Characterization And Functional Analysis Of An Immunity-inducing Protein From Magnaporthe Grisea

Plants have evolved sophisticated defense mechanisms that are activated in responseto pathogen attack.In addition to the basal immunity,plants also have an inducedimmunity,which is induced upon perception of exogenous general elicitors.Elicitorsthemselves have no inhibitory effect to pathogens,but can activate the defensemechanisms in plants.Recently,a class of microbe-derived proteinaceous elicitors hasattracted much attention because they have greater potential in developingenvironment-friendly agents for crop disease control.In this study,I identified a gene,MgSM1,encoding an immunity-inducing protein,from Magnaporthe grisea,the riceblast fungus.I studied in detail the immunity-inducing activity of MgSM1 and itspossible mechanism in regulation of defense response using Agrobacterium-mediatedtransient expression and transgenic approaches.Based on the sequence information of SM1 genes from other microbes,a cDNA thatencodes a SMl-like protein was cloned and identified from M.grisea and designagedas MgSM1.The MgSM1 gene encodes a 138 amino acid protein.The MgSM1 proteincontains four conserved cysteine residues and showed high level of similarity topreviously reported proteins of Cerato-platanin(CP) family.RT-PCR analysis revealedthat the MgSM1 gene is expressed during infection of rice plants,and is also expressedin cultures at different development stage ofM.grisea on artificial medium.To study the immunity-inducing activity of MgSM1 by Agrobacterium-mediatedtransient expression method,the MgSM1 ORF was cloned into pFLAG-CTS1,forminga recombinant MgSM1 protein with a FLAG tag fused to the C-terminal.The sequencefor the MgSM1-FLAG fusion was then inserted into pINDEX3 under control of aDEX-inducible promoter,yielding pINDEX3-MgSM1-FLAG.The immunity-inducingactivity of MgSM1 was analyzed in different plant species including tobaceo(Nicotiana tabacum,Nicotiana benthamiana),Arabidopsis thaliana,tomato(Solanumlycopersicon) and rice(Oryza sativa).Fully expanded leaves were first infiltrated withagrobacteria carrying pINDEX3-MgSM1-FLAG or empty vector pINDEX3 and thentreated with DEX to induce expression of the MgSM1 gene from the DEX-induciblepromoter 24 hr after agroinfiltration.Hypersensitive cell death was observed within 24hr after DEX induction in leaves of the tested plants that were agro-infiltrated with pINDEX3-MgSM1-FLAG but not in leaves that were agro-infiltrated with pINDEX3.In leaves of tobacco,tomato,Arabidopsis and rice plants,transient expression of theMgSM1 gene resulted in accumulation of reactive oxygen species(e.g.H_2O2 andsuperoxide anion),increased electrolyte leakage and elevated expression ofhypersensitive response marker genes.These results suggest that MgSM1 has acapacity to induce a typical HR in different plant species.To examine whether MgSM1 can induce defense response in plants,expression ofdefense genes and disease resistance in MgSM 1-treated plants were analyzed.RT-PCRanalyses showed that,after DEX treatment,transient expression of MgSM1up-regulated expression of PR genes not only in infiltrated leaves but also in upperuntreated leaves of tobacco,tomato,and Arabidopsis thaliana plants.Importantly,local expression of MgSM1 in the agro-infiltrated leaves of tomato and Arabidopsisthaliana plants led to enhanced disease resistance against Botrytis cinerea andPseudomonas syringae pv.tomato(Pst) DC3000 in upper untreated leaves.Theseresults demonstrate that MgSM1 has immunity-inducing activity to induce diseaseresistance in tomato and Arabidopsis plants through activation of defense-relatedgenes.To further study the function of MgSM 1 in regulation of plant defense response,theMgSM1 gene under control of the DEX-inducible promoter in pINDEX3 wasintroduced into Arabidopsis thaliana through floral dip transformation method.A totalof 15 individual transgenic lines were obtained and three independent lines with singlecopy of the transgene were identified through screening based on segregation ofantibiotic resistance marker.Without DEX treatment,the MgSM1-overexpressingtransgenic plants showed no difference in growth as compared with wild-type plants.However,expression of the MgSM1 gene in transgenic plants was detected after DEXtreatment,Significant growth suppression of the transgenic plants was observed andsome died after treatment with DEX at high concentrations.Disease resistance assaysindicated that the MgSMl-overexpressing plants enhanced resistance against PstDC3000,B.cinerea and Alternaria brassicicola after DEX treatment.Growth of PstDC3000 and B.cinerea in leaves of the MgSM1-overexpressing transgenic plants wassignificantly reduced as compared with those in leaves of the control plants.Lesionsizes caused by A.brassicicola on detached leaves of the MgSM1-overexpressingtransgenic plants were smaller than those of controls.RT-PCR analysis showed that,after DEX treatment,expression levels of some defense-related genes including PR1, PR5 and PDF1.2 were up-regulated in the MgSM1-overexpressing transgenic plants ascompared with those in control plants.Accumulation of hydrogen peroxide andsuperoxide anion in leaves of the MgSM1-overexpressing transgenic plants was alsoobserved after DEX treatment.These results indicate that MgSM1 can activateexpression of defense-related genes and thus enhance disease resistance againstdifferent pathogens.To explore the possible signaling pathway involved in MgSM1-mediated defenseresponse,the immunity-inducing activity of MgSM1 was compared in someArabidopsis mutants that are defect in well-established salicylic acid(SA) andethylene/jasmonci acid signaling pathways.Leaves of mutants(fls2-100,jar1-1,npr-1,NahG,ein2,bos1) and wild-type(Col-0) plants were agro-infiltrated withpINDEX3-MgSM1-FLAG or pINDEX3 and then treated with DEX to induceexpression of the MgSM1 gene from the DEX-inducible promoter.Levels ofhypersensitive cell death in the mutant plants were higher than in wild-type plants afterDEX treatment.Expression level of the PR genes and disease resistance against PstDC3000 and B.cinerea in NahG,npr1,ein2 and jar1-1 mutant plants were partiallyreduced as compared with those in the wild-type plants.These results suggest that theSA or JA/ET signaling pathways are required for the MgSMl-mediated defenseresponse.However,the expression level of PR genes and disease resistance against PstDC3000 and B.cinerea infls2-100 plants was similar to those in the wild-type plants.This result suggests that MgSM1 has receptor(s) other than FLS2 for itsimmunity-inducing activity in plants.To determine activity-determining region in the MgSM1 protein,four C-terminaltruncated constructs,including MgSM1-D1(a truncated MgSM1 with deletion of 18amin acids from C-terminal),MgSM1-D2(a truncated MgSM1 with deletion of 38amin acids from C-terminal),MgSM1-D3(a truncated MgSM1 with deletion of 66amin acids from C-terminal) and MgSM1-D4(a truncated MgSM1 with deletion of 84amin acids from C-terminal),were constructed into vector pINDEX3.Theimmunity-inducing activity of these truncated MgSM1 was tested in Arabidopsis usingAgrobacterium-mediated transient expression method.It was found that theMgSM1-D1 and MgSM1-D2 truncated proteins induced hypersensitive cell death andactivated expression of PR genes as did the full-length MgSM1 protein,and thatMgSM1-D3 and MgSM1-D4 lost completely the immunity-inducing activity.Theseresults indicate that the immunity-inducing activity of the MgSM1 protein exists in a region between 71-99 amino acids in MgSM1.Fine mapping of theactivity-determining sequence in MgSM 1 protein is still underway.