Xanthomonas axonopodis pv. citri (Xac) is a gram-negative bacterial phytopathogen that infects citrus. One possible virulence determinant is a chromosomally encoded Type IV Secretion System (T4SS), a multiprotein complex that spans the bacterial periplasm and both inner and outer membranes. The T4SS is used by some bacteria to secrete proteins and/or DNA to the extracellular milieu or the host interior. The model T4SS from Agrobacterium tumefaciens is made up of twelve structural proteins: VirB1-VirB11 and VirD4. The Xanthomonas T4SS is apparently incomplete because of the lack of a polypeptide with sequence similarity to VirB7. The aim of this project is the study of structure-function relationships in the Xanthomonas T4SS. Twenty-three T4SS protein-coding genes, including full-length proteins or domains, were cloned and the proteins were produced in different E. coli strains. Thirteen polypeptides were purified and some of them were submitted to structural, spectroscopic and protein-protein interaction studies. We used NMR to solve the solution structure of Xac2622_24-139 which consists of an unfolded N-terminal segment of ~30 residues followed by a globular domain. Xac2622_24-139 oligomerizes in fast exchange at the NMR time scale and interacts via its unfolded N-terminus with the VirB9 C-terminus (VirB9_154-255) in slow exchange. NMR analysis showed that VirB9_154-255 has a flexible structure in solution. However, this polypeptide undergoes a significant conformational modification in the presence of Xac2622,_24-139 and both proteins become rigid upon interaction. Xac2622 is the Xanthomonas VirB7, based on the chromosomal localization of its gene, predicted subcellular localization and protein interaction analysis. But surprisingly, unlike other VirB7 proteins, Xac2622 has an extra C-terminal folded domain whose topology and structure are strikingly similar to that of periplasmic domains found in outer membrane proteins of many bacterial Secretion Systems. Knockout of the xac2622 gene, however, does not affect the Xac virulence in orange leaf infection assays. The Xac2622 folded domain was also crystallized, and these crystals diffracted up to 1.0 Å resolution and belong to the space group C222₁. The preliminary refined model has R_factor of 0.121 and R_free of 0.147. Crystals of three other T4SS proteins have been obtained, but only one of them diffracted to high resolution (2.0 Å; space group C2). Xac2610 is a hypothetical protein whose gene is located in the T4SS locus, and its interactions were studied with VirB9, VirB11 and Xac2609, a putative T4SS substrate. The potential T4SS secretion signal is a conserved, approximately 115 residues, C-terminal domain found in the putative substrates of the Xanthomonas T4SS. This sequence mediates interactions with VirD4. We have characterized this domain from one substrate and it is mainly unfolded. This observation may have functional implications, as the substrates are unfolded before their secretion through the T4SS channel