No description available.

Mitochondrial alternative oxidase (AOX) plays a role in protecting plant cells against reactive oxygen species. The SA-inducible RNA-directed RNA polymerase 1 (RDR1), contributes to viral RNA degradation via RNA interference. Previous data suggested that these enzymes comprise separately regulated, redundant elements in SA-induced resistance to viruses. To test this hypothesis, I constructed transgenic tobacco<i> (Nicotiana tabacum) </i>and <i>N. benthamiana </i>plants compromised simultaneously in AOX function and RDR1 activity. Transgenic tobacco and <i>N. benthamiana </i>plants were characterised by measuring alternative respiratory pathway (AP) capacity and RDR enzyme activity. The resistance/susceptibility status of the transgenic plants was assessed by analysing <i>Tobacco mosaic virus</i> (TMV) accumulation in the chemically treated, directly-inoculated tissues. Antimycin A (AA)-induced resistance to TMV was inhibited in transgenic <i>N. benthamiana </i>with increased AP capacity, and SA- and AA-induced resistance was enhanced in transgenic <i>N. benthamiana </i>with decreased AP capacity. However, SA-induced resistance to TMV in directly-inoculated leaves was still unaffected in transgenic tobacco and <i>N. benthamiana </i>compromised in AOX function and RDR1 activity. This suggests that SA-induced resistance to viruses involves additional, unknown mechanisms. Surprisingly, SA can enhance RDR activity in transgenic 35S-<i>MtRDR1 N. benthamiana </i>but not wild-type and vector-control plants (natural mutants of RDR1). This SA-enhanced RDR activity resulted from increased MtRDR1 protein level, indicating the post-transcriptional regulation of MtRDR1 enzyme activity. SA-induced resistance to systemic movement was enhanced in transgenic 35S-<i>MtRDR1</i> <i>N. benthamiana </i>plants, suggesting that SA-induced increase in RDR1 activity plays a role in induced resistance to systemic movement of viruses. Basal resistance to viruses was studied in transgenic tobacco (<i>nn </i>or NN genotype) and <i>N. benthamiana </i>plants with modified AP capacity or RDR activity. Modification of AP capacity had no effect on TMV accumulation in HR lesions from transgenic tobacco plants overexpressing the <i>Aoxla</i> construct (<i>NN</i> background). Notably, transgenic <i>N. benthamiana </i>plants with increased AP capacity were more susceptibility to <i>Potato virus X </i>(PVX) than non-transgenic plants. This was seen in the transgenic plant with increased AP capacity that PVX accumulated to higher level in both directly-inoculated and systemic leaf tissues. It was also nearly discovered that transgenic 35S-<i>MtRDR1 N. benthamiana</i> plants were more resistant to <i>Potato virus Y </i>ordinary strain. The results suggest that altering AP capacity has effect on basal resistance to some viruses and confirms that RDR1 plays a role on basal resistance.

No description available.

No description available.

<i>Erwinia carotovora</i> is a Gram-negative plant pathogen affecting several commercially important crops such as potatoes and tobacco. Virulence in many strains of <i>E. carotovora</i> is controlled by the quorum sensing signalling molecule <i>N</i>-(3-oxohexanoyl)-L-homoserine lactone (OHHL) which accumulates as the bacteria multiply and activates exoenzyme and secondary metabolite production. Mutant strains lacking OHHL are avirulent, suggesting a preventative treatment for infection. It had previously been observed in <i>E. carotovora</i> that OHHL accumulates in a cell density dependent fashion until stationary phase when the concentration falls in an equally rapid manner. This work shows that the stationary phase decline in OHHL is due to alkalisation of the growth media and that growing in buffered media can prevent this decline. Functional genomics was used to explore the possibility that <i>E. carotovora</i> subsp. <i>atroseptica</i> might possess an OHHL degrading enzyme similar to AttM from <i>Agrobacterium tumefaciens</i>.  Four AttM orthologues were identified in the Eca strain 1043 genome, with one of these proteins (209-2), showing some degrading activity against synthetic HHL. Mutants were created in each of the orthologues but they had no effect on OHHL accumulation or exoenzyme production. The mutation in the <i>209-1</i> gene reduced motility, while mutation in the <i>91</i> gene reduced virulence in potato tubers. Further investigation using proteomic analysis found three of the mutants had reduced levels of Re1A, which is involved in regulation of the <i>attM</i> operon in <i>A. tumefaciens.</i> This study shows that E. carotovora would exhibit reduced virulence in basic environments due to increased AHL lability. Only one of the AttM orthologues identified possessed AHL degrading ability under the conditions tested but three did effect the expression of AttM, suggesting potential involvement in AHL regulation.

Fungicide resistance, whereby a mutation conferring reduced sensitivity to chemical control arises and spreads through a fungal population, severely inhibits the successful control of crop disease. Mathematical models play a vital role in assessing the risk of invasion of fungicide-resistant pathogens, and in the design of effective resistance management strategies. In this thesis, I investigate the factors affecting the invasion of resistance in heterogeneous crop environments. I develop a simple, nonlinear model for fungicide resistance which, improving on existing work, incorporates the dynamics of the host crop and quantities how the amount, decay and timing of a fungicide dose affect selection for resistance. The model structure is similar to those used to describe antibiotic resistance, and hence much of the analysis presented here applies more generally to drug and pesticide resistance. I identify a threshold for the invasion of resistance in terms of two key parameters, both of which are amenable to estimation in the field. These are the fitness of the resistant strain relative to the wild-type, and treatment efficacy (which summarises how control inhibits pathogen survival and reproduction). Using a discrete, stochastic formulation of the model, I demonstrate that this threshold is robust to the effects of demographic stochasticity, and estimate the probabilities of resistance pre-existing or emerging during treatment. In the final section of the thesis, I extend the simple model to examine the dynamics of multiple pathogen strains, the effects of seasonal disturbance to the host (through planting and harvesting) on persistence of the resistant pathogen, and how the scale of pathogen dispersal affects the spatial propagation of resistance.

The extracellular protease (PEP) of <I>Pyrenopeziza brassicae</I>, an economically important pathogen of oilseed rape and other brassicas, may be a significant factor in the development of the light leaf spot disease. The aim of this study was to further our understanding of PEP and the role of this enzyme in the disease process. Results of purification of PEP, monitored with a new microtite plate assay based on chromogenic protease substrates, are presented. The partial N-terminal sequence of the mature enzyme was reconfirmed and shown to share high levels of homology with fungal extracellular serine proteases of the subtilisin (S8) family. Internal amino acid sequence was also obtained from purified PEP. PEP was characterised by its inhibitor mixed trypsin/chymotrypsin specificity. PEP has a K<I><SUB>m</SUB></I> for a trypsin substrate that is the same as that for trypsin. In order to isolate the cDNA clone of the structural gene of PEP, a skimmed milk-induced cDNA library was screened with two separate probes. The first was a reverse transcription-polymerase chain reaction (RT-PCR) product amplified from total mycelial RNA with a primer designed from the N-terminal amino acid sequence of PEP and a poly-T primer. The second was a PCR product amplified from genomic DNA by primers designed from the PEP internal amino acid sequence. Although several cDNA clones were isolated by these approaches, none were identified as corresponding to PEP. Finally, antigens were identified in the epidermal cell walls of a light leaf spot susceptible cultivar of oilseed rape which may represent potential glycoprotein targets for PEP activity <I>in planta</I>. The antigens were recognised by antibodies that recognise the sugar moiety of turnip antigens, one of which is postulated to have a role in the progress of black rot disease caused by <I>Xanthomonas campestris </I>pv. <I>campestris</I> due to its susceptibility to degradation by an extracellular protease produced by that pathogen <I>in planta</I> (Davies 1996).

No description available.

The spread of mycelia through soil is the main process by which many economically important plant pathogens are transmitted between plants. Using mathematical modelling, this thesis provides a framework for investigating the interaction between soil structure and fungal growth, with a view to controlling this spread. Fungal dynamics are modelled at the scale of groups of hyphae by a stochastic cellular automaton (CA), with the cells of the CA corresponding to the network of pore species in the soil. The fungal model captures the characteristics of mycelial growth at this scale, whilst maintaining a level of mathematical tractability. The growth of mycelial fungi within and between patches is modelled by the rates of growth and quiescence, or transition to inactivity, together with structural parameters. The fungal model is analysed to identify key characteristics of the pore network which affect the morphology of the fungal colony. The soil-pore network is modelled as part of the entire soil structure. The pore space is abstracted to a network of connected patches of different sizes. The size and connections between these patches are converted into carrying capacities for hyphal colonisation and the level of connection between cells for subsequent implementation of the fungal model. The models are used to give insight on experimental data for soils of different bulk densities. The application of the soil and fungal models to this data allows the three-dimensional structure of the soils to be studied, and changes in the three-dimensional connectivity which contribute to changing hyphal density are identified. The results show that at low bulk densities there are few large, pore spaces with connections to distant pore spaces. This structure results in large sparse colonies. At high bulk densities, the pore space is made up of many smaller pore spaces which are connected to a few close pores. This structure results in small, dense colonies.

This study shows that 11 <i>Arabidopsis thaliana</i> ecotypes are susceptible to infection by two agronomically significant viruses of the genus <i>Polerovirus</i>. All ecotypes tested were, to varying degrees, susceptible to <i>Beet mild yellowing virus </i>(BMYV) and <i>Turnip yellows virus </i>(TuYV) though a closely related species, <i>Beet chlorosis virus </i>(BChV), was unable to establish detectable infection. <i>Agrobacterium tumefaciens </i>was used to transform <i>A. thaliana </i>with one of four constructs containing sequences corresponding to either the BMYV major capsid protein (<i>CP</i>) or putative movement protein (<i>MP</i>) genes in either full-length or 3’-truncated form. BMYV-derived sequences were under the constitutive <i>Cauliflower mosaic virus </i>35S promoter and linked to the <i>hpt</i> (hygromycin resistance) gene. T₁ plants were identified by selection on hygromycin and the presence of the viral gene sequences confirmed by PCR. Plants carrying full-length gene constructs were recovered with low frequency and exhibited abnormal phenotypic effects. Transgenic plants harbouring truncated <i>CP </i>and <i>MP</i> gene sequences arose with greater frequency; 16 and 12 independent lines were respectively identified. Primary transformants were grown to maturity under glasshouse conditions and allowed to self-pollinate. Analysis of T₂generation plants revealed four lines that displayed resistance; three harbouring the truncated <i>CP</i> construct and one carrying the truncated <i>MP</i> construct. Resistance was manifest as an amelioration of BMYV-induced symptoms and decreased susceptibility to disease correlated with decreased BMYV accumulation, as demonstrated by ELISA. <i>A. thaliana </i>has been established as a valuable model system with which to assess virus-derived transgenes for functionality. With respect to advances in transgenic design, which may afford protected plants effective viral immunity, a future strategy for engineering BMYV resistance is proposed. The prospects for commercial exploitation of pathogen-derived resistance technology by the sugar beet industry are also discussed.