Functional Characterizations And Mechanism Analysis Of Three Effectors In Phytophthora Sojae

Phytophthora sojae was identified as a fast-spreading and devastating root rotpathogen of soybean.The disease has threatened the yield of soybean in almost allsoybean production regions around the globe.P.sojae is an oomycete pathogen,whichform a distinct phylogenetic lineage of fungus-like eukaryotic microorganisms and relatedto diatoms.Chemicals applied in fungi are usually ineffective in controlling the diseasescaused by oomycetes.The high frequency of evolution on avirulence genes in P.sojaemakes resistance breeding using Rps gene not keep for long periods.Thus,the control ofthe soybean root rot disease is still a difficult and challenging problem which we currentlyfaces.Exploration of the pathogenic molecular mechanisms would provide a basis forpreventing the occurrence of this disease.Plants are resistant to pathogens because of thebasal resistance.To invade plant successfully,pathogens secrete a large amount of effectorsto host cell to modulate plant immune.Some of the effectors could suppress host defenceand promote pathogen infection,functioning as virulence factors;if the effectors isrecognized by host R genes,they would induce plant resistance and inhibit pathogeninfection,functioning as avirulence factors.Investigating the mechanism of the interactionbetween pathogens and plants through effectors would promote our understanding ofdisease occurrence regularity caused by pathogens in theory and promote the developmentof resistant varieties in practice.Here,we described the functions of 3 P.sojae cytoplasticeffectors in infection and explored the molecular pathogenesis respectively.The mainresults and conclusions are showed as follows:A P.sojae intracellular effector PsCRN108 targets host DNA to re-programexpression of plant HSP genes and plant defence.Here we identified 3Helix-hairpin-Helix(HhH)motif containing effectors from predicted P.sojae CRNeffectors,among which only PsCRN108 is up-regulated after infection,indicating apossibility on virulence.The signal peptide of PsCRN108 could mediate the secretion ofinvertase from yeast to the medium,indicating that PsCRN108 is a secreted protein.Thefull length of PsCRN108 could mediate the translocation of the C terminal of Avr1b indicating that it's an intracellular effector.Localization experiment demonstrated thatPsCRN108 was located in the haustoria when infection and accumulated in plant nuclear.These results demenstrated that PsCRN108 is secreted from haustoria after infection andcan be translocated to host nuclear.Silencing of PsCRN108 in P.sojae reduced thepathogenicity and the ability of suppressing host callose deposition while expression ofPsCRN108 in plant suppressed the plant resistance to P.capsici,indicationg thatPsCRN108 functions as a virulence factors by suppressing host immune.Digital GeneExpression(DGE)analysis of PsCRN108 transgenic Arabidopsis identified many HSPgenes which were down-regulated by PsCRN 108.PsCRN 108 could also suppress HSPgenes transcript in N.benthamiana and Giycine max.Silencing of Hsp90s in N.benthamiana lead to the suppression of plant resistance to P.capsici,indicating that HSP90gene was required for plant defence.The DNA binding activity test in vivo and in vitroindicated that PsCRN 108 could bind to the conserved element(HSE)in the promoterregion of HSP genes.Further investigation demonstrated that interaction ofPsCRN108-HSE inhibits plant heat shock factor(AtHsfAla)mediated HSE bindingactivity and HSP gene transcript.The substituted mutation of the predicted HhH motif inPsCRN108 leads to the fail in DNA binding activity,indicating that HhH motif is requiredfor PsCRN 108 mediated DNA binding activity.Both the substituted mutation of thepredicted HhH motif and the predicted nucleus localization signal(NLS)in PsCRN108 leadto the suppression of plant HSP gene transcript and plant immune,indicating that nuclearlocalization and DNA binding activity of PsCRN 108 were required for the suppression ofHSP gene transcript and plant defence.Taking these results together,we infered thatPsCRN 108 could bind to the conserved element(HSE)of plant HSP gene promoters andinhibit plant heat shock factor(AtHsfAla)mediated HSE binding activity and HSP genetranscript,thus suppress plant HSP mediated immune.This is the first report aboutommycete effector targetting to plant DNA.An unconventional secreted P.sojae effector PsIsc1 reduces plant salicylatebiosynthesis by hydrolyzing its precursor.Plant salicylate play a vital important role inplant defence to pathogens.Plants synthesize SA through two distinct enzymatic pathways.One is from chorismate via isochorismate(Strawn et al.,2007;Dempsey et al.,2011).Herewe identified a putative isochorismatase gene Pslscl in P.sojae.It can hydrolyzeisochorismate into 2,3-dihydro-2,3-dihydroxybenzoate(DDHB)and pyruvate,indicatingthe possible role of this gene in hydrolyzing the precursor of plant salicylate and suppressing plant salicylate mediated immune.Here we make a verification and analysis ofthis hypothesis.PsIscl gene is up-regulated after infection;indicating the relation topathogenicity.Silencing of Pslscl in P.sojae reduced the pathogenicity whileoverexpression of PsIscl enhanced the pathogenicity,indicating that Pslscl play a vitalimmportant role in pathogen infection.PsIscl silenced P.sojae lines enhanced the level ofSA and PR1 gene transcript in plant after infection indicating a potential role of PsIsc1 inreducing plant SA content.Localization experiment demonstrated that PsIsc1 was secretedfrom haustoria after infection and can be translocated to plant cell.Overexpression ofPsIsc1 in N.benthamiana reduced the SA level and PR1 gene transcript and suppressedplant defence.Enzyme activity assays and the substituted mutation assay demonstrated thatPsIsc1 could hydrolyze isochorismate and this activity is required for suppression of plantdefence.More importantly,this protein lacks signal peptide,but exhibits character ofunconventional secretion and thus was termed as unconventionally secreted effector.Takingthese results together,we infered P.sojae secreted isochorismatase effector throughunconventional secretion pathway and that disrupted the plant salicylate metabolismpathway by suppressing its precursor.Two RxLR avirulence genes in Phytophthora sojae determine soybeanRpslk-mediated disease resistance.Rpslk has been the most widely used Rps gene inagricultural soybean production,Here,we show that the products of two distinct but closelylinked RxLR effector genes are detected by Rpslk-containing plants,resulting in diseaseresistance.One of the genes is Avr1b-1,that confers avirulence in the presence of Rps1b.Three lines of evidence,including over-expression and gene silencing of Avr1b-1 in stable P.sojae transformants,as well as transient expression of this gene in soybean,indicated thatAvr1b-1 could trigger Rps1k-mediated defence response.Some isolates of P.sojae that donot express Avr1b are nevertheless unable to infect Rps1k plants.In those isolates,weidentified a second RxLR effector gene(designated Avrlk),located 5 kb away fromAvr1b-1.Silencing or overexpression of Avrlk in P.sojae stable transformants resulted inthe loss or gain,respectively,of the avirulence phenotype in the presence of Rpslk.Onlyisolates of P.sojae with mutant alleles of both Avrlb-A and Avrlk could evade perceptionby the soybean plants carrying Rps1k.Taking these results together,we infered that twoRxLR avirulence genes in Phytophthora sojae can both be recognized by host Rpslk geneand induce Rpslk mediated resistance and this may be the reason why Rpslk has been sowidely used in agriculture.Based on the results above,we concluded that pathogen secreted effectors to host cell and manipulate plant immune through sophisticated mechanism.The mechanisms behind that layes the groundwork for taking measures to control disease.