Research. Summary. Introduction

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1 Reserh The tomto CAROTENOID CLEAVAGE DIOXYGENASE8 (SlCCD8) regultes rhizosphere signling, plnt rhiteture n ffets reproutive evelopment through strigoltone iosynthesis Wouter Kohlen,, Ttsin Chrnikhov, Mihiel Lmmers, Toi Pollin, Peter Tóth,, Imrn Hier, Mrí J. Pozo, Ruu A.e Mg,6, Crolien Ruyter-Spir,, Hrro J. Bouwmeester,6 n Jun A. López-Ráez, Lortory of Plnt Physiology, Wgeningen University, Droevenlsesteeg, 678 PB, Wgeningen, The Netherlns; Deprtment of Plnt Breeing n Genetis, Mx Plnk Institute for Plnt Breeing Reserh, Crl-von-Linné-Weg, D-89, Cologne, Germny; Business Unit Biosiene, Plnt Reserh Interntionl, Droevenlsesteeg, 678 PB, Wgeningen, The Netherlns; Deprtment of Plnt Protetion, Slovk Agriulturl University, A. Hlinku, 9976, Nitr, Slovki; Deprtment of Soil Miroiology n Symioti Systems, Estión Experimentl el Ziín-Consejo Superior e Investigiones Científis (EEZ-CSIC), Prof. Alre, 88, Grn, Spin; 6 Centre for Biosystems Genomis, PO Box 98, 67 AB, Wgeningen, The Netherlns Author for orresponene: Hrro J. Bouwmeester Tel: Emil: hrro.ouwmeester@wur.nl Reeive: My Aepte: 7 July New Phytologist () oi:./j x Key wors: rusulr myorrhizl (AM) fungi, rotenoi levge ioxygense, Oronhee, plnt rhiteture, reproutive evelopment, strigoltones, tomto (Solnum lyopersium), xylem trnsport. Summry Strigoltones re plnt hormones tht regulte oth ove- n elowgroun plnt rhiteture. Strigoltones were initilly ientifie s rhizosphere signling moleules. In the present work, the tomto (Solnum lyopersium) CAROTENOID CLEAVAGE DIOXYGENASE 8 (SlCCD8) ws lone n its role in rhizosphere signling n plnt physiology ssesse y generting knok-own lines. Trnsgeni SlCCD8 plnts were generte y RNAi-meite silening. Lines with ifferent levels of strigoltone reution onfirme y UPLC-MS/MS were selete n their phenotypes investigte. Lines exhiiting reue SlCCD8 levels isplye inrese shoot rnhing, reue plnt height, inrese numer of noes n exessive ventitious root evelopment. In ition, these lines exhiite reproutive phenotypes suh s smller flowers, fruits, s well s fewer n smller sees per fruit. Furthermore, we show tht strigoltone loing to the xylem sp is possily restrite to oronhol. Infesttion y Phelipnhe rmos ws reue y 9% in lines with reltively mil reution in strigoltone iosynthesis n seretion while rusulr myorrhizl symiosis, pil ominne n fruit yiel were only milly ffete. This emonstrtes tht reution of strigoltone iosynthesis oul e suitle tool in prsiti wee mngement. Furthermore, our results suggest tht strigoltones re involve in even more physiologil proesses thn so fr ssume. Introution Strigoltones re group of rotenoi-erive plnt hormones (Mtusov et l., ; López-Ráez et l., 8). They were initilly ientifie s germintion stimulnts for root prsiti plnts of the Oronhee (Cook et l., 966; Bouwmeester et l., ) n pre-symioti signls inuing hyphl rnhing in rusulr myorrhizl (AM) fungi (Akiym et l., ; Besserer et l., 6; Bouwmeester et l., 7). Strigoltones hve een etete in the root extrts n exutes of oth monoot n iot plnt speies (Yoneym et l., 7; Golwsser et l., 8; Gomez-Roln et l., 8; López-Ráez et l., 8; Umehr et l., 8) n were ientifie to e the rnhing/tillering inhiiting signl (Gomez-Roln et l., 8; Umehr et l., 8). This grft-trnsmissile signl ws postulte to exist over yers go n to originte t lest to lrge extent from the roots (Beverige et l., 99; Npoli, 996; Turnull et l., ). However, intergrfting of hypootyl tissue is suffiient to restore shoot rnhing of strigoltone iosynthesis mutnts in pe n Ariopsis thlin (Ariopsis) to ner wiltype levels, initing tht in ition to roots other tissues n ontriute to the proution of strigoltones tht inhiit the outgrowth of xillry us (Foo et l., ). Nevertheless, strigoltones re trnsporte ropetlly to the prts of the plnt where they exert their funtion. This is supporte y the ft tht in oth Ariopsis n Solnum lyopersium (tomto) t lest one Ó The Authors New Phytologist () New Phytologist Ó New Phytologist Trust

2 Reserh New Phytologist strigoltone oronhol is present in the xylem (Kohlen et l., ). Reently, strigoltone ellulr exporter PhPDR ws ientifie in Petuni hyri (petuni). PhPDR elongs to the fmily of ABC trnsporters, n it is involve in strigoltone seretion into the rhizosphere. The ft tht it is lso expresse ner xillry us seems to suggest role in strigoltone loing to the xylem/poplst in the shoot s well ut how tht is involve in regulting shoot rnhing is still unler (Kretzshmr et l., ). Strigoltone proution is inue uner phosphte-limiting onitions in severl plnt speies (Yoneym et l., 7; López- Ráez et l., 8; Umehr et l., 8; Kohlen et l., ), likely to stimulte the estlishment of AM symiosis (Akiym et l., ; Bouwmeester et l., 7). More reently, it ws propose tht these elevte strigoltone onentrtions might lso serve n itionl funtion in plnt, s they oul e involve in reuing shoot rnhing uner these unfvorle onitions (Umehr et l., ; Kohlen et l., ). In greement with this, the onentrtion of oronhol in the xylem sp of Ariopsis is elevte uner phosphte efiieny (Kohlen et l., ). This oul explin why strigoltone iosynthesis in Ariopsis plnt speies whih is not myorrhize shows similr response to phosphte strvtion (Kohlen et l., ). In ition to regulting shoot rhiteture, novel iologil funtions for strigoltones re eing isovere t rpi pe, showing tht they ply roer role in plnt evelopment. A smll-moleule sreen ientifie severl puttive funtions for strigoltones in Ariopsis, rnging from see germintion to hypootyl elongtion (Tsuhiy et l., ). Strigoltones were lso ientifie s positive regultors of seonry stem growth y stimulting mium evelopment in severl speies (Agusti et l., ). In ition, strigoltones hve een shown to e involve in the regultion of plnt rhiteture elowgroun ffeting the root system rhiteture. It ws emonstrte tht mnipultion of the strigoltone onentrtion in the root either y muttion or exogenous pplition of the syntheti strigoltone nlog GR les to hnges in the primry root length, root hir evelopment n lterl root initition (Kolti et l., ; Kpulnik et l., ; Ruyter-Spir et l., ). Similrly, strigoltones were reently shown to repress ventitious root evelopment in Ariopsis n Pisum stivum (pe; Rsmussen et l., ). Interestingly, in most if not ll of these newly isovere strigoltone funtions ross-tlk with uxin seems to ply preominnt role (Bennett et l., 6; Agusti et l., ; Ruyter- Spir et l., ; Rsmussen et l., ). As mentione ove, strigoltones re erive from the rotenois n therefore elong to hemil lss lle the porotenois. Inee, reently 9-is--rotene ws ientifie s the sustrte for strigoltone iosynthesis (Aler et l., ). Moreover, three of the four enzymes previously emonstrte to e involve in their iosynthesis (Gomez-Roln et l., 8; Umehr et l., 8; Lin et l., 9; Kohlen et l., ) hve now een funtionlly ientifie (Aler et l., ). All-trns--rotene is first isomerise to 9-is--rotene y DWARF7, n is susequently sequentilly leve y New Phytologist () CAROTENOID CLEAVAGE DIOXYGENASE 7 (CCD7) n CAROTENOID CLEAVAGE DIOXYGENASE 8 (CCD8) to give rise to rltone (Aler et l., ). This porotenoi losely resemles strigoltones s it lrey hs the D-ring n enol ether rige hrteristi for strigoltones. Aler n o-workers hve lso postulte tht the fourth iosyntheti enzyme, with so fr unknown tlyti tivity the ytohrome P MORE AXILLARY GROWTH (MAX; Stirnerg et l., ; Booker et l., ) might tlyse the onversion of rltone to -eoxystrigol (Aler et l., ), propose to e the first rel strigoltone in the iosyntheti pthwy (Mtusov et l., ; Rni et l., 8). Strigoltone iosynthesis seems to e tightly regulte s it ws emonstrte tht espite inrese rotenoi umultion in the tomto mutnt high pigment- rkgreen (hp- g ) strigoltone iosynthesis n seretion into the rhizosphere were signifintly reue. As result, this mutnt ws less suseptile to the root prsiti plnt Phelipnhe egypthy (López-Ráez et l., 8). Also the Oronhe-resistnt n AM-efiient tomto mutnt Sl-ORT with milly rnhe phenotype ws shown to proue fewer strigoltones (Kolti et l., ). However, the gene unerlying Sl-ORT n its funtion in regulting strigoltone iosynthesis so fr remins unknown. This emonstrtes tht ontrolling strigoltone iosynthesis might e goo strtegy to ontrol these rop-mging prsiti wees s previously suggeste (Bouwmeester et l., ; López-Ráez et l., 9; Croso et l., ). Remrkly, AM symiosis in tomto lso les to reue germinting stimultory tivity for Phelipnhe rmos sees, reution use y erese in strigoltone proution n exution (López-Ráez et l., ). Tomto hs eome n importnt moel in strigoltone reserh n its mjor strigoltone omposition (solnol, oronhol n iehyro-oronhol isomers n ) hs een eluite (López-Ráez et l., 8,). Therefore, hving full insight into the strigoltone iosyntheti pthwy in tomto is vitl, s this woul mke it n exellent moel for omining oth nlytil n moleulr tools for strigoltone reserh in n gronomilly importnt rop. The first strigoltone iosyntheti gene hrterise in tomto ws SlCCD7 (Vogel et l., ), n the uthors emonstrte tht strigoltones lso regulte the outgrowth of xillry u in tomto. In the present stuy, we set out to lone the tomto SlCCD8 gene, n hrterise its role in strigoltone iosynthesis n tomto plnt evelopment using knok-own trnsgeni lines. Our results provie insight into possile new funtion for strigoltones in reproutive evelopment of tomto. Mterils n Methos Plnt mteril, growth onitions n hemils Sees of tomto (Solnum lyopersium L. v Crigell (LA7)) n the three inepenent trnsgeni SlCCD8 knok-own lines (L6, L, n L9) were surfe-sterilise in % soium hypohlorite ontining.% (v/v) Tween, rinse thoroughly with sterile wter n germinte for 8 h on moistene filter pper t C in rkness. For strigoltone nlysis, germinte Ó The Authors New Phytologist Ó New Phytologist Trust

3 New Phytologist Reserh sees were pre-grown on perlite for 7. Then, ll perlite ws remove from the roots n ten seelings per iologil replite were trnsferre to n X-strem eroponis system (Nutriulture, Lnshire, UK) operting on l moifie hlf-strength Hogln nutrient solution (López-Ráez et l., 8) s previously esrie (Liu et l., ). To inue strigoltone proution, 7 phosphte-strvtion stress ws pplie to the plnts y repling the nutrient solution y moifie hlf-strength Hogln solution without phosphte (López-Ráez et l., 8; Liu et l., ). Prior to exute olletion the nutrient solution ws refreshe in orer to remove ll umulte strigoltones. The exutes were ollete h lter; purifie n onentrte within h. Roots were quik frozen in liqui nitrogen n store t 8 C for further nlysis. For phenotypil nlysis, plnts were grown in soil-fille pots for 8 wk n irrigte with ml of tp wter twie week n one with ml of full-strength Hogln solution (Hogln & Arnon, 9). For omplementtion stuies n nlysis of ventitious root primori, pre-germinte tomto sees were trnsferre to n in vitro system using -ml hlf-strength MS meium supplemente with 9 Gmorg s B vitmin mix, % gr, n either (mok) or lm GR (syntheti strigoltone nlog). Cutting experiments were performe using the top primry stem noes of -wk-ol tomto seelings. All plnts were grown uner ontrolle onitions in glsshouse t with 6/8 h photoperio, / C, n 6% reltive humiity. Aitionl light ws provie when neee to hieve lmol m s minimum light intensity. IAA ws purhse from Sigm-Alrih (St. Louis, USA). GR ws kinly provie y Prof. r. Zwnenurg (Rou University, Nijmegen, The Netherlns). Cloning of the full-length SlCCD8 A -p prtil SlCCD8 oing sequene ws mplifie from tomto (v Moneymker) y reverse-trnsriptse polymerse hin retion (RT-PCR) using primers (forwr, -GCTGAGT GGCACGTACCTAA- ; reverse, -TCATCTTCTTCGGTT GCAC- ) esigne to highly onserve region of plnt CCD8s. The SlCCD8 - n -DNA ens were otine using the SMART RACE DNA Amplifition Kit (Clonteh, Mountin View, CA, USA; -RACE, -GCGTCCGATTCGATTTC- n RACE, -TCCTGCTTATTTAGGCAAG- ). The omplete SlCCD8 oing sequene (67 p) ws PCR mplifie from root DNA (forwr, -ATGGCTTCTTTTGCTCA TTCAG- ; reverse, -CTATTTCTTTGGAACCCAGC- ). Finlly, the mplifie DNA frgment ws moifie using the A-tiling proeure n lone into pgem-t Esy vetor (Promeg) oring to the mnufturer instrutions, n oth strins sequene. RNAi-meite silening of SlCCD8 n tomto trnsformtion The silening of SlCCD8 in tomto ws rrie out y mens of 9 p frgment plus the gtewy CACC iretionl loning sequene tht ws PCR mplifie using speifi primers (forwr, -CACCCAGGACAATGGCACATAGGT- ; reverse, -TCT AGGGTGTTCGGATCAA- ). The PCR frgment ws lone into the pentr/d vetor (Gtewy Tehnology; Invitrogen) n then introue into the inry estintion vetor phellsgte8 (Helliwell et l., ) y reomintion using the LR lonse II (Invitrogen). The phellsgte8::ccd8 RNAi onstrut ws trnsferre to Agroterium tumefiens strin LB n use to trnsform tomto (v Crigell) plnts s previously esrie (vn Roekel et l., 99). RNA isoltion n gene expression nlysis y rel time quntittive RT-PCR (qpcr) Totl RNA ws extrte using Tri-Regent (Sigm-Alrih St. Louis, USA) oring to the mnufturer s instrutions. The RNA ws trete with RQ DNse (Promeg, Mison, WI, USA), purifie through sili olumn using the NuleoSpin RNA Clen-up kit (Mherey-Ngel, Düren, Germny) n store t 8 C until use. For gene expression nlysis y rel-time quntittive PCR (qpcr) the icyler iq system (Bio-R Herules, CA, USA) ws use (Spinsnti et l., 6) using speifi primers. For the tomto elongtion ftor- (househol gene) SlEF: - GATTGGTGGTATTGGAACTGTC- n -AGCTTC GTGGTGCATCTC- ; for SlCCD7: -AGCCAAGAATTCG AGATCCC- n -GGAGAAAGCCCACATACTGC- ; for SlCCD8: -CCAATTGCCTGTAATAGTTCC- n -GC CTTCAACGACGAGTTCTC-. The first-strn DNA ws synthesize with lg of purifie totl RNA using the isript DNA Synthesis kit (Bio-R, Herules, CA, USA) oring to the mnufturer s instrutions. Three inepenent iologil replites were use n eh PCR retion ws one in triplite. Reltive quntifition of mrna level ws performe using the omprtive C t metho (Livk & Shmittgen, ). Vlues were normlise using the C t vlue for the tomto househol gene SlEF (Rotenerg et l., 6). Vlues were use to etermine the hnge in gene expression oring to the following lultion: folhnge = DðDCtÞ, where DC t = C t (trget) C t (househol) n D(DC t ) = DC t (tretment) DC t (ontrol). Strigoltone n uxin nlysis y multiple retion monitoring liqui hromtogrphy-tnem mss spetrometry (UPLC-MS/MS) Root exutes were purifie n onentrte s previously esrie (López-Ráez et l., 8, ) with some moifitions. Five liters of root exutes were loe onto pre-equilirte C8 olumn (GrePure C8-Fst mg ml ). Susequently, olumns were wshe with ml of eminerlise wter, n ml of % etone/wter. Strigoltones were elute with ml of 6% etone/wter. All exutes were ollete within h n store t C efore mesurements. Strigoltones were extrte from root mteril s previously esrie (López- Ráez et l., ). Xylem sp ws ollete n purifie s previously esrie (Kohlen et l., ). Anlysis n quntifition of strigoltones were performe using UPLC-MS/MS, s previously esrie (Kohlen et l., ). Auxin (IAA) ws Ó The Authors New Phytologist () New Phytologist Ó New Phytologist Trust

4 Reserh New Phytologist extrte, purifie n nlyse s previously esrie (Ruyter- Spir et l., ). Phelipnhe rmos germintion ssy n infetion stuy Germintion ssys with Phelipnhe rmos (L.) Pomel sees were onute s previously reporte y Mtusov et l. (). GR ( 9 M) n eminerlise wter were inlue s positive n negtive ontrols, respetively. To perform the P. rmos infetion ssy, -l pots were fille with soil:sn mixture ( : ) n sees were e following the proeure previously esrie y Kroshel () with some moifitions. P. rmos sees were sown in lyer of. m elow the surfe t ensity of mg pot. In orer to keep moist environment, pots were wtere ily with 6 ml of tp wter for. Then, --ol tomto seelings were introue into the pots n wtere for n itionl s esrie efore. Susequently, pots were irrigte with ml of tp wter twie week n one with ml of full-strength Hogln solution (Hogln & Arnon, 9). Emerge P. rmos shoots were ounte wk fter tomto plnting. Anlysis of AM oloniztion of SlCCD8 knok-own lines The AM fungus Glomus intrries N.C. Shenk & G.S. Sm (BEG ) ws mintine s soil:sn se inoulum ontining mix of iverse fungl propgules (spores, hyphe n hoppe myorrhizl roots). Tomto sees of the SLCCD8 knok-own lines n orresponing wil-type (v Crigell) were surfe-sterilise n germinte for on ontiner with sterile vermiulite t C in rkness. Susequently, iniviul seelings were trnsferre to.-l pots with sterile sn:soil ( : ) mixture. Pots were inoulte y ing % (v : v) G. intrries inoulum. The sme mount of soil:sn mix ut free from AM ws e to ontrol plnts. For eh tretment five replite plnts were use. Plnts were rnomly istriute n grown in glsshouse t /6 C with 6/8 h photoperio n 7% humiity n wtere three times week with Long Ashton nutrient solution (Hewitt, 966) ontining % of the stnr phosphorous onentrtion. Plnts were hrveste fter 8 wk of growth. Roots were stine with trypn lue (Phillips & Hymn, 97) n exmine using Nikon Elipse i mirosope (Nikon Corportion, Tokyo, Jpn) uner right-fiel onitions. The perentge of root length olonise y the AM fungus ws etermine y the griline intersetion metho (Giovnnetti & Mosse, 98). Surose nlysis y high-performne liqui hromtogrphy (HPLC) Smples were extrte n nlyse s previously esrie (Sergeev et l., ). Sttistil nlysis Dt for strigoltone n uxin ontent were sujete to one-wy nlysis of vrine (ANOVA) using GenStt for Winows (9 th New Phytologist () eition). To nlyse the results of germintion iossys, ANOVA fter rsine(squre root(x)) trnsformtion ws use. When pproprite, t were sujete to the Stuent s t-test. Results SlCCD8 loning n hrteriztion A serh of the ville tomto EST lirries file to ientify ESTs with homology to ny pulishe CCD8 sequene. Therefore, PCR-se pproh using primers esigne ginst highly onserve regions in known plnt CCD8s in omintion with RACE ws use to isolte the full-length oing sequene of the puttive tomto CCD8/MAX, herefter esignte s SlCCD8 (JF8). SlCCD8 hs n open reing frme (ORF) of 67 p (Supporting Informtion Tle S). A BLAST serh of the SlCCD8 sequene ws performe on the tomto genome (Bomrely et l., ) n the sequene ligne with -p region lote on hromosome 8. SlCCD8 is preite to ontin six exons (Fig. ). The ORF enoes 7 mino i protein (Tle S) with 89% n 66% homology to petuni CCD8/DAD (PhCCD8/DAD (Snowen et l., )) n Ariopsis CCD8/MAX (AtCCD8/MAX (Sorefn et l., )) proteins, respetively (Fig., Tle S). In phylogeneti lignment, SlCCD8 lustere losely together with PhCCD8/DAD in wht seems to onstitute su-le of iot CCD8s (Fig. ). Monoot CCD8s of mize, rie n sorghum lustere seprtely from iot CCD8s. SlCCD8 expression ws etete t low levels in ll plnt tissues. However, it ws primrily expresse in tomto roots n stems with the highest expression in roots (Fig. ). In orer to ress the iologil funtion of SlCCD8, n RNA interferene (RNAi) onstrut ontining speifi region of 9 p of the gene ws rete n introue into tomto (v Crigell) through Agroterium tumefiens-meite trnsformtion. Three inepenent SlCCD8 RNAi lines (L6, L n L9) isplying 6%, 9% n 97% reution in SlCCD8 mrna levels in the roots, respetively (Fig. ), were selete n propgte to T genertion. Trnsript levels of the other losely relte rotenoi levge ioxygense esrie in tomto SlCCD7 (Vogel et l., ) were not ffete in ny of the trnsgeni lines (Fig. S), emonstrting the speifiity of the RNAi onstrut for SlCCD8. Role of SlCCD8 in rhizosphere signling The onentrtions of ll strigoltones previously reporte in tomto (López-Ráez et l., 8,) were reue in the exutes of ll three trnsgeni lines ompre with wil-type plnts. In L6 strigoltone onentrtions were reue y % n in L n L9 y 9% (Fig. ), orrelting with the reution in SlCCD8 trnsript (Fig. ). Colonistion y the mutulisti AM fungus Glomus intrries in L6, L n L9 ws reue y 7%, % n 6%, respetively, ompre to wil-type plnts (Fig. ). This reution in AM symiosis orrelte to some extent with the erese in strigoltone exution (Fig. ). In ition, 9% reution in shoot emergene of Phelipnhe rmos ws oserve Ó The Authors New Phytologist Ó New Phytologist Trust

5 New Phytologist Reserh () () () Fig. Tomto (Solnum lyopersium) CCD8 (SlCCD8). () The postulte intron/exon struture for SlCCD8 ( nt). () Alignment of the puttive SlCCD8 mino i sequene with those from known CCD8 proteins; Sl, Solnum lyopersium; Ph, Petuni hyri; At, Ariopsis thlin. Ientil n similr mino is re she in lk n grey, respetively () Phylogeneti tree of known CCD, CCD7 n CCD8 nuleotie sequenes; Sl, Solnum lyopersium;ph, Petuni hyri; At, Ariopsis thlin; Ps, Pisum stivum; Os, Oryz stiv; Zm, Ze mys; Mt, Meigo truntul; S, Sorghum iolor (AtCCD (ATG6), OsCCD (Osg66), PhCCD (AY76), SlCCD (AY76), SlCCD (AY76), ZmCCD (GRMZMG7), AtCCD7 (ATG99), MtCCD7 (Metr7g8), OsCCD7 (Osg6), PsCCD7 (DQ6), PhCCD7 (FJ79878), SlCCD7 (GQ686), ZmCCD7 (GRMZMG867), AtCCD8 (ATG8), MtCCD8 (Metrg79), OsCCD8 (Osg66), OsCCD8 (Ost76), PsCCD8 (AY7), PhCCD8 (AY79), SCCD8 (Sg), SlCCD8 (JF8), ZmCCD8 (GRMZMG688). for ll three trnsgeni lines (Fig. ). Phelipnhe rmos see germintion using root exutes ws signifintly lower for ll trnsgeni lines (Fig. ), n this erese orrelte well with the reution in strigoltone exution (Fig. ). () SlCCD8 mrna levels (rel ve vlues) R S L F MG () SlCCD8 rele ve trnsript unne (%) WT L6 L L9 Fig. SlCCD8 trnsript umultion (normlise to the tomto (Solnum lyopersium) househol gene SlEF). () Reltive gene expression of SlCCD8 in tomto v Crigell (WT) in ifferent plnt tissues: R, root; S, stem; L, lef; F, flower; MG, mture green fruit. (n = ) () Reltive gene expression of SlCCD8 in the roots of v Crigell (WT) n three inepenent SlCCD8 RNAi lines (L6, L, n L9) The expression in wil-type tomto is set t % (n = ). Error rs represent mens ± SE. Brs with ifferent letters iffer signifintly t P <.. SlCCD8 knok-own lters strigoltone onentrtions in plnt Strigoltone onentrtions in root extrts of L6, L n L9 were lso nlyse. As in exutes, ler reution in the onentrtion of ll strigoltones ws etete for ll three trnsgeni lines (Fig. ). On verge, the strigoltone onentrtion in root extrts of L6, whih showe the milest reution in SlCCD8 expression, ws reue y %, wheres strigoltone onentrtions in L n L9 were reue y 9% n 9%, respetively (Fig. ). This erese in strigoltone proution ws lmost ientil to the reution oserve in the root exutes (Fig. ). As mentione ove, strigoltone proution is inue uner phosphte efiieny (Yoneym et l., 7; López-Ráez et l., 8), just s the onentrtion of oronhol in the xylem sp of Ariopsis (Kohlen et l., ). Inee, five-fol inrese in the onentrtion of oronhol ws lso etete in the xylem sp of phosphte-strve wil-type tomto plnts (Fig., Fig. S). The three knok-own lines L6, L n L9 showe ler reution in xylem sp oronhol onentrtions (6%, % n 8%, respetively) ompre with wil-type plnts (Fig. ). Interestingly, neither solnol nor ny of the Ó The Authors New Phytologist () New Phytologist Ó New Phytologist Trust

6 6 Reserh New Phytologist Strigoltone in exutes (Pek re g FW) P. rmos shoots per tomto plnt (#) 7 6 () () WT L6 L L9 WT L6 L L9 Solnol Diehyro Diehyro Oronhol P. rmos see germin on (%) iehyro-oronhol isomers were etete in the xylem sp. A seon strigoltone oronhyl ette ws lso etete in tomto xylem sp (Fig. S), ut its onentrtion ws too low to urtely quntify its reution in the trnsgeni lines or to ssess the effet of phosphte strvtion on the onentrtion of this ompoun in the xylem. No surose ws etete in ny of the xylem sp smples nlyse (Fig. S), showing the smples re not ontminte with phloem sp. Furthermore, hypootyls were lso nlyse for their strigoltone ontent, ut strigoltones were unetetle in these smples (Fig. S). These t onfirm tht the oronhol etete is from the xylem sp n not from ontminting phloem sp or hypootyl tissue () Myorhizl olonis on (%) 6 () WT L6 L L9 WT L6 L L9 GR MQ Fig. Strigoltone ontent of root exutes n rhizosphere intertion nlysis of tomto (Solnum lyopersium) v Crigell (WT) n three inepenent SlCCD8 RNAi lines (L6, L, n L9). () MRM-LC-MS/MS quntifition of the mjor tomto strigoltones (solnol, iehyro-oronhol isomers n, n oronhol) in root exutes oring to the pek re (n = ). () Perentge of root olonistion y the rusulr myorrhizl fungi Glomus intrries (n = 6). () Phelipnhe rmos infesttion ounte s emerge shoots per tomto plnt (n = 6). () P. rmos see germintion inue y root exutes. Syntheti strigoltone nlog GR ( 9 M) n eminerlise wter (MQ) re positive n negtive ontrol, respetively (n = ). Error rs represent mens ± SE. Brs with ifferent letter iffer signifintly; P <.. Effet of erese SlCCD8 expression on shoot rhiteture In orer to ssess the onsequene of the reue strigoltone onentrtions on shoot rhiteture, L6, L n L9 lines were grown in pots for 8 wks n their phenotypes ompre with wiltype plnts (Fig. ). All three knok-own lines were signifintly more rnhe, isplying.7-,.- n 7.-fol inrese in lterl shoot rnh numer, respetively (Fig. ), whih inversely orrelte with the level of SlCCD8 trnsript (Fig. ) n strigoltone proution (Figs, ). GR pplition omplemente the rnhing phenotype (Fig. ), further onfirming its reltion to strigoltone epletion. To get more etile insight into the effet of the reution in SlCCD8 expression on rnhing, the istriution of first- n seon-orer rnhes ws nlyse. In wil-type plnts. % of the primry stem noes were rrying visile lterl rnh, most of whih were shorter thn m (Fig. S). All SlCCD8 knok-own lines h signifintly (P <.) higher numer of rnhes of the first orer (Fig. S). Moreover, these rnhes were longer thn in the wil-type plnts (Fig. S). No rnhes of the seon orer were oserve in ny of the wil-type plnts t this stge of evelopment (Figs, S), wheres ll knok-own lines isplye multiple lterl rnhes of the seon orer (Figs e, S). The trnsgeni lines L6, L n L9 lso isplye reution in the primry stem height of 9%, % n 6%, respetively, ompre with wil-type plnts (Fig. f). In ition, the totl numer of noes in the trnsgeni lines inrese slightly, ut signifintly (P <.; Fig. g). SlCCD8 reution lters ventitious rooting In ition to these phenotypes, ll trnsgeni plnts, ut not the wil-type, isplye lrger numer of root primori n ventitious roots on their stems (Fig. 6,). The severity of this phenotype ws less pronoune in L6 thn in L n L9, orrelting with the stronger reution in strigoltone iosynthesis in these ltter two lines (Fig. ). Cuttings from the SlCCD8 knok-own lines proue %, 77% n 89% (L6, L n L9, respetively) more ventitious roots ompre with wiltype plnts (Fig. 6), phenotype tht oul e resue y GR pplition (Fig. 6). Moreover, pplition of GR lso reue the numer of ventitious roots (Fig. 6) n ventitious root primori (Fig. 6) in wil-type plnts. Conversely, IAA pplition le to n inrese in the numer of ventitious root primori (Fig. 6), while omine GR n IAA pplition restore this to ner untrete levels (Fig. 6). The free IAA onentrtion in the lower stem (first internoe ove the otyleons) ws.9-,.- n.-fol higher in L6, L n L9, respetively, thn in wiltype plnts (Fig. 6e), orrelting with the inrese in ventitious rooting. Effet of SlCCD8 knok-own on tomto reproutive evelopment An unexpete phenotype oserve in the SlCCD8 knok-own lines ws tht their flowers were signifintly smller thn in New Phytologist () Ó The Authors New Phytologist Ó New Phytologist Trust

7 New Phytologist Reserh 7 () 9 Solnol (). () 8 Diehyro Diehyro 7. Oronhol Strigoltones in root extrts (pek re g FW) 6 WT L6 L L9 Oronhol intomto xylem sp (ng h )... +Pi Pi WT L6 L L9 Fig. MRM-LC-MS/MS quntifition of strigoltone ontent in plnt of tomto (Solnum lyopersium) v Crigell (WT) n three inepenent SlCCD8 RNAi lines (L6, L, n L9). () Quntifition of the mjor tomto strigoltones (solnol, iehyro-oronhol isomers n, n oronhol) in root extrts of -wk-ol plnts oring to the pek re (n = ). () Quntifition of the effet of tretment with suffiient phosphte (+ Pi) n limiting phosphte ( Pi) on oronhol ontent in tomto xylem sp (n = ). () Quntifition of oronhol in tomto xylem sp of 8-wk-ol plnts (n = ). Error rs represent mens ± SE. Brs with ifferent letters iffer signifintly (P <.). Oronhol intomto xylem sp (pg h ) () () Numer of rnhes 8 6 WT L6 L L9 () WT L9 (e) () Numer of rnhes 8 6 WT L6 L L9 Fig. Anlysis of plnt rhiteture of tomto (Solnum lyopersium) v Crigell (WT) n three inepenent SlCCD8 RNAi lines (L6, L, n L9). () Photogrph of -wkol wil type n trnsgeni SlCCD8 RNAi line L9 plnts. () Averge numer of visile rnhes (st n n orer, > mm) on 8- wk-ol plnts (n = ). () The effet of GR on rnhing in -wk-ol plnts (n = ). Light grey rs, lm GR; rk grey rs, ontrol. () Close-up of 8-wk-ol wil-type primry rnh. (e) Close-up of 8-wk-ol L9 primry rnh, rrows inite seonry rnhes. (f) Averge min stem length of 8-wk-ol plnts (n = ). (g) Averge numer of noes per min stem of 8-wk-ol plnts (n = ). Error rs represent mens ± SE. Brs with ifferent letters iffer signifintly (P <.). (f) Primry stem length (m) 8 6 (g) WT L6 L L9 WT L6 L L9 Numer of noes wil-type plnts (Fig. 7). In orer to quntify this effet n eluite possile role of strigoltones in flower evelopment, the length of sepls, petls n nthers were mesure t nthesis. The verge length of ll these orgns ws signifintly (P <.) reue in ll three trnsgeni lines (Fig. 7). The imeter of the ovries ws lso signifintly reue (Fig. 7). This effet persiste throughout fruit evelopment (Fig. 7), leing to signifintly smller fruits in ll trnsgeni lines ompre with the wil-type in ifferent ripening stges, mture green ( DAP), reker n ripe re (Fig. 7e,f). Although fruit size of ll trnsgeni lines ws reue, only in L n L9 ws the totl fruit yiel slightly erese (Fig. 7g). Ó The Authors New Phytologist () New Phytologist Ó New Phytologist Trust

8 8 Reserh New Phytologist () m grient etween the ifferent tissues ws sent in the SlCCD8 knok-own fruits (Fig. 7i). () Aven ous root primori (No. per plnt) () Aven ous root primori (No. per plnt) WT L6 L L9 Aven ous roots (No. per plnt) WT L6 L L9 WT L6 L L9 Control GR IAA GR + IAA () (e) IAA (ng g DW) WT L6 L L9 As fruit size usully orreltes with see quntity (Mpelli et l., 978), the effet of SlCCD8 knok-own on see set ws investigte. A reution of out 6% in see quntity (Fig. 7h) n reution in see size (Fig. S6) ws oserve in ll three knok-own lines. Despite their reue numer n size, the qulity of the sees ppere to e unffete euse no ovious ifferenes in germintion rte were oserve in ny of these lines when germinte uner norml onitions (Fig. S7). Auxin plys n importnt role in severl strigoltone-relte phenotypes n it hs een shown to e one of the mjor regultors of fruit evelopment (Mpelli et l., 978; Gillspy et l., 99). Moreover, it hs reently een emonstrte tht uring fruit evelopment n uxin grient etween eveloping sees, plent n the perirp is estlishe uring phse III, with reltively high uxin onentrtions in the eveloping sees (Pttison & Ctlá, ). For this reson the onentrtion of free uxin ws ssesse in fruits hrveste uring this phse of fruit evelopment. Inee, in wil-type fruits.-fol higher onentrtion of IAA ws etete in the fruit prts ontining the sees ompre with the perirp (Fig. 7i). This uxin Fig. 6 Anlysis of ventitious root evelopment on tomto (Solnum lyopersium) v Crigell (WT) n three inepenent SlCCD8 RNAi lines (L6, L, n L9). () Photogrph of ventitious root primori on 8-wkol plnts. () Averge numer of visile ventitious root primori on -wk-ol plnts (n = ). () The effet of GR (light grey rs) on ventitious root evelopment in uttings (n = ). Drk grey rs, ontrol. () The effet of lm GR n. lm IAA on ventitious root evelopment in -wk-ol plnts (e) Conentrtion of free IAA in the lower stem (n = ). Error rs represent mens ± SE. Brs with ifferent letters iffer signifintly (P <.). New Phytologist () e e Disussion In the present stuy we ientifie n hrterise seon strigoltone iosyntheti gene in tomto SlCCD8 enoing rotenoi levge ioxygense. SlCCD8 showe highest homology to the petuni PhDAD/CCD8, elonging to iot sule within the CCD8 luster, lerly seprte from the monoots. SlCCD8 is expresse in ll tissues exmine, ut preominntly in root n stem tissue. This expression pttern is in greement with CCD8 expression in petuni, Ariopsis, pe n rie (Npoli, 996; Sorefn et l., ; Binrige et l., ; Arite et l., 7), initing onserve pttern ross plnt speies. In the Ariopsis mx/ccd8 mutnt, MAX/CCD7 expression ws shown to e ownregulte (Mshiguhi et l., 9). This feek seems not to e present in tomto s the expression of SlCCD7 ws not reue. It hs een reporte tht CCD7 is involve in proesses other thn strigoltone iosynthesis suh s the proution of myorriin in myotrophi plnts suh s tomto (Wlter et l., ). Therefore, the regultion of CCD7 my iffer etween tomto n other nonmyorrhizl plnt speies. By generting SlCCD8 knok-own lines with reution in strigoltone levels, we showe tht SlCCD8, through strigoltone iosynthesis, is involve in the regultion of multiple proesses relevnt for plnt physiology n rhizosphere signling. Strigoltones were initilly ientifie s germintion stimulnts for root prsiti plnts of the Oronhee (Cook et l., 966; Bouwmeester et l., ) n hyphl rnhing ftors for AM fungi (Akiym et l., ). As expete, reue strigoltone onentrtions in ll the SlCCD8 knok-own tomto lines resulte in reue AM olonistion (Fig.,). However, there ws nonliner orreltion etween AM olonistion n strigoltone reution, with the reution in myorrhiztion eing less severe thn the reution in strigoltone onentrtions. This is proly ue to the use of mixe inoulum ontining myelium, olonise roots n spores. Beuse strigoltones re hyphl rnhing ftors for AM fungi tht seem to e prtiulrly importnt in germinting spores, AM olonistion oul e more ompromise y strigoltone reution if only spores re use, s ws previously emonstrte (Kolti et l., ). In the present stuy mixe inoulum ws use s it more losely resemles the nturl sitution in the rhizosphere (Klironomos & Hrt, ). Interestingly, the infetion of ll SlCCD8 knok-own lines y P. rmos ws reue y 9% ompre with wil-type plnts (Fig. ). The oserve reution in emerging prsite shoots in L6 nnot e expline exlusively y its reue strigoltone exution (. 6%), lso onsiering tht germintion of P. rmos sees when using root exutes ws only reue y. %. Possily, the inrese shoot rnhing of the host n the ssoite resoure requirement inhiits P. rmos evelopment. Alterntively, it might e possile tht strigoltones proue y the host plnt re lso require in lter phses of the P. rmos lifeyle fter see germintion. Nevertheless, our results show tht mil reution in strigoltone exution oul e suffiient to Ó The Authors New Phytologist Ó New Phytologist Trust

9 New Phytologist Reserh 9 () WT L6 L L9 () Length of flower orgns (mm) WT L6 L L9 Sepl Petl Anthers (). Ovry imeter (mm) WT L6 L L9 Fig. 7 Anlysis of flowers, fruits n sees of tomto (Solnum lyopersium) v Crigell (WT) n three inepenent SlCCD8 RNAi lines (L6, L, n L9). () Photogrph of flowers t nthesis. () Flower orgns length (n = ). () Dimeter of un-pollinte ovries (n = ). () Dimeter of fruits t phse III tomto fruit evelopment ( DAP, ys fter pollintion; n = ). (e) Dimeter of mture green (MG) fruits ( DAP; n = ). (f) Photogrph of tomto fruits; mture green (MG), reker (BR) n mture re (MR). (g) Fruit yiel in grms fresh weight over -wk perio. (h) Numer of sees per fruit (n = ). (i) Auxin istriution t phse III ( DAP); PT, perirp tissue; RT, remining tissue. (n = ). Error rs represent mens ± SE. Brs with ifferent letter iffer signifintly (P <.). () (e) (f) Ovry imeter Phse III ( DAP, mm) 6 WT L6 L L9 Fruit imeter (MG, m) WT L6 L L9 (g) (h) (i) Tomto fruit yiel (g) Week Week 9 Week 8 6 PT RT Week 7 Week WT L6 L L9 WT L6 L L9 WT L6 L L9 Sees per fruit (ng g FW) WT L6 L L9 IAA (ng g FW) MG BR RR signifintly reue prsiti wee infetion without severely ompromising AM symiosis or pil ominne (Figs, ). Moreover, fruit yiel of L6 ws hrly ffete, even though initil fruit set ws elye (Fig. 7i). These finings mke strigoltone iosynthesis n ttrtive trget for ontrolling root prsitism, s previously postulte (Bouwmeester et l., ; López-Ráez et l., 9, ; Croso et l., ). However, more reserh is neee to further ssess the onsequenes of mil reution in strigoltone ontent on fruit qulity n prsiti wee resistne uner fiel onitions. Ó The Authors New Phytologist () New Phytologist Ó New Phytologist Trust

10 Reserh New Phytologist The onentrtions of strigoltones in root extrts in ll SlCCD8 knok-own lines were reue to the sme extent s in their exutes. This shows tht, s expete, strigoltone iosynthesis n not seretion is ompromise in these trnsgeni lines. All trnsgeni lines isplye reue primry stem height n n inrese in the totl numer of lterl rnhes. Both phenotypes inversely orrelte to some extent with the strigoltone onentrtions. Surprisingly, in roots of L the reution in SlCCD8 expression n strigoltone ontent were more severe thn expete onsiering the moerte inrese in lterl shoot outgrowth when ompre with L9. Interestingly, etter inverse orreltion with lterl rnhing ross the knok-own lines ws oserve with the onentrtions of oronhol present in the xylem sp (Fig. ). It seems tht the oronhol onentrtion in the xylem sp ws less ffete thn in the exutes, whih suggests preferentil loing into the xylem inste of seretion to the rhizosphere. The rtio of oronhol n the other strigoltones ws muh higher in root extrts thn in root exutes. Furthermore, neither solnol nor ny of the iehyro-oronhol isomers were etete in the xylem sp, wheres they were unntly present in root exutes n extrts (Figs, ). This suggests tht seletive mehnism of lolise strigoltone iosynthesis n/or trnsport ensures tht oronhol is less well serete into the rhizosphere n is preferentilly trnsporte through the xylem to the shoot. Therefore, initing tht this strigoltone (n/or its erivtives) might e the one tht is regulting shoot rnhing, while solnol n the iehyrooronhol isomers oul hve role s signling moleules in the rhizosphere (Fig. 8). As strigoltones lso o-regulte root system rhiteture (Kpulnik et l., ; Ruyter-Spir et l., ), it is possile tht ll re involve in this elowgroun funtion (Fig. 8). However, it is lso possile tht solnol n the iehyrooronhol isomers hve no funtion in the plnt n merely t s rhizosphere signling moleules. It ws previously propose tht solnol is erive from oronhol through iehyro-oronhol (Xie et l., ; Kohlen et l., ). Therefore, oronhol might ply oule role s the trnsmissile signl regulting root n shoot rhiteture, n s the preursor to signling moleules in the rhizosphere. When strigoltone proution ws ssesse uner phosphtelimiting onitions, strong inrese in ll tomto strigoltones ws etete in root extrts (Fig. S8), whih is in line with previous finings (López-Ráez et l., 8). In ition, we show tht phosphte limittion lso inue five-fol inrese in oronhol onentrtions in the xylem sp (Fig. ). Therefore, it seems tht this upregultion uner phosphte efiieny is more onserve tre s it hs lso een emonstrte in Ariopsis (Kohlen et l., ). A reution in min stem height ws lso oserve for ll trnsgeni lines, gin orrelting with xylem sp oronhol onentrtions. The reution in plnt height inversely orrelte to the inrese numer of shoot rnhes. This suggests tht perhps the oserve wrfism in the strigoltone-efiient knok-own lines is seonry effet of the inrese lterl shoot growth. However, the totl numer of internoes slightly inrese in ll trnsgeni lines (Fig. g). Our results show tht the New Phytologist () Xylem sp Root system rotenoi Rhizosphere D7 CCD7 CCD8 MAX -eoxystrigol oronhol oronhol iehyro-oronhol solnol Fig. 8 Propose moel for strigoltone movement in- n outsie the plnt. reution in plnt height in the SlCCD8 knok-own lines is ue to shorter internoes mye euse of inrese ompetition for nutrients etween the primry stem n the xillry rnhes. However, the inrese in noe numer suggests tht strigoltones re not only involve in the regultion of u outgrowth, ut possily lso in the timing of orgn evelopment. Aitionl reserh will e neee to stuy this in more etil. It hs lrey een shown tht strigoltones ply role in more plnt evelopment proesses thn just xillry u outgrowth (Tsuhiy et l., ; Kpulnik et l., ; Ruyter-Spir et l., ). Strigoltones were lso shown to o-regulte ventitious root evelopment in pe (Rsmussen et l., ). Our results orroorte this new funtion euse ll trnsgeni lines showe mrke inrese in ventitious root evelopment n rooting of uttings (Fig. 6). Interestingly, the inrese in ventitious rooting in the trnsgeni lines orrelte with elevte uxin onentrtions in the lower region of the stem, possily result of elevte trnsport s ws previously reporte (Bennett et l., 6). It is known tht uxin inues lterl root formtion (Klerk et l., 999), therefore it is plusile tht the oserve repressive effet of strigoltones on ventitious root formtion is onsequene of their ssume ontrol over uxin trnsport. However, it ws reently postulte tht the inrese in ventitious root initition in pe n Ariopsis is not ue to elevte uxin onentrtions ut to inrese uxin sensitivity (Rsmussen et l., ). The ft tht the omine pplition of IAA n GR suppresse the inrese in ventitious rooting inue y IAA (Fig. 6) seems to support this hypothesis. If so, the elevte IAA onentrtions etete in the lower prt of the stem might e seonry effet of reue uxin sensitivity. Nevertheless, our results inite tht link etween strigoltones n uxin exists in ventitious root initition. However, to etermine the preise unerlying mehnism of strigoltones-uxin ross-tlk in reltion to this phenotype further reserh will e neee. A puttive funtion for strigoltones in fruit evelopment ws lrey postulte s SlCCD7 is highly expresse in mture green n turning tomto fruits (Vogel et l., ), n the expression Ó The Authors New Phytologist Ó New Phytologist Trust

11 New Phytologist Reserh of ACCD7 n ACCD8 in Atinii hinensis (kiwifruit) ws shown to e reltively high in young fruits n sees (Leger et l., ). The petuni 8/ mutnt ws reporte to hve smller flowers (Snowen et l., ). In greement with these oservtions, we oserve tht in the SlCCD8 knok-own lines sepls, petls n nthers were smller thn in wil-type plnts (Fig. 7), suggesting tht strigoltone efiieny ffets flower evelopment. It might e tht this phenotype is the result of inrese ompetition for resoures in these highly rnhe plnts. However, the possiility tht strigoltones hve more iret role in ontrolling flower evelopment nnot e exlue. In ition to smller flowers, the fruit size in the trnsgeni lines ws lso reue, reution whih orrelte inversely with the strigoltone ontent in these lines. The reltive reution in fruit size ws similr to the reution in the imeter of un-pollinte ovries. This implies tht the initil reution in ovry size is not ompenste uring lter stges of fruit evelopment, ut lso tht the severity of the phenotype oes not inrese, whih woul e expete if the reue fruit size is only use y ompetition for resoures. Moreover, s fruit yiel ws not strongly reue in ny of the trnsgeni lines, resoure limittion seems not to e exlusively responsile for these reproutive phenotypes. It hs een reporte tht uxin proue in eveloping sees is require for fruit growth (Mpelli et l., 978). See numer, s well s see size in the trnsgeni lines were lerly reue ompre with wil-type plnts (Fig. 7g), n it is hene possile tht this reution resulting in lower uxin proution is responsile for the oserve reution in fruit size. However, it nnot explin the reution in ovry size efore pollintion, euse no uxin prouing sees re present t this stge. As mentione ove, ross-tlk etween strigoltones n uxin seems to ply n importnt role in severl strigoltone-relte phenotypes (Bennett et l., 6; Agusti et l., ; Ruyter-Spir et l., ; Rsmussen et l., ). It is plusile tht similr mehnism forms the sis for the oserve reproutive phenotypes, linking the effet of strigoltones on reproutive evelopment to the oregultion of uxin sensitivity n/or istriution. Auxin is known to e n importnt regultor of oth flower n fruit evelopment (Mpelli et l., 978; Gillspy et l., 99) n it ws emonstrte tht uxin iosynthesis loss-of-funtion mutnts isply severe reproutive efets inluing errnt flower morphology n mle/femle sterility (Cheng et l., 6; Gllvotti et l., 8). The reltive mil phenotypes oserve in ll trnsgeni lines re hene not onsequene of uxin loss-of-funtion, ut of uxin reue-of-funtion. However, more reserh will e neee to further explore this possile intertion etween strigoltones n uxin in reproutive evelopment. Overll, our results emonstrte tht SlCCD8 is require for strigoltone iosynthesis in tomto n therefore is ruil in regulting severl proesses in plnt evelopment n rhizosphere signling. We showe tht reltively smll reution in strigoltone seretion hs profoun impt on P. rmos infetion, wheres AM intertion, pil ominne n fruit yiel re only milly ffete, emonstrting tht strigoltone reution n e suitle strtegy ginst root prsiti wees. In ition, speifiity in strigoltone trnsport in n ex plnt is propose, s only oronhol is preferentilly loe into the xylem. Finlly, the phenotype of SlCCD8 knok-own plnts onfirms role for strigoltones in rnhing n ventitious root evelopment n hints t new role for strigoltones in reproutive evelopment. Further reserh is require to estlish whether this is iret or n iniret effet. Aknowlegements We knowlege Rlph Bours, Ronny Joosen, Jun Grí, Din Jmr n Frnel Verstppen for their tehnil support. GR ws kinly provie y Binne Zwnenurg (Deprtment of Orgni Chemistry, Rou University, Nijmegen, the Netherlns). Strigoltone stnrs use were kinly provie y Koihi Yoneym (Wee Siene Center, Utsunomiy University, Jpn). Phelipnhe rmos sees were kinly provie y Murizio Vurro (Instituto i Sienze elle Prouzioni Alimentri, Bri, Itly). We knowlege funing y the Netherlns Orgniztion for Sientifi Reserh (NWO; VICI grnt, n Equipment grnt, 8.8. to H.J.B.) n the Europen Commission (Intr-Europen Mrie Curie postotorl fellowship FP6-MEIF- CT-- to J.A.L-R., FP7-PIEF-GA-8-77 to P.T. n Reintegrtion Grnt PERG--7-7 to J.A.L-R.)., WK ws lso supporte y postotorl ontrt from the Mx Plnk Institute for Plnt Breeing Reserh. J.A.L-R. ws lso supporte y postotorl ontrt (JAE-Do) from the Spnish Reserh Counil (CSIC). This projet ws o-finne y the Centre for BioSystems Genomis (CBSG). Referenes Agusti J, Herol S, Shwrz M, Snhez P, Ljung K, Dun EA, Brewer PB, Beverige CA, Sieerer T, Sehr EM et l.. Strigoltone signling is require for uxin-epenent stimultion of seonry growth in plnts. Proeeings of the Ntionl Aemy of Sienes, USA 8: 7. Akiym K, Mtsuzki K-I, Hyshi H.. Plnt sesquiterpenes inue hyphl rnhing in rusulr myorrhizl fungi. Nture : Aler A, Jmil M, Mrzorti M, Bruno M, Vermthen M, Bigler P, Ghisl S, Bouwmeester H, Beyer P, Al-Bili S.. The pth from -rotene to rltone, strigoltone-like plnt hormone. Siene : 8. Arite T, Iwt H, Ohshim K, Mekw M, Nkjim M, Kojim M, Skkir H, Kyozuk J. 7. DWARF, n RMS/MAX/DAD ortholog, ontrols lterl u outgrowth in rie. Plnt Journl : 9 9. Binrige K, Sorefn K, Wr S, Leyser O.. Hormonlly ontrolle expression of the Ariopsis MAX shoot rnhing regultory gene. Plnt Journl : Bennett T, Sieerer T, Willett B, Booker J, Lushnig C, Leyser O. 6. The Ariopsis MAX pthwy ontrols shoot rnhing y regulting uxin trnsport. Current Biology 6: 6. Besserer A, Pueh-Pgès V, Kiefer P, Gomez-Roln V, Juneu A, Roy S, Portis J-C, Roux C, Bér G, Séjlon-Delms N. 6. Strigoltones stimulte rusulr myorrhizl fungi y tivting mitohonri. PLoS Biology : 9 7. Beverige CA, Ross JJ, Murfet IC. 99. Brnhing mutnt rms- in Pisum stivum (grfting stuies n enogenous inole--eti i levels). Plnt Physiology : Bomrely A, Men N, Tele IY, Buels RM, Strikler S, Fisher-York T, Pujr A, Leto J, Gosselin J, Mueller LA.. The Sol Genomis Network (solgenomis. net): growing tomtoes using Perl. Nulei Ais Reserh 9(Suppl ): D9 D. Ó The Authors New Phytologist () New Phytologist Ó New Phytologist Trust

12 Reserh New Phytologist Booker J, Sieerer T, Wright W, Willimson L, Willett B, Stirnerg P, Turnull C, Srinivsn M, Gor P, Leyser O.. MAX enoes ytohrome P fmily memer tht ts ownstrem of MAX/ to proue rotenoierive rnh-inhiiting hormone. Developmentl Cell 8: 9. Bouwmeester HJ, Mtusov R, Zhongkui S, Bele MH.. Seonry metolite signlling in host-prsiti plnt intertions. Current Opinion in Plnt Biology 6: 8 6. Bouwmeester HJ, Roux C, López-Ráez JA, Bér G. 7. Rhizosphere ommunition of plnts, prsiti plnts n AM fungi. Trens in Plnt Siene :. Croso C, Ruyter-Spir C, Bouwmeester HJ.. Strigoltones n root infesttion y plnt-prsiti Strig, Oronhe n Phelipnhe spp. Plnt Siene 8:. Cheng YF, Di XH, Zho YD. 6. Auxin iosynthesis y the YUCCA flvin monooxygenses ontrols the formtion of florl orgns n vsulr tissues in Ariopsis. Genes & Development : Cook CE, Whihr LP, Turner B, Wll ME, Egley GH Germintion of withwee (Strig lute Lour.): isoltion n properties of potent stimulnt. Siene : Foo E, Turnull CGN, Beverige CA.. Long-istne signling n the ontrol of rnhing in the rms mutnt of pe. Plnt Physiology 6: 9. Gllvotti A, Brzesh S, Mlomer S, Hll D, Jkson D, Shmit RJ, MSteen P. 8. sprse infloresene enoes monoot-speifi YUCCA-like gene require for vegettive n reproutive evelopment in mize. Proeeings of the Ntionl Aemy of Sienes, USA : 96. Gillspy G, Ben-Dvi H, Gruissem W. 99. Fruits: evelopmentl perspetive. Plnt Cell : 9. Giovnnetti M, Mosse B. 98. An evlution of tehniques for mesuring vesiulr rusulr myorrhizl infetion in roots. New Phytologist 8: 89. Golwsser Y, Yoneym K, Xie X, Yoneym K. 8. Proution of strigoltones y Ariopsis thlin responsile for Oronhe egypti see germintion. Plnt Growth Regultion : 8. Gomez-Roln V, Ferms S, Brewer PB, Pueh-Pges V, Dun EA, Pillot J-P, Letisse F, Mtusov R, Dnoun S, Portis J-C et l. 8. Strigoltone inhiition of shoot rnhing. Nture : Helliwell CA, Wesley SV, Wielopolsk AJ, Wterhouse PM.. Highthroughput vetors for effiient gene silening in plnts. Funtionl Plnt Biology 9: 7. Hewitt EJ Sn n wter ulture methos use in the stuy of plnt nutrition. Frnhm Royl, UK: Commonwelth Agriulturl Bureux. Hogln DR, Arnon DI. 9. The wter-ulture metho for growing plnts without soil. Cirulr. Cliforni Agriulturl Experiment Sttion 7:. Kpulnik Y, Delux P-M, Resnik N, Myzlish-Gti E, Wininger S, Bhtthry C, Séjlon-Delms N, Comier J-P, Bér G, Belusov E et l.. Strigoltones ffet lterl root formtion n root-hir elongtion in Ariopsis. Plnt : 9 6. Klerk G-J, Krieken Wv, Jong JC Review: the formtion of ventitious roots: new onepts, new possiilities. In Vitro Cellulr & Developmentl Biology. Plnt : Klironomos JN, Hrt MM.. Coloniztion of roots y rusulr myorrhizl fungi using ifferent soures of inoulum. Myorrhiz : 8 8. Kohlen W, Chrnikhov T, Liu Q, Bours R, Domglsk MA, Beguerie S, Verstppen FWA, Leyser HMO, Bouwmeester HJ, Ruyter-Spir C.. Strigoltones re trnsporte through the xylem n ply key role in shoot rhiteturl response to phosphte efiieny in nonrusulr myorrhizl host Ariopsis. Plnt Physiology : Kohlen W, Ruyter-Spir C, Bouwmeester HJ.. Strigoltones: new musiin in the orhestr of plnt hormones. Botny 89: Kolti H, Dor E, Hershenhorn J, Joel D, Weininger S, Lekll S, Sheltiel H, Bhtthry C, Elihu E, Resnik N et l.. Strigoltones effet on root growth n root-hir elongtion my e meite y uxin-efflux rriers. Journl of Plnt Growth Regultion 9: 9 6. Kolti H, LekKl SP, Bhtthry C, Myzlish-Gti E, Resnik N, Wininger S, Dor E, Yoneym K, Yoneym K, Hershenhorn J et l.. A tomto strigoltone-impire mutnt isplys errnt shoot morphology n plnt intertions. Journl of Experimentl Botny 6: New Phytologist () Kretzshmr T, Kohlen W, Ssse J, Borghi L, Shlegel M, Bhelier JB, Reinhrt D, Bours R, Bouwmeester HJ, Mrtinoi E.. A petuni ABC protein ontrols strigoltone-epenent symioti signlling n rnhing. Nture 8:. Kroshel J.. A tehnil mnul for prsiti wee reserh n extension. Dorreht, The Netherlns: Kluwer Aemi Pulishers. Leger SE, Jnssen BJ, Kruniretnm S, Wng T, Snowen KC.. Moifie CAROTENOID CLEAVAGE DIOXYGENASE8 expression orreltes with ltere rnhing in kiwifruit (Atinii hinensis). New Phytologist 88: 8 8. Lin H, Wng R, Qin Q, Yn M, Meng X, Fu Z, Yn C, Jing B, Su Z, Li J et l. 9. DWARF7, n iron-ontining protein require for the iosynthesis of strigoltones, regultes rie tiller u outgrowth. Plnt Cell :. Liu W, Kohlen W, Lillo A, Op en Cmp R, Ivnov S, Hrtog M, Limpens E, Jmil M, Smznik C, Kufmnn K et l.. Strigoltone iosynthesis in Meigo truntul n rie requires the symioti GRAS-type trnsription ftors NSP n NSP. Plnt Cell : Livk KJ, Shmittgen TD.. Anlysis of reltive gene expression t using rel-time quntittive PCR n the (T)(-Delt Delt C) metho. Methos : 8. López-Ráez JA, Chrnikhov T, Fernánez I, Bouwmeester H, Pozo MJ.. Arusulr myorrhizl symiosis ereses strigoltone proution in tomto. Journl of Plnt Physiology 68: López-Ráez JA, Chrnikhov T, Gómez-Rolán V, Mtusov R, Kohlen W, Vos RD, Verstppen FWA, Pueh-Pges V, Bér G, Muler P et l. 8. Tomto strigoltones re erive from rotenois n their iosynthesis is promote y phosphte strvtion. New Phytologist 78: López-Ráez JA, Chrnikhov T, Muler P, Kohlen W, Bino R, Levin I, Bouwmeester HJ. 8. Suseptiility of the tomto mutnt high pigment-g (hp-g) to Oronhe spp. infetion. Journl of Agriulturl n Foo Chemistry 6: López-Ráez JA, Kohlen W, Chrnikhov T, Muler P, Uns AK, Sergent MJ, Verstppen F, Bugg TDH, Thompson AJ, Ruyter-Spir C et l.. Does sisi i ffet strigoltone iosynthesis? New Phytologist 87:. López-Ráez JA, Mtusov R, Croso C, Jmil M, Chrnikhov T, Kohlen W, Ruyter-Spir C, Verstppen F, Bouwmeester H. 9. Strigoltones: eologil signifine n use s trget for prsiti plnt ontrol. Pest Mngement Siene 6: López-Ráez JA, Pozo MJ, Grí-Grrio JM.. Strigoltones: ry for help in the rhizosphere. Botny 89:. Mpelli S, Frov C, Torti G, Soressi GP Reltionship etween set, evelopment n tivities of growth regultors in tomto fruits. Plnt n Cell Physiology 9: Mshiguhi K, Sski E, Shim Y, Nge M, Ueno K, Nkno T, Yoneym K, Suzuki Y, Asmi T. 9. Feek-regultion of strigoltone iosyntheti genes n strigoltone-regulte genes in Ariopsis. Biosiene, Biotehnology, n Biohemistry 7: 6 6. Mtusov R, Rni K, Verstppen FWA, Frnssen MCR, Bele MH, Bouwmeester HJ.. The strigoltone germintion stimulnts of the plntprsiti Strig n Oronhe spp. re erive from the rotenoi pthwy. Plnt Physiology 9: 9 9. Npoli C Highly rnhe phenotype of the petuni - mutnt is reverse y grfting. Plnt Physiology : 7 7. Pttison RJ, Ctlá C.. Evluting uxin istriution in tomto (Solnum lyopersium) through n nlysis of the PIN n AUX/LAX gene fmilies. Plnt Journl 7: Phillips JM, Hymn DS. 97. Improve proeures for lering roots n stining prsiti n vesiulr-rusulr myorrhizl fungi for rpi ssessment of infetion. Trnstions of the British Myologil Soiety : 8 6, IN6 IN8. Rni K, Zwnenurg B, Sugimoto Y, Yoneym K, Bouwmeester HJ. 8. Biosyntheti onsiertions oul ssist the struture eluition of host plnt proue rhizosphere signlling ompouns (strigoltones) for rusulr myorrhizl fungi n prsiti plnts. Plnt Physiology n Biohemistry 6: Ó The Authors New Phytologist Ó New Phytologist Trust