The Regulatory Mechanism And Biological Function Analysis Of Adenine N6-Methylation(6mA) In Phytophthora Genomic DNA

Oomycetes,including Phytophthora,downy mildew,Pythium and many other pathogens of both animals and plants,can infect crops,forest,insects,fishes,human and other organisms.They greatly threaten food security and ecology safety.Oomycetes easily overcome environment change and drive successful Phytophthora adaptation on host immunity system through sequence polymorphism in important genetic loci.But more and more evidence proved that wild Phytophthora strains can be adapted to host plants by transcriptional polymorphism without sequence change.Epigenetic is the study of heritable phenotype changes without alterations in the DNA sequence,it participates in different biological processes.However,the epigenomes and their functions in oomycetes remain largely unknown.This research systematically studies DNA 6mA in Phytophthora infestans and Phytophthora sojae based on several methods.This study proved that N6-methyladenine/6mA is the major DNA methylation type,we drew 6mA methylome and dissected the regulatory mechanism of 6mA.The future insight into methylation broaden horizons of oomycetes variation and provide a starting point to further pathogenicity and fungicide resistance research.P.infestans and P.sojae genomic DNA methylation detection and methylome:DNA methylation is one of the most important parts of epigenetics,but it's largely unknown in Phytophthora.We found that there's no detectable 5-methylcytosine/5mC using UPLC-ESI-MS/MS.but 6mA exists indeed,and we quantified 6mA concentration.MeDIP-seq showed 6mA has a bimodal distribution near transcriptional start site,and negatively associated with gene expression.GO enrichment showed methylated genes are enriched in chromatin binding,DNA recombination,DNA metabolism,and enzyme activity.6mA mostly distributed(86%in P.infestans and 55%in P.sojae)in the intergenic region and enriched at LTR elements and DNA elements.6mA is enriched in gene sparse region.Fast evolved genes such as genes encode secreted proteins and RxLR effectors have higher 6mA level.This part suggested that 6mA is major methylation type in Phytophthora genome,and drew the first methylome.Distribution of 6mA,relationship with gene expression and identified methylated genes provide clues for eukaryotic DNA methylation and Phytophthora.Identification and molecular mechanisms of two Phytophthora 6mA methyltransferases:6mA was written by methyltransferases.We predicted novel and conserved 6mA methyltransferases in Phytophthora using bioinformatics.The methyltransferases are conserved in oomycetes and expanded in Phytophthora,DAMT3 in the ancestral gene.We didn't find 5mC methyltransferases using the same method and it partially explains that 5mC is undetectable.We established an in vitro enzyme activity assay and proved that PiDAMT1/2/3 and PsDAMT1/3 have enzyme activity,but PsDAMT2 doesn't,this result was similar to the result of Escherichia coli system assay.To explore the molecular mechanism of methyltransferases,we further screen interact proteins of 6mA methyltransferases by protein immunoprecipitation and mass spectrum on P.sojae over-expression strain.The mass spectrum result suggests that DAMTs can interact with each other and form polymers.GST pull-down assay supports our mass spectrum result.Mass spectrum also identified DEAD/DEAH box RNA helicase?D-isomer specific 2-hydroxyacid dehydrogenase?GTP-binding protein SARI and RAB family GTPase.We identified novel and expanded methyltransferases in Phytophthora,and they have enzyme activity and form polymers.Further studies on the cooperation of methyltransferases are needed.Moreover,many potential interactors are identified.We speculated that these methyltransferases are involved in 6mA regulation.6mA methyltransferases are required for methylation and pathogenicity in P.sojae:This part focuses on methyltransferases gene knockout and knockout mutants analysis.We got independent knockout mutants base on CRISPR/Cas9 of these three genes.6mA level of knockout mutants is reduced by UPLC-ESI-MS/MS detection.Mutants exhibit same growth rate on V8 medium.Oospore number of psdamtl and psdamt3 reduced but psdamt2 not.Pathogenicity of psdamtl and psdamt3 on Hefeng47 reduced,but psdamt2 not.We found 890 peaks(29.4%)lost in psdamt3 through MeDIP-seq,this partially explains the 6mA reduction.6mA level on DNA element,LTR elements and genes reduced in psdamt3.Knockout PsDAMT3 results in loss of the peak after TSS but not the peak before TSS,it suggest the complex regulatory system of 6mA methylation.Large scale gene differentially expressed in psdamt3,and differentially expressed genes are enriched in pathogenesis,enzyme activity,response to stimulus,metabolic and biosynthetic processes.We found effectors in differential expressed genes.These data suggest that psdamt3 mutant may either reduce the capability to infect the host or trigger stronger plant immune response and partially explains the impaired virulence phenotype of psdamt3.The length of 5' and 3'-flanking intergenic region from differential expressed genes were significantly longer than those from core genes and genes in gene dense region(GDR).The data indicated that genes more located in GSR tends to be affected in psdamt3.6mA colocalized with H3K9me3 and H3K27me3 and they regulated gene regulation:We identified H3 modifications before exploring the relationship of 6mA and histone methylation.We found conserved H3 proteins in Phytophthora.The H3 mass spectrum results show methylation,phosphorylation,acetylation,succinylation,crotonylation,2-hydroxyisobutyrylation,and other modifications.We confirmed H3K4me3?H3K9me3?H3K27me3 and H3K36me3 by western blot.We obtained whole genome H3K4me3?H3K9me3?H3K27me3 and H3K36me3 methylomes by ChIP-seq.Distribution of H3K9me3 and H3K27me3 have a similarity of 6mA,they both more distributed in intergenic regions and enriched in DNA elements and LTR elements.6mA co-localizes with H3K9me3 and H3K2 7me3.1267 6mA peaks intersected with H3K9me3 and H3K27me3 overlapped peaks,and 393 peaks intersected with these peaks.273 genes(69.5%)silenced,moreover,108 genes(27.4%)upregulated in infection stages,and most of them annotated as transposons.The result indicated 6mA colocalized with H3K9me3 and H3K27me3 and they regulated gene regulation corporately.