Abstract
On a global scale the impact and costs of plant diseases on agriculture is enormous, highlighting the importance of the research on this topic. Plant disease is the result of a compatible interaction between plants and adapted pathogens. The knowledge on the molecular mechanisms underlying compatibility or disease susceptibility is
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limited. The aim of this study was to identify Arabidopsis genes required for disease susceptibility to the oomycete Hyaloperonospora parasitica. We have undertaken a forward genetics approach to study susceptibility to downy mildew. Seeds of the susceptible Arabidopsis Ler eds1-2 line were mutagenised and twenty independent downy mildew resistant (dmr) Arabidopsis mutants were isolated of which eight were further characterized (described in chapter 2). Three dmr mutants, dmr1, dmr2 and dmr6, showed no induced expression of the defence-associated gene PR-1, and absence of programmed cell death and reactive oxidative intermediates, suggesting they are susceptibility mutants. In contrast, PR-1 expression was elevated in the dmr3, dmr4, and dmr5 mutants indicating that these mutants have an enhanced defence-response. The dmr1, dmr2, dmr5, and dmr6 mutants were still susceptible to other pathogens, such as the bacterium Pseudomonas syringae pv. tomato and the fungus Golovinomyces orontii. For two candidate susceptibility mutants, dmr1 and dmr6, the corresponding genes have been cloned (chapters 3 and 4). The DMR1 gene, At2g17265, was map-based cloned and found to encode for homoserine kinase (described in chapter 3). Homoserine kinase phosphorylates homoserine to phospho-homoserine. Amino acid analysis of the dmr1 mutants revealed high levels of homoserine that are absent in the parental line, Ler eds1-2. Infiltration of homoserine into Ler eds1-2 seedlings resulted in H. parasitica resistance. High levels of homoserine cause H. parasitica resistance in the dmr1 mutants via an as yet unknown mechanism. The DMR6 gene was found to encode for an oxido reductase (described in chapter 4). Oxido reductases catalyze the transfer of electrons from one molecule, the oxidant, to another, the reductant. For the DMR6 encoded oxido reductase no biological function has been demonstrated nor do we know the substrate and product of the predicted enzyme. DMR6 is locally up regulated during H. parasitica infection, in compatible and incompatible interactions. Other forms of biotic stress and abiotic stress result in an up regulation of DMR6. However, lack of DMR6, in the dmr6 mutants, results in resistance that is accompanied by the enhanced expression of a set of defence-associated transcripts, including DMR6. These results suggest a dual role of DMR6 during pathogen infection. The cloning of the DMR1 and DMR6 genes, which are required for H. parasitica susceptibility, has revealed novel ways to obtain disease resistance in plants, that have the potential to be applied in breeding for disease resistance. Future studies will reveal the molecular mechanisms that lead to loss of susceptibility to H. parasitica in the Arabidopsis dmr1 and dmr6 mutants.
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