Preliminary Research On The Dimorphic Transition Of Nomuraea Rileyi

As misuse of chemical pesticide during the production and application,Three Rs(Resistance,Residue,Resurgence)from pest control are becoming more and more serious.In recent years,biopesticide(including entomopathogenic fungi)has gradually become in a hot spot of research and application.Nomuraea rileyi is an entomopathogenous fungus existing widely,which infects noctuidae insects in the fields.Epidemic diseases of the incects could emerge while N.rileyi spreading,having potential biocontrol value.Dimorphic growth arises during natural infection or artifical culture of N.rileyi.The analysis on this phenomenon would contribute to its production and application,theoretically.Agrobacterium tumefaciens mediated transformation of this entomopathogenic fungus has succeeded developed in our lab,and its yeast cells are used as recipient cells in this method.The preparation method previously used for perpearing yeast cells cannot meet demand of ATMT,and needs to be improved urgently.In addition,the reaseach on dimorphic growth of N.rileyi will providing lage scale method of preparation of yeast cells for ATMA recipients requairment.Based on optimumed interspersing method of yeast cells for ATMT using conidium of N.rileyi in order to observe the process of dimorphic growth of N.rileyi on SMAY,investigate the molecular regulating mechanism of the dimorphic transition,found the key regulatory factor and verified it based on comparison transcriptomic analysis.The main research results obtained as follow:(1)Optimization of the method for massive perpearation of yeast cells as recipients for ATMTTo improve the output of the yeast cells,the method was improved.Conidia stored less than three months could be used for perpearing yeast cells.1% agar concentration was chosen,on account of extending yeast-cell growing period and prolonging harvest time window.Sow-inoculation was selected,since dispersing conidia more sufficiently,reducing inoculum density,more yeast-cell colonies growing out,and in the same stage.(2)Observation the dimorphic growth of N.rileyi on SMAYTo ensure sampling time of transcriptome,we inoculate the yeast cells of N.rileyi to the surface of SMAY,and observe the dimorphic growth.It was found that,0-12 hs is the yeast-cell growing stage,12-96 hs is the dimorphic transition stage,and 96-168 hs is the end of dimorphic transition stage.(3)Comparative transcriptome analysis of the dimorphic transition.of N.rileyiTo find the genes involved in dimorphic transition,we analyzed the transcriptomes through high-throughput sequencing to during the dimorphic growth.A total of 4.40 Gb of clean nucleotides comprising 20,165 sequences were obtained,and there were 20,919 sequences(about 71.83%)annotated.There were only 6,559 sequences concluded into 40 gene ontology(GO)functional categories,and 8,957 of these annotated sequences were mapped into 108 pathways after searching in the Kyoto Encyclopedia of Genes and Genomes pathway(KEGG)database.Further analyzation on the transcriptomic differences between the yeast-cell gorwth and hyphal formation was proceeded.We found 6,048 sequences expressing differentially,in total;2,023 of them were assigned into 25 GO classes and 2,931 unigenes differentially expressed were mapped into 108 KEGG pathways,with 5 enrichment pathways included.Subsequently,we examined these genes differently expressed during the dimorphic transition,in the enrichment pathways,and found that there were many pathways including RIM101,phosphatidylinositol synthesis participating in the dimorphic switch.To verify the result,we analyzed the expression of the unigenes using quantitative reverse transcription-PCR(RT-qPCR).The result shows that multiple pathways participate in the dimorphic transition,and pH might be the key factor affecting this phenomenon.This transcriptome research provides data for further study on the dimorphic switch of N.riley.(4)Conclusion and verification that pH is the key factor affecting the dimorphic transition.Based on the analyzation of transcriptome,we conclude that pH might be the key factor affecting the dimorphic transition,and verify it by ph gradient method.The results showed that the dimorphic transition accelerates under acidic conditions.N.rileyi yeast-cell growth stage extends,and the dimorphic transition delays under neutral and alkaline conditions.This conclusion can explain the dimorphic growth of N.rileyi during infection process to a certain degree.We conclude that,the dimorphic switch of N.rileyi is controlled by cell density and pH of the surroundings.It is also affected by the agar concentration or hardness of the culture medium.Main different up regulated expressing genes and enriching passways including RIM101,phosphatidylinositol synthesis participating in the dimorphic switch prossece.Since multiple pathways and genes participate in the dimorphic transition,further research needs to be done to show the regulatory mechanism.