Study On The Relationship Between HOG Pathway And Spore Production In Aspergillus Cristatu

Aspergillus cristatus is a dominant fungus in the fermentation of Fuzhuan tea and is also called “golden flower fungus” as it produces golden yellow cleistothecium.For a long time,the number of “golden flower fungus” has been used as one of the criteria for estimating the quality of Fuzhuan tea.Comparing with the phenomenon that the sexual and asexual spores are a mixture in Aspergillus nidulans,A.cristatus could produce sexual spores under hypotonic stress and asexual spores under hypertonic stress.So it could be a good material for studying the sporulation mechanism of filamentous fungi.Although previous studies have shown that osmotic stress could regulate the transition between sexual and asexual generations,it is necessary to further study this regulatory mechanism.It is well known that the HOG pathway is one of the main responses to osmotic stress in eukaryotes and exists widely in fungi.We speculate that there is a close connection between HOG pathway and sporulation in A.cristatus.In genome database of A.cristatus,SI65₀7698（Acpbs2）and SI65₀8279（Achog1）were homologous to key genes（pbs2,hog1）of HOG pathway through sequence alignment.The function of two genes is confirmed by the gene knockout,gene double knockout and gene complementation techniques.Their interaction and interaction sites between Acpbs2 and Achog1 were verified and analyzed by yeast two hybrid techniques.The downstream target genes Acptp2,3 and Acsko1 regulated by Achog1 were screened through transcriptome sequencing and pull-down combined with mass spectrometry.And the function of this two target genes was proved via gene knockout and gene complement techniques.Based on the above technologies,the relationship between HOG pathway and spore production of A.cristatus was explored.The main results of this study are as follows:（1）Acpbs2 and Achog1 participated in the regulating asexual sporulation,stress response and carbon metabolism in A.cristatus.Fifteen Acpbs2 knockout strains and their 12 complementary strains,and 12Achog1 knockout strains and their 22 complementary strains were obtained respectively by using gene knockout and complementary techniques.The time of conidial germination was delayed,and the conidial number was respectively decreased 12.5 times and 1.89 times in ΔAcpbs2 and ΔAchog1 strains under hypertonic stress.Acpbs2 and Achog1 participated in hypertonic stress.The tolerance of ΔAcpbs2 and ΔAchog1 strains to hypertonic stress was much higher than that of other Δpbs2 and Δhog1 strains in Aspergillus.Acpbs2 and Achog1 were involved in oxidative stress and alkaline p H stress.Deleting the two genes reduced the pigment produced by A.cristatus.In addition,Acpbs2 and Achog1 also participated in regulating carbon metabolism.It was the first report that pbs2 and hog1 played a role in pigment synthesis and carbon metabolism in Aspergillus.（2）Analysis of gene double knockout and interaction between Acpbs2 and Achog1The double knockout vector was constructed.Five ΔAcpbs2/ΔAchog1 strains were obtained by ATMT-mediated transformation.It was found that deleting simultaneously Acpbs2 and Achog1 inhibited the production of conidia.ΔAcpbs2/ΔAchog1 were more sensitive to hypertonic stress,high oxidative stress and alkaline p H.Additionally,the pigment produced by ΔAcpbs2/ΔAchog1 was increased.Acpbs2 could interact with Achog1 and the interaction sites were located in the PKc-like domain of Achog1.（3）Transcriptome anslysis of ΔAcpbs2,ΔAchog1 and wide type strainsRNA-seq was performed on the ΔAcpbs2,ΔAchog1 and wide type strains by using the Illumina Hi Seq 6000 platform.A total of 1448 differentially expressed genes were identified,among them,773 up-regulated and 715 down-regulated in ΔAcpbs2strains.A total of 1074 differentially expressed genes were screened,among them,480 genes up-regulated and 594 genes down-regulated in ΔAchog1 strains.Eighteen and 16 sporulation genes with significant differences were respectively found inΔAcpbs2 and ΔAchog1 strians.The genes related asexual sporulation were down-regulated.The functional enrichment analysis of GO and KEGG pathway revealed that differential genes were enriched in MAPK pathway in both knockout strains.Target gene SI65₀2513 was annotated to multiple locations in the MAPK pathway and located downstream of the hog1 gene in the HOG branch.（4）Analysis of proteins interacting with Ac Hog1The proteins interacting with Ac Hog1 under hypotonic and hypertonic stress were analyzed by pull-down combined with mass spectrometry in A.cristatus.Nine hundred and forty proteins and 2980 peptides were totally identified.Eight hundred and twenty-two differential proteins were screened,including 530 up-regulated proteins and 291 down-regulated proteins comparing WT-H（hypertonic stress）with WT-D（hypotonic stress）.Fourteen proteins participated in mycelial growth;Seventy proteins participated in stress response;Sixty proteins were related to the growth and development of fungi.The proteins related to conidial development only interacted with Ac Hog1 under hypertonic stress;Fourteen proteins involved in MAPK pathway.GO and KEGG enrichment analysis of the differential proteins showed that the proteins interacting with Ac Hog1 were mainly involved in the binding,antibiotic synthesis,carbon metabolism,fatty acid degradation of A.cristatus.New target proteins regulated by Ac Hog1 were also found,and the protein AA1E3BER4 located downstream of Hog1 was screened.The gene encoding the protein was SI65₀5898.（5）Functional analysis of target genes SI65₀2513 and SI65₀5898 regulated by Achog1SI65₀2513 gene is 2487 bp in length without intron,encodes 795 amino acids and contains a PTP-fungal protein family domain.SI65₀2513 gene was named Acptp2,3.A total of 7 gene deletion strains and 38 complementary strains were obtained by using gene knockout and complementary techniques.It was found that the number of ascospores and conidia were reduced respectively 4.4 times and 4.6times than that of wild type.The sensitivity of ΔAcptp2,3 to osmotic stress produced by sucrose and oxidative was increased,and the sensitivity to congo red and SDS was decreased.Acptp2,3 was involved in responding to neutral and acidic p H.Additionally,the pigmentation produced by ΔAcptp2,3 was also increased.SI65₀5898 gene is 1545 bp in length without intron,encodes 514 amino acids and contains a b ZIP superfamily domain.It was named Acsko1.A total of 9 gene deletion strains and 19 complementary strains were obtained by using gene knockout and complementary techniques.The number of ascospores and conidia were reduced respectively 3.81 times and 4.57 times than that of wild type.ΔAcsko1 was sensitive to osmotic produced by sucrose and sorbitol.The sensitivity of ΔAcsko1 to H2O2,congo red and SDS was increased.Acsko1 participated in response alkaline p H.The pigmentation produced by ΔAcsko1 knockout strain was increased.The functions of ptp2,3 and sko1 genes in Aspergillus were reported for the first time.