Identifying beneficial plant-microbial interactions using multi-omic approaches

内生菌是微生物，通常是细菌或真菌，它们生活在特定的植物组织中，不会引起疾病。许多内生菌已被证明可以促进植物的生长和恢复能力。以使用化肥和杀虫剂为基础的集约化育种计划忽视了植物与其微生物之间的相互作用，结果是许多有益的微生物相互作用已经被修改或从粮食作物中消失。然而，像芒属这样的生物能源作物在很大程度上是未被驯化的，因此保留了一个独特的、有益的微生物群落，代表着一种相对未被开发的资源。这个项目将研究这些内生菌的分子和代谢功能，从而提供关于植物-微生物相互作用的知识。这将影响生物肥料的发展，潜在地提高作物的抗逆性和产量，并减少生物燃料所需的农地数量。这种方法还可以转移到农作物上，通过操纵内生菌群落来提高产量，并减少对化肥的依赖。与其自由生活或致病的近亲相比，细菌内生细菌的基因组大小往往较小。寄主被认为提供了一个稳定的微环境。因此，进化上对内生生活方式的适应往往与较低的基因转移率和相对较少的可移动遗传元素相关。随着时间的推移，自由生活/致病生活方式所需的相对大量的遗传物质可能会丢失，导致代谢和信号通路改变的小而瘦的基因组。Farrar博士的实验室最近分离并对40多种内生菌进行了植物生长研究和全基因组测序，其中包括来自芒属营养组织和种子的内生菌，以及生长在盐碱和重金属环境中的植物。这些数据现在已经可用，斯温博士已经对这些数据进行了全基因组组装和初步分析。在这个项目中，我们将检验这一假设，即内生菌减少了为共生而优化的基因组，并对植物有好处，包括增加产量和/或对非生物胁迫的恢复能力。目的：1.新培养的内生菌与自由生活/致病亲缘关系的全基因组比较。2.利用激光辅助快速蒸发电离质谱仪(REIMS)对植物内生菌进行代谢组学分析。3.整合基因组和代谢数据集，以确定在内生菌中表达过多和表达不足的特定途径。4.植物在非生物胁迫和非生物胁迫下代谢途径与植物生长促进的关系。最初，该项目将完善基因组组装，包括战略性地执行额外的测序，以在需要的情况下产生更长的组装序列。一旦基因组组合被细化，将选择一个最佳的子集来研究基因组的修改/减少。内生菌基因组将与它们的野生和病原菌近亲进行比较，使用已经在公共储存库(如NCBI)中的序列。我们将研究在进化选择压力下染色体结构、操纵子结构、可移动元件和基因的差异(通过同义序列突变与非同义序列突变的比率来确定)，以深入了解作用于这些内生菌的不同进化力量。代谢组学实验将提供关于受这一过程影响的关键基因、酶和代谢途径的更多证据。此外，通过识别内生菌基因组中缺失的重要代谢途径，可以推断内生菌从寄主那里获得哪些代谢物；并且内生菌种群内的代谢串扰可以被阐明。最后，信息学预测将在平面实验中得到验证和改进。

Endophytes are microbes, usually bacteria or fungi, which live within specific plant tissues without causing disease. Many endophytes have been shown to boost plant growth and resilience. Intensive breeding programs, based around the use of chemical fertilizers and pesticides, have ignored the interactions between the plant and its microbes, with the result that many beneficial microbial interactions have been modified or lost from food crops. Bioenergy crops such as Miscanthus, however, are largely undomesticated and so retain a unique and beneficial microbial community, representing a relatively untouched resource. This project will investigate the molecular and metabolic functioning of these endophytes, thus providing knowledge on plant-microbe interactions. This will impact the development of biofertilisers, potentially increasing crop resilience and yield, and reducing the amount of agricultural land required for biofuels. The approach would also have transferable applications to agricultural crops, improving yields through manipulation of the endophyte community and reducing dependence on chemical fertilisers. Bacterial endophytes often have reduced genome sizes in comparison to their free-living or pathogenic relatives. It is thought that the host provides a stable microenvironment. As a result, evolutionary adaption to an endophytic lifestyle tends to correlate with low rates of gene transfer and relatively few mobile genetic elements. Over time, a relatively large quantity of genetic material required for a freeliving/pathogenic lifestyle may be lost, leading to small, lean genomes with altered metabolic and signalling pathways. Dr Farrar's laboratory have recently isolated and performed plant growth studies and whole genome sequencing on over 40 endophytes, including endophytes from Miscanthus vegetative tissues and seed, and from plants growing in saline and heavy metal environments. These data are available now, and Dr Swain has already performed whole genome assembly and preliminary analysis of these data. In this project we will test the hypothesis that endophytes have reduced genomes optimised for symbiosis and with benefits to the plant, including increasing yield and/or resilience to abiotic stresses. Objectives: 1. Whole genome comparisons of novel cultured endophytes with free-living/pathogenic relatives. 2. Metabolomic analysis of endophytes in culture and in planta using laser assisted rapid evaporative ionisation mass spectrometry (REIMS). 3. Integrating genomic and metabolomic data sets to identify specific pathways over- and underrepresented in endophytes. 4. Association of metabolic pathways with plant growth promotion in planta with and without abiotic stress. Initially, the project will refine the genome assemblies, including strategically performing additional sequencing to generate longer assembled sequences where required. Once the genome assemblies have been refined, an optimal subset will be selected to study the genome modification/reduction. The endophyte genomes will be compared to their wild and pathogenic relatives, using sequences already in public repositories such as the NCBI. Differences in chromosome structure, operon structure, mobile elements, and genes under evolutionary selection pressure (identified by the ratio of synonymous to nonsynonymous sequence mutations) will be investigated to gain insight into the different evolutionary forces acting on these endophytes. Metabolomics experiments will provide additional evidence about the key genes, enzymes and metabolic pathways impacted by this process. Moreover, by identifying important metabolic pathways that are missing in the endophyte genomes it will be possible to infer which metabolites the endophytes acquire from their host; and the metabolic cross-talk within the endophyte populations can be elucidated. Finally, informatics predictions will be validated and refined with in planta experiments.