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

海洋浮游植物共同贡献了地球一半以上的氧气，并负责海洋中90%的光合作用产生的碳，使其成为海洋食物网的基础。这一组的主要真核生物成员是硅藻，甲藻和附着植物。附着植物包括颗石藻，它们通过方解石基生物矿物细胞壁（颗石藻）贡献了海洋碳酸钙总产量的约三分之一，并对碳循环产生深远影响。它们的全球分布和相关活动非常广泛，以至于可以从地球轨道卫星上看到。尽管通过过去的实验研究，在这些生物体的生理学，钙化，光合作用和宿主-病毒相互作用获得了丰富的知识，有一个关键的差距，在理解的遗传和细胞机制控制的反应颗石藻在他们的环境变化。这一差距主要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方法，用于遗传操纵颗石藻，利用颗石藻细胞和在海上经常感染它们的病毒之间的基于脂质的相互作用。这项工作将独特地使未来的研究范围广泛的生理过程，从生物矿化到光合作用，病毒感染，使基本的洞察到关键的生物过程，影响这些全球分布和生态重要的生物体的遗传和生化控制。 Emiliania huxleyi是一种全球性的优势颗石藻，能够形成从太空可见的大花朵。它通过方解石基细胞壁（球石）的生物矿化作用贡献了海洋碳酸钙总产量的约三分之一，并深刻影响了海洋碳和硫循环。因此，它已成为一种模式海洋微真核生物，以了解生态生理学和钙化，光合作用，宿主-病毒相互作用的细胞机制，以及影响上层海洋生态和海洋地球化学的关键生物过程。然而，尽管基因组和转录组序列数据的可用性，无法操纵基因表达创建了一个严重的瓶颈，使用功能基因组学来了解环境变化如何影响这种生态相关的生物体。该项目旨在通过开发一种新的基于脂质的E. huxleyi，利用了几十年来对这些生物体中宿主-病毒相互作用的研究，这些研究揭示了E. huxleyi及其相关的球石病毒，EhVs。 具体目标是：1）产生用于由E驱动的报告基因过表达的表达构建体。huxleyi和EhV衍生启动子; 2）鉴定和纯化与EhV相关的新脂质; 3）开发用于将质粒表达构建体结合和/或包封在脂质体和EhV衍生病毒体（由病毒包膜糖蛋白和脂质组成的脂质体）中的方法;和4）在E.该奖项由EDGE计划和综合有机系统部的行为系统集群以及地球科学理事会的生物海洋学计划共同资助。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估来支持。

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.