Enhancing plant productivity using engineered microbes

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.