G C A T genes T A C G G C A T Article Molecular Cloning, Recombinant Expression and Antifungal Activity of BnCPI, a Cystatin in Ramie (Boehmeria nivea L.) YongtingYu1,*,† ID,GangZhang2,†,ZhiminLi1,YiCheng1,ChunshengGao1,LiangbinZeng1, JiaChen1,LiYan1,XiangpingSun1,LitaoGuo1andZhunYan1,* 1 InstituteofBastFiberCropsandCenterforSouthernEconomicCrops,ChineseAcademyof AgriculturalScience,Changsha410205,China;[email protected](Z.L.);[email protected](Y.C.); [email protected](C.G.);[email protected](L.Z.);[email protected](J.C.);[email protected](L.Y.); [email protected](X.S.);[email protected](L.G.) 2 CollegeofPharmacyandShaanxiProvincialKeyLaboratoryforChineseMedicineBasis& NewDrugsResearch,ShaanxiUniversityofChineseMedicine,Xi’an712406,China; [email protected] * Correspondence:[email protected](Y.Y.);[email protected](Z.Y.);Tel.:+86-731-8899-8585(Y.Y.) † Theseauthorscontributedequallytothiswork. Received:30July2017;Accepted:3October2017;Published:11October2017 Abstract: Phytocystatinsplaymultiplerolesinplantgrowth,developmentandresistancetopests andotherenvironmentalstresses. Aramie(BoehmerianiveaL.)phytocystatingene,designatedas BnCPI,wasisolatedfromaramiecDNAlibraryanditsfull-lengthcDNAwasobtainedbyrapid amplificationofcDNAends(RACE).Thefull-lengthcDNAsequence(691bp)consistedofa303bp openreadingframe(ORF)encodingaproteinof100aminoacidswithdeducedmolecularmassof 11.06kDaandatheoreticalisoelectricpoint(pI)of6.0. ThealignmentofgenomeDNA(accession No. MF153097) and cDNA sequences of BnCPI showed that an intron (~104 bp) exists in the codingregion. TheBnCPIproteincontainsmostofthehighlyconservedblocksincludingGly5-Gly6 at the N-terminal, the reactive site motif QxVxG (Q49V50V51S52G53), the L79-W80 block and the [LVI]-[AGT]-[RKE]-[FY]-[AS]-[VI]-x-[EDQV]-[HYFQ]-N(L22G23R24F25A26V27D28D29H30N31)block thatiscommonamongplantcystatins. BLASTanalysisindicatedthatBnCPIissimilartocystatins from Glycine max (77%), Glycine soja (76%), Hevea brasiliensis (75%) and Ricinus communis (75%). TheBnCPIwassubclonedintoexpressionvectorpSmart-IandthenoverexpressedinEscherichiacoli BL21 (DE3) as a His-tagged recombinant protein. The purified reBnCPI has a molecular mass of 11.4kDadeterminedbysodiumdodecylsulfatepolyacrylamidegelelectrophoresis(SDS–PAGE). Purified reBnCPI can efficiently inhibit the protease activity of papain and ficin toward BANA (Nα-benzoyl-L-arginine-2-naphthyamide),aswellasthemyceliumgrowthofsomeimportantplant pathogenic fungi. The data further contribute to our understanding of the molecular functions ofBnCPI. Keywords: BoehmerianiveaL.;BnCPI;cysteineproteaseinhibitors;intron;functionalexpression 1. Introduction Cysteineproteaseinhibitorsorcystatinsofplantoriginarecalledphytocystatins;theydisplay multiple functions in seed formation and germination, plant growth and development, as well as plantresistance/tolerancetobiotic/abioticstresses[1–3]. Foralmost30years,phytocystatinsandtheir rolesinplant–pestinteractionsattractedextensiveinternationalconcernandstudy. Adeeperand morenuancedunderstandingofphytocystatinswascreatedfromtheincreasingamountofdataand relatedresearch. Somephytocystatinscandirectlyimpairherbivoregrowth,asevidencedbyimpaired Genes2017,8,265;doi:10.3390/genes8100265 www.mdpi.com/journal/genes Genes2017,8,265 2of15 growthofthesepestswhenrearedonanartificialdietcontainingcystatinsortransgenicplantswith exogenous cystatin genes [4–6]. Cystatins of rice, maize and taro can increase plant resistance to variousphytopathogenicnematodes[7–9]. Manyphytocystatinscanalsodirectlyinhibitthegrowthof phytopathogenicfungi[2]. Somephytocystatins(suchasCeCPI,HvCPI-1)withantifungalactivity alsoinhibitthegrowthofnematodesandherbivores[7,10–12]. Transgenictobaccoover-expressing potatosporaminandtarocystatindisplayedincreasedresistancetobothinsectsandphytopathogens, including Helicoverpa armigera, Erwinia carotovora and Pythium aphanidermatum [13]. These reports supplydirect,substantialevidenceforthepotentialofphytocystatinsinimprovingplantresistance. The inhibition mechanism of phytocystatins on different pests has also been explored. Cysteine protease (CP) is a major digestive enzyme in many herbivores and nematodes; it plays an important role in reproduction, development, tissue invasion, pathogenesis and immune invasion [14–17]. The harmful effect of phytocystatins on nematodes and herbivores was thought to inhibit the activity of CP of these animals. However, the mechanism of growth inhibition of phytocystatinsonfungiisstillunclear. Someresearchershaveproposedthatitblocksindigenous proteinaseactivityoffungi[11,18],whereasothersfoundthattheinhibitionisnotassociatedwithits cysteineproteinaseinhibitoryproperties[10].Thesediscoveriesindicatethattheinhibitionmechanisms ofdifferentphytocystatinstowardfungiaredifferent. Ramie (Boehmeria nivea L.) is a perennial herbaceous plant of the family Urticaceae. It is also called “China grass” since it has been cultivated in China for over 6000 years [19]. Ramie is an importantnaturalfibercropplantedmainlyinChina,IndiaandotherSoutheastAsianandPacific Rim countries [20]. Traditionally, ramie was planted solely for harvesting bast fibers. Recently, ramie leaves and shoots have also been used as fodder for beef cattle and geese because of the plant’s high crude protein content [21]. Root lesion disease (RLD), a destructive root disease that iscausedbythenematodePratylenchuscoffeae,severelyimpairsthegrowthandyieldoframie[22]. The lack of knowledge on ramie RLD resistance mechanisms or genes severely hinders efforts in effectively breeding and utilizing nematode-resistant ramie. More recently, a ramie cystatin gene BnCPI (Cysteineproteaseinhibitor, Unigene11292)wasfoundtoberegulatedinP.coffeae-infected resistant ramie but not in a susceptible cultivar, which suggested that BnCPI may be involved in pestresistance[23]. InordertodecipherthebiologicalfunctionofBnCPI,itisveryessentialtoclone andcharacterizethegeneandactiveprotein. Thisstudyreportedthemolecularcloning,sequence analysis,recombinantexpressionandbiochemicalandantifungalactivitystudyofthisgeneandits correspondingprotein. 2. MaterialsandMethods 2.1. PlantGrowthandSampling Ramie varieties Qingdaye (QDY), Zhongzhu No.1 (ZZ1) and Heipidou (HPD), which were resistant,moderatelysusceptible,andsusceptibletoP.coffeae,respectively,wereusedinthisstudy. Seedlingswerepreparedwiththecuttingpropagationmethod[23]. Twoweekslater,whentheroots were about 20 cm length, roots of a single plant were sampled and immediately frozen in liquid nitrogenandstoredat−80◦C. 2.2. DNAandRNAExtractionandcDNASynthesis Roottissuesoframieina2mLEppendorftube(pre-chilledinliquidnitrogen)weregroundto finepowderusingaTissuelyser-24Multi-SampleTissueGrinder(ShanghaiJingxing,Shanghai,China). TotalDNAandRNAwereextractedusingaPlantDNAMiniKitandanEASYspinPlusTotalRNA Kit (both from Aidlab, Beijing, China), following the manufacturer’s protocol. A Nanodrop 2000 spectrophotometer(ThermoFisherScientific,Waltham,MA,USA)wasusedtomeasureDNAand RNAconcentration,andfirst-strandcDNAsamplesweresynthesizedfromabout1µgofthetotal RNAusingaRevertAidFirstStrandcDNASynthesisKit(ThermoFisherScientific). Genes2017,8,265 3of15 2.3. MolecularCloningoftheBnCPIGene A787-bpcDNAfragmentoftheBnCPIgenewasobtainedfromtranscriptomesequencingdata oframie[23]. Thesequencescontainedanopenreadingframe(ORF)-encodedramiehomologofthe cysteine protease inhibitor. Based on this sequence, 3(cid:48)-RACE (rapid amplification of cDNA ends) was performed to amplify the 3(cid:48)-end of this gene using a SMART™ RACE cDNA Amplification Kit(Clontech,MountainView,CA,USA)accordingtothemanufacturer’sguidelines. Twointernal gene-specificforwardprimers,cpi3-1(5(cid:48)-GATGGCGGTGTCAAGAAGGTTTACGA-3(cid:48))andcpi3-2 (5(cid:48)-AAGGTCTGGGAAAAGTTGTGGTTGAA-3(cid:48)),wereassignedfromtheknowncDNAfragment ofBnCPI.Briefly,first-strandcDNAwassynthesizedwith3(cid:48)-CDSprimerA,andthenamplifiedby PCR using cpi3-1 as a forward primer and UPM (Universal Primer A mix) as the reverse primer. Subsequently,theproductofthefirstroundofPCRwasdiluted50times,andusedasatemplatein thesecond-roundPCRamplificationusingcpi3-2andUPM.ThePCRproductswereseparatedon1% agarosegels,purified,clonedintopMD-18vectorandsequencedbyShanghaiSangonBiotechnology Co. Ltd. (Shanghai, China). Theresultingfull-lengthcDNAofBnCPI wasdepositedintheNCBI (NationalCenterforBiotechnologyInformation)GenBankwithaccessionnumberKT438742.1. 2.4. SequencesAnalysisandPhylogeneticAnalysis TheonlineORFfinderprogramofNCBI(https://www.ncbi.nlm.nih.gov/orffinder/)wasused tosearchtheentireORFsequencesofBnCPI,whiletheonlineBLASTprogramofNCBIwasusedto searchproteinhomology. Theoreticalmolecularweight(MW)andisoelectricpoint(pI)forBnCPIwere predictedusingtheExPASytool(http://web.expasy.org/compute_pi/);signalpeptidepredictionwas performedusingSignalP4.1(http://www.cbs.dtu.dk/services/SignalP/);andsecondarystructure waspredictedusingSOPMA[24]. TheconserveddomainwassearchedagainsttheNCBIConserved DomainDatabase[25]. Asetofprimers(cpif: 5(cid:48)-CGCAGAAAAGTAAAAGCA-3(cid:48) andcpir: 5(cid:48)-TCCACCAAAGAC GAA TGA-3(cid:48)) was assigned for PCR amplification of ORF fragments in different ramie cultivars. Primerscpifandcpirwerelocatedupstreamofthestartcodonanddownstreamofthestopcodonof theORF,respectively. PCRwasperformedinanETC-811PCRinstrument(Eastwin,Beijing,China). Thereactionmixturecontained25µLof2×TaqPCasterMix(Aidlab,Beijing,China),4µLofeach primer(10µmol/µL),2µLofgenomicDNAorcDNA(10µg/µL),andthetotalvolumewasadjusted to 50 µL with ddH O. The PCR amplification procedure consisted of 94 ◦C for 4 min, 35 cycles of 2 denaturationat94◦Cfor45s,annealingat50◦Cfor1min,andextensionat72◦Cfor1min,with final extension at 72 ◦C for 10 min. The PCR products were separated by electrophoresis on 1.5% agarosegelsandvisualizedbystainingwithGoldView(Applygen,Beijing,China). Ampliconswere sequenced by Tsingke Company (Tsingke, Beijing, China). BnCPI and phytocystatins from other plantsdownloadedfromtheNCBIwebsitewerealignedusingMEGA6.0software[26]. Theresulting alignmentwastrimmedandaphylogenetictreewasconstructedusingtheneighbor-joiningmethod with100bootstrapping. ThededucedaminoacidsequenceofBnCPIwasalsoalignedwiththoseof otherphytocystatinsavailableintheEMBL-EBIdatabaseClustalW2(http://www.ebi.ac.uk/Tools/ msa/muscle/),setatdefaultparameters. 2.5. ExpressionofaRecombinantBnCPIinEscherichiacoli TheORFofBnCPIcontainsmanyrarecodons(FigureS1a)thatwillimpairexpressionefficiency inarecombinantexpression. Genecodonoptimizationwasperformedbysynthesizingsequences encoding amino acids identical to those encoded by BnCPI but without rare codons (Figure S1b). ORFgenesynthesiswasperformedbytheTsingkeCo. (Tsingke,Beijing,China). Theoptimizedgene fragmentwasclonedintotheexpressionvectorpSmart-I(GeneralBiosystems,USA),whichcarried 6×His-tagandSUMOtofacilitatesolutionandpurificationoftherecombinantexpressedprotein. TherecombinantvectorwassubsequentlytransformedintoEscherichiacoliBL21(DE3). Positiveclones Genes2017,8,265 4of15 containingtherecombinantvectorwerescreenedonampicillin. Cellsofpositiveclonesweregrown at 37 ◦C in LB liquid medium with 220 rpm until OD600 reached 0.6–0.8. Subsequently, IPTG (isopropyl β-D-1-thiogalactopyranoside) was added to a final concentration of 0.5 mM to induce expressionofBnCPIat37◦Cfor4horat15◦Cfor16h. After culture, cells were collected by centrifugation, suspended in 25 mM Tris buffer (containing300mMNaCl,pH8.0),andsonicatedbyanultrasonicprocessorfor2min.Thefragmentized cell suspension was centrifuged at 12,000 rpm at 4 ◦C for 15min. The resultant suspension and precipitate were separated with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE)tochecktherecombinantexpressedBnCPI(reBnCPI).Then,recombinantproteinwas treatedwithSUMOproteasetoremovetheSUMOproteinandpurifiedbyaNi–NTAaffinitycolumn (GEHealthcare,Russellville,AR,USA)usinganÄKTA-primesystem(GEHealthcare). 2.6. ProteaseInhibitoryActivityAssay CysteineproteaseinhibitoryactivityofreBnCPItowardpapain(EC3.4.22.2),ficin(EC3.4.22.3) andbromelain(EC.3.4.22.32)(allfromSigma-Aldrich,St. Louis,MO,USA)weretestedusingamethod describedpreviously[15]. TheinhibitoryactivityofreBnCPIwasrecordedasresidualenzymeactivity in the presence of inhibitor. In the control treatment, equal volumes of the corresponding buffer wereusedinsteadofreBnCPI.Therewerethreereplicatesineachtreatmentandtheexperimentwas repeatedtwice. 2.7. AntifungalActivityAssayofreBnCPI Two pathogens of the ramie plant, Pythium vexans causing brown root rot [27] and Alternaria alternatacausingleafspot[28],aswellastwootherimportantplantpathogens(Fusariumoxysporum andBotrytiscinerea,isolatesofwhicharekeptinourlaboratory),wereusedforthegrowthinhibition assay. The invitro growth inhibition assays were performed as described by Pernas et al. [29]. Fungalstrainsweregrownonpotatodextroseagar(PDA)mediumfor3–7days. ForF.oxysporum, A. alternata and B. cinerea, spores were collected with 1/3 PDB (PDA medium without agar) and dilutedtoaconcentrationof104 spores/mL.ForP.vexans,hyphaewerecollectedwith1/3PDBin a2-mLEppendorftube,homogenizedanddilutedtoaconcentrationof104CFU/mL.ThereBnCPI (after filtration sterilization) was added to the suspension to produce a final concentration of 20–80 µg/mL. Fungicides carbendazim, jinggangmycin, fluazinam and mefenoxam were added tothesuspensionsofF.oxysporum,A.alternata,B.cinereaandP.vexans(finalconcentration50µg/mL), respectively,toserveaspositivecontrols. FungalsuspensionswithoutfungicidesandreBnCPIwere usedasnegativecontrols,andbuffer(25mMTris,300mMNaCl,pH8.0)usedtodissolvereBnCPIwas addedinsteadofreBnCPIorfungicides. Then,sporesorhyphaesuspensionof200µLwerecultivated on a sterile 96-well microtiter plate at 25 ± 1 ◦C for 48 h. Fungal growth was then monitored by measuringabsorbanceat492nm(Infinit200Pro,Tecan,Männedorf,Switzerland)andcheckedunder aNikonAZ100microscope(NikonCo.,Tokyo,Japan). Resultswereexpressedasthepercentageof relative growth in the absence of the reBnCPI. Each treatment was replicated three times, and the experimentwasrepeatedtwice. 3. Results 3.1. SequenceAnalysis ThecDNAsequenceoftheBnCPIgenewas691bpinlength,including123bpof5(cid:48) untranslated region(UTR),303bpofORFand265bpofthe3(cid:48) UTR.TheORFlikelyencodedadeduced100-residue aminoacid(AA)sequence(Figure1). Thematureproteinhadamolecularweightof11.06kDaand pIof6.0,aspredictedbytheExPASytool. NosignalpeptidewasfoundwhentheAAsequencewas analyzedwithSignalPsoftware.Aconserved-domaindatabase(CDD)searchshowedthatthededuced BnCPIproteinsequencewasidenticaltotheconservedcystatin-likedomain(CYdomain: cd00042). Genes2017,8,265 5of15 BnCPIcontainedmostofthehighlyconservedblocksthatareessentialforcysteineproteinaseactivity, Ginencelsu 2d0i1n7,g 8,t h26e5 G G(Gly5-Gly6),thereactivesitemotifQxVxG(Q49V50V51S52G53),theWresidue5(W of 8104) andthehighlyconserved[LVI]-[AGT]-[RKE]-[FY]-[AS]-[VI]-x-[EDQV]-[HYFQ]-Nblock(L22G23R24 (FF2i5gAu2r6eV 12)7. DTh28eD s2e9cHon30dNar3y1 )s(tFruigcuturere1 )o.fT thhee sceocdoendd aprryotsetirnu cmtuarienloyf cthoensciosdteedd opfr oαt-ehienlimceasi nalnydc orannsdisotemd coofilαs- h(Feilgicuerse aSn2da)r.a Tnhdeo mterctoiairlsy (sFtirguucrteurSe2 ap)r.edTihcetiotenr toiaf rtyhest rcuocdteudr epproretediinc tiionndiocafttehde tchoadt etdhep rsoptaecine sintrduicctautreed mthaaitntlhy ecospnataciensetdru αc-thuerleicmesa,i nβl-ytucronnst aaninde dβ-αsh-heeeltisc e(Fs,igβu-rtue rSn2sba).n dβ-sheets(FigureS2b). FFiigguurree 11.. NNuucclleeoottiiddee aanndd ddeedduucceedd aammiinnoo aacciidd sseeqquueenncceess ooff tthhee idideennttiiffiieedd ffuullll--lleennggtthh ccDDNNAA ooff BBnnCCPPII. . NNuucclleeoottiiddee iinn bboolldd tteexxtt iinnddiiccaatteess tthhee ssttaarrtt aanndd ssttoopp ccooddoonnss.. SSeevveerraall mmoottiiffss ooff pphhyyttooccyyssttaattiinnss wweerree ddeetteecctteedd iinn tthhiiss sseeqquueennccee,, ssuucchh aass GGGG,, LLAARRFFAAVV,,Q QVVVVSSGGa annddL LWW..T Thheer erespspecetcitviveem mootitfisfsa raereb booxexded.. Both the genome DNA and the cDNA sequences of BnCPI were amplified from different ramie BoththegenomeDNAandthecDNAsequencesofBnCPIwereamplifiedfromdifferentramie cultivars using the primers cpif and cpir (Figure 2a). Alignment of cDNA and the genome DNA cultivars using the primers cpif and cpir (Figure 2a). Alignment of cDNA and the genome DNA sequences indicated the presence of a 104 bp intron between the sequence encoding the LARFAV sequencesindicatedthepresenceofa104bpintronbetweenthesequenceencodingtheLARFAVmotif motif and the reactive site QxVxG (Figure 2b). The ORF sequences of BnCPI from the cultivars QDY andthereactivesiteQxVxG(Figure2b). TheORFsequencesofBnCPIfromthecultivarsQDYand and HPD were found to be identical, whereas two nucleic acids (positions 225 and 236) were HPDwerefoundtobeidentical,whereastwonucleicacids(positions225and236)wereidentifiedin identified in cultivar ZZ1 that differed from those in QDY and HPD (Figure 3a), while the encoded cultivarZZ1thatdifferedfromthoseinQDYandHPD(Figure3a),whiletheencodedaminoacids amino acids were identical (Figure 3b). wereidentical(Figure3b). (a) M 1 2 3 4 5 6 )) Genes2017,8,265 6of15 Genes 2017, 8, 265 6 of 14 (a) M 1 2 3 4 5 6 1337 bp 525 bp (b) FFigiguurere2 2.. ((aa)) EElelecctrtroopphhooreressisis ofo fPCPRC Ramapmlipflicifiactiaotnio fnrafgrmagemntesn otsf BonfCBPnIC fProImfr ocDmNcAD NanAd gaenndomgeen DomNeA DoNn A1.5o%n a1g.5a%rosaeg agreol.s Meg, eDl.NMA ,mDaNrkAerm IIaI;r k1‒e3r, IfIrIa;g1m-3e,nfrtas gammepnlitfsieadm fprolimfie DdNfrAo mof DZZN1A, HoPfDZZ a1n,dH QPDDY arnedspQecDtiYverleys;p e4c‒t6i,v eflrya;g4m-6e,nftrsa gammepnlitfsieadm fprloifime dcDfrNomA coDfN ZAZ1o,f ZHZP1D, HaPnDd aQnDdYQ, DrYes,preecstpivecetliyv; el(yb;) (bA)liAgnlimgnemnte onft goefngomeneo DmNeAD N(aAcce(sascicoens snioo.n MNFo1.53M09F71) 5a3n0d9 7cD) aNnAd (caDccNesAsio(anc nceos. sKioTn43N87o4.2)K sTe4q3u8e7n4c2e)s soeqf uBennCcPeIs forfoBmn rCaPmIifer o(cmulrtaivmaire. Q(cuDlYti)v. aTr.hQe sDhYa)d.eTdh neushclaedice dacnidusc lienidciaccaitdes ainn dinictraoten aonf 1in0t4r obnp oinf 1le0n4gbtph. inThleen sgetqhu.eTnhceesse eqnuceondciensge tnhceo dLiAngRFthAeVL mARoFtiAf Vanmd otthief arneadctthiveer esiatcet iQvexVsixteGQ axreV xuGndaerreliunnedde arnlidne ddoaunbdle duonudbelerluinnedde, rrleinspedec,trievseplyec. tively. Genes2017,8,265 7of15 Genes 2017, 8, 265 7 of 14 (a) (b) Figure 3. (a) Alignment of cDNA and (b) deduced amino acid of BnCPI from three ramie cultivars, Figure3. (a)AlignmentofcDNAand(b)deducedaminoacidofBnCPIfromthreeramiecultivars, QDY, HPD and ZZ1. Two nucleic acids (positions 225 and 236) in ZZ1 differ from QDY and HPD QDY,HPDandZZ1. Twonucleicacids(positions225and236)inZZ1differfromQDYandHPD (nucleic acid shaded). The deduced amino acids of three cultivars are identical. (nucleicacidshaded).Thededucedaminoacidsofthreecultivarsareidentical. 3.2. Homology Analysis of BnCPI 3.2. HomologyAnalysisofBnCPI Analysis of the BLAST results (11 May 2017) revealed that the deduced amino acid sequence of AnalysisoftheBLASTresults(11May2017)revealedthatthededucedaminoacidsequenceof BnCPI shared the highest identity (76%) with sequences of cystatin from Glycine max (accession nos. BnCPIsharedthehighestidentity(76%)withsequencesofcystatinfromGlycinemax(accessionnos. XP_003543365 and AAA97905) and Ricinus communis (accession no. XP_002523225), followed by XP_003543365 and AAA97905) and Ricinus communis (accession No. XP_002523225), followed by sequences of cystatin from Glycine soja (accession no KHN35444), G. max (accession no. sequencesofcystatinfromGlycinesoja(accessionnoKHN35444),G.max(accessionNo. NP_001239817) NP_001239817) and Hevea brasiliensis (accession no. ACZ02398), all of which shared 75% identity with and Hevea brasiliensis (accession No. ACZ02398), all of which shared 75% identity with BnCPI. BnCPI. These were followed by sequences of cystatin from Vigna unguiculata (accession no. These were followed by sequences of cystatin from Vigna unguiculata (accession No. CAA79954) CAA79954) and Cajanus cajan (accession no. XP_020236689), both of which shared 74% identity with andCajanuscajan(accessionNo. XP_020236689),bothofwhichshared74%identitywithBnCPI. BnCPI. AsshowninFigure4,alignmentofBnCPIwithsequencesofphytocystatinsfromotherplants As shown in Figure 4, alignment of BnCPI with sequences of phytocystatins from other plants confirmedthatBnCPIandR.communiscystatinweremostsimilar. BnCPIcontainedmostofthetypical confirmed that BnCPI and R. communis cystatin were most similar. BnCPI contained most of the featuresofotherphytocystatins,suchastheGGdoubletandLARFAVmotifintheN-terminalregion, typical features of other phytocystatins, such as the GG doublet and LARFAV motif in the N-terminal andthecentralsignaturemotifQxVxG(alsoastheactivesite),butitlackedthePWmotifcontainedin region, and the central signature motif QxVxG (also as the active site), but it lacked the PW motif someoftheothercystatinsfromplantsandanimals. Here,itwasrevealedbymultiplealignmentsthat contained in some of the other cystatins from plants and animals. Here, it was revealed by multiple thePWmotifwasreplacedbytheLWmotifintheBnCPIaminoacidsequences,whichisidenticalto alignments that the PW motif was replaced by the LW motif in the BnCPI amino acid sequences, thephytocystatinofpapaya(accessionNo. CAA50437)andragweed(accessionNo. AAA32672). which is identical to the phytocystatin of papaya (accession no. CAA50437) and ragweed (accession no. AAA32672). Genes2017,8,265 8of15 Genes 2017, 8, 265 8 of 14 FFiigguurree 44.. AAlliiggnnmmeenntt oof fththee ddeedduucecedd amaminino oacaicdi dsesqeuqeunecnec oefo BfnBCnPCIP (IA(LAGL3G83384374) 7w)iwthi tthhet hseeqsueqenuceensc oefs pohfyptohcyytsotcaytisntast i(nwsith(w mitohlemcuolalerc mulaasrs mofa assboouft 1a2b okuDta1) 2frokmD ao)thferor mplaontthse; rthpelya nartse; Htehveeya barraesilHieenvseisa HbrbaCsiPlieI n(sAisCHZ0b2C3P9I8)(,A MCaZn0ih2o3t9 e8s)c,uMlenantaih MoteeCscPuI l(eAntAaFM72e2C0P2I),( PAeAlaFrg7o2n2i0u2m), hPoerltaorrguomni uPmhChPoIr t(oAruBmG8P1h0C97P)I, T(AriBfoGliu81m0 9s7u)b,teTrrriafnoeliuumm TssuCbtPeIr r(aGnAeuUm21T0s3C7P),I R(uGmAeUx 2o1b0tu3s7i)f,oliRuus mReoxCoPbIt u(CsiAfolDiu2s14R4o1C), PMIe(dCicAagDo2 1sa4t4iv1a), MMseCdiYcaSg1o (AsaAtiZva98M79s1C),Y CSa1st(aAneAa Zsa9t8i7v9a 1C),YSC a(sCtaAnAea11s8a9t9iv)a, PCopYuSlu(uCpAhrAat1ic1a8 9P9e)C, PPIo (pAulGuXup2h8r1a3t9ic),a GPlyecCinPeI m(AaGx XG2m81C39P)I, G(KlyHciNne3m54a4x4G),m GClyPcIin(Ke HsoNja3 5G4a4C4)P,IG l(yKcHinNes3o5ja44G4a)C, PVIig(KnaH uNn3g5u4i4c4u)l,aVtai gVnauCunPgIu (iCcuAlaAta7V9u95C4P)I, R(CicAinAu7s 9c9o5m4m),uRnicisin RuscCcoPmI m(XuPn_is00R2c5C2P32I2(X5)P, _D00ia2n5t2h3u2s2 c5a),ryDopiahnytlhluuss cDaCry-oCpPhIynll u(sADACK-3C0P00In4)(,A AAraKb3id0o0p0s4i)s, tAhraalibaindao psAistCthYaSli1a n(aCAAtAC0Y3S9129()C, ACAar0i3ca9 2p9a),paCyaar icCaspt a(pCayAaAC5s0t4(3C7)A Aan5d0 43A7m)barnodsiaA marbtermosiisaiiaforltieam AisiaifColPiaI (AAaACAP3I2(6A7A2)A. 3T2h67e 2)i.deTnhteiciadl enatnidca lpaanrdtiapl-acrotinasle-crovnatsieornv arteiosnidrueessi daurees asrheadsheadd eind ibnlabclakc kanadn dggrraayy,, rreessppeeccttiivveellyy.. RReedd bbooxxeess sshhooww ccoonnsseerrvveedd mmoottiiffss ooff pphhyyttooccyyssttaattiinnss.. TThhee aalliiggnnmmeenntt wwaass ppeerrffoorrmmeedd bbyy CClluussttaallWW pprrooggrraamm aanndd wwaass sshhaaddeedd uussiinngg tthhee GGeennee DDoocc ssooffttwwaarree.. 3.3. Recombinant Expression of BnCPI The ORF sequence was optimized and subsequently introduced into the pSmart-I vector, then expressed in E. coli BL21 (DE3). Overexpression of BnCPI was induced by adding IPTG (final Genes2017,8,265 9of15 3.3. RecombinantExpressionofBnCPI Genes 2017, 8, 265 9 of 14 The ORF sequence was optimized and subsequently introduced into the pSmart-I vector, tchoennceenxtpraretisosne d0.5in mEM. )c otoli tBhLe 2m1e(dDiuEm3).. ExOpvreersesexdp rreescsoiomnbionfanBtn BCnPCIPwI awsaisn ndoutc seedcrbeyteda dtod itnhge lIiPqTuGid (mfineadliucmon. cBenactrteartiioaln c0e.l5lsm wMer)et ocothlleecmteedd biuym c.enEtxripfuregsasteiodnr,e hcoommboigneannizteBdn CbyP Iuwltarassnoontics eccrraesthe dantod tphuerliifqieudid bmy eadffiiunmity. Bcahcrtoemriaaltocgelrlaspwheyr. eAcfotlelre crteemdobvyincgen StrUifMugOa taionnd, fhuormthoegr epnuizriefdicbatyiounlt, rtahseo nreicsucrltaasnht apnrdotpeuinr iwfieads abnyaalyffizneidty ucshinrogm SDatSo–gPraApGhEy. eAlefctetrrorpemhoorveisnisg. SAU sMinOglea npdroftueritnh beranpdu rwifiictha taio mn,otlheecurelasru mltaansts porfo atebionuwt 1a1s.a4n kaDlyaz wedaus soibntgaiSnDeSd– (PFAigGuEree l5e)c, twrohpihcho rwesaiss. sAligshintlgyl ehpigrhoteeri nthbaann tdhew ditehdaumceodl epcruoltaerinm saiszse o(1f1a.b0o6u ktD1a1). 4fokrD BanCwPaIs (odbetsacirnibeded( Faibgouvree).5 T),hwe hreicshulwtsa isndsliicgahtetldy thhiagth seurctcheasnsfuthl epdroekdaurcyeodticp reoxtperiensssiizoen (o1f1 .B0n6CkPDI aw)faosr aBchniCePvIed(d. escribedabove). Theresultsindicatedthatsuccessfulprokaryoticexpression ofBnCPIwasachieved. FFiigguurree 55.. AAnnaallyyssiiss ooff rreeccoommbbiinnaanntt eexxpprreesssseedd BBnnCCPPII ((rreeBBnnCCPPII)) oonn 1122%% ssooddiiuumm ddooddeeccyyll ssuullffaattee ppoollyyaaccrryyllaammiiddee ggeell eelleeccttrroopphhoorreessiiss ((SSDDSS––PPAAGGEE)). .MM: :pprorotetienin mmarakrekre; r1;: 1r:eBrenBCnPCI PwIitwh iSthUMSUOM aOnda Hndis Htaigs t(abgef(obreef oernezeynmzyem deigdeisgteiosnti)o; n2) ;a2nadn d3: 3r:erBenBCnPCIP Iwwitihtohuotu tSSUUMMOO aanndd HHiiss ttaagg,, rreessppeeccttiivveellyy ((aafftteerr eennzzyymmee ddiiggeessttiioonn));; 44:: SSUUMMOO aanndd HHiiss ttaagg ((aafftteerr eennzzyymmee ddiiggeessttiioonn)).. AA pphhyyllooggeenneettiicc ttrreeee ((FFiigguurree SS33)) ccoonnssttrruucctteedd wwiitthh tthhee aammiinnoo aacciidd ((AAAA)) sseeqquueenncceess ooff BBnnCCPPII aanndd 5511 ootthheerr ccyyssttaattiinnss ffrroomm ddiiffffeerreenntt ppllaanntt ssppeecciieess rreevveeaalleedd tthhaatt tthhee BBnnCCPPII sseeqquueenncceess aarree mmoosstt cclloosseellyy rreellaatteedd ttoo ccyyssttaattiinn ffrroomm VV.. uunngguuiiccuullaattaa,, ffoolllloowweedd bbyy ccyyssttaattiinn ffrroomm VViiggnnaa rraaddiiaattaa,, GG.. mmaaxx aanndd GG.. ssoojjaa.. TThheessee rreessuullttss aarreei inna aggrreeeemmeennttw witithht thhoosseeg geenneerraatteeddb byyt htheeB BLLAASSTTr eresseeaarcrchha annaalylysissi.s. 33..44.. PPrrootteeaassee IInnhhiibbiittoorryy AAccttiivviittyy ooff rreeBBnnCCPPII TTood deetteerrmmiinneew whheetthheerr tthhee rreeccoommbbiinnaanntt BBnnCCPPII iinnhhiibbiittss ccyysstteeiinnee pprrootteeiinnaassee,, tthhee ppuurriififieedd rreeBBnnCCPPII ((≈≈1100 μµgg)) wwaass pprree--iinnccuubbaatteedd wwiitthh ppaappaaiinn,, ffiicciinn aanndd bbrroommeellaaiinn,, aanndd tthheenn tthhee rreessiidduuaall eennzzyymmee aaccttiivviittiieess oonn NNαα--bbeennzzooyyll--DDLL--aarrggiinniinnee ββ--nnaapphhtthhyyllaammiiddee ((BBAANNAA)) wweerree tteesstteedd aass aabboovvee.. FFoorr ppaappaaiinn aanndd fificciinn,, oonnllyy aabboouutt 1188..22%% aanndd 2299..00%% eennzzyymmee aaccttiivviittiieess rreemmaaiinneedd aafftteerr ttrreeaattmmeenntt,, rreessppeeccttiivveellyy,, wwhheerreeaass ffoorr bbrroommeellaaiinn,,r erBeBnCnCPIPsI hsohwoewdendo noob voiobuvsioiunhs ibinithioibnitaicotniv iatcyti(vFiitgyu r(eFi6g).uTreh e6s)e. dTahtaesien ddicaatate itnhdaitcpauter ifitheadt rpeuBrniCfiPedI hreaBsnstCroPnI ghainsh sitbriotnogry inefhfiebcittsooryn eefnfzeyctms eosnw enitzhycmysetse winiethp rcoytsetieniansee parcotitveiitnya,saen adctthivaittyfu, nanctdio tnhaalt efxupnrcetsiosinoanl ewxapsreascshiioenv ewda.s achieved. Genes2017,8,265 10of15 Genes 2017, 8, 265 10 of 14 Genes 2017, 8, 265 10 of 14 Figure 6.FiAgusrsea 6y. Aosfsaiyn hofi binihtiiobintioan catcitvivitityy ooff ppuruifrieifid eredBnrCePBIn tCowPaIrdt opwapaarind, fpicainp aanidn ,brfiomcienlaiann. Tdheb romelain. Theinhibiitnihoinbitwiona swaesx epxrperesssseedd aass rerseidsuidalu eanlzyemnez aycmtiveitya icnt itvheit pyreisnentche eofp BrneCsPeIn act e10o0 fμBg/nmCL,P aInda tN1α0-0µg/mL, benzoyl-DL-arginine β-naphthylamide (BANA) was used as substrate. Vertical bars represent andNα-besntaznodyarld-D dLe-vairagtiionnisn. eDβiff-enreanpt hstmhaylll almettiedrse o(Bn AbNarsA r)ewpreassenuts esidgnaifsicsauntb sdtirffaetree.ncVee artt ic5a%l bleavresl represent standard d(Deuvnicaatni'os nmsu.ltipDlei frfaenrgeen tetsts)m. all letters on bars represent significa nt difference at 5% level (Duncan(cid:48)sFmiguurleti p6.l eAsrsaany goef itnehsitb)i.tion activity of purified reBnCPI toward papain, ficin and bromelain. The 3.5. Antifungal Activity of reBnCPI inhibition was expressed as residual enzyme activity in the presence of BnCPI at 100 μg/mL, and Nα- 3.5. AntifungalAbAenntczitfouiyvnl-igDtayLl- oaarfcgtriivneBiintyne aCβsP-snaIaypsh wtheyrlea mpiedref o(rBmAeNdA w) itwha ds ifufeserden at sc osnucbesntrtartaet.i oVnesr toicfa pl ubrairfise dre rperBesneCntP I on four pstlaanndta prda thdoevgieantiiocn fsu. nDgifif e(Fre. notx ysmspaolrl ulmet,t eArs. aotner nbaatras, rBe.p crienseerneta saignndi fPic.a nvet xdainffse)r ceunclteu aret s5. %T hlee vreels ults Antifiunndigca(aDtleuadnc cttahinav'sti mtryeuBlatnispCslPea Iry ainsnghwei bteeistrete)d. ptheer fgorromwtehd owf ailtlh foduirf fpehryetnoptactohnogceennst, rbautti othnes ionhfipbiutorriyfi eefdfercte BnCPIon fourplantvparaietdh odgepeennidcinfug nogn ith(Fe .fouxnygaslp ospreucmie,s A(F.igautreer n7a).t aA,mBo.ncgi ntheerseea faonurd fuPn.gvie, xBa. ncsin)erceua lwtuarse ms.osTt heresults 3.5. Antifungal Activity of reBnCPI sensitive to reBnCPI. Concentrations of reBnCPI of 20 μg/mL (1.81 μM) slowed the growth rate of B. indicatedthatreBnCPIinhibitedthegrowthofallfourphytopathogens,buttheinhibitoryeffectvaried cinereAa ntoti 5fu4n%g oalf athcatitv eitxyh aibsistaeyds b wye Br.e c pineerrfeoar mcueldti vwaittehd dinif fmereednitu cmon wceitnhtorautti orenBsn oCf PpIu; raitf iceodn rceeBnntrCaPtiIo onns dependingofof ou8rn0 pμtlgha/nemt Lfpu a(7tnh.2go3ga μelnMsicp) ,fe tuhcneige Bis .( cF(iF.n oiexgreyuas prgoerrou7wm)t.,h A Ar.a matete owrnnaasgt ao,t nBhly.e c2sin1e%erfe ooaf ua trnhdaft u Pcn.u vlgteiixv,aanBtse.)d cc iiunnl tmeurreeedasi.u wTmha ews rimethsuoolusttst sensitive toreBnCPriIne.BdCnicCoaPtneIcd. eG tnhrotawrt atrhteiB ornantCesPs Ioo fif nFrh.e iobBxiytnespCdo PrtuhIme o gafrno2dw0 Athµ. a goltf/e ramnlal tLafo w(u1re .r8pe1 hryeµdtoMupcae)tdhs oblgoye wanbseo, dubtu t5th0 t%eh ega tri nao hcwiobntithcoernryta retaeftfieoocntf B.cinerea to 54% ofovtfha rraieetBdne CdxePhpIi ebonfid t8ein0d gμ bgo/nym tBLh;e. tchfuiannt egirsa,el atshpcee u5cl0iet%isv (gaFrtiogewudrthei n7in).hm Aibemitdiooinnug m( EthCwe5s0)ei tffhoorou turh tefusrene gtBwi, noBC .p PcaitnIh;eorageate nwcso awns acmse o8ns0tt rations of μsegn/msitLiv. eM toor reeoBvneCr,P tIh.e C ionnhcibeinttiorant ieofnfisc ioefn rceyB onfC rPeIB onfC 2P0I μ(agt/ m80L μ (g1/.m81L μ) Mw)a ss lsolwigehdtl yth hei gghroewr tthha rna tteh aotf oBf. 80µg/mL(7.23µM),theB.cinereagrowthratewasonly21%ofthatcultivatedinmediumwithout jciinngegreaan tgom 5y4%cin o (f5 t0h aμtg e/xmhLib)i. tIend t bhye Bca. scein oerf etah ceu rlotiovta rtoedt p inat mhoegdeinu mP. wveixthaonus,t grreoBwnCthP rIa; taets c foenllc ebnyt r5a0t%io nats reBnCPI.GroerfB o8n0wC μPtghI/ cmroaLnt c(ee7sn.2to3ra fμtiFMo.n)o,s x tohyfe s2 Bp0.o μcrigun/emmreLaa agnnrdodw A3t5h.% ar aaltttee c rwonnaacste aonntwrlayet i2ro1en%sr oeoffd 8tuh0a cμte gcd/umlbtLiyv, aiantebddio ciuant timn5ge0d t%hiuamta P tw. avitehcxooanunstc entration of reBnCPirsIe BonnofCt 8Pv0I.e rGµyr gos/wenmthsi Lrtiav;teet sh tooaf t FB.in soCx,yPtsIhp. oeFruo5mr0 a%anlld g fAoru.o arwl tfeutrhnnagtiia,n whheoirbwei ertveioderun, cet(hdEe bC yg5 ra0ob)wofutoht r5r0at%hte e atsd eae ccltoiwnnecoden ptarsaa tttihhoeno gens was 80 µg/mLco.of MnrceeoBnnrteCraoPtivIo oenfrs , 8o0tfh rμeegB/inmnCLhP;i Ibt ihniatctir oeisan,s teehdfe.fi 5 c0i%e ngcroywothf irnehBibnitCioPnI (E(aCt50)8 0forµ tgh/esme tLw)o wpaatshosgleignsh wtlays h80ig her than μg/mL. Moreover, the inhibition efficiency of reBnCPI (at 80 μg/mL) was slightly higher than that of thatofjinggangmycin(50µg/mL).InthecaseoftherootrotpathogenP.vexans,growthratesfellby jinggangmycin (50 μg/mL). In the case of the root rot pathogen P. vexans, growth rates fell by 50% at 50%atreBnCPIconcentrationsof20µg/mLand35%atconcentrationsof80µg/mL,indicatingthat reBnCPI concentrations of 20 μg/mL and 35% at concentrations of 80 μg/mL, indicating that P. vexans P.vexansisisn noott vveerryy sseennssitiitviev etot oBnBCnPCI. PFIo.rF aolrl afolulrf ofuunrgfiu, nhogwi,ehveorw, tehve egrr,otwhteh grraotew dtehclrinaetde dase cthlien edasthe concentratcioonncsenotrfartieoBnns CofP reIBinnCcPreI ainscereda.sed. Figure 7. Growth inhibition assays of four fungal pathogens by reBnCPI. Fo, Fusarium oxysporum; Aa, Alternaria alternata; Bc, Botytis cinerea; and Pv, Pythium vexans. reBnCPI was added to the fungal suspension to a final concentration of 0, 20, 40, 60 or 80 μg/mL. Fungicides carbendazim, jinggangmycin, fluazinam and mefenoxam were added to the suspension of Fo, Aa, Bc and Pv (final concentration 50 μg/mL), respectively, to serve as positive control. Vertical bars repre sent standard deviations. Different small letters on bars represent significant difference at 5% level (Duncan's Figure7. mGFiugrloutirwpel e7th .r Gainrnoghwe ittbehs iittn)i.ho inbitaiosns aasyssayos foff foouurr ffuunngagl aplatphaogthenosg beyn rseBbnyCPrIe. BFon, CFuPsaIr.iuFmo ,oxFyuspsoarruimu;m Aao, xysporum; Alternaria alternata; Bc, Botytis cinerea; and Pv, Pythium vexans. reBnCPI was added to the fungal Aa,Alternariaalternata;Bc,Botytiscinerea;andPv,Pythiumvexans.reBnCPIwasaddedtothefungal suspension to a final concentration of 0, 20, 40, 60 or 80 μg/mL. Fungicides carbendazim, suspensionjintoggaanfignmaylccion,n fclueanztirnaatmio anndo fm0e,f2en0o,x4a0m, 6w0eorer a8d0dµedg /tom thLe .sFuuspnegnsicioind eosf Fcoa,r Abae,n Bdca aznidm P,vj i(nfignagla ngmycin, fluazinamcaonncdenmtraetfioenn 5o0x μagm/mwL)e, rreespaedcdtiveedlyt, otot sheervseu ass ppeonsistiivoen coonftrFolo. ,VAerati,caBl cbaarns drepPrvese(nfitn satalncdoarndc entration 50 µg/mLd)e,vrieatsiponesc. tDivifefleyre,ntto smsearllv leettaesrs poons ibtairvse recpornesternotl .sigVneifrictaicnat ldbifaferrsenrcee parte 5s%en ltevsetla (nDduanrcdan'ds eviations. multiple range test). Differentsmalllettersonbarsrepresentsignificantdifferenceat5%level(Duncan'smultiplerangetest). The inhibitory effects that reBnCPI exhibited on the growth of these four phytopathogenic fungi were also checked under microscopy (Figure S4). In the negative control (without reBnCPI) treatments,allfourfungalspeciesgrewfast,withmoremyceliathanthatpresentwithBnCPIand