The proteome of Peronospora viciae, an obligate biotrophic pathogen that causes downy mildew of pea (Pisum sativum), was investigated using 2D gel electrophoresis and mass spectrometry. This provided data on changes in protein abundance during the sequential development of pre-invasion stages, from un-germinated conidia to germinated conidia with germ tubes and appressoria. Preliminary work developed reproducible methods for in vitro germination of conidia through to appressorium development. The method developed for in vitro conidia germination resulted in up to 65% of germinated conidia, of whi ch 35% fonned an appressorium. Six methods for protein extraction from the pre-invasion stages were compared by ID and 2D SDS-PAGE electrophoresis. The method chosen had relatively simple and non -toxic constituents (urea, thiourea, CHAPS and Tris-HCl), and would be appropriate for use in a disposable diagnostic device. Gels of extracted proteins showed over 700 protein spots following Coomassie blue staining of 2D gels and over 2000 protein spots on difference gel electrophoresis (DIGE) gels. A total of 21 proteins were identified from P. viciae using MALDI - and Q-TOF mass spectrometry analysis and searching against the MASCOT databases. Of the identified proteins, the majority (2 Hsp 70, a succinate dehydrogenase, an enolase, a catalase, a S-adenosyl-L-methioninedependent methyltransferase, a Hsp72, 2 actins, three GAPDH, a Fructose-bisphosphate aldolase, a fatty acid phospholipid synthesis protein, an uncharacterized aldolase, a Protein CpnIO and a NDK) decreased in relative abundance during spore germination, but with no further change on fonnation of appressoria. In contrast, the relative abundance of calmodulin and chitin synthase remained constant throughout pre-invasion development. However, calmodulin was not detected using western blotting whereas isoforms of actin and GAPDH were detected. Data are interpreted in relation to P. viciae pre-invasion biology, and the identification of key proteins and biomarkers as novel targets for control, pathogen detection and disease diagnosis.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:525211 |
| Date | January 2010 |
| Creators | Wandji, Josiane Laure Chuisseu |
| Publisher | University of the West of England, Bristol |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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