Identifying beneficial plant-microbial interactions using multi-omic approaches

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.