Infection of the resistant Arabidopsis ecotype Di-17 with Turnip Crinkle Virus (TCV) elicits hypersensitive response (HR), accompanied by increased expression of defense genes. HR to TCV is conferred by HRT, which encodes a coiled-coil (CC)-nucleotide-binding site (NBS)-leucine-rich repeat (LRR) class of resistance (R) protein. In contrast to HR, resistance requires HRT and a recessive locus designated rrt. Unlike most CC-NBS-LRR R proteins, HRT-mediated resistance is dependent on EDS1 and independent of NDR1. Resistance is also dependent on salicylic acid (SA) pathway and light. A dark treatment, immediately following TCV inoculation, suppresses HR, resistance and activation of a majority of the TCV-induced genes. To determine the genetic, molecular and biochemical basis of light-dependent defense pathway, we studied the role of various photoreceptors in HRT-mediated resistance to TCV, HRT protein levels and its localization. Interestingly, mutation in blue-light photoreceptors led to degradation of HRT via a proteasome-dependent pathway and resulted in susceptibility to TCV. Exogenous application of SA induced transcription of HRT, which restored HRT levels in some, but not all, mutant backgrounds. These results show that different photoreceptors function distinctly in maintaining post-transcriptional stability of HRT. In addition to photoreceptors, HRT also forms a complex with several other proteins, many of which participate in the RNA silencing pathway and are required for HRT-mediated resistance. Together, our results suggest that HRT forms a multi-protein complex and that HRT-mediated signaling involves reconstitution of this complex.

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.

Rhabdoviridae family viruses attacking the plant hosts were only described at the angiosperms. In this work, a gymnosperm rhabdoviridae infection was described for the first time ever ? specifically at Ginkgo biloba. Even though there were not observed any kinds of obvious infection symptoms on any of randomly chosen plant samples, through the molecular methods and detection primers the rhabdoviridae infection was proved at six of the plant samples. The acquired nucleotide and amino acid sequences, which were compared with the GenBank sequences, confirm the Rhabdoviridae family viruses occurrence. These given sequences demonstrated a certain analogy with a Strawberry crinkle virus assigned to the genus of Cytorhabdoviruses. The analyses proved a different reciprocal homology among the nucleotide sequences of the individual isolates, and in all likelihood an occurrence of two up to now unknown viruses in the Ginkgo biloba samples was proved for the first time. A definite categorization will be dependent on an acquisition and comparison of other sequences from the isolates genome and also on certain biological characteristics observation.

No description available.

In a quest to design novel deployable structures, flexible polyhedra provide interesting insights. This work follows the discovery of flexible polyhedra and aims to make flexible polyhedra more useful. The dissertation describes how flexible polyhedra can be made. The flexible polyhedra first considered in this dissertation have a rotational degree of freedom. The range of this rotational movement is measured and maximised in this work by numerical maximisation. All polyhedra are established computationally: an iterative solution method is used to find vertex coordinates; several clash detecting methods are described to define whether each rotational position of a flexible polyhedron is physically possible; then a range of motion is defined between occurrences of clashes at the two ends; finally, an optimisation tool is used to maximise the range of motion. By using these tools, the range of motion of two types of simplest flexible polyhedra are maximised. The first type is a series of flexible polyhedra generalised from the Steffen flexible polyhedron. The range of motion of this type is improved to double that of Steffen’s original, from 27° to 59°. Another type of flexible polyhedron is expanded from a model provided by Tachi. Based on the understanding of Steffen’s flexible polyhedron, optimisation parameters are carefully given. This new type has achieved a wider range of motion, so now the range of motion of flexible polyhedron is tripled to 80°. After enlarging the range of motion of the degree of freedom in the 1-dof systems, the dissertation found multiple degrees of freedom in one polyhedron. The multiple mechanisms can be even repetitive, so that an n-dof polyhedron is found. A polyhedron of two degrees of freedom is first presented. Then, a unit cell for any number of mechanisms is found. As a repetitive structure, a 3-dof polyhedron is presented. Finally, this work presents the possibility of configuring a flexible polyhedral torus and a closed polyhedral surface that is able to flex without the need to stop.