Study Of Differential Virulence In Scytalone Dehydratase-Coding Gene Bcscd1 Mutant Of Botrytis Cinerea

Botrytis cinerea is a typical necrotrophic phytopathogen.This fungus is a formidable nuisance of crop production and preservation because of its broad host range and severe crop spoilage,and therefore the close study of pathogenesis of B.cinerea is necessary to provide basic information for an efficient crop protection strategy.DHN(dihydroxynaphthalene)-melanin acts as an important virulence factor in many plant pathogens.Although the biosynthesis pathway of DHN-melanin has been elucidated,biological functions of the melanin synthesis-related compounds are still obscure.Scytalone dehydratase is one of the key enzymes involved in DHN-melanin biosynthesis.In our laboratory,we have previously obtained a knock-out mutant of the scytalone dehydratase-coding gene bcscdl.This mutant was less virulent to host plants.On the other hand,another two mutants,.polyketide synthase-coding gene(bcpks13)mutant and THN(tetrahydronaphthalene)reductase-coding gene(bcbrnl)mutant,showed increased virulence.Based on these results,we postulated that the melanin biosynthesis pathway and related intermediates would play a special role in pathogenesis of B.cinerea.To test thispostulation,in this study,the author attempted to analyze the differences in transcriptome,DHN-melanin related metabolites of the melanin deficient mutants and their host responses.The main results are listed below:1.To explore the influence of lacking bcscdl on gene expression of B.cinerea,the author used the transcriptome sequencing technique to compare between the wild type and?bcscd1 for differential gene expression analysis.The data showed that the differentially expressed genes in ?bcscd1 were mainly related to carbohydrate metabolism,phytotoxin(botrydial and botcinic acid)biosynthesis and enzymatic cell wall degradation.The results suggested that the lack of bcscdl decreased the expression of the genes involved in growth metabolism and virulence factors,and this could be strongly linked with reduced virulence expressed by ?bcscd1.2.To analyze the role of melanin biosynthesis intermediates in virulence expression by three mutants(?bcscd1,?bcpks13 and ?bcbrn1),the author extracted the metabolites from mycelia and culture filtrate and analyzed them by thin layer chromatography.The result showed that ?bcscd1 abundantly accumulated a compound that was not detectable in either the wild type or other two mutants.This compound was further purified by column chromatography and identified by 1HNMR and HRMS analyses.The results showed that the compound is scytalone,the substrate of BcSCD1 enzyme.3.To examine whether the intermediate compound scytolane accumulated in ?bcscd1 was vital in its growth and virulence expression,large amounts of scytolane was successfully obtained by optimizing the culture methodand extraction procedure.The results showed that the scytalone production was larger in static culture than in shaking culture,and was amplified by acidizing the culture filtrate to pH 2-3.Interestingly,the pigmentation of?bcpksl3 and ?bcbrn1 can be restored by adding scytalone.4.Treatment of B.cinerea spores with different concentration of scytalone caused both delayed spore germination and reduced germ tube elongation.Scytalone did not show any effect on plants,while the necrosis on various hosts was reduced by adding scytalone to spore suspension.And the expression level of BOA biosynthesis related genes were decreased.These results suggested that scytalone can decrease the virulence of B.cinerea,most probably due to its inhibitory effect on spore germination and hyphal growth.5.Defense response also plays an important role in plant-pathogen interactions.To analyze the relationship between the reduced virulence of ?bcscd1 and defense response of hosts,the model plant Arabidopsis thaliana was chosen as host.In comparison with the wild type,the Abcscdl induced no stronger defense responses as assessed by cell death,ROS production,lignin accumulation and expression of some defense genes(pdfl.2,bosl,pad3,pr1,nprl)tested by Q-PCR.The results suggested that the reduced virulence of ?bcscd1 was rarely due to the alternation of host defense system.In brief,the decreased virulence of ?bcscd1 was successfully elucidated by transcriptome analysis,bioactivity assay of melanin related metabolite scytalone and plant defense response analysis.The present work is the first report to reveal bioactivity of scytalone to fungal pathogen;in fact the compound can delay spore germination and suppress virulence on hosts;in fact,in many previous works,melanin was the only compound to be concerned,and the melanin related metabolites were little investigated.This study brings novel understanding of the importance of fungal DHN-melanin synthesis pathway and related metabolites,and may contribute to develop an efficient way to control B.cinerea in the future.