Collaborative Research: EDGE FGT: Functional Genomic Tools for Parasitic Nematodes and their Bacterial Symbionts

包括人类在内的所有动物都与微生物和寄生虫生活在一起，这些微生物和寄生虫可以促进健康并引起疾病。动物通过与微生物和寄生虫交流来阻止、启动、维持和消除这种联系的机制才刚刚开始被发现。由于这些机制在整个生物学中通常是保守的，因此可以使用模式动物（如蛔虫或线虫）进行研究，这些动物与微生物有关，本身就是寄生虫。该项目将在与特定细菌形成伙伴关系或共生关系的蛔虫身上开发新的实验工具。这些蛔虫和它们的细菌伴侣一起感染并杀死昆虫，把它们作为食物来源。开发新的工具来研究这个优雅的动物-细菌系统将有助于扩大我们对动物和细菌如何形成伙伴关系，以及它们如何共同寄生在其他动物身上的理解。在本项目中获得的工具和知识将迅速与参与药物发现、作物害虫的农业控制、寄生、传染病、有益微生物组功能和基础细胞、分子、发育和进化生物学研究的研究人员分享。作为这个项目的一部分，本科生将参与一个以发现为基础的微生物实验室，在那里他们将练习从环境中分离和识别新的杀虫蛔虫。青少年（K-12）和教育工作者将通过与《儿童科学杂志》的合作参与其中，该杂志将制定基本课程和主要研究成果摘要，供课堂使用。昆虫病原线虫Heterorhabditis属和Steinernema属分别与Photorhabdus属和Xenorhabdus属细菌共生。线虫-细菌共生对专性地寄生在昆虫身上，作为繁殖的营养来源，作为农业害虫的生物防治剂和新化合物的来源具有实用价值。昆虫病原对及其昆虫宿主是理解基本生物学原理的模型，包括进化和互惠和拮抗生物相互作用的分子和细胞基础。许多特性使该系统成为一个优秀的实验模型，包括易于操作和成本低，生成时间快，光学透明度高。虽然遗传技术已经发展为具有代表性的细菌共生伙伴和昆虫宿主，但到目前为止，无论是斯坦氏线虫还是异habditis线虫，都没有广泛采用的可靠的遗传修饰工具。这种无法询问线虫基因功能的情况阻碍了该系统的充分利用，从而对寄生、动物微生物组相互作用和其他领域产生急需的见解。在这里，我们建议利用最近分离的斯坦纳线虫，该线虫具有开发遗传工具的有希望的特征，包括雌雄同体生殖，适应长期冷冻，在琼脂细菌草坪上健康发育，对显微注射的弹性，以及对实验室昆虫和农业害虫的显著致病性。通过对线虫和共栖生物的基因组进行全面测序和开发遗传技术和工具，包括在线虫中进行CRISPR-Cas9基因组编辑，并为细菌创建一个排列的突变文库，我们的团队将帮助实现这个优雅的动物-微生物模型系统的全部潜力。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

All animals, including humans live in association with microbes and parasites that can promote health and cause disease. The mechanisms by which animals communicate with microbes and parasites to block, initiate, maintain, and dissolve such associations are just beginning to be uncovered. Because these mechanisms are often conserved across biology, they can be investigated using model animals such as roundworms, or nematodes, which associate with microbes and are themselves parasites. This project will develop new experimental tools in roundworms that have developed partnerships, or symbioses, with specific bacteria. Together these roundworms and their bacterial partners infect and kill insects, using them as a food source. Developing new tools to study this elegant animal-bacterium system will help expand our understanding how animals and bacteria form partnerships, and how they work together to parasitize other animals. The tools and knowledge gained in this project will be rapidly shared with the community of researchers involved in drug discovery, agricultural control of crop pests, and in the study of parasitism, infectious disease, beneficial microbiome function, and fundamental cell, molecular, developmental, and evolutionary biology. As part of this project, undergraduates will be involved in a discovery-based microbiology lab where they will practice isolating and identifying new insect-killing roundworms from the environment. Young people (K-12) and educators will be engaged through collaboration with the Science Journal for Kids, where a basic curriculum and summary of key research findings will be developed for classroom use. Entomopathogenic nematodes in the genera Heterorhabditis and Steinernema are mutualistically associated with bacteria in the genera Photorhabdus and Xenorhabdus, respectively. The nematode-bacterium symbiotic pair obligately parasitizes insects as a nutrient source for reproduction and has utility as a biological control agent for agricultural insect pests and as a source of novel compounds. The entomopathogenic pair and their insect hosts are models to understand fundamental biological principles, including the evolution and molecular and cellular basis of mutualistic and antagonistic organismal interactions. Numerous features make this system an excellent experimental model including ease and cost of husbandry, fast generation time, and optical transparency. Although genetic techniques have been developed for representative bacterial symbiotic partners and insect hosts, to date there have been no broadly adopted, reliable genetic modification tools in either Steinernema or Heterorhabditis nematodes. This inability to interrogate nematode gene function has hobbled full use of this system to yield much-needed insights into parasitism, animal microbiome interactions, and other areas. Here we propose to capitalize on a recently isolated Steinernema nematode that has promising characteristics for development of genetic tools, including hermaphroditic reproduction, amenability to long-term freezing, healthy development on agar bacterial lawns, resilience to microinjection, and significant pathogenicity to lab insects and agricultural pests. By fully sequencing the genomes and developing genetic techniques and tools for both nematode and symbiont, including CRISPR-Cas9 genome editing in the nematode, and creating an arrayed mutant library for the bacterium, our team will help realize the full potential of this elegant animal-microbe model system.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.