The type IV secretion system component VirB5 binds to the trans-zeatin biosynthetic enzyme Tzs and enables its translocation to the cell surface of Agrobacterium tumefaciens

McMaster University, Department of Biology, 1280 Main St. West, Hamilton, ON L8S 4K1, Canada; Ludwig-Maximilians-Universität, Department Biologie I, Bereich Mikrobiologie, Maria-Ward-Str. 1a, D-80638 München, Germany; Max-Planck Institute of Biochemistry, Protein Analysis, Am Klopferspitz 18, D-82152 Martinsried, Germany

^* To whom correspondence should be addressed. Email: baronc{at}mcmaster.ca.

	Abstract

VirB5 is a minor component of the extracellular T-pilus determined by the Agrobacterium tumefaciens type IV secretion system. To identify proteins that interact with VirB5 during the pilus assembly process, we purified it as a recombinant fusion protein and using a gel overlay assay, we detected a 26 kDa interacting protein in Agrobacterium cell lysates. The VirB5-binding protein was purified from A. tumefaciens and identified as the cytokinine-biosynthetic enzyme Tzs. The VirB5-Tzs interaction was confirmed using pulldown assays with purified proteins and the yeast two-hybrid system. Analysis of the subcellular localization in A. tumefaciens showed that Tzs was present in the soluble as well as the membrane fraction. Tzs was extracted from the membranes with the mild detergent dodecyl--D-maltoside in complexes of different molecular masses and this association was strongly reduced in the absence of VirB5. Using immuno-electron microscopy, Tzs was also detected on the Agrobacterium cell surface. A functional type IV secretion system was required for efficient translocation to the surface, but Tzs was not secreted into the cell supernatant. The fact that Tzs localizes on the cell surface suggests that it may contribute to the interaction of Agrobacterium with plants.