Study On The Mechanism Of Histone Chaperone ASF1 Activating The PI3K/AKT Signaling Pathway To Regulate Sexual And Asexual Development In Stemphylium Eturmiunum

Filamentous fungi are an important group of organisms,widely distributed in land,sea,air or soil.They have a key taxonomic status,deserving extensive focus scientifically.Most species of filamentous fungi reproduce asexually,while few species sexually.The evolution and sexual development in filamentous fungi are regulated by several sex genes,encoding mating type,pheromone,G protein,etc.The common Stemphylium eturmiunum,is one of the few species in Stemphylium that could produce sexual characteristics,serving as an important material for studying the mechanism of sexual reproduction in filamentous fungi.Histone H3/H4 molecular chaperone ASF1 was highly conserved from yeast to mammals,and could participate in the regulation of nucleosome assembly/disassembly and normal cell cycle progression.It also played an important role in gametogenesis,DNA replication/repair.However,ASF1 had been less studied in filamentous fungi.In this study,ASF1 was used the research object,and the mechanism of how to regulate sexual and asexual development in S.eturmiunum is deeply explored.In the present study,S.eturmiunum wild-type was used as the experimental object,and the histone chaperone ASF1 was first identified and cloned successfully by whole genome sequencing.Alignment of the S.eturmiunum ASF1 sequence with its homologous sequences from plants,animals,and other fungal species revealed that S.eturmiunum asf1 was most related to S.lycopersici asf1.The N-terminus sequences(1-154 aa)of ASF1 was highly conserved and contained the ASF1 hist chap superfamily functional domain,whereas the Cterminus was greatly varied.Seasf1 was knocked out and complemented by agrobacteriummediated fungal genetic transformation.In this study,the hyphae of wild-type strain grew vigorously.Matured conidia formed in three weeks,while asci and ascospores produced in five weeks.DAPI staining showed that the nuclei were evenly distributed in the mycelia of wild-type.However,the mycelial growth rate of ?asf1 was reduced compared with WT.The hyphal fusion was more common,perithecia and ascospores were no longer produced,and?asf1 showed sterility.The conidia of ?asf1 changed in shape from conventional oval to irregular.They produced from on the hyphae to on the hyphae tipcease and even changed into chlamydospores.The number of conidial septa was less.Most of them were aggregated,and the single spore became larger.The distribution of nuclei in the mycelium of ?asf1 was unevenly distributed,and some parts were concentrated.Subsequently,the phenotype observation of asf1 complementation transformants showed that the colony growth rate and mycelial growth were basically the same as those of the wild-type strain.DAPI staining showed that the nuclei were evenly distributed,which was basically the same as wild type.It was confirmed that the histone molecular chaperone ASF1 could regulate sexual and asexual growth and development in S.eturmiunum.To further reveal the mechanism by which histone molecular chaperone ASF1 regulated sexual and asexual development in S.eturmiunum,a comparative analysis of gene expression was carried out among Se?asf1,WT-vegetative and WT-sexual.The results showed that 3716 genes were differentially expressed between Se?asf1 and WT-sexual,of which 2342 genes were up-regulated and 1374 genes were down-regulated;3023 genes were differentially expressed between Se?asf1 and WT-vegetative,of which 1719 were up-regulated and 1304 were down-regulated;also,3094 were differentially expressed between WT-sexual and WTvegetative,of which 1343 were up-regulated and 1751 were down-regulated.GO enrichment analysis of differentially expressed genes from Se?asf1 and WT-vegetative,as well as Se?asf1 and WT-sexual,were performed.GO analyses found that many of these genes were involved in cellular processes,secondary metabolite development,organelle activity,and catalytic activity in S.eturmiunum,such as allantoate permease(Se10206),Lys M domaincontaining protein(Se03485)and vesicle coat complexes COPII(Se07693)and DJ-1(Se02026).Then,the four genes were silenced by RNAi,and the silencing efficiency of transformants of each gene was above 75%.Phenotypic observation of silencing transformants showed that Se03485,Se07693 and Se10206 had no effect on sexual and asexual development in S.eturmiunum.However,the phenotype of Sedj-1 silenced transformers changed significantly,the conidia and conidia grew slowly and became malformed,and asci were not produced.The results suggested that Sedj-1 might cooperate with Seasf1 and play a key role in the regulation of sexual and asexual development of S.eturmiunum.Further studies found that DJ-1 is an important upstream component of PI3K/AKT signaling pathway,and it is involved in regulating a variety of biological functions in mammals.By using yeast two-hybrid experiments,Pull down and Co-IP experiments at the transcript level and protein level,it was found that Sedj-1 and Se H4 showed down-and upregulation in the two Se?asf1 mutants,respectively,Sedj-1 and Seasf1 displayed down-and up-regulation in the two Si Se H4 lines,respectively,and Seasf1 and Se H4 showed downregulation in two Si Sedj-1 lines.Se ASF1 interacted with Se H4 or Se DJ-1,and Se H4 also interacted with Se DJ-1.The downstream protein Se GSK3 in the pathway was up-regulation in Sedj-1 silenced and Sepi3 k silenced transformants.Both Sepi3 k and Sedj-1 were downregulated in Sedj-1 silenced transformants.This suggests that Se DJ-1 is located upstream of the PI3K/AKT signaling pathway,and Se DJ-1 interacts with Se PI3 K and Se GSK3 in S.eturmiunum.How Se DJ-1 regulates sexual and asexual development of S.eturmiunum in PI3K/AKT signaling pathway was further investigated.Through gene silencing experiments,Sepi3 k silenced transformants could produce perithecia and conidia,but no asci and ascospores.At the same time,through overexpression experiments,it was found that Sepi3 k was overexpressed in Sedj-1 silenced transformants,Sedj-1 silenced transformants could restore the characteristics of sexual development.Sedj-1 was overexpressed in Sepi3 k silenced transformants,Sepi3 k silenced transformants also could restore the characteristics of sexual development.These results suggest that Se DJ-1 and Se PI3 K are not only two important members of PI3K/AKT signaling pathway,but also play the same function in regulating sexual development in S.eturmiunum.To further elucidate the mechanism by which Se DJ-1regulates sexual development,Sedj-1 was truncated and seven truncations were obtained.Se DJ-1-M6 was demonstrated as a key region for the interaction of Se DJ-1 with Se PI3 K by yeast two-hybrid and Co-IP experiments.Se DJ-1-M6 was overexpressed in Sepi3 k silenced transformants,and the Sepi3 k silenced transformants restored the sexual characteristics.The results proved that Se DJ-1-M6 is also a key functional region in regulating sexual development of S.eturmiunum.In conclusion,ASF1,a molecular chaperone of histone H4,could interact with DJ-1 and participate in PI3K/AKT signaling pathway to regulate sexual and asexual development in S.eturmiunum.The specific regulatory mechanism is as follows: ASF1 interacts with H4 and then translocates into nucleus through the nuclear pore.After getting into nucleus,Se ASF1-Se H4 combing with Se DJ-1 constitutes a new trimeric complex that plays a novel role for modulating sexual and asexual reproduction.Subsequently,Se DJ-1 also participates in PI3K/AKT signaling pathway for regulating sexual reproduction.Therefore,Se ASF1 can activate the PI3K/AKT signaling pathway to regulate sexual and asexual development.