Functional Analysis Of Nucleic Acid Demethylase ALKBH Genes Family In Phytophthora Sojae And Phytophthora Infestans

Phytophthora is a kind of filamentous eukaryote,which belongs to the oomycetes,and it is phytogenetically different from fungi.There are hundreds of Phytophthora species,which cause damage various food crops,horticultural crops,and ecosysticms.The soybean root rot caused by Phytophthora sojae is a devastating disease in soybean production.The potato late blight caused by Phytophthora infestans infecting potatoes caused Irish hunger in 1840.The potato late blight in China has a disease area of more than 60 million mu per year,which is one of the bottleneck factors restricting the development of China’s potato industry.The analysis of the epigenetic regulation mechanism on Phytophthora DNA and RNA is of great value for the design and development of new disease prevention and control strategies.The previous study of this group did not find the traditional 5-methycytosine(5mC)modification in the P.sojae and P.infestans genome at the detection level,but N6-methyladenine(6mA)modification was detected.The 6mA methyltransferase homologous sequence was present in both P.sojae and P.infestans.In vitro and in vivo experiments demonstrated that PsDAMT1,PsDAMT2 and PsDAMT3 are 6mA methyltransferases in P.sojae.The study also found that 6mA is enriched before the transcription start site(TSS)and is associated with low expression genes.Some pathogenicity-related genes are more active in 6mA demethylase mutants,indicating that 6mA modification is regulated.It has potential functions in the pathogenic variation of Phytophthora and in maintaining genomic stability.The ALKBH(Alkylation homologous)protein family exists in both eukaryotes and prokaryotes,and can catalyze DNA methylation and RNA demethylation,and participate in biological processes such as DNA damage repair.Human ALKBH 1 has been shown to be a DNA 6mA demethylase and a tRNA 1mA demethylase,and ALKBH5 and FTO are RNA m6A demethylases.It is unclear whether 6mA demethylase is present in Phytophthora and participates in the regulation of the pathogenic and reproductive processes of Phytophthora.This is also an important question to be answered in this paper.In this study,we focus on Phytophthora ALKBH genes family,and use CRISPR/Cas9 gene editing technology to knock out PsALKBH1,PsALKBH2,PsALKBH3,PsALKBH4,PsALKBH5,PsALKBH6 and PsALKBH7.Our goal is finding 6mA demethylases in Phytophthora and explore it’s biological function.Identification and sequence analysis of ALKBH genes family in Phytophthora sojae and Phytophthora infestans:The proven DNA 6mA demethylase,RNA m6A demethylase and 1mA demethylase are mostly ALKBH gene families and are widely found in prokaryotes and eukaryotes.By homologous sequence alignment,we found seven ALKBH homologs genes in both Phytophthora genomes.Through functional domain analysis,it was found that the above 14 genes all contain the 20G-FeII_Oxy superfamily functional domain.By sequence polymorphism analysis of PsALKBH1,PsALKBH2,PsALKBH3,PsALKBH4,PsALKBH5,PsALKBH6 and PsALKBH7 in four physiological races of P.sojae,it was found that PsALKBH1 has no sequence polymorphism,PsALKBH2/3/4/5/6/7 have different degrees of sequence polymorphism.There are different degrees of polymorphism in the six genes,indicating that PsALKBH1 is more conservative.Through genomic correlation analysis in P.sojae,P.infestans and P.capsici,It was found that PsALKBH3 exists in a collinearly conserved region of certain genes,suggesting that it may have relatively conservative functions in different species.Through analysis of RNA-seq data of different stages of P.sojae and P.infestans,it was found that these 14 genes were transcribed at different stages.Preparation and Obtainment of Phytophthora sojae ALKBH homologous knockout mutants:Due to the abnormality of PsALKBHl and PsALKBH7 gene models,we used CRISPR/Cas9 gene editing technology to knock out 5 genes including PsALKBH2,PsALKBH3,PsALKBH4,PsALKBH5 and PsALKBH6.We obtained 5 psalkbh2 homozygous mutants by sequence amplification and sequencing.We obtained 5 psalkbh3 homozygous mutants by sequence amplification and sequencing.We obtained 6 psalkbh4 homozygous mutants by sequence amplification and sequencing.We obtained 2 psalkbh5 homozygous mutants by sequence amplification and sequencing.We obtained 8 psalkbh6 homozygous mutants by sequence amplification and sequencing.Phenotype determination and analysis of psalkbh2,psalkbh3,psalkbh4,psalkbhS and psalkbh6 knockout mutants:Growth rate measurements indicated that the obtained ALKBH knockout mutations did not affect mycelial growth and colony morphology on artificial media.The pathogenicity assay on soybean yellowing seedlings showed that the virulence of psalkbh2,psalkbh3,psalkbh4,psalkbh5 and psalkbh6 mutants on susceptible soybeans was not significantly reduced.The quantitative determination of the number of sexual progeny-oospores in the psalkbh2 and psalkbh3 mutants showed a significant decrease in the oospores production of P.sojae,while the oospores production of psalkbh4,psalkbh5 and psalkbh6 mutants have no sisignificantly change.The above findings are important for further exploration of the biological function of the ALKBH genes family in Phytophthora,and point out the key directions for future research.