Selection of reference genes for expression analysis in the entomophthoralean fungus Pandora neoaphidis

ht t p : / / w w w . b j m i c r o b i o l . c o m . b r /

Genetics

and

Molecular

Microbiology

Selection

of

reference

genes

for

expression

analysis

in

the

entomophthoralean

fungus

Pandora

neoaphidis

Chun

Chen

a,∗

_,

_Tingna

_Xie

a

_,

_Sudan

_Ye

b

_,

_Annette

_Bruun

_Jensen

c

_,

_Jørgen

_Eilenberg

c

a_{ChinaJiliangUniversity,ZhejiangProvincialKeyLaboratoryofBiometrologyandInspection&Quarantine,Hangzhou310018,China} b_{ZhejiangEconomic&TradePolytechnic,Hangzhou310018,China}

c_{DepartmentofPlantandEnvironmentalSciences,UniversityofCopenhagen,DK1871FrederiksbergC,Denmark}

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Articlehistory:

Received4May2015 Accepted23August2015

AssociateEditor:AndréRodrigues

Keywords: Pandoraneoaphidis

Referencegenes QuantitativePCR

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Theselectionofsuitablereferencegenesiscrucialforaccuratequantificationofgene expres-sionandcanaddtoourunderstandingofhost–pathogeninteractions.Toidentifysuitable referencegenesinPandoraneoaphidis,anobligateaphidpathogenicfungus,theexpression ofthreetraditionalcandidategenesincluding18SrRNA(18S),28SrRNA(28S)andelongation factor1alpha-likeprotein(EF1),weremeasuredbyquantitativepolymerasechain reac-tionatdifferentdevelopmentalstages(conidia,conidiawithgermtubes,shorthyphaeand elongatedhyphae),andunderdifferentnutritionalconditions.Wecalculatedtheexpression stabilityofcandidatereferencegenesusingfouralgorithmsincludinggeNorm,NormFinder, BestKeeperandDeltaCt.Theanalysisresultsrevealedthatthecomprehensiverankingof candidatereferencegenesfromthemoststabletotheleaststablewas18S(1.189),28S(1.414) andEF1(3).The18Swas,therefore,themostsuitablereferencegeneforreal-timeRT-PCR analysisofgeneexpressionunderallconditions.Theseresultswillsupportfurtherstudies ongeneexpressioninP.neoaphidis.

©2015SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Pandora neoaphidis is one of the most important fungal pathogens of aphids and has great potential for use in biocontrol.1–4 _{Althoughthe lifecycle ofP.neoaphidis iswell}

∗ _{Correspondingauthor.}

E-mail:aspring@cjlu.edu.cn(C.Chen).

known,adetailedmolecularunderstandingofgene expres-sionduringtheinfectionprocessisstilllacking. Adiversity ofgenescanbeup-ordownregulatedduringhost–pathogen interactions,5 _{however,littleisknownabouttheexpression} ofreferencegenesinthisparticularentomophthoralean fun-gus.

http://dx.doi.org/10.1016/j.bjm.2015.11.031

1517-8382/©2015SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

severalfungi.8–11_{However,anumberofcriticalaspectsmust} beoptimizedforeffectiveqRT-PCRanalysis;theseincludethe efficiencyofRNAextraction,thequalityoftheRNA,the pres-enceofinhibitors,theefficiencyofthereversetranscription and the selectionofa suitable referencegene as an inter-nalcontrol.12_{Whilethemajorityofthesepotentialsourcesof} errorcanbeavoidedbyadheringcloselytostandardized pro-tocols,selectionofappropriatereferencegenesisfrequently thegreatestchallengebecauseitrequiresaspecies-specific solution.13,14 _{Anidealreference geneshouldhaveconstant} expressionacrossallsamplestobeinvestigated,regardlessof biotype,developmentalstageoranyotherbiologicalor exper-imentalvariability.

Selectionofaninappropriatereferencegenewithvariable expression,leadstoerroneouscalculationsoftheexpression oftarget genesand,therefore,incorrectassumptionsabout thefunctionofthosetargetgenes.15_{Identificationofsuitable} referencegenesisessentialforaccuratetranscriptexpression analysis.Severalstatisticalalgorithmshavebeendeveloped toidentifythemostsuitableinternalcontrolswiththeleast variabilityinexpression;thesealgorithmsarebasedon qRT-PCRdatafromagivensetofcandidategenesandrankputative referencegenesaccordingtotheirexpressionstability,thereby indicatingthebestreferencegeneorcombinationofreference genesforaccuratenormalization.Thefourmostcommonly usedalgorithmsforassessingtheappropriateness of refer-encegenesare geNorm,16 _NormFinder,17 _Best-Keeper18 _and Delta Ct.19 _{These software packagesare freely available to} downloadfromtheauthors’websitesandhavebeenwidely usedtoidentifysuitablereferencegenes.20,21

Inthisstudy,threeputativehousekeepinggenes(18S,28S

andEF1)wereevaluatedaspotentiallyreliablereferencegenes inP.neoaphidisusingqPCR.Fungalinfectionofthehostusually involvesmultipledevelopmentalstages.22Therefore,profiling gene expression inmultiple development stages is impor-tantforunderstandingthemechanismsofpathogenesis.We testedfourdevelopmentalstagesincludingconidia,conidia with germ tubes, short hyphae and long hyphae. Profiling geneexpressionundervariousnutritionalconditionsisalso aroutineapproachtostudygenefunction.Weevaluatedthe stabilityofgeneexpressioninthreedifferentnutrientmedia. Theexpressionstabilityofeachgeneinallsampleswas ana-lyzedusingthegeNorm,NormFinder,Best-KeeperandDelta Ctprograms,andthemostsuitablereferencegeneforaccurate normalizationwasselected.

Material

and

methods

Isolateandcultureconditions

P.neoaphidisisolateARSEF5403wasobtainedfromtheUSDA CollectionofEntomopathogenicFungalCultures,Ithaca,NY. ItwassubculturedonSEMA(Sabourauddextroseagar[SDA]

Propagulesatdifferentstagesofgerminationwereprepared. Mycelialmats from liquidculturewereproduced usingthe methoddescribedbyXuandFeng.24_{Primaryconidiaactively} dischargedfromthemycelialmatduringtheperiodofpeak sporulationwereharvestedinto0.01mol/Lsterilephosphate buffered saline (PBS) solution (130mmol/L NaCl, 7mmol/L Na2HPO4,3mmol/LNaH2PO4,pH7.3).25Theconidial

suspen-sionwasfilteredthroughglasswooltoremoveanymycelia and the conidia centrifuged at 4500rpmfor 10min before beinginoculatedintoGLENmediuminflasks26_ata concen-trationof1010_{conidiapermilliliterfollowedbyincubationat} 20◦Cand150rpmina12:12light:darkregimenfordifferent periodsoftime.2 _{Samplesweretakenafter∼0hforconidia,} after∼6hforconidiawithgermtubes,after∼12hforearly, shorthyphae(i.e.thelengthofgermtubewasamaximum of200␮m),andafter24hforelongatedhyphae(i.e.hyphae that exceeded200␮m).Ateach stagethe fungalstructures wereobservedmicroscopicallytoensuretheyhadattainedthe correctdevelopmentalstage(Fig.1).Samplesofeach develop-mentalstagewerecollectedaftervacuumfiltrationthrough Whatman54paperandstoredat−80◦_{CuntiltotalRNAwas}

isolated.Therewerethreereplicateflasksforeach develop-mentalstage,i.e.12flasksintotalweresetup.

Toprovidesamplesgrownunderdifferentnutritional con-ditions, themyceliumfrom halfaPetridish (agarmedium removedusingascalpel)wasaddedto100mLflasks contain-ing either30mLGLENmedium,26_{30mLofGrace’smedium} (Invitrogen,USA)or30mLofOS-SDBmedium(Sabouraud Dex-troseBroth[SDB;Difco,BD,USA]supplementedwith0.5%(v/v) sesameoiland0.1%(w/v)sugarestersoffattyacids[Emulsifier E473,CASNo.37318-31-3,LiuzhouGaotongFoodChemicals Co.Ltd.,China]).Flaskswereincubatedat20◦Cand150rpmin a12:12light:darkregimenforthreedays.2_{Myceliumsamples} grownunderdifferentnutritionalconditionswere collected byvacuumfiltrationthroughWhatman54paperandstored at−80◦_{CuntiltotalRNAwasisolated.Therewerethree}

repli-cate flasksforeach nutritional conditioni.e.nine flasksin total.

Candidategeneandprimerdesign

Nucleotide sequences of the 18S and 28S genes (Genbank accessionnumbersHQ677591.1andEF392405.1,respectively), were downloaded from Genbank (http://www.ncbi.nlm.

nih.gov/genbank/),whiletheEF1genesequencewasacquired

through degenerate PCR. Primer pairs for each gene were designed using the Primer Premier 5.0 program (Table 1). Specificity of primer pairs for each candidate gene was confirmed using melting curve analysis and agarose gel electrophoresis.

TotalRNAisolationandcDNAsynthesis

RNAwaspreparedforeachsampleusingtheRNeasyMiniKit (QIAGEN,Cat.No.74104,Mississauga,Canada),andgenomic

Fig.1–ViewsofP.neoaphidispropagules.Topreparepropagulesatdifferentdevelopmentalstages,primaryconidia producedbyamycelialmatwereincubatedinGLENbroth.Thecultureswerecentrifugedtoharvestconidiaat0h(a), conidiawithgermtubesat6h(b),earlyhyphaeat12h(c)andelongatedhyphaeat24h(d).Propaguleswerestainedwith lactophenolandobservedunderamicroscope.Scalebar:50␮m.

DNAwaseliminatedbyloadingRNase-freeDNaseIintothe fil-tercolumn(QIAGEN).Nucleicacidextractionwasperformed accordingtothemanufacturer’sinstructions.Thequalityof theRNAwascheckedbyelectrophoresison1%agarosegels torevealany contaminatinggenomicDNAandintactrRNA subunits28Sand18S.Nucleicacidconcentrationswere mea-suredusingaNanodrop1000(ThermoScientific,Wilmington, DE,USA).

First-strand cDNA was synthesized using random hex-amerprimersandSureStartTaqDNApolymeraseaccording tothemanufacturer’sinstructions(AgilentTechnologies,Cat. No.20820,Santa Clara,CA, USA).Toavoiderrorscaused by contamination with genomic DNA, all RNA samples were treated with RNase-free DNase I before reverse transcrip-tion(AgilentTechnologies,USA).OnemicrogramoftotalRNA wasused forcDNA synthesis; thecDNA was subsequently dilutedwithnuclease-freewater(QIAGEN)to20ng/␮L.The cDNA mixtureswere diluted 1:10 withnuclease-free water

(QIAGEN)andstoredat−20◦_{CforsubsequentquantitativePCR}

analysis. QuantitativePCR

Real time RT-PCR amplification mixtures (25␮L) contained 20ngtemplatecDNA,2×BrilliantIISYBRGreenmastermix buffer with fluorescein for dynamic well factor collection (12.5␮L; Agilent Technologies) and 400nmol/L each of the forwardandreverseprimers(EurofinsGenomics,Ebersberg, Bayern, Germany). The reaction was performed using a StratageneMx3000P®_{system.PCRwasaccomplishedaftera}

10minactivation/denaturation stepat95◦C,followedby40 cycles of30seach at95◦C,60seach at60◦Cand30seach at72◦C.Fluorescencewasdetectedateach polymerization step.TotestthePCRefficiencyofeachprimerpair,acDNA mixturecontainingequalamountsofcDNAfromallsamples wasusedasthetemplate.Theten-folddilutionseriesofthe

Table1–PrimersequencesofselectedreferencegenesforqRT-PCRinP.neoaphidis.

Gene Accessionnumber Primerpairs Ampliconsize(bp)

18S HQ677591.1 F:CAAACCCGAGCAATAGTC R:GTAGGAGCCCGATAGTAAA 179 28S EF392405.1 F:TCTTATCAGTCTCAGCACCTTG R:TCTGTCAATCCTTACTATGTCTGG 181 EF1 DQ275343.1 F:GACGCACTCCTTGTTGAT R:CCTTGCCACTCTGGTTCT 82

Statisticalanalyses

Thesampleswere dividedintotwogroups: different devel-opmental stages (12 samples) and different nutritional conditions (9 samples). The expression levels of the can-didate reference genes were determined from the cycle threshold(Ct)value,thenumberofPCRcyclesatwhichthe quantity of amplified targets reached a specific threshold levelofdetection. Theexpressionstabilityofthereference genes was evaluated using the four programs, geNorm,16 NormFinder,17_BestKeeper18_andDeltaCt19_{toanalyzeallthe} rawCtvaluesfromthetwogroups;inordertobalancethe rankingofthecandidatereferencegenesacrossthedifferent algorithms,expressionstabilitywasevaluatedintheprogram

http://www.leonxie.com/referencegene.php?type=reference,

to determine an overall comprehensive stability value for eachcandidatereferencegene.

Results

Expressionprofilingofcandidatereferencegenes

Thecalculatedexpressionlevels(Ctvalues)ofthethreegenes evaluatedvariedfrom10.65to28.75,whichrepresentsquite alargedegreeofvariation(Fig.2).The18Sgenewas iden-tified as the most abundant transcript in all samples; the meanCtvalueforthe18Sgenefromdifferent developmen-talstagesanddifferentnutritionalconditionswas12.62and 19.76,respectively.ThemeanCtvalueforthe28Sgenefrom differentdevelopmentalstagesanddifferentnutritional con-ditionswas15.67and23.74,respectively.Incontrast,themean CtvaluefortheEF1genefromdifferentdevelopmentalstages and different nutritional conditions was 24.25 and 25.65, respectively.

Cycle threshold (C

18

8

18S 28S EF1

Fig.2–MeanCtvalueofthecandidatereferencegenesat differentdevelopmentalstagesandunderdifferent nutritionalconditions.Errorbarsrepresentstandard deviations.nindicatesthenumberofsamples.

Candidategeneexpressionstabilityofdifferent developmentalstagesandunderdifferentnutritional conditions

UsingthealgorithmsgeNorm,NormFinderandDeltaCt,for developmentalstageandnutritionalconditions,the18Sgene wasthemoststableofthethreegenes,followedinrankorder bythe28SgeneandfinallytheEF1gene(Fig.3).However,using theBestKeeperalgorithm,the28Sgenewasmuchmorestable thanthe18SorEF1genes(Fig.3).

Specifically, using geNorm software analysis, the P. neoaphidis expression stability values at different develop-mentalstagesandunderdifferentnutritionalconditionsfor the three candidate reference genes(M value) were in the order:18S(0.457)>28S(0.534)>EF1(0.749)and18S(0.389)>28S

(0.557)>EF1(0.607),respectively.UsingNormFindersoftware analysis, theP. neoaphidis expressionstability valuesat dif-ferentdevelopmentalstagesandunderdifferentnutritional conditions for the three candidate reference genes (stable value)wereintheorder:18S(0.084)>28S(0.264)>EF1(0.509) and 18S (0.118)>28S (0.355)>EF1 (0.403),respectively. Using

18S

18S

18S

18S

28S

28S

28S

28S

EF1

EF1

EF1

EF1

18S

18S

18S

18S

28S

28S

28S

28S

EF1

Developmental stages geNorm Ranking order 1 Most stable 2 Intermediate stability 3 Least stable NormFinder BestKeeper Delta Ct Nutritional conditions

EF1

EF1

EF1

Fig.3–ComparisonoftherankinginexpressionstabilitycalculatedbygeNorm,NormFinder,BestKeeperandDeltaCt.The analysiswasperformedseparatelyforeachsamplegroupundertheconditionsexaminedandtherankingorderswere highlightedbydifferentgraytonesfromwhitetodark,darkergrayindicatesgreaterstability.

3 0 185 1.189 285 1.414

Comprehensive gene stability

≤= Most stable genes Least stable genes =≥

EF1 3 0.5 1 1.5 2 2.5

Fig.4–Comprehensiverankingofthecandidatereference genes.

DeltaCtsoftwareanalysis,theP.neoaphidisexpressionstability valuesatdifferentdevelopmentalstagesandunderdifferent nutritionalconditionsforthethreecandidatereferencegenes (stable value) were inthe order:18S (1.19)>28S (1.20)>EF1

(2.02)and18S(0.41)>28S(0.55)>EF1(0.59),respectively. Overallstabilityrankingofcandidatereferencegenes Therankingfromthecomprehensivegenestabilityanalysis ofcandidatereferencegeneswas18S(1.189)>28S(1.414)>EF1

(3.000)(Fig.4).The18Sgenecould,therefore,beselectedas themoststablereferencegenefornormalizationofqRT-PCR.

Discussion

qRT-PCRisbecomingmoreandmoreimportantasaresearch tooltoidentifyandcharacterizegenesthataredifferentially expressedduringdifferenttypesofgrowthand atdifferent developmentalstages.7_{Anappropriatereferencegeneorset} of reference genes are required for accurate gene expres-sionanalysisand,beforethisstudy,werenotavailableforP. neoaphidis.

DuetoalackofsequencedataforthegenusPandora,we selectedthreehousekeepinggenes(18S,28SandEF1)as can-didatereferencegenesforexpressionstudiesinP.neoaphidis

(Table 1).Wedid notincludethe beta-tublin(␤-tublin)

ref-erence gene because the primers we developed forit also amplifiedthehostaphid’scDNAwhichwouldprecludeitsuse ingeneexpressionstudiesoftheinteractionbetweenthe fun-gusandthehostaphid(unpublisheddata).Priortodoingthe qPCR,wecheckedthespecificityofthedesignedprimers; anal-ysisofthemeltingcurvesshowedanabsenceofdimersinthe PCRamplifications.

Endogenouscontrols commonlyinclude the use of sin-gle,constitutivelyexpressed,housekeepinggenes.However, theexpressionofasinglehousekeepinggenecanvary con-siderablybetween samples27–29 _{and may not, therefore, be} acceptablefornormalizationofreal-timeRT-PCR.Thisisthe reasonthatwewantedtoselectgeneswithreliableand accu-rate expression levels that were expressed consistently at differentdevelopmentalstages(primaryconidia,conidiawith germtubes,earlyhyphaeandelongatedhyphae)andunder

differentnutritional conditions(GLEN,Grace’smediumand OS-SDB).

Ifonlyonesoftwarepackage,withonealgorithm,isused to analyze the biological stability of expression in candi-datereferencegenesitisoftendifficulttoselecttheoverall bestreferencegene.30_{Therefore,inthisstudy,a} comprehen-siveonlinetoolbasedonfourdifferentalgorithms(geNorm, NormFinder, BestKeeper and Delta Ct) was adopted. The resultsfor geNorm,Normfinder and Delta Ctwere all very similar and showed that, of the three candidate reference genes,expressionofthe18Sgenewasthemoststable;this resultisconsistentwithanumberofotherreports.31,32 How-ever, thestabilitycalculatedbyBestKeeperwasdifferentto theotheralgorithms;the28Sgenewasmorestablethanthe

18Sgene,andbothweremorestablethantheEF1gene.The reasonforthismaybebecausetheBestKeeperalgorithmuses theoriginalCtvaluesasinputdata,sothecalculated statis-tics canindicate false differences.33,34 _{However,the values} forthecoefficientofvariation(CV)wereveryclose;0.77and 0.78,respectivelyforthedifferentdevelopmentalstages,0.45 and0.53,respectivelyforthedifferentnutritionalconditions. Therefore, weusedvaluesfromall fouralgorithmstorank candidatereferencegenestabilityunderalltheexperimental conditionsasacomprehensiveindicatorofthemeanstability ofgeneexpressionasdid;overallthe18Swasstillthemost stablereferencegeneintermsofexpression.

Throughstabilityanalysis,weidentifiedthatthe18SrRNA referencegenewassuitablefornormalization ofqPCRdata undermultipleexperimentalconditions.Ourstudywasbased onqPCR,sothefindingswillprovidedirectguidanceforfuture qPCRexperimentsonP.neoaphidis.Moreover,thesefindings mayalsobeusefulforNorthernblot andreverse transcrip-tion PCR, techniques that also require reference genes for normalization.8,35,36 _{Itisalso possiblethat, using a} combi-nation of two reference genes might further improve the reliabilityofgeneexpressionbyRT-qPCRofP.neoaphidis.

Ineukaryotes,thegenesmostconservedandmostwidely usedare thosethat encoderibosomalRNA(rRNA).37 _These genes encode small subunit ribosomal RNA (18S) and two largesubunitribosomalRNA(28S and5.8S),ina transcrip-tionunit,constitutingabout85–90%oftotalcellularRNA,and are veryuseful asinternalcontrols.38 _{Though 28Stogether} with18SareallprocessedfromasingleprecursorRNAafter transcriptionfromtherRNAcistron,28Sand18Sare synthe-sizedrespectivelybyRNApolymeraseIandRNApolymeraseII; thesearecompletelydifferenttomRNAsynthesisand inde-pendentfromit.Thus,thereareonlyrare changesinrRNA levelswhilevariousconditionsaffectmRNAexpression.As aclassichousekeepinggene, the18Sgenehasbeenwidely usedasareferenceforgeneexpressionanalysis39_;for exam-pleintheentomopathogenicfungusBeauveriabassiana,8,32,35

andotherspecies.9,40,41_{Forthisreasonwearenotsurprised} thatitisalsousefulforgeneexpressionstudiesofP.neoaphidis.

However,ourunderstandingofP.neoaphidisdevelopmentand metabolismisstilllimited,soitislikelythatfurtherworkon thestabilityofgeneexpressioninother,asyetnot-cloned, ref-erencegenessuchasGAPDHand␤-actin,willalsobeuseful. Furthermore,theuseofseveralreferencegeneswouldallow moreaccurateandreliablenormalizationofgeneexpression data.16

based gene expression analysis ofthe entomophthoralean fungusP. neoaphidis. Ourresults suggest that the 18Sgene wouldbethemostreliablefornormalizingtheexpression lev-elsinmostsampleseries.Theseresultsprovideafoundation forthemoreaccurateandwidespreaduseofRT-qPCRinthe analysisofgeneexpressioninP.neoaphidis.

Normalization is a major issue when analyzing gene expressioninresponsetovariousexperimentalconditions, anduseofincorrectnormalizationtargetscouldleadto erro-neousinterpretationofquantitativedata.Thepresentstudy considered the issue of development and nutrition, thus the findings can aid in understanding the mechanisms of developmentandmetabolism.Fortheissueoffungus/insect interactions, it is more complicated since the condition involves fungal propagules and host tissue. Thus, further experimentsarerequiredtoassessandvalidatemore refer-encegenessuitableforevaluatingfungus/insectinteractions.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

Thisstudy wasfunded by:the Natural ScienceFoundation ofChina(31461143030),theInternationalFoundationfor Sci-ence(C/4822-1),theScienceandTechnologyPlanningProject ofZhejiangProvince(2013C37087)andtheZhejiangNatural ScienceFoundation(LR12C03001).

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