Identification Of Effectors From Puccinia Striiformis F.sp.tritici And Functional Analysis Of Pst₁374

Puccinia striiformis Westend.f.sp.tritici Eriks?Pst?,as an obligate biotrophic fungi,causes destructive stripe rust disease in wheat worldwide.Resistance in wheat cultivars is usually overcome quickly by the rapid emergence of new virulent Pst races,which frequently leads to the epidemic of the disease.Pst can not be cultivated in vitro and lack the stable transformant system,which hinder the intensive researches on the pathogenetic mechanism of PST via the traditional genetic techniques.Thus it is difficult to effectively control the disease.To speed up deciphering the mechanism of Pst pathogenicity,based on the long-term research on wheat-Pst interaction,we completed the genome sequencing of Pst CYR32,which provide us abundant gene resources for the screening of PST effectors.In this study,we screened candidate effectors in the secretome of CYR32 and focused on one of the candidates,Pst₁374.The main results obtained in this study are listed as follows:1.From the secretome of Pst isolate CYR32,we selected 120 candidates that were rich in cysteine,highly induced in Pst haustoria or small proteins with the number of amino acids less than 300.By using Agrobacterium-mediated transient expression,five effector candidates were identified to have the ability to suppress of cell death caused by mouse BAX protein.The signal peptide was tested to be functional in yeast signal sequence trap assay.Of these five effector candidates,four candidates?Pst₁178,Pst₁374,Pst₁873 and Pst₂511?were highly induced during Pst infection in wheat,especially in 18 hours post inoculation.However,the transcript level of Pst₂468 was down-regulated during Pst infection.All of the five candidates were localized intracellular when transiently expressed in tobacco cells,indicating they were cytoplasmic effectors that translocated into host cells.By a bacterial Type III secretion assay,five candidates were delivered into wheat cells to test whether they could inhibit host immunity in their host system.All of the five candidates were able to suppress callose deposition which is a hallmark of PTI triggered by non-pathogenic Pseudomonas EtHAn?Effector-to-Host Analyzer?.In addition,delivery of each candidate could also depress the host ETI defense triggered by Pst avirulent isolate infection,which manifests as reduction in H2O2 accumulation and necrotic area.However the contributions of these five effectors were minor as the inhibition of ETI could rarely promote Pst growth anddevelopment.2.Pst₁374 was further studied in this research.Pst₁374 was able to enter into root tip cells without pathogen participation by root tip cell uptake assay.Moreover,by using the truncation mutants assay,the regions responsible for uptake was in the N-terminal 23-37 amino acids.The Y/F/WxC motif in this region was not responsible for translocation.Surprisingly,we found the ?-helices present in the uptake regions of Pst₁374 is amphipathic,which might related to effector uptake.3.Two wheat targets?TaClpS and TaTrxm?were screened out from wheat-Pst incompatible cDNA library and the interaction was confirmed both in the yeast two hybrid?Y2H?system and in vivo bimolecular fluorescence complementation?BiFC?assays in tobacco cells and the amino acid from 61 to 105 was responsible for the interaction.One function of TaTrxm is to unfold the formation of multimeric by reduction of disulfide bond among the molecules,suggesting that Pst₁374 possibly form multimeric during infection.We confirmed the interaction of Pst₁374 with itself both in Y2 H and BiFC assay.Bioinformatic analysis revealed that two other homologues of Pst₁374 were tandem arranged next to Pst₁374.Transient expression of PSTG₀1159 could also suppress BAX triggered cell death and interact with Pst₁374,TaClpS and TaTrxm.Together,we implied that Pst₁374 might form homomultimeric or heteromultimeric during Pst development.TaTrxm possibly reduced multimeric formation and activate or inactivate effector function to modulate host immunity.4.Previous studies have shown that thioredoxin could also involve in R protein resistance signaling by acting as guard protein.We used TaTrxm as bait to screen for resistantrelated proteins in wheat-Pst interaction.TaEDS1 was obtained and confirmed by Y2 H assay.EDS1 is an immunity node in dicots resistance to various pathogens.However the function of EDS1 in monocots was rarely reported.The expression of TaEDS1 was induced only in the incompatible interaction,indicating a role in wheat against Pst infection.Meanwhile,TaEDS1 was highly induced when treated by salicylic acid?SA?,but not in JA and ETH treated wheat plants.When TaEDS1 was silenced by the virus-induced gene silencing system,sporadic urediniospores were observed in wheat inoculated by CYR23 and higher density of urediospores were observed in wheat inoculated by CYR31.Histological observation showed more haustoria number and larger infection area both in the compatible and incompatible interaction,which implying a positive role in wheat defense against Pst.To find out the defense mechanism of TaEDS1,we screened for the targets of TaEDS1 both in the incompatible cDNA library and the cDNA library of CYR32 urediospore.The interaction of TaEDS1 and a catalase isozyme 1?TaCATi?has been confirmed both in theyeast two hybrid assay and BiFC in tobacco cells.In Pst,two secreted proteins which contain domain of polysaccharide deacetylase and another secreted protein which was predicted to have a lipase domain were confirmed to have the ability to interact with TaEDS1 in Y2 H assay.Collectively,we proposed that efectors might cooperatively target diverse nodes of plant defence signaling pathway to interfere plant multi-layer immunity.