Host-microbe systems in the ocean: identifying genetic determinants of Phaeobacter inhibens pathogenesis of the phytoplankton Emiliania huxleyi

微生物主导着海洋栖息地，这些看不见的生命形式负责推动维持地球上生命的生物地球化学循环。也许最重要的是浮游植物，因为这些微藻完成了地球上一半的光合作用，从大气中清除二氧化碳并产生氧气。其中一种无处不在且数量丰富的植物是埃米利亚尼亚花，它可以在与国家一样大的海洋中形成水华。除了通过光合作用固定碳之外，它还可以将碳矿化成覆盖其表面的方解石圆盘。当微藻死亡时，它们会掉到海底，在那里它们可以停留超过5万年。E.huxleyi的独特之处还在于，它使用生物产生的分子与海洋食物网中较高水平(鱼类和鸟类)和较低水平(原生动物、食草动物和病毒)相互作用。我们最近发现E.huxleyi还与细菌密切相互作用，细菌可以在宿主上致病，从而加速方解石的释放。其中一种细菌，Phaebacter Inhibens，通过激活其程序性细胞死亡途径(PCD)，通过其IV型分泌系统(T4SS)传递的效应分子来杀死E.huxleyi。众所周知，病毒还可以激活其PCD途径，而这种细胞裂解是微生物循环中的关键步骤。我们是第一个表明细菌也占据了这个利基的小组。通过对P.hihibens基因(转录组学)在杀死E.huxleyi时的所有RNA转录本进行测序，我们的目标是发现与浮游植物细菌致病相关的基因。然后，我们将进行适应性实验，其中&gt；200,000个突变体(RB-TnSeq)的文库将应用于它们的宿主E.huxleyi，那些在发病过程中被低估和超过的突变体可以被确定为适应性决定因素。我们还将使用这个突变文库来进一步表征从转录组和RB-TnSeq中鉴定的遗传网络和途径。这项研究计划将阐明这种新的病原体是如何针对E.huxleyi的，并将使我们能够第一个确定浮游植物病原体的遗传决定因素。

Microbes dominate marine habitats and these invisible life forms are responsible for driving the biogeochemical cycles that sustain life on our planet. Perhaps most important are phytoplankton, as these microalgae perform half of the photosynthesis on earth, removing carbon dioxide from the atmosphere and generating oxygen. One ubiquitous and abundant of these is Emiliania huxleyi, which can form blooms in the oceans as large as countries. Besides fixing carbon through photosynthesis, it can also mineralize it into calcite disks that cover its surface. These fall to the bottom of the ocean when the microalgae dies, where they can stay for over 50,000 years. E. huxleyi is also unique in that it uses biologically produced molecules to interact with higher (fish and birds) and lower (protist grazers and viruses) tropic levels within the marine food web. We have recently identified that E. huxleyi also closely interacts with bacteria, which can be pathogenic on their host and thereby accelerate release of calcite. One such bacterium, Phaeobacter inhibens, kills E. huxleyi by activating its programmed cell death pathway (PCD) through an effector molecule delivered through its type IV secretion system (T4SS). Viruses are also known to activate its PCD pathway and this cell lysis is a critical step in the microbial loop. We are the first group to show bacteria also occupy this niche. By sequencing all the RNA transcripts of P. inhibens genes (transcriptomics) when it is killing E. huxleyi, we aim to discover genes involved in bacterial pathogenesis of phytoplankton. We will then perform fitness experiments where a library of >200,000 mutants (RB-TnSeq) will be applied to their host, E. huxleyi, and those which are under and over represented during pathogenesis can be identified as fitness determinants. We will also use this mutant library to further characterize the genetic networks and pathways identified from the transcriptomics and RB-TnSeq in this pathogenic niche. This research proposal will elucidate how this novel pathogen targets E. huxleyi and will allow us to be the first to identify the genetic determinants of a phytoplankton pathogen.