Functional Research On The Mitogen-activated Protein Kinase Pathway In Conidiation And Mycoparasitism Of Coniothyrium Minitans

Sclerotinia sclerotiorum (Lib.) de Bary is a worldwide fungal pathogen causing serious diseases on many economically important crops. Coniothyrium minitans is an important biocontrol agent against Sclerotinia diseases. The conidiation of C. minitans is very important to its life cycle and mycoparasitism. Previous work had reported about the conidiation of C. minitans, however, the complex network that regulates the conidiation is still not clear. In this thesis, the conidiation deficient mutant ZS-1T1000, screened from T-DNA insertional mutagenesis library of ZS-1, was investigated. The flanking sequence of the T-DNA insertional site was amplified using TAIL-PCR, and based on the conidiation-related gene that blocked in the mutant ZS-1T1000, we investigated the functions of cell wall integrity MAPK pathway (CWI-MAPK) which participates in the mycoparasitism, conidiation and maintaining its cell wall integrity in C. minitans. Furthermore, we also analyzed the functions of CmSTE7, the homolog of STE7in the FUS3/KSS1MAPK pathway of Saccharomyces cerevisiae, in sclerotial mycoparasitism of C. minitans. Results achieved so far were summarized as following:a) The conidiation deficient mutant ZS-1T1000was blocked by single copy of T-DNA insertion, and T-DNA inserted in the gene CmBCK1which encode a protein kinase kinase kinase. The CmBCK1contains one intron at the3'terminal end, and the coding sequence (CDS) of CmBCK1is5361bp which encodes a1786aa protein. The T-DNA insertional site exists at5100bp, and destroys the conservative catalytic domain. Only one copy of CmBCK1exists in the genome of C. minitans, and the lost function of CmBCKl leading to the lack of pycnidium and conidiation, significantly reduced the ability of sclerotial parasitization, hypersensitive to the cell wall inhibitor and cell wall lyases, the autolytic aerial mycelia and reduced pigmentation noticeably in the mutant ZS-1T1000.b) Targeted disruption of the CmBCK1showed that the ΔCmBCK1mutant was uncapable of conidiation; autolytic mycelia in center of colony, decreased melanin production, hypersensitive to the cell wall inhibitor and cell wall lyases, and defective in the mycoparasitization, these deficiencies were similar to the mutant ZS-1T1000.c) From C. minitans, the Slt2homolog CmSlt2which downstream of BCK1in the CWI-MAPK pathway was obtained. The CmSlt2contains5introns, and the CDS of CmSlt2is1254bp which encodes a417aa protein. Similar to the CmBCK1mutant, Cmslt2disruption mutants lost the ability to produce the conidia, significantly decreased in the sclerotial parasitization, increased in the susceptibility to the cell wall inhibitor and cell wall lyases and defective in the pigmentation. These defective phenotypes were restored by the complementation of CmSlt2gene.d) Taken together, the results from CmBCK1and Cmslt2suggest that the CWI-MAPK pathway is essential for the conidiation, sclerotial mycoparasitism, pigmentation, and maintenance the stability of cell wall in C. minitans.e) CmSTE7, the homolog of STE7from the FUS3/KSS1MAPK pathway of S. cerevisiae, was obtained from C. minitans. The CmSTE7contains3introns, and the CDS of CmSTE7is1362bp which encodes a453aa protein. CmSTE7disruption mutants showed a white colony, failed to produce melanin (slight melanin produced in the PDA medium after long time incubation), failed to form pycnidium, increased the number of aerial hyphae and multibranches, and extremely decreased the ability of sclerotial parasitization. These results suggest that CmSTE7involves in sclerotial mycoparasitism, the formation of pycnidium and melanin production in C. minitans.The MAPK pathways involved in the regulation of the various development procedures in mycoparasite fungus C. minitans had been implicated in this thesis, the results suggested that CWI-MAPK and FUS3/KSS1MAPK pathway played critical roles in the conidiation and sclerotial mycoparasitism in C. minitans. These progeny could be a theoretical basis to elucidate the complex progress of the conidiation and sclerotial mycoparasitism in C. minitans, advance our knowledge of filamentous fungal biology and the molecular biology of pathogenicity, and also have important theoretical significance to the commercial usage of C. minitans as the biocontrol agent.