2012 Annual Report 1a.Objectives (from AD-416): To characterize genetic expression profiles of drought stressed tall fescue containing novel endophytes strains and verify gene expression profiles of selected genes. 1b.Approach (from AD-416): Tall fescue, KY31, containing six novel endophyte strains, plus the endophyte-free and KY31 with wild-type endophyte will be evaluated under different drought stress regimes to characterize differences in gene expression profiles and correlate these with phenotypic response to the treatments. The KY31 lines differ in their response to drought stress and the goal is to compare gene expression levels and identify putative genes that are over- or under-expressed in response to the stress experiments that correlate with increased drought tolerance. ARS is developing an expression profiling tool for tall fescue for analysis of whole genome expression comparisons. This will be used to compare expression of the different KY31 lines containing the different endophyte strains to determine the effect of gene expression, using real time PCR techniques, in drought tolerance response. Three replications comparing non-stressed (beginning of the experiment) and three replications of stressed material for each of the lines will be evaluated. RNA will be isolated from pseudostems of each line for each treatment for analysis. 3.Progress Report: Objectives: 1.) Isolate E+ and E- lines of Kentucky-31 tall fescue with endophyte from UA strains 4, 15, 16, 17, 18, 19, 20, 28 and 32, designated as seedlots KY4, KY15, KY16, KY17, KY18, KY19, KY20, KY28 AND KY32. 2.) Isolate Ribonucleic Acid (RNA) from plants subjected to water stressed experiments conducted to do transcriptional expression profiles. Accomplishments: We have generated endophyte-infected (E+) and un-infected (E-) genetically identical clone pairs for each of the nine seedlots above (number of clone pairs in parenthesis): [KY4 (11); KY15 (6); KY16 (8); KY17 (13); KY18 (6); KY19 (5); KY20 (8); KY28 (10); KY32 (14)]. Verification of the absence of ergot alkaloids has been done on all clone pairs. Polymerase chain reaction (PCR) analysis has been conducted using primers to genes involved in the fungal alkaloid biosynthetic pathways for lolines (lolA, lolC and lolD), peramine, (perA), ergot alkaloids (dmaW) and endo-diterpenes (itmF, itmG, itmJ, itmP and itmQ) and the respective alkaloids (lolines, peramine, and ergot alkaloids) assayed by Gas Chromatography-Mass Spectrometry (GC-MS). Plants derived from one clone pair originally from each of the wild type (WT) (common toxic), KY16 and KY19 endophytes have been increased and subjected to water withholding experiments in the greenhouse at the Forage-Animal Production Research Unit (FAPRU) location, and samples collected over a five day period for metabolite analysis and RNA isolation. Water withholding experiments have been conducted for all clone pairs for plants harboring the endophyte KY16 and similarly for plants harboring the endophyte KY19. Additional experiments are planned for clone pairs harboring the other endophytes. RNA isolated from plants harboring the WT common toxic endophyte has been used for transcriptome analysis using the Illumina RNA-Seq procedure. Originally the plans called for analysis of the expression profiles using a Lolium specific microarray platform, however due to the costs associated with the microarray platform and the reduction in costs using the Illumina RNA-Seq protocol, we have converted our transcriptomic analysis utilizing the later. Results for RNA-Seq experiments from pseudo stem sections for Day (D) 1, D2 and D3, crown and root tissues from D2 of the water withholding experiments described above have been returned from Iowa State Sequencing Facility and analysis of the results is ongoing. The results will be used to generate primers for Real Time-Quantitative PCR (RT-qPCR) comparisons once specific genes have been identified that are significantly differentially expressed in the E+ vs. E- clone pairs, and over the different time points and tissues.