EDGE CT: Virus-inspired, lipid-mediated transfection and genetic manipulation of the marine coccolithophore, Emiliania huxleyi

海洋浮游植物总共贡献了地球一半以上的氧气，并负责海洋中90%的光合作用衍生碳，使它们成为海洋食物网的基础。这一类中占主导地位的真核生物成员是硅藻、甲藻和触藻。触生生物包括球藻，它们通过方解石生物矿物细胞壁(球藻)贡献了海洋碳酸钙总产量的大约三分之一，并对碳循环产生了深远影响。它们在全球的分布和相关活动是如此广泛，以至于可以从地球轨道卫星上看到它们。尽管过去对这些生物的生理、钙化、光合作用和宿主-病毒相互作用的实验研究获得了丰富的知识，但在理解控制球虫对环境变化的反应的遗传和细胞机制方面存在着严重的差距。这一差距在很大程度上是由于长期缺乏科学工具来实验性地操纵基因表达和阐明关键基因的功能。该项目旨在开发一种新的基因操作方法，利用球虫细胞与在海上经常感染它们的病毒之间基于脂类的良好相互作用，对球虫生物进行遗传操作。这项工作将独特地使未来能够对从生物矿化到光合作用再到病毒感染等广泛的生理过程进行研究，从而从根本上洞察影响这些全球分布和生态重要生物的关键生物过程的遗传和生化控制。埃米利亚尼亚是一种在全球占主导地位的球石生物，能够形成从太空中可见的大型水华。它通过方解石细胞壁(球石)的生物矿化贡献了海洋碳酸钙总产量的大约三分之一，并对海洋碳和硫循环产生了深远的影响。因此，它已经成为研究钙化、光合作用、宿主-病毒相互作用的生态生理学和细胞机制，以及影响上层海洋生态和生物地球化学的关键生物过程的模式海洋微真核生物。然而，尽管基因组和转录序列数据是可用的，但无法操纵基因表达造成了使用功能基因组学来理解环境变化如何影响这种生态相关生物体的严重瓶颈。该项目旨在通过开发一种新颖的、基于脂质的转基因方法来解决这一差距，该方法利用了几十年来对这些生物中宿主-病毒相互作用的研究，揭示了E.huxleyi与其相关的球虫病毒(Ehvs)之间特定的脂质介导的相互作用。具体目标是：1)产生由E.huxleyi和EHV衍生启动子驱动的报告基因过表达的表达载体；2)鉴定和纯化与EHV相关的新脂类；3)开发一种将表达载体结合和/或包裹在脂质体和EHV衍生病毒体(由病毒包膜糖蛋白和脂类组成的脂质体)中的方法；和4)用报告基因构建在E.huxleyi中进行转基因实验。该奖项由EDGE计划和整合组织系统司的行为系统集群和地质科学局的生物海洋学计划共同资助。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。

Marine phytoplankton collectively contribute more than half of Earth's oxygen and are responsible for 90% of photosynthetically derived carbon in the oceans, making them the basis of marine food webs. The dominant eukaryotic members of this group are diatoms, dinoflagellates, and haptophytes. Haptophytes include coccolithophores, which contribute approximately one-third of the total marine calcium carbonate production through calcite-based biomineral cell walls (coccoliths) and profoundly impact carbon cycling. Their global distributions and associated activities are so extensive that they can be seen from Earth-orbiting satellites. Despite the wealth of knowledge gained through past experimental studies on the physiology, calcification, photosynthesis, and host-virus interactions in these organisms, there is a critical gap in understanding the genetic and cellular mechanisms controlling the responses of coccolithophores to changes in their environment. This gap is largely due to a chronic lack of scientific tools to experimentally manipulate gene expression and elucidate the function of key genes. This project aims to develop a novel approach for genetically manipulating coccolithophores leveraging the well-characterized lipid-based interaction between coccolithophore cells and viruses that routinely infect them at sea. This work will uniquely enable future studies on a wide range of physiological processes ranging from biomineralization to photosynthesis to virus infection, allowing for fundamental insight into the genetic and biochemical controls of key biological processes that impact these globally distributed and ecologically important organisms. Emiliania huxleyi is a globally dominant coccolithophore capable of forming large blooms visible from space. It contributes approximately one-third of the total marine calcium carbonate production through the biomineralization of calcite-based cell walls (coccoliths) and profoundly impacts the marine carbon and sulfur cycles. Consequently, it has emerged as a model marine microeukaryote to understand the ecophysiology and cellular mechanisms of calcification, photosynthesis, host-virus interactions, and key biological processes that impact upper ocean ecology and biogeochemistry. However, despite the availability of genomic and transcriptomic sequence data, the inability to manipulate gene expression creates a severe bottleneck in using functional genomics to understand how environmental changes impact this ecologically relevant organism. This project aims to address this gap by developing a novel, lipid-based transfection method in E. huxleyi that takes advantage of decades of research into host-virus interactions in these organisms that have revealed a specific lipid-mediated interaction between E. huxleyi and its associated Coccolithoviruses, EhVs. Specific goals are to: 1) generate expression constructs for the overexpression of reporter genes driven by E. huxleyi and EhV-derived promoters; 2) identify and purify novel lipids associated with EhVs; 3) develop a method for associating and/or encapsulating plasmid expression constructs in liposomes and EhV-derived virosomes (liposomes composed of viral envelope glycoproteins and lipids); and 4) perform transfection experiments in E. huxleyi with reporter gene constructs.This award was co-funded by the EDGE program and the Behavioral Systems Cluster in the Division of Integrative Organismal Systems and the Program in Biological Oceanography in the Directorate of Geosciences.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.