Characterisation of novel, fungal dsRNA viral elements associated with the Amasya cherry disease and Phlebiopsis gigantea: genome-wide mapping of hypersensitive sites in Drosophila melanogaster

Abstract

The thesis is divided into three, independent projects, two of which concern mycoviruses. Amasya cherry disease (ACD) is a disease of cherry trees (Prunus avium) first described in the Amasya region of Turkey in the 1950s. The causative agent of the disease is unknown. ACD is associated with twelve dsRNA species that are found in the tissue of infected trees. A similar pattern of dsRNA species has been observed in the tissue of the trees infected with the Cherry chlorotic rusty spot (CCRS) disease in Italy. In this project the five largest and the two smallest dsRNA species have been isolated and sequenced. Their origin, function and relation to the ACD disease are investigated. The ACD dsRNAs are also compared to the equivalent sized dsRNA species found in CCRS diseased tissue. A PCR-based diagnostic test for the detection of ACD in cherry tree leaf tissue based on the sequences of the associated dsRNAs has been developed and tested. Mycoviruses are found in a large number of fungi. There are two large, asymptomatic dsRNA species isolated from the economically important fungus Phlebiopsis gigantea that may be useful as a tool for the genetic manipulation of the fungus. As a prelude to these investigations the two related dsRNAs are sequenced and characterised in terms of structure, function and origin. The final project aims to establish a method for the creation of libraries containing fragments of Drosophila melanogaster DNA containing DNaseI hypersensitive sites (HS). The existing indirect end-labelling technique allows for the detection of HS only in a limited genomic area with limited accuracy of HS mapping and no sequence information. The creation of libraries containing clones with HS is a novel, high-throughput approach which can lead in the rapid, genome-wide mapping and detection of HS. This could assist in the study and characterisation of any given genomic area in terms of its three-dimensional DNA structure and in the actual, and the in silico, location of elements such as enhancers and promoters. D. melanogaster was chosen for the creation of the genomic HS library, its genome being sequenced and well characterised. The project presents the first map of the known HS in D. melanogaster.