Progress 10/01/06 to 09/30/12
Outputs
OUTPUTS: Objective 1: Genomes were sequenced from the following species of mutualistic symbiotic (endophyte) fungi (host grasses listed in parentheses): Epichloe amarillans (Agrostis hiemalis), E. brachyelytri (Brachyelytrum erectum), E. bromicola (Bromus erectus), E. elymi (Elymus virginicus; Virginia wild rye), E. festucae (Festuca and Lolium species; fescues and ryegrasses), E. glyceriae (Glyceria striata; foul managrass), E. poae (Poa nemoralis), E. typhina (Lolium perenne; perennial ryegrass), Neotyphodium aotearoae (Echinopogon ovatus), N. coenophialum (Lolium arundinaceum; tall fescue), and N. gansuense (Achnatherum inebrians). Several of these endophytes are documented to produce bioprotective alkaloids, and N. coenophialum in tall fescue pastures causes episodic livestock toxicity. Also sequenced was the genome of Periglandula ipomoeae from Ipomoea asarifolia (which inhabits a morning glory species and causes livestock toxicosis), and three ergot fungi (Claviceps species) that pose mycotoxin poisoning hazards in grains and forage. Objective 2: To analyze gene structures and expression, RNA was isolated from meadow fescue plants symbiotic with E. festucae, and random segments, each 82 bases long, were sequenced. In total, 63,677,626 segments were sequenced, of which 12,149,888 (nearly one billion bases) were from the endophytic fungus. Objective 3: In planta gene expression by E. festucae was compared for its benign state, in which it resembles mutualistic endophytes such as N. coenophialum, and its pathogenic state, in which it suppresses grass seed production. Also, gene expression was compared between wild-type and a mutant E. festucae, whereby the mutant was more pathogenic than mutualistic on the host grass. PARTICIPANTS: Christopher L. Schardl, PI: Garnered additional funds, and supervised the Research Analyst, Senior Research Analyst, and other researchers in the laboratory. Alfred D. Byrd, Research Analyst: Was primarily responsible for regulatory compliance and safety training, as well as equipment maintenance. Walter Hollin, Senior Research Analyst, was responsible for training laboratory researchers, laboratory management, equipment maintenance, maintaining laboratory stocks of reagents, culturing and preserving fungal endophytes, inoculating plants, and maintaining databases. Padmaja Nagabhyru (Postdoctoral Scholar) conducted drought tolerance tests on tall fescue plants, analyzed free sugars and amino acids in treated and untreated plants, and conducted RNA-seq analysis of plants with and without endophyte symbiosis. Simona Florea (Postdoctoral Scholar) helped sequence genomes and conducted genetic modifications of the tall fescue endophyte to alter its ergot alkaloid profile. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The epichloae (Epichloe and Neotyphodium species of fungi) inhabit cool-season grasses and can produce any of four different classes of alkaloids, two of which (ergot alkaloids and indole-diterpenes) can be deleterious to livestock, and two of which (lolines and peramine) are more specifically active against insects. The findings demonstrated that epichloae species and strains in a wide range of host grasses are almost always capable of producing alkaloids of one or more of these classes. The findings also led to new understandings of the genetic basis for diversity of alkaloids within each class, and among strains of fungal endophytes. This, in turn, led to the development of rapid molecular-genetic screens to determine which alkaloids may be produced by each endophyte, and the likely profiles of chemical forms in each alkaloid class. Furthermore, this knowledge allowed targeted elimination of key genes to specifically alter alkaloid profiles, resulting in a tall fescue endophyte strain that lacks the ergopeptine forms of ergot alkaloids thought to be particularly toxic to livestock. Similar approaches were used to generate mutant strains that can be used in future to test the roles of various ergot alkaloids and loline alkaloids in protection against insects and nematodes that parasitize forage grasses. Finally, gene expression and follow-on genetic studies have suggested specific endophyte genes that may determine the abilities of these fungi to form mutualistic symbioses with fescues and ryegrasses, which are important forage and turf grasses throughout the temperate world.
Publications
- Florea, S., Machado, C., Andreeva, K., Schardl, C.L., 2012. The Cre/lox system: a practical tool to efficiently eliminate selectable markers in fungal endophytes. Methods in Molecular Biology 824, 371-379. doi: 10.1007/978-1-61779-433-9_19
- Florea, S., Machado, C., Andreeva, K., Schardl, C.L., 2012. The Cre/lox system: a practical tool to efficiently eliminate selectable markers in fungal endophytes. In: Lorence, A. (Ed.), Recombinant Gene Expression: Reviews and Protocols, Third Edition. Humana Press; Springer Science+Busines Media, LLC, New York Dordrecht Heidelberg London, pp. 371-379.
- Iannone, L.J., Novas, M.V., Young, C.A., De Battista, J.P., Schardl, C.L., 2012. Endophytes of native grasses from South America: Biodiversity and ecology. Fungal Ecology 5, 357-363. doi: 10.1016/j.funeco.2011.05.007
- Iannone, L.J., Pinget, A.D., Nagabhyru, P., Schardl, C.L., De Battista, J.P., 2012. Beneficial effects of Neotyphodium tembladerae and Neotyphodium pampeanum on a wild forage grass. Grass and Forage Science 67, 382-390. doi: 10.1111/j.1365-2494.2012.00855.x
- Schardl, C.L., Young, C.A., Faulkner, J.R., Florea, S., Pan, J., 2012. Chemotypic diversity of epichloae, fungal symbionts of grasses. Fungal Ecology 5, 331-344. doi: 10.1016/j.funeco.2011.04.005
- Iannone, L.J., Mc Cargo, P.D., Giussani, L.M., Schardl, C.L., 2013. Geographic distribution patterns of vertically transmitted endophytes in two native grasses in Argentina. Symbiosis (in press).doi: 10.1007/s13199-012-0214-y
- Schardl, C.L., Young, C.A., Hesse, U., Amyotte, S.G., Andreeva, K., Calie, P.J., Fleetwood, D.J., Haws, D.C., Moore, N., Oeser, B., Panaccione, D.G., Schweri, K.K., Voisey, C.R., Farman, M.L., Jaromczyk, J.W., Roe, B.A., O'Sullivan, D.M., Scott, B., Tudzynski, P., An, Z., Arnaoudova, E.G., Bullock, C.T., Charlton, N.D., Chen, L., Cox, M., Dinkins, R.D., Florea, S., Glenn, A.E., Gordon, A., Gueldener, U., Harris, D.R., Hollin, W., Jaromczyk, J., Johnson, R.D., Khan, A.K., Leistner, E., Leuchtmann, A., Li, C., Liu, J., Liu, J., Liu, M., Mace, W., Machado, C., Nagabhyru, P., Pan, J., Schmid, J., Sugawara, K., Steiner, U., Takach, J., Tanaka, E., Webb, J.S., Wilson, E.V., Wiseman, J.L., Yoshida, R., Zeng, Z., 2013. Plant-symbiotic fungi as chemical engineers: multi-genome analysis of the Clavicipitaceae reveals dynamics of alkaloid loci. PLoS Genetics (accepted).
|
Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Genomes of several more Epichloe species and a Neotyphodium species (endophytes) symbiotic with grasses were sequenced, as was the genome of a related symbiont from the dicotyledonous plant Ipomoea asarifolia (from the morning-glory family, Convolvulaceae). This brought the total number of sequenced genomes from plant-associated clavicipitaceous fungi to 14: E. amarillans, E. brachyelytri, E. festucae (2 isolates), E. glyceriae, E. typhina (2 isolates), N. coenophialum, N. gansuense, Aciculosporium take, Claviceps fusiformis, C. paspali, C. purpurea, and P. ipomoeae. All of these genomes, except that of N. coenophialum, were annotated to predict genes, which were compared between species and isolates. Then, the alkaloid biosynthesis genes were identified and compared. Objective 2: This objective was completed previously. Objective 3: Deep sequencing of expressed mRNA was conducted on plants with and without endophyte, including ovaries of Italian ryegrass (Lolium multiflorum), and vegetative tillers of tall fescue (Lolium arundinaceum = Festuca arundinacea). The mRNA samples from tall fescue included both unstressed and drought-stressed plants, to monitor both endophyte and plant gene expression changes in response to stress. PARTICIPANTS: Christopher L. Schardl, PI: Garnered additional funds, and supervised the Research Analyst, Senior Research Analyst, and other researchers in the laboratory. Alfred D. Byrd, Research Analyst: Was primarily responsible for regulatory compliance and safety training, as well as equipment maintenance. Walter Hollin, Senior Research Analyst, was responsible for training laboratory researchers, laboratory management, equipment maintenance, maintaining laboratory stocks of reagents, culturing and preserving fungal endophytes, inoculating plants, and maintaining databases. Padmaja Nagabhyru conducted drought tolerance tests on tall fescue plants, analyzed free sugars and amino acids in treated and untreated plants, and conducted RNA-seq analysis of plants with and without endophyte symbiosis. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Objective 1: There were three gene clusters for the three alkaloid classes, ergolines, indole-diterpenes and lolines. In addition, the peramine biosynthesis gene was identified in several Epichloe and Neotyphodium species. A striking difference between the alkaloid gene clusters in Epichloe and Neotyphodium species and those in other Clavicipitaceae was that the former had very large blocks of repetitive DNA throughout each cluster. These repeats appeared to be involved in destabilizing genes in the clusters, leading to considerable variation in alkaloid profiles of different endophytes. Objective 2: Together with data from objective 3, the expressed sequence tags were mapped onto the E. festucae genome as a tool for annotating the endophyte genomes by refining the sets of gene models. Objective 3: Plant genes that responded to drought stress, and were upregulated to a greater extent in endophyte-symbiotic plants than in asymbiotic plants, included those for biosynthesis of the plant stress hormone, jasmonic acid, as well as genes for detoxification of reactive oxygen species.
Publications
- Fleetwood D.J., Khan A.K., Johnson R.D., Young C.A., Mittal S., Wrenn R.E., Hesse U., Foster S.J., Schardl C.L., Scott B. (2011) Abundant degenerate miniature inverted-repeat transposable elements in genomes of epichloid fungal endophytes of grasses. Genome Biology and Evolution 3:1253-1264. DOI: 10.1093/gbe/evr098.
- Florea S., Machado C., Andreeva K., Schardl C.L. (2011) Elimination of marker genes from transformed filamentous fungi by unselected transient transfection with a Cre-expressing plasmid, in: A. M. Pirttila and S. Sorvari (Eds.), Prospects and applications for plant-associated microbes. A laboratory manual, part B: fungi, Karhukopio OY, Turku, Finland. pp. 146-149.
- Steiner U., Leibner S., Schardl C.L., Leuchtmann A., Leistner E. (2011) Periglandula, a new fungal genus within the Clavicipitaceae and its association with Convolvulaceae. Mycologia 103:1133-1145. DOI: 10.3852/11-031.
|
Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Draft genome sequences were completed for eight genomes of fungi in the family Clavicipitaceae, including the tall fescue symbiont (endophyte), Neotyphodium coenophialum, two isolates of the closely related fungus, Epichloe festucae, three ergot fungi, Claviceps purpurea, Claviceps fusiformis, and Claviceps paspali, and two other plant-associated Clavicipitaceae, Aciculosporium take and a newly discovered symbiont of Ipomoea asarifolia. Deep sequencing was conducted on mRNA (RNA-seq) from unstressed tall fescue plants with and without the symbiont, N. coenophialum, and material was collected for metabolomic and RNA-seq analysis of plants with and without N. coenophialum that were subjected to time periods of 1-7 days without watering. PARTICIPANTS: PARTICIPANTS: Christopher L. Schardl, PI: Garnered additional funds, and supervised the Research Analyst, Senior Research Analyst, and other researchers in the laboratory. Alfred D. Byrd, Research Analyst: Was primarily responsible for regulatory compliance and safety training, as well as equipment maintenance. Walter Hollin, Senior Research Analyst, was responsible for training laboratory researchers, laboratory management, equipment maintenance, maintaining laboratory stocks of reagents, culturing and preserving fungal endophytes, inoculating plants, and maintaining databases. Padmaja Nagabhyru conducted drought tolerance tests on tall fescue plants, analyzed free sugars and amino acids in treated and untreated plants, and conducted RNA-seq analysis of plants with and without endophyte symbiosis. COLLABORATORS: Jerzy W Jaromczyk, University of Kentucky. Randy Dinkins, U.S. Department of Agriculture Agricultural Research. Service, Lexington, Kentucky. Anar K. Khan, AgResearch, New Zealand. Daniel G. Panaccione, West Virginia University. Jan Schmid, Massey University, New Zealand. Barry Scott, Massey University, New Zealand. Paul Tudzynski, University of Muenster, Germany. Carolyn Young, Samuel R. Noble Foundation, Oklahoma. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The genome sizes of the two Epichloe festucae genomes were each 35 Mb. One of the E. festucae genomes gave 8142 gene predictions, and the other gave 8832 gene predictions. The Neotyphodium coenophialum genome was 95 Mb, and preliminary analysis indicates that the fungus is triploid, in keeping with its previous characterization as an interspecific hybrid with three ancestors. Genome sizes of the other Clavicipitaceae ranged from 27 Mb to 59 Mb, but the numbers of genes in each were comparable to E. festucae. Comparisons of these genomes indicated that the endophytes have especially unstable regions containing genes for biosynthesis of alkaloids that may cause livestock toxicosis or protect the host plants from insects. This result helps account for the chemotypic diversity of natural endophyte isolates. The RNA-seq analysis of tall fescue indicated that the symbiont, N. coenophialum, induced higher expression of plant genes for ethylene biosynthesis and glutathione-S-transferase. These activities may precondition the plant to resist effects of water deficit, in part accounting for the beneficial effect of the symbiont on drought tolerance.
Publications
- Arnaoudova E, Haws DC, Huggins P, Jaromczyk JW, Moore N, Schardl CL, Yoshida R (2010) Statistical phylogenetic tree analysis using differences of means. Frontiers in Neuroscience 4: 47 (12 pp)
- Eaton CJ, Cox MP, Ambrose B, Becker M, Hesse U, Schardl CL, Scott B (2010) Disruption of signaling in a fungal-grass symbiosis leads to pathogenesis. Plant Physiology 153: 1780-1794
- Schardl CL (2010) The epichloae, symbionts of the grass subfamily Pooideae Annals of the Missouri Botanical Garden 97: 646-665
- Schardl CL, Chen F (2010) Plant defences against herbivore attack. In Encyclopedia of Life Sciences. John Wiley & Sons, Ltd.
- Zhang D-X, Nagabhyru P, Blankenship JD, Schardl CL (2010) Are loline alkaloid levels regulated in grass endophytes by gene expression or substrate availability? Plant Signaling & Behavior 5: 11
|
Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: Activities: A new DNA-sequencing technology was acquired by the University of Kentucky Advanced Genetic Technologies Center, which provides pyrosequencing with much higher fidelity than previously achieved. This platform was employed for sequencing genomes of Epichloe festucae and Claviceps paspali. The Epichloe festucae genome was sequenced to approximately 20-fold redundancy, the data were incorporated with previously obtained data to generate a new assembly. Gene models were generated based on gene prediction as well as previous sequencing of expressed sequence tags (ESTs). The assembly, gene models, and other annotations are published on a web site, www.endophyte.uky.edu/. Coding sequences for six E. festucae genes of interest were fused with coding sequences for green fluorescent protein, then introduced into E. festucae, which in turn was reintroduced into symbiosis with meadow fescue plants. These plants will be analyzed to identify the tissues where these proteins are expressed, and the cellular compartments to which they target. This project provided research experiences for six undergraduate interns and two graduate students. The assembled genome sequence was submitted to National Center for Biotechnology Information, as ADFL01000000, Epichloe festucae E2368, Whole Genome Shotgun Sequencing Project ID 42133. Also submitted were EST sequences from Epichloe festucae (34,701 sequences), Festuca pratensis (74,111 sequences), and Festuca arundinacea (19,377 sequences). PARTICIPANTS: INDIVIDUALS WHO WORKED ON THE PROJECT: Christopher L. Schardl, PI: Garnered additional funds, and supervised the Research Analyst, Senior Research Analyst, and other researchers in the laboratory. Alfred D. Byrd, Research Analyst: Was primarily responsible for regulatory compliance and safety training, as well as equipment maintenance. Walter Hollin, Senior Research Analyst, was responsible for training laboratory researchers, laboratory management, equipment maintenance, maintaining laboratory stocks of reagents, culturing and preserving fungal endophytes, inoculating plants, and maintaining databases. COLLABORATORS: Wayne E. Beach, University of Kentucky Mark L. Farman, University of Kentucky. Jerzy W Jaromczyk, University of Kentucky. Zhiqiang An, Merck and Co., Inc., Pennsylvania. Faith Belanger, Rutgers University, New Jersey. Randy Dinkins, U.S. Department of Agriculture Agricultural Research. Service, Lexington, Kentucky. Linda Johnson, AgResearch, New Zealand. Richard Johnson, AgResearch, New Zealand. Anar K. Khan, AgResearch, New Zealand. Daniel G. Panaccione, West Virginia University. Bruce A. Roe, University of Oklahoma. Jan Schmid, Massey University, New Zealand. Barry Scott, Massey University, New Zealand. Paul Tudzynski, University of Muenster, Germany. Christine Voisey, AgResearch, New Zealand. Carolyn Young, Samuel R. Noble Foundation, Oklahoma. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The Epichloe festucae sequence assembled into total of 7611 contiguous sequences (contigs) totaling 30,679,931 bp, with N50 contig size of 65,842 bp (that is, 50 percent of the total sequence was in contigs greater than or equal to this length). Of this, 87.3 percent (26,783,389 bp) was arranged in 842 scaffolds, with N50 scaffold size of 126,277 bp. The scaffolds included all contigs greater than 2000 bp in length. Gene prediction and EST data identified 10,216 putative genes, of which at least 1998 had splice variants based on the EST data. Expression of several E. festucae genes in various plant tissues and stages of fungal development was compared by quantitative polymerase chain reaction. Of these, six were expressed at more than 10-fold higher levels in asymptomatic inflorescences compared to tillers that were choked by the fungal fruiting structures (stromata). To test if any of these genes may be specifically involved in seed-transmission of the endophyte, expression levels in inflorescences were compared with those in asymptomatic vegetative tissues, and two were found to be 10-to-100-fold (or greater) more highly expressed in the inflorescences. One of these is predicted to encode a small secreted protein, suggesting that it may mediate interaction with host cells. The other appears to encode a ribonuclease of unknown function or localization.
Publications
- Florea S, Andreeva K, Machado C, Mirabito PM, Schardl CL (2009) Elimination of marker genes from transformed filamentous fungi by unselected transient transfection with a Cre-expressing plasmid. Fungal Genetics and Biology 46: 721-730.
- Schardl CL, Scott B, Florea S, Zhang D (2009) Epichloe endophytes: clavicipitaceous symbionts of grasses. pp 275-306 in Deising H, ed, The Mycota V Plant Relationships, 2nd ed., Berlin Heidelberg: Springer-Verlag.
- Zhang D-X, Nagabhyru P, Schardl CL (2009) Regulation of a chemical defense against herbivory produced by symbiotic fungi in grass plants. Plant Physiology 150: 1072-1082.
- Zhang D-X, Stromberg AJ, Spiering MJ, Schardl CL (2009) Coregulated expression of loline alkaloid-biosynthesis genes in Neotyphodium uncinatum cultures. Fungal Genetics and Biology 46: 517-530.
- Liu M, Panaccione DG, Schardl CL (2009) Phylogenetic analyses reveal monophyletic origin of the ergot alkaloid gene dmaW in fungi. Evolutionary Bioinformatics 5: 15-30
- Schardl CL, (2009) Fungal endophytes in Lolium and Festuca species. pp 285-298 in Yamada T, Spangenberg G, eds. Molecular breeding of forage and turf: The proceedings of the 5th international symposium on the molecular breeding of forage and turf. New York: Springer.
|
Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Objective 1. The genome of an endophyte will be sequenced and annotated: Annotation continued for the genome sequence of Epichloe festucae, a fungal symbiont (endophyte) of Lolium and Festuca spp. grasses, known for its ability to produce alkaloids (lolines and peramine) that protect the host grasses from insects, and for its ability to switch between a mutualistic state (benign infection) and pathogenic expression ('choke' disease). After removing redundant information, the assembled genome was 27.4 Mb. The data were released as a GBrowse database published on the web at www.endophyte.uky.edu. Annotations published on that site include predicted genes, unigenes, and gene models (described below), unigene and gene model blastx results (top hit and top hit with annotation), gene ontology (GO) terms from InterPro and blast2go, and endophyte genes from NCBI. Objective 2, Expressed-sequence-tag (EST) databases of endophytes and the host grasses will be produced: Sequencing was completed in the previous year. ESTs were identified by sequence location on the E. festucae genome, and assembled into 6450 unigenes. The unigene information was supplemented with gene predictions from the genome sequence, to generate 11,173 gene models. More than 200 of these were manually annotated. Previous signature sequencing results indicating genes up- or down-regulated in asymptomatically infected tissues versus diseased tissues was followed up by the more accurate method, real-time reverse-transcription-quantitative PCR analysis. Objective 3, Expression profiles of endophyte genes will be characterized in symbio. Relative expression levels of genes for loline alkaloid biosynthesis were measured in asymptomatic leaves including young and old leaf blades and leaf sheathes, and also in asymptomatic and diseased inflorescences. Also, an Epichloe festucae gene encoding a small, secreted protein that was highly upregulated in asymptomatic flowering tissues was cloned with its promoter and with its coding sequence fused to that of the reporter green fluorescence protein to study expression in planta. PARTICIPANTS: Christopher L. Schardl, PI: Garnered additional funds, and supervised the Research Analyst, Senior Research Analyst, and other researchers in the laboratory. Alfred D. Byrd, Research Analyst: Was primarily responsible for regulatory compliance and safety training, as well as equipment maintenance. Walter Hollin, Senior Research Analyst, was responsible for training laboratory researchers, laboratory management, equipment maintenance, maintaining laboratory stocks of reagents, culturing and preserving fungal endophytes, inoculating plants, and maintaining databases. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Change of knowledge: The availability of the genome sequences greatly enhances capabilities of researchers world wide to investigate the basic biology of grass endophytes, and to screen or tailor their genetics for the benefit of turf and forage grasses that possess these symbionts. High levels of expression of genes for four small, secreted proteins in asymptomatic inflorescences were confirmed by quantitative PCR. These genes were expressed at between 33 and 1400-fold higher levels in asymptomatic compared to diseased inflorescences. This result sheds new light on the grass-endophyte interaction, and suggests a surprising relationship of endophyte mutualism with plant infection by pathogenic fungi such as those that cause corn anthracnose and rice blast. Loline alkaloid biosynthesis genes (LOL) were also more highly expressed in asymptomatic inflorescences compared to diseased (choked) inflorescences. This result suggest that lolines are important in protecting developing host seeds. In related symbionts of meadow fescue, Neotyphodium uncinatum and Neotyphodium siegelii, loline alkaloid levels were 3-20-fold higher in young regrowth tissue after the plants were clipped, but there was little or no concomitant increase in LOL gene expression. Instead, loline levels appeared to be determined by the levels of amino acid precursors mobilized to the young tissues by the host plants. Understanding of this metabolic cross-talk will help to manage pasture and forage for optimal livestock productivity as well as grass stand longevity. Change of action: Deep signature sequencing followed by quantitative PCR analysis was judged to be a more precise and potentially more cost effective means of transcriptome analysis than was the originally proposed DNA microarray technique, and will be the method of choice in this project.
Publications
- Spiering, M.J., Faulkner, J.R., Zhang, D.-X., Machado, C., Grossman, R.B., Schardl, C.L., 2008. Role of the LolP cytochrome P450 monooxygenase in loline alkaloid biosynthesis. Fungal Genetics and Biology 45: 1307-1314.
- Schardl, C.L., Craven, K.D., Speakman, S., Stromberg, A., Lindstrom, A., Yoshida, R., 2008. A novel test for host-symbiont codivergence indicates ancient origin of fungal endophytes in grasses. Systematic Biology 57: 483-498.
|
Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Objective 1. The genome of an endophyte will be sequenced and annotated: The complete genome sequence was determined for Epichloe festucae, a fungal symbiont (endophyte) of Lolium and Festuca spp. grasses, known for its ability to produce alkaloids (lolines and peramine) that protect the host grasses from insects, and for its ability to switch between a mutualistic state (benign infection) and pathogenic expression ('choke' disease). A clone library was prepared with randomly sheared, size-selected (average 4 kb) inserts. Both insert ends of approximately 70,000 randomly picked clones were sequenced to give 4.2 x coverage. Also, the ends of more than 8,500 fosmid clones were sequenced. In addition, genomic DNA was sequenced by "454" pyrosequencing to 20 x coverage. The combined assembly of preassembled pyrosequencing reads and Sanger reads totaled 29.8 Mb, which was almost exactly the original estimate based on quantitative Southern blot. The sequence has been annotated and
databases are being assembled. Annotations include predicted genes, unigenes, and gene models (described below), unigene and gene model blastx results (top hit and top hit with annotation), GO terms from InterPro and blast2go, endophyte genes from NCBI, and repeats. The data are in a GBrowse website that was released in 2006 to an annotation consortium of 14 institutions worldwide, and is actively updated. General public release is imminent. Objective 2, Expressed-sequence-tag (EST) databases of endophytes and the host grasses will be produced: Normalized cDNA libraries were generated from meadow fescue-Epichloe festucae symbiota, including benignly infected tissues and tissues with choke disease. Sequences were determined from the 5'-ends of 67,755 cDNA clones. The 3'-end sequences of 37,154 of these clones were also determined. The E. festucae ESTs were identified by sequence location on the E. festucae genome, and assembled into 9,866 unigenes at 7,678 genome locations, with the
difference attributable to alternative mRNA splicing. The unigene information was supplemented with gene predictions from the genome sequence, to generate 11,565 gene models, which were then extensively annotated. Objective 3, Expression profiles of endophyte genes will be characterized in symbio: A massively parallel signature sequencing (MPSS) procedure was conducted on mRNA from 4 different symbiotum genotypes (each from a distinct grass seed with a distinct endophyte isolate), with 2 tissue types: choked inflorescences bearing fungal fruiting bodies (stromata), and benignly infected inflorescences. In total 456,986 high quality signature sequences were obtained: 209,823 from stromata and 247,163 from inflorescences. Out of these, 73,915 from stromata and 4362 from inflorescences mapped to a total of 7673 E. festucae gene models. The rest are expected to be plant cDNA reads, and their analysis awaits completion of plant unigene assembly and annotation. Statistical analysis
(Fisher's exact test, p < 0.01) indicated 138 endophyte genes that are upregulated in inflorescences, and 59 upregulated in stromata. The functions of these genes are under investigation.
PARTICIPANTS: Christopher L. Schardl Walter Hollin Alfred D. Byrd
TARGET AUDIENCES: Forage, turf, basic and applied plant pathologists
Impacts
Several important forage and turf grasses, such as tall fescue and perennial ryegrass, have symbiotic fungal endophytes that protect against insects and nematodes, and enhance tolerance of abiotic stresses such as drought. They are maintained indefinitely in host cultivars because of their remarkable ability to colonize all of the floral primordia, ovaries, and ultimately the embryos in host seeds without causing any negative effects. The common endophyte of tall fescue is important for stand longevity of this important forage grass, but also produces ergot alkaloids that are toxic to livestock, resulting in an estimated $1 billion in losses in U.S. livestock production. Novel endophytes recently introduced are nontoxic to livestock, but are not always efficiently seed transmitted. In addition, a related fungus, Epichloe typhina, causes choke disease, which greatly reduces seed yield, and is an emergent problem in orchard grass (Dactylis glomerata) in the Pacific
Northwest. This project focuses on interaction of grass hosts and their endophytes leading to seed transmission and various beneficial effects, as well as the mechanisms underlying choke disease. The endophyte model is Epichloe festucae, which, like the related tall fescue and perennial ryegrass endophytes, is seed transmissible and beneficial to host grasses, but which also can cause choke disease. The project involves discovery of endophyte and host genes, and studies of gene expression involved in communication between endophyte and host during both pathogenic and mutualistic interactions, as well in response to stresses. Such studies will help reveal mechanisms underlying beneficial effects of endophytes, as well as the basis of choke disease. The information will help in management of forage grasses and their endophytes for stress tolerance, establishment of stable, seed transmissible symbioses of beneficial endophytes with forage grass cultivars, and insights that may contribute
to control strategies for orchard grass choke disease.
Publications
- Hesse, U., Maynard, P., Macmil, S., Wiley, G., Andreeva, K., Beech, W.E., Arnaoudova, E., Willey, B.T., Puram, V.-G., Wiseman, J., Webb, J., Gill, L., Farman, M.L., Jaromczyk, J.W., Roe, B.A. and Schardl, C.L. 2007. The genome and the genes of Epichloe festucae. Pages 461-465 in New Zealand Grassland Association: Endophyte Symposium (A. Popay, and E. R. Thom, eds.). New Zealand Grassland Association, Christchurch, New Zealand.
|
|