Deciphering the functions of type III secretion system components from the plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria

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The Gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria is the causal agent of bacterial spot disease in pepper and tomato plants. Pathogenicity of X. campestris pv. vesicatoria depends on a type III secretion (T3S) system, which translocates effector proteins into eukaryotic cells. T3S systems consist of a membrane-spanning secretion apparatus, which is associated with an extracellular pilus-like appendage and a channel-like translocon in the eukaryotic plasma membrane. In the past funding period, we investigated the functions of HrcU and HrcV, which are inner membrane components of the T3S system from X. campestris pv. vesicatoria, and the predicted cytoplasmic ring protein HrcQ. The results of mutant approaches, localization and interaction experiments suggest that HrcU, HrcV and HrcQ serve as docking sites for secreted proteins and that the cytoplasmic domain of HrcU is involved in the control of T3S substrate specificity. In the present project, we propose to characterize the functions of the conserved HrcR, HrcS and HrcT proteins, which are predicted components of the T3S export apparatus in the inner membrane and are essential for T3S. Preliminary experiments revealed that HrcS forms protein complexes, which associate with the bacterial membranes. We plan to investigate a possible oligomerization of HrcR, HrcS and HrcT as well as interactions with components and substrates of the T3S system. Furthermore, we will identify functionally important protein regions by mutant approaches. In the second part of this project, we will investigate the function of HrpB7, which is conserved in Xanthomonas spp. but has not yet been functionally characterized. Mutant studies and biochemical approaches revealed that HrpB7 is essential for T3S and forms protein complexes, which associate with the bacterial membranes upon activation of the T3S system. We plan to identify HrpB7 interaction partners and to analyse the contribution of conserved amino acid motifs in HrpB7 to protein function and complex formation.