Characterization Of Pathogenesis Related Transcription Factors And Open Chromatin In Phytophthora Sojae

Soybean root rot caused by Phytophthora sojae is one of the destructive diseases in soybean production.The transcriptome of P.sojae changes during development and infection to adapt to specific physiological needs.Transcription factors can bind to specific cis-element sequences to change chromatin conformation and thus regulate gene transcription.Therefore,identification and functional study of transcription factors and cis-elements is helpful for exploring the transcriptional regulation of P.sojae infection,and provides theoretical basis for understanding the pathogenesis and searching for possible drug targets.To understand the regulation mechanism in P.sojae,we studied the function of P.sojae pathogenicity-related transcription factors by CRISPR/Cas9 knock-out and analyzed downstream genes by RNA-seq.We also identified infection-related open chromatin by ATAC-seq.Functional study of P.sojae bZIP family transcritption factors:P.sojae genome encodes 71 Basic Leucine Zipper(bZIP)family transcritption factors,including Phytophthora specific bZIPs with novel residues in bZIP domain.In order to study the function of bZIPs,we selected PsBZPc4,PsBZPc29,PsBZPc31 and PsBZPc58,which are up-regulated during infection stage.We analyzed the sequences of these bZIPs,and found PsBZPc4 and PsBZPc31 are conversed in Phytophthora,while the sequences of PsBZP29 and PsBZPc58 homologs change a lot.We knocked out PsBZPc4,PsBZPc29,PsBZPc31 and PsBZPc58 by CRISPR/Cas9,respectivly.We found mutation of these 4 bZIPs did not infulence development related phenotypes of P.sojae.Virulence test showed that PsBZPc29 mutants showed reduced virulence to soybean.PsBZPc29 involved in specific transcription regulation during root infection:P.sojae can infect different organs of soybean.In previous work,transcriptome date showed P.sojae can regulate different gene expression during root or leaf infection.Many genes showed root-specific or leaf-specific expression pattern.These organ specific genes may involve in coping with different structure and immunity of host organs.PsBZPc29 shows root infection-specific expression pattern.PsBZPc29 mutantion PsBZPc29 mutantion influenced the infection on soybean roots instead of leaf.In order to study root-specific genes regulated by PsBZPc29,we analyze transcrptome of PsBZPc29 mutants during root infection.60%root-specific genes and 20%leaf-specific genes were suppressed in the mutants.Expression of other 4 organ-specific TFs was also infulenecd by PsBZPc29 mutantion.The results showed that PsBZPc29 may be a key regulator in specific transcription during root infection.Moreover,many root-specific effectors were down-regulated in PsBZPc29 mutants.In order to study the function of root specific effectors regulated by PsBZPc29,we knocked out Ps128038(encoding a SCP protein),and Ps133344(encoding a YxSL[RK]effector),respectively.The mutants of two effectors showed no defect in root infection.By transient expression in Nicotiana benthamiana we found these two effectors cannot trigger cell death,but can suppress cell death triggered by INF1.The results indicated that root-specific effectors regulated by PsBZPc29 play roles in suppressing plant inmmunity.Transcription factor PsMADl invovled in transcriptional regulation during sporangia development and infection:MADS-box transcription factors are conserved in eukaryotes,which play a key role in transcriptioanl regulation during development.We found all oomycete genome we checked code only one MADS-box protein.These MADS-box TFs are conserved which may have a common fuction in oomycete.In previous study,we found MADS-box transcription factor PsMADl is essential for sporangia development and infection.To investigate the regulation of PsMAD1 during sporangia development and infection,we analyzed transcriptome of PsMAD1 mutants and wildtype during sporangia and infection stages.In sporangia samples,280 genes were differentially expressed in mutants,including some protein kinases and TFs.In infection samples,44 genes were differentially expressed in mutants,including 25 secreted proteins.The results indicated that PsMAD1 involve in transcriptional regulation during sporangia development and infection.Analysis of open chromatin during mycelia and infection stage in Phytophthora sojae:Gene expression is accompanied with chromatin conformation changes.Open chromatin is the region depleted with histone,which contains many cis-elements for regulator binding.Analysis of open chromatin benefits cis-elements identification.In order to study open chromatin changes during infection,we use ATAC-seq to analyze open chromatin in P.sojae vegetative growth mycelia and infection samples.9593 and 9350 peaks were identified in mycelia and infection sample,respectively.Among them 3644 and 3272 peaks were stage-specifc,indicated that chromatin conformation changed dramatically during the transition from vegetative growth to infection.In genome-wide,the enriched peaks in genome are concentreted in promoter region.By GO enrichment and function annotation analysis,we found infection specific open chromatin is associated with protein trafficking and hydrolase related genes.The result indicated open chromatin may involve stage specific transcriptioanl regulation.In this study,we knocked out transcription factors which are highly induced during infection and analyzed phenotype of the mutants.We found PsBZPc29 and PsMAD1 are essential for virulence,and identified down-stream genes regulated by PsBZPc and PsMAD1 by RNA-seq.On the other hand,we analyzed open chromatin of P.sojae during infection by ATAC-seq.The result showed that open chromatin was related to gene activation.We also found enriched motifs in open chromatin peaks contained transcription factors binding site.This study will help in unraveling transcriptional regulation mechism during P.sojae infection,and provide theoretical basis for control of P.sojae.