High-Throughput Quantitative Gene Expression System to Investigate Epichloë-Grass Symbiosis.

It is well established that the Neotyphodium and Epichloë fungal endophytes of grasses confer numerous benefits to their hosts.  However, the details of the interaction are largely unknown.  One of the outstanding questions regarding the plant-endophyte relationship is what factors contribute to the maintenance of a compatible interaction.  Previous studies have established that gene expression in the plant is altered in response to endophyte infection.  Based on our hypothesis that these changes are important for the maintenance of the symbiotic interaction, we have used SOLiD-SAGE (Sequencing by Oligonucleotide Ligation and Detection – Serial Analysis of Gene Expression) to obtain a global quantitative comparison of the transcriptomes of endophyte-free and endophyte-infected plants.  We have three samples in the comparison, all with the identical strong creeping red fescue plant genotype: 1) endophyte-free (S1139 E-); 2) infected with an endophyte originally from strong creeping red fescue (S1139 RC); and 3) infected with an endophyte originally from Chewings fescue (S1139 DE).  The SAGE libraries were prepared in replicates, and sequenced using ABI’s SOLiD instrument. Over 54 million SAGE tags were obtained, with between 4 and 10 million tags per replicate. In addition, we have supplemented the SAGE data with over 200 000 454 transcriptome sequences of the Epichloë festucae strain isolated from strong creeping red fescue, and the plant infected with that strain (S1139 RC).  The SAGE tags have been mapped to a reference database consisting of our 454 transcriptome sequences, in addition to Festuca and Epichloë sequences downloaded from NCBI. Analysis of the data will reveal 1) general plant transcriptional changes in response to fungal endophyte infection; 2) plant transcriptional changes that are specific to the infecting fungal endophyte genotype; and 3) relative transcript levels for fungal endophyte genes for two endophyte genotypes each infecting the same plant genotype.