EDGE: Genetic transformation of chytrid fungi

菊苣是一个由1000多种真菌组成的多样性群体，包括在淡水、海洋和土壤生态系统的生物学中发挥关键作用的生物。一些龟裂类物种在水生和土壤食物网中占据关键地位，而另一些龟裂类则感染并杀死昆虫、原生动物、无脊椎动物、两栖动物、植物和其他真菌。目前，多种壶类物种正在导致全球两栖动物种群数量的下降，包括对青蛙种群造成毁灭性影响的树突蝙蝠属(Batrachochytrium endrobatidis，Bd)，以及被认为可能对北美火蜥蜴造成同样影响的蝙蝠属蝙蝠属(Batrachochytrium salamandrivorans，Bsal)。尽管菊花真菌在全球生态系统中很重要，但还没有为任何种类的菊花真菌开发出基本的现代生物工具。由于缺乏分子遗传学方法，因此不可能直接测试有关脊椎动物如何致病以及致病原因的假设。该项目将开发和测试首个脊椎动物分子遗传学工具，并将促进不同科学界对这些工具的使用。该项目将通过利用快速和开放的数据和方案共享广泛传播工具，以及直接培训来自不同实验室的科学家，对许多科学界产生广泛影响。该项目致力于攻克游动孢子菌的现代遗传学的主要瓶颈：分子转化。虽然脊椎动物占据了关键的生态、进化和致病的生态位，但目前缺乏基本的分子工具是假说驱动的研究的主要障碍。该项目将通过开发两个主要真菌谱系中的一个物种的遗传转化方法来克服这一不足，这两个主要谱系是属于chytridimycota的重要病原菌Batrachochytrium endrobatidis(Bd)和用于研究世代交替和Blastocladimycota成员的大别样芽孢杆菌(Allmyces Macgynus)(Am)。目的1开发一套用于表型复制和染色体整合以及耐药和荧光蛋白标记的转化载体，以便于筛选成功的转化。目标2将在我们通过电穿孔在细胞内传递荧光化合物方面取得初步成功的基础上，以及在目标1中开发的载体的基础上，实现在卵子中的分子转化。目标3使用目标1和2中开发的工具来建立测试基因功能和更广泛地将基因与表型联系起来的能力。作为一个原则的证明，鞭毛运动性的特定组成部分，一个关键的龟裂生物学特征，将被作为击倒和功能干扰的目标，并对产生的表型进行评估。该奖项由通过基因组工具(EDGE)实现发现(EDGE)、综合生态生理学以及环境生物学部门内整合组织系统和进化过程计划中的共生、防御和自我识别计划的资金共同资助。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.