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

微生物主宰着海洋栖息地，这些看不见的生命形式负责推动维持我们星球上生命的生物地球化学循环。也许最重要的是浮游植物，因为这些微藻执行地球上一半的光合作用，从大气中去除二氧化碳并产生氧气。其中一个普遍存在和丰富的是Emiliania huxleyi，它可以在国家大小的海洋中形成水华。除了通过光合作用固定碳外，它还可以将碳矿化成覆盖其表面的方解石盘。当微藻死亡时，它们会落到海底，在那里它们可以停留超过50，000年。E. huxleyi的另一个独特之处在于，它利用生物产生的分子与海洋食物网中的较高（鱼类和鸟类）和较低（原生食草动物和病毒）热带水平相互作用。我们最近发现，E。huxleyi还与细菌密切相互作用，这些细菌可能对其宿主具有致病性，从而加速方解石的释放。一种这样的细菌，Phaeoprostenbens，杀死E。huxleyi通过其IV型分泌系统（T4 SS）递送的效应分子激活其程序性细胞死亡途径（PCD）。病毒也已知激活其PCD途径，并且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这个生态位的小组。通过对该菌在杀灭大肠杆菌过程中的所有RNA转录本进行测序（转录组学），huxleyi，我们的目标是发现参与浮游植物细菌致病的基因。然后，我们将进行适应性实验，其中将> 200，000个突变体（RB-TnSeq）的文库应用于它们的宿主E。huxleyi，以及那些在发病过程中表现不足和过度的，可以被确定为健身决定因素。我们还将使用这个突变体库来进一步表征从转录组学和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如何靶向E。huxleyi和将使我们成为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.