A systems biology based approach to functionally annotate and analyse the genome of the fish pathogenic oomycete Saprolegnia parasitica

Watermoulds, or oomycetes, contain some of the most devastating pathogens of animals and plants, causing enormous economic and environmental damage in natural and cultured ecosystems. The most destructive oomycete pathogens on fish are Saprolegnia parasitica and Saprolegnia diclina. These species are present in all fresh water habitats and represent a serious problem for the aquaculture industry, where it has been estimated that these oomycetes alone, kill over 10% of all hatched salmon and eggs. Watermoulds have several fungus-like characteristics but are not 'true fungi'. They belong to a group of organisms called the Stramenopiles or Heterokonts, which also include the golden-brown algae, kelp, and diatoms. Several clearly defined developmental stages are found in the life cycle of oomycetes that are not found in fungal pathogens, which makes them unique and it is becoming clear now that they have evolved different infection strategies compared to other parasites, fungal and bacterial pathogens. The asexual spore or sporangium is formed at the end of hyphal cells and can release many zoospores. These zoospores are able to swim as they have two flagella. Once the zoospore has produced a cell wall, it can germinate and produce infection hyphae. These hyphae can infect the skin and underlying tissue of the fish. Initially the head or the fins become infected and later the watermould is able to spread over the entire surface of the body. Disease is characterised by an external, cotton-like appearance with crescent-shaped or whorled patterns. Saprolegnia is able to cause cellular necrosis as well as dermal and epidermal damage. Infections do not appear to be tissue specific. Hyphae can also penetrate into the muscle and blood vessels of infected fish. In some cases infection takes place very rapidly and inflammatory responses in the fish appear to be absent. This has led several researchers to believe that S. parasitica is able to suppress the immune response in fish. In the current proposal, we will perform experiments that will help us to understand how this group of organisms cause disease. This in return will allow us to develop new sustainable ways of controling the disease. In order to analyse disease processes at the molecular level, it is necessary to first determine the coding regions of all genes of the pathogen. We can then use computers to identify genes likely to be involved in invading the fish and carry out experiments to see what the corresponding gene products (proteins) do. Furthermore we will use this information to see if the same genes in related organisms are used to attack both animals and plants. We will then investigate which genes are important for infection by studying when they are switched on during the development and infection cycles of the pathogen.

Watermoulds, or oomycetes, contain some of the most devastating pathogens of animals and plants, causing enormous economic and environmental damage in natural and cultured ecosystems. One of the most destructive oomycete pathogens on fish is Saprolegnia parasitica. It is endemic to all fresh water habitats and is a serious problem in the aquaculture industry, where it has been estimated to kill more than 10% of all hatched salmon. This application is to request funds to appoint a post doctoral researcher who would coordinate and help with the annotation, and perform comparative bioinformatics analysis on the new genome sequences of both S. parasitica and Saprolegnia diclina in relation to the available plant pathogenic oomycete genomes. In addition, a systems biology based approach will be followed to investigate gene and protein expression profiles during the key stages of the lifecycle of these pathogens, thereby linking the genome to its transcriptome and proteome. We will generate expressed peptide tags with two-dimensional liquid chromatography mass spectrometry, and 80M of 50b Illumina reads of cDNA, which will greatly aid the gene calling and annotation process of the Saprolegnia genome sequence. In addition, a global overview of the stage specific proteome of S. parasitica during the life cycle stages will be obtained by computer-assisted comparisons of label-free mass spectrometry runs, which will allow us to profile and quantify differentially expressed proteins. Furthermore, micro-array hybridisation experiments will be performed to investigate gene expression of Saprolegnia during the pre-infection and infection stages; with the aim to investigate which genes and pathways may be involved in pathogenicity and development. The availability of a complete genome sequence will allow us to perform an integrated set of experiments, which will provide us with essential data that will be exploited in disease management in the future.