Yosuke Umehara

Publications of Yosuke Umehara

• Establishment of a Lotus japonicus gene tagging population using the exon-targeting endogenous retrotransposon LORE1.

  Authors: Eigo Fukai, Takashi Soyano, Yosuke Umehara, Shinobu Nakayama, Hideki Hirakawa, Satoshi Tabata, Shusei Sato, Makoto Hayashi

  The Plant journal : for cell and molecular biology. 02/2012; 69(4):720-30.

  We established a gene tagging population of the model legume Lotus japonicus using an endogenous long terminal repeat (LTR) retrotransposon Lotus Retrotransposon 1 (LORE1). The population wasWe established a gene tagging population of the model legume Lotus japonicus using an endogenous long terminal repeat (LTR) retrotransposon Lotus Retrotransposon 1 (LORE1). The population was composed of 2450 plant lines, from which a total of 4532 flanking sequence tags of LORE1 were recovered by pyrosequencing. The two-dimensional arrangement of the plant population, together with the use of multiple identifier sequences in the primers used to amplify the flanking regions, made it possible to trace insertions back to the original plant lines. The large-scale detection of new LORE1 insertion sites revealed a preference for genic regions, especially in exons of protein-coding genes, which is an interesting feature to consider in the interaction between host genomes and chromoviruses, to which LORE1 belongs, a class of retrotransposon widely distributed among plants. Forward screening of the symbiotic mutants from the population succeeded to identify five symbiotic mutants of known genes. These data suggest that LORE1 is robust as a genetic tool.

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

  Authors: Tsuneo Hakoyama, Kaori Niimi, Takeshi Yamamoto, Sawa Isobe, Shusei Sato, Yasukazu Nakamura, Satoshi Tabata, Hirotaka Kumagai, Yosuke Umehara, Katja Brossuleit, Thomas R Petersen, Niels Sandal, Jens Stougaard, Michael K Udvardi, Masanori Tamaoki, Masayoshi Kawaguchi, Hiroshi Kouchi, Norio Suganuma

  Plant & cell physiology. 11/2011; 53(1):225-36.

  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 thatLegume 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.

  Authors: Hiroshi Kouchi, Haruko Imaizumi-Anraku, Makoto Hayashi, Tsuneo Hakoyama, Tomomi Nakagawa, Yosuke Umehara, Norio Suganuma, Masayoshi Kawaguchi

  Plant & cell physiology. 09/2010; 51(9):1381-97.

  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 inThe 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.

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

  Authors: Eigo Fukai, Yosuke Umehara, Shusei Sato, Makoto Endo, Hiroshi Kouchi, Makoto Hayashi, Jens Stougaard, Hirohiko Hirochika

  PLoS genetics. 01/2010; 6(3):e1000868.

  Transposable elements represent a large proportion of the eukaryotic genomes. Long Terminal Repeat (LTR) retrotransposons are very abundant and constitute the predominant family of transposableTransposable 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.

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

  Authors: Koji Yano, Satoshi Shibata, Wen-Li Chen, Shusei Sato, Takakazu Kaneko, Anna Jurkiewicz, Niels Sandal, Mari Banba, Haruko Imaizumi-Anraku, Tomoko Kojima, Ryo Ohtomo, Krzysztof Szczyglowski, Jens Stougaard, Satoshi Tabata, Makoto Hayashi, Hiroshi Kouchi, Yosuke Umehara

  The Plant journal : for cell and molecular biology. 07/2009;

  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 intoEndosymbiotic 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 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 formation of a large number of small bumps which contained no endosymbiotic bacteria. These phenotypic and genetic analyses, together with comparisons to 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 for growth and differentiation of nodules.

• TOO MUCH LOVE, a Root Regulator Associated with the Long-Distance Control of Nodulation in Lotus japonicus.

  Authors: Shimpei Magori, Erika Oka-Kira, Satoshi Shibata, Yosuke Umehara, Hiroshi Kouchi, Yoshihiro Hase, Atsushi Tanaka, Shusei Sato, Satoshi Tabata, Masayoshi Kawaguchi

  Molecular plant-microbe interactions : MPMI. 04/2009; 22(3):259-68.

  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 thatLegume 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.

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

  Authors: Eigo Fukai, Alicja Dobrowolska, Lene Madsen, Esben Madsen, Yosuke Umehara, Hiroshi Kouchi, Hirohiko Hirochika, Jens Stougaard

  Plant molecular biology. 10/2008;

  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 foundWe 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.

• A novel ankyrin-repeat membrane protein, IGN1, is required for persistence of nitrogen-fixing symbiosis in root nodules of Lotus japonicus.

  Authors: Hirotaka Kumagai, Tsuneo Hakoyama, Yosuke Umehara, Shusei Sato, Takakazu Kaneko, Satoshi Tabata, Hiroshi Kouchi

  Plant physiology. 04/2007; 143(3):1293-305.

  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 atNitrogen-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(-) 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.

• NUCLEOPORIN85 is required for calcium spiking, fungal and bacterial symbioses, and seed production in Lotus japonicus.

  Authors: Katsuharu Saito, Makoto Yoshikawa, Koji Yano, Hiroki Miwa, Hisaki Uchida, Erika Asamizu, Shusei Sato, Satoshi Tabata, Haruko Imaizumi-Anraku, Yosuke Umehara, Hiroshi Kouchi, Yoshikatsu Murooka, Krzysztof Szczyglowski, J Allan Downie, Martin Parniske, Makoto Hayashi, Masayoshi Kawaguchi

  The Plant cell. 03/2007; 19(2):610-24.

  In Lotus japonicus, seven genetic loci have been identified thus far as components of a common symbiosis (Sym) pathway shared by rhizobia and arbuscular mycorrhizal fungi. We characterized the nup85In Lotus japonicus, seven genetic loci have been identified thus far as components of a common symbiosis (Sym) pathway shared by rhizobia and arbuscular mycorrhizal fungi. We characterized the nup85 mutants (nup85-1, -2, and -3) required for both symbioses and cloned the corresponding gene. When inoculated with Glomus intraradices, the hyphae managed to enter between epidermal cells, but they were unable to penetrate the cortical cell layer. The nup85-2 mutation conferred a weak and temperature-sensitive symbiotic phenotype, which resulted in low arbuscule formation at 22 degrees C but allowed significantly higher arbuscule formation in plant cortical cells at 18 degrees C. On the other hand, the nup85 mutants either did not form nodules or formed few nodules. When treated with Nod factor of Mesorhizobium loti, nup85 roots showed a high degree of root hair branching but failed to induce calcium spiking. In seedlings grown under uninoculated conditions supplied with nitrate, nup85 did not arrest plant growth but significantly reduced seed production. NUP85 encodes a putative nucleoporin with extensive similarity to vertebrate NUP85. Together with symbiotic nucleoporin NUP133, L. japonicus NUP85 might be part of a specific nuclear pore subcomplex that is crucial for fungal and rhizobial colonization and seed production.

• A novel fix- symbiotic mutant of Lotus japonicus, Ljsym105, shows impaired development and premature deterioration of nodule infected cells and symbiosomes.

  Authors: Md Shakhawat Hossain, Yosuke Umehara, Hiroshi Kouchi

  Molecular plant-microbe interactions : MPMI. 08/2006; 19(7):780-8.

  Nitrogen-fixing symbiosis between legume plants and rhizobia is established through complex interactions between two symbiotic partners. To identify the host legume genes that play crucial roles inNitrogen-fixing symbiosis between legume plants and rhizobia is established through complex interactions between two symbiotic partners. To identify the host legume genes that play crucial roles in such interactions, we isolated a novel Fix- mutant, Ljsym105, from a model legume Lotus japonicus MG-20. The Ljsym105 plants displayed nitrogen-deficiency symptoms after inoculation with Mesorhizobium loti under nitrogen-free conditions, but their growth recovered when supplied with nitrogen-rich nutrients. Ljsym105 was recessive and monogenic and mapped on the upper portion of chromosome 4. The mutant Ljsym105 formed an increased number of small and pale-pink nodules. Nitrogenase (acetylene reduction) activity per nodule fresh weight was low but retained more than 50% of that of the wild-type nodules. Light and electron microscopic observations revealed that the Ljsym105 nodule infected cells were significantly smaller than those of wild-type plants, contained enlarged symbiosomes with multiple bacteroids, and underwent deterioration of the symbiosomes prematurely as well as disintegration of the whole infected cell cytoplasm. These results indicate that the ineffectiveness of the Ljsym105 nodules is primarily due to impaired growth of infected cells accompanied with the premature senescence induced at relatively early stages of nodule development. These symbiotic phenotypes are discussed in respect to possible functions of the LjSym105 locus in the symbiotic interactions required for establishment of the nitrogen-fixing symbiosis.

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

  Authors: Niels Sandal, Thomas Rørby Petersen, Jeremy Murray, Yosuke Umehara, Bogumil Karas, Koji Yano, Hirotaka Kumagai, Makoto Yoshikawa, Katsuharu Saito, Masaki Hayashi [......] Shingo Hata, Norio Suganuma, Hiroshi Kouchi, Shinji Kawasaki, Satoshi Tabata, Makoto Hayashi, Martin Parniske, Krzysztof Szczyglowski, Masayoshi Kawaguchi, Jens Stougaard

  Molecular plant-microbe interactions : MPMI. 02/2006; 19(1):80-91.

  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-microbeDevelopment 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.

• Analysis of promoter activity of the early nodulin Enod40 in Lotus japonicus.

  Authors: Mette Grønlund, Andreas Roussis, Emmanouil Flemetakis, Nicolette E M Quaedvlieg, Helmi R M Schlaman, Yosuke Umehara, Panagiotis Katinakis, Jens Stougaard, Herman P Spaink

  Molecular plant-microbe interactions : MPMI. 06/2005; 18(5):414-27.

  Our comparative studies on the promoter (pr) activity of Enod40 in the model legume Lotus japonicus in stably transformed GusA reporter lines and in hairy roots of L. japonicus demonstrate aOur comparative studies on the promoter (pr) activity of Enod40 in the model legume Lotus japonicus in stably transformed GusA reporter lines and in hairy roots of L. japonicus demonstrate a stringent regulation of the Enod40 promoter in the root cortex and root hairs in response to Nod factors. Interestingly, the L. japonicus Enod40-2 promoter fragment also shows symbiotic activity in the reverse orientation. Deletion analyses of the Glycine max (Gm) Enod40 promoter revealed the presence of a minimal region -185 bp upstream of the transcription start. Stable transgenic L. japonicus reporter lines were used in bioassays to test the effect of different compounds on early symbiotic signaling. The responses of prGmEnod40 reporter lines were compared with the responses of L. japonicus (Lj) reporter lines based on the LjNin promoter. Both reporter lines show very early activity postinoculation in root hairs of the responsive zone of the root and later in the dividing cells of nodule primordia. The LjNin promoter was found to be more responsive than the GmEnod40 promoter to Nod factors and related compounds. The use of prGmEnod40 reporter lines to analyze the effect of nodulin genes on the GmEnod40 promoter activity indicates that LJNIN has a positive effect on the regulation of the Enod40 promoter, whereas the latter is not influenced by ectopic overexpression of its own gene product. In addition to pointing to a difference in the regulation of the two nodulin genes Enod40 and Nin during early time points of symbiosis, the bioassays revealed a difference in the response to the synthetic cytokinin 6-benzylaminopurine (BAP) between alfalfa and clover and L. japonicus. In alfalfa and clover, Enod40 expression was induced upon BAP treatment, whereas this seems not to be the case in L. japonicus; these results correlate with effects at the cellular level because BAP can induce pseudonodules in alfalfa and clover but not in L. japonicus. In conclusion, we demonstrate the applicability of the described L. japonicus reporter lines in analyses of the specificity of compounds related to nodulation as well as for the dissection of the interplay between different nodulin genes.

• Plastid proteins crucial for symbiotic fungal and bacterial entry into plant roots.

  Authors: Haruko Imaizumi-Anraku, Naoya Takeda, Myriam Charpentier, Jillian Perry, Hiroki Miwa, Yosuke Umehara, Hiroshi Kouchi, Yasuhiro Murakami, Lonneke Mulder, Kate Vickers [......] Shusei Sato, Erika Asamizu, Satoshi Tabata, Makoto Yoshikawa, Yoshikatsu Murooka, Guo-Jiang Wu, Masayoshi Kawaguchi, Shinji Kawasaki, Martin Parniske, Makoto Hayashi

  Nature. 03/2005; 433(7025):527-31.

  The roots of most higher plants form arbuscular mycorrhiza, an ancient, phosphate-acquiring symbiosis with fungi, whereas only four related plant orders are able to engage in the evolutionary youngerThe roots of most higher plants form arbuscular mycorrhiza, an ancient, phosphate-acquiring symbiosis with fungi, whereas only four related plant orders are able to engage in the evolutionary younger nitrogen-fixing root-nodule symbiosis with bacteria. Plant symbioses with bacteria and fungi require a set of common signal transduction components that redirect root cell development. Here we present two highly homologous genes from Lotus japonicus, CASTOR and POLLUX, that are indispensable for microbial admission into plant cells and act upstream of intracellular calcium spiking, one of the earliest plant responses to symbiotic stimulation. Surprisingly, both twin proteins are localized in the plastids of root cells, indicating a previously unrecognized role of this ancient endosymbiont in controlling intracellular symbioses that evolved more recently.

• Cloning and expression analysis of a MAPKKK gene and a novel nodulin gene of Lotus japonicus.

  Authors: Natsuko Kinoshita, Yasuhiro Ooki, Yuichi Deguchi, Svetlana A Chechetka, Hiroshi Kouchi, Yosuke Umehara, Katsura Izui, Shingo Hata

  Bioscience, biotechnology, and biochemistry. 09/2004; 68(8):1805-7.

  We isolated a cDNA encoding mitogen-activated protein kinase kinase kinase alpha, designated LjM3Kalpha, from Lotus japonicus, a model legume. The gene was expressed constitutively in roots, rootWe isolated a cDNA encoding mitogen-activated protein kinase kinase kinase alpha, designated LjM3Kalpha, from Lotus japonicus, a model legume. The gene was expressed constitutively in roots, root nodules, and shoots. We also identified a novel nodulin gene, LjNUF, that shows specific expression in nodules. LjNUF resembles the C-terminal half of a hypothetical protein (pir//D85436), the N-terminal half of which is similar to a portion of mitogen-activated protein kinase kinase kinase gamma. Although LjNUF was predicted to be a secreted protein, its function remains to be clarified.

• Plant recognition of symbiotic bacteria requires two LysM receptor-like kinases.

  Authors: Simona Radutoiu, Lene Heegaard Madsen, Esben Bjørn Madsen, Hubert H Felle, Yosuke Umehara, Mette Grønlund, Shusei Sato, Yasukazu Nakamura, Satoshi Tabata, Niels Sandal, Jens Stougaard

  Nature. 11/2003; 425(6958):585-92.

  Although most higher plants establish a symbiosis with arbuscular mycorrhizal fungi, symbiotic nitrogen fixation with rhizobia is a salient feature of legumes. Despite this host range difference,Although most higher plants establish a symbiosis with arbuscular mycorrhizal fungi, symbiotic nitrogen fixation with rhizobia is a salient feature of legumes. Despite this host range difference, mycorrhizal and rhizobial invasion shares a common plant-specified genetic programme controlling the early host interaction. One feature distinguishing legumes is their ability to perceive rhizobial-specific signal molecules. We describe here two LysM-type serine/threonine receptor kinase genes, NFR1 and NFR5, enabling the model legume Lotus japonicus to recognize its bacterial microsymbiont Mesorhizobium loti. The extracellular domains of the two transmembrane kinases resemble LysM domains of peptidoglycan- and chitin-binding proteins, suggesting that they may be involved directly in perception of the rhizobial lipochitin-oligosaccharide signal. We show that NFR1 and NFR5 are required for the earliest physiological and cellular responses to this lipochitin-oligosaccharide signal, and demonstrate their role in the mechanism establishing susceptibility of the legume root for bacterial infection.

• Characterization and expression analysis of genes encoding phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxylase kinase of Lotus japonicus, a model legume.

  Authors: Tomomi Nakagawa, Tomoko Izumi, Mari Banba, Yosuke Umehara, Hiroshi Kouchi, Katsura Izui, Shingo Hata

  Molecular plant-microbe interactions : MPMI. 05/2003; 16(4):281-8.

  Phosphoenolpyruvate carboxylases (PEPCs), one form of which in each legume species plays a central role in the carbon metabolism in symbiotic root nodules, are activated through phosphorylation of aPhosphoenolpyruvate carboxylases (PEPCs), one form of which in each legume species plays a central role in the carbon metabolism in symbiotic root nodules, are activated through phosphorylation of a conserved residue by a specific protein kinase (PEPC-PK). We characterized the cDNAs for two PEPC isoforms of Lotus japonicus, an amide-translocating legume that forms determinate nodules. One gene encodes a nodule-enhanced form, which is more closely related to the PEPCs in amide-type indeterminate nodules than those in ureide-type determinate nodules. The other gene is expressed in shoots and roots at a low level. Both forms have the putative phosphorylation site, Ser11. We also isolated a cDNA and the corresponding genomic DNA for PEPC-PK of L. japonicus. The recombinant PEPC-PK protein expressed in Escherichia coli phosphorylated recombinant maize C4-form PEPC efficiently in vitro. The level of mRNA for PEPC-PK was high in root nodules, and those in shoots and roots were also significant. In situ hybridization revealed that the expression patterns of the transcripts for PEPC and PEPC-PK were similar in mature root nodules, but were different in emerging nodules. When L. japonicus seedlings were subjected to prolonged darkness and subsequent illumination, the activity of PEPC-PK and the mRNA levels of both PEPC and PEPC-PK in nodules decreased and then recovered, suggesting that they are regulated according to the amounts of photosynthates transported from shoots.

• cDNA cloning, mRNA expression, and mutational analysis of the squalene synthase gene of Lotus japonicus.

  Authors: Satomi Akamine, Kazuki Nakamori, Svetlana A Chechetka, Mari Banba, Yosuke Umehara, Hiroshi Kouchi, Katsura Izui, Shingo Hata

  Biochimica et biophysica acta. 05/2003; 1626(1-3):97-101.

  A full-length cDNA for squalene synthase was isolated from Lotus japonicus, a model leguminous plant. The transcript was abundant in roots, symbiotic root nodules, and shoots, in that order. In situA full-length cDNA for squalene synthase was isolated from Lotus japonicus, a model leguminous plant. The transcript was abundant in roots, symbiotic root nodules, and shoots, in that order. In situ hybridization revealed that the mRNA level is high in expanding root cells but low in dividing root tip ones. The transcript is also abundant in vascular bundles and the basal portions of mature nodules. L. japonicus squalene synthase has an unusual Asp residue near the active site, where mammalian enzymes have Gln, and replacement of the Gln by Glu has been reported to cause severe inactivation. Site-directed mutagenesis of the L. japonicus enzyme and assaying in vitro showed that this Asp residue can be substituted by not only Gln but also Glu, suggesting that the local structure of plant squalene synthases is different from that of mammalian enzymes.

• The Sym35 gene required for root nodule development in pea is an ortholog of Nin from Lotus japonicus.

  Authors: Alexey Y Borisov, Lene H Madsen, Viktor E Tsyganov, Yosuke Umehara, Vera A Voroshilova, Arsen O. Batagov, Niels Sandal, Anita Mortensen, Leif Schauser, Noel Ellis, Igor A Tikhonovich, Jens Stougaard

  Plant physiology. 04/2003; 131(3):1009-17.

  Comparative phenotypic analysis of pea (Pisum sativum) sym35 mutants and Lotus japonicus nin mutants suggested a similar function for the PsSym35 and LjNin genes in early stages of root noduleComparative phenotypic analysis of pea (Pisum sativum) sym35 mutants and Lotus japonicus nin mutants suggested a similar function for the PsSym35 and LjNin genes in early stages of root nodule formation. Both the pea and L. japonicus mutants are non-nodulating but normal in their arbuscular mycorrhizal association. Both are characterized by excessive root hair curling in response to the bacterial microsymbiont, lack of infection thread initiation, and absence of cortical cell divisions. To investigate the molecular basis for the similarity, we cloned and sequenced the PsNin gene, taking advantage of sequence information from the previously cloned LjNin gene. An RFLP analysis on recombinant inbred lines mapped PsNin to the same chromosome arm as the PsSym35 locus and direct evidence demonstrating that PsNin is the PsSym35 gene was subsequently obtained by cosegregation analysis and sequencing of three independent Pssym35 mutant alleles. L. japonicus and pea root nodules develop through different organogenic pathways, so it was of interest to compare the expression of the two orthologous genes during nodule formation. Overall, a similar developmental regulation of the PsNin and LjNin genes was shown by the transcriptional activation in root nodules of L. japonicus and pea. In the indeterminate pea nodules, PsNin is highly expressed in the meristematic cells of zone I and in the cells of infection zone II, corroborating expression of LjNin in determinate nodule primordia. At the protein level, seven domains, including the putative DNA binding/dimerization RWP-RK motif and the PB1 heterodimerization domain, are conserved between the LjNIN and PsNIN proteins.

• Cloning, functional expression, and mutational analysis of a cDNA for Lotus japonicus mitochondrial phosphate transporter.

  Authors: Kazuki Nakamori, Reona Takabatake, Yosuke Umehara, Hiroshi Kouchi, Katsura Izui, Shingo Hata

  Plant & cell physiology. 11/2002; 43(10):1250-3.

  A full-length cDNA encoding mitochondrial phosphate transporter was isolated from symbiotic nodules of Lotus japonicus, a model legume. The gene appeared to be single-copied and its transcript wasA full-length cDNA encoding mitochondrial phosphate transporter was isolated from symbiotic nodules of Lotus japonicus, a model legume. The gene appeared to be single-copied and its transcript was abundant in roots, symbiotic root nodules and shoots, in that order. The L. japonicus transporter has a conserved N-ethylmaleimide-reactive Cys residue. Site-directed mutagenesis and phosphate transport assaying in vitro showed that this Cys residue is preferable for the transport activity, although it can be replaced to some extent by Ser, but not by Ala.

• cDNA cloning, mRNA expression, and mutational analysis of the squalene synthase gene of Lotus japonicus

  Authors: Satomi Akamine, Kazuki Nakamori, Svetlana A. Chechetka, Mari Banba, Yosuke Umehara, Hiroshi Kouchi, Katsura Izui, Shingo Hata

  Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression.

  A full-length cDNA for squalene synthase was isolated from Lotus japonicus, a model leguminous plant. The transcript was abundant in roots, symbiotic root nodules, and shoots, in that order. In situA full-length cDNA for squalene synthase was isolated from Lotus japonicus, a model leguminous plant. The transcript was abundant in roots, symbiotic root nodules, and shoots, in that order. In situ hybridization revealed that the mRNA level is high in expanding root cells but low in dividing root tip ones. The transcript is also abundant in vascular bundles and the basal portions of mature nodules. L. japonicus squalene synthase has an unusual Asp residue near the active site, where mammalian enzymes have Gln, and replacement of the Gln by Glu has been reported to cause severe inactivation. Site-directed mutagenesis of the L. japonicus enzyme and assaying in vitro showed that this Asp residue can be substituted by not only Gln but also Glu, suggesting that the local structure of plant squalene synthases is different from that of mammalian enzymes.