Abstract
Plants are constantly exposed to micro-organisms including pathogens. They are resistant to the vast majority of potential pathogens due to a multilayered defence system. Plant disease susceptibility, on the other hand, is determined by virulence factors from the pathogen, their targets in the host and the successful suppression of plant
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defences. To gain insight into the molecular mechanisms underlying disease susceptibility, we study the interaction between the oomycete pathogen Hyaloperonospora arabidopsidis, causing downy mildew, and its host Arabidopsis. A forward genetic screen resulted in several downy mildew resistant (dmr) Arabidopsis mutants of which dmr1 and dmr6 were further characterized. Chapter 2 described the cloning of DMR1 that encodes for homoserine kinase (HSK), a key enzyme in the Aspartate pathway. Mutation of HSK in the dmr1 mutant resulted in accumulation of the amino acid homoserine. Exogenous application of homoserine proved to be sufficient to induce resistance against H. arabidopsidis. No known defence-related pathways were associated with the observed resistance, indicating homoserine accumulation triggers a novel form of plant resistance. HSK was further analysed in chapter 3 where a reverse genetics approach (TILLING) resulted in additional hsk alleles. One mutant accumulated homoserine and is resistant to downy mildew infection. Another mutant did not develop beyond the seedling stage suggesting HSK is an essential plant gene. We also identified HSK orthologs for a number of crop species making them candidates for mutagenesis to further explore dmr1-mediated resistance. Chapter 4 described the characterization of the DMR6 gene, coding for a 2-oxoglutarate Fe(II)-dependent oxygenase. Absence of a functional DMR6 gene resulted in resistance that requires a functional salicylic acid pathway. On the other hand, overexpression of DMR6 resulted in enhanced susceptibility towards biotrophic pathogens indicating DMR6 acts as negative regulator of defence. A 3-dimensional model was constructed of the DMR6 protein that facilitated in the identification of important residues in the substrate pocket. Chapter 5 described the family of DMR6-like oxygenases (DLOs) and their role in plant defence. Two Arabidopsis DLOs, identified through phylogenetic analysis, have a similar molecular function as DMR6. Also, we have identified DMR6 orthologs in several crop species that are able to complement the dmr6-mediated resistance. A DMR6/DLO-specific motif is identified that could be used for the identification of crop DMR6 or DLO proteins. The presence of this conserved motif could determine the specificity for a particular substrate that is shared between different species.
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