Expression of a Novel Antimicrobial Peptide Penaeidin4-1 in Creeping Bentgrass (Agrostis stolonifera L.) Enhances Plant Fungal Disease Resistance

Expression of a Novel Antimicrobial Peptide Penaeidin4-1 in Creeping Bentgrass (Agrostis stolonifera L.) Enhances Plant Fungal Disease Resistance ExpressionofaNovelAntimicrobialPeptidePenaeidin4- 1inCreepingBentgrass(AgrostisstoloniferaL.) EnhancesPlantFungalDiseaseResistance ManZhou,QianHu,ZhigangLi,DayongLi,Chin-FuChen?,HongLuo* DepartmentofGeneticsandBiochemistry,ClemsonUniversity,Clemson,SouthCarolina,UnitedStatesofAmerica Abstract Background:Turfgrassspeciesareagriculturallyandeconomicallyimportantperennialcrops.Turfgrassspeciesarehighly susceptibletoawiderangeoffungalpathogens.Dollarspotandbrownpatch,twoimportantdiseasescausedbyfungal pathogens Sclerotinia homoecarpa and Rhizoctonia solani, respectively, are among the most severe turfgrass diseases. Currently,turffungaldiseasecontrolmainlyreliesonfungicidetreatments,whichraisesmanyconcernsforhumanhealth andtheenvironment.Antimicrobialpeptidesfoundinvariousorganismsplayanimportantroleininnateimmuneresponse. Methodology/PrincipalFindings:Theantimicrobialpeptide-Penaeidin4-1(Pen4-1)fromtheshrimp,Litopenaeussetiferus hasbeenreportedtopossessinvitroantifungalandantibacterialactivitiesagainstvariouseconomicallyimportantfungal andbacterialpathogens.Inthisstudy,wehavestudiedthefeasibilityofusingthisnovelpeptideforengineeringenhanced diseaseresistanceintocreepingbentgrassplants(AgrostisstoloniferaL.,cv.PennA-4).TwoDNAconstructswereprepared containing either the coding sequence of a single peptide, Pen4-1 or the DNA sequence coding for the transit signal peptide of the secreted tobacco AP24 protein translationally fused to the Pen4-1 coding sequence. A maize ubiquitin promoter was used in both constructs to drive gene expression. Transgenic turfgrass plants containing different DNA constructsweregeneratedbyAgrobacterium-mediatedtransformationandanalyzedfortransgeneinsertionandexpression. In replicated in vitro and in vivo experiments under controlled environments, transgenic plants exhibited significantly enhancedresistancetodollarspotandbrownpatch,thetwomajorfungaldiseasesinturfgrass.ThetargetingofPen4-1to endoplasmicreticulumbythetransitpeptideofAP24proteindidnotsignificantlyimpactdiseaseresistanceintransgenic plants. Conclusion/Significance: Our results demonstrate the effectiveness of Pen4-1 in a perennial species against fungal pathogensandsuggestapotentialstrategyforengineeringbroad-spectrumfungaldiseaseresistanceincropspecies. Citation:ZhouM,HuQ,LiZ,LiD,ChenC-F,etal.(2011)ExpressionofaNovelAntimicrobialPeptidePenaeidin4-1inCreepingBentgrass(AgrostisstoloniferaL.) EnhancesPlantFungalDiseaseResistance.PLoSONE6(9):e24677.doi:10.1371/journal.pone.0024677 Editor:HanyA.El-Shemy,CairoUniversity,Egypt ReceivedMarch17,2011;AcceptedAugust18,2011;PublishedSeptember12,2011 Copyright: ß 2011 Zhou et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited. Funding:ThisprojectwassupportedbytheBiotechnologyRiskAssessmentProgramCompetitiveGrantno.2005-39454-16551,2007-33522-18489and2010- 33522-21656fromtheUSDANationalInstituteofFoodandAgriculture,aswellasgrantSC-1700315fromtheUSDANationalInstituteofFoodandAgriculture.Dr. Chin-FuChenwasatClemsonUniversitywhenthisworkwasaccomplished.Hiscurrentemployer,GreenwoodGeneticCenter,didnotprovidefundsnordidit playanyroleinthiswork.Thefundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript. CompetingInterests:Theauthorshavethefollowingcompetinginterests:severalauthors(HongLuo,ManZhou,QianHu)havemadeapatentapplicationfor work described in this manuscript (US patent application publication 2011/0078820 A1 published 31 March 2011). There are no products in development or marketedproductstodeclare.Thisdoesnotaltertheauthors’adherencetoallthePLoSONEpoliciesonsharingdataandmaterials. *E-mail:hluo@clemson.edu ?Currentaddress:CenterforMolecularStudies,GreenwoodGeneticCenter,Greenwood,SouthCarolina,UnitedStatesofAmerica tively, are among the most severe and frequently occurring Introduction diseases on turfgrass lawns in the summer [3,4]. Currently, Turfgrasses, agriculturally and economically important crop fungicides arecommonlyappliedtocontrolfungaldiseases.This species, are used worldwide for lawns of buildings, roadsides, raises concerns about the potential emergence of new pathogen athletic and recreational fields providing numerous benefits strainsasaresultofintensiveuseofchemicals[5–7].Resistanceto including reducing soil erosion, trapping dust and pollutants, somemajorclassesoffungicidessuchasbenzimidazoles,demethy- moderating temperature, safer playing grounds and beautifying lation inhibitors (DMIs), Qo respiration inhibitors (QoIs) and the environment [1,2]. There are more than 50 million acres ofdicarboximides (DCFs) has been detected in many phytopatho - turfgrassand16,000golfcoursesintheUSalone,andtheturfgrass genicfungispecies[8].Forexample,largescaleagriculturaluseof industryisamultibilliondollarbusinessannually[1,2].Turfgrass DMIssince1970shasledtotheemergenceofresistantgenotypes speciesarehighlysusceptibletoawiderangeoffungalpathogens. ofseveralphytopathogenicfungiimpactingdifferentcropandfruit Dollar spot and brown patch, two important diseases caused by species including turfgrass [6,8–10]. Similarly, benzimidazole- fungalpathogensSclerotiniahomoecarpaandRhizoctoniasolanirespec- resistant genotypes were also identified in Monilinia fruccticola, PLoSONE | www.plosone.org 1 September2011 | Volume 6 | Issue 9 | e24677 DiseaseResistanceinTransgenicTurfgrass Penn A-4 with enhanced resistance to two important fungal Penicillium expansum, Botrytis cinerea, Helminthosporium solani and pathogens,SclerotiniahomoecarpaandRhizoctoniasolaniasaresultof sclerotinia homoeocarpa. [6,8,11–14]. Therefore, the problem of emergent new resistant pathogen strains and the negative long- expressionofasyntheticpeptidegene,Pen4-1. termimpactsoffungicidesonhumanhealthandtheenvironment havebothdriventhesearchfornewalternativesforthecurrently Results used chemicals [5,15]. It is desirable that new cultivars be Productionandmolecularcharacterizationoftransgenic developedthatpresentsustainableresistancetoabroadrangeof pathogensandaresafefortheenvironmentorhumanconsump- creepingbentgrassplantsharboringthePen4-1gene tion[5,16]. TogeneratetransgenicplantsexpressingPen4-1andstudythe Antimicrobialpeptides(AMPs)foundinvariousorganismsplay role Pen4-1 plays in plant disease resistance, two chimeric DNA animportantroleininnateimmuneresponse[16–20],providing constructswerepreparedcontainingeitherthecodingsequenceof goodcandidatesforuseinplantsforenhanceddiseaseresistance. asinglepeptidePen4-1(Figure1a)ortheDNAsequencecoding AMPs are short sequence peptides with generally fewer than 50 forthetransitsignalpeptideofthesecretedtobaccoAP24protein translationally fused to Pen4-1 coding sequence (Figure 1b). A amino acid residues, most of which have antimicrobial activity maizeubiquitin(ubi)promoterwasusedinbothconstructstodrive against a broad spectrum of pathogens. They are a first line of Pen4-1 expression and an herbicide resistance conferring gene defenseinplantsandanimalsandresistanceagainstthemismuch namedbardrivenbytheCaMV35Spromoterwasincludedasa lessobservedcomparedwithcurrentantibiotics[16].AMPsfrom selectablemarkerforplanttransformation.Theoriginalnucleotide various sources have been demonstrated to confer resistance sequences of Pen4-1 were modified for plant-optimized codon against fungal and bacterial pathogens in an array of genetically usage, and chemically synthesized for use in chimeric gene engineered plant species, including Arabidopsis [21], tobacco [22– construction(Figure2). 31],rice[32–37],potato[38–44],tomato[45],cotton[46],pear Using Agrobacterium-mediated transformation of embryogenic [47], banana [22], ornamental crops, geranium (Pelargonium sp.) callusderivedfrommatureseedsandphosphinothricinselection, [48],Americanelm[49]andhybridpoplar[50,51]. we separately introduced the two chimeric gene constructs Penaeidins, a family of AMPs originally isolated from the (Figure1a,1b)intoacreepingbentgrass(A.stoloniferaL.)cultivar, haemocytes of penaeid shrimp, is considered to be a source of PennA-4,producingatotalof25independentT0transgeniclines compoundsthathavethepotentialtobeappliedinagricultureto transformedwiththeconstruct,p35S-bar/Ubi-Pen4-1,and5with deliver disease resistance to plants. Unlike vertebrates possessing the construct, p35S-bar/Ubi-AP24::Pen4-1. PCR amplification of theadaptiveimmunesystem,shrimponlyhaveaninnateimmune foreign genes using genomic DNA from transgenic plants system, among which are penaeidin antimicrobial peptides confirmedthepresenceoftransgenes(datanotshown).Southern [52,53]. Upon pathogen challenge to the host, the peptides are hybridization with a bar-specific probe revealed that all the releasedfromgranularhaemocytestotheplasmaandattachedto transgenic events contained one or two copies of transgene cuticles fighting microbial infection [53–56]. Penaeidins have a integration, most of which carried single copy insertions (see uniquetwo-domainstructureincludinganunconstrainedproline- example in Figure 1c). No significant difference in general plant richN-terminaldomain(PRD)andacysteine-richdomain(CRD) morphology,rootandshootdevelopmentaswellasoverallplant with a stable a-helical structure [53,57,58]. The complexity biomass was observed between transgenic and control plants inherent in this unique structure might have contributed to its withoutPen4-1gene. broad range of microbial targets, including primarily Gram positivebacteriaandfungi[53,54,58,59]. Thepenaeidinfamilyisdividedintofourclasses,designatedas Pen4-1expressionintransgenicplantsofcreeping 2, 3, 4 and 5. Each class displays a remarkable level of primarybentgrass sequence diversity [52,60]. Pen4-1, an isoform within the class Transgenic plants were further analyzed for Pen4-1 expression number4penaeidins(isoformnumber1)isisolatedfromAtlantic by Northern blot analysis. Hybridization of RNA samples from white shrimp (Litopenaeus setiferus). It contains 6 cysteine residues leaves revealed detectable Pen4-1 transcript, indicating transgene forming3disulfidebridgesandistheshortestisoforminpenaeidin expressioninallthetransgenicplants(seeexamplesinFigure1d). familywithalengthof47aminoacids.Itcaninhibitmultipleplant Moreover, all transgenic lines, regardless of Pen4-1 alone or pathogenicfungalspecies,includingtheB.cinera,P.crustosumand AP24::Pen4-1fusiongenebeingexpressedinplants,didnotappear F.oxysporum[53].ItisalsoeffectiveagainstGram-positivebacteria toshowsignificantdifferencesfromeachotherforPen4-1mRNA speciesincludingM.luteusandA.viriduans,andinhibitoryagainst accumulation (data not shown). Our efforts in detecting Pen4-1 Gram-negative bacteria, E. coli, at relatively high concentrations protein in plant extracts from turfgrass transgenic lines using a [52].Notably,Pen4-1caninhibitthegrowthofmultidrug-resistant polyclonal antibody raised against the selected region of Pen4-1 fungispecies:Cryptococcusneoforman(SteroformA,SteroformB,Steroform protein was unsuccessful (data not shown). This difficulty in C,SteroformD)andCandidaspp.(Candidalipolytica,Candidainconspicua, detectingPen4-1proteininturfgrassplantswasalsoencountered Candida krusei, Candida lusitaniae and Candida glabrata) [52]. when analyzing Pen4-1 production in Arabidopsis transgenic lines Compared to other classes of penaeidins, penaeidin class 4 has expressing the Pen4-1 gene (data not shown). Many attempts in shownahighlevelofpotencyagainstfungi[52].Additionally,the improvingproteinextractionandimmunoblottingusingcurrently unusual amino acid composition of Pen4-1, especially in the availablemethodologyandpublishedproceduresdidnotresultin proline-rich domain, may confer resistance to proteases [52]. satisfactory results. This difficulty in Western assay with Pen4-1 may result from poor retention of the protein by blotting TheseresultssuggestthatPen4-1isagoodcandidateforgenetic engineeringofenhanceddiseaseresistanceinplants.Thepresent membranes due to its small size and a highly positive charge. study investigates the feasibility of using the plant-optimized ProteasedegradationofPen4-1duringproteinextractioncouldbe another possibility, but is unlikely given the unusual amino acid nucleotide sequences encoding Pen4-1 from L. setiferus for engineering fungal pathogen resistance into perennial turfgrass composition of PRD Pen4-1 conferring resistance to proteases [52]. The same problem had been reported previously for other plants. We report the development of transgenic lines of a commercial creeping bentgrass (Agrostis stolonifera L.) cultivar, cv. plant-expressedsmallAMPs[27,32,42,46]. PLoSONE | www.plosone.org 2 September2011 | Volume 6 | Issue 9 | e24677 DiseaseResistanceinTransgenicTurfgrass Figure1.GenerationandmolecularanalysisofthetransgeniclinesexpressingPen4-1.(a)SchematicdiagramofthePen4-1expression chimericgeneconstruct,p35S-bar/Ubi-Pen4-1.Pen4-1geneisunderthecontrolofthemaizeubiquitinpromoter(Ubi)andlinkedtotheherbicide resistancegene,bar,drivenbytheCaMV35Spromoter.(b)SchematicdiagramoftheAP24::Pen4-1expressionchimericgeneconstruct,p35S-bar/Ubi- AP24::Pen4-1, in which the AP24::Pen4-1 fusion gene is under the control of the maize Ubi promoter. The CaMV35S promoter-driven bar gene is includedforherbicideresistance.(c)ExampleofSouthernblotanalysisofPen4-1expressiontransgenics.TwentymicrogramsofthegenomicDNA extracted from young leaves and digested with BamHI that cuts once within the T-DNA region was probed by a 440bp 32P-labelled bar gene fragment. Hybridization signals revealed were indication of copy numbers of transgene insertion. Lanes 1–6 were DNAs from representative transgenic creeping bentgrass plants. The negative control (WT) was BamHI-digested genomic DNA from a non-transformed wild-type plant. (d) Example of Northernblot analysis of Pen4-1 expressiontransgenics. Lanes 1-6 were total RNA from the same representative transgenic creeping bentgrassplantsusedforSouthernanalysisin(c).TwentymicrogramsofthetotalRNAextractedfromyoungleavesandprobedwitha 32 P-labelled Pen4-1genefragment.Thenegativecontrol(WT)wastotalRNAfromanon-transformedwild-typeplant. doi:10.1371/journal.pone.0024677.g001 Four representative transgenic lines harboring p35S-bar/Ubi- transgenic lines were clonally multiplied by vegetative propaga- tion. Evaluation of these plants under greenhouse conditions Pen4-1and5containingtheconstruct,p35S-bar/Ubi-AP24::Pen4-1 were used in the subsequent pathogen test experiments, all of showed that they performed very similarly in growth. Three whichcontainedasinglecopyintegrationofthetransgene.These groupsofcontrolplantswereusedforcomparisonwiththePen4- Figure2.NucleotideanddeducedaminoacidsequencesofthePen4-1gene.TheoriginalnucleotidesequencesofPen4-1weremodifiedfor plant-optimizedcodonusage.Thepredictedsingle-letteraminoacidsareshownabovethecodingsequence.Theaddedtranslationstopcodonis alsoindicated. doi:10.1371/journal.pone.0024677.g002 PLoSONE | www.plosone.org 3 September2011 | Volume 6 | Issue 9 | e24677 DiseaseResistanceinTransgenicTurfgrass 1-expressing transgenic lines in our pathogen test experiments. bothchallengedbythepathogeninreplicatedexperimentsunder They were untransformed plants either derived from seeds or acontrolledenvironment.Plantsineachpotwereinoculatedwith regeneratedfromtissuecultureandtransgeniclinesharboringthe 3 grams of rye seeds colonized by R. solani . Pen4-1-expressing same expression vector without the Pen4-1, but with a different transgenic lines all exhibited high resistance against pathogen foreign gene, which was not associated with plant response to infectionwithareducedlesiondiameterby30%-43%compared pathogenattack.Allcontrolplants,regardlessoftheirorigins,did to control plants 14 days after inoculation (Figure 4a, b and c). notshowsignificantdifferencesinmorphologyandgrowthaswell Statistical analysis of Wilcoxon test indicated that the disease asresponsetopathogeninfection. symptomsamongthedifferentPen4-1-expressingtransgeniclines were not significant (P.0.05) (Figure 4c), whereas a significant difference in disease development between control and Pen4-1- InplantaantifungalassayswithR.solani expressingtransgenicplantswasobserved(P,0.01)(Figure4c). R.solaniisasoil-bornefungusthatcausesbrownpatchdisease, Whenexposedtoaseconddoseofpathogeninfection,i.e.plants oneofthemostseverediseasesonturfgrasslawns.Toexaminethe ineachpotwereinoculatedwithadditional3gramsofryeseeds impactofPen4-1onplantresponsetoinfectionwithR.solani ,we colonizedbyR.solani14daysafterthefirstinoculation,thecontrol conducted experiments investigating plant disease resistance by plants suffered severe damage with 75% to 95% of them in the both in vitro and in vivo assays using detached leaves and whole potsbeingaffected twoweeksafter inoculation, whereas Pen4-1- plants,respectively. expressing transgenic lines were much less impacted with only ThedetachedleavesfromT0transgeniclinesexpressingPen4-1 around25%ofplantsinthepotsbeinginfected(Figure5a,b).The andthecontrolplantswereplacedon1%ofagarinPetridishes, disease ratings of the Pen4-1-expressing transgenic lines were and challenged by the pathogen using agar plugs infested with reduced by 41% to 44% compared to that of the control plants mycelium of R. solani isolate obtained from infected creeping (Figure5c).StatisticalanalysisofWilcoxontestindicatedthatthe bentgrass plants. Diseasesymptoms measuredas lesionsizewere disease development among the different Pen4-1-expressing documented at various times after inoculation. Compared to transgeniclineswasnotsignificant(P.0.05)(Figure5c). control plants, transgenic lines harboring either p35S-bar/Ubi- Pen4-1 or p35S-bar/Ubi-AP24::Pen4-1 exhibited dramatically enhanced disease resistance with a reduction in lesion length by InplantaantifungalassayswithS.homoeocarpa 42% to 48% fourteen days after inoculation (Figure 3a, b). Transgenic plants expressing Pen4-1 were also evaluated for Statistical analysis of Tukey’s Hornesly Significant Difference theirresistancetodollarspot,anotherimportantturfgrassdisease (Tukey’sHSD)indicatedthatthelesionsizereductioninPen4-1- causedbyS.homoeocarpa[61,62].Bothinvitroandinvivoassayswere expressingtransgenicplantswassignificant(P,0.01);whereas,no conductedtoexaminetheimpactofPen4-1onplantresponseto significant difference in lesion size was observed among Pen4-1- infectionwithS.homoeocarpa. Pen4-1- expressingtransgeniclines(P.0.05)(Figure3b). In vitro assays were conducted using leaves from T0 expressingtransgeniclinesandcontrolplants.Thedetachedleaves Plant performance in response to an R. solani infection was furtherevaluatedbyinvivoassaysusingthewholeplantsgrownin wereplacedon1%ofagarinPetridishes,andchallengedbythe pathogen using pots.ControlplantswithoutPen4-1andtransgeniclinesharboring S. homoeocarpa-infested agar plugs. Disease either p35S-bar/Ubi-Pen4-1 or p35S-bar/Ubi-AP24::Pen4-1 were symptoms measured as lesion size were documented at various Figure3.ResponseoftransgeniccreepingbentgrassplantsexpressingPen4-1toR.solaniinfection-invitroplantleafinoculation assay.(a)Thedetachedsecondexpandedleavesfromthetopofplantstolonswereusedforpathogeninoculationtest.Theimageshowsexample ofrepresentativeleavesfromalltestedPen4-1-expressingtransgenicplantswithasingletransgeneinsertion(TG,ontheright)andwild-typecontrol plants (WT, on the left) 14 days post-inoculation (DPI). Transgenic plants exhibited significant resistance to R. solani in comparison to wild-type controls.(b)Thedevelopmentofbrownpatchdiseasewasratedbymeasuringthelesionlengthoftheinfectedleaves2,8and14DPI.Statistical analysisofR.solaniinoculationtestwasconductedonwild-typecontrolplants(WT)andvarioustransgeniclinesharboringeitherp35S-bar/Ubi-Pen4- 1(TG1andTG2)orp35S-bar/Ubi-AP24::Pen4-1(TG3andTG4).Dataarepresentedasmeans6SE(n=10),anderrorbarsrepresentstandarderror. Asterisks(**or*)indicateasignificantdifferencebetweenPen4-1-expressingtransgenicandcontrolplantsatP,0.01orP,0.05byTukey’sHSDtest usingJMP9.0.0.ThePvaluesarelistedinTableS1. doi:10.1371/journal.pone.0024677.g003 PLoSONE | www.plosone.org 4 September2011 | Volume 6 | Issue 9 | e24677 DiseaseResistanceinTransgenicTurfgrass Figure 4. Response of transgenic creeping bentgrass plants expressing Pen 4-1 to R. solani infection - in vivo direct plant inoculationbioassayswithlowerdoseofR.solani.(a)Thefullydevelopedtransgenic(independenteventsTG1toTG4)andwild-type(WT) plants clonally propagated from individual stolons were grown and maintained in pots (15cm610.5cm) and inoculated with 3g of rye seeds colonized by R. solani. The image on the upper panel shows plants before pathogen infection. Example of plants from wild-type (WT) and representative transgenic lines harboring either p35S-bar/Ubi-Pen4-1 (TG1, TG2) or p35S-bar/Ubi-AP24::Pen4-1 (TG3 and TG4) two weeks after pathogeninoculation(14DPI)areshownonthebottompanel.Transgenicplantsexhibitedlessseverdiseasesymptomthanwild-typecontrols.(b)A closerlookofinfectedplantsshowingthedifferentlesionsizeofWTandTG.(c)Thedevelopmentofbrownpatchdiseasewasratedbymeasuring thelesiondiametersoftheinfectedleaves14DPI.StatisticalanalysisofR.solaniinoculationtestwasconductedonWTandvariousTGlines.Dataare presentedasmeans6SE(n=6),anderrorbarsrepresentstandarderror.Asterisks(**or*)indicateasignificantdifferencebetweentransgenicplants andwild-typecontrolsatP,0.01orP,0.05byWilcoxontestusingJMP9.0.0.ThePvaluesarelistedinTableS2. doi:10.1371/journal.pone.0024677.g004 Figure 5. Response of transgenic creeping bentgrass plants expressing Pen 4-1 to R. solani infection - in vivo direct plant inoculation bioassays with higher dose of R. solani. (a) Transgenic (TG) and wild-type (WT) plants were inoculated with a second dose of R.solani(3gofryeseedscolonizedbythepathogen)14daysafterthefirstinoculationwith3gofryeseedscolonizedbyR.solani.Theimageshows exampleofplantsfromwild-type(WT)andrepresentativetransgeniclinesharboringeitherp35S-bar/Ubi-Pen4-1(TG1)orp35S-bar/Ubi-AP24::Pen4-1 (TG3 and TG4) two weeks after the second pathogen inoculation. Transgenic plants exhibited much less sever disease symptom than wild-type controls.(b)AcloserlookofinfectedplantsshowingthedifferentlesionsizeofWTandTG.(c)Thedevelopmentofbrownpatchdiseasewasratedby visualestimationofthelesionpercentageoftheinfectedleaves14DPIusingtheHorsfall/Barrettscale.StatisticalanalysisofR.solaniinoculationtest wasconductedonWTandvariousTGlines.Dataarepresentedasmeans6SE(n=6),anderrorbarsrepresentstandarderror.Asterisks(**or*) indicateasignificantdifferencebetweentransgenicplantsandwild-typecontrolsatP,0.01orP,0.05byWilcoxontestJMP9.0.0.ThePvaluesare listedinTableS3. doi:10.1371/journal.pone.0024677.g005 PLoSONE | www.plosone.org 5 September2011 | Volume 6 | Issue 9 | e24677 DiseaseResistanceinTransgenicTurfgrass times after inoculation. Compared to control plants, transgenic economically and environmentally important perennial grass lines harboring either p35S-bar/Ubi-Pen4-1 or p35S-bar/Ubi- specieswithageneencodinganAMPfromtheclassfourisoform AP24::Pen4-1 all exhibited dramatically enhanced disease resis- oftheshrimppenaeidinfamilyforenhancedresistanceagainsttwo tancewithareductioninlesionlengthby40%to47%sevendays fungalpathogens.Therewasonlyonerecentstudyreportingthe afterinoculation(Figure6a,b).StatisticalanalysisofTukey’sHSD useofpenaeidinproteinforplantdiseaseresistance[63].Inthat indicated that the lesion size reduction in Pen4-1-expressing study,Np3andNp5,thetwoAMPsbelongingtoclass3and5of transgenic lines was significant (P,0.05), whereas no significant thepenaeidinfamilyfromChineseshrimp(Fenneropenaeuschinensis) difference in lesion size was observed among Pen 4-1-expressing [64,65] were engineered into rice and the four transgenic lines transgeniclines(P.0.05)(Figure6b). generatedwerereportedtoshowenhancedresistancetobacterial blight(Xanthomonasoryzae). Plant performance in response to S. homoeocarpa infection was Inthepresentstudy,thePen4-1genewithplant-preferredcodon furtherevaluatedbyinvivoassaysusingthewholeplantsgrownin usage was chemically synthesized for chimeric gene construction big pots. Control plants and transgenics harboring either p35S- and plant transformation. The 30 transgenic turfgrass lines bar/Ubi-Pen4-1 or p35S-bar/Ubi-AP24::Pen4-1 were both chal- constitutively expressing either the Pen4-1 gene (25) or the lenged by the pathogen in replicated experiments under a AP24::Pen4-1 fusion gene (5) all contained one or two copies of controlled environment. Pen4-1-expressing transgenic lines all the integrated transgene and were normal in morphology and exhibited high resistance against pathogen infection with disease development.Pen4-1expressionwasconfirmedatthetranscription ratings reduced more than 50% compared to various control level (Figure 1d). In planta disease resistance assays to compare plants9daysafterinoculation(Figure7a,b).Statisticalanalysisof transgeniclinesexpressingPen4-1andcontrolplantswithoutPen4-1 the Wilcoxon test indicated that disease development in all the for their response to two important turfgrass fungal pathogens Pen4-1-expressing transgenic lines was significantly delayed clearly demonstrated the effectiveness of this novel AMP in (Figure7b)andintherecoveryphase,transgeniclinesperformed rendering transgenic plants with significantly enhanced resistance muchbetterthancontrolplants(P,0.05).However,nosignificant tobothbrownpatchanddollarspotdiseases.Itisunlikelythatthe difference in disease resistance among Pen4-1-expressing trans- observed results would be attributed to disrupted genes or geniclineswasobserved(P.0.05)(Figure7b). regulatory sequences at the transgene integration site(s) since Pen4-1-expressing transgenic lines from independent transforma- Discussion tion events all show similar phenotypes and confer increased The results reported herein show that Pen4-1, one of the resistance to fungal pathogens, whereas transgenic control plants penaeidinproteinsisolatedfromAtlanticwhiteshrimp(Litopenaeus thatcontainthesameexpressionvector,butwithoutPen4-1,donot setiferus), when expressed in transgenic perennial grass plants, exhibit enhanced performance when subjected to pathogen confers antifungal traits. Transgenic creeping bentgrass plants infection.Italsoshouldbenotedthatinthecurrentresearch,T0 expressing Pen4-1 exhibited significantly enhanced resistance to transgenicplantswereclonallypropagatedandusedforpathoge- dollar spot and brown patch, the two major fungal diseases in nicity assays as previously reported in other studies on perennial turfgrasscausedbyS.homoecarpaandR.solanirespectively.Toour grasses [66]. Our earlier work studying transgene expression and knowledge, this is the first report of genetically engineering an transmission using the selectable marker, herbicide resistance Figure 6. Response of transgenic creeping bentgrass plants expressing Pen 4-1 to S. homoeocarpa infection - in vitro plant leaf inoculationassay.(a)Thedetachedsecondexpandedleavesfromthetopofplantstolonswereusedforpathogeninoculationtest.Theimage showsexampleofrepresentativeleavesfromalltestedPen4-1-expressingtransgenicplantswithasingletransgeneinsertion(TG,ontheright)and wild-type control plants (WT, on the left) 7 days post-inoculation (DPI). Transgenic plants exhibited significant resistance to S. homoeocarpa in comparisontowild-typecontrols.(b)Thedevelopmentofdollarspotdiseasewasratedbymeasuringthelesionlengthoftheinfectedleaves2,4and 7DPI.SignificantresistancetoS.homoeocarpabytransgenicplantswasobserved7DPIwhencomparedtowild-typecontrols.Statisticalanalysisof S.homoeocarpainoculationtestwasconductedonwild-typecontrolplants(WT)andvarioustransgeniclinesharboringeitherp35S-bar/Ubi-Pen4-1 (TG1 and TG2) or p35S-bar/Ubi-AP24::Pen4-1 (TG3and TG4). Dataare presentedas means 6 SE (n=10),and error bars represent standard error. Asterisks(**or*)indicateasignificantdifferencebetweentransgenicplantsandwild-typecontrolsatP,0.01orP,0.05byTukey’sHSDtestusing JMP9.0.0.ThePvaluesarelistedinTableS4. doi:10.1371/journal.pone.0024677.g006 PLoSONE | www.plosone.org 6 September2011 | Volume 6 | Issue 9 | e24677 DiseaseResistanceinTransgenicTurfgrass Figure7.ResponseoftransgeniccreepingbentgrassplantsexpressingPen4-1toS.homoeocarpainfection-invivodirectplant inoculation bioassays with higher dose of S. homoeocarpa. (a) The fully developed transgenic (TG) and wild-type (WT) plants clonally propagated from individual stolons were grown and maintained in pots (15cm610.5cm) and inoculated with 0.5g of rye seeds colonized by S.homoeocarpa.Theimageshowsexampleofplantsfromwild-type(WT)andrepresentativetransgeniclinesharboringeitherp35S-bar/Ubi-Pen4-1 (TG1)orp35S-bar/Ubi-AP24::Pen4-1(TG3)9daysafterpathogeninoculation(9DPI).Theplantsinthefrontrowareuninfectedcontrols.Transgenic plants exhibited significant disease resistance compared to wild-type controls. (b) The development of dollar spot disease was rated by visual estimationofthelesionpercentageoftheinfectedleaves3,5,7,9DPI,and21dayspost-recovery(DPR)usingtheHorsfall/Barrettscale.Statistical analysisofS.homoeocarpainoculationtestwasconductedonWTandvariousTGlines.Dataarepresentedasmeans6SE(n=6),anderrorbars representstandarderror.Asterisks(**or*)indicateasignificantdifferencebetweentransgenicplantsandwild-typecontrolsatP,0.01orP,0.05by WilcoxontestusingJMP9.0.0.ThePvaluesarelistedinTableS5. doi:10.1371/journal.pone.0024677.g007 conferring gene bar in creeping bentgrass had demonstrated that produced when Pen4-1 gene is introduced into the plant host Agrobacterium-mediatedtransformationofcreepingbentgrassledtoa genome,andthatitsbiologicalactivitymaintainedwhenproduced highfrequencyofasingle-copytransgeneinsertionthatexhibited intransgenicplants.AlthoughtheantifungalactivityofPen4-1has stable inheritance patterns. The inheritance and stability of been demonstrated by in vitro test of the synthesized protein [52, transgene were demonstrated in both greenhouse and field 53,57],itremainstobedeterminedwhetherornothigh-levelgene conditions[67–70].Currently,wearealsoconductingexperiments expression, efficient protein production, and correct folding or studying stable transmission of Pen4-1 into next generations and processingofthisproteincouldbeachievedinplanta.Wetherefore inheritanceoftheenhanceddiseaseresistancetraitbytheprogeny modified the coding sequence of Pen4-1 for monocot plant- of the primary transgenic plants. Data from this research will preferred codon usage. The same strategy has been used providefurthersupportfacilitatinglarge-scaleapplicationofPen4-1 previouslywhenintroducingothernon-plant-derivedAMPgenes inturfspeciesforplantprotection. in plants [32]. In our study, a RNA transcript of the codon- SincePen4-1originatesfromshrimp,thesuccessfuluseofthis optimized Pen4-1 gene was detected in all the transgenic lines. Although attemptsin detecting protein products ofthe Pen4-1 in proteininagriculturalbiotechnologyrequiresthatitbeefficiently PLoSONE | www.plosone.org 7 September2011 | Volume 6 | Issue 9 | e24677 DiseaseResistanceinTransgenicTurfgrass transgenic lines wereunsuccessful, transgenic plants all displayed TheinvitrotestsofPen4-1haverevealeditsresistancetoawide significantlyenhancedresistancetothetwomajorturfgrassfungal range of phytopathogens, of which many infect plant species diseasescomparedtowild-typecontrolsindicatingthatPen4-1was including rice, wheat, wine grapes, strawberry and other crop successfullyproducedintransformedcreepingbentgrassplants.It plants[52].Thecurrentstudywithcreepingbentgrassasatarget shouldbenotedthatnativeAMPgeneshavealsobeenreportedto speciesprovidestheveryfirstexampleofusingPen4-1forgenetic be expressed and function in other plant systems. For example, engineering of enhanced disease resistance in transgenic crop when introducing Aspergillus giganteus antifungal protein AFP into plants, pointing to the great potential of implementing similar rice plants, the translational efficiencies of transcripts originating strategies in other plant systems, especially in food crops for fromthenativeandcodon-optimizedAFPgeneappearedsimilar improvementofplantbioticstressresistance. in transgenic plants [32]. Similarly, the introduction of np3 and np5, two other AMP genes from Chinese shrimp in their native MaterialsandMethods formsintoriceledtoenhancedplantresistancetobacterialblight. SynthesisofPen4-1gene ThissuggestedtheproductionofactiveAMPsintransgenicplants The full sequence of Pen4-1 gene was obtained from PenBase althoughthepresenceoftheproteinproductsintransgenicplants [74]. The original nucleotide sequences of Pen4-1 encoding the wasnotdemonstrated[63].Furthertransgenicstudiestocompare maturePen4-1protein(47aminoacids)weremodifiedforplant- the original and codon-optimized forms of Pen4-1 gene for their optimizedcodonusage.Astopcodon(TAG)wasaddedtothe3’ proteintranslationefficiencieswouldfacilitateitsuseinotherplant endofthecodingsequence(Figure2).Themodifiedfullsequence systemstoachieveenhanceddiseaseresistance. of Pen4-1 was chemically synthesized by Integrated DNA Allpenaeidinspossessauniquetwo-domainstructureincluding Technology(Coralville,IA,USA),clonedinpZErO-2(Invitrogen, an unconstrained proline-rich N-terminal domain, PRD and a Carlsbad,CA,USA)andverifiedbysequencing. disulfide bond-stabilized cysteine-rich domain, CRD [57]. To ensureefficientdisulfidebondformationofthePen4-1producedin Constructionofplantexpressionvectors transgeniccreepingbentgrassplants,wepreparedachimericgene encodingafusionproteininwhichtheDNAsequencecodingfor TogeneratetransgenicplantsexpressingPen4-1andstudythe thetransitsignalpeptideofthesecretedtobaccoAP24proteinwas role Pen4-1 plays inplant disease resistance, twochimeric DNA translationallyfusedtothePen4-1codingsequence.Transitsignal constructs were prepared containing either the coding sequence peptides,suchastheonefromAP24,areknowntobecapableof of a single peptide Pen4-1 (Figure 1a) or the DNA sequence directing proteins into the endoplasmic reticulum, facilitating the coding for the transit signal peptide of the secreted tobacco formation of disulfide bonds [32]. Therefore, the AP24::Pen4-1 AP24 protein translationally fused to Pen4-1 coding sequence fusionproteinproducedinplantcellsshouldleadtomaturePen4-1 (Figure 1b).Bothconstructs wereprepared usingapSB11-based thatismorelikelytobecorrectlyfoldedthanthePen4-1protein Agrobacterium binaryvectorthatcontainsaselectablemarkergene aloneproducedintransgenicplants.However,inthepresentstudy, conferring antibiotic spectinomycin resistance for bacterial we did not observe dramatic differences in enhanced plant transformation [75]. resistancetothetwoturfgrassfungalpathogensbetweentransgenic The two plant expression vectors, p35S-bar/Ubi-Pen4-1 , and turfgrass lines expressing AP24::Pen4-1 fusion gene and those p35S-bar/Ubi-AP24::Pen4-1constructedinthisworkarepresented expressing Pen4-1 gene alone. One possible explanation could be in Figures 1a and b. Plasmid p35S-bar/Ubi-Pen4-1 (Figure 1a) that a minimal protein activity was enough to inhibit pathogen containedonlythesinglepeptidesequenceofthecodon-optimized gene, whereas plasmid infection;therefore,themethodsforpathogenicityassaysusedinthe Pen4-1 p35S-bar/Ubi-AP24::Pen4-1 (Figure 1b) contained a chimeric Pen4-1 gene with the DNA presentstudycouldnotdetecttherealdifferenceinproteinactivities sequence coding for the transit signal peptide of the secreted between the Pen4-1 alone and the AP24::Pen4-1 fusion protein expressed in transgenic plants. Another possibility would be that tobacco AP24 protein [76] being translationally fused to Pen4-1 although the mechanism of protein secretion is highly conserved codingsequence.Fortheirexpressioninturfgrass,bothPen4-1and throughthelivingworld[71],signalpeptidesfromoneorganismdo AP24::Pen4-1wereclonedbetweenthemaizeubipromoterandthe notalwaysfunctionefficientlywhenexpressedinanotherorganism nosterminator.Anherbicideresistanceconferringgenenamedbar [71–73].Thecorrectchoiceofthesignalpeptidewouldhaveagreat drivenbytheCaMV35Spromoterwasincludedinbothplasmids effectontheproductionoftheAMPs. asselectablemarkerforplanttransformation. Themulti-domainstructureandthefeatureofproteaseresistance Topreparep35S-bar/Ubi-Pen4-1,thesynthesizedPen4-1coding of this peptide may also play important role in bestowing more sequence (with added stop codon) was PCR amplified from flexibility to protein processing and determining protein activities pZErO-2:Pen4-1byprimersPen4-ATG:59-CGCGGATCCATG- [52].InmostcasesthepresenceofboththeCRDandthePRDare CACTCCTCCGGCTACACC-39(aBamHIrestrictionsiteanda importanttoconferthemaximalantimicrobialactivity.However,it start codon, ATG added in the 59 end were underlined and in hasbeendemonstratedthatthesinglePen4PRDaloneexhibiteda italic respectively) and Pen4R: 59-CGCGCATGCGAGCTCTA- similarlevelofantimicrobialactivitytothatofthefull-lengthPen4 GAGGTGGCAGCAGTCG-39 (an SphI and an Sac I restriction [53,57].ThisimpliesthatthedisulfidebondformationinPen4may sitesaddedinthe59endwereunderlined).Theamplifiedfragment notplayacriticalroleinitsantimicrobialability.Therefore,specific was digested with BamHI and SacI enzymes and ligated into the targetingofPen4-1toendoplasmicreticulumbytheAP24signal corresponding sites of p35S-bar/Ubi-GUS (Luo, unpublished peptidedidnotseemtoresultinenhancedproteinactivity.Itisalso results) to replace the gusA coding sequence. To prepare p35S- possiblethatpathogenattackwouldleadtodisruptionoftheplant bar/Ubi-AP24::Pen4-1 construct, the PCR amplified fragment of cells, releasing the peptides, which could then be oxidized in thePen4 -1 using Pen4F (59-CACTCCTCCGGCTACACC-39 ) and extracellular space to form the disulfide bond. Further studies Pen4R primers was treated with DNA polymerase I, large comparingPen4PRDaloneandthefull-lengthPen4intransgenic (Klenow) fragment (New England Biolabs, Beverly, MA, USA) plantsfortheirantimicrobialactivitieswouldhelpbetterunderstand intheabsenceofdNTP,thendigestedwithSphIandligatedinto theroledisulfidebondsplayindeterminingtheoverallactivityof theNcoI(blunt-endedwithKlenowinthepresenceofdNTP)-SphI Pen4proteins. sites of the plasmid pGEM-T-AP24 [32], resulting in pGEM-T- PLoSONE | www.plosone.org 8 September2011 | Volume 6 | Issue 9 | e24677 DiseaseResistanceinTransgenicTurfgrass AP24::Pen4-1 (data not shown). Upon verification of the correct with 4 volumes of acetone at 220uC for 30min. Proteins were sequence of the amplified Pen4-1 and its in-frame fusion to the pelleted, dried and dissolved in SDS-PAGE loading buffer AP24 signal sequence, the AP24::Pen4-1 chimeric gene was containing5%2-mercaptoethanol.Forproteinanalysis,30m gof released from pGEM-T-AP24::Pen4-1 by BamHI and SacI protein sample was loaded onto a 16% Tricine SDS-PAGE gel. digestions and ligated into the corresponding sites of p35S-bar/ SDS-PAGE was performed as described previously [79]. For Ubi-GUStoreplacethegusAcodingsequence.Thetwoconstructs western blot, protein was transferred from the SDS-PAGE gel weretransformedintoAgrobacteriumtumefaciensstrainLBA4404by onto Protran BA76 Nitrocellulose media (Whatman Inc., Piscat- electroporationforsubsequentplanttransformation. away,NJ,USA)usinganelectrophoresisblottingsystem(Bio-Rad, Hercules,CA,USA).TheProteintransferefficiencywasverified byusingPoceauS,andincubatedwith5%carnationnonfatdry Production,propagationandmaintenanceoftransgenic milk in TBST overnight. The blots were then probed with anti- turfgrassplants Pen4-1antibodydevelopedbyYenZymAntibodies,LLC(Burlin- A commercial genotype of creeping bentgrass (A. stolonifera L.) game, CA, USA), using the peptide HSSGYTRPLRKP- PennA-4wasusedforplanttransformation.Transgeniccreeping SRC, followed by adding the HRP-conjugated goat anti-rabbit bentgrasslinesharboringeitherp35S-bar/Ubi-Pen4-1orp35S-bar/ IgGsecondaryantibody(JacksonImmunoResearchLaboratories, Ubi-AP24::Pen4-1wereproducedbyAgrobacterium-mediatedtrans- Inc., West Grove, PA, USA) and incubation for 1hour at room formation of embryonic callus initiated from mature seeds temperature with shaking. The signals were detected by essentially as previously described [67]. Transgenic plants were incubation of membrane for 30 minutes at room temperature in grownincommercialpottingmixturesoil(Fafard3-BMix,Fafard the substrate working solution (4-Chloro-1-naphthol). After Inc., Anderson, SC, USA) and maintained in the greenhouse stopping the reaction by rinsing the membrane with water, the undera16-hourphotoperiodwithsupplementallightingat27uC membranewasphotographedimmediately. in the light and 25uC in the dark. Plants from individual trans- formationeventswereclonallypropagatedfromstolonsandgrown InvitroplantleafinoculationwithR.solaniand inpots(15cm610.5cm,DillenProducts,Middlefield,OH,USA) usingcommercialpottingmixturesoilaspreviouslydescribed[70]. S.homoeocarpa Propagated plants were maintained in greenhouse for 4 to 6 Transgenic plants were challenged with R. solani and S. monthswithregularfertilization,mowingandirrigation,andused homoeocarpa,whichrespectivelycausebrownpatchanddollarspot, forfurtheranalysis. the two most common fungal diseases in creeping bentgrass. Followingproceduresmodifiedfromthepreviousreports[43,80– 82], we grew the R. solani and S. homoeocarpa cultures on potato PlantDNAisolationandsouthernblotanalysis PlantgenomicDNAwasextractedaspreviouslydescribedusing dextroseagarat25uCfor3dayspriortoinoculationofdetached leavesunderasepticconditions.Thesecondexpandedleavesfrom the cetyltrimethyl ammonium bromide (CTAB) method [69]. thetopofplantstolonswereusedforinoculation.Tenleavesfrom After digestion of the DNA with BamHI according to supplier’s each of the transgenic lines and wild-type control plants were instruction (New England Biolabs, Beverly, MA, USA), DNAs randomlychosenforstudy.Theleavescutfromplantswerefirst wereelectrophoresedon0.8%agarosegels,transferredontonylon washed with 70% ethanol, and then rinsed with sterilized water. membranes (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, Theleaveswereputon1%ofagarinPetridishes(150615mm). USA), and hybridized to 32P-labelled DNA probes of bar. Anagarplug(d=3mm)infestedwithmyceliumofR.solaniorS. Hybridization was carried out in modified Church and Gilbert homoeocarpa was placed on the bottom of the midrib of each buffer at65uCfollowingthestandardprotocol[77].Hybridizing detached leaf for inoculation. The Petri dishes were put in a fragments were detected by exposure of the membrane on a lighted growth chamber under a 14/10h (day/night) photoperi- phosphor screen at RT overnight, and scanning on a Typhoon od. Temperature and relative humidity (RH) in the growth 9400phosphorimager. chamberwere28uCand70%.Thedevelopmentofbrownpatch disease was rated by measuring the lesion length of the infected RNAisolationandnorthernblotanalysis leaves 2 days, 8 days and 14 days post-inoculation. The develo- TotalRNAwasisolatedfromtheleavesoftransgenicandwild- pment of dollar spot disease was rated by measuring the lesion typecontrolplantsusingTrizolreagent(Invitrogen).RNAswere length on the infected leaves 2 days, 4 days and 7 days post- subjectedtoformaldehyde-containingagarosegelelectrophoresis, inoculation.Theexperimentwasrepeatedthreetimes. and transferred onto Hybond-N+ filters (GE Healthcare Bio- Sciences Corp.). The DNA fragment coding for the Pen4-1 gene InvivodirectplantinoculationwithS.homoeocarpaand was used as probe. Hybridization and membrane wash were performedfollowingthestandardprotocol[77]. R.solani ThepreparationoftheS.homoeocarpaandR.solaniculturesand theinvivoplantinoculationwithpathogenswereconductedbased WesternblotAnalysis Total proteins were extracted from leaves following two onthepreviouslyreportedprocedures[3,83,84].SelectedPen4-1- expressing transgenic lines based on molecular analysis were different procedures. The first was essentially as described by Fu evaluated for resistance to the infection of the two fungal et al. [78]. Two hundred mg of leaf tissue was ground, then pathogens in comparison to control plants that did not contain suspendedin500mlofproteinextractionbuffer[16PBS(pH7.4), 10mMEDTA,1mMPMSF,6mlproteaseinhibitorcocktailfor Pen4-1.Thegrassesweremowedpriortoinoculation.Theplants plantcellandtissueextracts(sigma),1%(v/v)b-mercaptoethanol, ineachpotweretheninoculatedwithpathogensbyapplying,in and 0.1% (v/v) Triton X-100]. The second protocol was as thecenterofthepot,approximately0.5gofryeseedscolonizedby S.homoeocarpaor3and6gofryeseedscolonizedbyR.solani. describedbyCocaetal.[32].Fourhundredmgofplantmaterial Plants inoculated with S. homoeocarpa (0.5g of colonized was ground in liquid nitrogen and homogenized in SDS PAGE loadingbufferwithout2-mercaptoethanolandincubatedat95uC inoculum)wereplacedinplastictrayscontaining4cmofdistilled for10min.Aftercentrifugation,thesupernatantwasprecipitated water, lightly misted with distilled water at 48h intervals to PLoSONE | www.plosone.org 9 September2011 | Volume 6 | Issue 9 | e24677 DiseaseResistanceinTransgenicTurfgrass maintain relative 100% humidity. The plastic trays were placed SupportingInformation inside a greenhouse set to maintain a diurnal cycle of 14h light TableS1 Pvaluesofinvitroplantleafinoculationassay and10hdark.Threetofourreplicatesofeachtransgeniclineor withR.solani. wild type control were used for evaluation. Disease severity was (DOCX) visuallyestimatedat3,5,7and9dayspost-inoculationusingthe Horsfall/Barrettscale[85].Ninedayslater,theplantsweremoved Table S2 P values of in vivo direct plant inoculation toagrowthroomfromthegreenhousetorecoverforthreeweeks. bioassayswithlowerdoseofR.solani. Temperaturesinthegrowthroomweremaintainedat22uCinthe (DOCX) light and 17uC in the dark. The inoculation experiment was Table S3 P values of in vivo direct plant inoculation repeatedthreetimes. bioassayswithhigherdoseofR.solani. PlantsinoculatedwithR.solani(3gryegrassseedscolonizedby (DOCX) the pathogen) were placed in plastic trays containing 4cm of TableS4 Pvaluesofinvitroplantleafinoculationassay distilledwater,lightlymistedwithdistilledwaterat48hintervals withS.homoeocarpa. to maintain humidity. The trays were placed inside a growth (DOCX) chambertomaintainadiurnalcycleof14hlightand10hdark. ThetemperatureandRHwere30uCand70%duringdaytime, Table S5 P values of in vivo direct plant inoculation and 24uC and 95% at night. After 14 days, disease severity was bioassayswithS.homoeocarpa. either rated by measuring the total distance from the point of (DOCX) inoculationtothefarthestpointofthelesionsextended,orvisually estimated using the Horsfall/Barrett scale [85]. The inoculation Acknowledgments experimentwasrepeatedtwice. WethankDr.BlancaSanSegundoforthegiftoftheplasmidpGEM-T- AP24, Dr. Martin Bruce and Dr. Hanafy Fouly for providing Sclerotinia Statisticalanalysis homoecarpaandRhizoctonisolanicultures,andforassistanceinpathogenicity Both in vitro plant leaf inoculation and in vivo direct plant assays, Dr. Guido Schnabel and Dr. William R. Marcotte for helpful inoculation tests were conducted using a randomized complete discussions and Dr. Gregory L. Reighard for critically reading the block design. Data were analyzed using JMPH 9.0.0 (2010 SAS manuscript. 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