Functional Analysis Of Polyketide Synthase Gene Bcpks12 In Sclerotial Melanin Biosynthesis Of Botrytis Cinerea

Botrytis cinerea is the major fungal pathogen causing detrimental gray mold disease in several dicotyledonous plant species,and the huge economic losses of agricultural production have been due to spoilage of important crop plants as a result of infection by this pathogen.Sclerotium is a resting structre to survive under adverse circumstances,which composed of interwoven vegetative hyphae aggregated with highly melanized rind.Melanin pigments,high molecular weight compounds produced by oxidative polymerization of phenolic precursors,are detected in numerous organisms including animals,plants and microbes.In some plant pathogenic fungi,melanin in the cell wall was shown to play an important role as a pathogenic factor,in addition to some functions for stress resistance.In spite of numerous precedent studies in these aspects,biological functions of melanin in fungal sclerotium have remained obscure.In the present study for master's thesis,therefore,the author attempted to generate polyketide synthase gene Bcpksl2 knock-out mutants using conventional molecular genetic methods and to clarify biological functions of sclerotial melanin in B.cinerea.The main results obtained are listed below:1.In the first experiment,the author confirmed that the treatment with tricyclazole(DHN-melanin biosynthesis inhibitor)significantly impeded the melanin synthesis of sclerotium,indicating that sclerotial melanin of this fungus was DHN-melanin.The present bioinformatics analysis showed that BcPKS12(Bcpksl2 gene transcript)shared high amino acid sequence homology with previously reported melanogenic polyketide synthases in other fungi,which conserved typical functional domains of polyketide synthase in fungal melanin synthesis.Additionally,the qPCR-based gene expression analysis demonstrated that the Bcpksl2 gene was highly expressed during the sclerotial development.Judging from these results,the author concluded that the Bcpksl2 gene encodes the target enzyme polyketide synthase,which was involved in the regulation of melanin synthesis in the sclerotia of B2 cinerea.2.The author replaced the target gene Bcpksl2 with the hygromycin resistance gene cassette by a homologous recombination method,confirmed successful transformation by some ordinary DNA and RNA methods,and finally designated two Bcpksl2 gene knock-out mutants as Abcpksl2-6 and Abcpksl2-14.3.To analyze transcriptional network of Bcpksl2,the author performed its functional analysis at the stages of vegetative growth and sclerotial development.In the vegetative growth,both mutants and wild type strains showed the similar mycelial growth,conidiation and pigment synthesis.4.The mutants showed the impediment of melanin synthesis in the sclerotial development,and importantly the sclerotial melanization of the mutants was restored when 1,8-DHN was supplemented.These results indicate the involvement of DHN-mediated melanin biosynthesis in the formation of sclerotia by B.cinerea.Additionally,the author indicated that the deletion of the Bcpksl2 gene did not affect the normal development and morphology of the sclerotium.5.As an additional achievement of this study,the author established an efficient scleroitial germination system to analyze the role of sclerotial melanin.Using this system,the author examined sclerotial development on different medium.Eventually,the sclerotium formation was shown to be affected by the kinds and concentrations of carbohydrates added to the medium.In addition,the author examined the sclerotia of the wild type and mutant strains for their germination rate under various stress conditions(presence of oxidative compounds,low temperature and dryness).The results indicated that germination rates of the sclerotia of two mutants were significantly lower than that of the melanized sclerotia of the wild type strain,suggesting that melanin could make the sclerotium resistant to various stresses.6.The author performed virulence assay using spores and sclerotium of wild type strain and mutants for inoculation of apple,green grapes and tobacco leaves and detected that all of the spores and sclerotium tested exhibited the similar radial lesions at the inoculation site of these plants.From these results,the author demonstrated that the presence or absence of melanin production in the screlotium had no obvious effect on its virulence expression.7.Due to the lack of melanin,the mutant strains deposited exudate drops on the surface of the sclerotia.This exudate promoted both spore germination and germ tube elongation of this fungus.In addition,the author conducted qualitative and quantitative analyses of the exudate and indicated the presence of reducing sugars and proteins as possible metabolites of the sclerotia.These results suggest that the accumulated melanin acts as the mechanical barrier to prevent the leakage of nutrients for the spores inside the sclerotia.In conclusion,the author succeeded in proving the involvement of the Bcpks12 gene in the biosynthesis of sclerotial melanin of B.cinerea.In addition,the author proved that melanin plays an important role in stress resistance of sclerotia.All of these results will provide an experimental basis for further clarifying developmental characteristics of B.cinerea and improving methods to control this fungal pathogen.