Detection of Nodule-Specific Polypeptides from Effective and Ineffective Root Nodules of Medicago sativa L.

Detection of Nodule-Specific Polypeptides from Effective and Ineffective Root Nodules of Medicago sativa L. S.

s. MOHAPATRA and A. PUHLER

Lehrstuhl fur Genetik, Fakultat fur Biologie, Universitat Bielefeld, Postfach 8640, D-4800 Bielefeld 1, FRG Received April 3, 1986 . Accepted April 14, 1986

Summary At least twelve polypeptides specific to the nodule tissue of Medicago sativa (alfalfa) were detected by the immunoblotting procedure. These polypeptides ranged in size from 12 Kd to 120Kd. Nodules induced by seven different Rhizobium meliloti strains showing various symbiotic phenotypes were analysed by immunoblotting for the presence of nodule-specific polypeptides. All of these mutant-induced nodules contained the majority of the nodule-specific polypeptides but at reduced concentrations compared to the wild type. Nodules which were induced by mutant isolate 0540 and lacked infection threads and bacteroid-filled cells, exhibited elevated levels of a 97Kd and 120Kd polypeptide. Mutants 101.45 and 2204 induced increased amounts of the 39 Kd and 120 Kd protein respectively. While strains 938 (defective in succinate utilization) and 3046 (nod+fix-) incited a nodule-specific polypeptides pattern similar to that of strain 0540, strains A34 (defective in heme synthesis) and 9-3 (nod+fix-) were similar to the wild type. Comparative analysis following in vivo labelling of root and nodule proteins by 35S_ methionine showed some nodule-specific polypeptides similar in molecular weight to those found by immunoblotting. Key words: Nodule-specific polypeptides, Nodulins, Immunoblotting, Medicago sativa, In vivo labelling, R. meliloti, Nitrogen fixation.

Introduction The temporal development of nitrogen-fixing root nodules comprises a multistep cascade of events that include adhesion of rhizobia to the root hair, root hair curling, infection thread formation, nodule initiation and development, bacteroid development and ultimately the commencement of nitrogen fixation (Rolfe and Shine, 1984). This requires a tightly co-ordinated «read-off» of genetic signals between both partners, the plant and the Rhizobium. Certain defect(s) in either the bacterial or plant genome may block this sequential interaction resulting in an arrested phenotype (Piihler et aI., 1985). Using random (Beringer et al., 1978; Simon et aI., 1983) and/or site-directed (Meade et aI., 1982) transposon mutagenesis as a tool, three major categories of bacterial mutants can be obtained: (i) mutants that are unable to initiate nodulation (Nod -), for example, the mutants which are defective in hair curling (Hac) or host specificity (Hsp -), (ii) mutants which are defective in their infection mechaAbbreviations: Kd = kilo dalton; SDS-PAGE = Sodium deodecyl sulfate polyacrylamide gel

electrophoresis.

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nism and produce modified nodule structures and (iii) mutants which induce ineffective but otherwise normal-looking nodules. While it is relatively easy to obtain T n5 induced Rhizobium mutants, the molecular characterisation of plant responses following infection by these mutants is more challenging, particularly in view of the limited knowledge of the plant's genetic contribution to the symbiosis (Carroll et ai., 1985). Legocki and Verma (1980) for the first time identified a group of plant proteins specific to soybean root nodules, called «nodulins» which were induced following Rhizobium infection. Analysis of cDNA clones made from nodule mRNA revealed five unique sequence species (Fuller et ai., 1983) specific for the nodule tissue. Two of the soybean nodulins namely leghemoglobin (Brisson et ai., 1982) and n-uricase (Nguyen et ai., 1985) were characterised in detail in terms of structure and function, while two other nodulin genes of unknown function, i.e. nodulin 23 (Maura et ai., 1985) and nodulin 24 (Katinakis and Verma, 1985), were sequenced. Nodule-specific polypeptides were also detected in other legumes namely pea (Bisseling et ai., 1983; Govers et ai., 1985), bean (Lara et al., 1983) and alfalfa (Lang-Unnasch and Ausubel, 1985). Using immunoblotting temporal changes were examined in the pattern of nodule-specific polypeptides after the nodules were induced by Tn5-induced Rhizobium mutants (Bisseling et ai., 1983, LangUnnasch and Ausubel, 1985). The work presented here focusses on the characterisation of the host responses in alfalfa nodules following invasion by R. meliloti. We used the immunoblotting technique (Brittner et al., 1980) to identify the plant-derived nodule specific proteins in wild type nodules. The presence of these polypeptides in alfalfa nodules was further confirmed by labelling of root and nodule polypeptides in vivo with 35S-methionine. Additionally, this communication reports evidence for the presence of these nodulespecific polypeptides in several mutant nodules including the nodules characterised by the lack of visible infection threads and intracellular bacteria. Materials and Methods Plant culture The alfalfa seeds were surface sterilized and germinated. 2 - 3 days old seedlings were transferred to Petri-dishes (99 mm diameter) containing modified Fahraeus medium (Carroll and Gresshoff, 1983) and were inoculated with 0.2 ml of mutant or wild type R. meliloti cultures at a density of about 108 cells/ml. These plates were incubated at 28°C for 4 to 5 weeks. The upper nodules were picked, immediately frozen in liquid N2 and stored at -70°C.

Strains used Strains used in this study are listed in Table 1. The strains were maintained in Ty slopes (Cannon, 1980) and grown to exponential phase in Ty liquid medium for all experiments.

Preparation ofsoluble proteins Nodule tissue was powdered with liquid N2 using a mortar and pestle. This powder, after addition of the grinding buffer (Imllgram of tissue) containing 25mM Tris-HCI pH 7.5, 20 mM KCI and 10 mM MgClz was further homogenised at 4°C. Following the removal by centrifugation of cell debris (200g, 10') and bacteroids (20,000g, 30'), the resulting supernatant

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Table 1: A list of R. meliloti strains used in this study. Strains

2204

101.45

938

A-34

3046

9-3

Genotype Symbiotic/biochemical characteristics

Reference or source

Induced nodules contain characteristically a meri-Puhler et al., 1985 stematic zone, a zone of invasion showing infection threads, early and late symbiotic zones containing bacteroid filled cells and the senescent zone. The bacteria are calcofluor positive (Signer, personal communication)*). Similar to 2011 concerning the induced nodule Wong et aI., 1983 phenotype. Puhler et aI., 1985 Induced nodules are white without infection threads and bacteroid-filled cells. The mutant strain was obtained after Tn5 mutagenesis and is calcofluor negative. Puhler et aI., 1985 Induced nodules are phenotypically similar to nodules induced by 0540. The mutant strain was obtained after Tn5 mutagenesis and is calcofluor negative. Induced nodules are phenotypically similar to (P. Muller, Univ. of nodules induced by 0540. The mutant strain Bielefeld) was obtained after Tn5 mutagenesis and is calcoflour positive. The induced nodule structure resembles phe(T. Engelke, Univ. of notypically to the wild type. The mutant strain Bielefeld) was obtained after Tn5 mutagenesis and is defective in succinate utilization. Phenotypically nodules are similar to the wild (G. Ditta, Univ. of type but of white appearance. The mutant California, La Jolla) strain was obtained after Tn5 mutagenesis and is deficient in heme synthesis. Induced nodules are bigger in size but similar (P. Muller, Univ. of Bielefeld) in structure compared to the wild type. The mutant strain was obtained after Tn5 mutagenesis and is calcofluor positive. Puhler et aI., 1985 Induced nodules are phenotypically similar to the wild type but possess an extensive senescent zone. The mutant strain was obtained after Tn5 mutagenesis.

*) Binding of calcofluor strain to Rhizobium indicates their ability to synthesize exopolysaccharides (EPS). was treated as soluble cytoplasmic protein extract. Root proteins were prepared similarly except that the preparation was centrifuged only once (20,000 g, 30'). Protein content of the extracts was estimated using a Bio-Rad protein estimating kit. The pellet containing the bacteroids was washed 3 to 4 times with grinding buffer and the bacteroids were lysed by boiling for 2 min in 1 % SDS, 10% (v/v) glycerol, 5% (v/v) (J-mercaptoethanol and the protein extract was clarified by centrifugation (10 min, 1000g). For bacterial proteins, R. meliloti strain 2011 was grown in Ty medium to exponential phase, centrifuged (10,000 rpm, 10 min). The pellet was resuspended in the grinding buffer and the proteins were obtained by centrifugation (10,000 rpm, 10 min) after sonication.

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Preparation of antibodies

To obtain the antiserum against soluble nodule proteins of alfalfa, rabbits were first injected subcutanously with 1 mg soluble proteins in Tris-HCI pH 7.5 and 0.9 % NaCI mixed with an equal volume of adjuvant (Difco Laboratories, Detroit). Three weeks later this was followed by intravenous injections of 3 - 5 mg of soluble proteins. After a further 2 weeks the rabbits were bled through the jugular ear veins and the antiserum was collected. This antiserum was titrated partially with proteins extracted from 1 to 3 weeks old uninfected roots grown on sterile sand and subsequently with proteins from bacteria. The antiserum (200 JLI) was incubated successively with 40, 60, 80 and 100 JLI of the root and the bacterial proteins (total amount of added antigen was 3 to 5 mg). Each incubation lasted 12 h at 4 °C followed by removal of immunoprecipitates by centrifugation (14,000 g, 10'). Immunoblotting

SDS-polyacrylamide gel electrophoresis was carried out on discontinuous slabs (Laemmli, 1970). After electrophoresis, the polypeptides were transferred electrophoretically to nitrocellulose filter (BA 85 Schleicher and Schiill), by a blotting apparatus built essentially as described by Brittner et al., 1980. Following blotting, the nitrocellulose filter was quenched in a buffer containing 0.Q1 M Tris-HCI pH 7.5, 0.35 M NaCI, 0.1 M phenylmethylsulfonylfluorid (PMSF) and 3 % (w/v) bovine serum albumin (BSA) for 5 h with slow shaking at room temperature. The filter was then incubated for 16 h at 4°C in an assay buffer (0.01 M Tris-HCI pH 7.5, 0.15M NaC!, 1 % (v/v) Triton X-100, 0.5% sodium-deoxycholate, 0.1 % (w/v) SDS, 0.1 M PMSF and 1 % (w/v) BSA containing the appropriate antiserum (20 JLl/ml of the buffer). Following the antibody reaction the filter was washed three times for 20 min each in the assay buffer without BSA, by shaking at 50 rpm in a gyratory shaker. Subsequently, the filter was incubated in the assay buffer containing I25I-Iabelled protein A (Amersham Corp., illinois) at about 106 cpm/ ml of buffer. Analysis of in vivo translation products

For in vivo translation, about 50 -100 mg of sand grown root (2 - 3 weeks old) and nodule (3 weeks old) tissue were used. The tissues were incubated separately in 100 JLI sterile water containing 100 JLCi 35S-methionine (Du Pont de Nemours GmbH, NEN Division Dreieich) on a rocker for 3 h at room temperature. To prevent bacteroidal protein synthesis, the labelling was carried out in presence of 200 JLg/ ml streptomycin. Streptomycin at this concentration was not detrimental to protein synthesis by the plant tissue but inhibited bacteroid protein synthesis. The proteins were extracted as described above and were analysed by SDS-polylacrylamide gel electrophoresis. In vivo labelled proteins were immunoprecipitated with the nodule antiserum essentially as described by Fuller et al. (1983). Protein molecular weight standards

Molecular weight standards for polyacrylamide gel electrophoresis were purchased from Bethesda Research Laboratories GmbH, West Germany and New England Nuclear (14C Protein size markers). The proteins included in these sets are: Cytochrome C (12.3 Kd), fJ-Iactoglobulin (18.4Kd), a-chymotrypsinogen (25.7Kd), ovalbumin (43 Kd), bovine serum albumin (68Kd), phosphorylase B (97.4 Kd) and myosin H (200 Kd).

Results L Detection of nodule-specific polypeptides in effictive and ineffective nodules by immunoblotting

Analysis by SDS-polyacrylamide gel electrophoresis of the soluble proteins from uninfected roots and matured alfalfa nodules showed that the majority of the poly-

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2

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3 ~120 ~97

~

73

~

48·5

~

39

~ ~

31 30

4

15

54 >

36 > 35 >

25 >

~ ~

14

12

Fig. 1: Autoradiograph of a blot of soluble proteins extracted from uninfected roots (lane 1) and nodules induced by strains 2011 (lane2), AK 631 (lane3) and 2204 (lane4). 50Jlg of proteins were electrophoresed in 12.5 % polyacrylamide gel and blotted onto nitrocellulose filter. The filter was incubated with alfalfa anti-nodule serum and then with 125I-Iabelled protein A. The filled arrows indicate the position of nodule-specific polypeptides, whereas open arrows indicate the root polypeptides. Using the nomenclature suggested at the 5th International Symposium on Nitrogen Fixation (Van Kammen, 1983), the nodule-specific polypeptides are indicated by N followed by their molecular weight in Kd.

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peptides were common to both tissues. In order to detect the polypeptides specific to the root nodule, the proteins from the root and nodule tissues were separated on SDS-polyacrylamide gels and transferred onto nitrocellulose filter. The filter was reacted sequentially with the partially titrated nodule antiserum and 125I-Iabelled protein A. The autoradiograph of this blot is shown in Fig. 1. When comparing the peptide pattern from roots (lane 1) with that isolated from nodules induced by wild type strain 2011, one recognises some common bands of 25, 35, 36 and 54 Kd, which are the result of the incomplete (partial) titration in the production of the anti-nodule serum. Other than these common bands at least 12 other nodule-specific peptides were visualised. These range in size from 12 to 120 Kd. A comparison with nodules induced by another wild type Rhizobium strain AK631 (see lane 3) gave the identical peptide pattern as that seen for strain 2011. This underlines the conclusion that host specific peptides were investigated here. In contrast, the profile of polypeptides from mutant strain 2204 induced nodules was slightly different. Two polypeptides of 73 and 120 Kd molecular weight (N73 and N120) that were present only in trace amounts in the wild type nodules (lane 2 and 3), were more abundant in the nodules induced by the mutant isolate 2204 (lane 4). Furthermore, in these mutant nodules the accumulation of three polypeptides (N 14, N 15 and N 31) was reduced and the 30 Kd polypeptide (N 30) was clearly undetectable. To test whether the obtained polypeptide pattern is contamined by bacterial polypeptides because of remnant bactria in the supernatant or the disruption of bacterial cells during homogenisation, plant soluble proteins of root nodules were probed with an antiserum raised against both components of the nitrogenase (obtained from T. Bisseling, Wageningen). The proteins from cultured bacteria and from bacteroids served as negative and positive controls, respectively. The results (data not shown) demonstrated that the nitrogenase polypeptides were present only in the bacteroid extract, but neither in the extracts of root nodules (streptomycin treated) nor in freeliving bacteria. To investigate whether the changes seen for mutant 2204 (nod+fix-) were correlated with its inability to induce a nitrogen fixing nodule, other non-fixing mutants of R. meliloti were examined for the presence of nodule-specific polypeptides. The properties of the analysed mutants are described in Table 1. Fig.2 respresents the autoradiograph of a blot containing polypeptides from the root (lane 2), the root nodules induced by the wild type strain 2011 (lane 1) and the mutant isolates 0540, 101.45, 938, A-34, 3046 and 9-3 (lanes 3 to 8). Many of the nodule-specific polypeptides identified in lane 2 of Fig. 1 were found at a reduced concentration in all of the mutant nodules examined. There were, however, differences among the mutants. For example, nodule-specific polypeptides observed in nodules induced by isolates 0540 (lane 3), 938 (lane 6) and 3046 (lane 7) exhibited in addition to others, one additional predominant polypeptide (N 97) which was only present in trace amounts in wild type nodules (see Fig. 1, lane2 and Fig.2, lane 1). Furthermore, these mutant nodules also contained the 120 Kd protein which was identified in nodules induced by mutant 2204. The nodules induced by the isolate 101.45 (lane 5), a 39 Kd polypeptide was amplified compared to root tissue. No marked differences from wild

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2

3

5

6

7

275

8

120 • 97 •

73 • 51. > 1.8·5 39

••

31

........, ~

•

25 > 21 • 19 •

Fig. 2: Autoradiograph of a blot of soluble alfalfa proteins from root nodules of alfalfa induced by R. meliloti strains 2011 (lane 1) and uninfected roots (lane2), 0540 (lane3), A-34 (lane4), 101.45 (laneS), 938 (lane 6), 3046 (lane 7) and 9-3 (lane 8). The mutant nodules were harvested after 3-4 weeks of inoculation. Soluble nodule proteins were separated on a 7.5-17.5% gradient polyacrylamide gel, and then blotted onto nitrocellulose. The blot was incubated with antiserum (different preparation from that used in Fig. 1) and 125I-protein A. For explanation of symbols see Fig. 1.

type in the pattern of nodule-specific polypeptides was observed in the nodules induced by strains A-34 (lane 4) and 9 - 3 (lane 8).

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1 2 3 4

Fig.3: Fluorogram of 35S-methionine in vivo labelled products from the uninfected lad old root (lanel), lad old root nodules (lane2), 30d old nodules (lane3) and bacteroids extracted from nodules used in lane 3 (lane 4).

II. In vivo labelling of root and nodule tissue

The immunoblotting procedure provides an easy way of assaying the nodules induced by mutant Rhizobium strains for the presence of nodule-specific polypeptides, but there are several drawbacks (Govers et aI., 1985; also see discussion). Additionally, with this procedure relatively fewer nodule-specific polypeptides were detected in alfalfa (Lang-Unnasch and Ausubel, 1985) if compared to pea (Bisseling et aI., 1983). We therefore used another in vivo technique (i.e. labelling the root and nodule polypeptides with 35S-methionine in vivo) to confirm the number of nodule-specific polypeptides that occur in the wild type nodule.

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The root and nodule tissue (10 and 30 d old) was collected and labelled in presence of 200 JLl/ml streptomycin and 100 JLei 35S-methionine. The proteins were extracted from the root, nodule and nodule bacteria, separated on SDS-PAGE and fluorographed (Fig.3). About 7 polypeptides specific to the nodule tissue are detectable (lane 3) which cannot be seen in the root tissue (lane 1). The most striking effect is the appearance of the 97Kd and a 120Kd protein band in lane3. These two bands could already be identified with the immunoblotting procedure in different mutant nodules (compare Fig. 1 and Fig.2). In Fig.3 it is clearly shown by the in vivo labelling method that these two nodule-specific proteins N 97 and N 120 are also produced in wild type nodules. In addition, it is of interest that they can hardly be seen in 10 day old nodules (lane2) but are very prominent in 30 day old ones (lane3). Lane4 in Fig. 3 demonstrates that labelling in presence of streptomycin drastically reduces the protein synthesis in the bacteroid fraction. The few protein bands in lane 4 might be contaminating plant polypeptides. None of the bacteroid-specific abundant polypeptides (for example nitrogenase) were observed. Discussion While R. meliloti has been studied in great detail in terms of nodulation and nitrogen fixation genes (Kondorosi et aI., 1985), the molecular genetic studies of its host plant is progressing rather slowly. In soybean (Legocki and Verma, 1980) and pea (Bisseling et aI., 1983) immunoblotting constituted the first step in the analysis of plant gene products involved in symbiotic nitrogen fixation. Lang-Unnasch and Ausubel (1985) used this technique to detect nodule-specific polypeptides in alfalfa. Furthermore, they examined polypeptide patterns of nodules induced by Rhizobium mutants defective in the nitrogen fixation (nif) phenotype. The results presented in this paper add to the general applicability of immunoassay procedure to detect nodule polypeptides and provide new information regarding pattern of nodule-specific polypeptides of additional ineffective Rhizobium mutants (see Table 1). The nodule antiserum used in this study was titrated by successive addition of root and bacterial antigen. The results presented in Fig. 1 show that the titration was not complete, though the bulk of root-specific antibodies was removed. Since exhaustive titrations may remove nodule-stimulated proteins, the partially titrated antiserum was used in this study. After a comparison of root and nodule extracts by immunoblotting, twelve nodule-specific polypeptides were detected. Their molecular weights closely match nine of the ten polypeptides detected by Lang-Unnasch and Ausubel (1985). The 66Kd bacterial polypeptide (Lang-Unnasch and Ausubel, 1985) that disappeared following titration of nodule antiserum with bacterial proteins, was also not detectable in our study. Slight differences in molecular weights of some polypeptides reported here and by Lang-Unnasch and Ausubel (1985) may be due to the differences in alfalfa ecotypes, rabbits or the physiological growth conditions used in these studies. For example, both inter- Ging et aI., 1982) and intra-specific (Holl et aI., 1983) variation involving qualitative changes in the leghemoglobin components is well documented. Lang-Unnasch and Ausubel (1985) observed one polypeptide (N 13) as

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leghemoglobin, whereas in our study (Fig. 1, lane 2 and lane 3), three polypeptides (N 12, N 14 and N 15) were detectable. All three bands may be leghemoglobin and! or its modification/breakdown products. Because only 12 nodule-specific polypeptides were detectable by immunoblotting it was suspected that the alfalfa nodule proteins may be less antigenic than those from pea. Therefore, the antibody independent in vivo labelling technique was used to confirm the number of detectable nodule-specific polypeptides. While the immunoblotting procedure visualizes proteins accumulated over time, labelling in vivo examines the polypeptides synthesized during the time course of the experiment. Furthermore, while using the latter technique it is possible to prevent bacteroidal protein synthesis by using proper antibiotics. Thus, it is more likely that nodule-specific polypeptides detected by this method are plant gene products. Since proteins may undergo major post-translational modifications it is difficult to compare the unlabelled versus labelled proteins by molecular weight alone. However, in this study some nodule-specific polypeptides were detected by in vivo labelling which were similar in molecular weight to the polypeptides found by immunoblotting. The antiserum used to immunoblot the mutant nodule polypeptides shared cross reactivity with root polypeptides. Despite this, some nodule-specific polypeptides could be detected from the mutant nodules. This finding has implications with regard to the plant response to the infection by Rhizobium. Of particular interest is the second category of bacterial mutants, namely isolates 0540, 2204 and 101.45, where the nodule-specific polypeptides are detectable despite the absence of visible infection threads and intracellular bacteria. Recently, there have been several reports of nodule organogenesis by atypical infection following the transfer of R. meliloti symbiotic genes to Agrobacterium tume/aciens (Wong et aI., 1983; Hirsch et aI., 1984, 1985; Truchet et al., 1984) or Escherichia coli (Hirsch et al., 1984). R. meliloti mutants 0540, 2204 and 101.45 induce nodules that appear to be similar in structure to the nodules induced by modified A. tume/aciens or E. coli strains. The lesions in strain 0540 and 2204 may therefore be responsible for «uncoupling» nodule morphogenesis from infection thread formation. Furthermore, two of these mutants 0540 and 2204 are calcofluor negative and are defective in exopolysaccharide synthesis (Muller et aI., 1985). Some of the genes controlling exopolysaccharide synthesis in R. meliloti have been identified. Whether or not they play a role in effective nodulation is currently being examined. Although these aspects of symbiosis are beyond the scope of this report, they deserve attention in view of the plant responses observed in root nodules induced by strains 0540 and 2204. Some of the nodule-specific polypeptides are synthesized in these nodules. It thus appears that neither infection thread formation nor the presence of bacteria within the host cells is essential for the induction of most nodule-specific polypeptides. The difference in the patterns of nodule-specific polypeptides from the root nodules induced by mutant strains 0540, 2204, 938, 3046, 101.45 are equally interesting. Though strains 2204 and 0540 shared a bacteroid free nodule morphology, their nodules had a 120 Kd polypeptide in common but differed in a 97 Kd polypeptide. More

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surprisingly, two other mutants (938 and 3046) incited also nodules that exhibited both the 97 and 120 Kd polypeptides. On the other hand, strain 101.45, which also produces bacteroid-free nodules, did not induce the 120 Kd and 97 Kd polypeptides. The nodules induced by this mutant contained a prominent 39 Kd polypeptide. The 97 Kd and 120 Kd polypeptides may be induced relatively early (between 10 and 30 days) in the nodule development and switched off following the commencement of nitrogen fixation. Since the antiserum was prepared from comparatively mature wild type nodules, from which soluble proteins were extracted for blotting experiment, these polypeptides were detectable only in trace amounts in these nodules. These were observed, however, in the in vivo labelled products from 30 day old nodule tissue. Moreover, these polypeptides were clearly seen in the nodules induced by bacterial mutants causing either incomplete nodule development (e.g. strain 0540), or a nodule phenotype comprising an extensive meristematic zone (e.g. isolates 3046 and 938). The 97Kd and 120Kd polypeptides may, therefore, be involved in nodule morphogenesis. Further work involving purification of these proteins to determine their localization in the nodule tissue will clarify this issue. Acknowledgements The authors wish to thank Dr. A. Radunz for his help in raising antibodies against alfalfa root nodule proteins and Mrs. S. Malmivaara for typing the manuscript. The nitrogenase antisera was a generous gift from Dr. T. Bisseling. S. S. Mohapatra was a recipient of the Alexander von Humboldt Research Fellowship. Dr. P. M. Gresshoff is thanked for advice and discussion. The experiments were financially supported by a grant from the Stiftung Volkswagenwerk. References BERINGER, J. E., J. BEYNON, A. V. BUCHANAN-WOLLASTON, and A. W. B. JOHNSTON: Transfer of drug-resistance transposon Tn5 to Rhizobium. Nature 276, 633-634 (1978). BISSELING, T., C. BEEN, J. KLUGKIST, A. VAN KAMMEN, and K. NADLER: Nodule-specific host proteins in effective and ineffective root nodules of Pisum sativum. EMBO J. 2, 961-966 (1983). BRISSON, N., A. POMBO-GENTILE, and D. P. S. VERMA: Organisation and expression of leghemoglobin genes. Can. J. Biochem. 60, 272-278 (1982). BRITTNER, M., P. KUPFERER, and C. F. MORRIS: Electrophoretic transfer of proteins and nucleic acids from slab gels to diazobenzyoxymethyl cellulose or nitrocellulose sheets. Anal. Biochern. 102,459-471 (1980). CANNON, F. c.: Genetic studies with diazotrophs. In: BERGERSEN, F. (ed.): Methods for evaluating biological nitrogen fixation, p. 367. John Wiley and Sons Ltd. (1980). CARROLL, B. J. and P. M. GRESSHOFF: Nitrate inhibition of nodulation and nitrogen fixation in white clover. Z. Pflanzenphysiol. 110,77-88 (1983). CARROLL, B. J., D. L. McNEIL, and P. M. GRESSHOFF: Isolation and properties of soybean [Glycine max (L.) Merr] mutants that nodulate in the presence of high nitrate concentrations. Proc. Nat!. Acad. Sci. (USA) 82, 4162-4166 (1985). FULLER, F., P. W. KUNSTNER, T. NAGUYEN, and D. P. S. VERMA: Soybean nodulin genes: analysis of cDNA clones reveals several major tissue-specific sequences in nitrogen-fixing root nodules. Proc. Natl. Acad. Sci. 80, 2594-2598 (1983). GOVERS, F., T. GLOUDEMANS, M. MOERMAN, A. VAN KAMMEN, and T. BISSELING: Expression of plant genes during the development of pea root nodules. EMBO J. 4, 861- 867 (1985). HIRSCH, A. M., K. J. WILSON, J. D. G. JONES, M. BANG, V. V. WALKER, and F. M. AUSUBEL: Rhi· zobium meliloti nodulation genes allow Agrobacterium tumefaciens and Escherichia coli to form pseudonodules on alfalfa. J. Bacteriol. 158, 1133-1143 (1984).

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