Masayoshi Kawaguchi

Publications of Masayoshi Kawaguchi

• 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.

• Strategy for shoot meristem proliferation in plants.

  Authors: Hironori Fujita, Masayoshi Kawaguchi

  Plant signaling & behavior. 11/2011; 6(11):1851-4.

  Shoot apical meristem (SAM) of plants harbors stem cells capable of generating the aerial tissues including reproductive organs. Therefore, it is very important for plants to control SAMShoot apical meristem (SAM) of plants harbors stem cells capable of generating the aerial tissues including reproductive organs. Therefore, it is very important for plants to control SAM proliferation and its density as a survival strategy. The SAM is regulated by the dynamics of a specific gene network, such as the WUS-CLV interaction of A. thaliana. By using a mathematical model, we previously proposed six possible SAM patterns in terms of the manner and frequency of stem cell proliferation. Two of these SAM patterns are predicted to generate either dichotomous or axillary shoot branch. Dichotomous shoot branches caused by this mechanism are characteristic of the earliest vascular plants, such as Cooksonia and Rhynia, but are observed in only a small minority of plant species of the present day. On the other hand, axillary branches are observed in the majority of plant species and are induced by a different dynamics of the feedback regulation between auxin and the asymmetric distribution of PIN auxin efflux carriers. During evolution, some plants may have adopted this auxin-PIN system to more strictly control SAM proliferation.

• Expression and functional analysis of a CLV3-like gene in the model legume Lotus japonicus.

  Authors: Satoru Okamoto, Tomomi Nakagawa, Masayoshi Kawaguchi

  Plant & cell physiology. 06/2011; 52(7):1211-21.

  Plant aerial parts are differentiated from stem cells that are located in the shoot apical meristem (SAM). CLAVATA3 (CLV3)-CLV1 is a well-known ligand-receptor pair, which functions in SAMPlant aerial parts are differentiated from stem cells that are located in the shoot apical meristem (SAM). CLAVATA3 (CLV3)-CLV1 is a well-known ligand-receptor pair, which functions in SAM maintenance. In Lotus japonicus, HYPERNODULATION ABERRANT ROOT FORMATION1 (HAR1) shows the highest similarity with CLV1 of all Arabidopsis receptor-like kinases (RLKs). However, HAR1 functions in the systemic regulation of root nodule development, but does not appear to function in SAM maintenance. Therefore, the gene that is responsible for SAM maintenance in L. japonicus is largely unknown. Here, we identified the L. japonicus CLV3-like (LjCLV3) gene as a counterpart of AtCLV3 and performed expression and functional analysis. LjCLV3 transcripts were detected in the central region of the shoot meristems. However, unlike AtCLV3, LjCLV3 expression was not detected in the epidermal layer, but in the inner layers of the shoot meristems. RNA interference (RNAi) of LjCLV3 caused enlargement of not only the SAM, but also the primary and secondary inflorescence meristems (IMs). Furthermore, LjCLV3-silenced plants exhibited fasciated stems and an increased number of flowers per peduncle. These results reveal that LjCLV3 is responsible for the maintenance of the SAM as well as the primary and secondary IMs.

• Reaction-diffusion pattern in shoot apical meristem of plants.

  Authors: Hironori Fujita, Koichi Toyokura, Kiyotaka Okada, Masayoshi Kawaguchi

  PloS one. 01/2011; 6(3):e18243.

  A fundamental question in developmental biology is how spatial patterns are self-organized from homogeneous structures. In 1952, Turing proposed the reaction-diffusion model in order to explain thisA fundamental question in developmental biology is how spatial patterns are self-organized from homogeneous structures. In 1952, Turing proposed the reaction-diffusion model in order to explain this issue. Experimental evidence of reaction-diffusion patterns in living organisms was first provided by the pigmentation pattern on the skin of fishes in 1995. However, whether or not this mechanism plays an essential role in developmental events of living organisms remains elusive. Here we show that a reaction-diffusion model can successfully explain the shoot apical meristem (SAM) development of plants. SAM of plants resides in the top of each shoot and consists of a central zone (CZ) and a surrounding peripheral zone (PZ). SAM contains stem cells and continuously produces new organs throughout the lifespan. Molecular genetic studies using Arabidopsis thaliana revealed that the formation and maintenance of the SAM are essentially regulated by the feedback interaction between WUSHCEL (WUS) and CLAVATA (CLV). We developed a mathematical model of the SAM based on a reaction-diffusion dynamics of the WUS-CLV interaction, incorporating cell division and the spatial restriction of the dynamics. Our model explains the various SAM patterns observed in plants, for example, homeostatic control of SAM size in the wild type, enlarged or fasciated SAM in clv mutants, and initiation of ectopic secondary meristems from an initial flattened SAM in wus mutant. In addition, the model is supported by comparing its prediction with the expression pattern of WUS in the wus mutant. Furthermore, the model can account for many experimental results including reorganization processes caused by the CZ ablation and by incision through the meristem center. We thus conclude that the reaction-diffusion dynamics is probably indispensable for the SAM development of plants.

• Two CLE genes are induced by phosphate in roots of Lotus japonicus.

  Authors: Sachiko Funayama-Noguchi, Ko Noguchi, Chie Yoshida, Masayoshi Kawaguchi

  Journal of plant research. 01/2011; 124(1):155-63.

  Genes of CLE (CLAVATA3/ESR-related) family encode peptide ligands that regulate plant development in response to external stimuli such as rhizobial infection and the nitrate application as well asGenes of CLE (CLAVATA3/ESR-related) family encode peptide ligands that regulate plant development in response to external stimuli such as rhizobial infection and the nitrate application as well as various internal stimuli. To investigate whether LjCLE gene(s) may involve in plant response to inorganic phosphate (Pi), we analyzed Pi responses of 39 LjCLE genes in hydroponically grown Lotus japonicus plants (ecotype Miyakojima 'MG-20'). Two LjCLE genes, LjCLE19 and 20, were up-regulated specifically and greatly in roots of L. japonicus by Pi addition to the hydroponic solution. When the external Pi level increased, expressions of LjCLE19 and 20 increased before the increase in the Pi content in plants. On the other hand, when the external Pi level decreased, the Pi content in plants decreased first, then expression levels of LjCLE19 and 20 decreased. Based on our results, we discuss the relationship between LjCLE19 and 20 and the tissue Pi levels in plants. This is the first report showing induction of specific CLE genes by phosphate.

• The receptor-like kinase KLAVIER mediates systemic regulation of nodulation and non-symbiotic shoot development in Lotus japonicus.

  Authors: Hikota Miyazawa, Erika Oka-Kira, Naoto Sato, Hirokazu Takahashi, Guo-Jiang Wu, Shusei Sato, Masaki Hayashi, Shigeyuki Betsuyaku, Mikio Nakazono, Satoshi Tabata, Kyuya Harada, Shinichiro Sawa, Hiroo Fukuda, Masayoshi Kawaguchi

  Development (Cambridge, England). 12/2010; 137(24):4317-25.

  In legumes, the number of symbiotic root nodules is controlled by long-distance communication between the shoot and the root. Mutants defective in this feedback mechanism exhibit a hypernodulatingIn legumes, the number of symbiotic root nodules is controlled by long-distance communication between the shoot and the root. Mutants defective in this feedback mechanism exhibit a hypernodulating phenotype. Here, we report the identification of a novel leucine-rich repeat receptor-like kinase (LRR-RLK), KLAVIER (KLV), which mediates the systemic negative regulation of nodulation in Lotus japonicus. In leaf, KLV is predominantly expressed in the vascular tissues, as with another LRR-RLK gene, HAR1, which also regulates nodule number. A double-mutant analysis indicated that KLV and HAR1 function in the same genetic pathway that governs the negative regulation of nodulation. LjCLE-RS1 and LjCLE-RS2 represent potential root-derived mobile signals for the HAR1-mediated systemic regulation of nodulation. Overexpression of LjCLE-RS1 or LjCLE-RS2 did not suppress the hypernodulation phenotype of the klv mutant, indicating that KLV is required and acts downstream of LjCLE-RS1 and LjCLE-RS2. In addition to the role of KLV in symbiosis, complementation tests and expression analyses indicated that KLV plays multiple roles in shoot development, including maintenance of shoot apical meristem, vascular continuity, shoot growth and promotion of flowering. Biochemical analyses using transient expression in Nicotiana benthamiana revealed that KLV has the ability to interact with HAR1 and with itself. Together, these results suggest that the potential KLV-HAR1 receptor complex regulates symbiotic nodule development and that KLV is also a key component in other signal transduction pathways that mediate non-symbiotic shoot development.

• The Clavata2 genes of pea and Lotus japonicus affect autoregulation of nodulation.

  Authors: Lene Krusell, Naoto Sato, Izumi Fukuhara, Bjørn E V Koch, Christina Grossmann, Satoru Okamoto, Erika Oka-Kira, Yoko Otsubo, Grégoire Aubert, Tomomi Nakagawa, Shusei Sato, Satoshi Tabata, Gerard Duc, Martin Parniske, Trevor L Wang, Masayoshi Kawaguchi, Jens Stougaard

  The Plant journal : for cell and molecular biology. 12/2010; 65(6):861-71.

  The number of root nodules developing on legume roots after rhizobial infection is controlled by the plant shoot through autoregulation and mutational inactivation of this mechanism leads toThe number of root nodules developing on legume roots after rhizobial infection is controlled by the plant shoot through autoregulation and mutational inactivation of this mechanism leads to hypernodulation. We have characterised the Pisum sativum (pea) Sym28 locus involved in autoregulation and shown that it encodes a protein similar to the Arabidopsis CLAVATA2 (CLV2) protein. Inactivation of the PsClv2 gene in four independent sym28 mutant alleles, carrying premature stop codons, results in hypernodulation of the root and changes to the shoot architecture. In the reproductive phase sym28 shoots develops additional flowers, the stem fasciates, and the normal phyllotaxis is perturbed. Mutational substitution of an amino acid in one leucine rich repeat of the corresponding Lotus japonicus LjCLV2 protein results in increased nodulation. Similarly, down-regulation of the Lotus Clv2 gene by RNAi mediated reduction of the transcript level also resulted in increased nodulation. Gene expression analysis of LjClv2 and Lotus hypernodulation aberrant root formation Har1 (previously shown to regulate nodule numbers) indicated they have overlapping organ expression patterns. However, we were unable to demonstrate a direct protein-protein interaction between LjCLV2 and LjHAR1 proteins in contrast to the situation between equivalent proteins in Arabidopsis. LjHAR1 was localised to the plasma membrane using a YFP fusion whereas LjCLV2-YFP localised to the endoplasmic reticulum when transiently expressed in Nicotiana benthamiana leaves. This finding is the most likely explanation for the lack of interaction between these two proteins.

• plenty, a novel hypernodulation mutant in Lotus japonicus.

  Authors: Chie Yoshida, Sachiko Funayama-Noguchi, Masayoshi Kawaguchi

  Plant & cell physiology. 09/2010; 51(9):1425-35.

  Nitrogen fixation in nodules that contain symbiotic rhizobial bacteria enables legumes to thrive in nitrogen-poor soils. However, this symbiosis is energy consuming. Therefore, legumes strictlyNitrogen fixation in nodules that contain symbiotic rhizobial bacteria enables legumes to thrive in nitrogen-poor soils. However, this symbiosis is energy consuming. Therefore, legumes strictly control nodulation at both local and systemic levels. Mutants deficient in such controls exhibit a range of phenotypes from non-nodulation to hypernodulation. Here, we isolated a novel hypernodulation mutant from the M(2) progeny derived from Lotus japonicus MG-20 seeds mutagenized by irradiation with a carbon ion beam. We named the mutant 'plenty' because it formed more nodules than the wild-type MG-20. The nodulation zone in the plenty mutant was wider than that in the wild type, but not as enhanced as those in other previously reported hypernodulation mutants such as har1, klv or tml of L. japonicus. Unlike these hypernodulation mutants, the plenty mutant developed nodules of the same size as MG-20. Overall, the plenty mutant exhibited a unique phenotype of moderate hypernodulation. However, a biomass assay indicated that this unique pattern of hypernodulation was a hindrance to host plant growth. The plenty mutant displayed some tolerance to external nitrates and a normal triple response to ethylene. Grafting experiments demonstrated that the root of plenty was responsible for its hypernodulation phenotype. Genetic mapping indicated that the PLENTY gene was located on chromosome 2.

• 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.

• NENA, a Lotus japonicus homolog of Sec13, is required for rhizodermal infection by arbuscular mycorrhiza fungi and rhizobia but dispensable for cortical endosymbiotic development.

  Authors: Martin Groth, Naoya Takeda, Jillian Perry, Hisaki Uchida, Stephan Dräxl, Andreas Brachmann, Shusei Sato, Satoshi Tabata, Masayoshi Kawaguchi, Trevor L Wang, Martin Parniske

  The Plant cell. 07/2010; 22(7):2509-26.

  Legumes form symbioses with arbuscular mycorrhiza (AM) fungi and nitrogen fixing root nodule bacteria. Intracellular root infection by either endosymbiont is controlled by the activation of theLegumes form symbioses with arbuscular mycorrhiza (AM) fungi and nitrogen fixing root nodule bacteria. Intracellular root infection by either endosymbiont is controlled by the activation of the calcium and calmodulin-dependent kinase (CCaMK), a central regulatory component of the plant's common symbiosis signaling network. We performed a microscopy screen for Lotus japonicus mutants defective in AM development and isolated a mutant, nena, that aborted fungal infection in the rhizodermis. NENA encodes a WD40 repeat protein related to the nucleoporins Sec13 and Seh1. Localization of NENA to the nuclear rim and yeast two-hybrid experiments indicated a role for NENA in a conserved subcomplex of the nuclear pore scaffold. Although nena mutants were able to form pink nodules in symbiosis with Mesorhizobium loti, root hair infection was not observed. Moreover, Nod factor induction of the symbiotic genes NIN, SbtM4, and SbtS, as well as perinuclear calcium spiking, were impaired. Detailed phenotypic analyses of nena mutants revealed a rhizobial infection mode that overcame the lack of rhizodermal responsiveness and carried the hallmarks of crack entry, including a requirement for ethylene. CCaMK-dependent processes were only abolished in the rhizodermis but not in the cortex of nena mutants. These data support the concept of tissue-specific components for the activation of CCaMK.

• Analysis of two potential long-distance signaling molecules, LjCLE-RS1/2 and jasmonic acid, in a hypernodulating mutant too much love.

  Authors: Shimpei Magori, Masayoshi Kawaguchi

  Plant signaling & behavior. 04/2010; 5(4):403-5.

  Legume plants tightly control the number and development of root nodules. This is partly regulated by a long-distance signaling known as auto-regulation of nodulation (AON). AON signaling involves atLegume plants tightly control the number and development of root nodules. This is partly regulated by a long-distance signaling known as auto-regulation of nodulation (AON). AON signaling involves at least two potential long-distance signals: root-derived signal and shoot-derived signal. However, their molecular characteristics and the mode of action remain unclear. In our recent study, we isolated a novel Lotus japonicus hypernodulating mutant too much love (tml). Based on several grafting experiments, we concluded that its causative gene TML functions as a receptor of the shoot-derived signal. This finding prompted us to ask how the candidates of the long-distance signal molecules, LjCLE-RS1/2 and jasmonic acid (JA), are affected in tml mutants. Expression analysis revealed that rapid induction of LjCLE-RS1/2 upon rhizobial inoculation is still intact in tml, supporting that TML plays a role in reception of the shoot-derived signal but not in generation of the root-derived signal. Furthermore, physiological analysis showed that JA, a candidate of the shoot-derived signal, can suppress tml hypernodulation. Therefore, contrary to the previous report, JA might not be a component of AON signaling.

• 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.

• Conservation of Lotus and Arabidopsis basic helixloop-helix proteins reveals new players in root hair development.

  Authors: Bogumil Karas, Lisa Amyot, Christopher Johansen, Shusei Sato, Satoshi Tabata, Masayoshi Kawaguchi, Krzysztof Szczyglowski

  Plant physiology. 09/2009;

  Basic helix-loop-helix (bHLH) proteins constitute a large family of transcriptional regulators in plants. Although they have been shown to play important roles in a wide variety of developmentalBasic helix-loop-helix (bHLH) proteins constitute a large family of transcriptional regulators in plants. Although they have been shown to play important roles in a wide variety of developmental processes, relatively few have been functionally characterized. Here we describe the map-based cloning of the Lotus japonicus ROOTHAIRLESS1 (LjRHL1) locus. Deleterious mutations in this locus prevent root hair development, which also aborts root hair-dependent colonization of the host root by nitrogen fixing bacteria. We show that the LjRHL1 gene encodes a presumed basic helix-loop-helix transcription factor that functions in a non-redundant manner to control root hair development in L. japonicus. Homology search and cross-species complementation experiments defined three members of the Arabidopsis thaliana bHLH protein family, At2g24260, At4g30980, and At5g58010, as functionally equivalent to LjRHL1. Curiously, At2g24260 and At4g30980 mRNA species accumulate independently from the known positive regulators of root hair cell fate, while all three genes act in a partially redundant manner to regulate root hair development in Arabidopsis.

• 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.

• Long-distance control of nodulation: Molecules and models.

  Authors: Shimpei Magori, Masayoshi Kawaguchi

  Molecules and cells. 03/2009; 27(2):129-34.

  Legume plants develop root nodules to recruit nitrogen-fixing bacteria called rhizobia. This symbiotic relationship allows the host plants to grow even under nitrogen limiting environment. SinceLegume plants develop root nodules to recruit nitrogen-fixing bacteria called rhizobia. This symbiotic relationship allows the host plants to grow even under nitrogen limiting environment. Since nodule development is an energetically expensive process, the number of nodules should be tightly controlled by the host plants. For this purpose, legume plants utilize a long-distance signaling known as autoregulation of nodulation (AON). AON signaling in legumes has been extensively studied over decades but the underlying molecular mechanism had been largely unclear until recently. With the advent of the model legumes, L. japonicus and M. truncatula, we have been seeing a great progress including isolation of the AON-associated receptor kinase. Here, we summarize recent studies on AON and discuss an updated view of the long-distance control of nodulation.

• Nod factor, nitrate-induced CLE genes that drive systemic regulation of nodulation.

  Authors: Satoru Okamoto, Erika Ohnishi, Shusei Sato, Hirokazu Takahashi, Mikio Nakazono, Satoshi Tabata, Masayoshi Kawaguchi

  Plant & cell physiology. 01/2009;

  Host legumes control root nodule numbers by sensing external and internal cues. A major external cue is soil nitrate, whereas a feedback regulatory system in which earlier formed nodules suppressHost legumes control root nodule numbers by sensing external and internal cues. A major external cue is soil nitrate, whereas a feedback regulatory system in which earlier formed nodules suppress further nodulation through shoot-root communication is an important internal cue. The latter is known as autoregulation of nodulation (AUT), and is believed to consist of two long-distance signals: a root-derived signal that is generated in infected roots and transmitted to the shoot; and a shoot-derived signal that systemically inhibits nodulation. In Lotus japonicus, the leucine-rich repeat receptor-like kinase, HYPERNODULATION ABERRANT ROOT FORMATION 1 (HAR1), mediates AUT and nitrate inhibition of nodulation, and is hypothesized to recognize the root-derived signal. Here we identify L. japonicus CLE-Root Signal 1 (LjCLE-RS1) and LjCLE-RS2 as strong candidates for the root-derived signal. A hairy root transformation study shows that overexpressing LjCLE-RS1 and -RS2 inhibits nodulation systemically and furthermore, that the systemic suppression depends on HAR1. Moreover, LjCLE- RS2 expression is strongly up-regulated in roots by nitrate addition. Based on these findings, we propose a simple model for AUT and nitrate inhibition of nodulation mediated by LjCLE-RS1, -RS2 peptides and the HAR1 receptor-like kinase.

• 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.

• 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.

• Morphological effects of sinefungin, an inhibitor of S-adenosylmethionine-dependent methyltransferases, on Anabaena sp. PCC 7120.

  Authors: Hironori Fujita, Kunihiko Syono, Yasunori Machida, Masayoshi Kawaguchi

  Microbes and environments / JSME. 01/2008; 23(4):346-9.

  Anabaena cells develop regular one-dimensional filaments through cell division in planes parallel to each other. A gcvP mutant displayed morphological defects such as filaments with sharp bendsAnabaena cells develop regular one-dimensional filaments through cell division in planes parallel to each other. A gcvP mutant displayed morphological defects such as filaments with sharp bends and/or branching and irregular cell clumps. The defects probably result from depletion of S-adenosylmethionine (AdoMet), because they were rescued by the application of methionine, an AdoMet precursor, and because sinefungin, a strong inhibitor of AdoMet-dependent methyltransferases, caused morphological abnormalities in wild-type Anabaena similar to those of the mutant. AdoMet-dependent methylation is involved in the spatial regulation of cell polarity in Anabaena.