Identification And Functional Characterizations Of A Novel PAMP PsXEG1 In Phytophthora

Phytophthora sojae was identified as a fast-spreading and devastating root rot pathogen of soybean.The disease caused by this pathogen has threatened the yield of soybean in almost all soybean production regions around the globe.P.sojae is an oomycete pathogen,which form a distinct phylogenetic lineage of fungus-like eukaryotic microorganisms,related to diatoms.Chemicals applied in fungi are usually ineffective in controlling the diseases caused by oomycetes.The high frequency of evolution on avirulence genes in P.sojae makes resistance breeding using Rps gene not keep for long periods.Thus,the control of the soybean root rot disease is still a difficult and challenging problem which we currently faces.New control measures with durable broad-spectrum resistance against P.sojae is the persisitant need for agricultural production,national economy development and the destinies of our state.Plants can detect microbial/pathogen-associated molecular patterns(MAMPs/PAMPs)via PRRs to trigger PAMP-triggered immunity(PTI)which protect themselves against pathogens.PAMPs were originally defined as highly conserved molecules that have an essential function in microbial fitness or survival within a class of microbes.PAMPs identified so far from oomycetes include ?-glucans,heptaglucoside,transglutaminase(Pep 13),cellulose-binding elicitor lectins(CBDs),elicitins and NLP.Nevertheless,the mechanisms that allow plants to perceive oomycetes as non-self and to defend against infection remain only partly known.Identification of new PAMPs in oomycetes and exploration of the perception behind them will provide the basis for understanding the plant resistance mechanism and disease resistance breeding.Here we make a systematic screening of PAMPs in P.sojae.We identified a novel PAMP PsXEG1 and we described the functions and explored the possible mechanism.The main results and conclusions are showed as follows:A Glycoside Hydrolase 12(GH12)PsXEG1 from Phytophthora sojae is a novel PAMP.We identified a novel apoplastic elicitor(PsXEG1)of cell death from the soybean pathogen P.sojae.A 55 kDa protein purified by HPLC from culture filtrates of P.sojae can induce cell death in Nicotiana benthamiana.Mass spectrometry of 55 kDa matched proteins identified several putative proteins.By verification,only Ps109681(PsXEG1)could induce cell death in N.benthamiana.PsXEG1 could also induce cell death in tomato,pepper and soybean indicating that PsXEG1 functions as a novel elicitor.Signal peptide is required for cell death-inducing activity in N.benthamiana indicating that PsXEG1 is an apoplastic elicitor.PsXEG1rec could produce reducing sugars from both xyloglucan and?-glucan,with somewhat higher activity toward xyloglucan,and the enzymatic activity of PsXEG1 is not required for cell death activity.PsXEG1 did not trigger cell death in NbSerk3-silenced N.benthamiana indicating PsXEG1 induced cell death depends on NbSerk3 mediated receptor recognition.PsXEG1can trigger cell death in LeEIX2-silenced tomatto indicating PsXEG1 induced cell death does not rely on LeEIX2 which means PsXEG1 is a novel elicitor different from EIX.Treatment of N.benthamiana with low concentration of PsXEG1rec enhanced the disease resistance indicating that plants recognized PsXEG1 to induce plant defence.PsXEG1 belongs to the Glycoside Hydrolase GH12 family,which is widely distributed across microbial taxa and can widely induce HR in oomycetes and fungi.N-Terminal Region(PsXEG120-53)of PsXEG1 is Recognized by Plant to Trigger Immunity.The widespread distribution of GH12 proteins across microbial taxa,and the host-specificity of GH12 HR activity both suggest a common molecular pattern associated with the elicitor activity.Here we identified the relatively conserved N-terminal 34 amino acids of PsXEG1(PXN1)which is necessary and sufficient to trigger defense responses.Alignment of GH12 protein sequences revealed three different regions(PsXEG120-53?PsXEG1211-241 and PsXEG1 117-159)that are well conserved in oomycetes,fungi,and bacteria.Two of the conserved regions were located adjacent to the two catalytic residues,while the third conserved sequence was located in the N-terminal region(resides 20 to 53).C-terminal deletions or N-terminal deletions of PsXEG1 revealed that the N-terminal region is essential for triggering cell death by PsXEG1,while the C-terminal regions are required for full activity.Synthetic PXN1(PsXEG120-53)peptide could trigger an oxidative burst,induction of defense gene transcription,and resistance to P.parasitica in N.benthamiana.Substituting the N-terminal region of PsXEG1 homology protein without HR-inducing activity with PXN1 makes those protein posess the HR-inducing activity,indicating that N-terminal region of PsXEG1 is essential for triggering HR.Structure predicts of PsXEG1 revealed residues 20 to 53 form a beta sheet on the surface of those GH12 proteins.Moreover,site-directed mutagenesis in N-terminas of PsXEG1 suggest that the aromatic amino acids and hydrophilic amino acids are important for for HR-inducing activity of PsXEG1.Therefore,our data support a possible role for PsXEG120-53 as a conserved epitopes in PsXEG1 recognized by plant.PsXEG1 is a Major Virulence Factor during Soybean Infection and is Recognized as a PAMP.The hydrolytic enzymes of pathogens are important contributors to plant pathogenesis through degradation of host macromolecules and in some cases through detoxification of secondary metabolites.To explore the possible contribution of PsXEG1 to P.sojae virulence,we determined the expression patterns of PsXEG1 during different stages of development including mycelia,zoospores,cysts and germinating cyst and at 12 time points of infection following inoculation of etiolated soybean hypocotyls with zoospores.PsXEG1 was expressed at high levels during very early infection stages(20 min to 2 h)then rapidly declined from 3 h on wards.To more directly establish the role of PsXEG1 during infection,we silenced and overexpressed PsXEG1 using transformation with an antisense or sense construct,respectively.Both silencing and overexpression of PsXEG1 reduced P.sojae virulence.ROS accumulation in and callose deposition response to infection of overexpression of PsXEG1 lines was substantially enhanced compared to the silencing,control lines.The hypersensitive response(HR)induced by PsXEG1 can be effectively suppressed by P.sojae effectors in N.benthamiana by Agrobacterium tumefaciens-mediated transformation(agroinfiltration)and in soybean by bombardment assays.Several of the RXLR effectors,especially Avh52,Avh62,Avh94 and Avh109 were strongly expressed during very early infection,with similar dynamics to PsXEG1.PsXEG1 contributes to P.sojae virulence,but that soybean recognizes the PsXEG1 to induce defense responses,which in turn can be suppressed by RXLR effector proteins.