Differential Mitochondrial Proteomic Analysis Of Cryphonectria Parasitica Strains EP155 And EP713

Cryphonectria parasitica and hypovirus is a model system for the study of the interaction between host and virms.In recent years,some researches shows that hypovirus infection can impact on the function of mitochondria of Cryphonectria parasitica.To clarify the affection of hypovirus on mitochondria,differential mitochondrial proteomic analysis of Cryphonectria parasitica strains EP155 and EP713 were carried out.The extraction and purification method of fungal mitochondria was optimized.Fungal hypha was grinded in liquid nitrogen then differential centrifugation combined with sucrose density gradient was used to isolate and purify mitochondria.The concentration rate of mitochodria was further confirmed by western blot.The parameters of two-dimensional electrophoresis were determined.The amount of 200 ?g proteins was loaded on 24 cm pH3?lONL IPG strip for isoelectrofocusing,SDS-PAGE and followed by silver stain.The gel maps with clear background,high resolution and good reproducibility were obtained.Ten protein spots were randomly selected for mass spectrometric analysis and nine got effective identification.The successful detection rate was 90%.The result of these identified proteins which all belong to characteristic mitochondrial proteins verified the purity of the obtained mitochondria.2D-DIGE technology was used to separate mitochondrial proteins of EP155 and EP713.The DeCyder 2D 7.0 software performed variation analysis of the obtained maps.A total of 41 protein spots(by fliter Av.Ratio>1.5 and ANOVA<0.05)were detected and identified as 30 unique proteins.Sixteen of these proteins were up-regulated and others were down-regulated in EP713 upon hypovirus infection.These proteins,according to their functions,were classified to be related with citric acid cycle and oxidative phosphorylation,ROS levels,proline and glutamic acid metabolism,transport and transshipment,valine,leucine,isoleucine catabolism pathway.In this study,a comprehensive and systematic differentially expressed protein map of mitochondrial profiles between the wild type strain EP155 and hypo virus-infected strain EP713 was drawn.It is a useful exploration to indicate the mechanism of mitochondrial hypovirulence.