Research Accomplishment Reports 2007

Genomics of Fungal Endophytes and Their Host Grasses

C.L. Schardl
Department of Plant Pathology

 

Project Description

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.

Impact

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.