Specific targeting of membrane nodulins to the bacteroid-enclosing compartment in soybean nodules

Rhizobium bacteroids in nodule cells are surrounded by the peribacteroid membrane (pbm), which is derived from the host plasma membrane during infection. The pbm was purified from R. japonicum 61A76-induced soybean nodules and analyzed by comparing it with the host cell plasma membrane for the presence of nodulins, nodule-specific plant proteins. Nodulins were found in pbm by reacting Western blots with a nodule-specific antiserum raised against the pbm. Peribacteroid fluid (the fluid enclosed in the pbm) was also found to contain several nodulins. The pbm nodulins were confirmed to be of plant origin by in vitro translation of poly(A)⁺ nodule mRNA followed by immunoprecipitation by the nodule-specific antiserum. Antibodies raised against a synthetic peptide corresponding to a repeated domain in nodulin-24, a pbm nodulin, and the nodule-specific pbm antiserum reacted exclusively with the pbm. The absence of pbm-nodulins in the plasma membrane suggests that the infected cells direct the intracellular transport of the pbm nodulins exclusively to this de novo synthesized subcellular compartment essential for symbiotic nitrogen fixation.     

Plant genes induced in the Rhizobium-legume symbiosis

Rhizobium, Bradyrhizobium and Azorhizobium can elicit the formation of N₂-fixing nodules on the roots or stems of their leguminous host plants. The nodule formation involves several developmental steps determined by different sets of genes from both partners, the gene expression being temporally and spatially coordinated. The plant proteins that are specifically synthesised during the formation and function of the nodule are called nodulins. The nodulins that are expressed before the onset of N₂ fixation are termed early nodulins. These proteins are probably involved in the infection process as well as in nodule morphogenesis rather than in nodule function. The nodulins expressed just before or during N₂ fixation are termed late nodulins and they participate in the function of the nodule by creating the physiological conditions required for nitrogen fixation, ammonium assimilation and transport. In this review we will describe nodulins, nodulin genes and the relationship between nodulin gene expression and nodule development. The study of nodulin gene expression may provide insight into root-nodule development and the mechanism of communication between bacteria and host plant.J.A. Muñoz and A.J. Palomares are with the Departamento de Microbiologia y Parasitología, Facultad de Farmacia, Universidad de Sevilla, 41012 Sevilla, Spain. P. Ratet is with the Institut des Sciences Végétales, CNRS, Avenue de la Terrasse, F-91198 Gif-sur-Yvette, Fance     

Root nodule development: origin, function and regulation of nodulin genes

The symbiotic root nodule, an organ formed on leguminous plants, is a product of successful interactions between the host plant and the soil bacteria, Rhizobium spp. Plant hormones play an important role in the genesis of this organ. The hormonal balance appears to be modulated by the signals produced by bacteria. Many host genes induced during nodule organogenesis and the symbiotic state have been identified and characterized from several legumes. These genes encode nodule-specific proteins (nodulins) which perform diverse functions in root nodule development and metabolism. Formation of a subcellular compartment housing the bacteria is essential to sustain the symbiotic state, and several nodulins are involved in maintaining the integrity and function of this compartment. The bacteroid enclosed in the perbacteroid membrane behaves as an 'organelle,'completely dependent on the host for all its requirements for carbon, nitrogen and other essential elements. Thus it seems likely that the nodulins in the peribacteroid membrane perform specific transport functions. While the function of a few other nodulins is known (e.g. nodulin-100, nodulin-35), a group of uncharacterized nodulins exists in soybean root nodules. These nodulins share structural similarities and seem to have been derived from a common ancestor. Induction of nodulin genes occurs prior to and independent of nitrogen fixation, and thus is a prelude to symbiosis. Although some of the early nodulin genes are induced prior to or during infection, induction of late nodulins requires endocytotic release of bacteria.     

Nodule-specific host proteins in effective and ineffective root nodules of Pisum sativum

Nodule-specific root proteins – so called nodulins – were identified in root nodules of pea plants by an immunological assay. Nodulin patterns were examined at different stages of nodule development. About 30 nodulins were detectable during development. Some were preferentially synthesized before nitrogen fixation started, whereas the majority were synthesized concomitantly with leghaemoglobin. Some of the nodulins were located within the peribacteroid membrane. Ineffective Rhizobium strains (a natural nod⁺fix^- and a pop ^-fix^-) appeared to be useful in studying the expression of nodulin genes. Synthesis of some nodulins was repressed in ineffective root nodules, indicating that nodulins are essential for the establishment of nitrogen fixation. In both types of ineffective root nodules, leghaemoglobin synthesis was not completely repressed. Low amounts of leghaemoglobin were always detected in young ineffective root nodules whereas in old nodules no leghaemoglobin was present.     

A novel RNA‐binding peptide regulates the establishment of the Medicago truncatula–Sinorhizobium meliloti nitrogen‐fixing symbiosis

Plants use a variety of small peptides for cell to cell communication during growth and development. Leguminous plants are characterized by their ability to develop nitrogen‐fixing nodules via an interaction with symbiotic bacteria. During nodule organogenesis, several so‐called nodulin genes are induced, including large families that encode small peptides. Using a three‐hybrid approach in yeast cells, we identified two new small nodulins, MtSNARP1 and MtSNARP2 (for small nodulin acidic RNA‐binding protein), which interact with the RNA of MtENOD40, an early induced nodulin gene showing conserved RNA secondary structures. The SNARPs are acidic peptides showing single‐stranded RNA‐binding activity in vitro and are encoded by a small gene family in Medicago truncatula. These peptides exhibit two new conserved motifs and a putative signal peptide that redirects a GFP fusion to the endoplasmic reticulum both in protoplasts and during symbiosis, suggesting they are secreted. MtSNARP2 is expressed in the differentiating region of the nodule together with several early nodulin genes. MtSNARP2 RNA interference (RNAi) transgenic roots showed aberrant early senescent nodules where differentiated bacteroids degenerate rapidly. Hence, a functional symbiotic interaction may be regulated by secreted RNA‐binding peptides.     

Nodulin gene expression in effective root nodules of white sweetclover (Melilotus alba Desr.) and in ineffective nodules elicited by mutant strains of Rhizobium meliloti

Fifteen nodulins and several nodule-stimulated gene productswere expressed in effective, nitrogen-fixing root nodules ofwhite sweetclover (Melilotus alba Desr. cv. U389), as determinedby two-dimensional gel electrophoresis of in vitro translationproducts. The number and gel position of eight leghaemoglobin(Lb) products, as well as a product tentatively identified asnodule-stimulated glutamine synthetase (GS), was similar toprevious reports of alfalfa (Medicago sativa L. cv. Iroquois)nodulins. Three mutants of Rhizobium meliloti, including anexoH mutant, a lipopolysaccharide mutant, and a nifH mutant,elicited ineffective sweetclover nodules blocked at empty (bacteria-free),partially infected, or fully infected stages of nodule development,respectively. In these ineffective nodules, the nodulin Nma30and nodule-stimulated NSTma42 were expressed early in development,while a group of four nodulins and two nodule-stimulated productswere intermediate in order of expression. Lb, GS and the latenodulin Nmal2a were expressed later, following infection. TheexoH mutant, Rm7154, appeared to be a leaky mutant, as a smallpercentage of the plants developed nitrogen-fixing nodules about4 weeks after inoculation. The sequential expression of a largenumber of nodulins and nodule-stimulated products, as well asthe availability of sweetclover nodulation mutants indicatesthat sweetclover is a useful diploid system for analysis ofhost genes essential to the Rhizobium/legume symbiosis. Key words: Nitrogen fixation, nodulation mutants, nodulins     

Nodulin gene expression in effective alfalfa nodules and in nodules arrested at three different stages of development

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 nifH gene of the nif operon. 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 in fix21-induced nodules. The genes for nodule-enhanced glutamine synthetase as well as for all the other nodulins are expressed in nodules induced by the nifH 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.     

Rhizobium nod genes are involved in the induction of two early nodulin genes in Vicia sativa root nodules

Nodulin gene expresison was studied in Vicia sativa (common vetch) root nodules induced by several Rhizobium and Agrobacterium strains. An Agrobacterium transconjugant containing a R. leguminosarum symplasmid instead of its Ti-plasmid, that was previously shown to form empty nodules on pea, induced nodules on Vicia roots in which nodule cells were infected with bacteria. In the Vicia nodules induced by this transconjugant, two so-called early nodulin genes were found to be expressed, whereas in the nodules formed on pea the expression of only one early nodulin gene was detected. In both cases the majority of the nodulin genes was not expressed.Apparently, an intracellular location of the bacteria is not sufficient for the induction of the majority of the nodulin genes. All nodulin genes were expressed in nodules induced by cured Rhizobium strains containing cosmid clones that have a 10 kb nod region of the sym-plasmid in common. Since in tumours no nodulin gene expression was found at all, the Agrobacterium chromosome does not contribute to the induction of nodulin genes. Therefore it is concluded that the signal for the induction of the expression of the two Vicia early nodulin genes is encoded by the nod-region, and the signal involved in the induction of all other nodulin genes has to be located outside the sym-plasmid, on the Rhizobium chromosome. The apparent difference in early nodulin gene expression between pea and Vicia is discussed in the light of the usefulness of Agrobacterium transconjugants in the study of nodulin gene expression.     

The roots of nodulins

Nodulin gene expression is an integral and highly specific part of the formation of nitrogen-fixing nodules on the roots of leguminous plants. Dependent on the time of expression during root nodule development, nodulin genes can be divided into early and late nodulin genes. A brief overview of the functions assigned to early and late nodulins is presented. We hypothesize that nodulin genes originate from regular plant genes that evolved to fit the regulatory and/or physiological constraints of symbiotic nitrogen fixation. Data on nodulins and nodulin genes, nodulation taxonomy and nodule development are evaluated in the light of this hypothesis.     

Nodulin gene expression during soybean (Glycine max) nodule development

In vitro translation products of total RNA isolated from soybean nodules at successive stages of nodule development were analyzed by two-dimensional gel electrophoresis. In that way the occurrence of over 20 mRNAs specifically transcribed from nodulin genes was detected. The nodulin genes could be divided into two classes according to the time of expression during nodule development. Class A comprises at least 4 nodulin mRNAs which are found when a globular meristem is present in the root cortex. These class A nodulin genes have a transient expression. Class B nodulin genes are expressed when the formation of a nodule structure has been completed. Bradyrhizobium japonicum nod ⁺ fix^-mutants, with large deletions spanning the nif H,DK region, still induced nodules showing normal expression of all nodulin genes, indicating that the nif H,DK region is not involved in the induction of nodulin genes. In nodules induced by Bradyrhizobium japonicum nod ⁺ fix^-mutant HS124 the bacteria are rarely released from the infection thread and the few infected cells appear to be collapsed. All class A and class B nodulin genes are expressed in HS124 nodules with the exception of 5 class B genes.     

Identification of “nodule-specific” host proteins (nodulins) involved in the development of Rhizobium-Legume symbiosis

Infection of legume roots with Rhizobium species results in the development of a root nodule structure in which the bacteria form an intracellular symbiosis with the plant. We report here that the infection of soybean (Glycine max L.) roots with Rhizobium japonicum results in the synthesis by the plant of at least 18–20 polypeptides other than leghemoglobin during the development of root nodules. Identification of these “nodule-specific” host polypeptides (referred to as nodulins) was accomplished by two-dimensional gel analysis of the immunoprecipitates formed by a “nodule-specific” antiserum with in vitro translation products of root-nodule polysomes that are free of bacteroidal contaminations. Nodulins account for 7–11% of the total ³⁵S-methionine-labeled protein synthesized in the host cell cytoplasm, and the majority of them are of 12,000–20,000 molecular weight. These proteins are absent from the uninfected roots, bacteroids and free-living Rhizobium, and appear to be coded for by the plant genes that may be obligatory for the development of symbiosis in the legume root nodules. Analysis of nodulins in ineffective (unable to fix nitrogen) nodules developed due to Rhizobium strains SM5 and 61A24 showed that their synthesis is reduced and their expression differentially influenced by mutations in rhizobia. Two polypeptides of bacterial origin were also found to be cross-reactive with the “nodule-specific” antiserum, suggesting that they are secreted by Rhizobium into the host cell cytoplasm during symbiotic nitrogen fixation.     

A white clover nodulin gene,dd23b,encoding a cysteine cluster protein,is expressed in roots during the very early stages of interaction with Rhizobium leguminosarumbiovar trifolii and after treatment with chitolipooligosaccharide Nod factors

An early nodulin cDNA, dd23b, was isolated from white clover root tissue by differential display RT-PCR. Its full-length sequence of 340 nucleotides encodes a predicted 72-amino-acid protein of molecular mass 8.3 kDa, with a polypeptide region containing cysteine pairs spaced in the manner of a cysteine cluster protein. This feature, which is shared by some other late and early nodulins from pea and broad bean, suggests a role in metal ion binding and membrane transport. Temporal and spatial expression patterns were determined during infection and nodulation by the homologous microsymbiont. No expression was found in unchallenged root tissue over a 7-day sampling period. Expression was first detectable in roots by RT-PCR 6 h post-inoculation with Rhizobium leguminosarum biovar trifolii, placing dd23b among the earliest nodulins to be detected to date. In root nodules, expression occurred primarily in the central symbiotic zone, but also in some host cells within the infection zone. Addition of purified wild-type chitolipooligosaccharide Nod factor to axenic white clover roots induced dd23b expression, providing further evidence for the role of this gene in the early plant response to infection by rhizobia. Electronic Publication     

Appearance and accumulation of nodulin mRNAs and their relationship to the effectiveness of root nodules

Summary Cloned cDNAs corresponding to mRNAs which accumulate in nitrogen-fixing root nodules of soybean (nodulin mRNAs) were used as probes to investigate the sizes, sequence relationships, tissue specificities and developmental accumulations of individual nodulin mRNA sequences. Northern blot analysis indicated that the NodB, NodC and NodD mRNA sequences are 1 150, 770, and 3 150 nucleotides long, respectively, which is consistent with the previously determined sizes of the hybrid-selected translation products (27 000, 24 000 and 100 000 MW, respectively). The NodA clones pNodA15 and pNodA25 hybridized to two mRNAs of lengths 1 600 and 1 100 nucleotides, indicating that they contain significant sequence homologies. However, increasing the hybridization stringency showed that the pNodA15 clone encodes the 1 600 nucleotide mRNA corresponding to the major NodA hybrid-selected translation product (44 000 MW) while pNodA25 encodes an mRNA of 1 100 nucleotides. The latter probably corresponds to one of two smaller (23 500 and 24 500 MW) in vitro translation products. RNA dot-blot hybridizations indicated that nodulin and leghemoglobin mRNAs began to appear and accumulate in Rhizobium infected root tissue very early (day 3 to 5) and reached fully induced levels by day 11. This accumulation was specific for nodule tissue (except for the NodD sequence) and preceded the accumulation of nitrogen fixation activity. Nodules produced by different effective Rhizobium strains accumulated similar levels of leghemoglobin and nodulin mRNAs while ineffective strains had a pleiotropic affect. While one ineffective strain (61A24) gave reduced levels of all these mRNAs, the other (SM5) gave levels which were nearly normal by the time nitrogen fixation activity should have reached its maximal level (day 17). Thus, leghemoglobin and nodulin genes are switched on soon after infection, prior to nodule morphogenesis, and the switch occurs prior to and is independent of nitrogen fixation activity.     

Nodules elicited by Rbizobium meliloti heme mutants are arrested at an early stage of development

Summary Heme-deficient mutants of Rhizobium and Bradyrhizobium have been found to exhibit diverse phenotypes with respect to symbiotic interactions with plant hosts. We observed that R. meliloti hemA mutants elicit nodules that do not contain intracellular bacteria; the nodules contain either no infection threads (empty nodule phenotype) or aberrant infection threads that failed to release bacteria (Bar^– phenotype). These mutant nodules expressed nodulin genes associated with nodules arrested at an early stage of development, including ENOD2, Nms-30, and four previously undescribed nodulin genes. These nodules also failed to express any of six late nodulin genes tested by hybridization, including leghemoglobin, and twelve tested by in vitro translation product analysis which are not yet correlated with specific cloned genes. We observed that R. meliloti leucine and adenosine auxotrophs induced invaded Fix^– nodules that expressed late nodulin genes, suggesting that it is not auxotrophy per se that causes the hemA mutants to elicit Bar^– or empty nodules. Because R. meliloti hemA mutants elicit nodules that do not contain intracellular bacteria, it is not possible to decide whether or not the Fix^– phenotype of these nodules is a direct consequence of the failure of R. meliloti to supply the heme moiety of hololeghemoglobin. Our results demonstrate the importance of establishing the stage in development at which a mutant nodule is arrested before conclusions are drawn about the role of small metabolite exchange in the symbiosis.     

The pea late nodulin gene PsNOD6 is homologous to the early nodulin genes PsENOD3/14 and is expressed after the leghaemoglobin genes

The pea late nodulin gene PsNOD6 has been cloned and sequenced. PsNOD6 is homologous to the pea early nodulin genes PsNOD3 and PsENOD14. In situ hybridization experiments showed that, like the PsENOD3 and PsENOD14 genes, the PsNOD6 gene is only expressed in the infected cell type. The PsNOD6 gene is first expressed at the transition of the pre-fixation zone II into the interzone II–III (the amyloplast-rich zone preceding the fixation zone III), whereas the early nodulin genes PsENOD3 and PsENOD14 are already induced in the pre-fixation zone II. Thus these nodulin genes encoding homologous proteins are induced at consecutive stages of nodule development.The expression of the late nodulin genes encoding leghaemoglobin precedes the expression of the late nodulin gene PsNOD6. Therefore these late nodulin genes have to be regulated by different mechanisms despite the fact they are expressed in the same cell type. This conclusion is consistent with the fact that PsNOD6 lacks one of the conserved regions occurring in the promoters of all other late nodulin genes studied.     

cDNA cloning and developmental expression of pea nodulin genes

A cDNA library prepared from pea nodule poly(A)⁺ RNA was screened by differential hybridization with cDNA probes synthesized from root and nodule RNA respectively. From the cDNA clones that hybridized exclusively with the nodule probe five clones, designated pPsNod 6, 10, 11, 13 and 14 and each containing unique sequences, were further characterized together with one leghemoglobin and one root-specific cDNA clone. In vitro translation of RNA selected by the pPsNod clones showed that the corresponding genes encode nodulins with molecular weights ranging from 5 800 to 19 000. During pea root nodule development expression of the five PsNod genes starts more or less concomitantly with the onset of nitrogen fixing activity in the nodules and the time course of appearance and accumulation of the nodulin mRNAs is similar to that of leghemoglobin mRNA. In ineffective pea root nodules expression of the PsNod genes is induced but the final accumulation levels of the mRNAs are markedly reduced to various degrees. The expression of another nodulin gene, designated ENOD2, was followed using a heterologous soybean cDNA clone as probe. In pea root nodules the ENOD2 gene is expressed at least five days before the PsNod and leghemoglobin genes, and in contrast to the PsNod mRNAs the concentration of the ENOD2 mRNA is the same in wild type and fix ^- nodules. The results described suggest that in root nodules several regulatory mechanisms exist which determine the final nodulin mRNA amounts accumulating in the root nodule.