We describe the first two-partner secretion system known to play a role in mutualistic plant–bacterial interactions, identified in the soil and rhizosphere-colonizing bacterium Pseudomonas putida KT2440. The genes coding for the two components of the system are organized in an operon, which we have named hlpBA. HlpA is a secreted protein that has similarities with iron-regulated haemolysins, while HlpB would be responsible for the activation and transport of HlpA across the outer membrane. Mutations in this novel two-partner secretion system result in reduced capacity to colonize corn seeds. When introduced in the rhizosphere, hlpA and hlpB mutants show no competitive disadvantage, but the number of cells attached to the root surface is reduced with respect to the wild type, suggesting this protein plays a role directly in the bacterial cell–root surface interaction. Under iron-limiting conditions, the presence of a truncated HlpA causes reduced viability and high levels of siderophore release. These data further strengthen our previous observations indicating the importance of iron acquisition for attachment of P. putida KT2440 to plant surfaces.