EDGE: Genetic transformation of chytrid fungi

壶菌是一个由1000多种真菌组成的多样化群体，包括在淡水、海洋和土壤生态系统中发挥关键作用的生物。虽然一些壶菌物种在水生和土壤食物网中占据关键位置，但其他壶菌感染并杀死昆虫、原生生物、无脊椎动物、两栖动物、植物和其他真菌。目前，多种壶菌物种正在导致全球两栖动物种群数量的下降，其中包括破坏青蛙种群的壶菌（Bd），以及被认为准备对北美蝾螈造成同样影响的壶菌（Bsal）。尽管壶菌在全球生态系统中的重要性，但基本的现代生物学工具尚未开发出任何一种壶菌。由于缺乏分子遗传方法，因此不可能直接测试关于壶菌如何以及为什么引起疾病的假设。该项目将开发和测试第一批壶菌分子遗传工具，并将促进不同科学界对这些工具的使用。通过使用快速和开放的数据和协议共享广泛传播这些工具，以及直接培训来自不同实验室的科学家，该项目将广泛影响许多科学界。本项目旨在克服主要的瓶颈，现代遗传学在游动孢子壶菌：分子转化。虽然壶菌占据了关键的生态、进化和致病生态位，但目前缺乏基本的分子工具是假说驱动研究的主要障碍。该项目将通过开发两种主要壶菌系（壶菌科的重要病原体Batrachochytrium dendroatidis （Bd)）和用于研究代际交替的巨生异酵母菌系（Am））各一种的遗传转化方法来克服这一缺陷。目的1开发一组用于外泌体复制和染色体整合的转化载体，以及耐药和荧光蛋白标记物，以促进成功转化的筛选。目标2将基于我们在细胞内通过电穿孔传递荧光化合物的初步成功，以及目标1中开发的载体，在壶菌中实现分子转化。目标3使用目标1和目标2中开发的工具来构建测试基因功能的能力，并更广泛地将基因型与表型联系起来。作为原理的证明，鞭毛运动的特定成分，壶菌生物学的一个关键特征，将被靶向敲除和功能干扰，并由此产生的表型评估。该奖项由通过基因组工具实现发现（EDGE）、综合生态生理学、综合有机体系统部门的共生、防御和自我识别项目以及环境生物学部门的进化过程项目共同资助。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。

Chytrids are a diverse group of over 1000 species of fungi, including organisms that play key roles in the biology of freshwater, marine, and soil ecosystems. While some chytrid species occupy key positions in aquatic and soil food webs, other chytrids infect and kill insects, protists, invertebrates, amphibians, plants, and other fungi. Currently, multiple chytrid species are causing global declines in amphibian population, including Batrachochytrium dendrobatidis (Bd) that is devastating frog populations, and Batrachochytrium salamandrivorans (Bsal) that is thought to be poised to do the same to North American salamanders. Despite the importance of chytrid fungi in global ecosystems, basic modern biological tools have not yet been developed for any species of chytrid fungus. This lack of molecular genetic methods makes it impossible to directly test hypotheses about how and why chytrids cause disease. This project will develop and test the first molecular genetic tools for chytrids and will foster the use of these tools by different scientific communities. The project will broadly impact many scientific communities, via wide dissemination of the tools using rapid and open data and protocol sharing, as well as direct training of scientists from diverse laboratories. This project seeks to overcome the main bottleneck to modern genetics in the zoosporic chytrid fungi: molecular transformation. Although chytrids occupy key ecological, evolutionary, and pathogenic niches, the current lack of basic molecular tools is a major impediment to hypothesis-driven research. The project will overcome this deficiency by developing methods for genetic transformation for one species from each of the two main lineages of chytrid fungi, Batrachochytrium dendrobatidis (Bd), an important pathogen belonging to the Chytridiomycota, and Allomyces macrogynus (Am), a system used to study alternation of generations and a member of the Blastocladiomycota. Objective 1 develops a panel of transformation vectors for episomal replication and chromosomal integration as well as drug resistance and fluorescent protein markers to facilitate screening for successful transformation. Objective 2 will enable molecular transformation in chytrids, building on our preliminary success in intracellular delivery of fluorescent compounds by electroporation, and the vectors developed in Objective 1. Objective 3 uses the tools developed in Objectives 1 and 2 to build capability for testing gene function and more broadly relating genotype to phenotype. As a proof of principle, specific components of flagellar motility, a key feature of chytrid biology, will be targeted for knock-down and functional interference and resulting phenotypes assessed.This award was co-funded by funds from Enabling Discovery through GEnomic Tools (EDGE), Integrative Ecological Physiology, and Symbiosis, Defense and Self-recognition programs in the Division of Integrative Organismal Systems and the Evolutionary Process Program within the Division of Environmental Biology.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.