Xanthomonas axonopodis pv. citri (Xac) is a gram-negative bacteria able to infect specific plant hosts and is the causal agent of the citrus canker a disease of significant importance to the citrus-related economy. Xac during the infective process macromolecular pathogenic factors are secreted and injected into the host cell via the type III secretion system (TTSS). The TTSS is coded by a group of ca. 25 genes, most of which are localized in a single chromosomal locus (hrp "hypersensibility response and pathogenicity"). Some products encoded by these genes are conserved in all TTSS and have homologs with core flagellar components. Previously, we used two hybrid analysis to identify uncharacterized protein-protein interactions involving components, substrates and regulators of the TTSS of Xac. One of the protein-protein interactions identified was that involving HrpB2 and the HrcU C-terminal domain. HrcU, a conserved member of the TTSS superfamily, is a bacterial inner membrane protein with a large cytoplasmic C-terminal domain. HrcU is a paralog of the flagellar protein FlhB and an ortholog of YscU from the TTSS of Yersinia pseudotuberculosis. FlhB and YscU both undergo site-specific proteolytic cleavage at a conserved Asn-Pro-Thr-His (NPTH) sequence, also found in Xac HrcU (residues 264-267). HrpB2 is secreted into the culture medium in an hrp-dependent manner and is essential for type III protein secretion in Xanthomonas campestris pv. vesicatoria. In this report, we characterize more fully the interaction between HrpB2 and the HrcU C-terminal domain by way of overlay, fluorescence spectroscopy and pulldown experiments using purified recombinant proteins. We observed that the recombinant protein, HrcU_207-357 suffered a cleavage in the conserved Asn264-Pro265-Thr266-His267 sequence. The HrcU_207-357 cleavage product (MW = 6,9kDa), was purified, analysed and identificated by Edman degadation, western blot and mass spectometry. The fragment corresponded to residues 207-264 (HrcU_207-264) and does not interact with HrpB2 protein. We mutated the conserved NPTH sequence (N264A, P265A and T266A). The mutated HrcU_207-357AAAH was not cleaved and recovered the ability to interact with HrpB2. Finally, we produced the recombinant protein HrcU_277-357 which corresponds to the sequence following the NPTH cleavage site. This fragment was found to interact with HrpB2. These results indicated that 1) the NPTH sequence in HrcU is not necessary for interaction with HrpB2 and 2) the site for interaction is located in the sequence after the NPTH site. In a manner similar to that observed for the ordered assembly of bacterial flagella, the cleavage of the C-terminal domain of HrcU protein may be an important step in the orderly assembly of the TTSS. The secretion of HrpB2 may be a prerequisite for the subsequent secretion of TTSS pilus subunits and effector molecules.