The oomycete Phytophthora parasitica has a worldwide distribution and is an economically important pathogen of more than 100 species4. RNA-seq analysis showed that one gene, PPTG_16698 has the 5th highest level of expression of all transport proteins in the zoospore stage, and is highly conserved throughout Phytophthora species. This project attempts to characterize the important biological role that PPTG_16698 plays in P. parasitica and other oomycetes. Three strategies have been implemented to accomplish this goal: growth analysis by heterologous expression in yeast, metabolite analysis in yeast, and construction of a GFP fusion protein to enable localization of the gene in oomycete hyphae by confocal microscopy.
Confocal microscopy is expected to confirm the vacuolar localization of this gene. If this gene is localized to a vacuolar membrane, then heterologous expression in yeast should result in differential accumulation of metabolites mobilized by this transporter. In preliminary growth assays, expression of this gene did not inhibit the growth of yeast. Therefore, expression of the gene does not result in sequestering of a growth-limiting metabolite. To determine whether expression of the transporter results in the accumulation of polyamines, which are organic compounds necessary for growth in eukaryotes, polyamine levels will be measured by dansylation of amines and separation by HPLC. Other metabolites will be assayed by liquid chromatography mass spectroscopy analysis.
Dr. Paul F. Morris
First Advisor Department
Dr. Howard Casey Cromwell
Second Advisor Department
Padula, Stephanie; Morris, Paul F. Dr; Cromwell, Howard Casey Dr.; Ariyaratne, Menaka; and Wagner, Andrew, "Characterization of a Novel Clade of Transporters in Phytophthora" (2015). Honors Projects. 173.
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