bacteria-free nodules elicited by the exoB mutant. ... concerted expression of bacterial and host plant genes. ... However, host cells differentiate in that they.
Plant Physiol. (1988) 88, 321-328 0032-0889/88/88/0321/08/$01.00/0
Nodulin Gene Expression in Effective Alfalfa Nodules and in Nodules 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*4 Department ofBiological Sciences, Wellesley College, Wellesley, Massachusetts 02181 ABSTRACT Nodulin gene expression was analyzed in effective and ineffective root nodules of alfalfa (Medicago sativa L. cv Iroquois) elicited by three different Rhizobium meliloti mutants: an exoB mutant having defective acidic exopolysaccharide that does not fluoresce on plates containing the fluorescent brightener Calcofluor, fix21, a spontaneous mutant that has defective lipopolysaccharide and is Calcofluor bright; and a Rhizobium mutant resulting from a Tn5 insertion in the niffI gene of the nifoperon. The ineffective nodules elicited by these various mutant rhizobia are blocked at different stages of nodule development and have unique phenotypes. A distinctive pattern of nodulin gene expression as determined by in vitro translations of total nodule RNA characterizes each nodule phenotype. Seventeen nodulins are found in effective nodules including five leghemoglobins. Only one nodulin gene is expressed in the bacteria-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 all the other nodulins are expressed in nodules induced by the niJH mutant. The expression of genes for the nodulins, including leghemoglobin, is independent of the nitrogen-fixing ability of the nodule and appears to correlate with the differentiation of densely cytoplasmic host cells in the nodule and, to some extent, with bacterial release from infection threads.
The development of symbiotic nitrogen-fixing root nodules resulting from the infection of leguminous plants by host-specific species of Rhizobium and Bradyrhizobium is dependent on the concerted expression of bacterial and host plant genes. The role of rhizobial genes in host specificity, induction of nodule development, and nitrogen fixation has been the subject of intensive study (21). However, the role of the eukaryotic host genes involved in symbiosis has only recently come under scrutiny. Classical genetic analysis of the host legume has demonstrated that plant genes play a part at every stage of nodule development (1 1). The plant proteins specifically formed as a result of the plantmicrobe interaction are termed "nodulins" (32). Nine nodulespecific proteins have been detected in extracts of alfalfa root
'Supported by National Science Foundation Grants PCM 83-16793 and DCB 87-03297. 2 Supported by a National Science Foundation Postdoctoral Fellowship in Plant Biology. Present address: Department of Botany, University of Rhode Island, Kingston, RI 02881; Formerly Joanna F. Hanks. 3Present address: Department of Plant Pathology, University of Georgia, 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 were reported in a second study (31). The identities of a few nodulins in various legume hosts have been established. However, the role of most nodulins in symbiosis, especially those involved in nodule development, remains unknown. Studies with some cloned nodulin genes have demonstrated that expression of these genes is not coupled to activation of the nitrogenase genes of Rhizobium (8) or to nitrogen-fixing ability (9). Moreover, neither heme secretion by bacteroids (1, 9) nor bacteroid development (1, 8, 9, 27) was required for expression of Lb5 and other nodulins. Studies on the timing of nodulin gene expression in pea suggests that at least three groups of nodulin genes are expressed differentially (9). The early nodulin, N-40', was expressed from the beginning of nodule development while a second early nodulin, N-68, appeared about 2 d later. Several nodulins, including Lb, were found immediately prior to the onset of symbiotic nitrogen fixation. Other nodulins, for example, 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 the timing of expression of nodule-specific genes in alfalfa. The acidic exopolysaccharide Rhizobium meliloti mutant (exoB) exhibits a specific pattern of resistance to rhizobial phages, is conditionally sensitive to a monoclonal antibody to the bacterial surface, and does not fluoresce under ultraviolet light on plates containing the fluorescent brightener Calcofluor (6). This mutant induces nodules that lack infection threads and are devoid of bacteria indicating that nodule morphogenesis can be uncoupled from bacterial invasion (6). Nodules induced by exo mutants were found to contain low levels of nodule-specific proteins but neither Lb (19) nor nodule-specific GS (5). Similarly, "tumor-like" nodules controlled by the plant gene in3, which have a phenotype similar to that of the exo mutant-induced nodules, exhibited little cross-reactivity with nodule-specific antiserum and contained no detectable Lb protein (31). In contrast, bacteroidcontaining yet ineffective nodules elicited by R. meliloti nifHI mutants (18) and those controlled by the plant gene in, (31) were found to produce Lb and other nodule-specific proteins. In this study, we examined the in vitro translation products of total RNA isolated from exoB and nimH-induced nodules, rather than in vivo proteins. In addition, we examined nodules induced by R. meliloti mutant fix21 (17). These have a phenotype that differs from previously described ineffective nodules of alfalfa. The nodules contain infection threads that in some nodules abort but in others release bacteria. The rhizobia become surrounded by peribacteroid membranes that rapidly degenerate, and they do not differentiate into elongate bacteroids as do the nifl mutant bacteria. However, host cells differentiate in that they
'Abbreviations: Lb, leghemoglobin; GS, glutamine synthetase; Nmr, neomycin resistant; Otr, oxytetracycline resistant; PMSF, phenylmethylsulfonyl fluoride. 321
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become densely cytoplasmic and also contain mRNAs for most of the nodulins, including Lb, found in effective nodules. Our goal was to determine the difference in nodulin gene expression between three classes of ineffective nodules: those totally devoid of bacteria (exoB-mutant nodules); those partially infected (fix21-mutant nodules); and nodules that are fully infected (nifl-Imutant nodules). MATERIALS AND METHODS Bacterial Strains. Wild-type Rhizobium meliloti SU47, exoB mutants Rm5093 (Nmr) (6) and Rm5078 (Otr), and mutant EJ312fix21 (Rm5 121) were generously provided by Ethan Signer and Ralph Clover (Massachusetts Institute of Technology). Rm5121 is a spontaneous R. meliloti mutant that is bright under ultraviolet illumination when grown on LB-Calcofluor but is altered in cell surface properties (17). Moving the fix21 allele from the EJ312 background into wild-type strain Rm 1021 results in rhizobia with multiple defects (R. Clover, personal communication). However, the strain used in this study (Rm5121) appears to have only a lipopolysaccharide defect. Mutant Rm1491 (nifr::Tn5) was a generous gift from Fred Ausubel (Massachusetts General Hospital). Nitrogen-fixing ability in the mutant-induced nodules was analyzed by acetylene reduction (13). Plant Material. Seedlings of Medicago sativa L. cv Iroquois inoculated with R. meliloti SU47 were grown in the greenhouse in flats of perlite and watered with nitrogen-free medium. Plants inoculated with mutant R. meliloti were grown on sterile agar slants as described previously (13). Nodules were generally harvested 21 d after inoculation, although occasionally at earlier and later times, frozen in liquid nitrogen, and stored at -70°C. Control root tissue was harvested after 2 weeks from uninoculated plants grown in the greenhouse on nitrate-supplemented medium. Nodules were excised from roots, fixed, and prepared for 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 LiCl precipitation as described previously (9). Approximately 2,ug of RNA were translated for h in a 25-,tL reaction using a rabbit reticulocyte lysate kit (New England Nuclear) so that a linear relationship between the amount of RNA added and the incorporation of [35S]methionine was established. The amount of radioactivity incorporated into translation products was estimated by precipitation of ,uL of the reaction mixture in 10% trichloroacetic acid on Whatmann 3MM paper as specified in the lysate kit.
Plant Physiol. Vol. 88, 1988
Antisera. Lb antisera were generously provided by Ton Bisseling (Agricultural University, Wageningen, The Netherlands) and Carroll Vance (University of Minnesota, St. Paul). Antiserum prepared 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 protein was separated on a two-dimensional gel as described above and transferred to nitrocellulose (30). Peroxidase-conjugated goat antirabbit secondary antibody and reagents were purchased from Bio-Rad (Immun-Blot kit) and used according to manufacturer's instructions.
RESULTS Gene Expression in Effective Nodules. To serve as a frame of reference for the analysis to follow of the nodulins found in the ineffective nodules, a brief description of nodulin gene expression in effective, wild-type-induced nodules is included. Numerous descriptions of the histology of effective alfalfa nodules have been published (see references in 13). Comparison of the two-dimensional pattern of in vitro translation products of RNA from nitrogen-fixing root nodules of alfalfa with that from roots indicated that a majority of the polypeptides observed were present in both tissues. However, at least 17 unique spots ranging from 14 to 64 kD were observed consistently on fluorographs from gels of translation products from nodule RNA and therefore represent the expression of presumed nodulin genes (arrowheads, Fig. 1B). Caution was exercised in identification of nodule-specific products, as many spots that first appeared to be nodule specific were seen at low levels upon longer exposures of root gels. Several root-specific translation products were also observed but will not be discussed (Fig. IA). The appearance of the nodulin messages was analyzed in wildtype induced root nodules harvested at 12, 19, and 26 d after inoculation. Although nodules harvested at 19 d exhibited the highest acetylene reduction activity (data not shown), 12-d-old nodules were pink and reduced acetylene. However, they were much smaller than the older nodules. Most of the nodulins indicated in Fig. iB, including Lb and GS. (see below), were detected at high levels at all ages analyzed. The only exception was N-17a, which was present in very low amounts 12 d after inoculation and reached highest concentration at 26 d (data Two-Dimensional Electrophoresis. The translation products not shown). were separated by two-dimensional electrophoresis (9) using a Glutamine Synthetase. Antiserum against GS from Phaseolus 12.5% polyacrylamide slab gel for the second dimension. The vulgaris, which reacts with both nodule-specific GS. and GSr first dimension isoelectric focusing gels contained 1.6% ampho- (found in both roots and nodules) (3), was used to identify the lines pH 5 to 8 and 0.4% ampholines pH 3 to 10. The basic end isozymes of GS in alfalfa. Two spots were identified as isozymes of the focusing gel was always placed on the left side of the of GS by immunoprecipitation of in vitro translated nodule RNA second dimension gel. Molecular weight standards from Bio-Rad with the GS antiserum (Fig. 2A). The spot to the left (N-46) is (Richmond, CA) or prestained standards from BRL (Gaithers- significantly enhanced in nodules and thus may represent a burg, MD) were run in the second dimension. Usually, 300,000 nodule-specific form of GS. The additional immunoprecipitated cpm incorporated into translation products were applied to the translation product located immediately to the right of N-46 is gel. Gels were treated with Enhance (New England Nuclear), also present in root translations and probably represents GSr. Leghemoglobin. Nodulins N-I 5a, N- 14a, N- 14b, N-iSb, and dried, and autoradiographed on Kodak XAR-5 x-ray film. Immunoprecipitation. Translation reactions were incubated N-15c were identified as Lb translation products based on immunoprecipitation with the Lb antisera (Fig. 2B). Five Lb prowith ,uL of antiserum as described (9), except that mg of Sepharose-Protein A (Pharmacia) were added in the final 3 h of teins of the same size and pI were detected by immunoblot incubation. The Sepharose-A-bound immunoprecipitates were analysis of nodule protein extracts with an Lb antiserum (Fig. pelleted gently in a microfuge and washed with buffer five times, 2C). Nodulins N- I 7b, N- I 7c, and N- I 7d (Fig. IB) also appeared until negligible counts per minute were detected in the superna- to be Lb translation products because these nodulins were imtant. The immunoprecipitate was eluted from the Sepharose-A munoprecipitated by both Lb antisera (see "Materials and Methin100 L of buffer containing 5 % 2-mercaptoethanol and 2% ods") (Fig. 2B). Jing et al.(16) have identified large antigenic Lb NaDoSO4 and was analyzed by two-dimensional electrophoresis. translation products similar to N- I 7b, N- I 7c, and N- I 7d in 1
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and Agrobacterium strains carrying the symbiotic plasmid of R. meliloti. Their results differ from ours in that no nodule-specific transcripts are detected in in vitro translations of RNA isolated from nodules induced by EJ355 and the Agrobacterium transconjugants. 2. We propose that rhizobial genes that are intact infix2 l and defective in exoB may express gene products that are important for the induction of the infected host cell type. Theflx2 l mutant induces nodules in which two host cell types have differentiated, i.e. the highly vacuolate, uninfected cell type and densely cyto-
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