The phenome analysis of mutant alleles in Leucine-Rich Repeat Receptor-Like Kinase genes in rice reveals new potential targets for stress tolerant cereals

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ContentslistsavailableatScienceDirect

Plant

Science

jo u r n al h om ep age :w w w . e l s e v i e r . c o m / l o c a t e / p l a n t s c i

The

phenome

analysis

of

mutant

alleles

in

Leucine-Rich

Repeat

Receptor-Like

Kinase

genes

in

rice

reveals

new

potential

targets

for

stress

tolerant

cereals

Anne

Dievart

a,∗

_,

_Christophe

_Perin

a

_,

_Judith

_Hirsch

b

_,

_Mathilde

_Bettembourg

a

_,

Nadège

Lanau

a

_,

_Florence

_Artus

a

_,

_Charlotte

_Bureau

a

_,

_Nicolas

_Noel

a

_,

_Gaétan

_Droc

a

_,

Matthieu

Peyramard

a

_,

_Serge

_Pereira

b

_,

_Brigitte

_Courtois

a

_,

_Jean-Benoit

_Morel

b

_,

Emmanuel

Guiderdoni

a

a_CIRAD,_UMR_AGAP,₃₄₃₉₈_Montpellier_cedex_5,_France

b_INRA,_UMR_BGPI,_{INRA-CIRAD-SupAgro,}_TA_54/K,_Campus_{International}_de_Baillarguet,₃₄₃₉₈_Montpellier_cedex_5,_France

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received15May2015

Receivedinrevisedform17June2015

Accepted22June2015

Availableonline29June2015

Keywords: Abioticstress Mutant LRRRLK Rice

a

b

s

t

r

a

c

t

Plantsareconstantlyexposedtoavarietyofbioticandabioticstressesthatreducetheirfitnessand per-formance.Atthemolecularlevel,theperceptionofextracellularstimuliandthesubsequentactivation ofdefenseresponsesrequireacomplexinterplayofsignalingcascades,inwhichprotein phosphoryla-tionplaysacentralrole.SeveralstudieshaveshownthatsomemembersoftheLeucine-RichRepeat Receptor-LikeKinase(LRR-RLK)familyareinvolvedinstressanddevelopmentalpathways.Wereport hereasystematicanalysisoftheroleofthemembersofthisgenefamilybymutantphenotypinginthe monocotyledonmodelplantrice,Oryzasativa.Wehavethentargeted176ofthe∼320LRR-RLKgenes (55.7%)andgenotyped288mutantlines.Positionoftheinsertionwasconfirmedin128lines correspond-ingto100LRR-RLKgenes(31.6%oftheentirefamily).Allmutantlinesharboringhomozygousinsertions havebeenscreenedforphenotypesundernormalconditionsandundervariousabioticstresses.Mutant plantshavebeenobservedatseveralstagesofgrowth,fromseedlingsinPetridishestofloweringand grainfillingundergreenhouseconditions.Ourresultsshowthat37oftheLRR-RLKricegenesarepotential targetsforimprovementespeciallyinthegenerationofabioticstresstolerantcereals.

1. Introduction

Nowadays,rice of Asianorigin (OryzasativaL.)is thestaple foodformorethanhalfofthehumanpopulation.Inlessthan40 years,theworld’spopulationispredictedtoreach9billion, rais-ingtheso-called“9-billion-people”issue[1].Forsustainablerice productionintheyearstocome,anumberofchallengesneedto beaddressedbytheentirericecommunitywiththecommongoal ofcreatingnewelitericevarieties[2,3].Largeeffortshavefocused inthelastyearstocompletesequencingofseveralOryzagenomes [4–10].Infunctional genomics,thechallengeisnow to

system-Abbreviations:LRR-RLK,leucine-richrepeatreceptor-likekinase;MS,Murashige

andSkoogmedium;DJ,DongJin;HW,HwaYoung;Z11,Zhonghua11;TNG,Tainung

67;FST,ﬂankingsequencetag.

∗ Correspondingauthorat:UMRAGAP,CIRAD,AvenueAgropolis,TAA108/03,

Bat3,Bureau51,34398Montpelliercedex5,France.

E-mailaddress:[email protected](A.Dievart).

aticallyassigna biologicalfunctiontoallgenesinthegenomes. Tohelpinthistask,thericecommunityworldwidehasstartedto shareeffortsinthelate90’stoproduceinsertionmutant collec-tionsrequiredforgenefunctionalanalyses[11,12].Thesemutant collectionsareavailableinseverallaboratoriesaroundtheworld: CSIROinAustralia[13],NIASinJapan[14],OSTIDinEurope[15], OTL in France [16], POSTECH [17] and PMBBRC [18] in Korea, RMDin China[19],TRIMinTaiwan[20],andUCD inUSA[21]. Thesemutantcollectionscontaininsertionlinescreatedwith T-DNA,Tos17,Ds,anddSpminsertsmutagens andtheengineered mutagensmayadditionallycarrygenetraps,enhancertrapsand/or activationtags.Theyhavebeengenerated indifferentcultivars: Nipponbare(NB), DongJin (DJ),HwaYoung (HW), Zhonghua 11 (Z11),Zhonghua15,Tainung67(TNG)andKitaake.Alltheselines are listed based on theirﬂanking sequence tags (FSTs)in two databases:RiceGE(http://signal.salk.edu/cgi-bin/RiceGE)and Ory-GenesDB(http://orygenesdb.cirad.fr).Intotal,∼225,000FSTsare preciselypositionedonthessp.japonicacv.Nipponbaresequence

http://dx.doi.org/10.1016/j.plantsci.2015.06.019

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Table1

ListofLRR-RLKgeneswithknownfunctionsinrice.

Accessionnumbers Names Orthologousrelationshipsdescribed Functions/comments References

Os11g36180 Xa21 ResistancetoXanthomonasoryzaepv.

oryzae

[61]

Os02g34790 Xoo-INDUCEDKINASE1(XIK1) PositivelyregulatesXA21-mediated

immunity

[62]

Os11g47000 Xa3/Xa26 Immunereceptorplayingthesame

roleasXa21

[63,64]

Os04g52780 OsFLAGELLINSENSING2(OsFLS2) AtFLS2 AsinArabidopsismediatesﬂagellin

perception

[65]

Os01g52050 OsBRASSINOSTEROIDINSENSITIVE1

(OsBRI1)

AtBRI1 Cellelongationandcelldivisionin

shoot

[66]

Os09g12240 OsBRI1-LIKE1(OsBRL1) AtBRL1 Cellelongationandcelldivisionin

shootandrootinconjunctionwith

OsBRI1

[67]

Os08g25380 OsBRI1-LIKE3(OsBRL3) AtBRL3 Cellelongationandcelldivisionin

shootandrootinconjunctionwith

OsBRI1

[67]

Os06g50340 FLORALORGANNUMBER1(FON1) AtCLAVATA1(AtCLV1) Regulatesﬂoralmeristemsize [68]

Os01g68870 MULTIPLESPOROCYTE1(MSP1) AtEXTRASPOROGENOUSCELLS/

EXCESSMICROSPOROCYTES1

(EXS/EMS1)

Necessarytorestrictthenumberof

cellsenteringintomaleandfemale

sporogenesisandtoinitiateanther

wallformation

[55]

Os02g10100 MSP-LIKE1(MSL1) AtEXTRASPOROGENOUSCELLS/

EXCESSMICROSPOROCYTES1

(EXS/EMS1)

Necessarytorestrictthenumberof

cellsenteringintomaleandfemale

sporogenesisandtoinitiateantherwall

formationinconjunctionwithMSP1

[69]

Os03g12730 BLASTRESISTANCE-RELATED(BRR1) AtBARELYANYMERISTEM(BAM1and

BAM2)

Involvedinblastresistance [70]

Os03g50810 OsTRANSMEMBRANEKINASE(OsTMK) fourmembersofthe

TRANSMEMBRANEKINASE(TMK)

subfamily

Roleinplantgrowth [71]

Os04g38480 OsSOMATICEMBRYOGENESIS

RECEPTOR-LIKEKINASE2(OsSERK2)

AtSERKs RequiredforbothXa21,Xa3/Xa26and

FLS2signalingand

bassinosteroid-regulatedplantgrowth

[72]

Os08g07760 OsSOMATICEMBRYOGENESIS

RECEPTOR-LIKEKINASE1(OsSERK1)

alsonamedOsBRI1-ASSOCIATED

KINASE1(OsBAK1)

AtSERKs Functionsinricedevelopment,

affectinggrowthandangleoflamina

joint;brassinosteroidsignaling?

[73–75]

highdegreeofsimilarity

butnotidenticalto

Os08g07760

BENZOTHIADIAZOLE-INDUCED

SOMATICEMBRYOGENESISRECEPTOR

KINASE1(BISERK1)

AtSERKs Up-regulateduponMagnaporthe

inoculation

[76]

Os08g34380 COMMISSURALVEINEXCESSIVE1

(COE1)

AtSERKs Responsibleforcommissuralvein

patternformationinrice

[77]

Os02g14120 DEFECTIVEINOUTERCELLLAYER

SPECIFICATION1(DOCS1)alsonamed

OsSERK-like4(OsSERL4)

Involvedintheproperdevelopmentof

rootoutercelllayers

[75,78,79]

Os02g40240 LEAFPANICLE2(LP2) Negativeregulatorindroughtresponse [80,81]

Os02g12440 GAMMA-RAYINDUCEDLRR-RLK1

(GIRL1)

Highlyinducedbygammairradiation,

byseveralabioticstresses(salt,

osmotic,andheat),byhormonal

treatmentwithsalicylicacidorabscisic

acid,butdownregulatedinresponseto

jasmonicacidtreatment

[82]

Os05g40770 OsRPK1 Asalt-respondingprotein,whose

expressionisalsoinducedbycold,

drought,andabscisicacid;affectsroot

architecturebynegativelyregulating

polartransportandaccumulationof

auxininroots

[83,84]

Os07g41140 RECEPTOR-LIKEPROTEINKINASE1

(RPK1)

AtRPK1 OverexpressionofbothArabidopsis

andriceRPK1receptorsinducesa

reductioninsalttolerencein

Arabidopsistransgenicplants

[85,86]

Os06g03970 STRESS-INDUCEDPROTEINKINASE

GENE1(OsSIK1)

Affectsstomataldensityinleaf

epidermisandplaysimportantrolesin

saltanddroughtstresses

[87]

Os04g48760 XIAO(“small”inChinese) Regulatesbrassinosteroidsignaling

andcelldivision

[88]

Os11g07225-like1and

Os11g07225-like2

25L1and25L2 SpeciﬁctowildOryzaruﬁpogonrice;

responsibleforthehigh

temperature-dependentexpressionof

hybridweakness

[89]

Os02g05980 LEUCINE-RICHREPEATRECEPTOR-LIKE

KINASE1(LRK1)

Clusterof8genes;LRK1presentin

Dongxiangwildrice,butabsentin

Guichao2;OverexpressionofLRK1

improvedquantitativeyield

components

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Mutant plants with phenotype = putaveLRR-RLKinvolved in condionalresponsetoabioc

stress

Phenotypingonmannitolandsaltmedium

Search ininternaonal

mutantcollecons

Linesselecon

Genotyping

Homozygous plants

PhenotypingonMS/2medium

Mutant plants with phenotype = putave LRR-RLK involved in

leaforrootgrowth Mutant plant w/o phenotype

Fig.1.Summarizedschematicrepresentationofourscreenstrategy.

(MSUv7.0inOryGenesDB)with∼125,000locatedinthe∼35,000 genicregions(i.e.,anaverageof3.6FSTs/locus)[12,22–25].

Leucine-RichRepeatReceptor-LikeKinases(LRR-RLKs)belongto thelargestsubfamilyamongtheReceptor-LikeKinase(RLK)genes [26–28].Thesereceptorsareimportantmediatorsofcell-to-cell communication to relay developmental cues and environmen-talstimuliortoactivatedefense/resistanceagainstpathogensin plants[29–33](forreviewsseealsothespecialissueofJIPB ded-icatedtoReceptor-Like Kinases inDec. 2013).In Arabidopsis,to date,afunctionhasbeenassignedto∼35%ofthe∼230LRR-RLK members.ThemoststudiedreceptorsareBRASSINOSTEROID INSEN-SITIVE1(BRI1),areceptorforthebrassinosteroidhormone[34]; ERECTA,apleiotropicregulatorofmanydevelopmentalprocesses and responses to biotic and abiotic stimuli [35–37]; CLAVATA1 (CLV1)controlling shootand ﬂoralmeristem homeostasis [38]; FLAGELLINSENSING2(FLS2),a geneparticipatinginthe percep-tionofthebacterialelicitorﬂagellinandEF-TURECEPTOR(EFR),the receptorofthebacterialelongationfactorTu(EF-Tu),whichboth conferbroad-spectrumbacterialresistanceinArabidopsis[39,40]; andreceptorsbelongingtotheSERKsubfamily(SERK1,SERK2,and SERK3),whicharedescribedasco-receptorsinmultiplesignaling pathways,notablyBRI1,FLS2,andEFRpathways[41–46].Therice genomehasbeenshowntocontain∼320LRR-RLKgenesanda func-tionhasbeenassignedtolessthan10%ofthem(Table1)[47,48]. Becauseoftheirmanyrolesindevelopmentalandstressresponses, LRR-RLKgenesarepromisingtargetsforcropimprovement[49].

In an attempt to identify new rice LRR-RLK genes involved in stress tolerance, we carried out a reverse genetic approach [50].Wegeneratedacollectionofhomozygousinsertionmutant linesfor∼35%ofthewholeLRR-RLKgenefamilywithout precon-ceivedideasaboutputativegenefunctions.Thesemutantplants have been screened in vitro for altered growth phenotypes at theseedlingstageundercontrolandabiotic(saltandmannitol) stressconditions.Welookedparticularlyformutantswith condi-tionaldevelopmentalphenotypesunderabioticstress.Ourstrategy issummarized in Fig.1. Our analysisrevealsnew uncharacter-izedLRR-RLKgenesputativelyinvolvedinabioticstressresponses. Thesegenesarepotentialtargetsforbreedingofsalt-and drought-tolerantcereals.

2. Materialandmethods

2.1. Plantmaterialandgenotyping

Accessionnumbersofthemutantlinestobegenotypedwere definedonOryGenesDB(http://orygenesdb.cirad.fr/).Seedswere orderedtoOTL,NIAS,Postech,RMD,andTRIM.Uponreceipt,when available,15–20T1orT2seedsweresowninthegreenhouse(28◦C, 60% humidity, 16:8 photoperiod). Some of these mutant lines havebeengenotypedbySouthernblottingasdescribedpreviously [51],othersbyaquickdirectPCRmethodfollowingmanufacturer instructions(Phire® PlantDirectPCRKit,Finnzymes).For South-ernblots,genomicDNAwasextractedfromleavesof4week-old plants.Briefly,tissueswerefreeze-driedovernightanddisrupted thenextdaywithamixermill.Powderwasmixedwith extrac-tion buffer (Tris–HCl 200mM (pH 7.5), EDTA 25mM (pH 8.0), 0.025%SDS,andNaCl25mM)andprecipitatedwithisopropanol. EightmicrogramsofgenomicDNAweredigestedwithrestriction enzymesandloadedona0.8%agarosegelforelectrophoresisat 25Vfor∼17–18h.DNAwastransferredonnylonmembraneand hybridizedwithradioactiveprobeslabelledbytherandom-prime method.ForSouthernprobesandPCR-basedgenotyping,primers weredesignedontheOryGenesDBwebsite (http://orygenesdb. cirad.fr/tools.html).ForSouthernblots,2probesweregenerated byPCR:agene-specificprobe(chosen,dependingonthe restric-tionenzymeused,tohybridizetoaDNAfragment<12kb)anda vector-specificprobe(HPTorTos17).ForlinesgenotypedbyPCR, weused2pairsofprimers.Thefirstpair,gene-specific,toamplifya DNAfragmentsurroundingtheinsertion;andthesecondone,using onegene-specificprimerandoneT-DNA-orTos17-specificborder primer.

2.2. Growthconditionsformutantscreen

In all experiments, 10 seeds of T2 or T3 plants were grownverticallyinsterilesquarePetridishes(Corning,431.301; 20cm×20cm) under controlled conditions (day/night temper-ature of 28/25◦C, a 12h photoperiod, and a light intensity of 500␮Em−2_s−1₎_as_described_previously_[52]_._Brieﬂy,_after steril-ization,theseedswere sownonsquarePetri dishescontaining 250mLofhalfstrengthMurashigeandSkoog(MS/2)solidmedium withtheradicleorienteddownwards.TheMS/2solidmediumwas composedof2.15gL−1ofMurashigeandSkoogmediumbasalsalt mixture(DuchefaBiochemie,M0221),75mgL−1 Murashigeand Skoogvitaminmixture(DuchefaBiochemie,M0409)and8gL−1 of agarosetype II (Sigma–Aldrich, A6877). For salt and manni-tolmedium,7gL−1 ofNaCl(120mM)and21.9gL−1ofmannitol (120mM),respectively,wereaddedtoMS/2mediumbefore auto-claving.After6daysofgrowth,thelengthsoftheseminalrootand secondleaf(i.e.,theleaffollowingtheﬁrstincompleteleaf)were recordedforeachofthe10plantlets.

3. Resultsanddiscussion

3.1. Morethan90%oftheLRR-RLKgenesareputativelytagged byoneinsertionininternationalcollections

Inthisstudy,weusedthemethodwedescribedpreviouslyto establishourLRR-RLKgeneset[53].Brieﬂy,thehmmsearch pro-gramwasruntoextractpeptidesequencescontainingbothLRRs andakinasedomain(datanotshown)[54].Werantheprogram ontheMSUversion7.0oftheNipponbaregenomeandcompared ourgenelistwiththeonepublishedpreviously[25,47,48].Wekept alistof316LRR-RLKgenesconsideredformutantanalysis(Suppl. Table1).Thesegenesareunequallydistributedonthe12

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chro-Table2

NumberofLRR-RLKgenesandclustersperchromosome.

Chromosome Sequence length(bp) Numberof non-TEa_genes Numberof LRR-RLKgenes Numberof LRR-RLKgenes perMb Numberof LRR-RLKgenes for1000 non-TEloci Proportionof LRR-RLKgenes per chromosome Nbofclusters Nbofgenesin clusters Meannumber ofgenesper cluster 1 43,270,923 5078 34 0.79 6.7 10.8 5 11 2.2 2 35,937,250 4143 43 1.20 10.4 13.6 7 26 3.7 3 36,413,819 4388 19 0.52 4.3 6.0 1 2 2.0 4 35,502,694 3419 23 0.65 6.7 7.3 2 8 4.0 5 29,958,434 3118 26 0.87 8.3 8.2 3 10 3.3 6 31,248,787 3236 36 1.15 11.1 11.4 5 20 4.0 7 29,697,621 3065 17 0.57 5.5 5.4 1 2 2.0 8 28,443,022 2762 25 0.88 9.1 7.9 3 10 3.3 9 23,012,720 2260 20 0.87 8.8 6.3 3 8 2.7 10 23,207,287 2298 15 0.65 6.5 4.7 3 6 2.0 11 29,021,106 2707 46 1.59 17.0 14.6 6 35 5.8 12 27,531,856 2443 12 0.44 4.9 3.8 1 2 2.0 Total 373,245,519 39,102 316 0.85 8.1 100 40 140 3.5

a_TE:_Transposable_elements.

mosomes,withchromosomes2,6and11comprising∼40%ofthe 316genes(Table2).Moreover,manyofthesegenes(140,44.3%) belongto40tandemduplicationclusters.Theseclusterscontain2 to13genes(Table2andSuppl.Table1).

WeusedtheOryGenesDBdatabasetoidentifyinsertionmutants availableininternationalcollections[23,24].Thissearchrevealed that(i)26outofthe316genes(8.5%)hadnoinsertion,(ii)among the290geneswithatleastonepredictedinsertion,thenumberof insertionspergeneswasonaverage8.47+/−0.75extendingfrom 1to156insertions(Fig.2(Box1)andSuppl.Table2).Thisnumber istwiceashighasthecurrentaveragenumberofinsertsavailable pergeneinthericegenome(3.6FSTs/locus),suggestingthatsome LRR-RLKgenesareinsertionhotspots.Toselectthemutantlines tobegenotyped,wegaveﬁrstprioritytomutantspresentinour owncollection(OTL).Wealsochoseinsertionsinthecodingregion orinthepromoterwithin200bpoftranscriptioninitiationwhen available.Weendedupwith288mutantlinespredictedtotag176 (55.7%)ofthe316LRR-RLKgenes.Theselineshavebeenidentiﬁed intheOTL,Postech,RMD,OSTID,UCD,TRIM,andNIAScollections (Suppl.Table1).

3.2. Generationofacollectionof128insertionlinesforLRR-RLK genes

Mutantplantssegregatingforthemutationswereidentifiedby SouthernblottingorPCRinthe288mutantlines(Suppl.Fig.1). Followingthislargescalecharacterization,weconcludedthat128 (44.4%) lines(in100(31.6%) LRR-RLK genes) displayedthe pre-dictedinsertion(Suppl.Table3).Fortheexcluded160lines,we havebeenunabletoconfirmthepresenceofthepredicted inser-tionintheLRR-RLKgenetagged.Amongtherearrangedlines,we identifiedbothhomozygousandheterozygousmutatedplantsin 94 lines, but only heterozygous plants in 34 lines (Fig. 2 (Box 2)).In33outofthese34heterozygouslines,thelownumberof plantsgenotypedcouldexplainthisresult.However,inoneline, AQYD06(Os11g47030.1),amongthe18plantsgenotyped,alladult plantswereheterozygous(13plants)orwildtype(5plants)for theinsertion(probability=3.8e−6).Thisobservationsuggeststhat thisinsertionmayaffectanessentialdevelopmentalprocess.We alsoobservedthatinlines3A-51728 (Os03g05140.1),1C-10702 (Os06g45020.1),2D-00806,and3D-02697(both withinsertions inOs04g15660.1),andANZE10(Os01g68870.1),allhomozygous plantsweresterile.Theseobservationssuggestthatthese muta-tionscouldbeinvolvedinreproductiveorgandevelopment.The lattergene,MSP1(Os01g68870.1),hasalreadybeendescribedin theliteraturefor itsfunctioninfloraldevelopment,particularly

inmaleandfemalesporogenesisandininitiationofantherwall formation(Table1)[55].

3.3. Sixmutantlinesareaffectedinleafand/orrootgrowthon controlmedium

For phenotyping,wefocusedparticularlyonthe89 linesfor whichweidentiﬁedhomozygousprogenyplants.Altogether,these linestagatotalof79genes,including70,8,and1genetaggedby 1,2,and3independentinsertions,respectively(Suppl.Table4). First,wesowed10homozygousseedsperlineonacontrolMS/2 mediuminPetridishes.Foreachplant,wescoredtheleaf2and seminalrootlengths6daysaftergermination(Suppl.Fig.2).In parallelandforcomparison,wealsoanalyzedthe5wildtype vari-eties(NB,DJ,HW,Z11,andTNG)ascontrols.Weobservedthat24 homozygousmutantlines(27%)showedastatisticallysigniﬁcant differenceinleaf2and/orrootlengthcomparedtotheirrespective varietalcontrols(Dunnetttest,p<0.05)(Fig.2(Box3)).

Toascertainthatthephenotypesobservedwerenotduetoother mutationssegregatingintheline,wefurthergrewonMS/2control mediumtheprogenyofeitherawildtypeor,whennowildtype wasavailable,ofanheterozygoussiblingofthesemutants(Figure2 (Box4)).Thissecondevaluationofthephenotypewasdoneforall 24linesexceptone(RGT6318inOs04g57630.1)forwhichweonly foundhomozygousplants.Bycomparingtheresultsobtainedin thesetwoexperimentsforthe23otherlines,weobservedthatthe phenotypeobservedin13outofthe23 (56.5%)ofthe homozy-gouslineswasalsoidentiﬁedintheirsiblings,suggestingthatthis phenotypewasduetoindependentmutationssegregatinginthe T2 progenyand nottothemutatedLRR-RLK gene studied.Rice insertionmutantcollectionshavebeenproducedthrough trans-formationofcalluscultures.Thepresenceofmutationsinduced bythisinvitrophasehasbeenwelldocumented[56–59].Thus, fromthisscreenoncontrolMSmedium,weconcludedthatamong the89 homozygouslines analyzed,14 (15.7%) harbored a phe-notypenotlinkedtothegene understudy(Suppl.Table4).For the10 otherlines,we comparedleaf2 and rootlengthsof the homozygousplantstothoseoftheirnull-segregantsiblings(Fig.2 (Box5)).Ourresultsshowedthatonly6lines(i.e.,6.7%ofthe89 lines)actuallyexhibitedaphenotypelinkedtotheLRR-RLK muta-tion(Student test,p<0.05).These6linespresented statistically differentphenotypesfromboththeirvarietalcontrolandtheir null-segregantsiblings.Forthe4otherlines,eveniftheirphenotype wasslightlystatisticallydifferentfromthevarietalcontrol,this dif-ferencewasnotstatisticallydifferentfromtheirazygoussiblings. The6LRR-RLKgenestaggedintheselinespresentedphenotypes

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LRR-RLKgenes: 316

No inseron: 26genes

Search in internaonal mutantcollecons

Mutantavailable 290genes Mutant selecon 288 lines (176 genes) Genotyping 128 lines (100 genes)

94lineswithhomozygousplants 34lineswithonlyheterozygousplants

1 line with putave phenotype 5lines in4genessterile 89lines in79genes

PhenotypingonMS/2medium

24lineswith phenotype

Phenotypingof WT siblingsonMS/2medium

13(+1) lineswith phenotype = notlinkedtoLRR-RLK mutaon

10 linesw/o phenotype

69lines in63genesw/ophenotype

ComparisonofHovs WTphenotypes

6lineswith diﬀerence = putaveLRR-RLK phenotype

4lineswithout diﬀerence

Phenotypingonstressmedium

37 lines w/o phenotype

32 lines with phenotype putavelylinked toLRR-RLK mutaon Box1 Box2 Box3 Box4 Box5 Box6

Fig.2.Detailedviewofeachstepofthemutantscreen.

inleaf 2or rootgrowth(Fig.3).Amongthe5 lines affectedin leaf2growth,3werelonger(Os01g60060.1,Os01g60670.1,and Os02g13410.1)and2shorter(Os01g07630.1andOs01g59570.1) than theirwild type siblings. Root speciﬁc growth phenotypes were noticedin only 1 line, which exhibited a decreased root length(Os03g16010.1).For2ofthese6genes(Os01g07630.1and Os03g16010.1), we had 2 mutant lines analyzed per gene but thephenotypewasonlyobservedinoneline.Intheselines,the positionandorientationoftheT-DNAsaddedtothevarietal back-groundoftheseinsertionsmayhaveimpactedthephenotypes. Finally,wenoticedthatamongthe6geneswithphenotypeson controlmedium,3(Os01g07630.1,Os01g59570.1,Os02g13410.1) werepartofgeneclusters.TheOs01g07630.1andOs01g59570.1 genesarepartofclustersof twogeneswithOs01g07560.1and Os01g59550.1,respectively.Themutantlinesgenotypedforthese geneswerenotrearranged.TheOs02g13410.1genebelongsto clus-ter2–5withtheOs02g13430.1andOs02g13510.1genes.Inthis clusterofthreegenes,amutantlineinOs02g13430.1wasalso phe-notypedbutwasnotsigniﬁcantlyaffectedinleaf2orrootgrowth. Thisresultcouldsuggestthatafterduplication,thesegeneshave perhapsdivergedintheirfunction.

0,5 0,6 0,7 0,8 0,9 1 1,1 1,2 1,3 1,4 Os03g16010.1_AIQA08

Os01g59570.1_1B-16634 Os01g07630.1_AESB06 Os01g60060.1_3A-02322 Os02g13410.1_4A-50082 Os01g60670.1_3A-11424 Root Leaf 2 Growth rao

Fig.3. Mutantlinesaffectedinleaf2androotgrowth.

Ratioofmeangrowthsofmutantandazygouscontrolsiblingsforleaf2androotat

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Fig.4. ResponsestomannitolandNaClstresses.

(A)Responsesofvarietalcontrols(NB,HW,DJ,TNG,andZ11)tomannitolandNaClstresses.Ratioofmeanlengthsofplantsgrownonstressmediumvs.MS/2control

mediumatday6.(B–D)Insertionlinesexhibitingmoreorlesspronouncedresponsesthantheirrespectivevarietalcontrolstoeithermannitolorsaltstressesineitherleaf

2orrootgrowth(B),tobothmannitolandsaltstressesineitherleaf2orrootgrowth(C),toeithermannitolorsaltstressesinbothleaf2orrootgrowths(D).(E)Insertion

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Fig.4.(Continued).

3.4. Conditionalphenotypesunderabioticstressesareobserved in32mutantlines

Forabiotic stressexperiments,we ﬁrstanalyzed the pheno-typesofwildtypeNB,DJ,HW,TNG,andZ11plants.Wegrewthese

seedlingsonMS/2mediumsupplementedwithmannitol(120mM) orsalt(NaCl120mM)inPetridishes.Wemeasuredtheleaf2and seminalrootsizes6 daysafter germination(Fig.4A and Suppl. Fig.3).OurresultsshowedthatNB,DJandHWvarietiesbehaved

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Table3

Phenotypesobservedincontrolandstressscreens.

MS/2 Mannitol NaCl BestblasthitonTAIR10forArabidopsishomolog

Os01g03370.1 RdSpm1931 + AT4G29990.1

Os01g05960.1 CLUSTER1-1 AQGE09 + AT3G47570.1

Os01g05980.1 ANUC12 + + AT3G47570.1

Os01g07630.1 OsSERL5 CLUSTER1-2 AESB06 – AT1G60800.1(AtNIK3)

Os01g12790.1 ASQG04 + AT3G47570.1

Os01g33110.1 CLUSTER1-3 2B-40306 + AT4G08850.1

Os01g59570.1 CLUSTER1-5 1B-16634 – AT4G29990.1

Os01g60060.1 3A-02322 + AT1G79620.1

Os01g60670.1 3A-11424 + AT3G56370.1(IRK)

Os01g65650.1 AQSC01 – AT1G72180.1

Os01g74550.1 03Z11UB50 + AT2G37050.1

Os02g05970.1 CLUSTER2-1 AWBF12 – – AT1G72300.1

Os02g09740.1 M0019987 – AT4G22130.1(AtSRF8)

Os02g11930.1 CLUSTER 2-3 ANUH08 – AT3G47570.1

Os02g13410.1 CLUSTER2-5 4A-50082 + AT5G25930.1

Os02g34790.1 XIK1 CLUSTER2-6 AFDG12 + AT4G08850.1

Os02g41890.1 RGT1990 + AT2G02220.1(AtPSKR1)

Os02g42370.1 AVEA09 + AT3G47570.1

Os03g16010.1 AIQA08 – AT1G31420.1(AtFEI1)

AHJA09 – –

Os03g21510.1 AQXC10 – AT5G58300.1

Os03g27990.1 AOZA02 – AT1G53730.1(AtSRF6)

Os03g50810.1 OsTMK M0020673 – AT1G66150.1(AtTMK1)

Os03g56250.1 CLUSTER3-1 4A-01282 + + AT4G39270.1

Os04g39650.1 AMRA05 + AT5G06940.1

Os05g16824.1 CLUSTER5-1 ALLD11 + + AT1G56130.1

Os06g12120.1 2C-30183 – AT2G13790.1(AtSERK4)

Os06g42800.1 AKZA01 – AT4G22130.1(AtSRF8)

Os07g04190.1 CLUSTER7-1 AUTH09 + + AT5G65700.1(AtBAM1)

Os08g07760.1 AOEH03 + AT1G34210.1(AtSERK2)

Os08g10300.1 CLUSTER8-1 AKAH05 – – AT1G56130.1

Os08g10310.1 RdSpm4649 – AT1G56130.1

Os08g10330.1 AGCB02 – – AT1G56130.1

Os08g40650.1 AMFA08 + + AT4G29990.1

Os09g15700.1 M0050181 – AT1G28440.1(AtHSL1)

Os11g07060.1 CLUSTER11-1 APSH09 + AT3G47570.1

Os11g07270.1 ARJF11 – AT3G47570.1

Os11g14420.1 3A-06965 + + AT5G48380.1(AtBIR1)

approximatelythesamewayonmannitolorNaClmedium,albeit withslightdifferences.Bothleafandrootlengthswerereduced underabioticstressescomparedtoMS/2mediumin30–50%and 20–40%respectiverangeswithvarietyspeciﬁcities.Interestingly,in TNGplants,rootswerelongeronmannitol-butnotonsalt- supple-mentedmediumwhereasreductionofleaflengthwascomparable underNaClandmannitol.ForvarietyZ11,wenoticedthatleaf2 sizewasmuchmoreaffectedonmannitolthanonNaCl.

Keepinginmindthesevarietalspecificitiesandintheaimof detectingconditionalstress-responsivegenes,weselectedthe69 homozygousmutant lines(corresponding to63 LRR-RLK genes) thatdidnotexhibitedaphenotypewhengrownoncontrolMS/2 medium(Fig.2(Box6)).Wegrewthemundermannitol(120mM) andsalt(120mM)stresses.Wethencomparedthemeasurements obtainedfor leaf2 androotswiththeirrespectivevarietal con-trolsgrownundersamestressconditions(Dunnetttest,p<0.05, Suppl.Table5).First,weobservedthat 37lines(53.6%)didnot present aphenotypein eitherof thetwo stressconditions. For thelines showingdifferences compared tothevarietal control, weanalyzedseparatelyleaf2androotphenotypesineachstress condition(Fig.4B–D).Somelinesexhibitedphenotypesfora spe-cific organand undera particularstress (Fig.4B).We scored9 and 6 lines affected in leaf growth on mannitol or salt com-paredtotheircontrols,respectively.Threelineswerespecifically affectedinrootgrowthonmannitolwith2(Os01g12790.1ASQG04 andOs01g75550.103Z11UB50)and1(Os02g09740.1M0019987) exhibitinglongerandshorterrootsthantheircontrols,respectively. On salt, we recorded only reduced growth of leaves, suggest-ingthat thesemutantlines wereallmoresensitivetosalt. We

alsoobservedoneline(Os01g05960.1AQGE09)withlongerroots. Six lines presented a comparable phenotype on mannitol and NaClmedia(Fig.4C). Amongtheselines,3lines(Os08g10300.1 AKAH05, Os03g16010.1 AHJA09, Os08g10330.1 AGCB02) and 2 lines(Os03g56250.14A-01282, Os11g14420.13A-06965) exhib-itedreducedorenhancedrootgrowth,respectively.Onlyoneline exhibitedlongerrootsonbothmedia(Os07g04190.1AUTH09).For geneOs03g16010.1,wehaveshownabovethatrootsoflineAIQA08 wereshorterthancontrolonMS/2medium.Underabioticstresses, anotherlinetaggingthisgene(AHJA09)hadshorter leaves.Our resultsalsoshowedthat 5 lineshad phenotypes affectingboth leavesandroots(Fig.4D).Forthe2linespresentingthesecombined phenotypes onNaCl, plants were smallerthan their respective controls (Os06g12120.1 2C-30183, Os02g05970.1 AWBF12). On mannitol,werecorded1linewithsmallerplants(Os03g50810.1 M0020673)and2lineswithbiggerplants(Os05g16824.1ALLD11, Os08g07760.1AOEH03).Interestingly,for2otherlinesanalyzed (Os01g05980.1ANUC12andOs08g40650.1AMFA08),weobserved differentalthoughconsistentphenotypesonmannitoland NaCl (Fig.4E).Indeed,leaveswerelongerthancontrolonlyonmannitol, androotswerelongeronlyonNaClmedium.Wealsocompared theresultsobtainedinthedifferentlinestaggingthesamegene. Forexample,2linescarriedallelicinsertsingeneOs03g21510.1 (AQXC10andAHQF09).Aleafphenotypewasobservedonlyon salt medium in one of these lines. Among the 3 lines tagging Os03g27990.1 (ATDG06, AOZA02, and ARMB09) only leaves of AOZA02plantshadareducedsizecomparedtowildtypeonsalt. AlltheseresultsaresummarizedinFig.5.

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Mannitol

Na

Cl

Leaf 2

Root

Os01g03370 Os01g33110 Os02g11930 Os02g34790 Os02g41890 Os02g42370 Os04g39650 Os09g15700 Os11g07060 Os01g12790 Os01g74550 Os02g09740 Os01g65650 Os03g21510 Os03g27990 Os06g42800 Os08g10310 Os11g07270 Os01g05960 Os03g16010 Os03g56250 Os08g10300 Os08g10330 Os11g14420 Os07g04190 Os03g50810 Os05g16824 Os08g07760 Os02g05970 Os06g12120 Os01g05980 Os08g40650

Fig.5.VenndiagramofLRR-RLKgenesputativelyinvolvedinconditionalabiotic

stressresponses.

4. Conclusion

Alltogether,theseresultsshowthatthescreenwehave per-formed is a ﬁrst step to establish a list of 37 LRR-RLK genes potentiallyinvolvedindevelopmentalandadaptiveabioticstress responses(Table3).Amongthegeneswithalreadydescribed func-tionsinrice,wehighlightedapotentialroleforOsTMKandXIK1in theresponsetomannitol(Table1andTable3).Interestingly,this reversegeneticsapproach hasalready beenperformedon root-expressedLRR-RLKsin Arabidopsis[60]. We havecompared our genelistwiththeonepublishedinthis studyforabioticstress responses.Inbothstudies,theputativeinvolvementofBAM1in abioticstressresponseshasbeennoticed.

Ourresultsalsoshowthatunrelatedmutationsare segregat-ingathighfrequencyinmutantlinecollections.Inconsequence, acarefulanalysisofsiblingplantshastobedonetotryto elim-inatemostoftheunrelatedmutations.Despiteoureffortstoget ridoftheseextramutations,somephenotypesdescribedinour manuscriptcouldbe,atleastinpart,duetotheseadditional muta-tions.Thus,ﬁnefunctionalanalysesarealsorequiredtoconﬁrmthe phenotypesobservedforallthesemutantlines.Nonetheless,our screenhasbeensuccessfulatidentifying37LRR-RLKgenesthatare linkedtogrowthphenotypeseitherundercontrolorabioticstress conditions.Theselineswillbefurtherinvestigatedthrough com-prehensivefunctionalanalyses.Furthermore,ourmutantcollection isalsoavailableforotherscreenstoinvestigatenewLRR-RLK func-tions.

Acknowledgements

Thisprojecthasbeensupportedbygrant#ANR-08-GENM-021 fromAgenceNationaledela Recherche(ANR,France),by Euro-peanCommissionFP6Projectno.015468CEDROME(Developing drought-resistant cereals to support efﬁcient water use in the Mediterraneanarea)andbyGenerationChallengeProgram“Rice Stressmutants”(Reversegeneticsystemstovalidatefunctionof stresstolerancegenes). WethankPr G.Anand MsS.An(KHU, Korea),DrM.J.Fan,S.M.YuandY.I.Hsing(AcademiaSinica,Taiwan), DrC.WuandL.Yan(HZAU,China),PrV.SundaresanandMsK. Gal-imba(UCDavis)formakingavailabletousseedsoftheinsertion linesusedinthisstudy.ThetechnicalassistanceofM.Portefaix,C.

Chaine,R.MichelandE.Lorenziniforseedserviceandgreenhouse supportsisgreatlyacknowledged.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,athttp://dx.doi.org/10.1016/j.plantsci.2015.06. 019

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