Modularization of the type III secretion system from the plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria for functional studies and protein delivery

• Applicants: Professorin Dr. Daniela Büttner; Dr. Sylvestre Marillonnet
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms ; Microbial Ecology and Applied Microbiology ; Cell Biology
• Term: since 2015
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 269267294

The Gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria uses a type III secretion (T3S) system to translocate effector proteins into plant cells. T3S systems are essential pathogenicity factors of most Gram-negative plant- and animal-pathogenic bacteria and consist of a membrane-spanning secretion apparatus, which is associated with an extracellular pilus-like appendage and a translocon in the eukaryotic plasma membrane. In the past funding period, we have generated a modular T3S system from X. campestris pv. vesicatoria by assembling all genetic elements from promoter and ORF modules using the Golden Gate-based modular cloning system MoClo. The resulting modular T3S gene cluster is functional in X. campestris pv. vesicatoria and allows the fast and efficient exchange of single genes or operons for functional studies and the insertion of reporter fusions. We used the modular T3S gene cluster to study the assembly of a fluorescent fusion of the predicted cytoplasmic (C) ring component HrcQ and showed that the efficient assembly of the C ring depends on the ATPase complex and the yet uncharacterized HrpB4 protein. Focus of the proposed project is the use and optimization of the modular T3S system for functional studies of T3S system components and protein delivery into plant cells. In the first part, we aim at the further characterization of the T3S system assembly by the analysis of fluorescent reporter fusions of cytoplasmic and membrane-associated components. Additional in vivo and in vitro interaction studies will help to specifically analyse the yet uncharacterized predicted cytoplasmic sorting platform of the T3S system. In the second part of the project, we will reengineer T3S system operons to avoid the presence of overlapping sequences in ORF modules and thus to facilitate functional studies of single genes. For this, we will use a modified version of the MoClo system which allows the insertion of ribosome binding sites (RBS) upstream of each ORF and thus the optimization of gene expression. This approach will also help to optimize T3S gene expression in other bacterial recipients in the third part of the proposed project, which will focus on the establishment of the modular T3S system as a protein delivery tool for both biotechnological approaches and fundamental research.

• DFG Programme: Research Grants