Comparative transcriptomics reveals context- and strain-specific regulatory programmes of Agrobacterium during plant colonization.

Agrobacterium is a genus of plant-associated bacteria capable of transferring DNA into host genomes to induce tumourigenesis. The process has been primarily studied in a few model strains, particularly C58, and developed into Agrobacterium-mediated transformation for genetic manipulation. However, the diversity of wild-type strains and their context-specific regulatory responses remains poorly characterized. Here, we evaluated five wild-type strains and identified 1D1108 as superior in tumourigenesis on legumes and transient transformation in Nicotiana benthamiana. Under in vitro virulence induction with acetosyringone, we identified 126 differentially expressed genes (DEGs) in 1D1108. Although the number of DEGs was comparable to those in C58 and the legume isolate 1D1609 under the same condition, only 22 DEGs, primarily within the vir regulon, were conserved, indicating extensive divergence among these Agrobacterium strains. Leaf infiltration of N. benthamiana revealed 1,134 DEGs specifically regulated in planta for 1D1108. These included genes involved in attachment, virulence regulation, type IV pilus, succinoglycan biosynthesis and diverse nutrient transporters, providing new evidence on expression regulation during colonization. Comparative analyses of in planta transcriptomes with C58 and Pseudomonas syringae DC3000 revealed distinct secretion systems required for pathogenesis, namely, type IV for Agrobacterium and type III for Pseudomonas, and only ~5-19% of DEGs were conserved. These limited transcriptomic overlaps underscore the importance of studying gene expression in strains and conditions directly relevant to the biological context, rather than relying on model systems. Together, this work reveals how environmental and host-associated cues shape transcriptional responses in plant-associated bacteria.