Carbon Metabolic Versatility of Agrobacterium tumefaciens in Tomato Roots and Galls

Agrobacterium tumefaciens is a facultative plant pathogen that forms galls on a wide range of hosts and persists in soil, roots and galls through extensive metabolic versatility. Here, we combined genome-wide transposon sequencing (TnSeq), metabolomics and reverse genetics to identify carbon utilisation pathways supporting A. tumefaciens fitness in tomato roots and galls. TnSeq screening across 21 carbon sources, representative of rhizosphere exudates, root metabolites and gall-derived compounds, identified conserved and substrate-specific fitness determinants, including central metabolic enzymes, transporters and previously uncharacterized catabolic genes. Comparison with in planta TnSeq data revealed environment-dependent metabolic requirements, highlighting fluxes through the Entner-Doudoroff pathway, the TCA cycle and gluconeogenesis. Quinic acid catabolism emerged as a major determinant of fitness specifically in galls, where this compound accumulated. Deletion of pcaC (ATU_RS21295/atu4541) impaired growth on quinic and protocatechuic acids and reduced competitiveness during gall colonisation, with no effect in roots. Together, this work provides a system-level framework for understanding how A. tumefaciens exploits plant-derived nutrients in host-associated environments.