Characterization of Phytophthora capsici ERG3 through complementation of a yeast ERG3 null mutant

Abstract

Phytophthora capsici is a major pest to a broad host of plants. It is a sterol auxotroph that can grow vegetatively without sterols but requires them for reproduction. Previous studies demonstrated the absence of a sterol biosynthesis pathway in Phytophthora spp. but observed Δ7 sterols and Δ5,7 sterols converted to Δ5-sterols. In this study a P. capsici gene (FL-PcERG3) with homology to Saccharomyces cerevisiae ERG3 was examined for potential C5 sterol desaturase (DES-5) activity through complementation of a yeast erg3 null mutant. A FLAG-tagged FL-PcERG3 was amplified out of genomic P. capsici DNA and expressed under either the S. cerevisiae ERG3 promoter or under the inducible Gal1 promoter. The FLAG-tagged FL-PcERG3 demonstrated expression in a western analysis. A HPLC/MS analysis confirmed the ergosterol pathway complementation of the yeast erg3 null mutant by the putative FL-PcERG3. Chromatographs from the HPLC/MS analysis of the FL-PcERG3 complemented yeast erg3 null mutants had peaks at the same relative retention time and mass (m/z = 379.5) as ergosterol. The combination of data collected in this thesis and information from previous studies suggests the FL-PcERG3 expresses a functional DES-5 in P. capsici. This is the first study to evaluate a putative ERG3 gene from a Phytophthora spp. and provides evidence for an abridged sterol pathway in Phytophthora spp.