No description available.

No description available.

No description available.

Primers were designed to reveal pararetrovirus-like sequences from potato (Solanum tuberosum) by PCR. One primer pair covered an area of the reverse transcriptase region, others covered the end of a transactivator and a repetitive region. The sequences obtained ranged from 800 to 1100 bp and had homology to known sequences of TPV (tobacco pararetrovirus) and TVCV (Tobacco vein clearing virus). Aligning the sequences from the potato genome gave a phylogenetic clustering into three different groups. Genomic Southern hybridization was used to confirm the results, with probes hybridizing to the full length of the digested DNA. In situ hybridization was used to localize different pararetrovirus-like sequences to the chromosomes of potato, which showed a low copy number sequence dispersed throughout the genome.;A diverse range of plant species was analysed with the pararetrovirus-like primers, and clones homologous to TPV and TVCV were obtained from a liverwort, a fern, tomato, tobacco, pea, rice and banana. These results were confirmed with genomic Southern hybridization, which also revealed pararetrovirus-like sequences in the DNA of other lower plants, Gymnosperms and Angiosperms.;Integrated pararetroviruses have been found to be a serious problem in banana breeding as micro-propagation may give rise to episomal viruses, reducing the yield considerably. The possibility of the widespread presence of pararetrovirus-like sequences in plants may have important implications for virus resistance, sudden pathogen outbreak and genomic evolution.

Chapter 2 reports the synthesis of liquid viruses by nanoscale engineering of the capsids of cowpea mosaic virus (CPMV) and tobacco mosaic virus (TMV). This was achieved by cationization of the capsid surfaces by EDC-mediated coupling of ethylenediamine to the capsid surfaces followed by electrostatic attachment of an anionic polyethylene glycol-based surfactant. Characterization by transmission electron microscopy (TEM), dynamic light scattering (DLS) and analytical ultracentrifugation (AUC) confirmed the existence of discrete CPMV/polymer surfactant nanoconjugates. Retention of the protein secondary structure was confirmed by Fourier-Transform infrared spectroscopy (FT-IR) and circular dichroism spectroscopy (CD). Rheological investigations were performed on the liquid virus material. A series of infection studies revealed that the modified polymer-surfactant/virus nanoconstruct remained infective to the plant, V. unguiculata. Furthermore, the surfactant-bound conjugates showed solubility in a range of organic solvents as well as increased thermal stability (cf wild-type CPMV). Chapter 3 discusses the preparation of monodisperse conducting polyaniline (PAni) nanofibres using polyferrocenylsilane-b-poly(2-vinyl pyridine) (PFS-b-P2VP) block copolymer micelles as templates. PFS-b-P2VP cylindrical micelles with narrow length distributions (Lw/Ln < 1.04) were prepared using a one-dimensional (1-0) self-seeding procedure. These micelles were subsequently used in the template-directed synthesis of monodisperse leucoemeraldine (LEB) PAni nanofibres with lengths of up to 1200 nm. The LEB-PAni nanofibres were then oxidized and doped to the conductive emeraldine salt (ES) oxidation state. The oxidation states of the PAni nanofibres were determined using ultraviolet-visible (UV - Vis) spectroscopy. Retention of the same lengths and narrow length distributions of the micellar templates throughout the templating and doping processes was confirmed by statistical analysis of the PAni nanofibres lengths from TEM micrographs. Preliminary experiments into the characterization of the conductive properties of the PFS-b-P2VP micellar templates, LEB-PAni nanofibres and ES-PAni nanofibres were carried out by scanning conductance microscopy (SCM).

Pepino mosaic virus (PepMV) is a destructive pathogen that poses a large threat to the tomato industry. Efforts to control its spread have failed and it now displays a global distribution. Its emergence has been characterised by the fast evolution of new necrotic strains and as of yet, the genetic basis for phenotypic aggression is unknown. Gaining an understanding of this is of the utmost importance in developing new control methods. Furthermore, natural known sources of resistance (R) genes are limited. The Rx gene from potato has shown to confer resistance when introduced as a transgene into the main crop of threat, tomato. However recent work suggests this resistance is easily overcome and highly unstable. The main aim of this project was to determine the genetic basis for the phenotypic aggression displayed by necrotic strains of PepMV, through the generation of chimeric PepMV infectious clones (ICs). Full-length ICs of both mild EU and aggressive CH2 forms of PepMV were generated, as well as chimeric viruses containing the 5' end of EU origin and either the 3' region (triple gene block and coat protein, CP) or CP of CH2 origin. Analysis of the phenotypes presented by these in a range of solanaceous indicator plants suggested that a pathogenicity determinant may reside in the CH2 CP region, and furthermore, this acted in a host specific manner. Additionally, during this investigation new mutants evolved from the constructed ICs, which displayed genotypic variation and altered pathogenicity. In combination this evidence shows that the genetic basis for phenotypic aggression of PepMV is highly complex and difficult to elucidate. The secondary aim of this project was to assess the stability of the Rx gene as a source of resistance against PepMV. The Rx-based resistance was found to be easily overcome by singular mutations occurring in the PepMV CP region, and would therefore not provide a suitable source of resistance to introduce into susceptible cultivars. Other RNA-based resistance strategies were also investigated. The generation of transgenic plants expressing full-length PepMV CP was found to have the potential to confer a high level of resistance to challenge with the cognate virus, as well as a mechanism based on RNA hairpin (HP) and viral co-inoculation.

No description available.

Field, glasshouse and laboratory experiments and field surveys were done in Western Australia to develop control strategies for various non-persistently aphid-borne viruses of grain legumes, celery and carrots and for the fungally-transmitted lettuce big vein disease (LBVD). In a range of legumes, extreme resistance to Alfalfa mosaic virus (AMV) was found in Vicia faba cv. Ascot, Hedysarum coronarium cv. Grimaldi, and Lens culinaris IL5480, to Cucumber mosaic virus (CMV) in Lathyrus cicera ATC80521, L. clymenum C7022, Ornithopus sativus cv. Cadiz, and V. sativus cv. Languedoc, and to Pea seedborne mosaic virus (PSbMV) in all accessions and varieties of Cicer arietinum and L. culinaris and some of L. sativus and most pasture legumes tested. No sources of extreme resistance to Carrot virus Y (CarVY) was found to carrot germplasm. The first reports of seed transmission of AMV in Vicia faba and of CMV in Pisum sativum, V. faba, V. narbonensis and eight pasture legumes were made. No evidence for the seed transmission of CarVY was found. Plants of V. faba infected early with AMV recovered while plants infected later incurred yield losses (up to 45%). In contrast, plants of C. arietinum infected with AMV when young were killed. L. culinaris plants infected with AMV and CMV suffered high yield losses (up to 90%) dependent on the age of plants when they were infected. Plants of lettuce infected with LBVD when young often failed to form ‘hearts’ but leaf symptoms were mild, whereas later infected plants had more severe leaf symptoms. Spatial patterns of disease spread were monitored and implications for disease control discussed for CarVY and LBVD. The incidence of CarVY in carrot crops was often high in a wide selection of varieties grown throughout Australia. CarVY was determined to have a wide host range within the Apiaceae family but not in other plant families. No evidence for reservoirs of CarVY was found in species other than carrot, despite extensive surveys of known Apiaceous hosts. CarVY was readily transmitted by a wide range of aphid species in a non-persistent manner. Control strategies for Celery mosaic virus (CeMV), by instigation of a ‘celery free period’ and LBVD by combining resistant lettuce varieties and plastic mulch were demonstrated. New control measures for AMV, CMV, PSbMV and CarVY were also discussed.

Many oomycetes, such as the notorious potato late blight pathogen Phytophthora infestans, have devastating effects on crops. Recent findings have implied that eukaryotic plant pathogens deliver effector proteins inside host cells to facilitate colonization by modulating plant defences. Although the translocation mechanisms remain unknown, in oomycetes this process depends on a short conserved amino acid sequence located near the signal peptide of many secreted proteins. This sequence, termed the RxLR motif, is strikingly similar to the core RxLxE/D/Q host cell targeting-signal that is found in virulence proteins from the malaria parasite Plasmodium falciparum. Common infection strategies have been described for these divergent pathogens, albeit one is a plant pathogen while the other infects human cells. In this thesis, stable transformation of P. infestans, combined with a validated assay based on the intracellular recognition of the RxLR effector, A vr3a, was used to study the specificity of effector translocation in oomycetes and to demonstrate the functional similarity between translocation motifs from Plasmodium and two distantly related oomycetes. While accumulating evidence shows that RxLR effectors are delivered into the host cell, the subcellular targeting of these proteins is still unclear. Throughout this PhD project, the difficulty of visualizing translocated effectors during infection was tackled by seeking alternative approaches allowing the detection of fluorescently- tagged effectors once delivered into the plant cell. A further key research question addressed in this thesis was whether the mechanism of translocation required pathogen-encoded proteins or a pathogen- induced environment. Purified fluorescent protein fusions of A vr3a were used to demonstrate that this plant pathogen effector may hold the intrinsic ability to traverse the plasma membrane of animal cells.