Recognition of a translocation motif in the regulator HpaA from is controlled by the type III secretion chaperone HpaB.

The Gram-negative plant-pathogenic bacterium is the causal agent of bacterial spot disease in pepper and tomato plants. Pathogenicity of depends on a type III secretion () system which translocates effector proteins into plant cells and is associated with an extracellular pilus and a translocon in the plant plasma membrane. Effector protein translocation is activated by the cytoplasmic chaperone HpaB which presumably targets effectors to the system. We previously reported that HpaB is controlled by the translocated regulator HpaA which binds to and inactivates HpaB during the assembly of the system. In the present study, we show that translocation of HpaA depends on the substrate specificity switch protein HpaC and likely occurs after pilus and translocon assembly. Translocation of HpaA requires the presence of a translocation motif (TrM) in the N-terminal region. The TrM consists of an arginine-and proline-rich amino acid sequence and is also essential for the function of HpaA. Mutation of the TrM allowed the translocation of HpaA in mutant strains but not in the wild-type strain, suggesting that the recognition of the TrM depends on HpaB. Strikingly, the contribution of HpaB to the TrM-dependent translocation of HpaA was independent of the presence of the C-terminal HpaB-binding site in HpaA. We propose that HpaB generates a recognition site for the TrM at the system and thus restricts the access to the secretion channel to effector proteins. Possible docking sites for HpaA at the system were identified by and interaction studies and include the ATPase HrcN and components of the predicted cytoplasmic sorting platform of the system. Notably, the TrM interfered with the efficient interaction of HpaA with several system components, suggesting that it prevents premature binding of HpaA. Taken together, our data highlight a yet unknown contribution of the TrM and HpaB to substrate recognition and suggest that the TrM increases the binding specificity between HpaA and system components.