Study On MAP Kinase Gene Crmapk And Phosphatase Gene CrSsd1 Regulating Mycoparasitism Of Clonostachys Rosea 67-1

Clonostachys rosea(syn.Gliocladium roseum)is a widely distributed mycoparasite that has shown great potential against various plant fungal diseases,and the whole genome sequencing has been completed.In our previous study,the transcriptome of a highly efficient C.rosea isolate 67-1parasitizing Sclerotinia sclerotiorum sclerotia was constructed,from which a mitogen-activated protein kinase(MAPK)encoding gene Crmapk that significantly upregulated expressed under the induction of sclerotia was obtained.The functions of Crmapk in C.rosea mycoparasitism and biocontrol activity were confirmed by gene knockout and complementation.MAPK is a kind of important protein kinase that involves in signal transduction in fungi,and in turn regulates fungal growth,sporulation,pathogenicity and differentiation through phosphorylation cascade.However,the mechanisms of MAPK in mycoparasitism are not clear yet.In this study,the yeast library of C.rosea was constructed and the interacting proteins of Crmapk were screened and identified by yeast two-hybrid system and bimolecular fluorescence complementation methods.The functions of two interaction proteins CIP2 and CIP11 to mycoparasitism were demonstrated,and ultimately,to reveal the molecular mechanism of Crmapk regulating fungal parasitism.In addition,a cell wall biogenesis protein phosphatase encoding gene Cr Ssd1 which differentially expressed in the transcriptome of the wild type 67-1 was obtained,and its functions to mycoparasitism and biocontrol activity were investigated by gene knockout and biological experiments.The main research results are as follows:(1)Subcellular localization of Crmapk in C.roseaThe fusion expression vector Crmapk-GFP was constructed and transferred into the wide type 67-1by the method of protoplast transformation,and the fusion mutants were obtained based on the resistance selection of G418 and PCR identification.Ultimately,Crmapk was observed to be localized in cytoplasm through laser confocal microscopy.(2)Construction of the yeast two hybrid library of C.rosea 67-1The total number of clones of the yeast library is 1.6×10~7 CFU/m L,the recombination rate is 96%and the main inserted fragment length is larger than 1 kb.The high quality yeast library of C.rosea was constructed and confirmed,which provides a reliable research basis for screening interaction proteins.(3)Screening and identification of the interaction proteins of CrmapkThe Crmapk-BD bait vector was constructed,which exhibited no self-activation and toxicity to yeast cells,and was used as bait to screen the Y2H library.60 candidate interaction genes were screened using bait vector Crmapk-BD and verified by Y2H method.Ultimately,42 differentially expressed genes might be the target of Fus3/Kss1-MAPK pathway based on the analysis of bioinformatics and transcriptome.Among these,the interaction between CIP2,CIP3,CIP11 and Crmapk were identified by Y2H and Bi FC techniques.(4)Functions of CIP2 and CIP11 in mycoparasitism of C.rosea to S.sclerotiorum sclerotiaThe expression levels of CIP2 and CIP11 in 67-1 were investigated during different stages of mycoparasitizing sclerotia by q RT-PCR and found both genes were upregulated in C.rosea throughout mycoparasitism.Biological functions of CIP2 and CIP11 were investigated by gene deletion and complementation.Results showed that,compared with the wide type 67-1,?CIP2 and?CIP11 not only had irregular morphology,significantly decreased conidiation,but also showed reduced antagonistic activity,mycoparasitism and biocontrol efficacy.In addition,they didn't show any significant difference to a series of stress.(5)Function of Cr Ssd1 in mycoparasitism of C.rosea to S.sclerotiorum sclerotiaqRT-PCR results indicated that the Cr Ssd1 gene was upregulated during different stages of C.rosea mycoparasitizing sclerotia.Functional analysis results showed that the Cr Ssd1 gene was essential for conidiation,responses to sorbitol and CR and involved in mycoparasitism and biocontrol efficacy,indicating that it plays diverse and essential roles in C.rosea.These results will provide new insight into the protein phosphorylation involved in mycoparasitism-associated mechanisms of C.rosea and facilitate to explore and utilize highly efficient biocontrol-related genes and increase control efficacy to plant diseases.