Development of functional gene characterisation methods for animal pathogenic oomyscetes

Saprolegnia parasitica and Aphanomyces invadans are oomycetes that infect fish. S. parasitica is responsible for devastating infections in fresh water fish and in particular salmonids. It is estimated that at least 10% of all hatched salmon die due to Saprolegniosis in fish farms, which represents significant financial losses and is a serious animal welfare concern. A. invadans causes Epizootic Ulcerative Syndrome (EUS) in fish living in fresh and brackish water mainly and is mostly found in Asian aquaculture. With the completion of the genome sequence for S. parasitica and A. invadans, the interest from sequencing has shifted to the understanding of gene function and regulation. In order to functionally characterize genes from these organisms an efficient mutagenesis or silencing method needs to be in place. However, within the group of oomycetes, gene transformation technology has been developed but its efficiency is, at present, limited to a restricted number of oomycete species. For that reason, genome editing systems for fish pathogenic oomycetes involving stable and transient silencing were attempted in this study. In Chapter 2 we describe an attempt for a stable transformation protocol using cyanamide as a positive selection marker. Four main transformation protocols were tried: PEG-mediated protoplast, heat shock, electroporation and Turbofectin. Electroporation was selected for further optimization. The optimum conditions chosen were: L15 as electroporation buffer, 0.2mm cuvette gap and multiple pulses at 200V with 20s intervals with incubation temperature at 18°C. Transformations were also conducted using protoplasts and zoospores, however, although a few protoplasts survived following cyanamide selection, the cyanamide gene was not detected in their genome which suggested that only false positives were obtained. A different approach of stable transformation was attempted with GFP as a selection marker in Chapter 3, together with the development of CRISPR/Cas9 genome editing system for fish pathogenic oomycetes. In order to achieve this, sgRNAs were designed to target selected genes. Four target genes HlyE (sprg_03140), CotH (sprg_04414), SpHtp1 (sprg_04985) and SpHtp3 (sprg_03573) were selected initially and cloned into pYF2.3G-ribo-sg RNA. Also, the Cas9 vector that was originally designed for Phytophthora sojae, a plant pathogenic oomycete, was optimized in terms of the nuclear localization signal (NLS) and the resistance marker. The nuclear localization signal is important for the import of Cas9 enzyme into the cell nucleus where it modifies the target gene, while the resistance marker enables the selection of antibiotic-resistant transformants of S. parasitica. Following transformation, it was revealed that Cas9 was expressed in the S. parasitica protoplast and was detected in the DNA of transformants. However, it also seems that the Cas9 enzyme is degraded in Saprolegnia. In Chapter 4 and Chapter 5, transient silencing using RNA interference (RNAi) was conducted but using different species of fish pathogenic oomycetes, S. parasitica and A. invadans, respectively. In Chapter 4, a hemolysin gene of S. parasitica SpHlyE1 (SPRG_03140) was functionally investigated by RNAi silencing and a subsequent infection assay with Galleria mellonella was performed. The dsRNA-treated group showed reduced levels of mRNA of hemolysin compare to the untreated group. The hemolysin-silenced lines showed a delayed and decreased mortality rate in the infection assay with G. mellonella. Given the simple applicability of RNAi-silencing combined with the infection assay, this is a relatively fast strategy for future functional characterisation of genes in S. parasitica. In Chapter 5, a silencing experiment using RNA interference was conducted to reduce the mRNA level of a target gene, AiLhs1 followed by an infection assay with Galleria mellonella. The silencing resulted in the phenotypic impairment of sporulation. The dsRNA-treated group showed reduced levels of mRNA and high survival rate in the infection assay with G. mellonella. Phenotypic observations in AiLhs1-RNAi silenced lines revealed reduced size of spores in the hyphae, fewer spores in clusters and lower number of swimming spores in the medium with slightly delayed sporulation. Our results suggest that AiLhs1 is required for successful sporulation which is critical for host colonising. Based on the studies that have been conducted for this thesis, a better understanding of two genes predicted to be involved in virulence was achieved. However, a protocol for stable transformation was not achieved and this is urgently needed to functionally characterize genes or to express other genes, such as reporter genes, in these animal pathogenic oomycetes. The present work has provided important preliminary data that will help to generate a stable transformation method for animal pathogenic oomycetes and possibly a gene editing system.