Molecular Biology and Biosynthesis of Lolines by Grass Endophytes

Neotyphodium和Epichloe被称为禾本科真菌内生菌，是一种系统的、母系传播的共生体，可保护寄主植物免受食草性侵害，并可增强耐旱性、抗病性、养分获取、生物量生产和竞争力。内生菌产生四种不同化学类别的生物保护生物碱。本项目涉及一类饱和的1-氨基吡啶生物碱（洛林及其相关化合物），这是一种高度杀虫的复杂杂环分子，在植物-内生菌共生中可达到总生物量的2%。可以想象，这些丰富的生物碱也参与了抗旱性或其他几种宿主适应性增强，但需要遗传学和分子遗传学研究来解决它们真正的生态和生理作用。因此，本项目的目的是阐明洛碱生物碱的遗传和生物合成途径，并为研究这些生物碱在植物适应性中的作用提供背景信息和工具。两个基因簇（lol）已经被鉴定出与内生植物新伤寒（Neotyphodium uncinatum）生产loine生物碱有关。此外，还测定了碱类生物碱的代谢前体。该项目的第一个目标是完成两个lol基因簇的DNA序列测定。下一个目标是利用分子遗传学技术寻求证实lol簇基因参与了loline生物碱的生物合成。第三个目标是通过合成用稳定同位素标记的假定中间体，将这些中间体喂给产生洛林生物碱的培养物，并使用质谱和核磁共振检测和鉴定纳入洛林的标记原子，来测试可能的洛林生物合成途径。该项目开发的信息和突变体将为未来详细研究草内生共生中的天然作用提供资源。禾本科植物与真菌内生菌的共生是一种保护性的共生关系，通过这种共生关系，内生菌可以抵御多种植物害虫和寄生虫。在保护寄主植物的真菌产物中，碱生物碱具有很强的杀虫作用，在共生植物中含量丰富，化学成分复杂。碱产生的途径尚不清楚，阐明这一途径将为共生微生物的酶机制和进化提供新的见解。该项目将结合分子生物学和化学方面的现代技术来确定碱石生产的步骤。研究中产生的真菌突变体将为未来的研究提供资源，以确定内生真菌-草共生中碱的具体作用和活性。研究课题涉及植物、真菌和食草动物之间的生态相互作用，以及利用天然产物对植物进行环境友好型生物保护的潜力。

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.