Type III secretion system diversity in the Pseudomonas fluorescens species complex.

Type III secretion systems (T3SSs) play important roles in mediating interactions between bacteria and eukaryotic hosts by delivering effectors into host cells, yet their genetic diversity and evolutionary dynamics among rhizosphere-associated members of Pseudomonas fluorescens species complex (PFSC) remain poorly understood. We conducted a genome-based evolutionary analysis of T3SSs across a global collection of PFSC strains to investigate their phylogenetic evolution and diversity. The 88 T3SS-carrying strains were assigned to seven PFSC subgroups and clustered into six phylogenetically distinct T3SS categories, with 19 strains representing 13 previously undescribed species, highlighting substantial taxonomic diversity. Phylogenetic reconstructions showed that T3SSs formed a monophyletic clade sister to flagellar systems, suggesting a shared evolutionary origin, whereas variable GC content, the presence of mobile genetic elements, and incongruence between T3SS and species phylogenies suggested that horizontal gene transfer (HGT) has played an important role in T3SS evolution within the PFSC. T3SS-harboring strains possessed diverse genomic features, including genes previously associated with plant-growth-promoting functions and encoded atypical T3SS-associated proteins, while representative Rsp I and Rsp II strains retained partial effector translocation activity. Together, these findings reveal extensive diversification of T3SSs in rhizosphere-associated PFSC strains through HGT and provide a phylogenomic framework for future functional studies.