Functional Analysis Of The Transcriptional Factors PsHSF1 And PsBZPc32 Involved In Oxidative Stress Response And Pathogenicity In Phytophthora Sojae

Phytophthora sojae is one of the most devastating pathogens causing damping off androot rot disease on soybean all over the world.Losses are estimated at $1?2 billion per yearworldwide.Phytophthora belongs to oomycetes,which are phylogenetically distinct fromfungi.Many fungicides which are efficient for fungi pathogen controlling are useless toPhytophthora for the biochemical pathways,infection mechanisms and cell structurecharacteristics are significant different between Phytophthora and fungus.Therefore,it willbe helpful to explore the mechanism of Phytophthora pathogenesis on molecular level forfinding out new drug targets for Phytophthora controlling and designing strategy for thePhytophthora disease management.Transcription factors are key element in the geneexpression regulation network during the development,stress response and pathogenicprocess in pathogens.Based on bioinformatics analysis and Phytophthora genetictransformation system,two transcription factors involved in oxidative stress response andpathogenicity are characterized,and their molecular regulation mechanism are explored.The bioinformatics prediction and transcription analysis of HSF encoding genesin Phytophthora sojae:Heat shock transcriptional factors(HSFs)are terminal componentsof signal transduction pathways related with the cell response to heat stress and quite a lotof chemical stressors.They recognize and bind to the heat stress elements(HSEs)andregulate the expression of down-stream genes so as to adapt the environmental stresses.Wepresent a genome-wide analysis of the HSF DNA-binding domain containing sequences inPhytophthora sojae,P.rammorum and P.infestans,and identified quite a large number of candidate HSF encoding genes,about 17?24,which is much more than that in fungigenomes and similar to that in plant genomes.The gene modes of HSF genes inPhytophthora were firstly corrected.Based on sequences and domains analysis andphylogenetic dendrogram,we found that several HSF genes cluster on the same scaffold inPhytophthora genomes.The orthologs in Phytophthora species share high similarity witheach other.They cluster in one clade which is far away from other clades of animals,plantsand fungi.This data further suggested the large family of HSF TFs in Phytophthora mightdue to the duplication of genes from the common ancestor of Phytophthora lineage.Furthermore,real-time RT-PCR was employed to detect the transcripts levels of all HSFs atasexual life stages,different infection stages and the conditions under oxidative stress in P.sojae.These results provide a new clue for exploring genes related to stress response,which is crucial to disease control and drug development.HSFs are involved in regulating oxidative stress response and suppression ofplant immunity in Phytophthora sojae:Reactive oxygen species(ROS)play a central rolein plant defense response.However,the mechanisms how Phytophthora pathogens adaptand counteract the plant-derived ROS are currently underexposed.We present agenome-wide analysis of ROS tolerance-related transcription factors(TFs)in P.sojae andseveral relatives.Through the transcripts levels detection of related genes in P.sojae,a heatshock transcription factor,PsHSF1,was identified to be highly up-regulated underoxidative stress and during cyst germination.PsHSF1 is required for oxidative stresstolerance and virulence in P.sojae.PsHSF1-silenced transformants were compromised tosuppress hydrogen peroxide accumulation and callose deposition nearby the infection sitesinduced by plant innate immunity.Addition of the ROS reducer,GSH,partially restored itsvirulence.Silencing of PsHSF1 resulted in reduced extracellular peroxidase and Iaccaseactivities and the expression of some peroxidase and laccase encoding genes are regulatedby PsHSF1.Silencing of PsLAC4 illustrated its significant contribution to the phenotype ofPsHSF1-silenced transformants.Our results identify PsHSF1 as a positive transcriptionfactor that regulates transcription of extracellular peroxidase and laccase genes which areimportant for ROS resistance and defense plant immunity in P.sojae.Functional analysis of transcription factor PsBZPc32 of phytophthora sojaedruing development and pathogenicity:Transcription factors(TFs)of the basic leucinezipper(bZIP),which is one of the largest and most diverse TFs families,are widely presentin eukaryotes,controlling development and stress responses.According to phylogenetic and transcriptional analysis of the bZIP transcription factors in P.sojae perversely,one of thesegenes,PsBZPc32,which is up-regulated at infection stages was selected for furtherfunctional analysis.PsBZPc32 is the only bZIP gene containing an extra domain,PASdomain,at downstream of the bZIP domain.To explore the function of PsBZPc32,fourPsBZPc32-silenced transformants were obtained by stable transformation.Silencing ofPsBZPc32 has no effect on hyphae growth,sporulation,zoospore production and mobility.However,cyst germination stage was severely affected.PsBZPc32-silenced transformantsshowed reduced cyst germination rate and much more abnormal germinated cyst withmulti-branched compared with the wild type stain.The pathogenicity of PsBZPc32-silencedtransformants was severely attenuated on susceptible soybean cultivar Hefeng47 in contrastto the wild type stain.Through microscopic observation of infection process,the mutantscould penetrate into epidermal cells of soybean etiolated seedlings,but the infected hyphaecouldn,t spread in plant tissue.Besides,quite a lot plant-derived ROS accumulated atinfection sites 10 hours after infection.Although the transcripts level of PsBZPc32 was notaffected by H2O2 treatment,PsBZPc32-silenced transformants exhibited reduced toleranceto oxidative stress.These results indicate that PsBZPc32 is an important regulator for cystsgermination,stress response and pathogenesis.Molecular mechanism of PsBZPc32 in regulation oxidative stress response andpathogenicity:The bZIP transcription factors play important roles in regulatory network ofgene expression during development and stresses response in eukaryotic.Previously,weidentified a bZIP transcription factor,PsBZPc32,which is involved in cysts germination,oxidative stress response and pathogenicity.Based on phosphorylation sites prediction,multiple potential phosphorylation sites were identified in PsBZPc32,two of which werefound in P.sojae phosphoproteomics data of IF3h stage.Under oxidative stress,PsBZPc32in cytoplasm exhibited multiple phosphorylation levels and transferred into the nucleusfrom cytoplasm.Further study explored that the two phosphorylation sites identified in P.sojae phosphoproteomics data of IF3h stage are required for PsBZPc32 nucleus localization.In the infection stages,several Avh genes were down-regulated in PsBZPc32-silencedtransformants.To know more information about the downstream genes of PsBZPc32,digital gene expression analysis was carried out in PsBZPc32-silenced transformants,PsBZPc32-overexpressed transformants and the wide type stain at IF3h stage.215 geneswere found to be differently regulated in PsBZPc32-silenced transformant andPsBZPc32-overexpressed transformant,including several group genes related with its phynotypes in PsBZPc32-silenced transformant.These data suggest that PsBZPc32 was regulated at post-translational level by changing phosphorylation states,altering subcellular localization;PsBZPc32 regulates the down-stream genes that directly contribute to its roles in the processes of pathogenicity,cysts germination and oxidative stress.