Investigation of Epichloë festucae -- strong creeping red fescue mutualistic and antagonistic interaction

Many cool-season grasses have symbiotic relationships with Epichloë (Ascomycota, Clavicipitaceae) fungal endophytes that reside in the intercellular spaces of the above-ground parts of host plants. The presence of the Epichloë endophytes is generally beneficial to host plants due to enhanced tolerance to biotic and abiotic stresses conferred by the endophytes. Many Epichloë spp. are asexual and those infections always remain asymptomatic. However, some Epichloë spp. have a sexual stage and produce macroscopic fruiting bodies (stromata) that envelop the developing inflorescences causing a syndrome termed “choke disease”. To better understand this antagonistic association, a transcriptome analysis of fungal and plant genes was performed to compare stroma tissue and asymptomatic inflorescence tissue of Epichloë festucae infected strong creeping red fescue (Festuca rubra subsp. rubra). Hundreds of fungal genes and over 10% of the plant genes were differentially expressed between the two tissue types. The differentially expressed fungal genes in the stroma tissue indicated a change in carbohydrate and lipid metabolism. Plant stress related genes were up-regulated in the stroma tissue suggesting the plant host was responding to the normally symbiotic fungal endophyte as a pathogen.Genome and transcriptome analyses are often the first steps for gene discovery followed by gene function studies. CRISPR/Cas technology is a powerful molecular tool to genetically modify genes of interest for further functional characterization of those genes. Here CRISPR/Cas9 approach was utilized to knockout an E. festucae antifungal protein gene (Efe-afpA), whereas fungal transformation relying on homologous recombination was unsuccessful due to the lack of long and unique flanking regions of this gene. The mutants lacking the Efe-afpA gene were shown to have impaired growth in culture, and therefore, unlikely to form a symbiotic relationship by infecting and systematically colonizing its plant host, strong creeping red fescue. CRISPR/Cas9 approach is highly effective and precise compared to conventional homologous recombination approach. More importantly, the CRISPR/Cas9 approach is more versatile and is not restricted by the availability of long and unique homologous flanking regions of the target gene. CRISPR/Cas9 enables functional characterization of many more Epichloë genes to study the Epichloë–grass symbiosis.Ph.D.Includes bibliographical reference