The Lotus japonicus nucleoporin GLE1 is involved in symbiotic association with rhizobia.

Nucleoporins are components of the nuclear pore complexes, channels that regulate the transport of macromolecules between the nucleus and cytoplasm. The nucleoporin GLE1 (GLFG lethal1) functions in the export of messenger RNAs containing poly(A) tails from the nucleus into the cytoplasm. Here we investigated a mutant of the model legume Lotus japonicus that was defective in GLE1, which we designated Ljgle1. The growth of Ljgle1 was retarded under symbiotic association with rhizobia, and the nitrogen-fixation activities of the nodules were around one-third of those in the wild-type plant. The growth of Ljgle1 was not substantialy recovered by supplemention of combined nitrogen. Nodules formed on the Ljgle1 were smaller than those on the wild-type and colored faint pink. The numbers of infected cells of nodules on the Ljgle1 were smaller than on the wild-type plant, and the former cells remained undeveloped. Rhizobia in the cells of the Ljgle1 exhibited disordered forms, and the symbiosome membrane was closely attached to the bacterial membrane. These results indicate that GLE1 plays a distinct role in the symbiotic association between legumes and rhizobia.

Function and evolution of a Lotus japonicus AP2/ERF family transcription factor that is required for development of infection threads.

Legume-rhizobium symbiosis is achieved by two major events evolutionarily acquired: root hair infection and organogenesis. Infection thread (IT) development is a distinct element for rhizobial infection. Through ITs, rhizobia are efficiently transported from infection foci on root hairs to dividing meristematic cortical cells. To unveil this process, we performed genetic screening using Lotus japonicus MG-20 and isolated symbiotic mutant lines affecting nodulation, root hair morphology, and IT development. Map-based cloning identified an AP2/ERF transcription factor gene orthologous to Medicago truncatula ERN1. LjERN1 was activated in response to rhizobial infection and depended on CYCLOPS and NSP2. Legumes conserve an ERN1 homolog, ERN2, that functions redundantly with ERN1 in M. truncatula. Phylogenetic analysis showed that the lineages of ERN1 and ERN2 genes originated from a gene duplication event in the common ancestor of legume plants. However, genomic analysis suggested the lack of ERN2 gene in the L. japonicus genome, consistent with Ljern1 mutants exhibited a root hair phenotype that is observed in ern1/ern2 double mutants in M. truncatula. Molecular evolutionary analysis suggested that the nonsynonymous/synonymous rate ratios of legume ERN1 genes was almost identical to that of non-legume plants, whereas the ERN2 genes experienced a relaxed selective constraint.

Isolation and phenotypic characterization of Lotus japonicus mutants specifically defective in arbuscular mycorrhizal formation.

Several symbiotic mutants of legume plants defective in nodulation have also been shown to be mutants related to arbuscular mycorrhizal (AM) symbiosis. The origin of the AM symbiosis can be traced back to the early land plants. It has therefore been postulated that the older system of AM symbiosis was partially incorporated into the newer system of legume-rhizobium symbiosis. To unravel the genetic basis of the establishment of AM symbiosis, we screened about 34,000 plants derived from ethyl methanesulfonate (EMS)-mutagenized Lotus japonicus seeds by microscopic observation. As a result, three lines (ME778, ME966 and ME2329) were isolated as AM-specific mutants that exhibit clear AM-defective phenotypes but form normal effective root nodules with rhizobial infection. In the ME2329 mutant, AM fungi spread their hyphae into the intercellular space of the cortex and formed trunk hyphae in the cortical cells, but the development of fine branches in the arbuscules was arrested. The ME2329 mutant carried a nonsense mutation in the STR-homolog gene, implying that the line may be an str mutant in L. japonicus. On the ME778 and ME966 mutant roots, the entry of AM fungal hyphae was blocked between two adjacent epidermal cells. Occasionally, hyphal colonization accompanied by arbuscules was observed in the two mutants. The genes responsible for the ME778 and ME966 mutants were independently located on chromosome 2. These results suggest that the ME778 and ME966 lines are symbiotic mutants involved in the early stage of AM formation in L. japonicus.

Positive and negative regulation of cortical cell division during root nodule development in Lotus japonicus is accompanied by auxin response.

Nodulation is a form of de novo organogenesis that occurs mainly in legumes. During early nodule development, the host plant root is infected by rhizobia that induce dedifferentiation of some cortical cells, which then proliferate to form the symbiotic root nodule primordium. Two classic phytohormones, cytokinin and auxin, play essential roles in diverse aspects of cell proliferation and differentiation. Although recent genetic studies have established how activation of cytokinin signaling is crucial to the control of cortical cell differentiation, the physiological pathways through which auxin might act in nodule development are poorly characterized. Here, we report the detailed patterns of auxin accumulation during nodule development in Lotus japonicus. Our analyses showed that auxin predominantly accumulates in dividing cortical cells and that NODULE INCEPTION, a key transcription factor in nodule development, positively regulates this accumulation. Additionally, we found that auxin accumulation is inhibited by a systemic negative regulatory mechanism termed autoregulation of nodulation (AON). Analysis of the constitutive activation of LjCLE-RS genes, which encode putative root-derived signals that function in AON, in combination with the determination of auxin accumulation patterns in proliferating cortical cells, indicated that activation of LjCLE-RS genes blocks the progress of further cortical cell division, probably through controlling auxin accumulation. Our data provide evidence for the existence of a novel fine-tuning mechanism that controls nodule development in a cortical cell stage-dependent manner.

The SNARE protein SYP71 expressed in vascular tissues is involved in symbiotic nitrogen fixation in Lotus japonicus nodules.

Soluble N-Ethylmaleimide Sensitive Factor Attachment Protein Receptor (SNARE) proteins are crucial for signal transduction and development in plants. Here, we investigate a Lotus japonicus symbiotic mutant defective in one of the SNARE proteins. When in symbiosis with rhizobia, the growth of the mutant was retarded compared with that of the wild-type plant. Although the mutant formed nodules, these exhibited lower nitrogen fixation activity than the wild type. The rhizobia were able to invade nodule cells, but enlarged symbiosomes were observed in the infected cells. The causal gene, designated LjSYP71 (for L. japonicus syntaxin of plants71), was identified by map-based cloning and shown to encode a Qc-SNARE protein homologous to Arabidopsis (Arabidopsis thaliana) SYP71. LjSYP71 was expressed ubiquitously in shoot, roots, and nodules, and transcripts were detected in the vascular tissues. In the mutant, no other visible defects in plant morphology were observed. Furthermore, in the presence of combined nitrogen, the mutant plant grew almost as well as the wild type. These results suggest that the vascular tissues expressing LjSYP71 play a pivotal role in symbiotic nitrogen fixation in L. japonicus nodules.

The integral membrane protein SEN1 is required for symbiotic nitrogen fixation in Lotus japonicus nodules.

Legume plants establish a symbiotic association with bacteria called rhizobia, resulting in the formation of nitrogen-fixing root nodules. A Lotus japonicus symbiotic mutant, sen1, forms nodules that are infected by rhizobia but that do not fix nitrogen. Here, we report molecular identification of the causal gene, SEN1, by map-based cloning. The SEN1 gene encodes an integral membrane protein homologous to Glycine max nodulin-21, and also to CCC1, a vacuolar iron/manganese transporter of Saccharomyces cerevisiae, and VIT1, a vacuolar iron transporter of Arabidopsis thaliana. Expression of the SEN1 gene was detected exclusively in nodule-infected cells and increased during nodule development. Nif gene expression as well as the presence of nitrogenase proteins was detected in rhizobia from sen1 nodules, although the levels of expression were low compared with those from wild-type nodules. Microscopic observations revealed that symbiosome and/or bacteroid differentiation are impaired in the sen1 nodules even at a very early stage of nodule development. Phylogenetic analysis indicated that SEN1 belongs to a protein clade specific to legumes. These results indicate that SEN1 is essential for nitrogen fixation activity and symbiosome/bacteroid differentiation in legume nodules.

How many peas in a pod? Legume genes responsible for mutualistic symbioses underground.

The nitrogen-fixing symbiosis between legume plants and Rhizobium bacteria is the most prominent plant-microbe endosymbiotic system and, together with mycorrhizal fungi, has critical importance in agriculture. The introduction of two model legume species, Lotus japonicus and Medicago truncatula, has enabled us to identify a number of host legume genes required for symbiosis. A total of 26 genes have so far been cloned from various symbiotic mutants of these model legumes, which are involved in recognition of rhizobial nodulation signals, early symbiotic signaling cascades, infection and nodulation processes, and regulation of nitrogen fixation. These accomplishments during the past decade provide important clues to understanding not only the molecular mechanisms underlying plant-microbe endosymbiotic associations but also the evolutionary aspects of nitrogen-fixing symbiosis between legume plants and Rhizobium bacteria. In this review we survey recent progress in molecular genetic studies using these model legumes.

Host plant genome overcomes the lack of a bacterial gene for symbiotic nitrogen fixation.

Homocitrate is a component of the iron-molybdenum cofactor in nitrogenase, where nitrogen fixation occurs. NifV, which encodes homocitrate synthase (HCS), has been identified from various diazotrophs but is not present in most rhizobial species that perform efficient nitrogen fixation only in symbiotic association with legumes. Here we show that the FEN1 gene of a model legume, Lotus japonicus, overcomes the lack of NifV in rhizobia for symbiotic nitrogen fixation. A Fix(-) (non-fixing) plant mutant, fen1, forms morphologically normal but ineffective nodules. The causal gene, FEN1, was shown to encode HCS by its ability to complement a HCS-defective mutant of Saccharomyces cerevisiae. Homocitrate was present abundantly in wild-type nodules but was absent from ineffective fen1 nodules. Inoculation with Mesorhizobium loti carrying FEN1 or Azotobacter vinelandii NifV rescued the defect in nitrogen-fixing activity of the fen1 nodules. Exogenous supply of homocitrate also recovered the nitrogen-fixing activity of the fen1 nodules through de novo nitrogenase synthesis in the rhizobial bacteroids. These results indicate that homocitrate derived from the host plant cells is essential for the efficient and continuing synthesis of the nitrogenase system in endosymbionts, and thus provide a molecular basis for the complementary and indispensable partnership between legumes and rhizobia in symbiotic nitrogen fixation.

Genetics of symbiosis in Lotus japonicus: recombinant inbred lines, comparative genetic maps, and map position of 35 symbiotic loci.

Development of molecular tools for the analysis of the plant genetic contribution to rhizobial and mycorrhizal symbiosis has provided major advances in our understanding of plant-microbe interactions, and several key symbiotic genes have been identified and characterized. In order to increase the efficiency of genetic analysis in the model legume Lotus japonicus, we present here a selection of improved genetic tools. The two genetic linkage maps previously developed from an interspecific cross between L. japonicus Gifu and L. filicaulis, and an intraspecific cross between the two ecotypes L. japonicus Gifu and L. japonicus MG-20, were aligned through a set of anchor markers. Regions of linkage groups, where genetic resolution is obtained preferentially using one or the other parental combination, are highlighted. Additional genetic resolution and stabilized mapping populations were obtained in recombinant inbred lines derived by a single seed descent from the two populations. For faster mapping of new loci, a selection of reliable markers spread over the chromosome arms provides a common framework for more efficient identification of new alleles and new symbiotic loci among uncharacterized mutant lines. Combining resources from the Lotus community, map positions of a large collection of symbiotic loci are provided together with alleles and closely linked molecular markers. Altogether, this establishes a common genetic resource for Lotus spp. A web-based version will enable this resource to be curated and updated regularly.

The sulfate transporter SST1 is crucial for symbiotic nitrogen fixation in Lotus japonicus root nodules.

Symbiotic nitrogen fixation (SNF) by intracellular rhizobia within legume root nodules requires the exchange of nutrients between host plant cells and their resident bacteria. Little is known at the molecular level about plant transporters that mediate such exchanges. Several mutants of the model legume Lotus japonicus have been identified that develop nodules with metabolic defects that cannot fix nitrogen efficiently and exhibit retarded growth under symbiotic conditions. Map-based cloning of defective genes in two such mutants, sst1-1 and sst1-2 (for symbiotic sulfate transporter), revealed two alleles of the same gene. The gene is expressed in a nodule-specific manner and encodes a protein homologous with eukaryotic sulfate transporters. Full-length cDNA of the gene complemented a yeast mutant defective in sulfate transport. Hence, the gene was named Sst1. The sst1-1 and sst1-2 mutants exhibited normal growth and development under nonsymbiotic growth conditions, a result consistent with the nodule-specific expression of Sst1. Data from a previous proteomic study indicate that SST1 is located on the symbiosome membrane in Lotus nodules. Together, these results suggest that SST1 transports sulfate from the plant cell cytoplasm to the intracellular rhizobia, where the nutrient is essential for protein and cofactor synthesis, including nitrogenase biosynthesis. This work shows the importance of plant sulfate transport in SNF and the specialization of a eukaryotic transporter gene for this purpose.

cDNA macroarray analysis of gene expression in ineffective nodules induced on the Lotus japonicus sen1 mutant.

The Lotus japonicus sen1 mutant forms ineffective nodules in which development is arrested at the stage of bacterial differentiation into nitrogen-fixing bacteroids. Here, we used cDNA macroarray systems to compare gene expression in ineffective nodules induced on the sen1 mutant with gene expression in wild-type nodules, in order to identify the host plant genes that are involved in nitrogen fixation. Macroarray analysis coupled with Northern blot analysis revealed that the expression of 18 genes was significantly enhanced in ineffective sen1 nodules, whereas the expression of 30 genes was repressed. Many of the enhanced genes encoded hydrolase enzymes, such as cysteine proteinase and asparaginase, that might function in the early senescence of sen1 nodules. By contrast, the repressed genes encoded nodulins, enzymes that are involved in carbon and nitrogen metabolism, membrane transporters, enzymes involved in phytohormone metabolism and secondary metabolism, and regulatory proteins. These proteins might have a role in the establishment of nitrogen fixation. In addition, we discovered two novel genes that encoded glutamate-rich proteins and were localized in the vascular bundles of the nodules. The expression of these genes was repressed in the ineffective nodules, which had lower levels of nitrogenase activity.

Large-scale analysis of gene expression profiles during early stages of root nodule formation in a model legume, Lotus japonicus.

Gene expression profiles during early stages of formation of symbiotic nitrogen-fixing nodules in a model legume Lotus japonicus were analyzed by means of a cDNA array of 18,144 non-redundant expressed sequence tags (ESTs) isolated from L. japonicus. Expression of a total of 1,076 genes was significantly accelerated during the successive stages that represent infection of Mesorhizobium loti, nodule primordium initiation, nodule organogenesis, and the onset of nitrogen fixation. These include 32 nodulin and nodulinhomolog genes as well as a number of genes involved in the catabolism of photosynthates and assimilation of fixed nitrogen that were previously known to be abundantly expressed in root nodules of many legumes. We also identified a large number of novel nodule-specific or enhanced genes, which include genes involved in many cellular processes such as membrane transport, defense responses, phytohormone synthesis and responses, signal transduction, cell wall synthesis, and transcriptional regulation. Notably, our data indicate that the gene expression profile in early steps of Rhizobium-legume interactions is considerably different from that in subsequent stages of nodule development. A number of genes involved in the defense responses to pathogens and other stresses were induced abundantly in the infection process, but their expression was suppressed during subsequent nodule formation. The results provide a comprehensive data source for investigation of molecular mechanisms underlying nodulation and symbiotic nitrogen fixation.

Expression of genes encoding late nodulins characterized by a putative signal peptide and conserved cysteine residues is reduced in ineffective pea nodules.

Five nodulin genes, PsN1, PsN6, PsN314, PsN335, and PsN466, with reduced expression in ineffective nodules on the pea (Pisum sativum) mutant E135 (sym13) were characterized. They encode small polypeptides containing a putative signal peptide and conserved cysteine residues and show homology to the nodulins PsENOD3/14 and PsNOD6. For each gene, multiple bands were detected by genomic Southern analysis. Northern analysis showed that all five genes were expressed exclusively in nodules and that their temporal expression patterns were similar to that of the leghemoglobin (Lb) gene during nodule development. Their transcripts were localized predominantly from the interzone II-III to the distal part of nitrogen-fixing zone III in effective nodules, resembling the Lb gene. However, transcripts in ineffective E135 nodules were localized in narrower regions than those in the effective nodules. These results indicate that these nodulins are abundant in pea nodules and that their successive expression during nodule development is associated with nitrogen-fixing activity.