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而不会引起疾病。许多内生菌已被证明可以促进植物生长和恢复力。基于使用化肥和农药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改变或从粮食作物中消失。然而，生物能源作物如芒草，在很大程度上是未驯化的，因此保留了独特而有益的微生物群落，代表了相对未受影响的资源。该项目将研究这些内生菌的分子和代谢功能，从而提供植物-微生物相互作用的知识。这将影响生物肥料的发展，可能增加作物的适应性和产量，并减少生物燃料所需的农业用地。这种方法也可以应用于农作物，通过操纵内生菌群落提高产量，减少对化肥的依赖。细菌内生菌与其自由生活的或致病的亲属相比通常具有减小的基因组大小。据认为，宿主提供了稳定的微环境。因此，内生生活方式的进化适应往往与基因转移率低和相对较少的移动的遗传因素相关。随着时间的推移，自由生活/致病性生活方式所需的相对大量的遗传物质可能会丢失，导致小而瘦的基因组改变代谢和信号通路。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，包括来自芒属植物营养组织和种子的内生菌，以及生长在盐水和重金属环境中的植物。这些数据现在已经可用，Swain博士已经进行了全基因组组装并对这些数据进行了初步分析。在这个项目中，我们将测试内生菌减少了共生优化的基因组并对植物有益的假设，包括增加产量和/或对非生物胁迫的恢复力。目的：1.新培养的内生菌与自由生活/致病亲属的全基因组比较。2.使用激光辅助快速蒸发电离质谱法（REIMS）对培养物和植物中的内生菌进行代谢组学分析。3.整合基因组和代谢组学数据集，以确定内生菌中过度和不足的特定途径。4.代谢途径与植物生长促进在植物与非生物胁迫的关联。最初，该项目将完善基因组组装，包括战略性地进行额外测序，以在需要时生成更长的组装序列。一旦基因组组装已经被精炼，将选择最佳子集来研究基因组修饰/减少。内生菌基因组将与它们的野生和致病亲属进行比较，使用已经在公共存储库（如NCBI）中的序列。染色体结构，操纵子结构，移动的元素，和进化选择压力下的基因（确定的同义与非同义序列突变的比例）的差异将进行调查，以深入了解不同的进化力量对这些内生菌。代谢组学实验将提供有关受此过程影响的关键基因，酶和代谢途径的额外证据。此外，通过鉴定内生菌基因组中缺失的重要代谢途径，将有可能推断内生菌从其宿主获得哪些代谢物;并且可以阐明内生菌群体内的代谢串扰。最后，信息学预测将验证和完善与在植物实验。

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.