The long term objective of this proposal is to clarify the cellular role of cyclophilins. Cyclophilins are present in a wide range of cell types suggesting that they play an essential role in cell biology. Cyclophilins have recently been implicated in a number of processes including apoptosis, signal transduction, protein stabilization (chaperones), immunosuppression and pathogenesis of a variety of organisms (i.e. Leishmania and HIV-1). The specific goals of this research are to: 1) verify the interaction between the VirD2 protein of Agobacterium tumefaciens and two cyclophilins of Arabidopsis thaliana, 2) demonstrate that this interaction is important in tumorigenesis, 3) examine the interaction of these cyclophilins with VirD2 and other host proteins in an effort to define the cellular role of host cyclophilins and 4) investigate the involvement of cyclophilins in Agrobacterium-mediated transformation of yeast. Affinity chromatography and in vivo across- linking analysis will confirm the specific interactions between VirD2 and cyclophilins previously defined using yeast two-hybrid analysis. Cyclosporin A inhibition antisense RNA analysis will be used to examine the importance of this interaction in tumorigenesis initiated by agrobacterium. Site and region specific mutagenesis will be used in concert with forward and reverse yeast two-hybrid analysis to identify VirD2 residues essential to this interaction and to construct suppressor mutations in cyclophilins will be identified in an effort to elucidate host transport or signal transduction pathways linked to cyclophilins. Further work will utilize yeast as a tractable genetic model in which to examine the effect of cyclophilin disruptions on transformations.
