Research Accomplishment Reports 2007

Gene Expression Profiles in a Grass-Endophyte Symbiosis

C.L. Schardl
Department of Plant Pathology

 

Project Description

The ascomycete, Epichloe festucae, is an endophyte of Festuca and Lolium spp. grasses, and has several characteristics that make it an attractive model. This fungus grows systemically and intercellularly throughout the life of its host plant, but on each reproductive tiller the fungus either infects benignly and transmits clonally in seeds, or produces its sexual state (stroma) and chokes inflorescence development. The endophyte switches between mutualistic and antagonistic states, is seed transmissible, has a sexual state amenable to genetic analysis, and is rich in bioprotective alkaloids.

Our working hypothesis is that choke and benign infection are associated with different gene expression profiles. Research objectives are:

1. Generate normalized cDNA libraries of asymptomatic and symptomatic inflorescences from endophyte-plant symbiota.

2. Sequence cDNA 5'-ends from normalized libraries.

3. Profile expression patterns of sequences.

4. Validate regulation of selected genes by using quantitative reverse transcription polymerase chain reaction (qRT-PCR).

RNA was isolated from meadow fescue-E. festucae symbiotum tissues at various developmental stages. RNA from benignly infected inflorescences was used for production of a normalized inflorescence cDNA library. Similarly, RNA from choked inflorescences was used for a normalized stroma library. A total of 108,497 sequences (expressed sequence tags = ESTs) were obtained from the normalized cDNA libraries. The ESTs assembled into 25,533 plant unigenes and 9,866 unigenes from E. festucae. The E. festucae unigenes represented 7,678 distinct genes plus intron splice variants. A comparison of fungal gene expression in asymptomatic inflorescences and chokes was conducted by massively parallel signature sequencing (MPSS), and verified by qRT-PCR.

Impact

Numerous fungal genes were found to be differentially expressed when comparing asymptomatic and choked inflorescences. A Fisher's exact test indicated 257 fungal genes that were significantly upregulated in the asymptomatic inflorescences, and 70 fungal genes significantly upregulated in choked inflorescences. Several secondary metabolism gene clusters appeared to be up-regulated in choked inflorescences, and asymptomatic inflorescences had much higher expression of several small, secreted protein genes, a protease gene, and genes for plant cell wall and plant cell matrix degradation. The number and types of genes upregulated in asymptomatic tissues was surprising because many homologues are associated in other fungi with pathogenicity to plants or animals. These results suggest that plant-fungus mutualistic symbiosis may share some mechanisms in common with pathogenicity in other plant-fungus interactions.

Publications

Sullivan, T.J., Rodstrom, J., Vandop, J., Librizzi, J., Graham, C., Schardl, C.L. and Bultman, T.L. 2007. Symbiont-mediated changes in Lolium arundinaceum inducible defenses: evidence from changes in gene expression and leaf composition. New Phytologist 176:673-679.

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.