nodulin gene expression in effective alfalfa nodules and in

Plant Physiol. (1988) 88, 321-3280032-0889/88/88/0321/08/$01.00/0

Nodulin Gene Expression in Effective Alfalfa Nodules and inNodules Arrested at Three Different Stages of Development'

Received for publication November 30, 1987 and in revised form March 18, 1988

JOANNA H. NORRIS2, LISA A. MACOL3, AND ANN M. HIRSCH*4Department ofBiological Sciences, Wellesley College, Wellesley, Massachusetts 02181

ABSTRACT

Nodulin gene expression was analyzed in effective and ineffective rootnodules of alfalfa (Medicago sativa L. cv Iroquois) elicited by threedifferent Rhizobium meliloti mutants: an exoB mutant having defectiveacidic exopolysaccharide that does not fluoresce on plates containing thefluorescent brightener Calcofluor, fix21, a spontaneous mutant that hasdefective lipopolysaccharide and is Calcofluor bright; and a Rhizobiummutant resulting from a Tn5 insertion in the niffI gene of the nifoperon.The ineffective nodules elicited by these various mutant rhizobia areblocked at different stages of nodule development and have uniquephenotypes. A distinctive pattern of nodulin gene expression as deter-mined by in vitro translations of total nodule RNA characterizes eachnodule phenotype. Seventeen nodulins are found in effective nodulesincluding five leghemoglobins. Only one nodulin gene is expressed in thebacteria-free nodules elicited by the exoB mutant. Other nodulin genes(leghemoglobin and nine others) are expressed infix2l-induced nodules.The genes for nodule-enhanced glutamine synthetase as well as for allthe other nodulins are expressed in nodules induced by the niJH mutant.The expression of genes for the nodulins, including leghemoglobin, isindependent of the nitrogen-fixing ability of the nodule and appears tocorrelate with the differentiation of densely cytoplasmic host cells in thenodule and, to some extent, with bacterial release from infection threads.

The development of symbiotic nitrogen-fixing root nodulesresulting from the infection ofleguminous plants by host-specificspecies of Rhizobium and Bradyrhizobium is dependent on theconcerted expression of bacterial and host plant genes. The roleof rhizobial genes in host specificity, induction of nodule devel-opment, and nitrogen fixation has been the subject of intensivestudy (21). However, the role of the eukaryotic host genes in-volved in symbiosis has only recently come under scrutiny.Classical genetic analysis of the host legume has demonstratedthat plant genes play a part at every stage of nodule development(1 1).The plant proteins specifically formed as a result of the plant-

microbe interaction are termed "nodulins" (32). Nine nodule-specific proteins have been detected in extracts of alfalfa root

'Supported by National Science Foundation Grants PCM 83-16793and DCB 87-03297.

2 Supported by a National Science Foundation Postdoctoral Fellow-ship in Plant Biology. Present address: Department of Botany, Universityof Rhode Island, Kingston, RI 02881; Formerly Joanna F. Hanks.

3Present address: Department of Plant Pathology, University ofGeor-gia, Athens, GA 30602.

4 Present address: Department of Biology, University of California,Los Angeles, CA 90024.

nodules in one study (18), while at least 19 nodulins werereported in a second study (31). The identities of a few nodulinsin various legume hosts have been established. However, the roleof most nodulins in symbiosis, especially those involved innodule development, remains unknown.

Studies with some cloned nodulin genes have demonstratedthat expression of these genes is not coupled to activation of thenitrogenase genes of Rhizobium (8) or to nitrogen-fixing ability(9). Moreover, neither heme secretion by bacteroids (1, 9) norbacteroid development (1, 8, 9, 27) was required for expressionof Lb5 and other nodulins. Studies on the timing of nodulin geneexpression in pea suggests that at least three groups of nodulingenes are expressed differentially (9). The early nodulin, N-40',was expressed from the beginning of nodule development whilea second early nodulin, N-68, appeared about 2 d later. Severalnodulins, including Lb, were found immediately prior to theonset of symbiotic nitrogen fixation. Other nodulins, for exam-ple, N-2 1, were expressed later in development (9).We have studied three classes of ineffective root nodules

arrested at different stages of development to determine thetiming ofexpression ofnodule-specific genes in alfalfa. The acidicexopolysaccharide Rhizobium meliloti mutant (exoB) exhibits aspecific pattern of resistance to rhizobial phages, is conditionallysensitive to a monoclonal antibody to the bacterial surface, anddoes not fluoresce under ultraviolet light on plates containingthe fluorescent brightener Calcofluor (6). This mutant inducesnodules that lack infection threads and are devoid of bacteriaindicating that nodule morphogenesis can be uncoupled frombacterial invasion (6). Nodules induced by exo mutants werefound to contain low levels ofnodule-specific proteins but neitherLb (19) nor nodule-specific GS (5). Similarly, "tumor-like" nod-ules controlled by the plant gene in3, which have a phenotypesimilar to that of the exo mutant-induced nodules, exhibitedlittle cross-reactivity with nodule-specific antiserum and con-tained no detectable Lb protein (31). In contrast, bacteroid-containing yet ineffective nodules elicited by R. meliloti nifHImutants (18) and those controlled by the plant gene in, (31) werefound to produce Lb and other nodule-specific proteins.

In this study, we examined the in vitro translation products oftotal RNA isolated from exoB and nimH-induced nodules, ratherthan in vivo proteins. In addition, we examined nodules inducedby R. meliloti mutant fix21 (17). These have a phenotype thatdiffers from previously described ineffective nodules of alfalfa.The nodules contain infection threads that in some nodules abortbut in others release bacteria. The rhizobia become surroundedby peribacteroid membranes that rapidly degenerate, and theydo not differentiate into elongate bacteroids as do the niflmutant bacteria. However, host cells differentiate in that they

'Abbreviations: Lb, leghemoglobin; GS, glutamine synthetase; Nmr,neomycin resistant; Otr, oxytetracycline resistant; PMSF, phenylmeth-ylsulfonyl fluoride.

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become densely cytoplasmic and also contain mRNAs for mostof the nodulins, including Lb, found in effective nodules. Ourgoal was to determine the difference in nodulin gene expressionbetween three classes of ineffective nodules: those totally devoidof bacteria (exoB-mutant nodules); those partially infected(fix21-mutant nodules); and nodules that are fully infected (nifl-I-mutant nodules).

MATERIALS AND METHODS

Bacterial Strains. Wild-type Rhizobium meliloti SU47, exoBmutants Rm5093 (Nmr) (6) and Rm5078 (Otr), and mutantEJ312fix21 (Rm5121) were generously provided by Ethan Signerand Ralph Clover (Massachusetts Institute of Technology).Rm5121 is a spontaneous R. meliloti mutant that is bright underultraviolet illumination when grown on LB-Calcofluor but isaltered in cell surface properties (17). Moving the fix21 allelefrom the EJ312 background into wild-type strain Rm 1021 resultsin rhizobia with multiple defects (R. Clover, personal commu-

nication). However, the strain used in this study (Rm5121)appears to have only a lipopolysaccharide defect. MutantRm1491 (nifr::Tn5) was a generous gift from Fred Ausubel(Massachusetts General Hospital). Nitrogen-fixing ability inthe mutant-induced nodules was analyzed by acetylene reduc-tion (13).

Plant Material. Seedlings of Medicago sativa L. cv Iroquoisinoculated with R. melilotiSU47 were grown in the greenhousein flats of perlite and watered with nitrogen-free medium. Plantsinoculated with mutant R. meliloti were grown on sterile agar

slants as described previously (13). Nodules were generally har-vested 21 d after inoculation, although occasionally at earlierand later times, frozen in liquid nitrogen, and stored at -70°C.Control root tissue was harvested after 2 weeks from uninocu-lated plants grown in the greenhouse on nitrate-supplementedmedium. Nodules were excised from roots, fixed, and preparedfor microscopy as described previously (13).RNA Isolation and in Vitro Translation. Total RNA was

isolated from 0.2 g of nodule tissue or 3.0 g of root tissue by LiClprecipitation as described previously (9). Approximately 2,ug ofRNA were translated for1 h in a 25-,tL reaction using a rabbitreticulocyte lysate kit (New England Nuclear) so that a linearrelationship between the amount of RNA added and the incor-poration of [35S]methionine was established. The amount ofradioactivity incorporated into translation products was esti-mated by precipitation of1 ,uL of the reaction mixture in 10%trichloroacetic acid on Whatmann 3MM paper as specified inthe lysate kit.Two-Dimensional Electrophoresis. The translation products

were separated by two-dimensional electrophoresis (9) using a

12.5% polyacrylamide slab gel for the second dimension. Thefirst dimension isoelectric focusing gels contained 1.6% ampho-lines pH 5 to 8 and 0.4% ampholines pH 3 to 10. The basic endof the focusing gel was always placed on the left side of thesecond dimension gel. Molecular weight standards from Bio-Rad(Richmond, CA) or prestained standards from BRL (Gaithers-burg, MD) were run in the second dimension. Usually, 300,000cpm incorporated into translation products were applied to thegel. Gels were treated with Enhance (New England Nuclear),dried, and autoradiographed on Kodak XAR-5 x-ray film.

Immunoprecipitation. Translation reactions were incubatedwith10,uL of antiserum as described (9), except that10 mg ofSepharose-Protein A (Pharmacia) were added in the final 3 h ofincubation. The Sepharose-A-bound immunoprecipitates were

pelleted gently in a microfuge and washed with buffer five times,until negligible counts per minute were detected in the superna-tant. The immunoprecipitate was eluted from the Sepharose-Ain100 L of buffer containing 5% 2-mercaptoethanol and 2%NaDoSO4 and was analyzed by two-dimensional electrophoresis.

Antisera. Lb antisera were generously provided by Ton Bissel-ing (Agricultural University, Wageningen, The Netherlands) andCarroll Vance (University of Minnesota, St. Paul). Antiserumprepared against GS was the generous gift of Julie Cullimore(University of Warwick, Coventry, U.K.).Immunoblots. Nodules or roots were ground to a powder in

liquid nitrogen, dissolved in a minimal volume of 50 mM Tris(pH 7.4), 1 mm EDTA, 50 mm NaCl, 0.5% deoxycholate, 0.1%NaDoSO4, and 0.2 mm PMSF (18), and microfuged for 10 min.A sample of the supernatant fraction containing 70 ,ug of proteinwas separated on a two-dimensional gel as described above andtransferred to nitrocellulose (30). Peroxidase-conjugated goatantirabbit secondary antibody and reagents were purchased fromBio-Rad (Immun-Blot kit) and used according to manufac-turer's instructions.

RESULTS

Gene Expression in Effective Nodules. To serve as a frame ofreference for the analysis to follow of the nodulins found in theineffective nodules, a brief description of nodulin gene expressionin effective, wild-type-induced nodules is included. Numerousdescriptions of the histology of effective alfalfa nodules have beenpublished (see references in 13).Comparison of the two-dimensional pattern of in vitro trans-

lation products of RNA from nitrogen-fixing root nodules ofalfalfa with that from roots indicated that a majority of thepolypeptides observed were present in both tissues. However, atleast 17 unique spots ranging from 14 to 64 kD were observedconsistently on fluorographs from gels of translation productsfrom nodule RNA and therefore represent the expression ofpresumed nodulin genes (arrowheads, Fig. 1B). Caution wasexercised in identification of nodule-specific products, as manyspots that first appeared to be nodule specific were seen at lowlevels upon longer exposures of root gels. Several root-specifictranslation products were also observed but will not be discussed(Fig. IA).The appearance of the nodulin messages was analyzed in wild-

type induced root nodules harvested at 12, 19, and 26 d afterinoculation. Although nodules harvested at 19 d exhibited thehighest acetylene reduction activity (data not shown), 12-d-oldnodules were pink and reduced acetylene. However, they weremuch smaller than the older nodules. Most of the nodulinsindicated in Fig. iB, including Lb and GS. (see below), weredetected at high levels at all ages analyzed. The only exceptionwas N-17a, which was present in very low amounts 12 d afterinoculation and reached highest concentration at 26 d (datanot shown).Glutamine Synthetase. Antiserum against GS from Phaseolus

vulgaris, which reacts with both nodule-specific GS. and GSr(found in both roots and nodules) (3), was used to identify theisozymes of GS in alfalfa. Two spots were identified as isozymesofGS by immunoprecipitation of in vitro translated nodule RNAwith the GS antiserum (Fig. 2A). The spot to the left (N-46) issignificantly enhanced in nodules and thus may represent anodule-specific form of GS. The additional immunoprecipitatedtranslation product located immediately to the right of N-46 isalso present in root translations and probably represents GSr.

Leghemoglobin. Nodulins N-I 5a, N- 14a, N- 14b, N-iSb, andN-15c were identified as Lb translation products based on im-munoprecipitation with the Lb antisera (Fig. 2B). Five Lb pro-teins of the same size and pI were detected by immunoblotanalysis of nodule protein extracts with an Lb antiserum (Fig.2C). Nodulins N- I 7b, N- I 7c, and N- I 7d (Fig. IB) also appearedto be Lb translation products because these nodulins were im-munoprecipitated by both Lb antisera (see "Materials and Meth-ods") (Fig. 2B). Jing et al.(16) have identified large antigenic Lbtranslation products similar to N- I 7b, N- I 7c, and N- I 7d in

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GENE EXPRESSION IN DEVELOPMENTALLY ARRESTED ALFALFA NODULES

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FIG. 1. Identification of nodulin gene products. Autoradiographs of two-dimensional gels of in vitro translation products from total RNA isolatedfrom (A) uninoculated roots and (B) effective alfalfa root nodules induced by wild-type R. meliloti SU47. Nodulins (arrowheads) and nodule-stimulated (nst) products (short arrows) are indicated by their molecular weight in kilodaltons. The corresponding locations of nodulins (open circles)and nodule-enhanced GS (open box) as well as three spots present on all gels (long arrows) are labeled to aid in orientation.

alfalfa nodules as products of incompletely processed LbmRNAs. Likewise, three RNA products corresponding to thesize of the unspliced precursor RNA and RNAs lacking one ortwo introns were detected in total RNA of soybean using se-quences from the third intron as probes (23). The alfalfa nodu-lins, N- 17b, N- 17c, and N- 17d, were not detected in total solubleprotein extracts of nodules (Fig. 2C).

INEFFECTIVE NODULES

Mutant exoB-Derived Nodules. Nodules induced by the spon-taneous, exopolysaccharide (exoB) mutant EJ355 have beendescribed previously (6). Moving the exoB355 allele into the R.meliloti 1021 background resulted in rhizobia that were capableof forming some shepherd's crooks on alfalfa roots, albeit 3 to 4d later than wild-type R. meliloti (data not shown). This is incontrast to the originally described EJ355 mutant, which did notinduce 360° curling of root hairs. Like EJ355, Rm5093 orRm5078 induced anomalous root hair deformation and nodulesthat were devoid of infection threads (Inf) and intracellularbacteria. The distinctive pattern of nodule histology was evidentin that the central mass of tissue was separated from the nodulecortex by an endodermis and vascular bundles were positionedperipherally. However, in the case of mutant exoB-derived nod-ules, the central mass of tissue was uninfected.When in vitro translation products of total RNA from mutant

exoB-derived nodules were analyzed, only one nodule-specifictranslation product (N-38) was observed (Fig. 3A). In additionto N-38, there were also two nodule-stimulated spots: Nst-57,found in both effective nodules and roots, and Nst-28, foundfrequently as a complex of spots and significantly enhanced in

nodules elicited by other exopolysaccharide mutants (20) (datanot shown). Transcripts for other nodulins, including those forLb or nodule-enhanced GS, were not detected in nodules inducedby exoB mutants.Nodules Induced by fix2l Mutants. R. meliloti EJ312 fix2l1

mutants caused delayed shepherd's crook formation and inducedthe development of small, white, ineffective nodules within 3weeks after inoculation. They were consistently Fix- as measuredby acetylene reduction assay. The nodules originated from theroot inner cortex, developed a meristem, and had peripheralvascular bundles (Fig. 4A). The majority of nodules examined,although devoid of released bacteria, contained infection threads(Fig. 4A-C). Rhizobia were present within these threads, whichwere misshapen and appeared to end blindly within the nodulecortex. In addition, rhizobia were observed within intercellularspaces, frequently in association with an aborted infection thread(data not shown).

In nearly 40% of thefix21-mutant-induced nodules examined(16 of the 41 nodules sectioned serially), rhizobia were releasedfrom infection threads that penetrated into the central tissue ofthe nodule. The host cells ahead of the infection thread becamedensely cytoplasmic and, in some cases, infected with rhizobia(Fig. 4B, C). These infection threads were narrow in diameterand resembled those observed in wild-type induced nodules.However, the rhizobia in these infected cells did not differentiateinto elongate bacteroids (Fig. 4C). The phenotype of the mutantfix21-induced nodules is thus intermediate between that of mu-tant exoB- and mutant nif1-elicited nodules.We observed that release from infection threads in mutant

fix21-derived nodules appeared to be normal. The rhizobia were

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tant R. meliloti contained elongate bacteroids. The bacteroidsdifferentiated to a stage evaluated by ultrastructural criteriacomparable to wild-type nitrogen-fixing bacteroids, i.e. they haveheterogeneous cytoplasm (13). However, they did not fix nitrogenbecause of a lesion in the ni.I- gene. The host cells also differ-entiated in that they contain measureable levels of Lb and othernodule-specific proteins (19). However, the levels are less thanthose found in effective nodules, most likely because there is asmaller infected zone in nodules induced by nifzI mutant bacteriadue to premature nodule senescence.Measurement of nodulin gene expression by in vitro transla-

tions of total RNA demonstrated that all the nodulins found inwild-type RNA translations were present, although some werefound at reduced levels (Fig. 3C). The translation product iden-tified as a nodule-enhanced GS appeared as a doublet that wascomparable in level and appearance to that observed in transla-tions ofRNA isolated from wild-type-induced nodules (Fig. SC).

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FIG. 2. Immunoprecipitation of (A) GS and (B) Lb from in vitrotranslation products from total RNA isolated from effective alfalfa rootnodules. Nodulins are indicated by open triangles. C, immunoblot of Lbproteins from a nodule extract gel transferred to nitrocellulose. Only theupper (A) or lower portion (B, C) of the two-dimensional gel is shown.

surrounded by peribacteroid membranes and elongated 2 to 2.5times more than bacteria in the infection thread. However, theydid not fully differentiate into mature bacteroids. In addition,peribacteroid membranes were pulled away from the rhizobia(arrows, Fig. 4C).Most nodulin messages were expressed in the ineffective, mu-

tant fix21-induced nodules. The major nodulin in these RNAtranslations was N-38 (Fig. 3B). It was difficult to determine thelevel ofexpression of nodule-enhanced GS (N-46, see above; boxin Fig. 3B) because spots near this position (Fig. 5A,C, arrow-

heads) were also observed in root RNA translations with longerexposure and higher magnification (Fig. 5B). However, the faintspots seen in the root (to the left of the arrow) differed inisoelectric point from N-46. N-46 RNA from wild-type-inducednodules appeared to translate as a doublet (double arrowheads,Fig. 5A). The spot, indicated by an arrow (Fig. 5, all panels), waspresent in both roots and nodules and immunoprecipitated withGS antiserum (Fig. 2A, termed GSr above). In translations ofRNA isolated from nodules elicited byfix2 1 mutants, no intensespot (or doublet) was found at the position of N-46, and thespots observed more closely resembled those found in the rootgel (cef Fig. SD, SB).The Lb translation products, N- 14a, N- 14b, N- 1 5a, N- 1 Sb,

and N-15c, were present at low levels in translations of totalRNA from flx2 1 nodules (Fig. 3B). This may in part be due tothe fact that the infected zone in these nodules is significantlysmaller than that found in wild-type-induced, effective nodules.N- 17c and N-1 7d, which were immunoprecipitated by Lb anti-serum (Fig. 2B, above), were also present.The late nodulin N- 1 7a was detected'only in the oldest mutant-

induced nodules analyzed (38 d after inoculation). This nodulinexhibited the highest level of expression in wild-type-inducednodules 26 d after inoculation.Mutant nifil-Induced Nodules. Nodules elicited by nih-I mu-

DISCUSSION

Ineffective nodules induced by mutant rhizobia are useful forelucidation of the steps involved in nodule development. Wehave analyzed gene expression in ineffective nodules elicited bythree different R. meliloti mutants and have compared thisexpression with that found in wild-type, effective root nodules.We have identified at least 17 nodule-specific translation prod-

ucts in effective root nodules of alfalfa, indicating that a relativelylarge group of host plant genes is expressed specifically duringnodule development. Because alfalfa nodules exhibit a develop-mental continuum from meristem to senescent end, nodulinsinvolved in all aspects of symbiosis can be detected in effectivenodules at any age. On the other hand, altered nodule develop-ment by infection with various R. meliloti mutants can becorrelated with the pattern of expression of nodule-specific tran-scripts. Our investigation of the in vitro translation products inthe different mutant-induced nodules has led us to the followingconclusions:

1. We propose that the nod genes of R. meliloti trigger theexpression of nodulin N-38 and several nodule-stimulated spotsthat are prominent in translations ofRNA isolated from noduleselicited by exopolysaccharide mutants. Previously, we concluded(6) that exo genes are not essential for the induction of nodulestructure and thus, the nod genes, which are intact in the exo

mutants, are the best candidates for initiating nodule develop-ment. We had found earlier (14) that Agrobacterium tumefacienstransconjugants carrying the R. meliloti nod genes induce bac-teria-free nodules on alfalfa roots, suggesting that the nod genesinitiate a cascade of developmental events leading to noduleorganogenesis. Dudley et al. (4) have found that the primary andearliest effect of Rhizobium nod gene action is plant cell activa-tion and that Nod- mutants are unable to elicit cell divisions inthe root cortex. The exoB-induced nodules exhibit a phenotypesimilar to the Agrobacterium transconjugants carrying R. melilotinod genes, i.e. they have peripheral vascular bundles, a noduleendodermis, and a nodule cortex, but only one cell type is presentwithin the central zone, the uninfected cell. Although noduleselicited by A. tumefaciens carrying only R. meliloti common nodgenes, nodDABC, have not been examined for nodulin geneexpression, nodules induced by Agrobacterium transconjugantscarrying nod and hsn genes have been investigated. The transla-tion product observed on fluorographs is in a position equivalentto N-38 of the exo-elicited nodules (3a). However, the structureof the latter nodules has not been determined.

Similar conclusions have been reached by Govers et al. (10),who found that pea nodules induced by A. tumefaciens transcon-jugants carrying the R. leguminosarum symbiotic plasmid ex-

pressed the early nodulin ENOD2 (7) and that Vicia noduleselicited by the same transconjugant contained ENOD2 and an

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Recently, Lullien et al. (22) have examined plant gene expres-sion in nodules elicited by wild-type R. meliloti, mutant EJ355,

FIG. 3. Nodulin gene products in ineffective nodules. Autoradi-ographs of two-dimensional gels of in vitro translation productsfrom total RNA isolated from nodules induced by (A) exoB mutantRm5O78, (B) mutant EJ312 flx21, and (C) mutant Rml491(niHI::Tn5). Nodulins (arrowheads), nodule-stimulated (nst) prod-ucts (short arrows), the location of nodule-enhanced GS or N-46(open box), and three spots (long arrows) for orientation are labeledto correspond with Figure 1. N-38 in (C) has been more clearlyresolved on other fluorographs.

and Agrobacterium strains carrying the symbiotic plasmid of R.meliloti. Their results differ from ours in that no nodule-specifictranscripts are detected in in vitro translations of RNA isolatedfrom nodules induced by EJ355 and the Agrobacteriumtransconjugants.

2. We propose that rhizobial genes that are intact infix2 l anddefective in exoB may express gene products that are importantfor the induction of the infected host cell type. Theflx2 l mutantinduces nodules in which two host cell types have differentiated,i.e. the highly vacuolate, uninfected cell type and densely cyto-

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<4 infection thread (it) is present within the interior of the nodule. Rhizobia (r)are found on the outer surface. B, light micrograph of infected host cells. Hostcells have small or large vacuoles (v), are densely cytoplasmic, and containbacteria (b) released from infection threads (it). C, transmission electron

ON micrograph of infected host cells. Three host cells separated by cell walls (cw)are evident. Bacteria (b) are released from infection threads (it) but do notelongate. Mitochondria (mi), plastids (p), and other cell organelles are present.Bar, 1 gm.

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plasmic cells with small vacuoles and large nuclei. The latteroccasionally are infected with rhizobia infix2 1-induced nodules.The phenotype ofnodules induced byfix2 1 mutants represents

a stage in nodule development hitherto undescribed for alfalfa.Studies from Ethan Signer's laboratory (17) indicate that thefx21 mutant has defective lipopolysaccharide. LPS mutants ofR. phaseoli induce nodules that are devoid of bacteria, that lackLb and that do not differ in protein profile from roots as judgedby SDS-PAGE analysis (26). However, these nodules have notbeen analyzed for nodulin transcripts.

3. Moreover, we conclude that the initiation of infectionthreads alone is not sufficient for the induction of the latenodulins in alfalfa. Evidence for the uncoupling of infectionthread formation and for induction of the later nodulins comes

from studies of nodules formed by exoH (20) and exoA mutantsas well as by Agrobacterium transconjugants carrying pSyma (J.H. Norris et al., in preparation). These nodules contain infectionthreads that are confined to the superficial cells of the noduleand bacteria are not released from the threads. Only the N-38message and increased levels of the nodule-stimulated mRNAsas well as an mRNA that hybridizes to ENOD2 from peas (A.M. Hirsch, unpublished results) are present.

In contrast, nodules induced by mutantfix21 rhizobia expressLb and other nodulin genes. Infection threads differentiate be-yond the superficial cells into the central tissue of these nodules.This suggests that (a) differentiation of host cells into two differ-ent nodule cell types and/or (b) release of rhizobia into thedensely cytoplasmic cell is required for expression of nodulins,

326 NORRIS ET AL.

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FIG. 5. Expression of nodule-enhanced GS in (A) nodules inducedby wild-type strain RmSU47, (B) uninoculated roots, (C) nodules inducedby mutant Rm 1491, and (D) nodules induced by mutant EJ312 fix2 1.Only the enlarged area of the autoradiograph of in vitro translationproducts of total RNA surrounding N-46 (GS, Fig. 2A) is shown. Thedoublet corresponding to N-46 is indicated by two arrowheads. Anadditional product that also immunoprecipitated with the GS antisera(Fig. 2A), possibly a root form of GS, is indicated with a long arrow inall panels.

with the exception of N-38, which is expressed in "empty"nodules. We cannot distinguish between these two possibilitiesbecause it is difficult to be certain that these events are reallyuncoupled in fix21-induced nodules. Alfalfa nodules formed byfix2 1 R. meliloti exhibit a range of phenotypes including nodulesdevoid of bacteria but containing aborted infection threads andhaving few internal, densely cytoplasmic cells as well as noduleswith infected cells carrying released rhizobia. This "leakiness" inphenotypic expression may be related in part to the fact thatalfalfa is genetically heterogeneous or to some cryptic defect inthe fix21 mutant. However, the lack of correlation betweeninfection thread initiation and nodulin gene expression is asurprising result because thread formation would seem to be asignificant event in terms of nodule development as it marks oneof the earliest stages of bacterial invasion.

4. Likewise, we conclude that bacteroid differentiation is notrequired for expression of the later nodulin genes including Lbbecause bacteria do not differentiate into elongate bacteroids infix21-induced nodules. A similar conclusion has been reportedfor soybean nodules by Morrison and Verma (25). However,released fix2 1 bacteria are slightly more differentiated than theBradyrhizobium japonicum T8- 1 mutant.

5. Our data indicate that GS (perhaps a nodule-specific form)is absent in nodules lacking elongate bacteroids (mutant fix2 I-

elicited) and present in nodules containing differentiated bac-teroids (mutant nifH-induced). Although our results do notdiscriminate between a nodule-enhanced or nodule-specific GS,we have observed that the levels and appearance of translationproducts corresponding to GS are comparable between wild-typeand ni-H-mutant-derived nodules. In contrast, the pattern ofspots in the GS region derived from mutantflx2 1 nodules moreclosely resembles the pattern observed for roots. Northern blot

analysis of mRNAs from the mutant-induced nodules using ahomologous clone for GS. (2) as a probe most likely would beinconclusive because the mRNAs for the different isozymes ofGS have the same molecular weight and would cross-hybridizewith the cDNA clone (29).

Recently, a nodule-specific clone ofGS has been identified inalfalfa nodules using an oligonucleotide probe corresponding tothe 3' nontranslated region (5). Thus, in alfalfa and bean (2),nodule-specific forms of GS have been identified. In contrast,nodule-specific GS clones have not been identified in pea (29)or in soybean (12). In the latter, a root GS gene is responsiblefor elevated levels of glutamine synthetase in soybean nodulesand appears to be regulated by ammonia. However, Dunn et al.(5) have found that GS. is expressed in ineffective alfalfa nodulesinduced by nifA, nifH, fixA, and ntrA mutants but not in thoseelicited by exoA mutant R. meliloti; they suggest that GSnexpression may be under developmental rather than ammoniacontrol. GS. appears to be induced later in alfalfa nodule devel-opment and may be correlated with the presence ofdifferentiatedbacteroids rather than nitrogen-fixing ability.

In summary, we conclude, similar to what has been reportedfor pea nodules (9), that there are at least two and perhaps threephases of nodulin induction in alfalfa. The first phase, an earlynodulin phase, is most likely triggered by the Rhizobium nodgenes and, in alfalfa, results in the induction of N-38. N-38 maybe a marker for nodule morphogenesis and, in particular, theuninfected cell type. So far, no cDNA that hybrid selects N-38has been found (R. Dickstein, personal communication). This isalso true for N-40' in pea nodules (T. Bisseling, personal com-munication) and hence the nature of this polypeptide is un-known. Another early nodulin gene, ENOD2, found in soybean(7) encodes a protein that is high in prolines, presumably astructural protein. This protein may be related to the P4 proteinfound in soybeans (15) or to other structural proteins likeextensin (28).

Perhaps the "first wave" of nodulin gene expression (N-38,ENOD2), elicited by rhizobial nod genes, results in structuralchanges in the host cell walls and/or in different metabolicpathways and/or changes in endogenous hormone levels. Therhizobial genes that are important for eliciting the "second wave"of nodulin gene expression are not clearly defined by this orother studies. Our results with exoB and exoH mutants (the latterlack a succinylated exopolysaccharide [20]) suggest that intactexopolysaccharide is important for the further development ofinfection threads and for the induction of later nodulins. Thefix21 mutants are Exo+ and induce nodules that contain tran-scripts for the later nodulins including Lb. Thus, intact exo-polysaccharide seems to be essential in some undetermined wayfor further nodule development and nodulin expression.

Acknowledgments-We thank Ethan Signer, Ralph Clover, and Fred Ausubelfor R. meliloti strains; Ton Bisseling, Carroll Vance, and Julie Cullimore forantisera; Carol Smith for the microscopic work; Gretchen Kuldau for technicalassistance; and Kay Leland for the photography. We also are grateful to EthanSigner, Ton Bisseling, and Kathy Dunn for valuable discussions and to Mac Friedand Amala Reddy for comments on the manuscript.

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