Study On The Function And Transport Mechanism Of Melanin Synthesis Intermediate Scytalone In Botrytis Cinerea

Botrytis cinerea is a widespread fungal pathogen causing gray mold in both pre-and post-harvest fruits and vegetables.However,the effective control of this pathogen has been extremely difficult,partially because of production of sclerotia that exhibit resistance to various adversities in environment.DHN melanin,which is accumulated on the surface of conidia and sclerotia of B.cinerea,has been believed to be the substance that can endow the fungi with environmental tolerance.Indeed,most pathogenic fungi accumulate melanin at specific stages of their life cycle.In spite of an increased importance of melanin production,however,the metabolic regulation mechanism and biological effects of intermediate substances involved in the melanin biosynthesis pathway has remained obscure.To solve this problem,the direct gene-deletion technique has been developed in some fungi;in fact,this method was applied to B.cinerea to evaluate the roles of the intermediates in morphological development and virulence expression by specifically blocking the genes involved in the melanin biosynthesis pathway.The purpose of our research was to further analyze the role of intermediate products and subcellular localization of the related enzymes in B.cinerea.For this purpose,the present research program was designed to specially analyzes the function and transport mechanism of intermediate products by molecular genetic techniques and cytology.The results obtained are summarized as follows:1.Melanin-deficit mutant ?bcscd1 was obtained by homologous recombination.This mutant produced lower number of conidia on hyphae and hypogenetic sclerotia,and its colony color was orange.In the ?bcpks12 melanin deletion mutant,the sclerotia were albinic,and the production of conidia and germination of sclerotia showed no different in comparison to the wild type.From these results,the author considered that the hypogenetic sclerotial germination and decreased spore production in the ?bcscd1mutant was not caused by melanin deficiency.It maybe related to the special intermediate products accumulated in the mutant.2.To analyze which substance in ?bcscd1 has inhibitory effect on the germination and sporulation of sclerotia,we extracted this compound from culture filtrate of ?bcscd1and added it to conidial suspension of the wild type and observed its germination.The spore germination rate and germ tube length were decreased by the addition of scytalone.In addition,the inhibition was alleviated when tricyazole(Bc BRN inhibitor)was adding in ?bcscd1 sclerotia.These results indicated that the accumulation of scytalone inhibits the germination of spores and sclerotia.Apparently,scytalone seemed to be toxic to B.cinerea.3.In this experiment,the author attempted to explain the mechanism of avoiding self-poisoning by scytalone that was involved in the melanin biosynthesis pathway of B.cinerea.Firstly,the author examined the subcellular localization of scytalone dehydratase(Bs SCD1)and found that Bc SCD1 was mainly located in the cell wall.This result suggested that scytalone was secreted to cell wall to avoid its toxicity and to catalyze it to the next intermediate of the pathway.Secondly,the author indicated that BRN reductase catalyzing scytalone from its precursor T4 HN localized in the endosomes and the intermediate product(scytalone)was accumulated in the endosome.These results indicated that scytalone was transported using endosome system,by which fungal cell avoided cytotoxicity of scytalone.4.In order to complete the mechanism of melanin synthesis and transportation in B.cinerea,the author examined subcellular localization of other enzymes(Bc PKS12,Bc PKS13 and Bc YHG1)involved in the melanin synthesis.Fluorescence microscopic data indicated that the target enzymes mentioned above were located in small endocellular peroxisome-like vesicles.Finally,the author deduced the possible model for melanin synthesis and transport: acetyl-Co A or malonyl-Co A is transformed to T4HN(first intermediate)by Bc PKS12,Bc PKS13 and Bc YHG1 in peroxisomes;T4HN is transported to the endosome to generate scytalone(second intermediate)by Bc BRN reductase;scytalone is transported to cell wall to generate T3HN(third intermediate)by Bc SCD1;T3HN is transformed to vermelone(fourth intermediate)by Bc BRN reductase located in the cell wall,and vermelone is changed to 1,8-DHN melanin(final product)by Bc SCD1,which aggregates on the cell wall.In conclusion,through the present study,the author clearly demonstrated the inhibitory effect of scytalone,an intermediate product of melanin synthesis in B.cinerea,on conidia production and sclerotia germination.Besides,the author clarified that scytalone was secreted to call wall by the endosome transportation system,such that fungal cells can avoid its cytotoxicity.This study also proposed the possible involvement of peroxisome and endosome participate in melanin synthesis,providing a theoretical basis for further study on intracellular division and transport of other secondary metabolites or related toxins in B.cinerea.