Proteomic Study Of Mutation Strain UV57 And Function Study Of Prb1 And Sahh Gene In Chestnut Blight Fungus

Cryphonectria parasitica is a pathogenic fungus causing the devastating chestnut blight.Cryphonectria hypovirus 1(CHV1)was a class of positive strand RNA virus obligate parasitic in the cytoplasm of Cryphonectria parasitica.We gain a mutant UV57 that it dose not support CHV1 replication by the method of UV irradiation on the Cryphonectria parasitica transgenic strain CN2(carry CHV1).UV57 was characterized by a slower growth rate,dry intolerance,fewer aerial hyphae,fewer orange pigmentation,an absence of pycnidia and conidiation,and an apparent reduction in virulence.Differential proteomic analysis between wild-type strain EP155 and mutant UV57 has been made by two dimensional liquid chromatographic fractionation.83 protein UV absorption peaks were detected which show more than 2 fold changes on the base of EP155:35 up-reg?lated and 48 down-reg?lated.These differentially expressed proteins were implied to have functions in metabolisms of energy,methylation,amino acid,tricarboxylic,acid cycle,pyruvate and MAPK(promoting mitogen-activated protein kinase)signaling pathway.We also found a number of mitochondrial proteins also differentially expressed.The prbl gene coding for PRB1(Subtilisin-like protease B)in Cryphonectria parasitica.Differential proteomic analysis revealed that PRB1 was down-regulated by 2-fold in mutant UV57 compared to that in EP155 strain.qRT PCR analysis revealed that the mRNA accumulation of prbl was also down-regulated by 4.38-fold compared to that in EP155 strain.We knocked out the sahh gene from the genome of strain CP80 and obtained ?prbl strains.Paired inoculation of hypovirus-infected strain EP713 with mutant ? prbl strains resulted in anastomosis.Agarose gel analysis of replicative viral double-stranded RNA(dsRNA)isolated from the hypovirus-converted EP713/?prbl strains revealed that the accumulation level of CHV1-EP713 viral dsRNA was decreased comparable with that observed for strain EP713.The prbl gene may be a replication or maintenance related gene of the CHV1-EP713 virus.The sahh gene coding for SAHH(S-adenosylhomocysteine hydrolase,AdoHcyase)in Cryphonectria parasitica.Hypovirus regulation the sahh,the transcriptional level of sahh was up-regulated in the hypovirus-infected strain EP713.We used prokaryotic expression to obtain and purify a soluble recombinant SAHH protein with S-adenosylhomocysteine hydrolase activity.The hydrolytic enzyme activity exhibited Michaelis-Menten kinetics,with K,of 15.6 ?M and Vmax of 0.25?mol/min/mg for SAH.To study the role of SAHH in Cryphonectria parasitica,we knocked out the sahh gene from the genome of strain CP80.Here we report that deletion of sahh result in increasing the levels of intracellular concentrations of S-Adenosylhomocysteine(SAH)and S-Adenosylmethionine(SAM),reducing the level of Adenosine(ADO),dramatically increasing the ratio of SAH/SAM,and remarkably increasing levels of transcripts that encode key components(adenosine kinase,methionine adenosyltransferase and an 0-methyltransferase)of the methylation pathway.The ?sahh knockout mutant was characterized by a slower growth rate,fewer aerial hyphae,loss of orange pigmentation,an absence of pycnidia and conidiation,and an apparent reduction in virulence.Transcriptional analysis revealed that deletion of sahh reduced the levels of transcript encoding the virulence gene cypl and genes(cpgal,cpgbl cpgcl and stel2)encoding key components(coding for G?,G?,G? and Stel2,respectively)of multiple G-protein signaling pathways.These results speculated that SAHH-mediated effects on intracellular methylat ion affect other phenotypes,include the multiple G-protein signaling pathways,thus affect the virulence of Cryphonectria parasitica.