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.

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.

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.

Sandal et al. MPMI 4 INTRODUCTIONGenetic analysis and application of genetic approaches in the model legume Lotus japonicus (Handberg and Stougaard 1992) has progressed rapidly. Several key genes important for symbiosis with mycorrhizal fungi, root nodule development and other developmental processes have been identified using molecular genetics. The developmental regulators Nin (Schauser et al. 1999) and Pfo (Zhang et al. 2002) were isolated by transposon tagging while map-based cloning led to the molecular characterisation of Har1, SymRK, Nfr1, Nfr5, Castor and Pollux involved in autoregulation, Nod-factor signal perception or signal transduction (Schauser et al. 1999, Krusell et al. 2002 Nishimura et al. 2002a;Stracke et al. 2002;Radutoiu et al. 2003;Madsen et al. 2003; Imaizumi-Anraku et al. 2005). Genetic loci required for the early stages of endosymbiosis have attracted particular interest. Diallelic crosses together with phenotypical studies defined seven loci, SymRK, Nup133, Castor, Pollux, Sym6, Sym15,Sym24, in the common pathway required for both rhizobial and mycorrhizal symbiosis (Kistner et al. unpublished data) and map-based cloning of these loci has been accomplished or is advancing rapidly. A similar interest and effort is now emerging for genetic dissection of nodule organogenesis and function using the Fix -mutants arrested at various stages of nodule development or impaired in nodule function. Cloning of the Sst1 sulfate transporter required in functional root nodules is a first example (Krusell et al. 2005).Continuous isolation of new plant mutant lines is important for completing the genetic dissection of symbiosis and so far six independent mutant populations have been obtained by chemical (EMS) mutagenesis (Perry et al. 2003;Szczyglowski et al. 1998; Webb et al. unpublished data; Gresshoff et al. unpublished data), four populations after T-DNA or transposon insertion mutagenesis (Thykjaer et al. 1995;Schauser et al. 1998;Webb et al. 2000; Gresshoff et al. unpublished data), one population made with fast neutrons (Gresshoff et al. unpublished Umehara and Kouchi (unpublished data). All in all more than 400 symbiotic Lotus mutant lines were identified by screening in these populations and more are likely to follow. Assignment to complementation groups is next logical step in order to determine the number of loci involved, identify all alleles that contribute to phenotypic characterisation of mutants and genotyping of loci. However, diallelic crossing is a relatively slow process where progress is determined by generation time and slowed by a continuously increasing number of individual crosses necessary to keep up with mutant isolation programs. Given the number of symbiotic mutant lines already available and considering the time used to define seven complementation groups with a total of 26 alleles constituting the common pathway (Kistner et al. unpublished data), this approach is unlikely to encompass all alleles in near future. Detection of alleles in already cloned genes ...

Nitrogen-fixing symbiosis of legume plants with Rhizobium bacteria is established through complex interactions between two symbiotic partners. Similar to the mutual recognition and interactions at the initial stages of symbiosis, nitrogen fixation activity of rhizobia inside root nodules of the host legume is also controlled by specific interactions during later stages of nodule development. We isolated a novel Fix 2 mutant, ineffective greenish nodules 1 (ign1), of Lotus japonicus, which forms apparently normal nodules containing endosymbiotic bacteria, but does not develop nitrogen fixation activity. Map-based cloning of the mutated gene allowed us to identify the IGN1 gene, which encodes a novel ankyrin-repeat protein with transmembrane regions. IGN1 expression was detected in all organs of L. japonicus and not enhanced in the nodulation process. Immunoanalysis, together with expression analysis of a green fluorescent protein-IGN1 fusion construct, demonstrated localization of the IGN1 protein in the plasma membrane. The ign1 nodules showed extremely rapid premature senescence. Irregularly enlarged symbiosomes with multiple bacteroids were observed at early stages (8-9 d post inoculation) of nodule formation, followed by disruption of the symbiosomes and disintegration of nodule infected cell cytoplasm with aggregation of the bacteroids. Although the exact biochemical functions of the IGN1 gene are still to be elucidated, these results indicate that IGN1 is required for differentiation and/or persistence of bacteroids and symbiosomes, thus being essential for functional symbiosis.

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SUMMARYThe nitrogen-fixing symbiosis of legumes and Rhizobium bacteria is established by complex interactions between the two symbiotic partners. Legume Fix -mutants form apparently normal nodules with endosymbiotic rhizobia but fail to induce rhizobial nitrogen fixation. These mutants are useful for identifying the legume genes involved in the interactions essential for symbiotic nitrogen fixation. We describe here a Fix -mutant of Lotus japonicus, apn1, which showed a very specific symbiotic phenotype. It formed ineffective nodules when inoculated with the Mesorhizobium loti strain TONO. In these nodules, infected cells disintegrated and successively became necrotic, indicating premature senescence typical of Fix -mutants. However, it formed effective nodules when inoculated with the M. loti strain MAFF303099. Among nine different M. loti strains tested, four formed ineffective nodules and five formed effective nodules on apn1 roots. The identified causal gene, ASPARTIC PEPTIDASE NODULE-INDUCED 1 (LjAPN1), encodes a nepenthesin-type aspartic peptidase. The well characterized Arabidopsis aspartic peptidase CDR1 could complement the strain-specific Fix -phenotype of apn1. LjAPN1 is a typical late nodulin; its gene expression was exclusively induced during nodule development. LjAPN1 was most abundantly expressed in the infected cells in the nodules. Our findings indicate that LjAPN1 is required for the development and persistence of functional (nitrogen-fixing) symbiosis in a rhizobial strain-dependent manner, and thus determines compatibility between M. loti and L. japonicus at the level of nitrogen fixation.

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.