(387d) Engineering Sucrose Exchange in the Rhizosphere

The plant root microbiome is a nexus for carbon exchange between plants and their environment. Root-exuded carbon supports the colonization, growth, and phytobeneficial activities of root-colonizing bacteria (rhizobacteria), which in turn exert substantial influence on soil carbon sequestration, plant health/disease resistance, and food and bioenergy crop productivity. While boosting exudate exchange between plants and target rhizobacteria could enable breeding and design of beneficial plant microbiomes, the molecular, genetic, and physiological design rules of exudate exchange remain poorly defined. As primary metabolites (sugars, organic acids, and amino acids) constitute the bulk of root exudates and are key carbon sources for rhizobacterial growth, they are attractive targets for metabolic engineering of root microbiomes. The disaccharide, sucrose, is particularly noteworthy as an exudate target, given its role as the primary plant photosynthesis product and high abundance within the root environment. Since many rhizobacterial chassis do not natively catabolize sucrose, we hypothesized that transgenic plants could be paired with engineered rhizobacterial strains to selectively boost transkingdom exchange and utilization of this sugar. This was tested by introducing genetic circuitry within Arabidopsis thaliana and Pseudomonas putida KT2440 as model plant and bacterial species, respectively. Using high throughput hydroponic growth assays and live imaging microscopy, we interrogated how modulated expression of sucrose transporters in Arabidopsis paired with sucrose-utilizing P. putida impacts bacterial fitness in the root environment relative to non-utilizing wild-type strains. Metabolomics analyses was also used to understand how root exudates drive P. putida catabolite preferences, which enabled strain engineering for optimal sucrose utilization. Collectively, these results inform how plant and bacterial synthetic biology strategies can be leveraged to engineer root microbiomes and potentially applied to broader exudate targets.