Detection, promoter walking and analysis of malus defence-related genes

Abstract

The experimental work in this thesis is based around the central concept of developing methods for the analysis of defence-related genes in apple. Three areas of research are described: (1) an examination of transient expression systems in mature apple Ieaf tissue, (2) the detection and gene expression profiles of candidates for a marker gene for the hypersensitive response and (3) the development of a novel genome walking system to isolate the promoter regions of these candidate marker genes. The initial main aim of this thesis at the outset was to develop a high- throughput transient expression assay system that would enable rapid analysis of gene function in apple, in particular genes involved in pathogen defence. Many apple pathogens will only grow on apple and often the full defence response is observed only in mature leaves. For these reasons, the transient expression systems tested, used this particular tissue. The ultimate aim of this research was to conduct transient assays for disease resistance genes (R genes) and fungal avirulence (av^ genes. Two different transient expression methods were examined for this purpose: biolistics, and Agrobacterium-mediated transformation. Successful pathogen defence in plants is marked by a necrotic reaction around the pathogen called the hypersensitive response (HR). In order to confirm this response in a transient R gene or avτgene assay, an indicator gene that is induced specifically during the (HR) was sought. Putative apple homologues of the tobacco HR-specific gene Hsr203J were identified in both the HortResearch EST database and GenBank. In addition a strategy for detection of further homologues using degenerate primers was developed. A total of four candidate MXd Hsr203J genes were eventually identified, and members of this family were subjected to genetic and phylogenetic analysis. The transcription profile of these candidate HR-specific genes were analysed by real time quantitative RT PCR for their response to the apple pathogen Venturia inaequalis in both resistant and susceptible accessions. A putative functional orthologue of the tobacco Hsr203Jgene was identified. The promoter regions of this gene (and another candidate Hsr203J homologue) were isolated by a novel single step genome walking method that was developed for this purpose. Quantitative PCR was used to determine the optimal reaction conditions for this process and determine the efficiency of the single restriction/ligation reaction that is crucial to this procedure.