Susceptibility of Arabidopsis to the downy mildew pathogen Hyaloperonospora parasitica : A reverse genetics approach

Abstract

The research described in this thesis was performed to gain insight into disease susceptibility in the model system Arabidopsis thaliana – Hyaloperonospora parasitica. The aim was to identify host genes that support the infection process. For this, global expression profiling using DNA microarrays was combined with reverse genetics approaches. A particular focus was on the compatible interaction, i.e. when the pathogen is able to colonise the host and reproduce. As the downy mildew pathogen H. parasitica, like many other biotrophic pathogens, forms haustoria during the interaction with the host, it was expected that host genes encoding membrane-associated proteins are particularly important as they possibly act at the host pathogen interface. Unraveling processes occurring at this interface would greatly enhance our understanding of compatible plant-pathogen interactions and disease susceptibility in general. To focus on membrane-related processes the initial studies were on transcripts encoding membrane-associated proteins. Comparing a MBP fraction to a free polysomal fraction via microarrays led to the identification of transcripts encoding membrane-associated and secreted proteins as determined by bioinformatic analysis. Unfortunately, when applied to H. parasitica-infected Arabidopsis plants, analysis of MBP fractions provided only limited information on disease-associated transcripts. Additional microarray data is presented that shows that gene-expression profiles of compatible and incompatible Arabidopsis – H. parasitica interactions are very similar. A limited number of 17 genes turned out to be particularly interesting as they were only induced during the compatible interaction and not during the incompatible interaction. A subset of these genes is associated with responses to abiotic stress and their promoters showed an overrepresentation of dehydration-responsive elements (DRE). Other compatible specific (CS) genes are not induced by abiotic stress and most of them do not contain these promoter elements. Among the CS genes are several ethylene responsive factors (ERFs), especially the ones from clade IIIb of the ERF family. These factors could be the early responders to compatible downy mildew growth, either directly or indirectly. These CS genes could be differentially expressed as a physiological consequence of pathogen infection. What the exact stress factors are that provoke this is unclear, but is highly interesting as compatible specific gene-expression is not described before in Arabidopsis. An alternative reverse genetics approach provides the screen of a subset of T-DNA lines in leucine-rich repeat receptor-like kinase encoding genes. One mutant, rlk902-1, was found to be resistant to downy mildew. However, microarray studies revealed that, in this line, a region of 84 kb showed downregulation which appeared to be linked to the observed resistance. In addition, resistance was shown not to be caused by the mutated gene itself. Another mutation in the genome or a combination of downregulation of genes in the 84 kb region could be responsible for resistance. Although “true” susceptibility genes from Arabidopsis have not been found in this study, the finding of compatible-specific and pathogen-associated expression patterns is promising. They provide a first step in unraveling the molecular processes that occur during infection of susceptible plants.