Research Accomplishment Reports 2009

Genomics of Fungal Endophytes and Their Host Grasses

C.L. Schardl
Department of Plant Pathology

 

Non-Technical Summary

Epichloe species and closely related Neotyphodium species are common symbionts of many temperate turf, forage and wild grasses, and provide a variety of fitness enhancements such as increased stress tolerance, as well as resistance to nematodes and insects. In addition, the Epichloe species, but not Neotyphodium species, sometimes can cause disease on the host plants. A survey of endophyte and host plant genes will enable studies of gene expression in response to each other and to stresses, as well as during pathogenic or mutualistic phases. Such studies will help reveal mechanisms underlying beneficial effects of endophytes, as well as the basis of fungal pathogenicity and suppression of pathogenicity.

2009 Project Description

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).

2009 Impact

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.

2009 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.