of North America annual meeting. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Results of the LolO study will be submitted for publication. Objective 2. Further complementation of the EAS1 exgenic strain with several ergot alkaloid biosynthesis genes will be conducted to determine the why the pathway terminates at an unexpected early intermediate, chanoclavine I and accumulates high levels of ergotryptamine. Sufficient seeds of tall fescue with exgenic endophytes and controls (wild-type endophyte and no endophyte) will be raised for future studies of effects on grazing livestock and agronomic traits of the plant.

Impacts
What was accomplished under these goals? Objective 1. An intriguing aspect of the loline alkaloid structure is the ether bridge, and previous investigation suggested that the ether bridge may be formed by direct oxygenation of two unactivated carbons, C2 and C7 of 1-acetamidopyrrolizidine, in a process involving the a non-heme iron 2-oxoglutarate-dependent dioxygenase designated LolO. In a collaboration with structural biochemists at Pennsylvania State University and a chemist at the University of Kentucky, the activity of LolO was tested. The enzyme was capable of oxidizing both C2 and C7, probably in that order. Isotopologues of 1-acetamidopyrrolizidine and L-aspartate were synthesized with positional deuterium labels and used, respectively, for in vitro LolC assays and for application to fungal fermentation cultures to determine which positions retained the deuterium labels and which lost them in biosynthesis of N-acetylnorloline. The results indicated that the hydrogen atom in the endo position at C2 is abstracted in the initial step of LolO activity. Objective 2. The EAS1 exgenic strain of E. coenophiala was complemented with a wild-type lpsB gene, which restored ergovaline production. Genome sequence results indicated that no exogenous genes were present in the exgenic strain, and the only exogenous DNA was a 45-bp oligonucleotide introduced by the procedure adjacent to the telomere repeat array at the end of the chromosome from which the EAS1 gene cluster had been removed to render the strain unable to produce ergovaline. Plants and seeds with the exgenic strain of Epichloe coenophiala (= Neotyphodium coenophialum) that lacks the toxin, ergovaline, are being increased in preparation for tests of their effects on animals.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Florea, S., T.D. Phillips, D.G. Panaccione, M.L. Farman, and C.L. Schardl. 2016. Chromosome-end knockoff strategy to reshape alkaloid profiles of a fungal endophyte. G3: Genes - Genomes - Genetics 6:2601-2610. doi: 10.1534/g3.116.029686.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Mulinti, P., S. Florea, C.L. Schardl, and D.G. Panaccione. 2016. Modulation of ergot alkaloids in a grassendophyte symbiosis by alteration of mRNA concentrations of an ergot alkaloid synthesis gene. Journal of Agricultural and Food Chemistry 64:4982-4989. doi: 10.1021/acs.jafc.6b01604.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Saikkonen, K., C.A. Young, M. Helander, and C.L. Schardl. 2016. Endophytic Epichlo� species and their grass hosts: from evolution to applications. Plant Molecular Biology 90:665-675. doi: 10.1007/s11103-015-0399-6.


Progress 10/01/14 to 09/30/15

Outputs
Target Audience:We targeted fellow agricultural and biological researchers through peer-reviewed publications in Mycologia and Toxins, and through scientific meetings such as the International Symposium on Fungal Endophytes in Grasses, the Mycological Society of America annual meeting, and the Fungal Genetics Conference. We also consulted with representatives of AgResearch U.S.A. and Grasslanz Technology Ltd., which are companies that specialize in development and marketing of forage grass seeds. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?The PI was the editor of aspecial issue of the journal Toxins (Basel) about ergot-alkaloid biosynthesis, effects on grazing livestock, and beneficial roles (such as in pharmaceutical production). In addition to an introduction, the PI coauthored a review, "Genetics, genomics and evolution of ergot alkaloid diversity." Another paper taxonomically described the endophyte, Epichloe inebrians, which is responsible for ergot alkaloid poisoning of animals that graze drunken horse grass, which is associated with grassland degradation in western China. Finally, a contribution to Current Protocols in Microbiology details the state-of-the-art in detection, isolation and culture of Epichloe species, endophytes of cool-season grasses. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: The LolO protein will be produced in an Escherichia coli expression system, and tested on 1-acetamidopyrrolizidine to determine its activity and, therefore, its role in formation of the ether bridge of loline alkaloids. Objective 2. Two more years are required to increase tall fescue plants and seeds with the exgenic strain of Epichloe coenophiala that lacks ergovaline, in preparation for tests in animal systems. Genome sequencing will be conducted on the exgenic strain to determine if any exongenous DNA remains in the genome. Complementation with the wild-type lpsB gene and other ergot alkaloid biosynthesis genes will be conducted to confirm the reason that the exgenic strain lacks ergovaline, and to determine the why the pathway terminates at an unexpected early intermediate, chanoclavine I.

Impacts
What was accomplished under these goals? Objective 1. A very unusual aspect of the loline alkaloid structure is the ether bridge, in which an oxygen atom links two carbon atoms, both in unactivated positions. Previous identification of 1-acetamidopyrrolizidine as the immediate precursor to loline alkaloids suggested that the ether bridge may be formed by direct oxygenation of the two unactivated carbons, C2 and C7, of this precursor. Molecular genetic analysis indicates that the predicted non-heme iron 2-oxoglutarate-dependent dioxygenase, LolO, is involved in that process, and a collaboration was initiated with structural biochemists at Pennsylvania State University and a chemist at the University of Kentucky to test the activity of LolO. Objective 2. Plants and seeds with the exgenic strain of Epichloe coenophiala (= Neotyphodium coenophialum) that lacks the toxin, ergovaline, are being increased in preparation for tests of their effects on animals.

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Chen, L., X.Z. Li, C.J. Li, G.A. Swoboda, C.A. Young, K. Sugawara, A. Leuchtmann, and C.L. Schardl. 2015. Two distinct Epichloe species symbiotic with Achnatherum inebrians, drunken horse grass. Mycologia 107:863-873. doi: 10.3852/15-019.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Young, C.A., C.L. Schardl, D.G. Panaccione, S. Florea, J.E. Takach, N.D. Charlton, N. Moore, J.S. Webb, and J. Jaromczyk. 2015. Genetics, genomics and evolution of ergot alkaloid diversity. Toxins (Basel) 7:1273-1302. doi: 10.3390/toxins7041273.
  • Type: Book Chapters Status: Published Year Published: 2015 Citation: Florea, S., C.L. Schardl, and W. Hollin. 2015. Detection and isolation of Epichloe species, fungal endophytes of grasses. Current Protocols in Microbiology 38:19A.11.11-19A.11.24. doi: 10.1002/9780471729259.mc19a01s38.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Schardl, C.L. 2015. Introduction to the Toxins special issue on ergot alkaloids. Toxins (Basel) 7:4232-4237. doi: 10.3390/toxins7104232.


Progress 10/01/13 to 09/30/14

Outputs
Target Audience:We targeted fellow agricultural and biological researchers through peer-reviewed publications in Phytochemistry and PLoS One, and through scientific meetings such as the national meeting of the American Phytopathological Society, and the Gordon Research Conference on Fungal Biology. We also consulted with representatives of AgResearch U.S.A. and Grasslanz Technology Ltd., which are companies that specialize in development and marketing of forage grass seeds. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Impact: Tall fescue, the most widely planted pasture and forage grass in the U.S.A., is particularly dominant in mid-latitudes of the eastern half of the contiguous U.S. However, in this "fescue belt" the grass is subjected to drought and other stresses, and the demonstrated ability of tall fescue to tolerate such stresses is significantly enhanced by a symbiotic fungus (endophyte) that grows throughout its above-ground tissues. Common strains of the endophyte, Epichloe coenophiala (formerly named Neotyphodium coenophialum) produce several alkaloids, which are molecules that typically affect invertebrate or vertebrate animals. Lolines and peramine, for example, deter or kill insects that feed on the plant, whereas ergovaline (an ergot alkaloid) affects vertebrates. The ergovaline effects on livestock range from reproductive problems to dry gangrene of hoves, but most often manifest as loss of appetite and poor weight gain. Losses to the U.S. beef industry due to toxicity of the ergovaline-producing endophyte of tall fescue have been estimated at US$ 600 million. Past research on the endophyte has led to breakthroughs in techniques to genetically manipulate these fungi and to replace existing strains with novel or genetically altered strains. Furthermore, clusters of genes for ergot alkaloid and loline alkaloid biosynthesis have been identified, opening the door to detailed studies of these genes and alkaloids, which in turn can facilitate tailoring the endophyte for use in forage tall fescue. Objective 1. Identify genes and enzymes for key steps in loline alkaloid biosynthesis by epichloid fungi (endophytes) symbiotic with grasses. Three genes were characterized and roles confirmed for steps leading to a variety of loline alkaloids. The lolO gene encodes an oxygenase that converts 1-acetamidopyrrolizidine (AcAP) into the first loline alkaloid, N-acetylnorloline (NANL). In the process of elucidating the role of lolO, AcAP was discovered and found to be the pathway end product in some endophytes of native grasses such as Canadian wild rye (Elymus canadensis) and long-awned wood grass (Brachyelytrum erectum). Similarly, NANL is the end product in some other native grasses such as foul managrass (Glyceria striata) and some lines of Canadian wild rye. The enzyme encoded by lolN catalyzes conversion of NANL to norloline, which is then N-methylated by the enzyme encoded by lolM to give loline and N-methylloline (NML). A plant enzyme converts loline to N-acetylloline, and the fungal LolP monooxygenase converts NML to N-formylloline (NFL). NAL and NFL are the most abundant alkaloids in the tall fescue with common strains of E. coenophiala and are well characterized protectants against invertebrates. Objective 2. Alter and eliminate ergot alkaloid biosynthesis by the tall fescue endophyte, Neotyphodium coenophialum. In the continuing effort to eliminate ergovaline production by the common strain of Epichloe coenophiala, this strain was subjected to transient introduction of a DNA molecule that was designed to specifically eliminate one of two sets (loci) of ergot alkaloid genes. Cultures of E. coenophiala having been treated in this manner are predicted to produce lysergic acid, but none of the toxic lysergic acid derivatives such as ergovaline. These cultures have been reintroduced into tall fescue plants to permit their chemical analysis. If they lack ergovaline as predicted, but retain characteristics such as peramine and loline alkaloid production and positive effects on drought tolerance, these cultures will be useful as bioprotectants that can be introduced into tall fescue without risk to livestock health.

Publications

  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Pan J, Bhardwaj M, Faulkner JR, Nagabhyru P, Charlton ND, Higashi RM, Miller A-F, Young CA, Grossman RB, Schardl CL (2014) Ether bridge formation in loline alkaloid biosynthesis. Phytochemistry 98: 60-68. doi 10.1016/j.phytochem.2013.11.015
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Pan J, Bhardwaj M, Nagabhyru P, Grossman RB, Schardl CL (2014) Enzymes from fungal and plant origin required for chemical diversification of insecticidal loline alkaloids in grass-Epichlo� symbiota. PLoS ONE 9: e115590. doi 10.1371/journal.pone.0115590
  • Type: Book Chapters Status: Published Year Published: 2014 Citation: Schardl CL, Young CA, Moore N, Krom N, Dupont P-Y, Pan J, Florea S, Webb JS, Jaromczyk J, Jaromczyk JW, Cox MP, Farman ML (2014) Genomes of plant-associated Clavicipitaceae. Advances in Botanical Research 70: 291-327. doi 10.1016/B978-0-12-397940-7.00010-0


Progress 10/01/12 to 09/30/13

Outputs
Target Audience: The target audiences reached in this reporting period were research scientists, extension specialists, and students. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? One postdoctoral scholar and one graduate student were involved in the project. Both presented their work at the 2013 joint meeting of the American Phytopathological Society and the Mycological Society of America. Based on her work on this project, the graduate student is first author on a paper in press in the journal Phytochemistry. An undergraduate research intern majoring in education also participated in this research. How have the results been disseminated to communities of interest? Results have been disseminated as part of two published research papers and a published review, all in peer reviewed journals. In addition, the graduate student and project director each presented oral talks at the 2013 joint meeting of the American Phytopathological Society and the Mycological Society of America, the postdoctoral scholar presented a poster at the same meeting, and the project director presented an invited talk at the Gordon Research Conference on Mycotoxins and Phycotoxins, a seminar at the University of Massachussets, Amherst, and a seminar at the University of Kentucky. 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 fungal endophyte of the forage grass, tall fescue, provides the grass protection from stresses such as drought, from invertebrates such as insects and nematodes, and from small and large grazing animals. Protection from invertebrates and small mammals is agriculturally beneficial, but toxicity to livestock is a problem. The endophyte produces chemicals called alkaloids as part of the plant defense. Among these, all (and especially lolines) are active against invertebrates, but complex ergot alkaloids also affect mammals. A complex ergot alkaloid, ergovaline, is particularly toxic to livestock. It is crucial to understand the importance of different alkaloids, and to identify endophytes or genetically alter them for the most beneficial alkaloid profiles. In this reporting period, individual genes and groups of genes were eliminated, and in some cases, replaced in order to test their roles in production of lolines or ergot alkaloids. Several different endophytes were used for these experiments because their natural variation in alkaloid gene contents helped facilitate this strategy to clearly determine what genes were important for which steps in the alkaloid biosynthetic pathways. Objective 1: Out of 11 genes associated with loline alkaloid production, results allowed probable functions to be assigned to four genes, and a previously proposed function was disproven for a fifth gene. Objective 2: The genome of the tall fescue endophyte, Neotyphodium coenophialum, was sequenced. Two homologous clusters of genes were identified (designated EAS1 and EAS2), of which EAS1 had 11 ergot alkaloid biosynthesis genes, and EAS2 had copies of all 11 genes but with one inactivated by natural mutations. All of the EAS1 genes were eliminated, and the resulting eas1 mutant strain was introduced into tall fescue plants. Plants with this mutant lacked ergovaline as expected, but surprisingly accumulated only chanoclavine and its precursor, while lacking other simple ergot alkaloids that were expected. This suggests that additional EAS2 genes were defective or not expressed. Material generated in Objective 1 will be useful for detailed studies of the importance of each form of loline alkaloids in protection and productivity of the plant. Different forms of lolines may protect the grass plant from different invertebrate pests. Similarly, material generated in Objective 2 will allow studies of the relative importance of simple ergot alkaloids compared to ergovaline in effects on livestock. Taken together, the results will help direct the search for optimally beneficial alkaloid profiles of forage grass endophytes.

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2013 Citation: Pan, J., M. Bhardwaj, J.R. Faulkner, P. Nagabhyru, N.D. Charlton, R.M. Higashi, A.-F. Miller, C.A. Young, R.B. Grossman, and C.L. Schardl. 2013. Ether bridge formation in loline alkaloid biosynthesis. Phytochemistry (in press).
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Schardl, C.L., S. Florea, J. Pan, P. Nagabhyru, S. Bec, and P.J. Calie. 2013. The epichloae: alkaloid diversity and roles in symbiosis with grasses. Current Opinion in Plant Biology 16:480-488. doi: 10.1016/j.pbi.2013.06.012.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Schardl, C.L., C.A. Young, U. Hesse, S.G. Amyotte, K. Andreeva, P.J. Calie, D.J. Fleetwood, D.C. Haws, N. Moore, B. Oeser, D.G. Panaccione, K.K. Schweri, C.R. Voisey, M.L. Farman, J.W. Jaromczyk, B.A. Roe, D.M. O'Sullivan, B. Scott, P. Tudzynski, Z. An, E.G. Arnaoudova, C.T. Bullock, N.D. Charlton, L. Chen, M. Cox, R.D. Dinkins, S. Florea, A.E. Glenn, A. Gordon, U. Gu?ldener, D.R. Harris, W. Hollin, J. Jaromczyk, R.D. Johnson, A.K. Khan, E. Leistner, A. Leuchtmann, C. Li, J. Liu, J. Liu, M. Liu, W. Mace, C. Machado, P. Nagabhyru, J. Pan, J. Schmid, K. Sugawara, U. Steiner, J. Takach, E. Tanaka, J.S. Webb, E.V. Wilson, J.L. Wiseman, R. Yoshida, and Z. Zeng. 2013. Plant-symbiotic fungi as chemical engineers: multi-genome analysis of the Clavicipitaceae reveals dynamics of alkaloid loci. PLoS Genetics 9:e1003323. doi: 10.1371/journal.pgen.1003323.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Schardl, C.L., C.A. Young, J. Pan, S. Florea, J.E. Takach, D.G. Panaccione, M.L. Farman, J.S. Webb, J. Jaromczyk, N.D. Charlton, P. Nagabhyru, L. Chen, C. Shi, and A. Leuchtmann. 2013. Currencies of mutualisms: sources of alkaloid genes in vertically transmitted epichloae. Toxins 5:1064-1088. doi: 10.3390/toxins5061064.
  • Type: Journal Articles Status: Accepted Year Published: 2013 Citation: Leuchtmann, A., C.W. Bacon, C.L. Schardl, J.F. White, and M. Tadych. 2014. Nomenclatural realignment of Neotyphodium species with genus Epichlo�. Mycologia (in press).