VIB seminar location:Blankenberge, Belgium date:9-10 March 2006
Plants have developed various mechanisms of defense against invading pathogenic organisms. One example is the production of antifungal molecules, namely osmotin, by the tobacco plant. Osmotin belongs to the Plant Pathogenesis-related (PR) protein family 5, and it has been shown to have antifungal activity both in vitro and in vivo (Yun, 1997).
An important feature of osmotin, and PR-5 in general, is that they share similarities with antimicrobial peptides that are known components of the related innate immune response in plants and animals. Osmotin is able to induce programmed cell death in the model yeast Saccharomyes cerevisiae. The resulting apoptosis occurs by intracellular accumulation of reactive oxygen species (Narasimhan, 2001). Osmotin seems to function upstream of the cAMP/PKA pathway via activation of Ras2. Osmotin has recently been shown to specifically bind to a plasma membrane protein encoded by PHO36 that is located upstream in the signalling pathway (Narasimhan, 2005). PHO36 is involved in lipid metabolism and zinc ion homeostasis.
Mammalian cells possess a functional but not structural homolog of the yeast PHO36 in respect to binding osmotin. It is a receptor for binding the hormone adiponectin, thereby regulating lipid and sugar metabolism (Narasimhan, 2005).
An osmotin-binding receptor in Candida albicans which is able to mediate apoptosis could be an interesting target for antifungal treatment. Blast searches with PHO36 against the genome of Candida albicans resulted in one specific ORF with 37% homology. Interestingly this receptor contains seven transmembrane domains. A homozygote knockout of the respective gene has been constructed in the diploid strain replacing the original alleles consecutively with a sequence carrying the auxotrophic marker gene URA3 in a URA3 deletion background. After each step the capability to form Ura3p was removed from the insert hisG-URA3-hisG by selecting the cells for the spontaneous loss on 5-fluororotic acid remaining only one copy of hisG in the former proteins CDS. The knockouts were confirmed by PCR diagnosis and Southern blot. As a first step we will investigate the resulting morphology in different growth conditions, localize the protein within the cell and determine the cell sensitivity against osmotin and functional analogs. Comparative studies are intended to show a potential protein function upstream of the RAS2-cAMP/PKA pathway as indicated for Saccharomyes cerevisiae.
Narasimhan, M. L., et al. (2005) Molecular Cell 17: 171-180.
Narasimhan, M. L., et al. (2001) Molecular Cell 8: 921-930.
Yun, D. J., et al. (1997) Proceedings of the National Academy of Sciences USA 94: 7082-7087.