Neotyphodium and Epichloe species, known as fungal endophytes of grasses, are systemic, maternally transmitted symbionts that protect their host plants from herbivory, and can enhance drought tolerance, disease resistance, nutrient acquisition, biomass production, and competitiveness. The endophytes produce bioprotective alkaloids of four distinct chemical classes. This project concerns one such class, the saturated 1-aminopyrrolizidine alkaloids (loline and related compounds), which are highly insecticidal, complex heterocyclic molecules, and can reach levels of 2% total biomass in plant-endophyte symbioses. It is conceivable that these abundant alkaloids are also involved in drought tolerance or any of several other host fitness enhancements, but genetic and molecular genetic investigations are needed to address their true ecological and physiological roles. Thus, the goals of this project are to elucidate the genetics and biosynthetic pathways of the loline alkaloids, and to develop background information and tools to study the roles of these alkaloids in plant fitness. Two gene clusters (lol) have been identified that are associated with loline alkaloid production by the endophyte species, Neotyphodium uncinatum. In addition metabolic precursors of loline alkaloids have been determined. The first objective in this project is to complete DNA sequence determinations for the two lol gene clusters. The next objective is to utilize molecular genetic techniques to seek confirmation that the lol cluster genes are involved in loline alkaloid biosynthesis. The third objective is to test possible biosynthetic pathways for lolines by synthesizing putative intermediates labeled with stable isotopes, feeding these to loline alkaloid-producing cultures, and employing mass spectrometry and nuclear magnetic resonance to detect and identify the labeled atoms incorporated in the lolines. The information and mutants developed in this project will be a resource for future studies detailing the natural roles of lolines in grass-endophyte symbioses.
Symbioses of grasses with fungal endophytes are protective mutualisms, whereby the endophytes ward off a variety plant pests and parasites. Among the fungal products involved in protecting host plants are the loline alkaloids, which are highly insecticidal, abundant in the symbiotic plant, and chemically complex. The pathway of loline production is unknown, and elucidating this pathway will provide new insights into enzyme mechanisms and evolution of symbiotic microorganisms. This project will employ a combination of modern techniques in molecular biology and chemistry to identify the steps in the loline production. Fungal mutants that are produced in the study will be a resource for future research to determine the specific roles and activities of lolines in endophyte-grass symbioses. The research topic is relevant to the ecological interactions of plants, fungi and herbivores, and also to the potential use of natural products for environmentally friendly biological protection of plants.
