Interplay between SpaO variants shapes the architecture of the type III secretion sorting platform.

utilizes a virulence-associated type III secretion system (T3SS) to inject bacterial effectors directly into host cells. Central to this machinery is the sorting platform (SP), a cytosolic assembly whose core scaffolding protein, SpaO, is produced in two isoforms: a full-length (SpaO) and a shorter variant (SpaO) comprising the C-terminal 101 residues of SpaO. Although SpaO is evolutionarily conserved across type III secretion systems, its precise function has remained elusive. Here, we combined a sensitive, real-time translocation assay with site-directed photo-crosslinking to elucidate the role of SpaO in SPI-1 T3SS. We found that while SpaO is not absolutely required for effector secretion, its absence significantly dampens T3SS-mediated protein delivery. Further biochemical and structural probing revealed that SpaO is a structural component of the sorting platform, arranged as a homodimer associated to SpaO via an N-terminal "docking motif." This interaction occurs while SpaO is associated with other SP components, supporting a model in which SpaO is integrated into the SP pods alongside SpaO, OrgA, and OrgB. Collectively, these findings show that SpaO, while not strictly essential, functions as a critical structural component of the sorting platform, providing new insights into how and related bacteria assemble and maintain these specialized protein-injection systems.