RNA interference-inducing hairpin RNAs in plants

scientific

reportscientificreport

RNAinterference-inducinghairpinRNAsinplants

actthroughtheviraldefencepathway

AdrianaF.Fusaro1,LouisaMatthew1,NeilA.Smith1,ShaunJ.Curtin1,2,JasminaDedic-Hagan1,3,GeoffA.Ellacott1,JohnM.Watson1,Ming-BoWang1,ChrisBrosnan4,BernardJ.Carroll4&PeterM.Waterhouse1+

1CSIRO

PlantIndustry,Canberra,Australia,2CharlesSturtUniversity,WaggaWagga,Australia,3AustralianNationalUniversity,

Canberra,Australia,and4TheUniversityofQueensland,StLucia,Australia

RNAinterference(RNAi)iswidelyusedtosilencegenesinplantsandanimals.ItoperatesthroughthedegradationoftargetmRNAbyendonucleasecomplexesguidedbyapproximately21nucleo-tide(nt)shortinterferingRNAs(siRNAs).Asimilarprocessregulatestheexpressionofsomedevelopmentalgenesthroughapproximately21ntmicroRNAs.PlantshavefourtypesofDicer-like(DCL)enzyme,eachproducingsmallRNAswithdifferentfunctions.Here,weshowthatDCL2,DCL3andDCL4inArabidopsisprocessbothreplicatingviralRNAsandRNAi-inducinghairpinRNAs(hpRNAs)into22-,24-and21ntsiRNAs,respectively,andthatlossofbothDCL2andDCL4activitiesisrequiredtonegateRNAiandtoreleasetheplant’srepressionofviralreplication.WealsoshowthathpRNAs,similartoviralinfection,canengenderlong-distancesilencingsignalsandthathpRNA-inducedsilencingissuppressedbytheexpressionofavirus-derivedsuppressorprotein.ThesefindingsindicatethathpRNA-mediatedRNAiinplantsoperatesthroughtheviraldefencepathway.

Keywords:Dicer-like;siRNA;signal

EMBOreports(2006)7,1168–1175.doi:10.1038/sj.embor.7400837

genes(Wesleyetal,2001).TheplantspeciesArabidopsisthalianahasfourDicer-like(DCL)proteinsthatproducedifferentlysizedsmallRNAs,whichdirectasuiteofgene-silencingpathways.DCL1producesmicroRNAs(Parketal,2002),DCL2generatesbothstress-relatednaturalantisensetranscriptshortinterferingRNAs(siRNAs;Borsanietal,2005)andsiRNAsagainstatleasttwoviruses(Xieetal,2004;Akbergenovetal,2006),DCL3producesapproximately24ntsiRNAsthatdirectheterochromatinformation(Xieetal,2004)andDCL4generatesbothtrans-actingsiRNAs,whichregulatesomeaspectsofdevelopmentaltiming,andsiRNAsinvolvedinRNAinterference(RNAi;Dunoyeretal,2005;Gasciollietal,2005;Xieetal,2005).ToobtainfurtherdetailsofthepathwaysinvolvedinRNAiandvirusdefence,weexaminedthesizeandefficacyofsmallRNAsengenderedbymanyRNAi-inducinghpRNAs,twodistinctvirusesandaviralsatelliteRNA(Sat)indifferentsingleandmultipledcl-mutantArabidopsisbackgrounds.

RESULTSANDDISCUSSION

ExaminingthesiRNAprofilesofmorethan20differenthpRNAconstructsinwild-type(WT)Arabidopsis,targetingeitherendo-genesortransgenes,showedthatthepredominantsizeclassisusuallyaround21nt,withasmallerproportionaround24ntRNAs(Fig1B).ToexaminehpRNA-derivedsiRNAsindclmutants,anhpRNAconstruct(hpPDS),regulatedbytheArabidopsisRubiscosmallsubunit(SSU)promoter,wasmadethattargetedthephytoenedesaturase(PDS)gene.SilencingPDScausesaphotobleachedphenotypeinplants.ThehpPDSconstructwastransformedintoWTplantsandintoplantsthatwerehomozygousmutantsfordcl2,dcl3ordcl4-1;ithasalreadybeenshownthatRNAiandsiRNAproductionisuncompromisedinthestrongestDCL1hypomorphicmutantavailable—dcl1-9(Finneganetal,2003).TheprimaryWTanddcl2transformantsshowedsimilardegreesofphotobleaching,anddcl3transformantsshowedextremephotobleaching(Fig1C).ThisshowsthatneitherDCL2norDCL3activityisrequiredforRNAiandsuggeststhattheabsenceofDCL3activityenhancessilencing.Interestingly,theago4mutantbackgroundhasalsobeenreportedtoenhancehpRNA-mediatedsilencing(Zilbermanetal,2004).BothDCL3

&2006EUROPEANMOLECULARBIOLOGYORGANIZATION

INTRODUCTION

TransformingplantswithhairpinRNA(hpRNA)constructs(Fig1A)hasbeenusedtodiscoverorvalidatethefunctionsofasteadilyincreasingnumberofgenes.Inseveralcases,thehpRNA-transformedplantsphenocopynullmutantsofthetarget

CSIROPlantIndustry,POBox1600,Canberra,AustralianCapitalTerritory2601,Australia

2SchoolofWineandFoodSciences,CharlesSturtUniversity,LockedBag588,WaggaWagga,NewSouthWales,2678,Australia3

SchoolofBiochemistryandMolecularBiology,AustralianNationalUniversityCanberra,AustralianCapitalTerritory2601,Australia4

DepartmentofBiochemistryandMolecularBiology,TheUniversityofQueensland,Queensland4071,Australia+

Correspondingauthor.Tel:þ610262465365;Fax:þ610262465000;E-mail:peter.waterhouse@csiro.au

Received11May2006;revised15September2006;accepted18September2006;publishedonline13October2006

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