Screening Of Haustorium Effector Factors Of P.striiformis F.sp. Tritici Using Wheat Protoplasts And Its Functional Verification

Wheat stripe rust is caused by Puccinia striiformis f.sp.tritici（Pst）,which is one of the most widespread and harmful fungal diseases in the world.When stripe rust outbreaks,it can cause serious losses of wheat yield and even crop failure.Breeding disease-resistant wheat varieties is the main mean of controlling traditional wheat production.However,with the continuous variation of stripe rust races,disease-resistant wheat varieties lose their resistance,which poses a continuous threat to the global wheat cultivation.Therefore,it is of great significance to analyze the pathogenic mechanism of the effector factors in the interaction between stripe rust fungus and wheat,in order to prevent and control wheat stripe rust.In this study,by analyzing the PAMP-Triggered Immunity（PTI）reaction process triggered by pathogenesis-related model molecules in wheat,elicitors that can induce PTI reaction in wheat were identified.Further more,the PTI regulatory pathway in wheat was analyzed by transcriptome sequencing,with the report genes of PTI reaction in wheat being identified.On the basis of Stripe rust sucker transcriptome sequencing and genome resequencing,the effect factors of stripe rust were screened in wheat protoplasts for the first time,and the pathogenic factors were verified.The role of pathogenic effect factors in the process of wheat infection was clarified.In this study,a new method for screening wheat stripe rust effect factors was proposed,which laid a foundation for revealing the molecular mechanism of interaction between stripe rust and wheat and providing important theoretical basis for the control of wheat stripe rust.The main findings are as follows:1.Determination of PTI Reaction elicitors in Wheat.Histological observation,by aniline blue staining and DAB staining,showed that flg22 and chitin could induce callose accumulation and hydrogen peroxide outbreak in wheat leaves,which indicate that they could stimulate PTI reaction in wheat.Moreover,chitin-induced PTI reaction in wheat was stronger than that of flg22.Results above revealed that flg22 and chitin were identified as the elicitors of PTI reaction in wheat.2.Identification of Reporter Genes for PTI Response in Wheat.RNA-Seq was used to analyze the differentially expressed transcriptomes of wheat leaves treated with H₂O,flg22 and chitin.And q RT-PCR was used to verify the report genes,indicating that the PR1 family gene Ta Pr-1-14 could be used as one to detect PTI reaction in wheat at the transcriptional level.Ta Pr-1-14 provides a convenient method for studying PTI reaction in wheat at the cellular level.3.A New Method for Screening Stripe Rust Effectors in Wheat Protoplasts.The expression of PSTha5a23 in wheat protoplasts could inhibit the transcription of PTI report gene Ta Pr-1-14.And the expression of Puccinia striiformis pathogenic effector protein in wheat protoplasts could inhibit the transcription of PTI report gene Ta Pr-1-14.The results showed that it was feasible to identify pathogenic effectors by protoplast.This method has the advantages of short test period,more economical,and can screen dozens of candidate proteins at the same time.Using protoplast screening method provides a more efficient screening and identification method for analyzing the interaction between stripe rust and wheat.4.Screening and identification of candidate effector proteins of stripe rust.On the basis of genome re-sequencing and reseach on haustorium transcriptome of stripe rust fungus,50 candidate effector factors were identified by bioinformatics analysis.In those50 candidate genes,39 candidate effectors were successfully cloned.Through screening haustorium effector factors from wheat protoplasts,three effector proteins that could inhibit PTI reaction of wheat protoplasts were identified.Then,Host-Induced Gene Silencing（HIGS）was used to verify that PSEC17,a secretory protein of stripe rust organs,was a pathogenic effector protein.5.Functional Study of Pathogenic Effective Protein PSEC17.The function of PSEC17 gene of wheat stripe rust pathogenic effector protein was studied.The gene encoded 257 amino acids without PFAM structure,of which 19 amino acids functioning as signal peptide at N terminal.The effector protein PSEC17 was located in the cytoplasm and nucleus of wheat protoplasts.PSCE17 protein inhibited the upstream immune signal transduction of MAPK,resulting in PTI response induced by chitin.PSCE17 may have a high transcriptional level after the formation of haustorium in host cells.PSCE17 may participate in the process of inhibiting PTI immune signal transduction in wheat and promote the successful infection of wheat leaves by stripe rust.