The mycoparasitic oomycete Pythium oligandrum is homothallic, producing an abundance of thick-walled spiny oospores in culture. After mining a cDNA sequence dataset, we identified a family of genes that code for small tyrosine rich (Pythium oligandrum small tyrosine rich (PoStr)) proteins. Sequence analysis identified similarity between the PoStr proteins and putative glycine-rich cell wall proteins from the related plant pathogenic oomycete Pythium ultimum, and mating-induced genes from the oomycete Phytophthora infestans. Expression analysis showed that PoStr transcripts accumulate during oospore production in culture and immunolocalisation indicates the presence of these proteins in oogonial and oospore cell walls. PoStr protein abundance correlated positively with production of oogonia as determined by antibiotic-mediated oogonia suppression. To further characterise the role of PoStr proteins in P. oligandrum oospore production, we silenced this gene family using homology-dependent gene silencing. This represents the first characterisation of genes using gene silencing in a Pythium species. Oospores from silenced strains displayed major ultrastructural changes and were sensitive to degradative enzyme treatment. Oogonia of silenced strains either appeared to be arrested at the mature oosphere stage of development or in around 40 % of the structures, showed a complete suppression of oospore formation. Suppressed oogonia were highly vacuolated and the oogonium wall was thickened by a new inner wall layer. Our data suggest PoStr proteins are probably integral structural components of the normal oospore cell wall and play a key role in oospore formation. (C) 2013 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Bibliographical noteOur work is supported by the University of Aberdeen (PvW), the Biotechnology and Biological Sciences Research Council (BBSRC) (LGB, PvW), the Royal Society (PvW), the Total Foundation (PvW), the EU (LGB) via a Marie Curie Intra European Fellowship (FP7-PEOPLE-2010-IEF; CBOP) and the Natural Environment Research Council (NERC) via the Strategic Ocean Funding Initiative Initiative (award NE/F012705/1) (LGB, PvW) and an NERC PhD-studentship (NH). Funding sources had no role in experimental design, analysis, interpretation of data, or in the preparation of this manuscript. Plasmid pTOR was a gift from Felix Mauch.
- gene Silencing
- oomycete sexual reproduction
- oospore formation
- appressorium formation