Forest Pathology. 2017;e12354. wileyonlinelibrary.com/journal/efp  | 1 of 9https://doi.org/10.1111/efp.12354

© 2017 Blackwell Verlag GmbH

Received:25October2016  |  Accepted:8April2017DOI: 10.1111/efp.12354

O R I G I N A L A R T I C L E

The distinct roles of Argonaute protein 2 in the growth, stress responses and pathogenicity of the apple tree canker pathogen

H. Feng1 | M. Xu1 | Y. Liu1 | X. Gao1 | Z. Yin1 | R. T. Voegele2 | L. Huang1

1College of Plant Protection and State Key LaboratoryofCropStressBiologyforAridAreas,NorthwestA&FUniversity,Yangling,China2InstitutfürPhytomedizin,UniversitätHohenheim,Stuttgart,Germany

CorrespondenceLiliHuang,CollegeofPlantProtectionandState Key Laboratory of Crop Stress Biology forAridAreas,NorthwestA&FUniversity,Yangling,China.Email: huanglili@nwsuaf.edu.cn

Funding informationNationalNaturalScienceFoundationofChina,Grant/AwardNumber:31501591;ChinaPostdoctoralScienceFoundationSpecialFunding,Grant/AwardNumber:2016T90953;DoctoralScientificResearchFoundationofNorthwestA&FUniversity

Editor: S. Woodward

SummaryValsa mali (V. mali),thecausalagentofappletreeValsacanker,severelydamagesappleproduction,amajoreconomiccropinChina.Todate,ourunderstandingofthemo-lecular mechanisms associated with the pathogenicity of V. mali isstill limited.RNAinterferenceparticipatesinvariousbiologicalprocessesinmulticellularorganisms.TheArgonaute proteins (AGOs),which are a core component of the RNA interferencesystem,playkeyrolesinvegetablegrowth,environmentalresponsesandfungalpath-ogenicity.Previously,transcriptomeanalysisrevealedthattheAGO2gene(VMAGO2) was up- regulated during V. maliinfection,suggestingthatVMAGO2 plays a potential roleinpathogenicity.Inthisstudy,weinvestigatedthepotentialrolesofVMAGO2 in thegrowth,stressresponsesandpathogenicityofV. mali. VMAGO2 was isolated and found to be orthologous to AGO2 of Neurospora crassa and Fusarium graminearum. Real- time quantitative PCR analysis showed that VMAGO2 expression was 3.4- fold higher than that of the control (mycelium) at 24 hr post- inoculation (hpi). Six positive VMAGO2mutantsweregeneratedusingdouble-jointPCRandPEG-mediatedtrans-formation. Deletion of VMAGO2 did not result in any obvious phenotypic change whencomparedwiththatofwild-typestrain03-8.Furthermore,thecolonialmorphol-ogy was not obviously affected when the mutants were subjected to osmotic and pH stresstreatments.However,theknockoutmutantsdidnotgrowonPDAwith0.05%H2O2.Moreimportantly, infectionassaysshowedthattheaveragelesiondiameter/lengthresultingfrommutantinfectionswas34.8%and19.8%smalleronappleleavesandtwigs,respectively,thanthoseresultingfromwild-typeinfections.Allsixpositivemutantsshowedaconsistentphenotype,andthedefectsofthemutantswerefullycomplementedbyre-introducingtheWTVMAGO2 allele. Our results demonstrated that VMAGO2 plays an important role in H2O2 tolerance and the pathogenicity of V. mali.

1  | INTRODUCTION

AppletreeValsacanker,whichiscausedbyValsa mali,aweakparasiticpathogen,isseverelyrestrictingthedevelopmentoftheappleindustryinChina(Cao,Guo,Li,Sun,&Chen,2009;Wang,Zang,Yin,Kang,&Huang,2014).InvestigatingthepathogenesisofV. mali is important for developing a resistance breeding strategy and for identifying new fun-gicidetargetsites;however,themolecularmechanismsunderlyingthe

pathogenesis of V. maliarecomplexandnotfullyunderstood.Althoughrecent analyses of the V. mali genome and transcriptome informa-tionhaveprovided ameans to explore thepathogenesis, and somepathogenesis- related genes have been identified and characterized (Ke etal.,2014;Lietal.,2015;Yin,Zhu,Feng,&Huang,2016;Yinetal.,2015),theRNAinterference(RNAi)pathwayofV. mali is still unknown.

RNAi,which ismediatedby small regulatoryRNAsanda familyofribonucleo-proteincomplexesorRNA-inducedsilencingcomplexes

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(RISCs), is a powerful mechanism of gene silencing that underliesmany aspects of eukaryotic biology (Pratt &MacRae, 2009). ThereareseveralRNAprocessingpathwaysthatcanleadtothegenerationofvarioussmallRNAs (sRNAs), suchas short interferingRNAs (siR-NAs), microRNAs (miRNAs), transfer RNA-derived fragments (tRFs)andqiRNA(Agrawaletal.,2003;Bartel,2004;Jöchletal.,2008;Leeetal.,2009).ThesesRNAscanonlyguidegenesilencingwhentheyareboundtoArgonauteproteins(AGOs)throughasimilarbase-pairingmechanismwiththeirtargetRNAtranscripts(Kim,Heo,&Kim,2010).Therefore,studyingthefunctionsofAGOsmayleadto insights intopotentialnewrolesofRNAi.

AsthecorecomponentsofRISC,AGOproteinscontainfourdo-mains: theN-terminal,PAZ,MIDandPIWI functionaldomains.ThePAZandPIWIdomains are the twomain structural featuresof theAGOproteins (Joshua-Tor,2004).ThemainfunctionofthePAZdo-main is to identify and combine with the two overhanging nucleotides atthe3′endofthesmalldouble-strandedRNA,whilethefunctionofthePIWIdomainistoprocessmRNAsasitcontainsacatalyticcentre(Jinek&Doudna,2009;Yuanetal.,2005).

Argonaute proteins have been shown to play significant rolesin regulating gene expression, chromosome structure and func-tion, andvirus defencemechanisms (Hutvagner& Simard, 2008).In Drosophila,AGO1 andAGO2 can participate in the generationofmiRNAs and siRNAs, respectively (Okamura, Ishizuka,&Siomi,2004). In plants, manyAGOs have been reported to be involvedin resistance to different pathogens (Agorio&Vera, 2007;Duan,Fang,&Zhou,2012;Ellendorff,Fradin,deJonge,&Thomma,2009;Mallory&Vaucheret,2010).AGOproteinshavebeenhighlycon-served throughout evolution and are frequently present in eukary-otes.Similarly,AGOproteinsarealsopresent in thevastmajorityoffilamentousfungi(Hu,Stenlid,Elfstrand,&Olson,2013;Nunes,Sailsbery, & Dean, 2011). Previously, the function of AGOs infungi has been primarily assessed based on phenotypic changes and changes in the sRNA levels in theAGO-knockout strains. Ina model fungal system such as Neurospora crassa, qde-2 (quell-ing deficient- 2) and SMS-2 (suppressor of meiotic silencing- 2) are known to be involved in silencing meiosis, and qde-2 is also as-sociatedwith the generation of qiRNAs andmilRNAs (Lee, Pratt,McLaughlin, &Aramayo, 2003; Lee etal., 2009).Among the fourAGOsinCryphonectria parasitica,onlyAGO2isinvolvedininducingRNAsilencing-mediatedantiviraldefenceandpromotingviralRNArecombination(Sun,Choi,&Nuss,2009).Finally,nutritionalstress-induced vegetative development and autolysis were significantly affected when the ago-1 gene was deleted in Mucor circinelloides,indicating that ago-1 may play a role in the response to environmen-talsignals(Cervantesetal.,2013).

Based on the published genome sequence of V. mali,threeAGOs(VMAGO1,VMAGO2 and VMAGO3) appeared to be present in this fungus(Yinetal.,2015).Interestingly,transcriptomeanalysisrevealedthat VMAGO2 was highly induced during V. mali infection (Ke etal.,2014),suggestingapotentialregulatoryroleinthepathogeninfectionprocess.Onthebasisofthisfinding,thisstudywasdesignedtofur-ther investigate the potential roles of VMAGO2inthegrowth,stress

responses and pathogenicity of V. mali.TheobjectiveofthisstudywastoprovideinsightsintothepotentialmechanismsofsRNA-mediatedregulation of V. mali infection.

2  | MATERIALS AND METHODS

2.1 | Fungal strains and vector

The wild-type strain 03-8 (WT) of V. mali was provided by the Laboratoryof IntegratedManagementofPlantDiseases,CollegeofPlant Protection, Northwest A&F University, Yangling, Shaanxi, PRChina. Strainsweremaintainedonpotatodextrose agar (PDA)me-diumat25°Cinthedark.ForDNAextraction,thestrainsweregrownin liquid potato dextrose broth (PDB)medium. The fungal genomicDNA was extracted using the CTAB method (Möller, Bahnweg,Sandermann, & Geiger, 1992). The vector pBIG2RHPH2-GFP-GUSwas used for gene deletion.

2.2 | Characterization of the VMAGO2 sequence

TheVMAGO2 gene sequence was identified from the available V. mali genomedatabase.TheVMAGO2 gene sequences were deposited in GenBankunderaccessionnumberKT191022.Thedomainarchitec-ture was analysed using InterProScan (http://www.ebi.ac.uk/cgi-bin/iprscan/).Aphylogenetic analysisof thededucedprotein sequenceand the corresponding sequences of other species was performed usingtheDNAMAN,version6.0.

2.3 | Generation of ΔVMAGO2 mutants

The double-joint PCR approach was used for vector construc-tion to generate VMAGO2 deletion mutants (Yu etal., 2004). Thestrategy consisted of replacing VMAGO2 with the hygromycin- phosphotransferase (hph) cassette, which was amplified fromPHIG2RHPH2-GFP-GUSusingprimersofHYG-FandHYG-R(TableS1). The upstream and downstream flanking sequences were am-plified with primers of VMAGO2-1F/2R and VMAGO2-3F/4R, re-spectively (Table S1). The amplified fragments (upstream flankingsequence,hph and downstream flanking sequence) were fused using double-jointPCR(Yuetal.,2004).PCRproduction,amplifiedwiththenestedprimerpairVMAGO2-CF/CRusingthelinearhph cassette as template,wasconcentratedandtransformedtotheprotoplasmoftheWTstrainusingthePEG-mediatedtransformationmethoddescribedbyGao,Li,Ke,Kang,andHuang(2011).AsinglehyphafromacolonyofeachtransformantwasplacedinthecentreofaPDAmediumplateusingthesinglehyphaseparationmethod.Afterculturingfor3daysinthedarkat25°C,thestrainsweretransferredtoPDAmediumplatescontaining 50 μg/ml of hygromycin and 100 μg/ml of cephalosporin for three generations of continuous culture to positively select for hygromycin- resistant transformants. Hygromycin- resistant transfor-mantswerescreenedbyPCRusingtheVMAGO2-5F/6RandH852/H850 primers, and confirmed using the VMAGO2-1F/H855R andH856F/VMAGO2-4Rprimers(TableS1,FigureS1).Putativemutants

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werefurtherconfirmedbySouthernblotanalysis.ThegenomicDNAofWTandΔVMAGO2 mutants was isolated and digested using the Xho I and Pst Irestrictionenzymes.TheHYG probe was amplified with theH850/H852primers,andtheVMAGO2 probe was amplified with theVMAGO2-5F/6Rprimers. The detailed procedure for Southernblotwasfollowedtothemanufacturer’sinstructionsoftheDIGDNALabelingandDetectionKitII(Roche,Mannheim,Germany).

2.4 | Observations of the colonial morphology of mutants and WT strains

Mycelial agar discs (5- mm- diameter) of the ΔVMAGO2 and ΔVMAGO2- C mutant strains and theWTstrainwereplaced in the centreofPDAmediumplatesandculturedinthedarkat25°C.AllthestudieswereconductedintriplicatewiththreePetridishesineachrepetition.Thecolonial morphology was photographed after 2 days of incubation.

2.5 | Measurement of stress responses

To determine the sensitivity of the strains to osmotic stress,5- mm- diameter mycelial agar discs of the ΔVMAGO2 and ΔVMAGO2- C mutantstrainsandtheWTstrainwereplacedonPDAplatescontain-ingdifferentconcentrationsofNaCl(0.1,0.5or1.0M)orKCl(0.5,1.0or1.5M).After7daysofincubation,themorphologyofthecolonieswasexaminedandphotographed.TodeterminethesensitivityofthemutantsandWTtodifferentpHlevels,thecoloniesweregrownonPDAplateswithdifferentpHvalues(pH2–11)at25°Cinthedark.After2daysofincubation,thecolonieswerephotographedandthediametersofthecoloniesweremeasured.Todeterminethesensitiv-ity of the strains to oxygen radicals, 5-mm-diameter mycelial agardiscs of the ΔVMAGO2 and ΔVMAGO2-CmutantstrainsandtheWTstrainwereplacedonPDAplatescontaining0.05%H2O2 and cultured inthedarkat25°C.Thediametersandmorphologiesofthecolonieswere examined on the third and seventh day. All the studieswereconductedintriplicate,withthreePetridishesineachrepetition.Thestatistical significancewas determined using the protected Fisher’sleast significant difference (LSD) test (p < .05) or the Student’s t test (p<.05)usingSPSS18software.

2.6 | Pathogenicity test

“Fuji”apple(Malus domesticaBorkh.cv“Fuji”)leavesandtwigswereused for assaying the pathogenicity of the ΔVMAGO2mutants.ThepathogenicitytestwasconductedasdescribedbyWei,Huang,Gao,Ke,andKang(2010).Leaveswerewashedwithtapwater,immersedin0.6%sodiumhypochloritefor3minandthenrinsedthreetimeswithsterile water. Moistened cotton was wrapped around the basal parts ofthepetiolestokeepthemwet.Foursmallwoundsweremadeoneachleafusingasterileneedle.Thewoundswereinoculatedbyplac-inga5-mm-diameterPDAcultureblockontoeachofthefourwounds.Theleaveswereplacedinaplasticbox,whichwasimmediatelycov-eredwithvinylfilmtoretainthehumidity,andincubatedinthedarkat25°C.Thetwigsusedinthepathogenicityassayswerewashedwith

tapwater,immersedin1%sodiumhypochloritefor10minandthenrinsedthreetimeswithsterilewater.Thetopendofeachtwigwassealed with wax before inserting the end in sand in a plastic basin. Eachtwigsegmentwaswoundedusingaflat iron (5-mm-diameter),andtheninoculatedbyplacinga5-mmPDAcultureblockontoeachwound.Then, thebasinwas immediatelycoveredwithvinyl film toretainthehumidityandincubatedinthedarkat25°C.Thelesiondi-ametersweremeasured,andthestatisticalsignificanceofdifferenceswasdeterminedasdescribedabove.Threereplicatesoftheinfectionassays were performed with six leaves/twigs in each replicate.

2.7 | RNA isolation, cDNA synthesis and qRT- PCR

AppletissueinfectedwiththeWTstrainwassampledat0and24hpi(hourspost-inoculation).ThetotalRNAofeachsamplewasextractedusingTrizolReagent(Invitrogen,Carlsbad,CA)accordingtotheman-ufacturer’s instructions, andDNA contaminationwas prevented byDNaseItreatment.First-strandcDNAwassynthesizedusinganRT-PCRsystem(Promega,Madison,WI,USA)accordingtothemanufac-turer’s instructions. Quantitative PCR amplifications were performed usingaCFX96Real-TimeSystem(Bio-Rad)withSYBRGreenIchem-istry(Invitrogen).TheV. mali housekeeping gene G6PDH was used as an internal reference control to determine the relative gene expres-sion levels of VMAGO2(Yinetal.,2013).Therelativeexpressionlevelof VMAGO2 was calculated as the fold change in inoculated plants versus mock- inoculated plants at the same time point using the com-parative 2−ΔΔCTmethod(Livak&Schmittgen,2001).Threebiologicalreplicateswereperformedforeachexperiment.TheprimersusedarelistedinTableS1.

2.8 | Generation of the VMAGO2 complementary strain

TheentireVMAGO2 gene fragment and its whole promoter sequence (~1,500bp)wereamplifiedfromgenomicDNAusingtheprimerpairsVMAGO2-CM-F/R (TableS1)andcloned intopFL2using theyeastgaprepairapproach,asdescribedbyZhou,Li,andXu(2011).Fusionconstructswereconfirmedbysequencing,andplasmidsweretrans-formed into the corresponding deletion mutants by PEG-mediatedtransformation(Gaoetal.,2011).ThetransformantswereconfirmedbyPCR.Thephenotypeofthecomplementarystrainswasdeterminedusing the methods described above.

3  | RESULTS

3.1 | Protein sequence and phylogenetic tree analysis

Based on the genome information of V. mali, the VMAGO2 gene sequence was obtained. InterProScan analyses showed that the deduced VMAGO2 protein contained three domains, known asDUF1785(adomainusuallyfound inArgonauteproteinsthatoftenco-occurswith thePIWIdomain),PZA (anevolutionarily conserved

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domainofArgonauteproteins,whoseprimaryfunctionistobindthe3′ endof smallRNAs) andPIWI (adomain found in theArgonautefamilyof relatedproteins,whose function isdouble-stranded-RNA-guidedhydrolysis of single-strandedRNA) (Figure1a).As expected,thephylogenetic analysis suggested that theVMAGO2 is clusteredwithAGOsinfungiandalsoappearstoshareacommonancestorwithAGOsinplantsandanimals(Figure1b).

3.2 | VMAGO2 was upregulated during pathogen infection

To investigate how VMAGO2 is regulated in V. mali during the in-fection process, qRT-PCR was used to analyse its transcript levelchanges.TheanalysisshowedthatVMAGO2 expression was 3.4- fold higherthanthatofthecontrol(mycelium)at24hpi(Figure2).

3.3 | Construction of VMAGO2 deletion mutants

Asshown inFigure3, thepositivedeletionmutantswere identifiedand confirmed via screening using PCR and Southern blot hybridiza-tion.WhenweamplifiedtheORFsegmentofVMAGO2,noproductwas amplified using the ΔVMAGO2mutantDNA(Figure3b;Lane1),but the hph segmentwasamplified (Figure3b; Lane2).Anobviousband could also be seen when the segment was amplified using the primersofVMAGO-1F/H855RandH856F/VMAGO2-4R(Figure3b;Lanes3and4).Allputativeknockoutmutantswerefurtherverifiedby Southern blot hybridization. When hybridized with an hphprobe,nohybridizationsignalwasobservedfortheWTstrainanda4.0kbXho I band was observed for the ΔVMAGO2 mutant. When hybridized with a VMAGO2fragment,a4.5kbPst I band was observed for the WT,whilenocorrespondinghybridizationsignalwasobservedfortheΔVMAGO2mutant(Figure3c).Theseresultsindicatedthatwegener-ated six positive VMAGO2 knockout mutants.

3.4 | VMAGO2 is not essential for Valsa mali vegetative growth

To investigatewhetherVMAGO2 plays a critical role in the growth and development of V. mali,thegrowthrateandthecolonyandhy-phal morphology of the mutants (△VMAGO2 and △VMAGO2- C) andWTwere compared. The colonialmorphologies of themutantandWTstrainswerenotobviouslydifferent,andthegrowthrateof

F IGURE  1 Domains and homologous analysisoftheVMAGO2protein.(a)ThededucedVMAGO2proteincontainsthreedomains,DUF1785,PAZandPIWI,whichwerepredictedatthe259–319,370–466and512–930aminoacidsites,respectively.(b)AphylogenetictreeofVMAGO2proteins constructed with the multiple alignmentprogramDNAMANusingtheAGOproteinsequencesfromplants,animalsandfungi.VMAGO2proteinswereclusteredwithAGOsfromfungiandsharedacommonancestorwithAGOsinplantsandanimals.TheGenBankaccessionnumbers and the species names of the organisms within the clade are shown

Vlas mali AGO-21,007aa DUF1785

259–319PAZ370–466

Piwi512–930

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F IGURE  2 TherelativeexpressionofVMAGO2 during the infection process of Valsa mali.TheexpressionprofileofVMAGO2 at 24 hpi (hours post- inoculation) compared with that of the control (mycelium) using reference gene G6PDHfornormalization.Verticalbars represent the standard deviations of the means of three independent replicates

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ΔVMAGO2 andWTwere2.662cm/dayand2.591cm/day, respec-tively(Figure4).Allsixpositivemutantsexhibitedasimilarphenotype(detailed data not shown).

3.5 | Deletion of VMAGO2 increases the sensitivity of Valsa mali to 0.05% H2O2

TheresponseoftheVMAGO2mutantsandWTtoosmoticstresseswasalsoevaluated.After7days incubationat25°C,wefoundthatthe growth of ΔVMAGO2andWTwasseverelyrestrictedunderthetreatment conditions and that the colony diameters were similar (Figure5a). The responseof theΔVMAGO2 mutant to different pH levels(pH2–11)wasalsoexamined.Afterincubationfor2days,boththe ΔVMAGO2mutantandWTexhibitednormalgrowthunderacidconditions, butneitherof themshowednormal growthunder alka-lineconditions. Ingeneral, knockoutof theVMAGO2 did not result in an altered response to pHwhen compared to theWT response(Figure5b).TheresponseofVMAGO2 mutants to oxidative stress was also evaluated. In the presence of 0.05%H2O2, vegetative growthof ΔVMAGO2 was severely restricted (the mutant showed almost no growth),whilethecomplementarystrainshowednormalgrowthwhencomparedwiththatoftheWT(Figure6a,b).Theseresultssuggestedthat VMAGO2 may be important for oxidative stress responses.

3.6 | The VMAGO2 gene is critical for pathogen virulence on apple leaves and twigs

TotestwhethertheVMAGO2 mutants showed similar virulence de-fects, a pathogenicity assaywas performed using apple leaves andtwigs of “Fuji.” The infection assay results showed thatΔVMAGO2 wassignificantlylessvirulentonbothleavesandtwigsthantheWTstrain (Figure7a,b). Furthermore, the average lesion diameter onleaves and twigs inoculated with the ΔVMAGO2strainwas34.8%and19.8%smaller,respectively,thanthelesionscausedbytheWTstrain(Figure7c,d).Thecompromisedpathogenicitycouldberecoveredbycomplementation of the ΔVMAGO2 mutants with VMAGO2. These results suggested that VMAGO2 plays a positive role during the V. mali infectionprocess.Unravelling thedetailedmechanismwillbeakeyaim of future studies.

4  | DISCUSSION

Argonaute proteins are ubiquitous in plants and animals, com-moninmanyfungiandprotists,andpresent insomearchaea (Pratt&MacRae, 2009). In this study, we identified VMAGO2 inV. mali. VMAGO2hasdomainsthataretypicalofAGOproteins.Evolutionary

F IGURE  3 Construction of ΔVMAGO2mutants.(a)AphysicalmapoftheVMAGO2 genomic region and gene replacement constructs. (b) PCR detection of ΔVMAGO2usingfourpairsofprimers.M:DL2000.Lane1:productamplifiedby5F/6RtodetecttheVMAGO2. Lane 2: product amplifiedbyH852/H850todetecttheinsertionofHYG.Lane3andlane4:productsamplifiedby1F/H855RandH856F/4R,whichwereusedtoensurehomologousrecombinationupstreamanddownstream,respectively.(c)SouthernblotdetectionofΔVMAGO2.GenomicDNAofWTand ΔVMAGO2 strains was isolated and digested with the restriction enzymes Xho I and Pst I,respectively.TheDNAwashybridizedwithHYG probes and VMAGO2fragmentswith4.0and4.5kbbands,respectively

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treeanalysisrevealedthatVMAGO2iscloselyhomologoustofungalAGOs.Indeed,thesRNApathwaysappeartoberelativelyconservedinfungi,andtheAGOproteinshavebeenidentifiedinseveralfungi,particularlyinascomycetes(Nunesetal.,2011).

PreviousstudieshaveshownthatdisruptionsofAGOscanaf-fect the vegetative growth and responses to environmental signals of M. circinelloides and Colletotrichum higginsianum(Campo,Gilbert,& Carrington, 2016; Cervantes etal., 2013). In our study, whenVMAGO2wasknockedout,thecolonymorphologyofthemutants,and their responses to saline ion stresses and different pHs were notsignificantlydifferenttothoseoftheWT.Onepossibleexpla-nation for this is that the functions of VMAGO genes are redundant. Aprevious studyhas shown that theCaenorhabditis elegansAGOproteins ALG-1 and ALG-2 have overlapping functions (Grishoketal., 2001). Indeed, differentAGOsbelonging to the same genefamilycanhavespecialanduniquefunctions.Forexample,ofthefourAGOproteins in humans, onlyAGO2 is a catalytically activeslicer(Liuetal.,2004).

As plant pathologists, our main interest in this study was thepathogenicity contributed by VMAGO2.Anaccumulationofhydrogenperoxide in host plant tissues is considered to be an early sign of a plant–pathogen interaction (Bolwell, 1999). Hydrogen peroxide notonlyworksasasignaltoactivatetheresistancereactionsoftheplant,but also kills pathogens directly when it has accumulated to a certain level(Torres,Jones,&Dangl,2006).Inthisstudy,wefoundthattheexpression of VMAGO2 was highly induced during the V. mali infection process.More importantly,ΔVMAGO2 showed a greater sensitivity to hydrogen peroxide stress and a significantly decreased pathoge-nicity compared with VMAGO2.Furtherinvestigationsareneededto determine whether the decreased pathogenicity is related with the increased sensitivity to hydrogen peroxide.

During the interaction between the pathogen and the host plant,sRNAsplaypivotalrolesbyregulatingthecorrespondingtar-getgenes.TheRNAipathwayhasbeenshowntoplayanimportantrole in the plant response to pathogen infection (Huang,Yang,&Zhang,2016;Voinnet,2008).A recent study reported that fungal

F IGURE  5 Responsesofmutantsandwild-type(WT)toosmoticstressesanddifferentpHvalues.(a)Seven-day-oldculturesofWTandΔVMAGO2growingonpotatodextroseagar(PDA)mediasupplementedwith0.1,0.5or1.0MNaClorwith0.5,1.0or1.5MKClat25°Cinthedark.(b)Two-day-oldculturesofWTandVMAGO2growingonPDAmediawithpHvaluesrangingfrom2to11at25°Cinthedark

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sRNAswere able to affect the host RNAi pathways by targetingthehostAGOgenes,whichsuppressedtheplantimmuneresponse(Ellendorffetal.,2009;Weibergetal.,2013). In fungi, thegener-ationofsRNAs iscomplexandvaries indifferentspecies (Nicolás&Ruiz-Vázquez,2013).AGOsmayalsobeinvolvedinthegenera-tionofsRNAs(Leeetal.,2009).Althoughithasnotbeenconfirmed

whetheranAGOproteincouldperformitsbiologicalfunctionsbyinteractingwith other proteins as a common coding protein, themain role ofAGOproteins is to combine sRNAs.Thus, in furtherstudies,wewill isolatethesRNAscombinedbyVMAGO2,andex-plore the molecular functions of VMAGO2 in the regulatory mech-anismsofsRNAs.

F IGURE  6 Defects of mutants and wild-type(WT)responsetohydrogenperoxidestress.(a)Three-andseven-day-oldculturesofWT,ΔVMAGO2 and ΔVMAGO2- C growing on potato dextrose agar(PDA)mediumcontaining0.05%H2O2 at 25°C in the dark. (b) Colony diameter of theWT,ΔVMAGO2 and ΔVMAGO2-C.Thediameter of the colony was determined usingthecrossingmethod.TheBarsindicates the standard deviation of the meanofthreereplicates.Thestarsabovethe bars indicate significantly different means (least significant difference [LSD] test,p < .05)

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F IGURE  7 Pathogenicity of mutants andwild-type(WT).(aandb)“Fuji”apple(Malus domesticacv“Fuji”)leavesandtwigsshowing the pathogenicity of the mutants relativetotheWT.Thecontrolleavesandtwigs (CK) were inoculated with sterile potatodextroseagar(PDA)blocks.(candd)Thelesiondiameterofappleleavesandtwigs3daysafterinoculation.Thebarindicates the standard deviation of the meanofthreereplicates.Thestarsabovethe bars indicate significantly different means (least significant difference [LSD] test,p<.05).Theinfectionassayswererepeated eight times with three biological replicates

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Insummary,theV. maliAGO2genewasidentified,andcharacteri-zation of its biological functions indicates roles related to the hydrogen peroxide stress response and the pathogenicity of V. mali. Our results provide an insight into the roles of VMAGO2 in V. mali,especiallytheregulation of pathogenicity by V. malisRNAs.

ACKNOWLEDGEMENTS

WethankDrFengmingSongforprovidingthegenedeletionvector.ThisworkwasfinanciallysupportedbytheNationalNaturalScienceFoundationofChina(No.31501591),TheChinaPostdoctoralScienceFoundationSpecialFunding(2016T90953)andtheDoctoralScientificResearchFoundationofNorthwestA&FUniversity.

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How to cite this article:FengH,XuM,LiuY,etal.ThedistinctrolesofArgonauteprotein2inthegrowth,stressresponsesand pathogenicity of the apple tree canker pathogen. For Path. 2017;00:e12354. https://doi.org/10.1111/efp.12354