Differentiation and Pathogenicity within the Saprolegniaceae : Studies on Physiology and Gene Expression Patterns in Saprolegnia parasitica and Aphanomyces astaci

Sammanfattning: Saprolegnia parasitica and Aphanomyces astaci are parasitic water moulds belonging to the Oomycetes. Despite their importance as parasites they are very little studied at the molecular level and the work described in this thesis was aimed at increasing the molecular knowledge of these organisms by cloning and characterising genes of potential importance for reproduction and pathogenicity.Stage-specific transcripts from Saprolegnia parasitica were isolated by differential display RT-PCR. One of the markers, puf1 encodes a putative mRNA binding protein which may be involved in post-transcriptional regulation of gene expression. S. parasitica puf1 is expressed exclusively in spore cysts that have not been determined for germination or repeated zoospore emergence indicating that the cyst stage has two phases, of about equal duration, which are physiologically and transcriptionally distinct. A similar expression pattern is observed in Aphanomyces spp. with different regulation of spore development and in the transcript is detected in both primary and secondary cysts.A putative chitinase AaChi1, was cloned from the crayfish plague fungus, Aphanomyces astaci. Analysis of chitinase activity and AaChi1 expression showed that chitinase in A. astaci is constitutively expressed in growing and sporulating mycelia, but absent in zoospores, a pattern which reflects the infectious life cycle of A. astaci. This expression pattern is conserved between the four known genotypes of A. astaci, in contrast to saprophytic and fish-pathogenic Aphanomyces spp. Genetic and physiological analysis were conducted on five strains of Aphanomyces, isolated from suspected outbreaks of crayfish plague in Spain and Italy. The strains are not virulent against freshwater crayfish, and RAPD PCR and ITS sequence analysis show that they are unrelated to the crayfish plague fungus, A. astaci.