|Prometheus - PROteoME of Diplodia corticola, the cork oak paTHogEn fungUS|
Ana Cristina Esteves
Programme - PTDC/AGR-CFL/113831/2009
Execution dates - 2011-03-01 - 2014-02-28 (36 Months)
Funding Entity - FCT
Funding for CESAM - 164000
Total Funding - 164000
Proponent Institution - Universidade de Aveiro
Universidade de Ghent
Phytopathogenic fungi are a worldwide problem, since they damage crops quite severely. Specifically in the Iberian Peninsula and the Mediterranean Basin (MB) an important problem caused by phytopathogenic fungi has been recognised. Diplodia corticola is a fungus pathogenic of Quercus suber , the cork oak, involved in several diseases responsible for the decline of the cork oak forest. In the Atlantic and West Mediterranean countries and particularly in Portugal the cork transformation industry is of great economical importance. Recent data refer that in Portugal there are hundreds of companies related to cork with an estimated cork production of over than 550 million euros. Nevertheless, Q. suber is threatened and it was given a protection status due to fungal diseases, namely, disease caused by D. corticola. The main question arising from the 'cork oak problem' is: how can we control the infection caused by D. corticola? Answering this question implies determining the infection cycle of this pathogen. It is widely recognised that secreted proteins are of particular relevance when studying fungal pathogenicity since these molecules trigger the ongoing interaction with the plant host . Therefore, to answer this question we intend to get information on the fungal proteins involved in infection cycle. Therefore, Prometheus intends answering to:- Which proteins are expressed by D. corticola that allow it to enter and spread throughout the plant? - Are there any proteins specific to the most virulent species/strains? We will use several strains of D. corticola, with several degrees of virulence in order to conclude on the proteins involved on the infection cycle used by this organism. For that, we will determine the D. corticola protein map. This is, to our knowledge, the first proteomic exploratory study of this organism. Furthermore, we will determine the differential proteome of the fungus upon infection by 2-D Fluorescence Difference Gel Electrophoresis (DIGE) followed by MALDI-mass spectroscopy, and database searching. For filamentous fungi, we can estimate the presence of thousands proteins per cell and therefore we will focus not only on the secretome but also on subproteomes. Combining classic biochemical fractionation techniques for the enrichment of particular subcellular structures (nucleus, mitochondria and cytoplasm) with the large-scale identification of proteins by mass spectrometry provides a powerful methodological approach that makes possible the discovery and dissection of the 'subcellular proteome' . In this way, it will be possible to identify the proteins of D. corticola that are up- or down regulated during the infection process and study the dynamic changes at the subcellular level. Protein secretion plays an important role in filamentous fungi and frequently a broad spectrum of structural proteins and enzymes, the majority of which are hydrolytic, is secreted by filamentous fungi. This ability has been widely exploited by the biotechnology industry for the production of enzymes. Therefore, a selection of enzymes presumptively involved in plant infection or with biotechnological application will be made; those enzymes will be purified and characterised functional and structurally. In brief, this proteomic study D. corticola will be a useful basis for exploring the proteins involved in the infection cycle, which will in turn provide new targets for crop diagnosis and focused fungicide design. The identification and study of the complete pool of secreted proteins may lead to new control measures for fungal diseases, impacting the plant yields and the agricultural profits. This project will also contribute to the training and development of research competences of young investigators.
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