Comparative Genome And Transcriptome Research Of Phytophthora Sojae

Soybean root rot caused by Phytophthora sojae can result in severe yield loss to soybean and threaten global food security of soybean,causing losses of $2 billion per year worldwide.Although outwardly resemble fungi in their growth habits and nutritional strategies,Phytophthora sojae belongs to oomycetes which have closer relationship with photosynthetic brown algae and diatoms.This makes Phytophthora and ture fungi differ in many respects including structure,metabolic pathways and infection mechanisms,so many fungicides have no effect on Phytophthora.Therefore,the study on molecular mechanisms of Phytophthora pathogenesis lay the foundation for developing new strategies and technologies for desease control,and is of great significance for the guaranteeing soybean production in China.With the continuous development of high-throughput sequencing technology and gradual reduction of cost,more and more plant pathogen genomes have been sequenced,which has promoted the progress of research on the pathogenesis of plant pathogens.Through the genome sequencing of many plant pathogenic oomycetes,people found that Phytophthora genomes contains hundreds of effector-coding genes,which play a very important role in tge process of Phytophthora infection.The effectors can subvert host immunity through a variety of mechanisms,so as to promote the infection of Phytophthora.Therefore,studying the variation,evolution and mechanisms of transcriptional regulation can help us to further understand Phytophthora and design candidate targets for the development of new fungicide.In this study,we did the comparative genomics and transcriptomic analysis of Phytophthora sojae to study the sequence variation characteristics,structural basis and transcription polymorphism at different temporal and spatial dimensions.In addition,we identified potential transcriptional regulators of effector genes by using transcriptome analysis.Comparative genomics analysis among four representative isolates of Phytophthora sojae reveals genes under evolutionary selection:We compared sequence data among four isolates of P.sojae,which represent its four major genotypes.These isolates exhibited>99.688%,>99.864%,and>98.981%sequence identities at genome,gene,and non-gene regions,respectively.One hundred and fifty-three positive selection and 139 negative selection candidate genes were identified.Between the two categories of genes,the positive selection genes were flanked by larger intergenic regions,poorly annotated in function,and less conserved;they had relatively lower transcription levels but many genes had increased transcripts during infection.Genes coding for predicted secreted proteins,particularly effectors,were overrepresented in positive selection.Several RxLR effector genes were identified as positive selection genes,exhibiting much stronger positive selection levels.In addition,candidate genes with presence/absence polymorphism were analyzed.This study provides a landscape of genomic variation among four representative P.sojae isolates and characterized several evolutionary selection-affected gene candidates.The results suggest a relatively covert two-speed genome evolution pattern in P.sojae and will provide clues for identification of new virulence factors in the oomycete plant pathogens.Bioinformatic analysis of secondary structure of oomycete RxLR effector proteins and the relationship to sequence variation:Through the secondary structure prediction of the putative RxLR proteins,we found that the C-terminus of the majority of these RxLR proteins,irrespective of the presence of W/Y/L motifs,contains abundant short alpha-helices.Since a large-scale experimental determination of protein structures has been difficult to date,results of the current study extend our understanding on the oomycete RxLR effectors in protein secondary structures from individual members to the entire family.Moreover,we identified less alpha-helix-rich proteins from secretomes of several oomycete and fungal organisms in which RxLRs have not been identified,providing additional evidence that these organisms are unlikely to harbor RxLR-like proteins.Therefore,these results provide additional information that will aid further studies on the evolution and functional mechanisms of RxLR effectors.Organ-specific expression profiling of Phytophthora sojae transcriptome:Phytophthora can infect multiple organs of host plant.The different organs of a plant are significantly different in structure,metabolism,and defense response,however,the mechanisms on how these pathogens regulate for organ-specific infection were largely unknown.Phytophthora sojae is the agent of root and stem rot of soybeans,it is also capable of infecting soybean leaves.We compared the transcriptomes of P.sojae during its infection in soybean roots and leaves,and found dramatic transcriptional change of hundreds of genes.These genes were enriched in families of carbohydrate-active enzyme,secreted protein,oxidoreductase-related proteins,and transporter,which were proposed be associated to the response of pathogen to different plant cell structure components and defense stresses.We knocked out PsBZPc29,a bZIP transcription factor-coding gene,in P.sojae using CRISPR/Cas9 system.The mutants displayed a significant defect in root-rather than in leaf-infection,which was consistent with its specific expression pattern.Many differentially regulated genes in the mutants were overlap with the identified organ-specific genes,indicating that PsBZPc29 may function in transcriptional regulation for the organ-specific infection of P.sojae.Our results provided an important support to the conclusion that transcriptional regulation are essential for successful infection and host environment adaptability of Phytophthora pathogens.Geneme-wide identification and analysis of long non-coding RNAs in Phytophthora sojae:Numerous long non-coding RNAs(lncRNAs)identified and characterized in mammals,plants,and fungi have been found to play critical regulatory roles in biological processes.However,little is known about the role of IncRNAs in oomycete plant pathogens.We used strand-specific RNA sequencing to generate a computational pipeline to identify IncRNAs in Phytophthora sojae,a model oomycete plant pathogen.In total,940 IncRNAs with 1,010 isoforms were identified from RNA-seq data obtained from four representative stages of P.sojae.The IncRNAs had shorter transcript lengths,longer exon lengths,fewer numbers of exons,lower GC content,and higher minimum free energy values compared to protein-coding genes.IncRNAs in P.sojae exhibited low sequence conservation among oomycetes species and P.sojae isolates.Transcription data indicated that P.sojae lncRNAs tended to be transcribed in a stage-specific manner;representative IncRNAs were validated by semi-quantitative reverse transcription polymerase chain reaction.P.sojae IncRNAs were concentrated in gene-sparse regions,and the lncRNAs were associated with secreted protein and effector coding genes.The neighboring genes of IncRNAs encoded various effector family members,and RNA-seq data revealed a correlation in the transcription level of IncRNAs and their neighboring genes.In addition,we analysed P.sojae IncRNAs by using the organ-specific transcriptome data and revealed transcriptional dynamic of IncRNAs between different organs.Our results provide the first comprehensive identification of IncRNAs in oomycetes and suggest a potential association between IncRNAs and effector genes.Summary:We used high-throughput sequencing and bioinformatic method to study Phytophthora sojae genome and transcriptome,and found that the genes coding for predicted secreted proteins,particularly effectors,had high level of sequence polymorohism and were under positive selection.By predicting the protein secondary structure of RxLR proteins,we found the structural basis of their sequence variations.Hundreds of organ-specific genes of P.sojae had been identified during infecion on different organs and we also identified long non-coding RNAs of P.sojae by using RNA-seq data.The lncRNAs may play important role in regulation of P.sojae genes.