Analysis Of Interaction Between SsHADV-1 And Sclerotinia Sclerotiorum By RNA-seq

Sclerotinia sclerotiorum(Lib.)de Bary is an important fungal pathogen which could cause serious damage to diverse crops.However,methods which could efficiently controll this disease have not been developed yet.Mycoviruses are viruses that infect diverse fungi.An increasing number of novel mycoviruses have been found in S.sclerotiorum.The infection of some mycovirus alters eharacteristies of the host fungi,including hypovirulence,slow growth and abnormal colony development.Previously,a circular ssDNA virus,named Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1(SsHADV-1),was isolated and identified from hypovirulent strain DT-8 of S.sclerotiorum.SsHADV-1 is the first DNA mycovirus that was found to infect fuigi and confers hypovirulence.DT-8VF is a virus-free culture derived by hyphal tipping of strain DT-8,and showed a normal phenotype of S.sclerotiorum.DNA mycovirus-host interaction system may provide an opportunity to identify determinants that change host phenotypes and to understand the molecular basis of fungal biology.Moreover,genomic sequences of pathogenic fungi are available and facilitate deciphering virus-host interaction at the molecular level.Here,we apply high-throughput sequencing(RNA-Seq)to understand the molecular basis of interaction.The primary results are shown below:1.Analysis of gene expression changes of S.sclerotiorum in response to SsHADV-1 infection.Hyphae of strain DT-8 and DT-8VF which were taken from dynamic colonies were collected for RNA extractions.Three biological replicates for DT-8 and DT-8VF samples were made in order to determine whether a change is biologically significant.Comparative transcriptome profiling of S.sclerotiorum in response to SsHADV-1 infection was conducted by RNA-Seq.A total 928 genes of DT-8 were differentially expressed comparing to DT-8VF,including 255 down-and 673 up-regulated genes.2.Gene ontology(GO)analysis showed that differentially expressed genes enriched in regulation of nitrogen and carbon utilization,melanin biosynthetic process,amino acid transmembrane transport and so on.KEGG pathway enrichment analysis revealed that up regulated genes enriched in pathways of nitrogen metabolism,homologous recombination,non-homologous end-joining and DNA replication;Down-regulated genes enriched in pathways of pyruvate metabolism,earbon metabolism,starch and sucrose metabolism and peroxisome.Further computational analysis using the database,STRING,which detects predicted protein interactions,identified that predicted protein interaction is involved in diverse complex of DNA repair,membrane transporters and Cytochrome P450.3.The transcriptional profile of host genes related to pathogenicity were evaluated by reverse transcription quantitative real-time polymerase chain reaction(qRT-PCR).We observed that many genes encoding CWDEs:PGs,cellulases,hemicellulases and proteases genes were evidently down-regulated in strain DT-8 based on the data.Ss-oahl gene,which encodes an oxaloacetate acetylhydrolase,required for oxalic acid accumulation was down-regulated in DT-8.pH value of culture solution of DT-8 was higher than that of strain DT-SVF during 1?7 days.However there was no dramatic difference in quantitative detection of oxalic acid by high performance liquid chromatography(HPLC).Most Laccase genes and other genes related to metabolism of melanins were dramatically up regulated in strain DT-8,which play a role in strengthening fungal survival ability such as removing active oxygen free radicals effectively.There were no significant differences of gene expression involving in MAPK pathway,cAMP-dependent pathway and calcium-signaling pathway between strain DT-8 and DT-8VF.3.Identification and analysis of anti-virus genes in virus-host interaction.RNA silencing is one of the major mechanisms for antiviral immunity.Genes encoding DICER and ARGO protein were found up-regulated in strain DT-8.Host cell damage response plays a vital impact on viral replication and viral recombination.Key factors of repair pathway provide promising inhititor target for viral disease treatment.The genes in DNA repairing pathway were dramatically up-regulated in strain DT-8.Non-homologous end-joining(NHEJ)pathway is important in response to DNA damage repair.More interestingly,the genes of NHEJ were totally obviously up regulated in strain DT-8.Mycovirus is likely to evolve an efficient system to up-regulate host genes for its own replication and this deserved furture investigation.We applied diverse analysis-softwares with different reference value of pathway within fungi,and found NHEJ is the only one enriched pathway.4.RNAi silencing vector of SsKU70 and SsKU80 were constructed,and then transformed to S.sclerotiorum strain Ep-1PNA367 by ATMT method.Comparsion to the strain Ep-1PNA367,the expression level of SsKU70 and SsKU80 declined significantly in silencing transformants.Transformants of SsKU70 and SsKU80 showed no significant difference in colony morphology,growth rate and pathogenicity.Transmission of SsHADV-1 from strain DT-8 in SsKu70/Ss-K180 silencing strains revealed that virus replication of SsKu70/Ss-Ku80 transformants were not stable comparing with strain Ep-1PNA367.More work about important role of NHEJ in viral replication need to be verified.5.NHEJ Inhibitor(vanillins)treatment had a significant influence on hypha growth.We inoculated SsKu70/Ss-Ku80 transformants,DT-8 and DT-8VF on PDA plates amended with different concentrations of NHEJ Inhibitor.The higher the concentration of NHEJ Inhibitor solution,the slower the growth rate were and the later sclerotium formed.