Tsuneo Hakoyama

Publications of Tsuneo Hakoyama

• 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.

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

  Authors: Tsuneo Hakoyama, Kaori Niimi, Hirokazu Watanabe, Ryohei Tabata, Junichi Matsubara, Shusei Sato, Yasukazu Nakamura, Satoshi Tabata, Li Jichun, Tsuyoshi Matsumoto, Kazuyuki Tatsumi, Mika Nomura, Shigeyuki Tajima, Masumi Ishizaka, Koji Yano, Haruko Imaizumi-Anraku, Masayoshi Kawaguchi, Hiroshi Kouchi, Norio Suganuma

  Nature. 11/2009; 462(7272):514-7.

  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 diazotrophsHomocitrate 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.

• 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.

• 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.

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

  Authors: Norio Suganuma, Atsuko Yamamoto, Ai Itou, Tsuneo Hakoyama, Mari Banba, Shingo Hata, Masayoshi Kawaguchi, Hiroshi Kouchi

  Molecular plant-microbe interactions : MPMI. 12/2004; 17(11):1223-33.

  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 macroarrayThe 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.

• Transcriptional response of soybean suspension-cultured cells induced by Nod factors obtained from Bradyrhizobium japonicum USDA110.

  Authors: Tsuneo Hakoyama, Tadashi Yokoyama, Hiroshi Kouchi, Ken-ichi Tsuchiya, Hisatoshi Kaku, Yasuhiro Arima

  Plant & cell physiology. 12/2002; 43(11):1314-22.

  Genes responding to Nod factors were picked up by the application of a differential display method for soybean suspension-cultured cells. Forty-five cDNA fragments derived from such genes wereGenes responding to Nod factors were picked up by the application of a differential display method for soybean suspension-cultured cells. Forty-five cDNA fragments derived from such genes were detected. Seven fragments (ssc1-ssc7) were successfully cloned. The putative product of genes corresponding to ssc1 was estimated to be a disease-resistance protein relating to the induction of the plant defense response against pathogens, and that corresponding to ssc7 was a sucrose transporter. Amino acid sequences deduced from full-length cDNA corresponding to ssc2 and ssc4 were investigated, and it was shown that these polypeptides were equipped with a leucine zipper motif and with phosphorylation sites that were targeted by tyrosin kinase and cAMP-dependent protein kinase, respectively. In a differential display experiment, the transcriptional levels of three genes corresponding to ssc2, ssc3 and ssc5 were estimated to be up-regulated at 6 h after initiation of the treatment and the remaining four were estimated to be down-regulated. However, transcription of the genes corresponding to all ssc was clearly repressed within 2 h after initiation of the treatment. Five of them were restored to their transcriptional level 6 h after initiation of the treatment, although the others were repressed throughout the experimental period.