Improved antifungal activity of barley derived chitinase I gene that overexpress a 32 kDa recombinant chitinase in Escherichia coli host

h ttp : / / w w w . b j m i c r o b i o l . c o m . b r /

Bacterial,

Fungal

and

Virus

Molecular

Biology

Improved

antifungal

activity

of

barley

derived

chitinase

I

gene

that

overexpress

a

32

kDa

recombinant

chitinase

in

Escherichia

coli

host

Nida

Toufiq,

Bushra

Tabassum

,

Muhammad

Umar

Bhatti,

Anwar

Khan,

Muhammad

Tariq,

Naila

Shahid,

Idrees

Ahmad

Nasir,

Tayyab

Husnain

UniversityofthePunjab,CentreofExcellenceinMolecularBiology,BaigLahore,Pakistan

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received2November2016 Accepted16May2017

Availableonline31October2017 AssociateEditor:FernandoAndreote

Keywords:

Antifungalactivityassay BarleychitinaseclassI

Escherichiacoliexpression Recombinantchitinaseprotein

a

b

s

t

r

a

c

t

Agriculturalcropssuffermanydiseases,includingfungalandbacterialinfections,causing significantyieldlosses.Theidentificationandcharacterisationofpathogenesis-related pro-teingenes,suchaschitinases,canleadtoreductioninpathogengrowth,therebyincreasing toleranceagainstfungalpathogens.Inthepresentstudy,thechitinaseIgenewasisolated fromthegenomicDNAofBarley(HordeumvulgareL.)cultivar,Haider-93.TheisolatedDNA wasusedastemplatefortheamplificationofthe∼935bpfull-lengthchitinaseIgene.Based onthesequenceoftheamplifiedgenefragment,classIbarleychitinaseshares93%amino acidsequencehomologywithclassIIwheatchitinase.Interestingly,barleyclassI chiti-naseandclassIIchitinasedonotsharesequencehomology.Furthermore,theamplified fragmentwasexpressedinEscherichiacoliRosettastrainunderthecontrolofT7promoter inpET30avector.Recombinantchitinaseproteinof35kDaexhibitedhighestexpression at0.5mMconcentrationofIPTG.Expressedrecombinantproteinof35kDawaspurifiedto homogeneitywithaffinitychromatography.Followingpurification,aWesternblotassayfor recombinantchitinaseproteinmeasuring35kDawasdevelopedwithHis-tagspecific anti-bodies.Thepurifiedrecombinantchitinaseproteinwasdemonstratedtoinhibitsignificantly theimportantphytopathogenicfungiAlternariasolani,Fusariumspp,Rhizoctoniasolaniand

Verticilliumdahliaecomparedtothecontrolatconcentrationsof80␮gand200␮g.

©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

Introduction

Agricultural crops havegreat economic importance world-wide,andwiththepopulationincrease,thecropsproduced arenotsufficienttofeedthehumanpopulation.Additionally,

∗ _{Correspondingauthor.}

E-mail:[email protected](B.Tabassum).

the agricultural crops are attacked by multiple pathogens includingbacterial,viralorfungalcausingreductioninboth quantityandqualityoftheoverallyield.Almost26–30%ofthe yieldlossesforsugarbeet, cottonandwheatare causedby fungalpathogensalone.1_{Moreover,fungicause35%,39%and} 40% ofthedamageinmaize,potatoandrice,respectively.1 Fungi,suchasFusariumsolaniandFusariumoxysporum,arethe causeofwiltinganddampingoffdiseases,whereasAlternaria alternatecausesleafspotinvariousagriculturallyimportant

https://doi.org/10.1016/j.bjm.2017.05.007

1517-8382/©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

crops.2_{Rhizoctoniasolanicausesmanydiseasesincludingblack} scurfinpotatoes,3_{rootrotinsugarbeet}4_{andsheathblightin} rice.5_{Similarly,Verticilliumdahliaeisthecauseofverticillium} wiltinmanyplantsincludingpotato,6,7_cotton,8_peppermint9 andothers.

Thefirststructureofthefungusthatcomesintocontact withthe host(plantsinthe caseofphytopathogenicfungi) is its cell wall. Chitin,the mainconstituent of the fungal cellwall,isahomopolymerofN-acetylglucosamineunits.In nature,plantspossessnaturaldefencesystemsagainstfungal attack.Certainplantsproduceantifungalproteinstodefend againstfungalgrowth.10 _{Isolatingtheseantifungalproteins} andinsertingthemintodifferentplantsincreasesresistance againstfungal pathogens in these newhost plants. These proteinsincludechitinasesandglucanases.11Chitinasesare presentinalmostallorganismsincludingbacteria,12_fungi,13 insects,14_plants15_{andmammals.Chitinasesactonchitinas} theirsubstrateandhydrolyseittomono-andoligomers.16_A chitinaseisclassifiedasanendochitinaseifitbreakschitin ininternalsitesbyactingrandomly;oranexochitinase,ifit breaksthechitinfromeithernonreducingorreducingends.17 Theyhavedifferentrolesindifferentorganismsbutinplants chitinasesare mainlyinvolvedin resistanceagainstfungal pathogens.18 _{Barley possesses endochitinases (class I and} classII)thatcanbeusedtocontrolfungalpathogens.19

Escherichiacoliisthemostwidelyusedbacterialexpression systemforproducingheterologousproteins.20_{Toobtainahigh} yieldofrecombinantprotein,thegeneisusuallyexpressed atits highestpossiblelevel. Thepurposeofthis studywas to isolate chitinase I gene from Barley and express it in anE.coliexpressionsystemtorevealitsantifungalactivity againstfoureconomicallyimportantphytopathogenicfungi;

Alternariasolani,Fusariumspp,R.solaniandV.dahliae.

Materials

and

methods

Plantmaterial

Barley(HordeumvulgareL.)varietyHaider-93growninCEMB fieldwasusedassourcematerialforisolationofchitinaseI gene.ThededucedsequenceofchitinaseIgenewasusedto obtaintheaminoacidsequenceofthegene,whichwas fur-therusedforalignmentand constructionofaphylogenetic tree.

Geneamplificationandaminoacidsequencehomology studies

Genomic DNA was extracted from young Barley leaves using Genomic DNA Purification kit (Thermoscien-tific, K0512). The 935bp chitinase I gene was amplified with 5-AGAGCGTTCGTGTTGTTCG-3 forward and 5 -CTGTAGCAGTCGAGGTTGTTG-3 reverse primer in a PCR reactionmixturecomprisedof2␮Lof10×PCRbuffer,1.5mM MgCl2, 0.1mM dNTPs, 1pmol each of forward and reverse

primers,50ngDNAand2unitsofTaqpolymerase (Thermo-scientific).ThePCRamplificationwascarriedoutunderthe followingconditions: initialdenaturationat95◦Cfor5min, followedby35cyclesofamplification(94◦Cfor45s,61◦Cfor

45s,and 72◦C for45s) witha finalextension of10minat 72◦C.Theamplifiedfragmentwasanalysedon0.8%agarose gel by electrophoresis. The amplified fragment of ∼935bp was cloned into the pCR 2.1 vector (Invitrogen, K4500-01) for sequencing. The Barley chitinaseclone was sequenced through the facility at Macrogen (Korea), and edited and analysed using the BioEdit tool. The sequences of other plantchitinasesusedforcomparisonweredownloadedfrom the National Centre of Biotechnology Information (NCBI). Multiple sequence alignment of amino acid sequences of plant chitinase retrieved from GenBank and our deduced sequence wasdoneusingClustalW(Megav6)software.A phylogenetic tree wasconstructedusing Neighbourjoining method and the reliability was measured by boot strap analysiswith1500trialsusing Megav6software.The pro-tein families of barley chitinase class I and class II were compared using conserved domain database, CDD, using

http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi.

Constructionofaprokaryoteexpressionvectorand expressionofrecombinantchitinase

A∼935bpchitinaseIgenewasligatedinpET30a(Addgene) expressionvectorhavingN/C-terminalHis-tagsequenceand expressedunderT7promotertocreateapET-chiIconstruct. TheligationwastransformedintocompetentcellsofE.coli

throughtheheatshockmethodasdescribedbyFrogerand Hall.21_{ThepET-chiIconstructDNAwasmixedwith50␮Lof} chemicallycompetentE.coliDH5␣cellsandafterashort incu-bationonice,heatshockwasgivenat42◦Cfor45sandplaced backonice.SOCmediawasaddedandthetransformedcells wereincubatedat37◦Cfor30minwithagitation.

Then, the mix was spread onto LB media plates with kanamycinforantibioticselectionandincubatedat37◦Cfor 16–18h. Positiveclones were confirmedthrough restriction digestion with EcoR1.For expression analyses,the pET-chiI construct was transformed in E. coli host Rosetta. E. coli

Rosettacellsweretransformedwiththeprokaryotic expres-sion vector pET-ChiI encoding the chitinase I protein and growninshakingculture(220rpm,37◦C)inLBsupplemented with50␮g/mLkanamycintoanODof0.6at600nm.Protein expressionwasinducedbytheadditionof0.5mMisopropyl- ␤-d-thiogalactopyranoside(IPTG,Sigma)for3h(220rpm30◦C). Induced cells were harvested by centrifugation at 4000×g

for 10min at 4◦C. The pellet was weighed, resuspended in 1× phosphate saline buffer (1:10W/V) and centrifuged at 4000×g for 10min. Lysozyme (4mg/mL) was added in the pellet and keptat roomtemperature for20–30minfor complete cell lysis. The lysate was sonicated (20min at 16W) and then cleared by centrifugation at 14,000×g for 15minat4◦C.

Purificationwasperformedat4◦CinsideaKelvinator chro-matographyrefrigeratorusinga16/20XKcolumnwithvolume of20mLandheightof15mLwhichwaspackedwithChelating FastFlowSepharoseadsorbent(GEHealthcare;catalogueNo. 17-0575-01).Thecolumnwaschargedwith0.2MNiCl2

solu-tion.Bindingbuffer(500mMNaCl,20mMsodiumphosphate buffer, pH7.4) was added tobalance the Sepharose adsor-bent. The supernatant containing the protein was filtered through0.45␮mmembranetoremovecelldebris.Thefiltered

A

B

C

Fig.1–AmplificationandcloningofchitinaseIgeneinTAvectorandinE.coliexpressionvectorpET30a.(A)Lane1:1kb DNAladder;lanes2–4:samplesshowingamplificationof∼935bpofchitinaseIgene.(B)Restrictiondigestionofplasmid pTA:chiIharbouringchitinaseIgeneclonedinpCR2.1vectorwithEcoRIenzyme.Lanes1–2:pTA:chiIDNAsamples;M represent1kbDNAladder.Thereleaseof∼3.9kbDNAfragmentrepresentpCR2.1vectorwhile∼935bpdepictchitinase gene.(C)RestrictiondigestionofpET-chiIrecombinantplasmidwithEcoRItoconfirmligationofchitinasegeneinE.coli expressionvector.LaneM:1kbDNAladder,lanes1–3:pET-chiIplasmidDNA.Thereleaseof∼5.4kbDNAfragment representpET30avectorwhile∼935bpdepictchitinasegene.

protein supernatant was then loaded onto the column at a linear flow rate of 150cm/h and the time for complete loadingwas calculated.Washing was performed first with wash solution 1 (20mM imidazole+bindingbuffer, pH 7.4) and then with wash buffer 2 (40mM imidazole+binding buffer, pH 7.4) such that all of the unbound solutes and other proteincontaminants werecompletelyremoved. Elu-tionoftheHis-tagrecombinantchitinaseIproteinwasdone byprovidingstep-wiseincreasingconcentrationsof imidaz-ole (ThermoFisher Scientific; catalogue No. AC30187-0010). Five concentrations of elution buffer were prepared from 70mM to 150mM. Five fractions (1mL each) for each elu-tionwereselectedandanalysedbyBradfordassaytocheck theproteinconcentration.Ateachstep,fractionswere ana-lysedbySDS-PAGE on 12%polyacrylamide gel and stained with Coomassie blue (ThermoScientific). Protein size was determinedusingPerfectProteinTM_{Markers(cat#926-98000}

WesternSure®). Recombinant protein was fractionated by SDS-PAGEon12% gelusingaMiniProteanII tetracell (cat #1658004;BioRad).Theresolvedpurifiedrecombinantprotein wastransferredtoPVDF(polyvinylidenedifluoride)membrane (cat # RPN303E; Amersham) using a Trans-blot Cell (Bio-Rad)blockedwith5%non-fatdrymilkwithHis-tagspecific antisera. Recombinant chitinase was detected with rabbit polyclonalHis-tagantiserum(1:5000dilution;cat#sc-804; SantaCruz).Anappropriateantibodyconjugatedwith perox-idasewasusedindetectionwiththeECLsystem(Amersham Biosciences).

Antifungalactivityassay

Thephytopathogenic fungiA. solani,Fusarium spp,R.solani

andV.dahliaewereprovidedbytheInstituteofAgricultural Sciences,UniversityofthePunjabLahore-Pakistan. Antifun-gal assays of purified recombinant chitinase protein were performed on the four phytopathogenic fungi by employ-ingcylinderplatemethod.21_{Forthis,threewellsweremade} onPDA(potatodextroseagar)mediumbythecylinderplate method.ThePDAblockcontainingmyceliaofthetestfungi wasinoculatedinthecentreofthepetriplates(23◦C;48h). When the colonysizeincreasedto3–4cm indiameter,the wellswerefilledwithdifferentconcentrationsofpurified chiti-naseprotein;80␮g,200␮g,andcontrol.Forthecontrolwell, recombinantchitinaseproteinwasplacedinboilingwaterfor 5minandpouredintothe respectivewell.Theplates were thenincubatedat23◦Cfor48h.Zoneofinhibitioncreatedby therecombinantchitinaseproteinwasmarkedandmeasured incomparisonwithnegativecontrol.

Results

PCRamplificationandaminoacidsequencecomparisonof chitinasegenes

A∼935bpchitinaseIgenewassuccessfullyamplifiedfrom genomic DNA ofbarley using specific primers (Fig. 1A). A

Table1–Aminoacidsequenceidentityofbarley chitinasewithotherplantchitinases.

Plantspecies Chiclass GenBankAccessionNo. Identity(%)

Secalecereal I Q9FRV1 95

Triticumaestivum II AAX83262 93

Brachypodium distachyon

I XP003563256.2 88

Triticumaestivum I AAR11388.1 80

Triticumaestivum III BAB82473.1 80

Secalecereal I AAG53609.1 80

Festuca arundinacea

I ACJ23248.1 76

Elaeisguineensis I XP010941404.1 72

Oryzasativa I CAA82849.1 72

Oryzasativa I BAA03751 72

Gossypium hirsutum

I CAA92277 72

Acaciakoa I AFY08283.1 71

Solanum lycopersicum

I CAA78845.1 70

Nicotianatabacum I AAB23374 69

Pisumsativum I CAA45359.1 69

Canavalia ensiformis

II CAA07413.1 67

Oryzasativa II BAA31997 59

Solanum tuberosum

II Q43835 58

Hordeumvulgare II AGS38341.1 58

Solanum lycopersicum

II CAA78846.1 57

Cucumismelo II AAF69836.1 57

Heterodera glycines I AAN14979.1 57 Chenopodium amaranticolor IV BAA22966 42

Vitisvinifera IV AAB65776 42

Oryzasativa IV BAA19793 41

Zeamays IV P29022 40

Triticumaestivum II AAD28730 40

Zeamays I ACX37090.1 40

Triticumaestivum IV AAD28733 39

Gossypium hirsutum VII Q7X7Q1 39 Arabidopsis thaliana IV NP191010 38

sharpanddefinedfragmentcanbeseeninFig.1A.Toconfirm insertion, plasmid DNA was subjected torestriction diges-tionbyEcoRI, generatingtwofragments;a∼3.9kbfragment representingpCR2.1vectoranda∼935bpfragment represent-ingchitinaseIgene(Fig.1B).The935bpofChiIencodeda polypeptideof318aminoacids.BLASTandNCBIsearchusing theaminoacidsequenceindicatedthatithashighdegreeof sequencesimilaritywithproteinsencodingclassIchitinaseof

Secalecereal,chitinasesofclassI,II&IIIofwheat(Triticum aes-tivum),andclassIchitinaseofotherplantsincludingFestuca arundinacea, Elaeis guineensis, Oryza sativa, Gossypium hirsu-tum,Acaciakoa,Solanumlycopersicum,buthasalowdegreeof sequencesimilaritywithclassIVchitinaseofwheat(T. aes-tivum)and Arabidopsisthaliana,andclassVIIchitinaseofG. hirsutum(Table1).Moreover,it showed58%identity witha classIIchitinaseofHordeumvulgare(Fig.2).Itshighest iden-tityiswithclassIchitinaseofS.cereal,at95%(Fig.2).Wheat chitinaseclassII(AAX83262)doesnothaveachitinasebinding

domainandhasonlyacatalyticsitebutbarleyclassIchitinase has93%aminoacidsequencesimilaritytoitwhilein compar-ison,barleyclassIchitinasehasonly58%similaritytobarley chitinaseclassII(AGS38341.1)(SupplementaryFigure1).

Expressionofrecombinantchitinaseproteininprokaryote hostandpurification

Amplified∼935bpchitinaseIgenewasclonedbehindT7 pro-moter infusion with a His-tagin a prokaryoteexpression system.Recombinantclone,pET-chiI,uponrestriction diges-tion releasetwosizespecificfragments:a∼5.4kbfragment representingpET30avectoranda∼935bpfragment represent-ingthechitinaseIgene(Fig.1C).Expressionofthe∼35kDa recombinantchitinaseproteinwasoptimisedusingSDS-PAGE anditwasfoundthat0.5mMIPTGwasbestforrecombinant protein expression(Fig. 3A). Theexpressionwas lowest at 0.2mMconcentrationofIPTGwhilehighestat0.5mM con-centrationofIPTGcomparedtothecontrol,non-transformed Rosetta cells where recombinant protein is not expressed. Subsequently, the recombinant chitinaseproteinwas puri-fied through affinity chromatography and a thick band of ∼35kDawasobservedinelutedfractionsobtainedat125mM and 150mM imidazole concentration (Fig. 3B). The puri-fied recombinant proteinfractions were electrophoretically transferred onto PVDF (polyvinylidene fluoride) membrane (Millipore, USA) and incubated with polyclonal anti-His-HRPantibody(SantaCruz,UK).Anapparentlyhomogeneous band of approximately 35kDa was detected using Chemi-luminescentHRPSubstrate(Millipore)(Fig.3C).

Antifungalinhibitionactivityassays

Purified recombinant chitinaseprotein, measuring∼35kDa when applied on four important phytopathogenic fungi, exhibitedstronginhibitoryeffectonthemycelialgrowthofthe subjectedfungi.Significantgrowthretardationinthemycelial growthofA.solani,FusariumsppandV.dahliaewasobserved at200␮gconcentrationofpurifiedchitinaseproteinwhilethe zoneofinhibitionwaslessaroundthewellswhere80␮gof the purifiedproteinwasapplied.Inthe controlsample,no inhibitoryeffectwasmeasured(Fig.4).InR.solani,less inhi-bition was observed at80␮gconcentration of the purified proteincomparedto othertested fungiwhileat200g con-centration, azone ofinhibitioncan beseen (Fig.4). Itcan beconcludedthatrecombinantchitinaseprotein,when over-expressedexhibitsstrongandsignificantantifungalpotential. Hyphalgrowthoftheinoculatedfungiwasrestrictedtoa par-ticularzoneofinhibition.

Discussion

Chitinasesbelongtogroupofpathogenesis-related(PR) pro-teinsinplants22–25_{andseveralofthemhavebeenemployed} to enhancethe toleranceofthe plant towardsaparticular pathogen throughgenetic transformation technologies.26,27 OuraimwastoisolateaplantbasedchitinaseIgene,over expressitintoaprokaryoteexpressionvectorunderT7 pro-moter and reveal the antifungal potential of the purified

Timetree Original Tree M6: Tree Explorer

File Image Subtree View Compute Caption Help

0.04 0.10 0.14 0.02 0.14 0.15 0.16 0.17 0.19 0.20 0.41 0.39 0.18 0.16 0.16 0.12 0.01 0.77 0.13 0.07 0.01 0.13 0.02 0.06 0.07 0.15 0.17 0.14 0.07 Displaying Timetree

Oryza sativa (II) BAA31997 Hordeum vulgare (II) AGS38341.1 Solanum tuberosum (II) q43835 Solanum lycopersicum (II) CAA78846.1 Cucumis melo (II) AAF69836.1 Pipum sativum (I) CAA45359.1 Canavalia ensiformis (II) CAA0713.1 Nicotiana tabacum (I) AAB23374 Solanum lycopersicum (I) CAA78845.1 Elaeis guineensis (I) xP 010941404.1 Gossypium hirsutum (I) CAA92277 Oryza sativa (I) CAA82849.1 Acacia koa (I) AFY08283.1 Brachypodium distachyon (I) XP 003563256.2 Triticum aestivum (II) AAX83262 Hordeum vulgare (I) Secale cereal (I) Q9FRV1 Festuca arundinacea (I) ACJ23248.1 Triticum aestivum (I) AAR11388.1 Triticum aestivum (III) BAB82473.1 Gossypium hirsutum (VII) Q7X7Q1 Triticum aestivum (II) AAD28730 Triticum aestivum (IV) AAD28733 Chenopodium amaranticolor (IV) BAA22966 Vitis vinifera (IV) AAB65776 Arabidopsis thalina (IV) NP 191010 Oryza sativa (II) BAA19793 zea mays (IV) P29022 zea mays (I) ACX37090.1 Momordica charantia (V) AAM18075 Heterodera glycines (I) AAN14979.1

Fig.2–PhylogeneticanalysisofaminoacidsequencesofHordeumvulgarederivedchitinaseIgeneincomparisonwith otherplantchitinases.Thebranchlengthindicatesthepercentageofsequencesimilarity.Chitinaseclassandaccession numberismentionedagainstparticularcropplant.

A

B

C

Fig.3–ExpressionanalysisofrecombinantchitinaseproteinexpressedinE.colihostRosetta.(A)SDS-PAGE(12%) representingexpressionofa∼35kDarecombinantchitinaseproteinoptimisedatvariousconcentrationsofIPTG.Lane1: celllysatefromun-inducedcolonyofRosetta,lane2:celllysatefromtransformedRosettainducedwith0.2mMIPTG,lane3: celllysatefromtransformedRosettainducedwith0.3mMIPTG,lane4:celllysatefromtransformedRosettainducedwith 0.4mMIPTGandlane5:celllysatefromtransformedRosettainducedwith0.5mMIPTG,lane6:pre-stainedproteinmarker, (B)SDS-PAGEfractionatingpurifiedfractionsofrecombinantchitinaseprotein.Lane1:totalcelllysatefromun-transformed Rosetta,lanes2–3:purifiedfractionofrecombinantchitinaseproteinsample,lane6:pre-stainedproteinmarker,(C) Westernblotofpurifiedrecombinantchitinaseprotein.TheproteinwasdetectedthroughHistagspecificantibodiesusing ECLsystem.Lanes1–2:purifiedfractionof35kDachitinaseprotein.

recombinantproteinagainstfourimportantphytopathogenic fungi.

In our study, we selected a plant chitinaseas they are usuallyendo-chitinasesandcontainlysozymeactivity.While, incontrast,the bacterial chitinasesare exo-chitinases and hydrolysethechromogenictrisaccharideanaloguepNPP (p-nitrophenyl-P-D-N, N-diacetylchitobiose) as the substrate. Additionally, plant chitinase I is reported to exhibit high antifungal activity compared to other plant chitinases.28 Barley-derived chitinasegenes havebeen used toenhance fungalresistanceinmanycropslikebrassica,19_wheat,29_rice30

andcarrot.31_{ThechitinaseIgeneisevolutionarilyconserved} and is present in both monocot and dicot plants such as cocoa,32_potato33_andrice.34_{ThechitinaseIgenealsohasa} chitinbindingdomain,whichisabsentinclassIIchitinase andhencemakeitsuperiorinantifungalpotential.

Wedocumentedaninterestingfindingthatbarley-derived chitinaseIgeneand IIgenenotonlydiffersinpresenceof chitinbindingdomainbutalsodonotsharesequence homol-ogy.Rather, barleychitinaseclassIshares 93% aminoacid sequencehomologywithchitinaseclassIIofwheat. Further-more, we employed pET expression system forexpression

200 µg 80 µg 200 µg 200 µg 80 µg 80 µg 200 µg 80 µg C C Alternaria solani C Fusarium spp C Verticillium dabilon Rhizoctonia solani

Fig.4–Antifungalactivityassayofpurifiedrecombinantchitinaseproteintowards(A)Alternariasolani,(B)Fusariumspp.,(C) Verticilliumdahliae,(D)Rhizoctoniasolani.Eachselectedfunguswassubjectedtotwoproteintreatments,80␮gand200␮g

alongwithcontrol.Theletter‘C’representcontrolthatcontainsheatdeactivatedpurifiedchitinaseprotein.

studiesofchitinaserecombinantproteinaspreviouslydone byLiuandNaismith.35_{Thisisthemostwidelyusedsystem} forthe cloning andin vivoexpressionofrecombinant pro-teinsinE.coli.Thereasonbehindthisisthehighselectivity ofthepETsystem’sbacteriophageT7RNApolymeraseforits cognatepromotersequences,thehighlevelofactivityofthe polymeraseandthehightranslationefficiencymediatedby theT7gene10translationinitiationsignals.36_Weemployed IPTGasaninducingagentasitisreportedtoprovidebetter induction.37

Ourantifungalresult showedthat 80␮gofrecombinant chitinaseproteinhasaclearzoneofinhibitionwhile200␮g oftheproteinhasanincreasedzoneofinhibition.Thereare reports38_{thatricechitinasehadnosignificanteffectonfungi} testedat100␮gandhadonlysubtleeffectatahigherdoseof 300␮g.KirubakaranandSakthivelusedE.colistrainBL-21as hostandpET28a+vectorfortheoverexpressionofchitinaseI geneandanalyseditsantifungalactivityagainstdifferent fun-galpathogensandreportedthat300␮gofenzymeactivityhas broadspectrumanti-fungalinhibition.22_Thisfinding demon-stratesthatourchitinaseisolatehasmuchbetterinhibitory effectontestfungiatlowdosesthanisolatesusedinother studies.

Inconclusion, Chitinasesthat cleave chitinpolymers in thefungalcellwallareusedtoincreaseresistancetofungal pathogens.Andforthispurpose,adequateantifungalactivity ofthechitinaseismandatory.Therefore,screeningchitinase withhighantifungalactivityisimportanttomeettheneedto

improveresistanceagainstphytopathogensbygene manip-ulation.Attemptshavebeenmadebyseveralresearchersto identifynewchitinasegeneswithenhancedantifungal poten-tial. Like Liuet al.expressedtwo chitinasegenes,LbCHI31 andLbCHI32fromLimoniumbicolorinE.coliBL21strainand it hassignificant inhibitionagainstA.alternate.39 _Ina simi-larstudy,Itohetal.overexpressed acellsurface-expressed chitinasefromPaenibacillusinE.coli.40_{Inanotherstudy,} horse-tailderivedchitinasegenewasoverproducedinE.coli.41_Ina similarstudy,achitinasegene(pcht28),isolatedfrom Lycoper-siconchilensewastransformedtostrawberryanditsexpression was checked against V. dahliae. Itshowed moreresistance againstthesefungicomparedtocontrol,untransformed.This suggests pcht28 playsarole indefence againstfungal dis-ease caused by V. dahliae.42 _{Mishraet al. transformed the} Trichoderma endochitinasegene into guava and measured itsinhibitoryactionagainstF.oxysporum,thecauseof fusa-riumwilt.43_{Inourfindings,overexpressionofchitinaseIgene} resultsinelevatedchitinolyticactivity,inductionofexpression oftherecombinantchitinasegeneandincreasedinhibitionfor fungalphytopathogens.Alltheseresultscollectivelyindicate thatthechitinaseIgenederivedfrombarleycanbeusefulin geneengineeringagainstplantdiseases.

Conflicts

of

interest

Appendix

A.

Supplementary

data

Supplementarydataassociatedwiththisarticlecanbefound, intheonlineversion,atdoi:10.1016/j.bjm.2017.05.007.

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e

s

1. OerkeE-C,DehneH-W.Safeguardingproduction—lossesin

majorcropsandtheroleofcropprotection.CropProt.

2004;23(4):275–285.

2. ElhamidMA,MakboulH,SedikM,IsmailI,IbrahimM.

Cloning,expressionandantifungalactivityofan

endochitinasegenederivedfrombarley(Hordeumvulgare).

ResJAgricBiolSci.2010;6(3):356–363.

3. KumarA,RajK.Evaluationofdifferentfungicidesagainst

blackscurfofpotatocausedbyRhizoctoniasolani.IndianJ

PlantProt.2016;44(1):110–115.

4. ZhangX,DongB,ZhouH,XieJ,HaoJJ.Firstreportof

RhizoctoniasolaniAG4-HG-IinfectingsugarbeetinChina.

PlantDis.2015.

5. ZhengA,LinR,ZhangD,etal.Theevolutionandpathogenic

mechanismsofthericesheathblightpathogen.Nat

Commun.2013;4:1424.

6. AshrafA,RaufA,AbbasMF,RehmanR.Isolationand

identificationofVerticilliumdahliaecausingwiltonpotatoin

Pakistan.PakJPhytopathol.2012;24(2):112–116.

7. NairPVR,WiechelTJ,CrumpNS,TaylorPWJ.Firstreportof

VerticilliumtricorpuscausingVerticilliumwiltinpotatoesin

Australia.PlantDis.2015.

8. WoodwardJE,KeelingJW,WheelerTA,DeverJK,KellyCM,

AlbersCR.ImpactofVerticilliumwiltoncottonfiberquality.

Phytopathology.vol.103,No.6.StPaul,MN,USA:Amer

PhytopathologicalSoc;2013:161.

9. FletcherRS,SlimmonT,KottLS.Environmentalfactors

affectingtheaccumulationofrosmarinicacidinspearmint

(MenthaspicataL.)andpeppermint(MenthapiperitaL.).Open

AgricJ.2010;4:10–16.

10.YanJ,YuanSS,JiangLL,YeXJ,NgTB,WuZJ.Plantantifungal

proteinsandtheirapplicationsinagriculture.ApplMicrobiol

Biotechnol.2015;99(12):4961–4981.

11.HegedusN,MarxF.Antifungalproteins:morethan

antimicrobials?FungalBiolRev.2013;26(4):132–145.

12.GomaaEZ.ChitinaseproductionbyBacillusthuringiensisand

Bacilluslicheniformis:theirpotentialinantifungalbiocontrol.J Microbiol.2012;50(1):103–111.

13.HartlL,ZachS,Seidl-SeibothV.Fungalchitinases:diversity,

mechanisticpropertiesandbiotechnologicalpotential.Appl

MicrobiolBiotechnol.2012;93(2):533–543.

14.ArakaneY,MuthukrishnanS.Insectchitinaseand

chitinase-likeproteins.CellMolLifeSci.2010;67(2):201–216.

15.GroverA.Plantchitinases:geneticdiversityand

physiologicalroles.CritRevPlantSci.2012;31(1):57–73.

16.StoykovYM,PavlovAI,KrastanovAI.Chitinase

biotechnology:production,purification,andapplication.Eng

LifeSci.2015;15(1):30–38.

17.Garcia-FragaB,DaSilvaAF,López-SeijasJ,SieiroC.

Functionalexpressionandcharacterizationofachitinase

fromthemarinearchaeonHalobacteriumsalinarumCECT395

inEscherichiacoli.ApplMicrobiolBiotechnol.

2014;98(5):2133–2143.

18.IslamR,DattaB.Diversityofchitinasesandtheirindustrial

potential.IntJApplRes.2015;1:55–60.

19.ChhikaraS,ChaudhuryD,DhankherOP,JaiwalPK.

CombinedexpressionofabarleyclassIIchitinaseandtypeI

ribosomeinactivatingproteinintransgenicBrassicajuncea

providesprotectionagainstAlternariabrassicae.PlantCell

TissueOrganCult.2012;108(1):83–89.

20.MakinoT,SkretasG,KangTH,GeorgiouG.Comprehensive

engineeringofEscherichiacoliforenhancedexpressionofIgG

antibodies.MetabEng.2011;13(2):241–251.

21.FrogerA,HallJE.TransformationofplasmidDNAintoE.coli

usingtheheatshockmethod.JVisExp.2007;(6):253.

22.KirubakaranSI,SakthivelN.Cloningandoverexpressionof

antifungalbarleychitinasegeneinEscherichiacoli.Protein

ExprPurif.2007;52(1):159–166.

23.BenitezT,RincónAM,LimónMC,CodónAC.Biocontrol

mechanismsofThrichodermastrains.IntMicrobiol.

2010;7(4):249–260.

24.ElHadramiA,AdamLR,ElHadramiI,DaayfF.Chitosanin

plantprotection.MarDrugs.2010;8(4):968–987.

25.HermosaR,ViterboA,ChetI,MonteE.Plant-beneficial

effectsofTrichodermaandofitsgenes.Microbiology.

2012;158(1):17–25.

26.JungSC,Martinez-MedinaA,Lopez-RaezJA,PozoMJ.

Mycorrhiza-inducedresistanceandprimingofplant

defenses.JChemEcol.2012;38(6):651–664.

27.EbrahimS,UshaK,SinghB.Pathogenesisrelated(PR)

proteinsinplantdefensemechanism.SciAgainstMicrob

Pathog.2011;2:1043–1054.

28.AhmedNU,ParkJ-I,SeoM-S,etal.Identificationand

expressionanalysisofchitinasegenesrelatedtobioticstress

resistanceinBrassica.MolBiolRep.2012;39(4):3649–3657.

29.KolosovaN,BreuilC,BohlmannJ.Cloningand

characterizationofchitinasesfrominteriorspruceand

lodgepolepine.Phytochemistry.2014;101:32–39.

30.HassaneinSE,Abdel-TawabFM,FahmyEM,etal.Molecular

assessmentofchitinaseactivityintransgenicwheat.EgyptJ

GenetCytol.2016;38(2).

31.SinghD,HaicourR,SihachakrD,RajamMV.Expressionof

ricechitinasegeneintransgeniceggplantconfersresistance

tofungalwilts.IndianJBiotechnol.2015;14:233–240.

32.HuangX,WangJ,DuZ,ZhangC,LiL,XuZ.Enhanced

resistancetostriperustdiseaseintransgenicwheat

expressingthericechitinasegeneRC24.TransgenicRes.

2013;22(5):939–947.

33.MaximovaSN,MarelliJ-P,YoungA,PishakS,VericaJA,

GuiltinanMJ.Over-expressionofacacaoclassIchitinase

geneinTheobromacacaoL.enhancesresistanceagainstthe

pathogen,Colletotrichumgloeosporioides.Planta.

2006;224(4):740–749.

34.SinghHR,DekaM,DasS.Enhancedresistancetoblister

blightintransgenictea(Camelliasinensis[L.]O.Kuntze)by

overexpressionofclassIchitinasegenefrompotato

(Solanumtuberosum).FunctIntegrGenomics.2015;15(4):461–480.

35.YamamotoT,IketaniH,IekiH,etal.Transgenicgrapevine

plantsexpressingaricechitinasewithenhancedresistance

tofungalpathogens.PlantCellRep.2000;19(7):639–646.

36.LiuH,NaismithJH.Asimpleandefficientexpressionand

purificationsystemusingtwonewlyconstructedvectors.

ProteinExprPurif.2009;63(2):102–111.

37.RamosCRR,AbreuPAE,NascimentoALTO,HoPL.A

high-copyT7Escherichiacoliexpressionvectorforthe

productionofrecombinantproteinswithaminimal

N-terminalHis-taggedfusionpeptide.BrazJMedBiolRes.

2004;37(8):1103–1109.

38.FaustG,StandA,Weuster-BotzD.IPTGcanreplacelactosein

auto-inductionmediatoenhanceproteinexpressionin

batch-culturedEscherichiacoli.EngLifeSci.2015;15(8):824–829.

39.LiuZ,HuangY,ZhangR,DiaoG,FanH,WangZ.Chitinase

genesLbCHI31andLbCHI32fromLimoniumbicolorwere

successfullyexpressedinEscherichiacoliandexhibit

recombinantchitinaseactivities.SciWorldJ.2013.

40.ItohT,SugimotoI,HibiT,etal.Overexpression,purification,

chitinaseChiWwithtwocatalyticdomains.BiosciBiotechnol Biochem.2014;78(4):624–634.

41.SakiI,OnagaS,OhnumaT,FukamizoT,TairaT.Purification,

cDNAcloning,andcharacterizationofLysM-containing

plantchitinasefromhorsetail(Equisetumarvense).Biosci

BiotechnolBiochem.2015;79(8):1296–1304.

42.ChalaviV,TabaeizadehZ,ThibodeauP.Enhancedresistance

toVerticilliumdahliaeintransgenicstrawberryplants

expressingaLycopersiconchilensechitinasegene.JAmSoc

HorticultSci.2003;128(5):747–753.

43.MishraM,JalilSU,MishraRK,KumariS,PandeyBK.Invitro

screeningofguavaplantletstransformedwithendochitinase

geneagainstFusariumoxysporumf.sp.psidii.CzechJGenet