Local and systemic N signaling are involved in Medicago truncatula preference for the most efficient Sinorhizobium symbiotic partners

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1 Reserh Lol nd systemi N signling re involved in Medigo truntul preferene for the most effiient Sinorhizoium symioti prtners Gisèle Lguerre 1,2,3,4,5, Krine Heulin-Gotty 1,2,3,4,5, Brigitte Brunel 1,2,3,4,5, Agnieszk Klonowsk 1,2,3,4,5, Antoine Le Quéré 1,2,3,4,5, Psl Tillrd 6,7,8,9, Yves Prin 1,2,3,4,5, Jen-Clude Cleyet-Mrel 1,2,3,4,5 nd Mr Lepetit 1,2,3,4,5 1 INRA, USC 1242, Symioses Tropiles et Méditerrnéennes, F-34 Montpellier, Frne; 2 IRD, UMR 113, Symioses Tropiles et Méditerrnéennes, F-34 Montpellier, Frne; 3 CIRAD, UMR 113, Symioses Tropiles et Méditerrnéennes, F-34 Montpellier, Frne; 4 SupAgro, UMR 113, Symioses Tropiles et Méditerrnéennes, F-34 Montpellier, Frne; 5 UM2, UMR 113, Symioses Tropiles et Méditerrnéennes, F-34 Montpellier, Frne; 6 INRA, UMR 54, Biohimie et Physiologie Moléulire des Plntes, F-34 Montpellier, Frne; 7 CNRS, Biohimie et Physiologie Moléulire des Plntes, F-34 Montpellier, Frne; 8 SupAgro, Biohimie et Physiologie Moléulire des Plntes, F-34 Montpellier, Frne; 9 UM2, Biohimie et Physiologie Moléulire des Plntes, F-34 Montpellier, Frne Author for orrespondene: Gisèle Lguerre Tel: Emil: gisele.lguerre@supgro.inr.fr Reeived: 5 Deemer 211 Aepted: 21 Mrh 212 doi: /j x Key words: dpttive response, Medigo truntul, nitrogen fixtion, nodule development, prtner hoie, sntion, Sinorhizoium, systemi N signling. Summry Responses of the Medigo truntul Sinorhizoium intertion to vrition in N 2 -fixtion of the teril prtner were investigted. Split-root systems were used to disriminte etween lol responses, t the site of intertion with teri, nd systemi responses relted to the whole plnt N sttus. The lk of N quisition y hlf-root system nodulted with nonfixing rhizoium triggers ompenstory response enling the other hlf-root system nodulted with N 2 -fixing prtners to ompenste the lol N limittion. This response is medited y stimultion of nodule development (numer nd size) nd involves systemi signling mehnism relted to the plnt N demnd. In roots o-infeted with poorly nd highly effiient strins, prtner hoie for nodule formtion ws not modulted y the plnt N sttus. However, the plnt N demnd indued preferentil expnsion of nodules formed with the most effiient prtners when the symioti orgns were funtionl. The response of nodule expnsion ws ssoited with the stimultion of symioti plnt ell multiplition nd of teroid differentition. A generl model where lol nd systemi N signling mehnisms modulte intertions etween Medigo truntul nd its Sinorhizoium prtners is proposed. Introdution Leguminous plnts disply the pity to quire gseous nitrogen (N 2 ) through the estlishment of symioti ssoition with soil teri lled rhizoi whih proliferte nd differentite inside root nodules. The mutul enefit of this ssoition is sed on nutritionl exhnges etween plnts nd teri: tmospheri N 2 is fixed y rhizoi nd relesed s NH 4 + to the plnt, wheres the host plnt provides C metolites to the teri. However, in soil, the roots re often sujeted to stresses tht temporlly nd lolly limit N quisition, espeilly N 2 fixtion whih is highly sensitive to environmentl ftors. In ddition, legumes re usully nodulted y indigenous rhizoil popultions displying geneti polymorphism ssoited with vriility in N 2 fixtion effetiveness. Plnt growth will therefore depend on its ility to offset this N defiit y inresing nitrogen quisition pity in unstressed root prts. New Phytologist Ó 212 New Phytologist Trust Regultions responsile for the djustment of symioti N quisition pities to the N demnd of the whole plnt hve een hrterized in the model legume Medigo truntul (Mt) using split-root systems (Ruffel et l., 28; Jeudy et l., 21). When nodulted plnts re supplied y minerl N, systemi N stiety mehnism represses speifi N 2 fixtion tivity (SNA) of the symioti orgns, djusting their N quisition pities to the N demnd of the whole plnt (Ruffel et l., 28). By ontrst, plnt N limittion does not trigger upregultion of nodule SNA nd therefore results in redution of N quisition pity of the whole plnt in the short term (few dys). Nevertheless, N 2 -fixing plnts exhiit developmentl ompenstory responses to N defiit whih ompenste the lol N limittion in the long term (> 2 wk; Jeudy et l., 21). Indeed, oth nodule formtion nd nodule expnsion re stimulted y systemi N signling of N defiit. These responses of nodule development to systemi N signling hve een ssoited with erly hnges in 437

2 438 Reserh New Phytologist C-metolite llotion towrd N 2 -fixing roots t the expense of the ineffiient ones. Autoregultion of nodultion (AON) is systemi feedk repression ontrolling the nodule numer nd the nodultion zone on the roots (see reviews of Ok-Kir & Kwguhi, 26 nd Reid et l., 211). Crosstlk etween AON nd plnt N signling hs een suggested, euse inhiition of nodultion y minerl N is suppressed in AON mutnts, nd the repressive AON n e, t lest prtilly, relesed y the N demnd of the whole plnt in Mt (Jeudy et l., 21). However, the response of mture nodule expnsion to the plnt N demnd ws shown to e independent of this AON regultory iruit. Medigo truntul forms elongted indeterminte nodules with persistent meristemti tivity in whih teri undergo differentition proess from free-living reprodutive teri to terminlly differentited stte resulting in nonreprodutive, polyploid nd elongted teroids (Mergert et l., 26). Histologilly, indeterminte nodules of Medigo spp. re orgnized into five zones (Vsse et l., 199; Timmers et l., 2, Munoury et l., 21): the pil meristemti zone I, the infetion zone II in whih the teri re relesed from infetion threds nd oth prtners differentite progressively, the onstntly growing N 2 -fixing zone III, nd the senesent zone IV. In older nodules, n dditionl zone V ws desried. It onsists of senesed plnt ells tht re reinvded y nondifferentited teri, relesed from the remining infetion threds, whih n e regrded s sprophytes. Medigo truntul interts speifilly with two losely relted speies, Sinorhizoium meliloti nd S. medie (Rome et l., 1996). Geneti diversity within these speies results in vriility for N 2 -fixtion effetiveness (Mhdhi et l., 25; Heth & Tiffin, 27; Rngin et l., 28; Terpolilli et l., 28). To sueed in nodultion in ompetition with other omptile rhizoi, prtiulr rhizoium must pss different steps, from rhizosphere oloniztion to root infetion nd nodule formtion. Seletion of rhizoil genotypes mong omptile rhizoi present in soil hs een desried (Bromfield et l., 1995; Hrtmnn et l., 1998; Lguerre et l., 23). It hs een proposed tht the plnt hs developed mehnisms to promote nd mintin mutulisti intertions with the most enefiil prtners, ssuming tht the host is le to disriminte mong nodules sed on effiieny in N 2 fixtion nd to preferentilly support the most effiient ones (see reviews of Simms & Tylor, 22; Oono et l., 29). Pre-infetion prtner hoie refers to the seletion y the plnt of the most effiient prtner efore the formtion of funtionl nodules. Post-infetion prtner hoie refers to the ility of the plnt to llote resoures preferentilly to the most effiient nodules. However, there is so fr only little experimentl support for suh mehnisms. Some studies hve provided indiret evidene suggesting pre-infetion prtner hoie (Heth & Tiffin, 29; Shs et l., 21) ut other studies filed to vlidte this theory (Simms et l., 26; Shs et l., 21). Interestingly, the reent disovery of plnt trnsription ftor from the legume speies Phseolus vulgris, NF-YC1, tht influenes teril ompetitiveness for nodultion possily ording to N 2 fixtion effetiveness (Znetti et l., 21) opens new perspetives to deipher the moleulr sis of prtner hoie. Severl experiments with plnts o-infeted y multiple prtners vrying in symioti performne provided evidene for post-infetion prtner hoie s reveled y differene in nodule size nd or in teril numer per nodule (Singleton & Stokinger, 1983; Simms et l., 26; Shs et l., 21). However, Heth & Tiffin (29) did not find evidene of post-infetion prtner hoie y Mt in similr experiment. Suppression of N 2 -fixtion tivity y Ar:O 2 tretments resulted in rpid inhiition of development of nonfixing nodules (Singleton & vn Kessel, 1987; Kiers et l., 23, 26; Jeudy et l., 21; Oono et l., 211). These results suggest lol ontrol of nodule development y N 2 fixtion tivity, whih is onsistent with the onept of post-infetion prtner hoie. The growth inhiition of nodules pled in n Ar:O 2 environment ws ssoited with redution of the numer of reprodutive teri oth within nodules hroring either reprodutive or nonreprodutive teroids, whih hs een proposed s plnt sntion ginst defetive prtners (Kiers et l., 23, 26; Oono et l., 211). However, no evidene of plnt sntioning leding to differenes in teril fitness ws deteted when pirs of nonfixing nd fixing strins were used in split-root systems (Mro et l., 29; Gury-Rngin et l., 21). Up to now, whole plnt responses to vritions in N supply indued y nodultion with teril prtners vrying for N 2 - fixtion effiieny hve een poorly investigted. In this study, this question ws ddressed using speifi experimentl systems llowing the disrimintion etween lol effets, t the site of intertion with the teri, nd responses relted to the whole plnt N sttus nd whole plnt N demnd. Both funtioning nd development of the symioti strutures were investigted. Nturl vriility existing within olletion of rhizoi omptile with Mt ws exploited. Intertion with ineffiient teri nd rtifiil suppression of fixtion y Ar:O 2 tretments were ompred. We lso ddressed the question of the ility of the plnt to promote nodultion with the most enefiil prtner ording to its inresed N demnd, oth t the level of initil prtner hoie (suess of nodule oupny in o-inoulted plnts) nd of nodule growth. Our findings support mjor role of plnt N signling in mutulisti intertions etween Mt nd its Sinorhizoium prtners. Mterils nd Methods Bteril strins The teril strins used in this study re listed in Tle 1. Bteri were grown t 28 C for 2 24 h in yest mnnitol (YM) liquid medium or for 2 4 d on YM gr (Vinent, 197). Plnt growth onditions nd experimentl design The Medigo truntul Gertn. Jemlong A17 ws used in this study. The plnts were grown either under erted hydroponi onditions in tnks (two to four replites, five to seven plnts per tnk) in limti hmer s previously desried (Ruffel New Phytologist Ó 212 New Phytologist Trust

3 New Phytologist Reserh 439 Tle 1 Bteril strins used nd origin Speies Strin Medigo host of origin nd or relevnt hrteristis Geogrphi origin Referene S. meliloti M. stiv Austrli Rosenerg et l. (1981) S. meliloti 211 TH2.3 derivtive, Btut et l. (1985) Nod + Fix ), fixj::tn5 S. meliloti TII7 M. truntul Tunisi Mhdhi et l. (25) S. meliloti WSM122 M. oriulris Greee Terpolilli et l. (28) S. meliloti ml2 M. truntul Frne Rngin et l. (28) S. meliloti ml6 M. truntul Frne Rngin et l. (28) S. meliloti ml7 M. truntul Frne Rngin et l. (28) S. meliloti ml8 M. truntul Frne Rngin et l. (28) S. medie md2 M. truntul Frne Rngin et l. (28) S. medie M. truntul Frne Rngin et l. (28) et l., 28), or in pots filled with perlite sustrte (three replites, three plnts per pot) nd wtered weekly with n N-free nutrient solution (Moreu et l., 28) in glsshouse under nturl light (23 28 C during the dy nd 18 2 C during the night). Plntlets were inoulted with teril ell suspensions (. 1 7 per plnt). In hydroeroponi onditions, plnts were first grown in medium supplemented with.5 mm of KNO 3.In stndrd tnks, fter 1 2 wk of growth, the medium ws hnged to N-free nutrient solution nd the roots were then inoulted. In pot onditions, plnts were inoulted t sowing. In ddition to stndrd tnks, split-root systems (two to four replites, five plnts per system) were used. After 1 wk of growth, the root tips were ut to promote root rnhing. After 1 2 wk, the root systems were seprted into two prts, eh side eing instlled in seprte omprtment filled with the sme N-free sl nutrient solution. Different tretments iming to limit the N intke to one side of the root system were pplied. Short-term lolized N-limittion tretments were pplied on nodulted roots 21 dys fter inoultion (DAI). To remove the N soure (N 2 ) from the treted omprtment, ontinuous flow of 8% rgon:2% O 2 ws pplied s previously desried (Ruffel et l., 28). For long-term lolized N-tretments, roots were instlled in split-root systems prior inoultion. One side ws then inoulted with the non-n 2 -fixing strin (Fix ) ) 211 TH2.3, nd the other side with N 2 -fixing strins (Fix + ), either s single inoulnts or using mixture of strins, oth side eing exposed to the sme nutrient solution s given ove. The nutrient solutions were renewed weekly. Mixed inoulnts nd strin identifition A preliminry experiment ws performed in order to estimte the rtio of teril ells in mixed inoulnts of S. medie nd S. meliloti to get. 5% of nodules formed with eh strin. The reltionship etween the rtio of teril ells in mixed inoulnts nd the resulting rtio of nodules formed y eh strin ws estlished y inoultion of seven different : rtios (from.1 : 99.9 to 99.9 :.1) to plnts grown in pots. Nodules were olleted 36 DAI, nd rhizoil strins were isolted from rushed nodules (96 per tretment) s desried previously (Depret & Lguerre, 28). The strins nd ould e relily disriminted sed on differene in olony morphology on YM gr medium. The following reltionship ws found (r 2 =.97), P n nd P i eing the proportion of -formed nodules nd ells in the inoulnts, respetively: P n ¼ :18 Log e ðp i Þþ:97 Therefore, this model predited tht 1 : 9 rtio of : ells in the inoulum should led to n equl proportion of nodules formed with eh strin. A similr pproh ws used for mixtures of strins nd Fix ). The two strins were disriminted sed on the streptomyin resistne (1 lg ml )1 ) of the Fix ) strin. The following reltionship (R 2 =.57) ws found: P n ¼ :16 Log e ðp i Þþ1: No event of o-infetion within single nodules ws deteted with oth types of mixed inoulnts. Plnt hrvesting nd mesurements Mesurements of 15 N 2 fixtion lsting 1 min were performed on freshly exised nodulted roots s desried in Ruffel et l. (28). This previous study reported tht mesurements of 15 N 2 fixtion on exised nodulted roots were equivlent to those otined on intt plnt roots. All plnt orgns were olleted, nd nodules were ounted. Dry weights were determined fter oven drying t 8 C for 48 h. In some experiments, freshly olleted nodules were snned for further imge nlysis nd used for miroiologil mesurements. Nodules were lso onserved in Bouin s fixtive for histologil nlysis. Nodule size ws estimted either from the men nodule DW or nondestrutively y imge nlysis on freshly olleted nodules. A signifint (R 2 =.76) liner reltionship etween nodule DW (mg) nd projeted surfe re (S in m 2 ) of nodule ws otined nd is s follows: DW = 3.95 S The N nutrition index (NNI) ws lulted s desried in Moreu et l. (28) to quntify plnt N nutrition level s the rtio etween shoot N onentrtion nd %N, whih is the miniml N onentrtion llowing mximum iomss prodution, estimted s %N = 8.1 Shoot DW ).1. A NNI vlue of 1 is onsidered s optiml. New Phytologist Ó 212 New Phytologist Trust

4 44 Reserh New Phytologist Bteril ell ounting nd histologil methods Single freshly olleted nodules were surfe disinfeted nd refully rushed in sterile wter. For ounting of ultivle teril ells, nodule suspensions were serilly diluted in 1-fold dilutions nd plted on YM gr pltes. Nodule suspensions were kept frozen in 12% v v glyerol t ) 2 C for diret ell ounting y light mirosopy using ell ounter slides (KOVA Ò Glssti Ò Slide) ording to the mnufturer s instrution (Hyor Biomedil GmH, Kssel, Germny). Bteril ells of length 2 lm were onsidered s elongted differentited teroids ording to Mergert et l. (26). Bteril ells of 1 2 lm-long were onsidered s reproduile undifferentited ells, whih ws onfirmed y omprison of diret ounting nd ounting of ultivle ells. For nodule histologil nlysis, semi-thin (7 lm) longitudinl setions of prffin-emedded nodules were stined with.1% (w v) toluidine lue in 1% orx solution, oserved nd photogrphed using n Olympus Provis mirosope. Sttistil nlyses Dt nlysis y ANOVAs nd orreltion ws performed using XLSTAT softwre v (Addinsoft Ô, Pris, Frne). Mens were lssified using post ho Lest-Signifint Differene (LSD) Fisher test. Log 1 trnsformtions of dt were used for nodule surfe re nd numer of ells per nodule nd the logit trnsformtion for proportions. Results Nitrogen quisition y Mt vries ording to the N 2 -fixtion pity of the symioti prtner The teril vriility for N quisition y the symioti ssoition ws investigted y sreening olletion of nine S. meliloti nd S. medie strins from vrious origins whih were hosen to represent the known rnge of vrition in N 2 -fixtion tivity in symiosis with Mt Jemlong A17 (Tle 1 nd referenes therein). This olletion inluded the referene strin S. meliloti whih hs een reported to e poorly effetive in N 2 -fixtion with Mt Jemlong A17 (Mhdhi et l., 25). The effiieny of N 2 fixtion on the sis of nodule onstrution ost, expressed s SNA, ws mesured ut onsidered n pproximtion. Although the strin-dependent vritions in N umulted in shoot g )1 nodule DW re to some extent the result of their overll symioti effiieny, the preise evlution of N 2 fixtion effiieny, whih is the rtio of enefit to ost, requires diret mesurements of ron llotion to nodule s well s respirtory loss of ron (Oono & Denison, 21). Signifint positive liner reltionships were found etween SNA, N intke, plnt N ontent, plnt iomss, nd nodule size (Fig. 1; Supporting Informtion Fig. S1 nd Tle S1), showing tht the teril strin strongly ffets plnt N quisition, whih is limiting ftor of plnt growth in these experimentl onditions. Nodule numer ws negtively relted to nodule size. The vriility nd rnking of strins for plnt growth effet were μmol N g 1 nodule DW h ml7 TII7 onfirmed with older plnts (51 DAI) grown in stndrd pot onditions (Supporting Informtion Tle S2). Plnt N nutrition levels were estimted y NNI vlues (s defined in Moreu et l., 28). The NNI vlues vried from.4 to.8 mong the different symioti ssoitions (Tles S1, S2), the theoretil optiml vlue to fulfill the plnt N requirements eing 1. Bsed on its overll higher symioti performne inluding higher SNA, the S. medie strin ws seleted for further omprtive studies with the referene strin. Effet of the teril prtner on the plnt response to lolized N limittion Responses of plnts inoulted with or to lolized N limittion were hrterized using plnts grown in split-root systems. Adpttion to N defiit ws hrterized y ompring N quisition nd developmentl responses of N- limited nd ontrol plnts. Short-term N limittion tretments (4 d) were pplied 21 DAI on nodulted hlf-root systems y repling ir with mixture of 8% Ar nd 2% O 2 for 4 d (Fig. 2). The response to plnt N defiit of the nodulted hlf-root side tht remined supplied y ir (Fix + ) ws investigted. The whole plnt N intke y the N-limited plnts ws 54% nd 41% lower thn in the ontrol plnts with nd, respetively (Fig. 2). Consequently, shoot N ontent ws lso redued in N-limited plnts y 42% nd 22% with nd, respetively (Fig. S2). As expeted, nodule SNA in ontrol Fix + hlf-roots nodulted y ws higher thn in -nodulted hlf-roots. These tivities remined unhnged in response to the N defiit tretment (Fig. 2) inditing the sene of ompenstory inrese of SNA under suh onditions. However, nodule expnsion ws stimulted with in response to N defiit (23% higher thn the ontrol plnts; Fig. 2d). ml8 ml2 ml6 md2 WSM122 y = 1.8x R² = DW per plnt (mg) Fig. 1 Vriility of whole plnt iomss nd N 2 -fixtion-speifi tivity of nodules ording to the rhizoil prtner nd orreltion etween the two prmeters (19 d fter inoultion). Eh point is the men vlue for eh strin (n = 6). LSD vlues re 34.9 nd 53.6 for plnt dry weight (DW) nd speifi nodule tivity, respetively. The sttistil omprison of mens is given s supplementry dt in Tle S1. New Phytologist Ó 212 New Phytologist Trust

5 New Phytologist Reserh 441 () () Control N-limited Air Air Ar:O 2 1 Fig. 2 Effet of the teril strin on responses of N 2 -fixing plnt to short-term (4 d) lol N limittion indued y suppression of N 2 from the root tmosphere y Ar:O 2 tretment of nodulted roots on one side of split-roots systems shown in (). The N intke y the whole plnt (), speifi nodule tivity (), nd men nodule size (d) of nontreted hlf-root systems were ompred etween the ontrol (drk grey rs) nd N-limited plnts (light grey rs). Hlf-root systems exposed to the sme lol environment during the tretment (entrl omprtment) were used for omprison. Vlues re the mens of 1 replites. Identil letters show tht the men vlues re not signifintly different y LSD (P.5). Plnt N intke μmol N g 1 plnt DW h 1 () Speifi nodule tivity μmol N g 1 nodule DW h 1 (d) Nodule size (μg DW per nodule) did not lter the ility of the teri to form nodules, the nodule numer per hlf-root eing equivlent to tht in ontrol hlf-roots nodulted y the prentl strin (dt not shown). The Fix ) nodules were white nd smller thn those formed with in ontrol plnts. The response to plnt N defiit of the nodulted hlf-root side tht remined supplied y ir (Fix + ) ws investigted. For eh Fix + strins, the N-limited nd the ontrol plnts displyed similr iomsses (Fig. 3) nd N ontents despite the sene of effiient N 2 -fixtion in Fix ) hlf-roots (Tle S3). The N-limited plnts were le to fully ompenste for the lol N limittion indued y ineffetive nodultion in hlf-root systems (Fix ) sides) y inresing the N quisition pity of the hlf-root systems of the Fix + sides (62 64% higher thn in ontrol roots; Tle S3). This long-term ompenstory response resulted from strong inrese of nodule iomss in Fix + roots explined y n inrese of oth nodule numer nd men nodule size (Fig. 3) rther thn from djustment of nodule SNA. Biomss of Fix + roots ws lso inresed (Tle S3). Both symioti ssoitions displyed qulittively similr responses, ut these responses were more effiient with thn with. Although the inrese in nodule numer in Fix + roots ws omprle for ll strins (33 37% higher thn in ontrol roots), the stimultion of nodule expnsion in Fix + roots ws stronger with thn with (7% nd 3%, respetively). The Fix + nodules of N limited plnts were distriuted into two lsses of size. The smllest nodules displyed size similr to tht of nodules of ontrol plnts (Fig. 4). They were proly young nodules formed de novo in response to N limittion. The nlysis of nodule struture (zontion nd teril infetion) showed tht the iggest nodules, proly representing the expnded nodules, displyed penut shpe (Fig. 4,d). This shpe suggests two wves of nodule development s if, t some point, the pereption of N defiieny used n elertion of nodule development. This response ws muh stronger in nodules formed with (Fig. 4d) thn in those formed with (Fig. 4). The expnsion of nodules orresponded to signifint inrese of the N 2 -fixing zone III surfe nd of the numer of symioti plnt ells, while we did not reord suh signifint responses in elongted nodules formed with. Long-term N limittion tretments (3 d) were pplied y inoulting one side of split-root systems (Fix ) side) with Fix ) (fixj) mutnt of, the other side (Fix + ) eing inoulted with or (Fig. 3). In Fix ) roots, the fixj muttion New Phytologist Ó 212 New Phytologist Trust Effet of the N sttus of the plnt on the symioti prtner preferene The influene of the plnt N sttus on initil prtner hoie, s reveled y suessful nodultion, ws investigted y inoultion

6 442 Reserh New Phytologist () () Entire plnt DW (mg per plnt) () (d) Nodule DW (mg per hlf-root) No of nodules per hlf-root (e) Nodule size (μg DW per nodule) Control of plnts grown in hydro-eroponi onditions with mixture of nd ells. In preliminry experiment where the two strins were o-inoulted in different ell rtios, ws found to more ompetitive for nodultion thn in N-limited Fix + Fix + Fix Fig. 3 Effet of the Fix + teril strin pplied s single inoulnt on responses of N 2 -fixing plnts to long-term (3 d) lol N limittion indued y nodultion of hlf-root systems with Fix ) strin in split-roots systems shown in (). Asene of N 2 -fixtion tivity ws onfirmed for the Fix ) roots. () Entire plnt iomss. Developmentl responses were ompred etween the ontrol (drk grey rs) nd N-limited plnts (light grey rs) on the hlf-root systems exposed to the sme lol environment during the tretment (entrl omprtment): () nodule iomss, (d) numer of nodules, (e) men nodule size. Vlues re the mens of 1 replites. Identil letters show tht the men vlues re not signifintly different y LSD (P.5). sene of minerl N fertiliztion (see Mteril nd Methods). A 1 : 9 rtio of : ells in the inoulum ws hosen to otin n equl proportion of nodules formed y eh teril strin. The effet of.2 or 2 mm KNO 3 supplies to the plnts on the : nodule rtio ws investigted. The ontrsted N regimes resulted in signifint differenes in plnt iomss, plnt N ontent, nd NNI (Fig. 5). The plnt N sttus lso hd strong effet on nodule formtion s the numer of nodules ws four-fold higher in the.2 mm KNO 3 tretment (Fig. 5). Nevertheless, the identifition of the nodulting teri nd the quntifition of the proportion of nodules formed with eh strin in these ssys showed tht the N regimes tested here did not signifintly ffet the initil prtner hoie. Indeed, 63% nd 53% of nodules were formed y for 2 nd.2 mm KNO 3 tretments, respetively (differenes no sttistilly signifint t the.5 proility level). The plnt N regime hd n importnt effet on nodule expnsion s nodule size ws greter in those plnts supplied with.2 mm KNO 3 thn in those supplied with 2 mm KNO 3 (Fig. 6). This effet ws stronger for nodules thn for nodules (inrese in size of 162% nd 58%, respetively). Interestingly, nodule expnsion resulted in highly signifint stimultion of the multiplition nd elongtion of differentited teroids in nodules, ut not in signifint inrese in numers of ultivle teri per nodule (Fig. 6 d; Tle 2). Additionl split-root experiments were designed to disriminte etween the effets of the lol nd systemi N signlings. The system used ws similr to the one desried ove for investigtion of long-term N-limittion exept tht mixture of : ws used insted of single inoulnts (Fig. S3). The sene of systemi effet of the N sttus of the plnt on initil prtner hoie ws onfirmed s Fix + roots of N-limited nd ontrol plnts displyed identil : nodule rtios (Tle S4). Agin, the stimultion of nodule expnsion in response to systemi N signling ws higher with thn with, nd the expnsion of nodules ws ssoited with signifint inrese of the men length of teroids nd of the numer of teroids per nodule. The numer of ultivle teri per nodule did not vry mong tretments nd mong teril strins (Tle S4). In hlf-roots nodulted y the Fix ) strin, the men numer of undifferentited teri per nodule (5 1 5 ells) nd teroid length (5 lm) were equivlent to those otined in Fix + nodules of ontrol plnts. However, the men numer of teroids per Fix ) nodule ws severely ffeted y the fixj muttion (men vlues of nd ells New Phytologist Ó 212 New Phytologist Trust

7 New Phytologist Reserh 443 () () (d) () Fig. 4 Nodule struture (3 d fter inoultion) in plnts grown in split-root systems (see Fig. 3) nd inoulted either with or on hlf-root systems. Semi-thin longitudinl nodule setions were stined with toluidine lue nd oserved y light mirosopy. () in ontrol plnts, () in N-limited plnts, () in ontrol plnts, (d) in N-limited plnts. 1, meristem; 2, infetion nd differentition zone II; 3, nitrogen fixtion zone III. Brs, 2 lm. for the Fix ) nodules nd the Fix + nodules of ontrol plnts, respetively). All together these dt suggest tht the plnt does not fvor the est prtner t erly stges of nodultion in response to n inrese in N demnd. At lter stges, systemi signling of N defiit might stimulte the development of nodules ording to the teril strin in symioti orgns. The preferentil growth stimultion of nodules in o-inoulted plnts is not likely to e t the expense of nodules euse the growth of nodules in these onditions ws not repressed in response to N defiit (Tle S4). New Phytologist Ó 212 New Phytologist Trust Role of sptil heterogeneity of N provision on the root system in the plnt response to N defiit In the split-root experiments desried in this study, vritions in the N sttus of the plnt were otined y lolly suppressing N 2 -fixtion in nodulted hlf-root systems (Ar:O 2 tretment or nodultion y Fix ) rhizoil strin). It remined unler whether the glol plnt N defiit or the imlne in N provision etween sptilly seprted root systems is suffiient y itself to trigger the plnt ompenstory response. In order to ddress this issue, the split-root plnts in whih the Fix ) nodulted roots

8 444 Reserh New Phytologist () Entire plnt iomss mg DW per plnt () Shoot N ontent mg.g -1 DW () No of nodules per plnt mm.2 mm KNO 3 tretment 2 mm.2 mm KNO 3 tretment 2 mm.2 mm KNO 3 tretment Fig. 5 Effet of the plnt N sttus of N 2 -fixing plnts supplied with 2 or.2 mm KNO 3 nd inoulted with mixture of strins nd on plnt iomss (), shoot N ontent (), nd nodultion (). Men vlues of N nutrient index (NNI) were of.89 nd.73 for plnts fed with 2 mm nd.2 mm KNO 3, respetively. The mesurements were rried out 2 d fter inoultion. Vlues re the mens of 12 replites. Identil letters show tht the men vlues re not signifintly different y LSD (P.5). were sptilly seprted from the Fix + () nodulted roots were ompred with plnts inoulted with mixture of nd the Fix ) strin on the whole root system (Fig. 7,). The dt from the split-root experiment re those given ove (Fig. 3; Tle S3). Control plnts inoulted with single inoulnts were lso inluded in the omprison. In o-infeted plnts, the nodules formed y eh strin were homogeneously distriuted on the whole root system (see Fig. S4), 2% of the nodules eing formed y the Fix ) strin. A wek expnsion (+15%) of nodules ws oserved in these plnts y omprison with the ontrol plnts (Fig. 7d). However, the whole plnt iomss in these plnts ws redued y 32% y omprison with the ontrol plnts (Fig. 7) showing tht the response to N defiit ws not s effetive thn in split-root plnts. Although 5% of nodules were Fix ), the split-root plnts were still le to fully ompenste the N defiit y speifilly stimulting expnsion of nodules on the Fix + hlf-root systems. This result strongly suggests tht the effetiveness of the ompenstory response to N defiit of split-root plnts ws dependent on the imlne of root intke within the root system due to sptil seprtion of the Fix ) nodules. The suppression of suh disrete distriution of nd Fix ) nodules drstilly redued plnt ility to ompenste effiiently the N defiit, proly euse the systemi response to the N defiit ws muh less effetive when diluted on the whole root system. The plnts tht were o-inoulted with the mixture of nd the Fix ) strin were lso ompred with the Fix ) ontrol plnts (Fig. 7). As expeted, the sene of N 2 fixtion in the Fix ) plnts resulted in severe N limittion nd redued plnt growth (Fig. 7). The size of the Fix ) nodules ws smller in these plnts thn in the o-infeted plnts (Fig. 7d). The presene of Fix + nodules on the root system of o-infeted plnts improved plnt N nutrition, whih proly mde possile the expnsion of the Fix ) nodules. This result indites tht the Fix ) nodules enefited lolly from more resoure llotion in plnts tht were supplied in N y the nodules. Similr results were otined using mixtures of nd, the growth of nodules enefiting from the ourrene of nodules on the root system (Tle S5). Disussion In this study, we hve hrterized the N nutrition of Mt Jemlong A17 in symiosis with vrious Sinorhizoium strins. The plnt growth nd the N quisition rte of these ssoitions were orrelted to SNA, showing tht, even with the most effetive prtner, N 2 fixtion remins the limiting ftor of plnt growth in our onditions. Symioti N nutrition ws lwys suoptiml ompred with plnts supplied with minerl N, onsistent with previous reports on Mt Jemlong A17 (Ruffel et l., 28; Moreu et l., 28; Suliemn & Shultze, 21). Whether the suoptiml N quisition is generl phenomenon within the Mt-Sinorhizoium ssoitions or is speifilly relted to the Jemlong A17 line, remins to e investigted. We show tht the plnt responses to prtil nodultion with Fix ) strin involve whole plnt N signling mehnism(s). N defiit resulting from the Fix ) nodules in hlf-root system triggered ompenstory response tht stimultes the N quisition y the other hlf-root system when nodulted with Fix + terium. This response ws le in the long term to fully ompenste the N defiit of the whole plnt. The inresed N quisition ws relted to the stimultion of nodule development y systemi N signling. Both nodule initition nd nodule expnsion in the Fix + side were instrumentl to this response with oth strins nd. However, there ws no ompenstory inrese in SNA in response to N defiit even with the most effetive strin (Fig. 2; Tle S3). This result is onsistent with the ssumption tht SNA would lwys e t its mximum in oth N-limited nd ontrol plnts s previously proposed (Ruffel New Phytologist Ó 212 New Phytologist Trust

9 New Phytologist Reserh 445 () Surfe re per nodule (mm 2 ) Fig. 6 Effet of the teril strin on nodule development ording to the N sttus of N 2 -fixing plnts supplied with 2 mm KNO 3 (drk grey rs) or.2 mm KNO 3 (light grey rs) nd inoulted with mixture of strins nd. The mesurements were rried out 2 d fter inoultion: () men nodule size, () men numer of ultivle teri per nodule, () men numer of differentited teroids (length of ells > 2 lm) per nodule, (d) men length of differentited teroids. Vlues re the mens of individul mesurements of 4 8 nodules, with mesurements on 6 1 individul teroids per nodule. Identil letters show tht the men vlues re not signifintly different y LSD (P.5). () 5. No of ultivle ells per nodule 1 5 () No of differentited ells per nodule (d) 6. Length of teroids (μm) et l., 28; Jeudy et l., 21). Tken together, the present dt indite tht the model of regultion of symiosis y the plnt N demnd initilly sed on the effet of rtifiil Ar:O 2 tretments New Phytologist Ó 212 New Phytologist Trust on the Mt- ssoition (Jeudy et l., 21) lso works for more effiient symioti ssoition. Our study shows tht the plnt response to the vriility of the teril prtner n e explined y this model. Our dt suggest tht the plnt proly ddresses systemi N signl(s) in response to N limittion t the sle of individul root undles tht re glolly pereived s effiient or ineffiient rther thn t the sle of individul nodules. The effiieny of the dpttive response of the whole plnt to prtil suppression of N 2 fixtion ws relted to the sptil seprtion of the N limittion within the root system. When Fix + nd Fix ) nodules were homogeneously distriuted on the roots of o-infeted plnts, the plnt responded y inresing the size of the Fix + nodules. However, this response ws not gret enough to fully ompenste the N defiit used y the Fix ) nodules. These results re onsistent with erly work y Singleton & Stokinger (1983) reporting ompenstory expnsion of Fix + nodules in n equivlent experiment with soyen, ut lso negtive reltionship etween N quisition nd the proportion of Fix ) nodules. Our study suggests tht in sene of imlned N provision etween root undles, the systemi response to the N defiit is diluted on the whole root system, whih limits the effiieny of the response. Erlier work y Jeudy et l. (21) hs shown tht the ompenstion of lol suppression of N 2 fixtion y Ar:O 2 tretments ws ssoited with preferentil llotion of ron metolites to hlf-root systems tht remined effiient for N 2 fixtion t the expense of the ineffiient ones. Therefore, plusile explntion for our results ould e tht the plnt n neither disriminte etween effetive nd ineffetive strutures nor llote its limited resoures only to the effetive ones in sene of sptil seprtion of these strutures. Moreover, this ssumption is lso onsistent with our results, suggesting tht the less effetive prtner enefits indiretly from the most effetive ones of more resoures. This phenomenon would ontriute to the persistene of ineffetive rhizoi in soils. Although plnts ssoited with either or shred the sme generl response to lol N limittion, this response ws more effiient with thn with. This result ws prtly expeted euse the higher SNA in nodules would led y itself to higher level of N quisition even if nodule iomss hd similrly inresed with oth strins. However, the stronger expnsion of nodules ontriuted dditionlly to inrese the N quisition level. Histologil nlyses of nodules indited tht the numer of symioti plnt ells ws inresed in response to systemi N signling, suggesting

10 446 Reserh New Phytologist Tle 2 Mtrix of orreltions mong nodule sizes, numers of ultivle teri nd differentited teroids, nd teroid length 2 d fter inoultion in N 2 -fixing plnts o-infeted with nd nd supplied with 2 or.2 mm KNO 3 2 mm KNO 3.2 mm KNO 3 Vrile (1) (2) (3) (4) (1) (2) (3) (4) Nodule size (1) 1 ) * 1.43*.7***.61** No of ultivle ells per nodule (2).77*** 1 ).39 ).45* ).8 No of teroids per nodule (3) ***.65*.2 1.7*** Bteroid length (4).54* ).3.74** 1 Vlues ove nd elow digonl re Person orreltion oeffiients otined for strins nd, respetively. Men vlues over strin KNO 3 tretments (n = for ; n = for ). Log 1 trnsformed vlues. Correltions re signifint t *, P.5; **, P.1; ***, P.1 respetively. tht nodule meristemti tivity nd differentition of symioti plnt ells re stimulted in mture nodules. No evidene for suh stimultion ws seen with. These dt re onsistent with previous work suggesting n influene of the teril prtner on the nodule meristemti tivity (Sgn & Gresshoff, 1996; Tirihine et l., 2; Lguerre et l., 27). The preferentil response of nodule expnsion ws lso orrelted to stimultion of teroid differentition nd elongtion. In ontrst to nodule expnsion, stimultion of nodule formtion in response to systemi N signling remined quntittively equivlent with oth strins, inditing tht this proess ws proly not influened y the teril prtner in these onditions. This result is in greement with geneti study inditing tht the responses of nodule formtion nd nodule expnsion to systemi N signling re proly medited y distint pthwys (Jeudy et l., 21). The mehnisms underlying the strin-dependent responses of mture nodule development to systemi N signling deserve further investigtion. The ft tht strin- dependent differenes were oserved on o-infeted roots where nd nodules or Fix ) nd Fix + nodules were in viinity nd were likely to reeive the sme phloem llotion indites tht nodule speifi ftors hve n importnt role in determining lolly the response of nodule development to systemi signling. The reltion t the sle of the individul nodule etween the rtes of N 2 fixtion nd of stimultion of nodule growth in response to systemi N signling suggests possile role of nodule SNA in the ontrol of nodule expnsion. However, dditionl trits my e involved nd it remins to e eluidted whether they re determined y the host plnt or nd y the teril prtner within the symioti orgn. In ddition to systemi signling resulting in stimultion of development of effetive symioti strutures in response to lol N limittion, N 2 fixtion my exert lol ontrol on nodule development. Severl studies on diverse legume speies hve supplied evidene for rpid inhiitory effet of Ar:O 2 tretments on nodule growth t the site of pplition (Singleton & vn Kessel, 1987; Jeudy et l., 21; Oono et l., 211), inluding t the sle of individul nodules (Kiers et l., 23). It hs een proposed tht the plnt develops mehnisms to lolly restrit the development of nodules formed with ineffetive teri (Kiers et l., 23; Simms & Tylor, 22; Oono et l., 29, 211). In the long term, Ar:O 2 tretments resulted in derese of the numer of ultivle teri in nodules, whih ould e ssoited with erly nodule senesene, in legumes forming nodules hroring either reprodutive or nonreprodutive teroids (Kiers et l., 23, 26; Oono et l., 211). This result ws interpreted s host sntion towrds the less enefiil prtners. However, in the present study, lthough the sene of N 2 fixtion resulted in restrition of nodule development, we did not find evidene of sntion towrds the Fix ) nodules sed on ounting of reprodutive undifferentited teri. This oservtion is in greement with previous reports on nlogous experiments (Mro et l., 29; Gury-Rngin et l., 21). Also, we did not oserve differene in teril fitness etween nd nodules. Nevertheless, the sene of sntion in our experimentl onditions my e relted to the developmentl stge of the 4-wk-old nodules nlysed whih did not yet show symptoms of senesene. Indeed, it hs een reported tht the multiplition of undifferentited rhizoil ells in Medigo nodules ws ffeted y the Fix ) muttion only in senesent plnt ells reinvded y sprophyti undifferentited teri in nodule zone V (Timmers et l., 2; Munoury et l., 21). An impt of sntion t the ltest stge of nodule growth, when reproduile teri will e relesed in the soil, would e onsistent with funtion tht tends to limit the multiplition nd the dispersion of ineffetive rhizoi. Our study does not provide evidene tht the plnt N demnd influenes the initil prtner hoie, t lest etween nd. Our results lerly show tht the plnt N sttus hs no effet on their ompetitive suess in nodultion suggesting tht, whtever the mehnisms ehind ompetitiveness for nodultion, they opertes in sme wy under different N sttus. Consistently, systemi N signling lso stimulted nodule formtion similrly for oth strins (Fig. 3). This result is in greement with erlier o-inoultion studies with Fix + nd Fix ) strins showing tht N 2 -fixtion per se does not influene the suess of nodule formtion (e.g. Johnston & Beringer, 1976; Amrger, 1981). In onlusion, oth lol nd systemi signling mehnisms re likely to ontriute onomitntly to the whole plnt dpttion to flutution of its root N supply due to vritions of effetiveness of its teril prtners. We propose generl model where oth mehnisms modulte the intertions etween the New Phytologist Ó 212 New Phytologist Trust

11 New Phytologist Reserh 447 () () Control () (d) Entire plnt DW (mg per plnt) Surfe re per nodule (mm 2 ) Control Fix Co-infeted ontrol N-limited Fix host plnt nd its symioti prtners. The whole plnt N demnd triggers systemi ontrols regulting nodule development nd funtioning. When the plnt N demnd is fully stisfied (suffiient ddition of minerl N), n N stiety systemi repression of N 2 fixtion tivity opertes (Ruffel et l., 28). However, when Co-infeted Control + Fix Fix Fig. 7 Effet of homogenous o-infetion y nd the Fix ) strin of the whole root system on plnt nd nodule development. The mesurements were rried out 26 d fter inoultion: () Split-roots systems with sptilly seprted nd Fix ) nodulted roots used for omprison (56% of nodules t the whole plnt level). () Experimentl design used for plnts homogenously o-infeted on the whole root system (8% of nodules) inluding ontrol plnts mono-infeted with nd the Fix ) strin. A 3 : 7 rtio of :Fix ) ells ws used s inoulnts. () Entire plnt iomss (men vlues of 16 replites), (d) men nodule size sed on individul mesurements of nodules nd 7 12 Fix ) nodules. nd Fix ) nodules from o-infeted plnts (light grey rs) were ompred with nodules of their respetive ontrol (drk grey rs) (mono-infeted plnts). Identil letters indite tht the men vlues re not signifintly different y LSD (P.5). Fix-ontrol the plnts rely exlusively on N 2 fixtion, this repression is not tive euse under suh onditions the plnt N nutrition is suoptiml. In these plnts, more severe whole plnt N defiit triggers the N defiit systemi stimultion of nodule formtion nd expnsion. The integrtion y the plnt of effetiveness of root N quisition in this proess is likely to e t the sle of the root undle rther thn t the sle of the individul nodule. Together, N stiety nd N defiit ehve s systemi feedk mehnisms where whole plnt N ssimiltes modulte N quisition pity nd development of the symioti strutures. These regultory loops re tightly integrted with the AON repression mehnism whih is involved in the repression of nodultion y N stiety signling, nd prtilly relesed under N defiit (Jeudy et l., 21). In ddition to systemi signling, there re lso lol ontrols operting t the individul nodule level. First, the expnsion of effetive symioti strutures t mture stges is preferentilly stimulted y the N defiit systemi signling. Seond, low N 2 -fixtion tivities result in repression of growth of the symioti strutures nd likely in derese of teril fitness when teri re relesed in the environment. This nodulespeifi mehnism might inlude the plnt sntion initilly desried y Kiers et l. (23). Whether growth stimultion of effetive nodules nd growth repression of ineffetive ones re prtiulr spets of generl mehnism oupling nodule growth nd nodule tivity t the lol level, or elong insted to speifi mehnisms deserve further studies. Aknowledgements We thnk Céile Gury-Rngin for useful dvie on rhizoil strin seletion. We re grteful to Nïm Rezkllh for help with miroiologil work, nd to Guy Ruiz nd Mr Boursot for tehnil ssistne in the plnt experiments. We lso thnk Eri Girud nd Mihel Lerun for helpful omments on the mnusript. This work ws funded y Agropolis Foundtion nd INRA. Referenes Amrger N Competition for nodule formtion etween effetive nd ineffetive strins of Rhizoium meliloti. Soil Biology nd Biohemistry 13: Btut J, Terzghi B, Ghérrdi M, Huguet M, Terzghi E, Grnerone AM, Boistrd P, Huguet T Loliztion of symioti fix region on Rhizoium meliloti psym megplsmid more thn 2 kiloses from the nod-nif region. Moleulr nd Generl Genetis 199: Bromfield ESP, Brrn LR, Whetroft R Reltive geneti struture of popultion of Rhizoium meliloti isolted diretly from soil nd from nodules of lflf (Medigo stiv) nd sweet lover (Melilotus l). Moleulr Eology 4: Depret G, Lguerre G. 28. Plnt phenology nd geneti vriility in root nd nodule development strongly influene geneti struturing of Rhizoium leguminosrum iovr viie popultions nodulting pe. New Phytologist 179: Gury-Rngin C, Gri M, Bén G. 21. Prtner hoie in Medigo truntul-sinorhizoium symiosis. Proeedings of the Royl Soiety of London Series B, Biologil Sienes 277: Hrtmnn A, Girud JJ, Ctroux G Genotypi diversity of Sinorhizoium (formerly Rhizoium) meliloti strins isolted diretly from soil nd from New Phytologist Ó 212 New Phytologist Trust

12 448 Reserh New Phytologist nodules of lflf (Medigo stiv) grown in the sme soil. FEMS Miroiology Eology 25: Heth KD, Tiffin P. 27. Context dependene in the oevolution of plnt nd rhizoil mutulists. Proeedings of the Royl Soiety of London Series B, Biologil Sienes 274: Heth KD, Tiffin P. 29. Stilizing mehnisms in legume rhizoium mutulism. Evolution 63: Jeudy C, Ruffel S, Freixes S, Tillrd P, Sntoni AL, Morel S, Journet E-P, Du G, Gojon A, Lepetit M et l. 21. Adpttion of Medigo truntul to nitrogen limittion is modulted vi lol nd systemi nodule developmentl responses. New Phytologist 185: Johnston AW, Beringer JE Mixed inoultions with effetive nd ineffetive strins of Rhizoium leguminosrum. Journl of Applied Bteriology 4: Kiers ET, Rousseu RA, Denison RF. 26. Mesured sntions: legume hosts detet quntittive vrition in rhizoium oopertion nd punish ordingly. Evolutionry Eology Reserh 8: Kiers ET, Rousseu RA, West SA, Denison RF. 23. Host sntions nd the legume-rhizoium mutulism. 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Eukryoti ontrol on teril ell yle nd differentition in the Rhizoium legume symiosis. Proeedings of the Ntionl Ademy of Sienes, USA 13: Mhdhi H, Jer M, Limm F, Huguet T, Aouni ME. 25. Intertion etween Medigo truntul lines nd Sinorhizoium meliloti strins for symioti effiieny nd nodule ntioxidnt tivities. Physiologi Plntrum 124: Moreu D, Voisin AS, Slon C, Munier-Jolin NG. 28. The model symioti ssoition etween Medigo truntul v Jemlong nd Rhizoium meliloti strin 211 leds to N-stressed plnts when symioti N 2 fixtion is the min N soure for plnt growth. Journl of Experimentl Botny 59: Ok-Kir E, Kwguhi M. 26. Long-distne signling to ontrol root nodule numer. Current Opinion in Plnt Biology 9: Oono R, Anderson CG, Denison RF Filure to fix nitrogen y non-reprodutive symioti rhizoi triggers host sntions tht redue fitness of their reprodutive lonemtes. Proeedings of the Royl Soiety of London Series B, Biologil Sienes 278: Oono R, Denison RF. 21. Compring symioti effiieny etween swollen versus nonswollen rhizoil teroids. Plnt Physiology 154: Oono R, Denison RF, Kiers ET. 29. Controlling the reprodutive fte of rhizoi: how universl re legume sntions? New Phytologist 183: Rngin C, Brunel B, Cleyet-Mrel J-C, Perrineu M-M, Bén G. 28. Effets of Medigo truntul geneti diversity, rhizoil ompetition nd strin effetiveness on the diversity of nturl Sinorhizoium spp. ommunity. Applied nd Environmentl Miroiology 74: Reid DE, Ferguson BJ, Hyshi S, Lin YH, Gresshoff PM Moleulr mehnisms ontrolling legume utoregultion of nodultion. Annls of Botny 18: Rome S, Fernndez MP, Brunel B, Normnd P, Cleyet-Mrel JC Sinorhizoium medie sp. nov., isolted from nnul Medigo spp. Interntionl Journl of Systemti Bteriology 46: Rosenerg C, Boistrd P, Dénrié J, Csse-Delrt F Genes ontrolling erly nd lte funtions in symiosis re loted on megplsmid in R. meliloti. 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An empiril test of prtner hoie mehnisms in wild legume rhizoium intertion. Proeedings of the Royl Soiety of London Series B, Biologil Sienes 273: Singleton PW, Stokinger KR Compenstion ginst ineffetive nodultion in soyen. Crop Siene 23: Singleton PW, vn Kessel C Effet of lolized nitrogen vilility to soyen hlf-root systems on photosynthte prtitioning to roots nd nodules. Plnt Physiology 83: Suliemn S, Shultze J. 21. The effiieny of nitrogen fixtion of the model legume Medigo truntul (Jemlong A17) is low ompred to Medigo stiv. Journl of Plnt Physiology 167: Terpolilli JJ, O Hr GW, Tiwri RP, Dilworth MJ, Howieson GH. 28. The model legume Medigo truntul A17 is poorly mthed for N 2 fixtion with the sequened mirosymiont Sinorhizoium meliloti 121. New Phytologist 179: Timmers ACJ, Soupène E, Auri M-C, de Billy F, Vsse J, Boistrd P, Truhet G. 2. Sprophyti intrellulr rhizoi in Alflf nodules. Moleulr Plnt-Miroe Intertions 13: Tirihine L, de Billy F, Huguet T. 2. Mtsym6, gene onditioning Sinorhizoium strin-speifi nitrogen fixtion in Medigo truntul. Plnt Physiology 123: Vsse J, de Billy F, Cmut S, Truhet G Correltion etween ultrstruturl differentition of teroids nd nitrogen fixtion in lflf nodules. Journl of Bteriology 172: Vinent JM A mnul for the prtil study of root-nodule teri. Oxford, UK: Blkwell Sientifi Pulitions Ltd. Znetti ME, Blno FA, Beker MP, Bttgli M, Aguilr OM. 21.AC suunit of the plnt nuler ftor NF-Y required for rhizoil infetion nd nodule development ffets prtner seletion in the ommon en-rhizoium etli symiosis. Plnt Cell 22: Supporting Informtion Additionl supporting informtion my e found in the online version of this rtile. Fig. S1 Signifint orreltions etween prmeters of plnt N quisition, plnt iomss, nd nodule trits for plnts grown under hydro-eroponi onditions. Fig. S2 Effet of the teril strin on shoot N ontent in N 2 -fixing plnts sumitted to short-term lol N limittion. New Phytologist Ó 212 New Phytologist Trust