Enhancing plant productivity using engineered microbes

农业系统的生产力和复原力面临多重挑战，包括气候变化、日益增长的需求以及对更可持续的化肥来源的需求。微生物-植物相互作用的生物技术潜力已被用于农艺领域。内生菌是生活在植物内部组织中而不会引起疾病的微生物，能够调节植物的发育，提高植物的抗逆性和抗病能力，抑制病原菌的毒力和竞争植物的发育，并将土壤中的养分输送到植物中。因此，利用植物生长促进细菌(PGPB)作为减少化肥、杀菌剂、杀虫剂和除草剂的替代品受到越来越多的关注。该项目的目标是在不对寄主植物进行遗传操作的情况下，设计植物内生关系，但开发细菌合成生物学模式生物。为此，对根际革兰氏阴性菌(假单胞菌)之间的相互作用进行研究。CT364)和拟南芥植物将进行研究和工程。该菌株是最近由其中一位主管分离出来的，其定殖和刺激拟南芥生长的能力以及PGP的其他特征，如磷的溶解和植物激素和铁载体的合成和释放得到了证实。该项目有三个不同的目标，将在不同的实验室轮换完成。第一个目标是研究细菌基因组学，以确定哪些基因组特征与内生生活方式和植物生长促进有关或参与。根据该菌株的测序读数，基因组已被注释，揭示了该菌株的主要特征。之后，进行了系统发育研究、基因组挖掘和比较分析，揭示了大量潜在的PGP性状。此外，还概述了其他内生菌和病原菌假单胞菌之间的相似和差异，以突出可能解释生态位专一性的一组独特的基因。第二个目标是开发该菌株的分子工具箱，以优化培养条件、细菌转化、细菌基因组工程、细菌特性、植物接种程序以及表征可重复生物工程所需的接种剂、报告基因、复制起点和可选标记等潜在安全风险。在这一阶段结束时，将根据体内/体外确认的菌株特征和潜在的可修改特性来建立未来和确定的研究和工程方法，以便在植物上创造效益。第三个目标是研究植物(A.thaliana)对菌株定殖(P.sp.CT364)和工程菌株所产生的变化。为此，将描述短期信号和长期发育的生理和形态变化。因此，将容易和快速地设计出一种模式植物/内生菌关系，为植物寄主引入新的功能，并探索植物与其微生物之间的分子关系。CT364基因组为深入了解植物生长促进和保护的分子、生理生化特性提供了信息。转录学、代谢组学、诱变学和基因组工程的结合将允许为假定的基因和途径分配新的功能，评估菌株的代谢潜力，并增强和创造促进植物受益的细菌特性。这些发现可以制定策略，从使用内生细菌来改善植物健康和可持续应用于农业的生物量中获利。

The productivity and resilience of agricultural systems face multiple challenges including climate change, increasing demand and the need for more sustainable sources of fertilizers. The biotechnological potential of microbe-plant interactions have been purposed in the agronomic area. Endophytes, microbes that inhabit internal tissues of plants without causing disease, are able to modulate plant development, increase plant stress tolerance and disease resistance, suppress virulence in pathogens and development of competitor plant species and carry nutrients from the soil into plants. As a result, there is a growing interest in using plant growth-promoting bacteria (PGPB) as an alternative to reduce fertilizers, fungicides, insecticides and herbicides.The objective of this project is to engineer plant endophytic relationships without genetically manipulating the host plant but developing a bacterial synthetic biology model organism. For that purpose, the interactions between a gram negative rhizospheric bacteria (Pseudomonas sp. CT364) and the plant Arabidopsis Thaliana will be studied and engineered. This strain was isolated recently by one of the supervisors and the ability to colonize and stimulate A. thaliana growth was confirmed as well as other PGP features like phosphorous solubilization and plant hormone and siderophore synthesis and release. The project has three different objectives which will be completed in different laboratory rotations. The first objective is to study the strain bacterial genomics in order to define which genomic features are related or involved in the endophytic lifestyle and plant growth enhancement. From the sequencing reads of the strain the genome has been annotated revealing the main traits of the strain. After that, a phylogenetic study, genome mining and a comparative analysis have been performed unravelling a high number of potential PGP traits. Moreover, similarities and divergences between other endophyte and pathogen Pseudomonas strains were outlined to highlight the unique set of genes that may possibly account for specificity in niche occupation. The second objective is based into develop a molecular toolkit of the strain in order to optimize culture conditions, bacterial transformation, bacterial genome engineering, bacterial characterization, plant inoculation procedures as well as characterizing safety potential risks as inoculant, reporter genes, origins of replication and selectable markers required for reproducible bioengineering. At the end of this stage, the future and definitive research and engineering method will be stablished depending on the in vivo/vitro confirmed strain features and the potentially modifiable traits in order to create a benefit in plants. The third objective is to examine the plant (A. thaliana) responses and signalling to strain colonization (P. sp. CT364) and the changes created by the engineered strains. For that, short-term signalling and long-term developmental physiological and morphological changes will be characterized. As a result, a model plant/endophyte relationship will be readily and rapidly engineered to introduce new functionality to the plant host and to probe the molecular relationships between plants and their microbiome.Overall, the Pseudomonas sp. CT364 genome provides information to deeper understand the molecular, physiological and biochemical characteristics of plant growth promotion and protection. A combination of transcriptomics, metabolomics, mutagenesis and genome engineering will allow assigning new functions to putative genes and pathways, assess the metabolic potential of the strain and enhance and create bacterial traits that promote a benefit to the plant. These findings can develop strategies to profit from the use of endophytic bacteria to improve plant health and biomass applicable in agriculture in sustainable way.