The presence of turnip crinkle virus (TCV) in Arabidopsis thaliana plants has previously been shown to suppress the ability of these plants to produce a hypersensitive response (HR) upon inoculation with pathogens that would normally elicit this defense response. The ecotype Colombia-0 was examined using wildtype TCV and non-pathogenic strains of Pseudomonas syringae pv. glycinea Race 4 containing virulence genes avrRpt2, avrRpm1 and avrRps4. Transgenic lines of A. thaliana that express the TCV proteins p8, p9 or CP were also examined in an attempt to determine if these proteins play a role in suppression of the HR. Crosses of these transgenic lines were made in order to determine if binary combinations of these proteins were sufficient for HR suppression. In addition, assays were completed to determine if the inhibition of the HR correlated with suppression of resistance to the virulent Pseudomonas syringae pv. maculicola ES4236 avrRpt2 growth in the plant. Finally, PR-1 protein expression was inspected by visual and quantitative GUS reporter gene assays to determine if TCV also played a role in inhibition of the plants ability to develop systemic acquired resistance (SAR).

Replication of genetic material is the most important and central process during the viral life cycle. Most RNA viruses assign one or more proteins translated from their own genome for replicating genomic RNAs. Understanding the various biochemical activities of these replication proteins is the aim of this dissertation research. The replicase proteins of Turnip crinkle virus (TCV) and Tomato bushy stunt virus (TBSV) were selected for this study. Both viruses have small, messenger-sense, single-stranded RNA genomes. Replicase proteins p28/p88 of TCV and p33/p92 of TBSV- were expressed and purified from E. coli as N-terminal fusions to maltose binding protein. In vitro assays revealed that the recombinant p88 has RNA-dependent RNA polymerase (RdRp) and RNAbinding activities. Deletion of the N-terminal p28 domain in p88 resulted in a highly active RdRp, while further deletions at both N- and C-terminal ends abolished RdRp activity. Comparison of p88, the N-terminal p28-deletion mutant of p88 and a TCV RdRp preparation obtained from infected plants revealed remarkable similarities in RNA template recognition and plus and minus strands synthesis. Contrary to recombinant TCV RdRp activities under similar experimental conditions. p33 preferentially binds to singlestranded (ss) RNA with positive cooperativity in vitro. The RNA binding activity was mapped to arginine/proline-rich motif (RPR-motif) at the C-terminus of p33 and the corresponding sequence in p92. The non-overlapping C-terminal domain of p92 also contained additional RNA-binding regions that flank the conserved RdRp motifs on both sides. Cooperative RNA binding by p33 suggested inter-molecular interactions between p33 monomers. Indeed the yeast two-hybrid and surface plasmon resonance assays revealed interactions between p33 and p33 and also between p33 and p92. The sequence involved in the protein-protein interactions was mapped to the C-terminal region in p33, proximal to RPR-motif. Within this region, mutations introduced at two short stretches of amino acid residues were found to affect p33:p33 and p33:p92 interactions in vivo and also decreased the replication of a TBSV-defective interfering RNA in yeast, a model system, supporting the significance of these protein interactions in tombusvirus replication.