.,/y ,.),, -, y,;>- ' /--=qg---; 'i- #f. c, i, .< <* i-- CRW I 13' -&, m3# LRj- --- No : f6.gqv. il-'! B.P i395 DAKAH ; .

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1 - #f. c,, l 'lrtll 2\lr~e~uldÎ ululugy Y: W Y - JD (Yb/) (0 Martnus Njhoff Publshers, Dordrcch! - Prnted n the Netherlands c Plant gene expresson n effectve and neffectve root nodules of alfalfa (Medcago satva) V. Lullen, D. G. Barker, P. de Lajude and T. Huguet Laboratore de Bologe Moléculare des Relatons Plan#es-Mcroorga/stnes, NRA-CNRS, BP 2 7, F Castanet-Tolosan Cedes, France Keceved 23 March 1987; accepted n revsed form 14 July 1987 Key words: ìn vtro translaton, leghemoglobn, noduln gene expresson, Nortlern analyss, Rhzobumalfalfa symboss Abstract Expresson of plant genes nvolved n t he symboss between alfalfa (Medmgo wrw) and Rl:obutn mellofl has been studed by comparng root and root nodule mrna populatons. Ttvo-dmcnsonal gel electrophoretr separaton of the n vtro translaton products of polya+ RNA solated from ether roots or effectve roo1 nodules has allowed us to dentfy thrteen nodule-specfc translaton products, ncludng those correspondng to the leghemoglobns (Lb). These translaton products, representng putatve noduln nrnas, are frst detected between 9 and 12days after noculaton, a result whch has been confrmed for Lb nrna by Northern blot tng and hybrdzaton wth a Lb cdna probe. Analyss of three dfferent types of neffectve root nodules arrested n dfferent stages of development has led to the followng conclusons. () The transcrpton of eleven nodule-specfc genes, ncludng the Lb genes, s ndependent of ntrogen-fxng actvty. () Dfferentaton oftheprmarynodulestructuredoes not requrethe transcrptonofany of thesegenes but can becorre1a:ed wth a dramatc reducton n the level of at least fve transcrpts present n the root. () There s enhanced expresson of certan plant genes n the case of nodules elcted by an Agrobacterum stran carryng the symbotc plasmd of R. mellot. '! ntroducton Legumnous plant speces can develop symbotc assocatons wth ntrogen-fxng bactera of the genus Rhzobum. Ths nteracton leads to the formaton of specalzed organs, root nodules, and nvolves the Co-dfferentaton of both symbotc partners. Con- n the developmett of thc ntrogen-fxng nodulcs. Protens specfc to the nodule that are encoded by the plant genome have been termed nodulns 115, 27). Amongst the few nodulm whose functons have so far been dentfed are the usygen-bndng protens leghemoglobn (Lb) [ l] and the nodulc-:pccfc forms of two enzymes nvolved n ammonun assderable progress has been made n recent years -- smlaton, urcase [3] and glutamne - -- synthetase - [S, --_-._ --- towards dentfyng bacteral genes nvolved% ths pocess [2, 201. Whlst parallel studes on the host plant have advanced nore slowly, both genetc [61 and molecular [5, 291 approaches have ncvertheless under lned the essental rolc playcd by plant genes n the symbotc relatonshp between alfalfa (Medcago sutvo) and Rhzobum mellot, Lang- Unnasch [41 and Vance (261, usng mmunologkal technques, haw reported respectvelv the detecton.,/y-- 1 y,;>- ' /--=qg---; % - *,, DE k?-y* date - 4'5 / /m :. << '.. -- CRW 13' -&, Cc''?. t *e, '- l-'! B.P 395 DAKAH ; '- \ 7'6). 1: 3, -9 f m3# LRj-?2.1J,;6 -: \* /r,/..-.< <* ' -. --, - Qf"OM Fonds Documentare \.,. e; \ ' - 7 \, -. 3,.),, -, 'a ' 1989 Cote 2 No : f6.gqv /,3

2 RNA 470 of nne and nneteen nodule-spec fc polypeptdes n vvo. We have used an alternatve and complementary methodology by whch plant gene expresson durng nodule development has been studed at the mrna level. Thrteen nodule-spec fc plant mrna translatonal actvtes, ncludng those for the Lb famly, have been detected by n vtro translaton of nodule polya + RNA, followed by two-dmensonal (2-D) ge electrophoretc separaton of the translaton products. Furthermore, we have followed the knetcs of Lb mrna nducton by means of Northern blottng and hybrdzaton wth a Lb cdna probe. Fnally, we have compared the expresson of the genes encodng nodule-specfc polypeptdes n mature effectve nodules wth that n three dfferent types of nodules elcted by non ntrogen-fxng bactera. We conclude that, whlst all except two of the alfalfa nodule-specfc plant genes that we have detected are expressed ndependently of ntrogenase synthess, ther expresson s not a prerequste for nodule organogeness. Materal and methods Plant materal Cuttngs from an alfalfa clone (Medcago satva, cv. hybrd 11 x 8, from Dr Dattée, Orsay, France) were' grown n aeroponc condtons at 20 C, wth a relatve humdty of 75% and a 16 h lght/8 h dark photoperod. The composton of the nutrtve soluton was a modfcaton of that descrbed by Cok et al. [7]: 5.5 mmpotassumphosphate(ph 7.0), 0.52mM K2SO4, 0.25 mm MgS04, mm CaC,, 50 pm FeS04, 50 pm Na,EDTA, 30 pm H3BO3, 10 pm MnS04, 0.7 pm ZnS04, 0.2,pM CuS04, 1 pm Na2Mo04, 0.04 pm COQ, 5 mm NH4N03. Two days before noculaton, the plant medum was changed for a low-ntrogen nutrtve soluton (0.5 mm NH4N0,). The ntrogen-fxng actvty of the nodules was measured by the acetylene reducton tve of RCR 2011 [22]. R. mellot 1354 s a Sm' Nod+ Fx- dervatve of RCR 2011 carryng a %5 nserton n the nfa regulatory gene [23]. R. mellol EJ355 s a spontaneous acdc exopolysacchardedefcent mutant of a SmC RT dervatve of RCR [lo]. The Agrobactehun stran (GM19013 (pgm27)) s an Ery' Cmr dervatve of C58, cured of ts T plasmd, and carryng the symbotc plasmd (psym) of RCR 2011 [24]. Extracton of RNA Roots, noculated roots and hand-pcked root nodules were stored n lqud ntrogen. Frozen tssue was ground n a mortar to a fne powder under lqud ntrogen. To g fresh weght of tssue, 2 ml 0.2 M Trs-HC1 (ph 7.2), 20 mm EDTA, 100 mm NaC, 1 Yo SDS and 2 ml pheno1:chloroform:somyl alcohol (24:24:1) were added. After shakng and centrfugaton of ths suspenson, the aqueous phase was reextracted three tmes wth 2 ml pheno1:chloroform:somyl alcohol and then twce wth the same volume of ch1oroform:soamyl alcohol (24: 1). Total RNA was precptated overnght at -20 C n the presence of 0.3 M sodum acetate and 70% ethanol. The polya+ RNA was purfed by passng twce through an olgodt cellulose column (Collaboratve Research) [ 191. method [25]. - --_ - d-k-asdescrbed-by For the soelectrc focusng 1.6% ampholnes ph 5-7 and Bacteral strans The Nod" Fx+ R. mellor stran s a Rf derva- n vtro translaton and gel electrophoress n vtro translaton of polya + was carred out n a rabbt retculocyte lysate system (Genoft) and polypeptdes were labeled wth (35S) methonne (Amersham). Typcally 1 pg of polya+ RNA was translated durng 30 mn at 30 O C n a 5O-pl ncubaton mxture contanng 1 mc/ml (3sS) methonne. Approxmately 3 x O' cpm of n vtro translaton products was analysed by two-dmensonal gel elec- 0.4% ampholnes ph (L.K.B.) were used and SDS electrophoress was performed n 12.5% polyacrylamde slab gels. After proten fxaton the gels were dred, treated wth Amplfy (Amersham) and t

3 l ~ f 5 f 1 t [ exposed at -70 C to Kodak X-Omat S flm for fluorography [6]. mmunoprecptaton The translaton products were mmunoprecptated, accordng to Fuller et al. [ll], wth rabbt antsera rased aganst ether alfalfa or Sesbana rostrata purfed leghemoglobn preparatons (kndly gven by Dr Bogusz, Dakar, Senegal) and separated by2-dgel electrophoress. Northern blottng and hybrdzaton Ten pg of total RNA was separated under denaturng condtons n 1.2% agarose gels contanng 6% formaldehyde. After eletrophoress the RNA was blotted onto Genescreen membrane (New England Nuclear Corp.) accordng to manufacturer's nstructons. The membrane was hybrdzed, at 42"C, n the presence of 50% formamde and 10% dextran sulfate as descrbed n the Genescreen protocol. After hybrdzaton the flter was washed at 65 C n 2xSSC (0.3 M NaC, 0.03 M sodum ctrate, ph 7.0), 1% SDS, then at room temperature n O.lxSSC, and exposed for autoradography. Lb mrnas were dentfed usng a plasmd (pnl154) consstng of a 600-bp long alfalfa Lb cdna nsert cloned n- the Pst of pbr322 (confrmed by sequencng; manuscrpt n preparaton). For hybrdzaton, a 100-bp fragment of the nsert was 32Pradolabeled usng the random olgodeoxynucleotde prmng method of Fenberg and Yogelsten [9]. Results n vtro translaton of mrnas from.effectve root nodules The expresson of plant genes durng nodule development has been analysed by 2-D gel electrophoretc separaton of the n vtro translaton products of polya+ RNA. Ths technque has allowed us to detect polypeptdes n the range 10 to 100 kda molecular mass n a reproducble manner. The comparson of the 2-D pattern of. translaton products from mature effectve root nodules, elcted by R. mellot 2011, wth that obtaned from unnoculated alfalfa roots (Fg. 1A and 1B) shows that whlst the majorty of polypeptde spots are present n the patterns of both roots and nodules, there are, however, a number of qute clear dfferences. Approxmately ffteen root polypeptdes show reduced levels n the nodule pattern, and at least fve of these, wth apparent molecular masses of 22 kda; 23 kda, 32 kda, 33 kda, 39 kda, are undetectable even after lengthy exposure of the fluorograph. On the other hand, ten polypeptdes are more abundant n the 2-D gel pattern of the n vtro translated nodule mrnas compared wth that from root. n addton, our data show unambguously that thrteen polypeptdes are specfc to the effectve nodule and hence mght represent the expresson of noduln genes. Eght of these (four major and four mnor spots wth molecular masses of around 15 kda) have been dentfed as leghemoglobns (Lb) because they can be mmunoprecptated by an, alfalfa ant-lb serum (Fg. 1C). Furthermore, an antserum rased aganst Lbs from the tropcal legume - gesbanarostratashows cross reactvty wth all eght. of these alfalfa polypeptdes (data not shown). The approxmate molecular masses of the other 5 nodulns are 19 kda, 20 kda, 32 kda, 43 kda, 60 kda. Expresson of noduln genes durng the development of effecfve root nodules Under our partcular growth condtons, alfalfa nodules frst appear 1 week after noculaton wth R. mellot2011, but are too small to be hand-pcked untl approxmately 1 week later. Thus, n order to compare noduln gene expresson throughout nodule development we were oblged to work wth nodulated root materal nstead of solated nodules. The resultng dluton of the nodule-specfc mrnas led us to study the nducton of Lb gene expresson by means of Northern hybrdzaton analyss and makng use of an alfalfa Lb cdna clone (see Materal 1 l ' '.L

4 c 472?. 7 PH PH k Da %:.....

5 , 473 and methods). For ths purpose, total RNA was extracted from roots of M. satva whch had been harvested at regular ntervals up to 28 days followng noculaton wth R. mellotì Fgure 2A shows that Lb mrnas (rangng from 650 to 700 bp n sze) are absent n unnoculated roots, and frst appear around 9 days after noculaton. There s a dramatc ncrease n Lb mrna levels durng the followng 3 days, and ths correlates well wth the observed onset of ntrogen fxaton around days after noculaton. The 2-D gel patterns of n vtro translated polya+ RNA extracted from nodulated roots had confrmed these results for the Lbs, but- the other nodulns are barely detecta6le even 12 days after noculaton (data not shown). Because of the dluton of the sgnal we are unable to deduce the precse moment durng nodule development when these other genes are nduced. Root nodules dt$mtve n a late stage of ther development Havngestablshed that certan alfalfa genes aresubject to programmed expresson durng the establshment of the ntrogen-fxng nodule, we wanted to examne f ths expresson could be correlated wth that of Rhzobum symbotc genes. For ths we used a Rm2011 Rm1354 '. days ' O 2 6 9, 12 19, ' : after noculaton. :,.*!. kb. f-,.., "',,;..' 4 4, :::.. '.. -:.., , _:,. 1 ",. L... A Fg. 2. Autoradograph rpresentng Northern blot of total RNA solated from noculated roots at dfferent stages durng the development of (A) effectve nodules nduced by R. mellot201 1 (Rm2011)and (B) neffectvenodulesnduccd by RM1354. The probe used for hybrdzaton was a 32P labeled fragment of the nsert of pnl154, an alfalfa Lb cdna clone. B." + Fg.. Fluorographs'of 2-D gels of n vfm translaton pp&&,!&e:& wlh (%) methonne, from polya+ RNA solated from (A) effectve alfalfa root nodules nduced by R. m+wof 2011, 5 days after noculaton, (B) unnoculated alfalfa roots, and (D) neffectve ' f(bm1314), 28 daysafter noculaton. n(c)then vrro translaton products from effectve root nodule-plya+ RNA were mmunoprecptated wth alfalfa ant-lb serum and theprecptate was then separated on a 2-D gel. Crcles ndcate the postons of root polypeptdes whch decrease n nodules, - the postons of those whch ncrease and t the nodule-specfc polypeptdes. The nodule-specfc polypeptdes not detected n Rml354-nduced nodules are marked by *. L ndcates polypeptdes mmunoprecptated wth the alfalfa ant-lb serum. Molecular mass markers ncluded Whnethylated 19 lactoglobuln (18.4 kda), (Y chymotrypsnogen (25.7 kda), ovalbumn (43 kda), bovne serum albumn (68 kda) and phosphorylase B (97.4 kda) alfalfa root nodules nduced-by thc-ntxngulm V..*

6 474 mutant of R. mellot (Rm1354) whch carres a 'h5 nserton n the nfa regulatory gene requred for ntrogenase expresson n Rhzobum [23]. Plant cells n nodules elcted by ths mutant contan released bactera as n the case of effectve nodules, but there s no ntrogen fxaton. The 2-D gel electrophoretc pattern of n vtro translaton products of polya+ RNA solated from these neffectve nodules s very smlar to that from nodules nduced by R. mellot 2011 (Fg. 1D).n partcular, the levels of the same fve root polypeptdes are agan dramatcally reduced, and the levels of certan others are ncreased. The Lbs and al1 except two of the other nodulns (19 kda, 20 kda) are also present n these neffectve nodules, and ths shows that, for the most part, the nducton of nodul ì gene expresson s ndependent of ntrogen-fxng actvty. Snce the Lbs (see below) and certan of the other noduln mrnas are present at sgnfcantly lower levels n nodules nduced by ths mutant, the. fluorograph shown n Fg. 1D has been exposed for consderably longer than that shown n Fg. 1A (effectve nodules). Ths was necessary n order to confrm the absence of the two nodulns wth molecular masses of 19 kda and 20 kda. The four spots whch mgrate just behnd certan of the Lbs n Fg. 1D are not addtonal polypeptdes. We have found that. slow runnng of the second dmenson gves rse to' frequent smearng and occasonal spot doublng n the low molecular mass regon of the gel. Northern hybrdzatm analyss (Fg. 2B) reveals that the appearance of Lb mrnas occurs roughly at the same tme as n effectve nodules; however, the level of ths mrna s approxmately 4-fold lower than n effectve nodules. Root nodules defectve n early stages of ther development Snce the majorty of noduln genes are stll expressed n the nodules elcted 'by the ntrogenasedefcent mutant we extended our studes to neffectve nodules arrested n earler stages of development. The frst of these s nduced by a spontaneous acdc exopolysaccharde-defcent mutant of R. mellot (EJ355[lo]) and the second by an Agrobacrerum stran, cured of ts T plasmd but carryng the symbotc plasmd (psym) of R. mellot 2011 (GM19013 (pgm27) [24]. n these nodules there there are no nfecton threads and bacteral penetraton s ntercellular. The central tssue of the nodule s therefore devod of bactera and we should thus be able to dstngush between those molecular events nvolved n nodule organogeness and those concerned wth the dfferentaton and functonng of both the bactera and the central tssue of the host n the nodule. Apparently none of the nodule-specfc transcrpts can be detected n nodules elcted ether by the exopolysaccharde-defcent mutant. of Rhzobum or the Agrobacterum stran carryng the symbotc plasmd of R. mellot2011, as shown n Fgs. 3A and 3B. Furthermore, even wth the hghly senstve Northern hybrdzaton technque we were unable to detect Lb mrna n ether of these two types of neffectve nodule (data not shown). However, t s mportant to note that, n common wth effectve nodules, thc formaton of these two types of neffectve nodules s accompaned by a reducton n the levels of'the same fve mrnas descrbed prevously, whch are normally present n roots. Fnally, n the 2-D gel pattern derved from nodules nduced by the Agrobacterum stran carryng thepsym of R. mellot, numerous polypeptde spots show an ncreased ntensty and n partcular those wth apparent molecular masses of 40 kda anld 70 kda (Fg. 3B). Ths response has not been observed for any other type of nodule that we have studed and does not occur when roots are n contact, wth the Agrobacterum reference stran lackng the symbotc plasmd (results not shown). Dscusson n studyng the symbotc assocaton between-alfalfa and Rhzobum mellot, we have chosen an approach n whch gene expresson s analysed at the transcrptonal level by means of n vtro translaton of nodule polya" RNA followed by 2-D gel electrophoress of the translaton products. Only mrnas of plant orgn should be present n the

7 ph PH r.. A C % '! +j $ -a:; jj - gyp , 9 B $ 4 UA Fg D gel analyss of n vfm translaton products from polya+ RNA solated from (A) neffectve nodules formed by a spontaneous acdc uropolysaccharde-defcent mutant of R. mellot (EJ355), 28 days after noculaton and (8) neffectve nodules formed by an Agrobucrerumstrdncng thesymbotc plasmd of R. me//of2011 (GM9013) (pgm27). 28 daysafter noculaton. For crclesand b, see Fg. 1. Rectangles ndcate translaton products whch specfcally ncrease n nodules elcted by the Agrobucterum/pSym stran. polya+ RNA fracton and hence all nodule-specfc polypeptdes detected n ths manner should be derved from noduln genes. Ths method has allowed us to detect ndrectly thrteen plant mrnas whch are present n effectve nodules but not n unnoculated roots. The SDS gel electrophoretc mobltes of the polypeptdes resultng from n vtro translaton of these nodule-specfc mrnas are dffcult to correlate wth those descrbed for alfalfa protens detected n vvo usng nodule-specfc antbodes [14, 261. These -dffer- - - ences can probably be accounted for by the fact that these latter studes were unable to dscrmnate between plant- and Rhzobum-encoded protens, and secondly, that some of these protens may be subject to post-translatonal modfcaton. Nevertheless, eght of the low molecular mass nodulns can be dentfed as Lbs because they were mmunoprecptated by an ant-alfalfa Lb serum, and also wth an antserum rased aganst the Lbs of the dstantly related legume Sesbuna rostrotu, %ken together, the n vvo and n vtro approaches suggest that there are fewer hghly expressed nodulns n alfalfa than n ether pea or soybean, for whch the detecton of twenty to thrty nodule-specfc polypeptdes has been-reported [4, 151. The pregse sgnfcan- - ths observaton s at present unclear and may smply reflect the need for technques of greater senstvty to reveal addtonal alfalfa nodulns. n the studes carred out on the pea-r....

8 . t legumnosarum symboss, t has been possble to classfy nodule-specfc genes between those few whch are transcrbed relatvely early n nodule development and the large majorty whose expresson broadly concdes wth that of the Lb genes [12]. The noduln genes that we have descrbed for alfalfa all fall nto the latter category and wll therefore be termed late noduln genes. Because we can detect all except two of the nodulns, ncludng Lbs, n neffectve alfalfa nodules elcted wth a ntrogenasedefcent stran of R. mellot (defectve n the nfa regulatory gene) we conclude that ther expresson s ndependent of ntrogen-fxng actvty. These results agree wth those reported for both pea and soybean, usng varous Nod+ Fx- mutants of R. legumnosarum and R. japoncum respectvely [ 12, 281, Furthermore, by means of an al'falfa Lb cdna probe, we have shown that the level of Lb mrna s sgnfcantly lower n these neffectve nodules as compared wth normal ntrogen-fxng nodules. Ths s consstent wth the reducton n the amount of Lb protens found n alfalfa nodules nduced by the same mutant [14, 301. Alfalfa root nodules are elongated and cylndrcal n shape, and beng ndetermnate n character, contan at all tmes regons n varous stages of symbotc development [18]. Hence, the lower than normal expresson' of Lb genes n the ntrogenase-defcent neffectve nodules could smply be the result of a reducton n the proportonal sze of the zone n whch these genes are actvely transcrbed. Ths may be correlated wth the cytologcal observaton that these nodules contan bactera blocked n an mmature stage of ther normal dfferentaton (G. Truchet, personal communcaton). Alternatvely, the nablty to establsh a normal ntrogen-fxng symboss wth the bactera may lead to mpared nducton of Lb gene expresson or perhaps to an ncreased degradaton of Lb transcrpts. We have examned two other types of neffectve nodules formed when alfalfa s noculated ether wth an acdc exopolysaccharde mutant OY R. melloy, Oran Agrobacterum stran carryng the R. mellot symbotc plasmd. n both cases, root nodule development s perturbed.at a relatvely early stage because there are no nfecton.threads and as a consequence bactera are not released nto the host cells. Snce these nodules do not contan detectable levels of any of the noduln mrnas dentfed by us n effectve nodules, the process of nodule organogeness s apparently ndependent of the presence of these partcular nodulns. Smlar results have also been presented for pea nodules nduced by Agrobacterum carryng the symbotc plasmd (psyml) of R. legumnosarum, n whch the only noduln transcrpt detectable corresponds to a gene, ENOD2, whch s expressed early n nodule development [13]. Thus the symbotc plasmd of Rhzobum n the Agrobacterum genetc background s not suffcent to nduce late noduln gene expresson n the host, and so one must conclude that chromosomal or other plasmd genes of Rhzobum are ether drectly or ndrectly nvolved n ths process. Furthermore, a comparson of the three types of neffectve nodules descrbed n ths paper suggests that there may be a correlaton between late noduln gene expresson and the release of bactera from the nfecton threads nto the plant host cells. Clearly, addtonal types of mutants wll have to be studed n order to throw more lght on the nterdependence of these two events. n parallel wth the nducton of the nodulespecfc mrnas we have also observed a strkng reducton n the expresson of certan root genes durng the development of the ntrogen-fxng alfalfa nodule. We have confrmed that ths effect s confned to the nodule by carryng out a parallel analyss of portons of the root whch are not, nodulated (results not shown). Furthermore, the same root mrnas are undetectable n all three of the neffectve nodule types that we have examned. Thus, the change n the levels of transcrpton of these partcular root genes s presumably one of the.events assocated wth nodule organogeness, and unlke late noduln gene expresson appears to be ndependent of the presence of released bactera n the central tssue of the nodule. On the other hand, an effect that has only been observed n the case of nodules formed by the Agrobacterum carryng the symbotc plasmd s a large ncrease n expresson of certan plant genes dfferent from those nvolved n normal nodule formaton. Snce contact wth the Agrobacterum stran alone does not elct the same reacton, one possble _. -.. _.j.,."...

9 L 477 explanaton for ths effect s that the ntercellular localzaton of ths normally non-symbotc bacterum [U] nduces a defensve reacton on the part of the plant. Such a possblty s now beng further examned n ths nteracton by studyng the expresson Óf varous plant genes known to be mplcated n the general defense response. Acknowledgements We would lke to thank P. Bostard, J. DCnar6, G. Truchet and P. Yot for helpful comments durng the preparaton of the manuscrpt and C. Govn for her' secretaral assstance. V.L. holds a grant and D.G.B. a postdoctoral fellowshp from the French Mnstry of Research and Technology. References 1. Appleby CA: Lcghemoglobn and Rhzobum respraton. n: Brggs WR (ed.) Ann Rev Plant Physol 35: (1984). 2. Ausubel FM: Developmental genetcs of the Rhzobumflegumc symboss. n: Losck R, Shapro L (eds) Mcrobal Development. Cold Sprng Harbor, NY: Cold Sprng Harbor Laboratory (1984) pp ' Bergmann H, Predde E, Verma DPS: Noduln 35: a subunt of specfc urcase (urcase 11) nduced and localzed n the unnfectcd cells of soybean nodules. EMBO J 2: (1983). 4. Bsselng T, Been C, Klugkst J, Van Kammen A, Nadler K Nodule-specfc host protens n effectve and neffectve root nodules of Psum sutvum. EMBO J (1983). 5. Bsselng T, Govers F. Stekema W dentfcaton of protens and ther m-rnas nvolved n the establshment of an effectve symboss. n: Mfln BJ (ed) Oxford Surveys of Plant Molecular and Cell Bology, Vol., Clarendon Press, Oxford (1984) pp Bonner WM, Laskey RA: A flm detecton method for trtum-labeled protens and nuclec acds n polyacrylamde gels. Eur J Bochem (1974). 7. Coc Y, Tendlle, Lesant C: La nutrton azotte du tournesol (Helonthusonnuus): acton sur le rendement et la composton bochmque de la grane. Agrochmca 16: (1972). 8. Cullmore JV, bra M. Lea PJ, Mfln BJ: Purfcaton and propertes of two forms of glutamne synthetase from the plant fracton of Phaseolus root nodules. Planta 157: (1983). 9. Fenbcrg AP, Volgelsten B: A technque for radolabelng DNA restrcton endonuclease fragmenu to hgh specfc actvty. Anal Bochem 132: 6-13 (1983). 10. Fnan TM, Hrsch AM, hgh JA, Johanscn E, Kuldau A, Decgan S, Walker Oc. Sgner ER Symbotc mutants of. Rhzobum mellot that uncouple plant from bacteral dfferentaton. Cell 40: (1985). 11. Fuller F, Knstncr PW, Nguyen T, Verma DPS: Soybean noduln genes: analyss of cdna clones reveals several major tssue-specfc sequences n ntrogen-fng root nodules. Proc Nat1 Acad Sc USA (1983). 12. Govers FG, Gloudemans T. Moerman M. Van Kammen A, Bsselng '? Expresson of plant genes durng the development of pea root nodules. EMBO J (1985). 13. Govers F, Moerman M, Downe JA, Hwykaas P, Franssen HJ, Louwcrse J, Van Kammen A, Bsseng'? Rhzobum nod genes are nvolved n nducng an early noduln gene. Nature 323: (1986). 14. Lang-Unnasch N. Ausubel FM: Nodule-specfc polypeptdes from effectve alfalfa root nodules and from neffectve nodules lackng ntrogenase. Plant Physol 77: (1985). S. Legock RP, Verma DPS: ndentfcaton of nodule specfc host protens (nodulns) nvolved n the development of Rhzobum-legume symboss. Cell (1980). 16. Nutman PS Heredtary host factors affectng nodulaton and ntrogen fxaton. n: Gbson AH, Newton WE (eds) Current Perspectves n Ntrogen Fxaton. Elsever, Amsterdam (1981) pp O'Fafrell PH: Hgh resoluton two-dmensonal electrophoress of protens. J Bo1 Chem (1975). 18. Paau AS, Cowles JR: Development of bacterods n alfalfa (Medcago solva) nodules. Plant Physol 62: ' (1978). 19. Rochax JD, Malnoe PM: Use of DNA-RNA hybrdzaton for locatng chloroplast genes and for estmatng the sze and abundance of chloroplast DNA transcrpts, n: Edelman et al. (eds) Methods n Chloroplast Molecular Bology. Elsever Bomedcal Press (1982) pp Rolfe BG, Shne J: Rhzobum - Legumnosue symboss: The bacteral pont of vew. n: Verma DPS, Hohn TH (eds) Genes nvolved n Mcrobe Plant nteracton, Sprnger- Verlag, Venna, New York (1984) pp Rosenberg C, Bostard P. DCnart J. Casse-Delbart F Genes controllng early and late functons n symboss are located on a megaplasmd n Rhzobum mellot. Mol Gen Genet 184: (1981). 22. Sengupta-Gopalan C, Ptas JW: Expresson of nodulespecfc glutamne synthetase genes durng nodule development n soybean. Plant Mol Bo1 7: (1986). 23. Szeto WW. Zmmerman JL, Sundarcsan V, Ausubel FM: A Rhzobum mellot symbotc regulatory gene. Cell 36: (1984). 24. Truchet G, Rosenberg C, Vasse J, Jullot JS, Camut S, Denar6 J: Ttansfer of Rhzobum mellot psym genes nto Agrobacterum lumefacens: Host specfc nodulaton by atypcal nfecton. J Bact 157: (1984). -.

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