Characters And Functional Analysis Of Effector Protein Pt2567 In Puccinia Triticina

Wheat leaf rust caused by the obligate parasitic fungus Puccinia triticina Eriks.（Pt）seriously threatens the safe production of wheat worldwide.However,due to the fast frequency of virulence mutations and the diversification of variability of the pathogenic,the rust resistance of the wheat varieties continues to be overcome.Studying the pathogenic mechanism of Pt is of great significance for understanding the variation of pathogenic fungi and the rational use of resistant varieties.The specific nutrient structure of Pt is haustorium which secretes a large number of effector proteins to host cell,and creates conducive to the growth of pathogens environment by inhibiting,modifying and regulating the physiological and metabolic processes of the host.Therefore,effector become an important arms for leaf rust to cause disease.In this study,the transcriptome data of the spore dormancy,germination,and infection interactions between the Pt races KHHT,JHKT,THSN and the susceptible host Thatcher were analyzed.A total of 635 candidate secreted effector proteins（CSEPs）were screened by bioinformatics,one of these genes,Pt2567,was selected for research to analyze the functional characteristics.The specific research results are as follows:1.The sequence structure characteristics of Pt2567 were clari fiedThe online software SignalP v4.1,TargetP v1.1,TMHMM v2.0 and EffectorP v2.0 were used to predict the effector.It was found that Pt2567 contained a signal peptide,was located in the secretory pathway,and did not contain a transmembrane domain.EffectorP predicted that it would have 94%probability is a effector protein.Analysis of the coding sequence revealed that it contained 169 amino acids,including one cysteine,and did not contain any known motif and domain.Sequence alignment in NCBI revealed that Pt2567 was a putative protein with 100%identity to PTTG₂8625.2.Pt2567 can inhibit PCD induced by BAXPt2567 was cloned by PCR,and pGR107:Pt2567 vectors were constructed.The positive and negative controls was the Avrlb and the empty vector pGR107,respectively.And co-injected with BAX results on Nicotiana benthamiana showed that Pt2567 could effectively inhibit PCD inducted by BAX.3.Analysis the expression characteristics of Pt2567Real-time fluorescence quantitative（qRT-PCR）technology was used to analyze the expression of Pt2567 at different time points after the interaction between Pt and susceptible hosts.It was found that the expression level of this gene was the highest at 36h and 60h after interaction,and the overall expression was up-regulated in the early and middle stage of the interaction between wheat and Pt,which was consistent with the expression level of transcriptome sequencing.4.Analysis the polymorphism of Pt2567 in different virulence Pt strainUsing nine strains of Pt with different virulence to carry out intraspecies polymorphism analysis of the effector protein Pt2567.The result showed that all the 702 bp fragments can be amplified in the tested strains.By sequencing and using MEGA6 software for sequence alignment,revealed that the sequence contains three exons,two introns,and a total of four amino acid site mutations,which are threonine（T）and isoleucine（I）,refined Leucine（R）and leucine（L）,tyrosine（Y）and aspartic acid（D）,isoleucine（I）and valine（V）respectively,showed low polymorphism levels.5.Determine the subcellular localization of Pt2567Subcellular localization of Pt2567 in tobacco cells and wheat protoplast was conducted on N.benthamiana and wheat protoplast by constructing the pRG107:Pt2567 vector with GFP fluorescent labeling,and using pRG107:GFP as a control,revealed that the fusion-expressed protein was localized inside of host cells to effect.6.Demonstrated the virulence and avirulence of Pt2567 in different hostsBy constructing pRG107:Pt2567 expression vector and using Agrobacterium tumefaciens transient injection method to deliver Pt2567 into 37 near（single）gene lines.And it was found that this gene can specifically cause hypersensitive response（HR）in TcLr28,indicating that Pt2567 may be a candidate avirulent gene for Lr28.Then the bacterial three type secretion system and Pseudomonas fluorescens strain EtHAn were used to overexpress Pt2567 in different wheat varieties,through construing of pEDV6:Pt2567 expression vector,using pEDV6:avrRpt2 and pEDV6:dsRED as controls.According to the accumulation of callose deposition,we determined that the gene can inhibit PTI in Thatcher,ETI in TcLr19,showing virulent functions in Thatcher and TcLr19 respectively,and can enhance PTI and ETI in TcLr28,and exert avirulent functions.7.Prove the avirulence function of Pt2567 to Lr28 through HIGSThe host-induced gene silencing technology（HIGS）mediated by BSMV was used to silence Pt2567 in the near-isogenic line TcLr28 by inoculating with avirulence strains（KHHT）to Lr28.The analysis revealed that the response type of TcLr28 changed from"0" to "4" after Pt2567 was silenced.Histological observation revealed that at 120 hour post inoculation,compared with the control group BSMV:00,a large number of mycelium appeared in TcLr28 that silenced Pt2567,indicating that Pt2567 played avirulent function in the infection of TcLr28 by wheat leaf rust fungus.This study showed that the wheat leaf rust is rich in a large number of effector proteins and is produced during the infection interaction stage between host and pathogen.The Pt2567 sequence structure has certain characteristics,and plays a role inside the host cell,and has both virulent or avirulent functions.It was determined that Pt2567 played avirulent role in the infection of TcLr28 by Pt,and may be a candidate avirulent gene for Lr28.The results laid a foundation for studying the pathogenic mechanism of wheat leaf rust.