The success of eukaryotic oomycete pathogens depends largely on effector proteins, molecules that manipulate or interfere with host defence mechanisms in the extracellular space or inside their host cells. One economical important oomycete parasite is the fish pathogen Saprolegnia parasitica, which is the causal agent of saprolegniosis. S. parasitica is responsible for devastating losses in the aquaculture industry worldwide. In order to effectively fight S. parasitica it is crucial to understand its pathogenicity strategies. With the focus on cytoplasmic effector proteins, the genome of S. parasitica was screened in the present study for secreted proteins that possess Ricin-B-like lectin domains or potential N-terminal RxLR-motifs. A list of more than 30 potential effector candidates was compiled and gene expression analysis was performed with RNAseq. Possible functions of the candidate effectors are discussed. SPRG_21856.2 (Ricin-B-like lectin candidate) and SpHtp3 (RxLR candidate) were selected for further analysis. Although two different expression systems, Escherichia coli and Pichia pastoris, were used, recombinant protein production of SPRG_21856.2 was not successful. Possible reasons and alternative overexpression methods are discussed. For SpHtp3 recombinant protein production was successful. Functional characterisation revealed that SpHtp3 is an active nuclease capable of destroying both DNA and RNA molecules. Furthermore, SpHtp3 was found to specifically enter fish cells in an O-sulfate and pH-dependent manner. Interestingly, the potential N-terminal RxLR-motif of SpHtp3 was not required for translocation. In support of that finding also PsHtp3, a Phytophthora sojae homolog of SpHtp3 that lacks an RxLR-sequence, was capable of translocating into fish cells. After translocation SpHtp3 localised in small vesicles from which it was only released when the host cells were under direct attack by S. parasitica. SpHtp3 is the first protein described from an oomycete that exhibits both self-translocation and nuclease activities combined in one molecule. Potential functions and future applications are discussed.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:606469 |
| Date | January 2014 |
| Creators | Löbach, Lars |
| Publisher | University of Aberdeen |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=211033 |
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