Characterization Of Phytophthora N~6-methyladenine (m~6A) Reader Protein And Functional Analysis

Phytophthora is a filamentous eukaryote belonging to the Oomycota of the chromists,but the phylogenetic studies showd that it is very different from fungi.There are hundreds of species of Phytophthora,most of which are phytopathogen,which can infect various crops,horticultural plants and trees communities.Among them,soybean root rot caused by Phytophthora sojae is a devastating disease in soybean production all over the world.Phytophthora infestans causes potato late blight,which struck potato crops and caused the Irish famines in 1840s.The annual area late blight disease happened in China is more than 2 million hectare,which is one of the reemerging threats limit the development of the potato industry in China.At present,such diseases are mainly controlled by planting cultivars contained resistence genes and fungicides,but the rapid mutations of Phytophthora make it difficult to prevent and control the disease.The analysis of the pathogenic mechanism of Phytophthora plays an important role in understanding the process of disease occurrence and prevention,and the clarification the epigenetic regulation mechanism of Phytophthora could provide potential valuable guidance and new control strategies for prevent Phytophthora diseases.m6A modification is the most common and abundant modification within eukaryotic mRNAs.m6A can participate in various life processes of eukaryotic organisms,and has universal and important biological significance.m6A modification components include methyltransferases,demethylases and reader proteins.The YTH domain protein has been identified as the first type of m6A reader protein,which can affect translation efficiency by affecting mRNA metabolism,such as affecting mRNA degradation,stability and nuclear export,thereby participating in various life processes.At present,the types,contents and distribution of Phytophthora DNA methylation(6mA)have been reported in detail,while the modification on RNA is poorly understood.In order to further analyze the epigenetic modification network of Phytophthora,m6A on the Phytophthora mRNA was investgated in this study.This project focused on the YTH domain-containing protein of P.sojae and P.infestans,which are two important plant pathogens.We have studied whether the YTH protein is involved in the infection process of Phytophthora,and identified whether it is the m6 reader protein.The main contents are as follows:Bioinformatics was utilized for bioinformatic prediction analyzing and identification YTH proteins in P.sojae and P.infestans.YTH domain-containing proteins were found in 11 oomycete species including Phytophthora,Saprolegnia and Pythium by homologous sequence alignment.There are two potential m6A reader proteins in P.sojae and P.infestans,named as PsYTH1 and PsYTH2 in P.sojae and PiYTH1 and PiYTH2 in P.infestans.Phylogenetic analysis shows that PsYTH1 and PiYTH1 are homologous protein,and are closer to the human intracellular YTH protein in the genetic distance,while PsYTH2 and PiYTH2 are closer in phylogenic analysis,and are closer to human nuclear YTH proteins.Domain analysis showed that PsYTH1 and PiYTH1 contain only one conserved YTH domain,while PsYTH2 and PiYTH2 contain a YTH domain,they also contain a DCD domain(Development and cell death domain)related to development and cell death,and four zinc finger domains.Analysis of key catalytic residues showed that YTH domain containing proteins of P.sojae and P.infestans contain conserved catalytic residues related to m6A recognition and binding.Analysis of the expression profile of PsYTH1 and PsYTH2 showed that the genes were up-regulated in the early stages of infection.The above results indicate that the oomycete contains conserved YTH domain proteins,which are putative m6A reader proteins.Knocking out and complement of PsYTH1 by transformation system in P.sojae were applied into further exploration of PsYTH1 function.The CRISPR/Cas9 system was used to knock out the PsYTH1 gene of P.sojae.Three knockout mutants were verified by PCR and sequencing:T2,T10 and T16.The phenotypes of the three knockout mutants were identified,and the growth rate of the knockout mutants was significantly reduced.Zoospores infection of soybean hypocotyl showed that the pathogenicity of the knockout mutants was significantly reduced.Samples of soybean hypocotyl infected after 48 hours were performed coomassie blue staining and microscopic observation.The wild-type infected mycelium showed a long,straight intercellular crossing state,while knockout mutants mycelium entangled near the infection site.To further verify the function of the PsYTH1,in-situ complement of the knockout mutant T16 was performed.PCR and sequencing confirmed that three complementary mutants R1,R35 and R42 were obtained.The growth rate of complementary mutants was tested and found to partially rescue the mutants phenotype.Pathogenicity testing found no difference between the complement mutants and the knockout mutant.The above results indicate that PsYTH1 gene of P.sojae is indeed involved in its vegetative growth and infection process.Detect the Histone H3K27me3 modification levels of psythl knockout mutants.ChIP-seq data of P.sojae showed that H3K27me3 was deposited on the PsYTH1 gene.In order to understand whether there is a certain relationship between the histone methylation and RNA methylation modification of Phytophthora,the overall H3K27me3 modification level of the psythl knockout mutant protein was tested.The mycelium protein of wild-type(P6497)and psyth1 knockout mutants were extracted,and western blot analysis was performed with specific antibody H3K27me3.There was no significant difference in H3K27me3 modification levels.It has been reported that the overall H3K27me3 modification level of mycelium protein decreased when knocked out Pssu(z)12 in P.sojae.Therefore,the expression level of PsYTH1 gene in wild type of P.sojae and pssu(z)12 knockout mutants were detected,but we found no difference.The above results indicate that there is no evidence that the PsYTH1 gene is related to the histone H3K27me3.We need more experimental evidence to explain the relationship between these two modifications.Expression of recombinant PsYTH1 protein in vitro and detection of its nucleic acid binding function.It was found that PsYTH1 does participate in the infection process of P.sojae.In order to verify whether the function of m6A reader protein effect pathogenic process,the protein was tested for nucleic acid binding ability after being expressed through the prokaryotic expression system.The E.coli prokaryotic expression system was used to express the PsYTH1 protein and its enzymatic mutant protein PsYTH1-m.The mutation site of the mutant was the m6A recognition and binding site reported in human YTHDF proteins previously.The empty vector protein was used as a negative control.After purifying the proteins by AKTA system,it was proved that PsYTH1 does bind to RNA by nuclease digestion agarose gel migration experiment.To further verify whether the single-stranded RNA probe with m6A modification is more likely to be bound,the EMSA experiment was performed.The above results indicate that it is necessary to further verify whether the PsYTH1 protein binds to the single-stranded RNA probe with m6A modification.