Genome-wide Identification Of Splicing Factors And Alternative Splicing In Phytophthora Sojae

Phytophthora sojae infects soybean,causing soybean root rot,seriously affecting soybean yield and quality.Phytophthora sojae is an oomycete pathogen,which is close to algae in evolution and far from fungi.Therefore,traditional fungal control methods have limited effect on Phytophthora sojae,and it is important to learn the molecular mechanisms of Phytophthora sojae development and infection.With the development of sequencing technology and bioinformatics,transcriptome has become an important tool to analyze the pathogenesis mechanisms of P.sojae.In this paper,transcriptome sequencing technology was used to analyze the transcriptome data of P.sojae at different stages.It is hoped that the information of alternative splicing in Phytophthora sojae can be identified by transcriptome,which will provide data supporting subsequent research.In addition,splicing factors regulate the splicing of mRNA and the process of alternative splicing.However,splicing factors in oomycetes and fungi have not been systematically identified.Therefore,candidate splicing factors of oomycetes and fungi were identified from the whole genomes to provide data supporting subsequent splicing factor analysis.The main research contents are as follows:Identification of alternative splicing in Phytophthora sojae transcriptome:The full-length transcriptomes were analyzed for P.sojae at of the stages of zoospores,mycelia,and 3 hours post infection.A total of 13409 full-length transcripts were identified,including 6545 known transcripts and 6864 novel transcripts（including 1577 novel loci）.The results showed that there were some mis-annotated gene models which actually contain two or more genes.The expression pattern between or among the neighboring genes showed a positive correlation in general,indicating that they might have functional correlation.A computational pipeline was used to identify 2160 long non-coding RNAs（LncRNAs）form 13409 full-length transcripts.Comparing with other mRNAs,LncRNAs had fewer introns and shorter average length.Moreover,we identified 1803 alternative splicing events and identified some genes that gain or lost domains after alternative splicing.In addition to the full-length transcriptome,we continue to compare the RNA-seq data between P.sojae wild type strain and two obtained MADS-box gene（Ps_MAD1）knock-out mutants.The analysis identified the differentially regulated alternative splicing events found at different stages between Ps_MAD1 knockout mutants and wild type.The expression levels of alternative splicing genes affect by Ps_MAD1 were high at stage of mycelium,sporangium and 3 hours post infection.Gene ontology（GO）enrichment analysis showed that these AS genes are highly enriched in cellular protein metabolic process and toxin transport at IF stage.Total,the result illustrated that Ps_MAD1 could affect the development of sporangia and infection of P.sojae by regulating the genes of alternative splicing.Genome-wide identification of RNA splicing factors from plant pathogenic oomycetes and fungi:Splicing factors belong to a class of regulatory proteins involved in pre-RNA splicing.It can be divided into small nuclear ribonucleo proteins（snRNP）and non-snRNP.The common splicing factors include SR protein family,hnRNP,and others.We collected HMM models of RNA splicing related domains from PFAM database and used these models to identify the proteins which contained these domains in 17 oomycetes,6 fungi,and 8 plants.According to the distribution of proteins with certain domains in different species,some splicing factor domains enriched in oomycetes were identified,e.g.,PPR_long,Torus,PRP38,and zf-CCCH₂.All the candidates of PRP38 splicing factor in 17 oomycetes and 6 fungi were identified.Among the 39 candidates,we found that each fungal species has one PRP38 splicing factor while each oomycete has two PRP38 splicing factors,except Phytophthora kernoviae.According to evolutionary tree,nearly all PRP38 splicing factors can be divided into two groups,i.e.Group A and Group B.Group B is specific to oomycetes.The majority of the members in Group A had only one PRP38 domain,while those of Group B had multiple domains.There are 13 members in Group B have TUDOR domain which is related to methylation.Analysis of conserved motif implied that the PRP38 splicing factor in oomycete and fungi within the same group may have similar function.In addition,an identified motif,namely motif2,were widely existed in PRP38 splicing factor.Although all the candidate proteins contained PRP38 domain,they had different predicted other motifs,and some motifs were specific to Group A or Group B.The results will provide a basis for further studies of PRP38 splicing factors.In summary,alternative splicing in P.sojae transcriptome and candidate RNA splicing factors encoded by P.sojae genome were identified by bioinformatics,the results provide data for further studies of biological significance of alternative splicing in P.sojae development and infection,and for further studies of biological functions and RNA splicing factors and their regulatory mechanisms.