Both incompatible and compatible rhizobia inhabit the intercellular spaces of leguminous root nodules.

In addition to rhizobia, many types of co-existent bacteria are found in leguminous root nodules, but their habitats are unclear. To investigate this phenomenon, we labeled Bradyrhizobium diazoefficiens USDA122 and Bradyrhizobium sp. SSBR45 with Discosoma sp. red fluorescent protein (DsRed) or enhanced green fluorescent protein (eGFP). USDA122 enhances soybean growth by forming effective root nodules, but SSBR45 does not form any nodules. Using low-magnification laser scanning confocal microscopy, we found that infected cells in the central zone of soybean nodules appeared to be occupied by USDA122. Notably, high-magnification microscopy after co-inoculation of non-fluorescent USDA122 and fluorescence-labeled SSBR45 also revealed that SSBR45 inhabits the intercellular spaces of healthy nodules. More unexpectedly, co-inoculation of eGFP-labeled USDA122 and DsRed-labeled SSBR45 (and vice versa) revealed the presence of USDA122 bacteria in both the symbiosomes of infected cells and in the apoplasts of healthy nodules. We then next inspected nodules formed after a mixed inoculation of differently-labeled USDA122, without SSBR45, and confirmed the inhabitation of the both populations of USDA122 in the intercellular spaces. In contrast, infected cells were occupied by single-labeled USDA122. We also observed Mesorhizobium loti in the intercellular spaces of active wild-type nodules of Lotus japonicus using transmission electron microscopy. Compatible intercellular rhizobia have been described during nodule formation of several legume species and in some mutants, but our evidence suggests that this type of colonization may occur much more commonly in leguminous root nodules.

Characterization of photosynthetic Bradyrhizobium sp. strain SSBR45 isolated from the root nodules of Aeschynomene indica.

We isolated a novel strain of Bradyrhizobium sp., SSBR45, from the nodulated roots of Aeschynomene indica and labeled it with Discosoma sp. red fluorescent protein (dsRED) or enhanced green fluorescent protein (eGFP) and determined its draft genomic sequence. The labeled SSBR45 stimulated the growth of A. indica markedly on a nitrogen-free medium, as observed by visualizing the fluorescent root nodules. The nodulated roots also exhibited high acetylene reduction activities. The SSBR45 genome included genes involved in nitrogen fixation, photosynthesis, and type IV secretion system; however, it did not consist of canonical nodABC genes and type III secretion system genes. SSBR45, a novel species of the genus Bradyrhizobium, consisted of an average nucleotide identity and average amino acid identity of 87% and 90%, respectively, with the closest strain B. oligotrophicum S58.

Loss-of-function of ASPARTIC PEPTIDASE NODULE-INDUCED 1 (APN1) in Lotus japonicus restricts efficient nitrogen-fixing symbiosis with specific Mesorhizobium loti strains.

The 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.

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.

A thaumatin-like protein, Rj4, controls nodule symbiotic specificity in soybean.

Soybeans exhibit a nitrogen-fixing symbiosis with soil bacteria of the genera Bradyrhizobium and Ensifer/Sinorhizobium in a unique organ, the root nodule. It is well known that nodulation of soybean is controlled by several host genes referred to as Rj (rj) genes. Among these genes, a dominant allele, Rj4, restricts nodulation with specific bacterial strains such as B. elkanii USDA61 and B. japonicum Is-34. These incompatible strains fail to invade the host epidermal cells as revealed by observations using DsRed-labeled bacteria. Here, we describe the molecular identification of the Rj4 gene by using map-based cloning with several mapping populations. The Rj4 gene encoded a thaumatin-like protein (TLP) that belongs to pathogenesis-related (PR) protein family 5. In rj4/rj4 genotype soybeans and wild soybeans, we found six missense mutations and two consecutive amino acid deletions in the rj4 gene as compared with the Rj4 allele. We also found, using hairy root transformation, that the rj4/rj4 genotype soybeans were fully complemented by the expression of the Rj4 gene. Whereas the expression of many TLPs and other PR proteins is induced by biotic/abiotic stress, Rj4 gene expression appears to be constitutive in roots including root nodules.

The transcription activation and homodimerization of Lotus japonicus Nod factor Signaling Pathway2 protein.

The legume-rhizobia symbioses lead to the formation of a novel adaptive complex organ, termed the root nodule, which arises from cortical cell division and rhizobial infection in the root. Lipochitin oligosaccarides, Nod-Factors (NFs) secreted by rhizobia, are responsible for the onset of nodule development. Here we describe the characterization of Lotus japonicas, Nod factor Signaling Pathway2 (LjNSP2) protein that belongs to the plant GRAS family of transcription factors. Yeast two-hybrid analysis indicates that LjNSP2 alone has a transcription-stimulating ability and for this the SH2(src-homology2)-like domain, vital for function of STAT proteins is required. The ADG4 (the activation domain of GAL4)-LjNSP2 fusion coupled with BDG4 (the DNA binding domain of GAL4)-LjNSP2 increased the expression level, whereas the ADG4-Ljnsp2-1 mutant fusion did not, indicating that LjNSP2 interacts with itself to form a homodimer and this depends on the SH2-like domain. Based on the evidence, we discuss the action of LjNSP2, compared with that of the family of animal-specific STAT transcription factors, which induce developmental programmes in response to external stimuli.

Nodule inception directly targets NF-Y subunit genes to regulate essential processes of root nodule development in Lotus japonicus.

The interactions of legumes with symbiotic nitrogen-fixing bacteria cause the formation of specialized lateral root organs called root nodules. It has been postulated that this root nodule symbiosis system has recruited factors that act in early signaling pathways (common SYM genes) partly from the ancestral mycorrhizal symbiosis. However, the origins of factors needed for root nodule organogenesis are largely unknown. NODULE INCEPTION (NIN) is a nodulation-specific gene that encodes a putative transcription factor and acts downstream of the common SYM genes. Here, we identified two Nuclear Factor-Y (NF-Y) subunit genes, LjNF-YA1 and LjNF-YB1, as transcriptional targets of NIN in Lotus japonicus. These genes are expressed in root nodule primordia and their translational products interact in plant cells, indicating that they form an NF-Y complex in root nodule primordia. The knockdown of LjNF-YA1 inhibited root nodule organogenesis, as did the loss of function of NIN. Furthermore, we found that NIN overexpression induced root nodule primordium-like structures that originated from cortical cells in the absence of bacterial symbionts. Thus, NIN is a crucial factor responsible for initiating nodulation-specific symbiotic processes. In addition, ectopic expression of either NIN or the NF-Y subunit genes caused abnormal cell division during lateral root development. This indicated that the Lotus NF-Y subunits can function to stimulate cell division. Thus, transcriptional regulation by NIN, including the activation of the NF-Y subunit genes, induces cortical cell division, which is an initial step in root nodule organogenesis. Unlike the legume-specific NIN protein, NF-Y is a major CCAAT box binding protein complex that is widespread among eukaryotes. We propose that the evolution of root nodules in legume plants was associated with changes in the function of NIN. NIN has acquired functions that allow it to divert pathways involved in the regulation of cell division to root nodule organogenesis.

Rj (rj) genes involved in nitrogen-fixing root nodule formation in soybean.

It has long been known that formation of symbiotic root nodules in soybean (Glycine max (L.) Merr.) is controlled by several host genes referred to as Rj (rj) genes, but molecular cloning of these genes has been hampered by soybean's complicated genome structure and large genome size. Progress in molecular identification of legume genes involved in root nodule symbiosis have been mostly achieved by using two model legumes, Lotus japonicus and Medicago truncatula, that have relatively simple and small genomes and are capable of molecular transfection. However, recent development of resources for soybean molecular genetic research, such as genome sequencing, large EST databases, and high-density linkage maps, have enabled us to isolate several Rj genes. This progress has been achieved in connection with systematic utilization of the information obtained from molecular genetics of the model legumes. In this review, we summarize the current status of knowledge of host-controlled nodulation in soybean based on information from recent studies on Rj genes, and discuss the future research prospects.

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.

From defense to symbiosis: limited alterations in the kinase domain of LysM receptor-like kinases are crucial for evolution of legume-Rhizobium symbiosis.

Nitrogen-fixing symbiosis between legumes and rhizobia is initiated by the recognition of rhizobial Nod factors (NFs) by host plants. NFs are diversely modified derivatives of chitin oligosaccharide, a fungal elicitor that induces defense responses in plants. Recent evidence has shown that both NFs and chitin elicitors are recognized by structurally related LysM receptor kinases. Transcriptome analyses of Lotus japonicus roots indicated that NFs not only activate symbiosis genes but also transiently activate defense-related genes through NF receptors. Conversely, chitin oligosaccharides were able to activate symbiosis genes independently of NF receptors. Analyses using chimeric genes consisting of the LysM receptor domain of a Lotus japonicus NF receptor, NFR1, and the kinase domain of an Arabidopsis chitin receptor, CERK1, demonstrated that substitution of a portion of the αEF helix in CERK1 with the amino acid sequence YAQ from the corresponding region of NFR1 enables L. japonicus nfr1 mutants to establish symbiosis with Mesorhizobium loti. We also showed that the kinase domains of two Lotus japonicus LysM receptor kinases, Lys6 and Lys7, which also possess the YAQ sequence, suppress the symbiotic defect of nfr1. These results strongly suggest that, in addition to adaptation of extracellular LysM domains to NFs, limited alterations in the kinase domain of chitin receptors have played a crucial role in shifting the intracellular signaling to symbiosis from defense responses, thus constituting one of the key genetic events in the evolution of root nodule symbiosis in legume plants.

Function of GRAS proteins in root nodule symbiosis is retained in homologs of a non-legume, rice.

Root nodule (RN) symbiosis in legumes shares genes involved in the early signaling pathway with more ancient arbuscular mycorrhiza (AM) symbiosis, which is widespread in higher plants. The non-legume homologs of such genes have been well documented to be not only essential for the AM symbiosis in non-legume mycorrhizal plants but also functional in the RN symbiosis in legume plants. In contrast, it has not been investigated in detail whether RN symbiosis-specific genes, which are not essential for AM symbiosis, are functionally conserved in non-legumes. Two GRAS-domain transcription factors, NSP1 and NSP2, have been shown to be required for RN symbiosis, but not for AM symbiosis. In this study, we demonstrated that their homologs, OsNSP1 and OsNSP2, from rice are able to fully rescue the RN symbiosis-defective phenotypes of the mutants of corresponding genes in the model legume, Lotus japonicus. Our results indicate that some of the genes essential for RN symbiosis conserve their functions in homologs from non-legumes, which do not nodulate.

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.

A dominant function of CCaMK in intracellular accommodation of bacterial and fungal endosymbionts.

In legumes, Ca(2+)/calmodulin-dependent protein kinase (CCaMK) is a component of the common symbiosis genes that are required for both root nodule (RN) and arbuscular mycorrhiza (AM) symbioses and is thought to be a decoder of Ca(2+) spiking, one of the earliest cellular responses to microbial signals. A gain-of-function mutation of CCaMK has been shown to induce spontaneous nodulation without rhizobia, but the significance of CCaMK activation in bacterial and/or fungal infection processes is not fully understood. Here we show that a gain-of-function CCaMK(T265D) suppresses loss-of-function mutations of common symbiosis genes required for the generation of Ca(2+) spiking, not only for nodule organogenesis but also for successful infection of rhizobia and AM fungi, demonstrating that the common symbiosis genes upstream of Ca(2+) spiking are required solely to activate CCaMK. In RN symbiosis, however, CCaMK(T265D) induced nodule organogenesis, but not rhizobial infection, on Nod factor receptor (NFRs) mutants. We propose a model of symbiotic signaling in host legume plants, in which CCaMK plays a key role in the coordinated induction of infection thread formation and nodule organogenesis.

Derepression of the plant Chromovirus LORE1 induces germline transposition in regenerated plants.

Transposable elements represent a large proportion of the eukaryotic genomes. Long Terminal Repeat (LTR) retrotransposons are very abundant and constitute the predominant family of transposable elements in plants. Recent studies have identified chromoviruses to be a widely distributed lineage of Gypsy elements. These elements contain chromodomains in their integrases, which suggests a preference for insertion into heterochromatin. In turn, this preference might have contributed to the patterning of heterochromatin observed in host genomes. Despite their potential importance for our understanding of plant genome dynamics and evolution, the regulatory mechanisms governing the behavior of chromoviruses and their activities remain largely uncharacterized. Here, we report a detailed analysis of the spatio-temporal activity of a plant chromovirus in the endogenous host. We examined LORE1a, a member of the endogenous chromovirus LORE1 family from the model legume Lotus japonicus. We found that this chromovirus is stochastically de-repressed in plant populations regenerated from de-differentiated cells and that LORE1a transposes in the male germline. Bisulfite sequencing of the 5' LTR and its surrounding region suggests that tissue culture induces a loss of epigenetic silencing of LORE1a. Since LTR promoter activity is pollen specific, as shown by the analysis of transgenic plants containing an LTR::GUS fusion, we conclude that male germline-specific LORE1a transposition in pollen grains is controlled transcriptionally by its own cis-elements. New insertion sites of LORE1a copies were frequently found in genic regions and show no strong insertional preferences. These distinctive novel features of LORE1 indicate that this chromovirus has considerable potential for generating genetic and epigenetic diversity in the host plant population. Our results also define conditions for the use of LORE1a as a genetic tool.

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.

CERBERUS, a novel U-box protein containing WD-40 repeats, is required for formation of the infection thread and nodule development in the legume-Rhizobium symbiosis.

Endosymbiotic infection of legume plants by Rhizobium bacteria is initiated through infection threads (ITs) which are initiated within and penetrate from root hairs and deliver the endosymbionts into nodule cells. Despite recent progress in understanding the mutual recognition and early symbiotic signaling cascades in host legumes, the molecular mechanisms underlying bacterial infection processes and successive nodule organogenesis are still poorly understood. We isolated a novel symbiotic mutant of Lotus japonicus, cerberus, which shows defects in IT formation and nodule organogenesis. Map-based cloning of the causal gene allowed us to identify the CERBERUS gene, which encodes a novel protein containing a U-box domain and WD-40 repeats. CERBERUS expression was detected in the roots and nodules, and was enhanced after inoculation of Mesorhizobium loti. Strong expression was detected in developing nodule primordia and the infected zone of mature nodules. In cerberus mutants, Rhizobium colonized curled root hair tips, but hardly penetrated into root hair cells. The occasional ITs that were formed inside the root hair cells were mostly arrested within the epidermal cell layer. Nodule organogenesis was aborted prematurely, resulting in the formation of a large number of small bumps which contained no endosymbiotic bacteria. These phenotypic and genetic analyses, together with comparisons with other legume mutants with defects in IT formation, indicate that CERBERUS plays a critical role in the very early steps of IT formation as well as in growth and differentiation of nodules.

Too much love, a root regulator associated with the long-distance control of nodulation in Lotus japonicus.

Legume plants tightly control the development and number of symbiotic root nodules. In Lotus japonicus, this regulation requires HAR1 (a CLAVATA1-like receptor kinase) in the shoots, suggesting that a long-distance communication between the shoots and the roots may exist. To better understand its molecular basis, we isolated and characterized a novel hypernodulating mutant of L. japonicus named too much love (tml). Compared with the wild type, tml mutants produced much more nodules which densely covered a wider range of the roots. Reciprocal grafting showed that tml hypernodulation is determined by the root genotype. Moreover, grafting a har1 shoot onto a tml rootstock did not exhibit any obvious additive effects on the nodule number, which was further supported by double mutational analysis. These observations indicate that a shoot factor HAR1 and a root factor TML participate in the same genetic pathway which governs the long-distance signaling of nodule number control. We also showed that the inhibitory effect of TML on nodulation is likely to be local. Therefore, TML may function downstream of HAR1 and the gene product TML might serve as a receptor or mediator of unknown mobile signal molecules that are transported from the shoots to the roots.

Divergence of evolutionary ways among common sym genes: CASTOR and CCaMK show functional conservation between two symbiosis systems and constitute the root of a common signaling pathway.

In recent years a number of legume genes involved in root nodule (RN) symbiosis have been identified in the model legumes, Lotus japonicus (Lotus) and Medicago truncatula. Among them, a distinct set of genes has been categorized as a common symbiosis pathway (CSP), because they are also essential for another mutual interaction, the arbuscular mycorrhiza (AM) symbiosis, which is evolutionarily older than the RN symbiosis and is widely distributed in the plant kingdom. Based on the concept that the legume RN symbiosis has evolved from the ancient AM symbiosis, one issue is whether the CSP is functionally conserved between non-nodulating plants, such as rice, and nodulating legumes. We identified three rice CSP gene orthologs, OsCASTOR, OsPOLLUX and OsCCaMK, and demonstrated the indispensable roles of OsPOLLUX and OsCCaMK in rice AM symbiosis. Interestingly, molecular transfection of either OsCASTOR or OsCCaMK could fully complement symbiosis defects in the corresponding Lotus mutant lines for both the AM and RN symbioses. Our results not only provide a conserved genetic basis for the AM symbiosis between rice and Lotus, but also indicate that the core of the CSP has been well conserved during the evolution of RN symbiosis. Through evolution, CASTOR and CCaMK have remained as the molecular basis for the maintenance of CSP functions in the two symbiosis systems.

Transposition of a 600 thousand-year-old LTR retrotransposon in the model legume Lotus japonicus.

We have identified a new Ty3-gypsy retrotransposon family named LORE2 (Lotus retrotransposon 2) and documented its activity in the model legume Lotus japonicus. Three new LORE2 insertions were found in symbiotic mutant alleles isolated from a plant population, established by tissue culture mediated transformation of the L. japonicus Gifu accession. Low transcriptional and transpositional activities of LORE2 in cultured cells suggested that the LORE2 transpositions identified in the three symbiotic mutants occurred in intact plants, not in callus. Tracing of the transpositional events identified two active LORE2 members in Gifu. One of them named LORE2A possesses a deletion in its coding region and polymorphisms between intraelemental LTRs. LORE2A is thus an aged element, estimated as 600 thousand years old. Our findings indicate that plant genomes carry more cryptic LTR retrotransposons, i.e., aged yet active, than estimated before, and that these cryptic elements may have contributed to plant genome dynamics, for example, the burst of transpositions reported in several plant species.