Wednesday, November 15, 2006

Microarray Comparison of Tall Fescue Endophyte-Infected and Endophyte-Free Gene Expression.

Randy Dinkins, ARS-USDA-FAPRU, "N220C Ag Sci. Center, North", Lexington, KY 40546-0091, United States of America

Many grasses have mutualistic symbioses with fungi of the family Clavicipitaceae.  Tall fescue (Festuca arudinacea = Lolium arundinaceum) can harbor the obligate endophyte, Neotyphodium coenophialum that are asexually propagated and transmitted via host seeds.  The endophyte receives shelter and nutrients from the host plant and in turn produces anti-herbivory compounds targeted towards insects and mammals.  In addition, plants with the endophyte are more stress tolerant, although the mechanism for this tolerance is unknown.  In an effort to begin to dissect the host plant endophyte cross-talk, global gene expression was analyzed using the Affymetrix Wheat Genome Array GeneChip® and Barley1 Genome Array GeneChip®.  Total RNA was isolated from pseudostems of known endophyte-infected and endophyte-free plants and tested in triplicate.  Overall only 14-15% and 17-18% of the probe sets were called present on the wheat and barley chips, respectively.  These results are somewhat lower than expected based on the relatedness between fescue, wheat and barley.  However, a number of probe sets were observed to be differentially expressed greater than two-fold (P<0.01) between the endophyte-infected and endophyte-free plants.  PCR primers were designed to a number of fescue EST’s with homology to sequences on the GeneChip arrays and tested to verify the expression profile observed in the microarray experiments.  Initial results using forty primer pairs have shown that some primers gave results similar to the expected, including a number of wheat probe sets that contain fungal sequences.  In some cases no differences were observed between the endophyte-infected and endophyte-free, and it is unknown whether this is indicative of the particular gene or allele that was used compared to what was detected on the array.  Some of the primers did not produce a PCR product and additional primers are being synthesized to verify these results.  Experiments are ongoing and results will be presented.