Molecular Biology and Biosynthesis of Lolines by Grass Endophytes

禾本科内生真菌Neotyphodium和Epichidium是一类系统性的、母系传播的共生菌，它们能保护寄主植物免受草食性害虫的侵害，并能增强植物的耐旱性、抗病性、营养吸收、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性生物碱。该项目涉及这样的一类，饱和1-氨基吡咯里西啶生物碱（洛林和相关化合物），这是高度杀虫，复杂的杂环分子，并可以达到植物内生菌共生体中总生物量的2%的水平。可以想象，这些丰富的生物碱也参与耐旱性或任何其他几个主机健身增强，但需要遗传和分子遗传学研究，以解决其真正的生态和生理作用。因此，本项目的目标是阐明黑麦草碱生物碱的遗传和生物合成途径，并开发背景信息和工具来研究这些生物碱在植物适应性中的作用。两个基因簇（lol）已被确定为与黑麦草碱生物碱生产的内生菌物种，钩孢Neotyphodiumuncinatum。此外，还测定了黑麦草碱生物碱的代谢前体。本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确认lol簇基因参与了黑麦草碱生物碱的生物合成。第三个目标是测试可能的生物合成途径，通过合成推定的中间体标记的稳定同位素，这些饲料的黑麦草碱生物碱生产文化，并采用质谱和核磁共振检测和识别的标记原子纳入黑麦草碱。本项目中开发的信息和突变体将成为未来研究的资源，详细说明了黑麦草在草内生真菌共生中的自然作用。草与真菌内生菌的共生是保护性互惠关系，内生菌借此抵御各种植物害虫和寄生虫。参与保护宿主植物的真菌产物中有黑麦草碱生物碱，其具有高度杀虫性，在共生植物中丰富，并且化学成分复杂。黑麦草碱的产生途径尚不清楚，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。该项目将采用分子生物学和化学的现代技术相结合，以确定在黑麦草碱生产的步骤。在这项研究中产生的真菌突变体将是未来研究的资源，以确定内生菌-草共生体中的特定作用和活性。该研究主题与植物，真菌和食草动物的生态相互作用有关，也与天然产品用于环境友好的植物生物保护的潜在用途有关。

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.