Functional Analysis And Pathogenic Mechanisms Of Histone Demethylase BcJar1 In Botrytis Cinerea

Botrytis cinerea is a necrotrophic plant pathogenic fungus with a wide range of hosts in the world.It can infect more than 1,000 plant species and seriously threatens the sustainable development of fruits,vegetables,flowers,wine and other industries and causes huge economic losses.As a model species of plant pathogenic fungi,molecular mechanisms and pathogenesis of the fungal epigenetic factors remains largely unknown.Jar1,a histone H3 lysine 4 demethylase,regulates gene expression via controlling the abundance and level of H3K4 methylation.It plays an important role in a variety of physiological processes and is highly conserved in all eukaryotic organisms.Homologous genes of Jar1 are highly expressed in various human malignant tumors and have been used as potential drug targets to control cancers.The homologous genes regulate the normal growth and development of plants.Little is known about BcJAR1,a gene encods Jar1 in B.cinerea,in regualtation of development and virulence of the pathogen.We analyzed the regulatory mechanisms of BcJAR1 in the fungual pathogenesis by investigating the functions of BcJAR1 in growth,development and virulence of B.cinerea.Our findings are listed as follows:1.BcJar1 was verified as a histone H3 Lysine 4 demethylase via bioinformatics,heterogenous expression of yeast and Western blot analyses.BcJar1 is a histone demethylase containing JmjC domain and belongs to the JARID/KDM5 subfamily.cDNA of BcJAR1 gene was constructed into yeast expression vector by homologous recombination,and transformed into yeast mutant ?Scjhd2 that is sensitive to salt stress.Our results showed that ?Scjhd2 containing BcJAR1 could completely restore the growth deficiency of the yeat mutant under salt stress.Western blot analysis indicated that the level of H3K4 trimethylation(H3K4me3)in B.cinerea BcJAR1 deletion mutants(?Bcjar1)was higher than that of the wild-type(WT)B05.10 strain and complemented transformants in the stage of vegetative growth.2.We obtained BcJAR1 deletion mutants and complemented transformants via A.tumefaciens-mediated transformation(ATMT)approaches.Resistance screening,PCR,qPCR and Southern analysis were used to verify the mutants and complemented transformants.3.BcJAR1 played crucial roles in pathogenesis of B.cinerea as demonstrated by pathogenicity and phenotypic analyses.The roles mainly include,but are not limited to,the following aspects:? The ?Bcjar1 mutants lost their ability to cause disease.When conidia suspension and mycelia plugs were inoculated on the leaves of different hosts and the ?Bcjar1 mutants were nonpathogenic;? The mutants failed to produce infection cushions,and the number of appressoria formed by the mutant strains dramatically reduced;? The mutants were more sensitve to environmental stesseses.Compared to the WT(B05.10)and complemented strains,the ?Bcjar1 mutants grew faster under salt and osmotic stress(NaCl,KCl,1 M each),whereas the growth of the mutants were significantly decreased under H2O2 stess,cell wall synthesis inhibits stress(SDS,CR),hypoxia stress(CoCl2);? The yield of mutants' conidia was decreased.Conidia and sclerotia morphology of the mutants were affected,both conidia and sclerotia of the mutants became smaller than the wildtype strain;? The growth of the mutants were not affected.Complemented strains restored these phenotypic defects.4.BcJAR1 affects Septin enrichment and assembly.Diaphragm filaments can be formed by polymerization of several Septin protein monomers and can be further assembled into diaphragm microfilaments,which have a variety of structures,such as ring,cage,fiber bundle,grid and hourglass.Diaphragm microfilaments play an important role in physiological processes such as cytokinesis.Covalent modification of histones,such as SUMO,phosphorylation and ubiquitination,had been proved to regulate Septin assembly.In this study,the vector containing BcSep4-GFP was transformed into the mutant ?Bcjar1 and wild type B05.10(WT)by ATMT.Compared with the control,Septin assembly was disordered and enrichment was decreased in BcSep4-GFP-tagged ?Bcjar1.5.The H3K4me2 and H3K4me3 levels played an important role in the formation of infection cushion through western blot analysis.The H3K4me2/3 level of the mutants were significantly increased at early and late stages of infection cushion formation.6.BcJAR1 was involved in the production of ROS and affected the formation of the infection structures by regulating the ROS signal transduction.Meanwhile,cAMP promotes appressorium formation that is independent of BcJAR1.7.BcJar1 regulates the expression of disease-related genes through transcriptome data analysis.In the process of infection cushion formation,about 2/3 of the genes related to ROS production are down-regulated,which will affect the reproductive growth and pathogenic development of the mutant.8.BcSet1,which is a H3K4 methyltransferase of B.cinerea,is involved in the regulation of pathogenicity of B.cinerea.Its functional domain N-SET and SET play an important role in regulation of pathogenicity of B.cinerea by BcSet1.The pathogenicity of BcSET1 knockout mutants and functional domains(N-SET and SET)was significantly reduced.In summary,we demonstrate that BcJar1 is a histone H3K4 demethylase that is highly conserved in evolution,BcJAR1 regulates the expression of genes related to fungal pathogenesis,ROS level and Sep4 proper enrichment and assembly by regulating the level of H3K4 methylation.Thus BcJAR1 plays pleiotropic roles in the nutrition,pathogenic development and toxicity of B.cinerea.Briefly,BcJar1 controls the vegetative growth by regulating the H3K4me3 level,and controls the pathogenic development and toxicity by regulating the H3K4me2/me3 level.We preliminarily discussed the function of H3K4 methyltransferase BcSet1 in regulating the pathogenicity of B.cinerea,and its domain N-SET and SET played an important role in the function of BcSet1.Our work provides novel insights into histone demethylase mediation of fungal development and pathogenesis.