Proteomic Study Of The Interplay Between Hypovirus And Chestnut Blight Fungus, Cryphonectria Parasitica

Hypovirus is unencapsidated dsRNA virus. The hypovirulence and phenotype variation of the chestnut blight fungus, Cryphonectria parasitica happens by hypovirus CHV1infection. The reports about proteomics of C. parasitica are limited at the moment, although the molecular biology research of hypovirus/C. parasitica system are in-depth study. This thesis included several aspects which revealed the interaction between hyporirus and the pathogen fungus in proteomic level.According to the characteristics of filamentous fungi, the extraction of tots protein was improved. Differential proteomic analysis between wild-type strai EP155and hypovirulent strain EP713has been made by two dimensional liqu chromatographic fractionation. ProteoVue and DeltaVue software analyzed at build two-dimensional simulation of gel patterns with good reproducibility.2protein UV absorption peaks were detected which show more than2fold changes the base of EP155:209up-regulated and87down-regulated. The same prot samples were used on2D-gels and71differential expressed proteins were fou The variation tendency was consisitent with the previous report.An optimized protein extraction method suitable for the preparation of secre proteins of a phytopathogenic filamentous fungus, C. parasitica, was developed, evaluated by SDS-PAGE and two-dimensional gel electrophoresis (2-DE), method was both reliable and comprehensive in obtaining secreted proteins wide range of molecular weight and pI, when the fungus was cultured in the complete liquid medium from one to seven days. A total of137unique proteins MW ranging from10to150kDa and pI ranging from pH3to8on2-DE gels identified by tandem mass spectrometry. By annotation, these proteins are m cell-wall-degrading enzymes, virulence factors and components involve plant-pathogen interactions. Of which,20proteins got a signal peptide and proteins were predicted to be involved in known secretory pathways by Fungal Secretome Database (FSD). Through knockout of high abundant secreted gene, we found2differential expressed proteins in secretome which belong to ubiquitin and a hypothetical protein.To expand our understanding of virus-host interactions, the vesicle proteins of different strains were analyzed. Thirty-three differentially expressed protein spots of vesicle protein were found in response to the virus infection. These proteins were implied to have functions in metabolisms of protein, nucleic acid, and carbohydrate, signal transduction events, cellular transport, response to stress and oxidation/reduction. The result indicated that the virus infection did not affect the function of transport vesicle but greatly changed transport efficiency. Four virus protein spots displayed in virus-infect vesicle protein samples. The identified peptides all came from ORF B of hypovirus CHV1and located in the scope of p48by mass spectrum analysis. We supposed that after autoproteolysis of NH2-terminal portion of ORF B to produce p48, the viral protein, p48may lead the viral ORF B replication and translation, then went through post-translational modification to produce protein isoforms with different functions.