Haruko Imaizumi-Anraku

Publications of Haruko Imaizumi-Anraku

• Rhizobial and Fungal Symbioses Show Different Requirements for Calmodulin Binding to Calcium Calmodulin-Dependent Protein Kinase in Lotus japonicus.

  Authors: Yoshikazu Shimoda, Lu Han, Toshimasa Yamazaki, Rintaro Suzuki, Makoto Hayashi, Haruko Imaizumi-Anraku

  The Plant cell. 01/2012; 24(1):304-21.

  Ca(2+)/calmodulin (CaM)-dependent protein kinase (CCaMK) is a key regulator of root nodule and arbuscular mycorrhizal symbioses and is believed to be a decoder for Ca(2+) signals induced by microbialCa(2+)/calmodulin (CaM)-dependent protein kinase (CCaMK) is a key regulator of root nodule and arbuscular mycorrhizal symbioses and is believed to be a decoder for Ca(2+) signals induced by microbial symbionts. However, it is unclear how CCaMK is activated by these microbes. Here, we investigated in vivo activation of CCaMK in symbiotic signaling, focusing mainly on the significance of and epistatic relationships among functional domains of CCaMK. Loss-of-function mutations in EF-hand motifs revealed the critical importance of the third EF hand for CCaMK activation to promote infection of endosymbionts. However, a gain-of-function mutation (T265D) in the kinase domain compensated for these loss-of-function mutations in the EF hands. Mutation of the CaM binding domain abolished CaM binding and suppressed CCaMK(T265D) activity in rhizobial infection, but not in mycorrhization, indicating that the requirement for CaM binding to CCaMK differs between root nodule and arbuscular mycorrhizal symbioses. Homology modeling and mutagenesis studies showed that the hydrogen bond network including Thr265 has an important role in the regulation of CCaMK. Based on these genetic, biochemical, and structural studies, we propose an activation mechanism of CCaMK in which root nodule and arbuscular mycorrhizal symbioses are distinguished by differential regulation of CCaMK by CaM binding.

• The genotype of the calcium/calmodulin-dependent protein kinase gene (CCaMK) determines bacterial community diversity in rice roots under paddy and upland field conditions.

  Authors: Seishi Ikeda, Takashi Okubo, Naoya Takeda, Mari Banba, Kazuhiro Sasaki, Haruko Imaizumi-Anraku, Shinsuke Fujihara, Yoshinari Ohwaki, Kenshiro Ohshima, Yoshimichi Fukuta, Shima Eda, Hisayuki Mitsui, Masahira Hattori, Tadashi Sato, Takuro Shinano, Kiwamu Minamisawa

  Applied and environmental microbiology. 07/2011; 77(13):4399-405.

  The effects of the Oryza sativa calcium/calmodulin-dependent protein kinase OsCCaMK genotype (dominant homozygous [D], heterozygous [H], recessive homozygous [R]) on rice root-associated bacteria,The effects of the Oryza sativa calcium/calmodulin-dependent protein kinase OsCCaMK genotype (dominant homozygous [D], heterozygous [H], recessive homozygous [R]) on rice root-associated bacteria, including endophytes and epiphytes, were examined by using a Tos17 rice mutant line under paddy and upland field conditions. Roots were sampled at the flowering stage and were subjected to clone library analyses. The relative abundance of Alphaproteobacteria was noticeably decreased in R plants under both paddy and upland conditions (0.8% and 3.0%, respectively) relative to those in D plants (10.3% and 17.4%, respectively). Population shifts of the Sphingomonadales and Rhizobiales were mainly responsible for this low abundance in R plants. The abundance of Anaerolineae (Chloroflexi) and Clostridia (Firmicutes) was increased in R plants under paddy conditions. The abundance of a subpopulation of Actinobacteria (Saccharothrix spp. and unclassified Actinosynnemataceae) was increased in R plants under upland conditions. Principal coordinate analysis revealed unidirectional community shifts in relation to OsCCaMK gene dosage under both conditions. In addition, shoot length, tiller number, and plant weight decreased as the OsCCaMK gene dosage decreased under upland conditions. These results suggest significant impacts of OsCCaMK on both the diversity of root-associated bacteria and rice plant growth under both paddy and upland field conditions.

• 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.

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

  Authors: Teruyuki Hayashi, Mari Banba, Yoshikazu Shimoda, Hiroshi Kouchi, Makoto Hayashi, Haruko Imaizumi-Anraku

  The Plant journal : for cell and molecular biology. 07/2010; 63(1):141-54.

  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 isIn 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.

• 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.

• 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.

• CYCLOPS, a mediator of symbiotic intracellular accommodation.

  Authors: Koji Yano, Satoko Yoshida, Judith Müller, Sylvia Singh, Mari Banba, Kate Vickers, Katharina Markmann, Catharine White, Bettina Schuller, Shusei Sato, Erika Asamizu, Satoshi Tabata, Yoshikatsu Murooka, Jillian Perry, Trevor L Wang, Masayoshi Kawaguchi, Haruko Imaizumi-Anraku, Makoto Hayashi, Martin Parniske

  Proceedings of the National Academy of Sciences of the United States of America. 01/2009;

  The initiation of intracellular infection of legume roots by symbiotic rhizobia bacteria and arbuscular mycorrhiza (AM) fungi is preceded by the induction of calcium signatures in and around theThe initiation of intracellular infection of legume roots by symbiotic rhizobia bacteria and arbuscular mycorrhiza (AM) fungi is preceded by the induction of calcium signatures in and around the nucleus of root epidermal cells. Although a calcium and calmodulin-dependent kinase (CCaMK) is a key mediator of symbiotic root responses, the decoding of the calcium signal and the molecular events downstream are only poorly understood. Here, we characterize Lotus japonicus cyclops mutants on which microbial infection was severely inhibited. In contrast, nodule organogenesis was initiated in response to rhizobia, but arrested prematurely. This arrest was overcome when a deregulated CCaMK mutant version was introduced into cyclops mutants, conferring the development of full-sized, spontaneous nodules. Because cyclops mutants block symbiotic infection but are competent for nodule development, they reveal a bifurcation of signal transduction downstream of CCaMK. We identified CYCLOPS by positional cloning. CYCLOPS carries a functional nuclear localization signal and a predicted coiled-coil domain. We observed colocalization and physical interaction between CCaMK and CYCLOPS in plant and yeast cell nuclei in the absence of symbiotic stimulation. Importantly, CYCLOPS is a phosphorylation substrate of CCaMK in vitro. Cyclops mutants of rice were impaired in AM, and rice CYCLOPS could restore symbiosis in Lotus cyclops mutants, indicating a functional conservation across angiosperms. Our results suggest that CYCLOPS forms an ancient, preassembled signal transduction complex with CCaMK that is specifically required for infection, whereas organogenesis likely requires additional yet-to-be identified CCaMK interactors or substrates.

• Gibberellin controls the nodulation-signaling pathway in Lotus japonicus.

  Authors: Takaki Maekawa, Makoto Maekawa-Yoshikawa, Naoya Takeda, Haruko Imaizumi-Anraku, Yoshikatsu Murooka, Makoto Hayashi

  The Plant journal : for cell and molecular biology. 01/2009;

  Summary Root nodule formation is regulated by several plant hormones, but the details of the regulation of the nodulation-signaling pathway are largely unknown. In this study, the role of gibberellinSummary Root nodule formation is regulated by several plant hormones, but the details of the regulation of the nodulation-signaling pathway are largely unknown. In this study, the role of gibberellin (GA) in control of root nodule symbiosis was investigated at the physiological and genetic levels in Lotus japonicus. Exogenous application of biologically active GA, GA(3), inhibited the infection thread formation and nodule formation, which was counteracted by application of a biosynthesis inhibitor of GA, Uniconazol P. Nod factor-induced root hair deformation was severely blocked in the presence of GA, which was phenocopied by nsp2 mutants. The number of spontaneous nodules triggered by gain-of-function mutation of calcium/calmodulin-dependent kinase (CCaMK) or the lotus histidine kinase 1 (LHK1) was decreased upon GA addition. Moreover, overexpression of the gain-of-function mutation of L. japonicus SLEEPY1, a positive regulator of GA signaling, resulted in a reduced nodule number without other aspects of root development being affected. These results indicate that higher GA signaling levels specifically inhibit the nodulation-signaling pathway. Nod factor-dependent induction of NSP2 and NIN was inhibited by exogenous GA. Furthermore, cytokinin-dependent induction of NIN was suppressed by GA. From these results, we conclude that GA inhibits the nodulation-signaling pathway downstream of cytokinin, possibly at NSP2, which is required for Nod factor-dependent NIN expression. These results clarify the roles of GA in the nodulation-signaling pathway and in relation to the cytokinin signaling pathway for nodulation in L. japonicus.

• Arbuscular Mycorrhiza-Specific Signaling in Rice Transcends the Common Symbiosis Signaling Pathway.

  Authors: Caroline Gutjahr, Mari Banba, Vincent Croset, Kyungsook An, Akio Miyao, Gynheung An, Hirohiko Hirochika, Haruko Imaizumi-Anraku, Uta Paszkowski

  The Plant cell. 12/2008;

  Knowledge about signaling in arbuscular mycorrhizal (AM) symbioses is currently restricted to the common symbiosis (SYM) signaling pathway discovered in legumes. This pathway includes calcium as aKnowledge about signaling in arbuscular mycorrhizal (AM) symbioses is currently restricted to the common symbiosis (SYM) signaling pathway discovered in legumes. This pathway includes calcium as a second messenger and regulates both AM and rhizobial symbioses. Both monocotyledons and dicotyledons form symbiotic associations with AM fungi, and although they differ markedly in the organization of their root systems, the morphology of colonization is similar. To identify and dissect AM-specific signaling in rice (Oryza sativa), we developed molecular phenotyping tools based on gene expression patterns that monitor various steps of AM colonization. These tools were used to distinguish common SYM-dependent and -independent signaling by examining rice mutants of selected putative legume signaling orthologs predicted to be perturbed both upstream (CASTOR and POLLUX) and downstream (CCAMK and CYCLOPS) of the central, calcium-spiking signal. All four mutants displayed impaired AM interactions and altered AM-specific gene expression patterns, therefore demonstrating functional conservation of SYM signaling between distant plant species. In addition, differential gene expression patterns in the mutants provided evidence for AM-specific but SYM-independent signaling in rice and furthermore for unexpected deviations from the SYM pathway downstream of calcium spiking.

• 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.

  Authors: Mari Banba, Caroline Gutjahr, Akio Miyao, Hirohiko Hirochika, Uta Paszkowski, Hiroshi Kouchi, Haruko Imaizumi-Anraku

  Plant & cell physiology. 11/2008;

  In recent years a number of legume genes involved in the root nodule (RN) symbiosis have been identified from the model legumes, Lotus japonicus (Lotus) and Medicago truncatula. Among them, aIn recent years a number of legume genes involved in the root nodule (RN) symbiosis have been identified from 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 provide not only 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 the RN symbiosis. Through evolution, CASTOR and CCaMK have remained as the molecular basis for the maintenance of CSP functions in the two symbiosis systems.

• Requirement for Mesorhizobium loti ornithine transcarbamoylase for successful symbiosis with Lotus japonicus as revealed by an unexpected long-range genome deletion.

  Authors: Elina Mishima, Atsuko Hosokawa, Haruko Imaizumi-Anraku, Katsuharu Saito, Masayoshi Kawaguchi, Kazuhiko Saeki

  Plant & cell physiology. 04/2008; 49(3):301-13.

  With the original aim of surveying the role of exopolysaccharide (EPS) in Lotus-Mesorhizobium symbiosis, we carried out Tn5 mutagenesis of Mesorhizobium loti and obtained 32 mutants with defects inWith the original aim of surveying the role of exopolysaccharide (EPS) in Lotus-Mesorhizobium symbiosis, we carried out Tn5 mutagenesis of Mesorhizobium loti and obtained 32 mutants with defects in EPS biosynthesis. One of the mutants, HIA22, formed pseudonodules and failed to fix nitrogen with Lotus japonicus. However, complementation analysis unexpectedly revealed that the potential gene with the locus tag, mll2073, interrupted by Tn5 was responsible for neither normal EPS synthesis nor symbiosis. Further analysis uncovered that HIA22 had a genome deletion of approximately 20 kbp, resulting in the loss of two separate genes responsible for EPS biosynthesis and symbiosis. One gene with the locus tag, mll5669, was needed to synthesize normal EPS that fluoresced on medium containing Calcofluor and encoded a homolog of O-antigen acetyl transferase in Salmonella typhimurium. A specific mutant of mll5669, EMB-B58, successfully fixed nitrogen when infected onto L. japonicus. Another gene, mlr5647, was needed to establish fully functional nodules and encoded ornithine carbamoyl transferase [ArgF (EC 2.1.3.3)], which participates in arginine biosynthesis. A specific mutant of mlr5647, EMB-Y2, showed arginine auxotrophy and formed infection threads, but the nodules formed by this strain had few infected cells filled with bacteroids. These mutant phenotypes were complemented by supplementation of arginine or citrulline to bacterial or plant medium. EMB-Y2 represented a novel class of rhizobial arginine auxotrophs with symbiotic deficiency, and its phenotypes indicated that sufficient supply of citrulline or its derivative is essential for successful infection or for a stage in the infection process in Lotus-Mesorhizobium symbiosis.

• Isolation and characterization of arbuscules from roots of an increased-arbuscule-forming mutant of Lotus japonicus.

  Authors: Keishi Senoo, Zakaria Solaiman, Satoki Tanaka, Masayoshi Kawaguchi, Haruko Imaizumi-Anraku, Shoichiro Akao, Akiyoshi Tanaka, Hitoshi Obata

  Annals of botany. 01/2008; 100(7):1599-603.

  BACKGROUND AND AIMS: Previous methods for isolation of arbuscules from mycorrhizal roots are time-consuming, complex and expensive. Therefore, a simple, rapid and inexpensive method for the isolationBACKGROUND AND AIMS: Previous methods for isolation of arbuscules from mycorrhizal roots are time-consuming, complex and expensive. Therefore, a simple, rapid and inexpensive method for the isolation of metabolically active arbuscules from plant root of an increased-arbuscule-forming mutant of Lotus japonicus (Ljsym78-2) is described. METHODS: Roots of the L. japonicus mutant plants Ljsym78-2 colonized by Glomus sp. were separated from soil, washed with water, immersed in CaSO(4) before being cut into 5-mm pieces and homogenized with a Waring blender at 6000 rpm for 30 s. The arbuscules were purified by separation from plant tissues with a 50-mum nylon mesh, finally collecting on a 30-mum nylon mesh. Enzyme histochemical staining showed that the collected arbuscules had succinate dehydrogenase, alkaline phosphatase and acid phosphatase activities. KEY RESULTS AND CONCLUSIONS: The enzymic activity of the arbuscules was not affected after the isolation process. The establishment of this simple, rapid and inexpensive method for the isolation of metabolically active arbuscules will be useful to clarify the biochemical processes occurring in nutrient exchange at the arbuscular interface.

• 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.

• Positional cloning identifies Lotus japonicus NSP2, a putative transcription factor of the GRAS family, required for NIN and ENOD40 gene expression in nodule initiation.

  Authors: Yasuhiro Murakami, Hiroki Miwa, Haruko Imaizumi-Anraku, Hiroshi Kouchi, J Allan Downie, Masayoshi Kawaguchi, Shinji Kawasaki

  DNA research : an international journal for rapid publication of reports on genes and genomes. 01/2007; 13(6):255-65.

  Rhizobia-secreted Nod-factors (NFs) are required for nodulation. In the early developmental process of nodulation, a large number of changes occur in gene expression. Lotus japonicus nsp2 mutantsRhizobia-secreted Nod-factors (NFs) are required for nodulation. In the early developmental process of nodulation, a large number of changes occur in gene expression. Lotus japonicus nsp2 mutants isolated from Gifu B-129 ecotype have defects in nodule initiation and display non-nodulating phenotype. Here, we describe positional cloning of LjNSP2 as a component of the nodulation-specific signaling pathway. LjNSP2 was mapped near the translocation site of chromosome 1 where the recombination is severely suppressed. To circumvent this problem, we introduced Lotus burttii as an alternative crossing partner in place of L. japonicus Miyakojima. The development of the high-resolution map using a total of 11 481 F2 plants, in combination with newly developed DNA markers and construction of BAC library, enabled us to identify the gene responsible for mutant phenotype. LjNSP2 encodes a putative transcription factor of the GRAS family that constitutes a subfamily with Medicago truncatula NSP2. LjNSP2 was expressed in roots and early nodules, but strongly suppressed in matured nodules. The expression analysis of NIN and LjENOD40-1 genes in Ljnsp2 mutants indicates that LjNSP2 functions upstream of these genes. These results suggest that LjNSP2 acts as a transcription factor to directly or indirectly switch on the NF-induced genes required for nodule initiation.

• [Molecular genetics of signal perception and transduction in plant-microbe symbioses]

  Authors: Makoto Hayashi, Haruko Imaizumi-Anraku, Masayoshi Kawaguchi

  Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme. 09/2006; 51(9):1030-7.

• New nodulation mutants responsible for infection thread development in Lotus japonicus.

  Authors: Koji Yano, Myra L Tansengco, Taihei Hio, Kuniko Higashi, Yoshikatsu Murooka, Haruko Imaizumi-Anraku, Masayoshi Kawaguchi, Makoto Hayashi

  Molecular plant-microbe interactions : MPMI. 08/2006; 19(7):801-10.

  Legume plants develop specialized root organs, the nodules, through a symbiotic interaction with rhizobia. The developmental process of nodulation is triggered by the bacterial microsymbiont butLegume plants develop specialized root organs, the nodules, through a symbiotic interaction with rhizobia. The developmental process of nodulation is triggered by the bacterial microsymbiont but regulated systemically by the host legume plants. Using ethylmethane sulfonate mutagenesis as a tool to identify plant genes involved in symbiotic nodule development, we have isolated and analyzed five nodulation mutants, Ljsym74-3, Ljsym79-2, Ljsym79-3, Ljsym80, and Ljsym82, from the model legume Lotus japonicus. These mutants are defective in developing functional nodules and exhibit nitrogen starvation symptoms after inoculation with Mesorhizobium loti. Detailed observation revealed that infection thread development was aborted in these mutants and the nodules formed were devoid of infected cells. Mapping and complementation tests showed that Ljsym74-3, and Ljsym79-2 and Ljsym79-3, were allelic with reported mutants of L. japonicus, alb1 and crinkle, respectively. The Ljsym82 mutant is unique among the mutants because the infection thread was aborted early in its development. Ljsym74-3 and Ljsym80 were characterized as mutants with thick infection threads in short root hairs. Map-based cloning and molecular characterization of these genes will help us understand the genetic mechanism of infection thread development in L. japonicus.

• Deregulation of a Ca2+/calmodulin-dependent kinase leads to spontaneous nodule development.

  Authors: Leïla Tirichine, Haruko Imaizumi-Anraku, Satoko Yoshida, Yasuhiro Murakami, Lene H Madsen, Hiroki Miwa, Tomomi Nakagawa, Niels Sandal, Anita S Albrektsen, Masayoshi Kawaguchi, Allan Downie, Shusei Sato, Satoshi Tabata, Hiroshi Kouchi, Martin Parniske, Shinji Kawasaki, Jens Stougaard

  Nature. 07/2006; 441(7097):1153-6.

  Induced development of a new plant organ in response to rhizobia is the most prominent manifestation of legume root-nodule symbiosis with nitrogen-fixing bacteria. Here we show that the complexInduced development of a new plant organ in response to rhizobia is the most prominent manifestation of legume root-nodule symbiosis with nitrogen-fixing bacteria. Here we show that the complex root-nodule organogenic programme can be genetically deregulated to trigger de novo nodule formation in the absence of rhizobia or exogenous rhizobial signals. In an ethylmethane sulphonate-induced snf1 (spontaneous nodule formation) mutant of Lotus japonicus, a single amino-acid replacement in a Ca2+/calmodulin-dependent protein kinase (CCaMK) is sufficient to turn fully differentiated root cortical cells into meristematic founder cells of root nodule primordia. These spontaneous nodules are genuine nodules with an ontogeny similar to that of rhizobial-induced root nodules, corroborating previous physiological studies. Using two receptor-deficient genetic backgrounds we provide evidence for a developmentally integrated spontaneous nodulation process that is independent of lipochitin-oligosaccharide signal perception and oscillations in Ca2+ second messenger levels. Our results reveal a key regulatory position of CCaMK upstream of all components required for cell-cycle activation, and a phenotypically divergent series of mutant alleles demonstrates positive and negative regulation of the process.

• 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.

• 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.

• Pollen development and tube growth are affected in the symbiotic mutant of Lotus japonicus, crinkle.

  Authors: Myra L Tansengco, Haruko Imaizumi-Anraku, Makoto Yoshikawa, Shingo Takagi, Masayoshi Kawaguchi, Makoto Hayashi, Yoshikatsu Murooka

  Plant & cell physiology. 06/2004; 45(5):511-20.

  The symbiotic mutant of Lotus japonicus, crinkle (crk), exhibits abnormal nodulation and other alterations in the root hairs, trichomes, and seedpods. Defective nodulation in crk mutant is due to theThe symbiotic mutant of Lotus japonicus, crinkle (crk), exhibits abnormal nodulation and other alterations in the root hairs, trichomes, and seedpods. Defective nodulation in crk mutant is due to the arrested infection thread growth from the epidermis into the cortex. Here, we describe that crk is also affected in male fertility that causes the production of small pods with few seeds. Under in vitro conditions, pollen germination and tube growth were markedly reduced in the crk mutant. A swollen tip phenotype with disorganized filamentous actin (F-actin) was observed in the mutant pollen tubes after prolonged in vitro culture. During pollen development, the striking difference noted in the mutant was the small size of the microspores that remained spherical. Histological examination of ovule development, as well as outcrosses of the mutant as female to wild type as male, showed no evidence of abnormality in the female gametophyte development. Based on these findings, the Crk gene, aside from its role in the infection process during nodulation, is also involved in male gametophyte development and function. Therefore, this gene represents a connection between nodule symbiosis, polar tip growth, and other plant developmental processes.