; R o o t E n t r y F g 5 @ C o m p O b j b W o r d D o c u m e n t O b j e c t P o o l :z 5 :z 5 F Microsoft Word 6.0 Document MSWordDoc Word.Document.6 ; Oh +' 0 $ H l D h R:WWUSERTEMPLATENORMAL.DOT marcia steinberg marcia steinberg @ _ 5 @ e = e { n " " " " " " " L L L L L d n L C x | | | | | | | # ? e V T 4 " | | | | | | " " | x | | | | " | " | 6 > " " " " | | 4 | 9506144 Banta The plant pathogen Agrobacterium tumefaciens causes a disease in which crown gall tumors form on susceptible plants. The disease results from a complex interaction that is initiated when the bacterium binds to the plant cell surface at a wound site. The bacterium has the unusual ability to transfer a fragment of DNA (the T DNA) into a host plant cell and deliver the DNA to the nucleus, where it becomes part of the host plant's genome. Extensive molecular characterization over the past several years has helped elucidate the process of T DNA production within the bacterium, as well as the mechanism by which a molecule of bacterial origin is directed to a specific compartment in a eukaryotic cell. One of the fun damental aspects of this plant pathogen interaction that remains shrouded in mystery is the actual transport process mediating export of the T DNA from the bacterium and delivery into the plant cell. A multiprotein channel or pore complex has been hypothesized to allow passage of the T DNA (and its associated proteins) through the two membranes that surround the bacterium. Several candidate proteins have been identified that could make up such a transport apparatus; these proteins are encoded by the virB operon on the large tumor inducing (T) plasmid that carries many of the virulence associated genes (as well as the T DNA itself). Recent results from a number of labs including our own have shed some light on the localization and other characteristics of many of the VirB proteins; these observations support a model in which several VirB proteins interact to form a T DNA transport pore, while others many function as chaperones and/or sources of energy for the translocation event. The proposed studies are designed to determine which VirB proteins interact to form oligomeric complexes. Genetic analyses will take advantage of point mutations in the virB10 gene that result in a loss of tumor formation; potentially interacting species will be identified by screening for compensatory changes in a second virB gene that can suppress the avirulent phenotype of the original mutant. A second genetic approach will utilize the "dihybrid" strategy, in which proteins or protein fragments are tested for their ability to bring together two domains of a yeast transcriptional activator such as the GAL4 protein. These genetic studies will be complemented by biochemical experiments designed to explore the composition of VirB protein containing aggregates. Together, these studies should provide some insight into the mechanism by which a large polar molecule such as the T DNA and associated proteins is transported across the two apolar membranes that enclose the Agrobacterium cell. Molecular dissection of the structure and function of the putative transfer apparatus will, in turn, help elucidate the processes and interactions mediating assembly of what is thought to be an elaborate membrane associated complex. %%% The soil bacterium Agrobacterium tumefaciens infects susceptible plants, resulting in unregulated cell division and crown gall tumor formation. This disease can be thought of as "plant cancer" in that, as with human tumors, cells are multiplying at an inappropriate time and/or place. Infection by Agrobacterium involves the movement of a piece of DNA (the T DNA) from the bacterium into the host plant cell. This unusual interaction is the only known naturally occurring example of DNA transfer from one kingdom (bacterial) to another (plant). We are especially interested in the mechanism by which a large water loving molecule of DNA is able to cross the oily membrane that surrounds the bacterial cell. T DNA processing and transfer are mediated by a number of bacterial virulence (Vir) proteins. One group of Vir proteins, the VirB proteins, are believed to play a direct role in allowing the T DNA to exit the bacterial cell, most likely by interacting to form an opening in the bacterial membrane through which the T DNA is transferred. We plan to test this hypothesis, using both genetic and biochemical approaches, by studying defective VirB proteins that do not permit T DNA movement, and by isolating complexes of VirB proteins. *** ; @ ....()()))()() ;
