Summary Erwinia amylovora is a Gram-negative bacterium responsible for fire blight, a necrotic disease affecting plants of the Rosaceae family. E. amylovora virulence is dependent on a functional type III secretion system. To date, four proteins have been shown to travel through this secretion system: HrpN, HrpW, HrpA, and DspA/E. Next to dspA/E, dspB/F encodes a small acidic protein sharing features similar to those of type III secretion chaperones described in animal systems. Here, we show that DspA/E was not secreted in a dspB/F background, while other known type III secreted proteins (HrpN, HrpW, and HrpA) remained secreted to wild-type level. A functional copy of dspB/F introduced in trans in a dspB/F background restored DspA/E secretion. Further analysis showed that DspA/E was not detected in a dspB/F background. This effect was post-transcriptional as the expression of a dspA/E::uidA transcriptional fusion was not reduced in a dspB/F background. Affinity blot analysis experiments demonstrated that DspB/F specifically interacts with DspA/E in vitro. Therefore, DspB/F acts as a specific DspA/E chaperone and DspB/F prevents intrabacterial DspA/E degradation. dspB/F mutants were found to retain some pathogenicity to pear seedlings. This phenotype contrasts with the non-pathogenic phenotype of the dspA/E mutant and suggests that the DspA/E protein still transits through the type III secretion machinery in a dspB/F mutant even though it is not detected in vitro.