Functio Analysis Of Melanin Synthesis Related Genes In Botrytis Cinerea

Melanin is a polymer formed by oxidative polymerization of phenolic or indolic compounds in various organisms,including bacteria,fungi,plants and animals.Melanin which accumulated on the cell wall of appressoria has been considered to be an important virulence factor in most plant pathogens by enhancing the turgor pressure to mechanically penetrate the host plant epidermis.The DHN pathway is conserved among filamentous ascomycetes,and there are three key enzymes in this pathway:polyketide synthase,reductase and dehydratase.Melanin synthesis deficient mutants in those pathogens which rely on strong mechanical pressure from appressoria showed impaired virulence.Botrytis cinerea is considered one of the most important fungal plant pathogens,causing gray mold disease in more than 200 plant species,and has become the worldwide second most aggressive pathogen.B.cinerea has flexible strategies and secretes many virulence factors during infection process rather than produces typical appressoria of rice blast pathogen Magnaporthe oryzae,which accumulates high turgor pressure to drive mechanical penetration through the host epidermis.B.cinerea also could produce melanin during growth development.However,the exact role of these melanin biosynthesis enzymes in the pathogenesis of B.cinerea remains to be elucidated.This work investigated the functions of the melanin synthesis-related genes bcpks13,bcbrn1and bcscdl of B.cinerea by acquiring related mutants(?bcpks13,Abcbrnl and Abcscd1)and comparing the phenotype of growth and virulence.RNAseq analysis was conducted to explain the regulation mechanisms of melanin synthesis pathway in this pathogen.Based on this study,the functions of melanin synthesis-related genes were described in B.cinerea.The summary of the results is listed below:1.During different fungal developmental stages,the peak transcript levels of melanin synthesis related genes bcpks13,bcbrnl and bcscdl occurred at the late hyphal maturation stage and sporulation stage(approximately 7 dpi).These results indicated that the bcpksl3,bcbrnl and bcscdl probably play important roles during the hyphae maturation and sporulation.Two mutants of each gene(bcpks13,bcbrnl and bcscd1)were obtained respectively.Pigmentation of each mutant showed significant difference.The Abcpksl3 did not accumulate obvious pigment and showed white colony.The ?bcbrnl showed orange pigment accumulation,while the Abcscdl accumulated reddish pigment.These results showed that the three genes were required in melanin biosynthesis pathway and the mutants lacking each gene maybe accumulate different intermediate metabolite.2.Deletion of melanin synthesis related genes affected the growth,sporulation and sclerotia formation.The ?bcbrn1 and ?bcpks13 mutants showed an increased growth rate and hyphal branching pattern,without conidia and sclerotia production in vitro.The Abcscdl mutant showed slow growth rate and normal conidia and sclerotia production.However the spore morphology of Abcscdl mutant was changed and spore germination was delayed.These results indicated that the melanin synthesis related genes(bcpks13,bcbrnl and bcscdl)play vital roles during the vegetative and neproductive growth process.3.Mutants of melanin related genes exhibited different virulence.The ?bcpks13 and Abcbrnl mutants showed increased virulence inoculated on apple,pepper and tobacco leaves,while Abcscdl mutant showed decreased virulence.Both of the?bcpks13 and Abcbrnl mutants quickly produced invasive mycelia during infection process when inoculated on the onion epidermal layers and rapidly induced more ROS production when inoculated on the tobacco leaves.The secretion of known virulence factors,including hydrolytic enzyme secretion activities and acidification capabilities,were enhanced in the Abcpksl3 and Abcbrnl mutants,but decreased in the Abcscdl mutant in vitro.These results indicated that the melanin synthesis related genes(bcpks13,bcbrnl and bcscdl)had different regulating functions on virulence in B.cinerea.4.In order to explore the reason of different growth and virulence phenotype among the three mutants(?bcpks13,Abcbrn1 and ?bcscd1),comparison of different genes expression of the three mutants was conducted by RNAseq analysis.The results showed that the main differences among the three mutants(?bcpks13,?bcbrnl,?bcscdl)were embodied in various metabolic pathways,including glycometabolism,amino acid metabolism,fatty acid metabolism,TCA,ROS metabolism and secondary metabolic pathways.Differentially expressed genes in ?bcpks13(up regulated),?bcbrn1(up regulated)and Abcscdl(down regulated)involved in glycometabolism account for the largest proportion.In addition,genes involved in virulence factors,transcription factors,extracellular protein secretion and secondary metabolites synthesis related also changed.Thus,in melanin synthesis pathway,when the two key steps catalyzed by the polyketide synthase(coding by bcpksl3)and reductase(coding by bcbrnl)were blocked,the metabolic activities involved in vegetative growth and virulence were enhanced,whereas when the third key step catalyzed by thedehydratase(coding by bcscdl)was blocked,the metabolic activities involved in growth and virulence were decreased.These results indicated that the generation regulated functions of melanin synthesis related genes exhibited a complex role during the growth development and infection process in B.cinerea.Taken together,melanin synthesis related genes played diverse roles during the growth development and virulence in B.cinerea.Our results provided a new understanding about the function of melanin synthesis related genes and would be benefit for exploitation of any feasible application of this knowledge to control the crop damages caused by this pathogen.