MF研究者総覧

研究者データベース

詳細
中野　亮平(ナカノ　リヨウヘイ)
理学研究院　生物科学部門　形態機能学分野
教授
researchmap個人ページ
Last Updated :2023/12/05

基本情報

所属

  • 理学研究院　生物科学部門　形態機能学分野

職名

  • 教授

学位

  • 博士（理学）（2012年03月 京都大学）

ORCID ID

J-Global ID

研究キーワード

  • ER body   グルコシノレート   植物微生物相互作用   植物マイクロバイオータ   

研究分野

  • ライフサイエンス / 植物分子、生理科学 / 植物微生物相互作用

職歴

  • 2023年04月 - 現在 北海道大学 大学院理学研究院 教授
  • 2019年01月 - 2023年03月 マックスプランク植物育種学研究所 植物微生物相互作用部門 独立研究員
  • 2013年04月 - 2018年12月 マックスプランク植物育種学研究所 植物微生物相互作用部門 博士研究員
  • 2013年07月 - 2015年06月 独立行政法人日本学術振興会 海外特別研究員
  • 2012年04月 - 2013年03月 京都大学 大学院理学研究科 生物科学専攻 特定研究員（科学研究）
  • 2009年04月 - 2012年03月 独立行政法人日本学術振興会 特別研究員（DC1）

研究活動情報

論文

  • Arpan Kumar Basak, Anna Piasecka, Jana Hucklenbroich, Gözde Merve Türksoy, Rui Guan, Pengfan Zhang, Felix Getzke, Ruben Garrido-Oter, Stephane Hacquard, Kazimierz Strzałka, Paweł Bednarek, Kenji Yamada, Ryohei Thomas Nakano
    The New phytologist 2023年09月28日 
    Endoplasmic reticulum (ER) bodies are ER-derived structures that contain a large amount of PYK10 myrosinase, which hydrolyzes tryptophan (Trp)-derived indole glucosinolates (IGs). Given the well-described role of IGs in root-microbe interactions, we hypothesized that ER bodies in roots are important for interaction with soil-borne microbes at the root-soil interface. We used mutants impaired in ER bodies (nai1), ER body-resident myrosinases (pyk10bglu21), IG biosynthesis (myb34/51/122), and Trp specialized metabolism (cyp79b2b3) to profile their root microbiota community in natural soil, evaluate the impact of axenically collected root exudates on soil or synthetic microbial communities, and test their response to fungal endophytes in a mono-association setup. Tested mutants exhibited altered bacterial and fungal communities in rhizoplane and endosphere, respectively. Natural soils and bacterial synthetic communities treated with mutant root exudates exhibited distinctive microbial profiles from those treated with wild-type (WT) exudates. Most tested endophytes severely restricted the growth of cyp79b2b3, a part of which also impaired the growth of pyk10bglu21. Our results suggest that root ER bodies and their resident myrosinases modulate the profile of root-secreted metabolites and thereby influence root-microbiota interactions.
  • Jana Ordon, Julien Thouin, Ryohei Thomas Nakano, Ka-Wai Ma, Pengfan Zhang, Bruno Huettel, Ruben Garrido-Oter, Paul Schulze-Lefert
    2023年04月20日
  • Ryohei Thomas Nakano
    Plant & cell physiology 62 11 1645 - 1647 2021年12月10日
  • Katarzyna W Wolinska, Nathan Vannier, Thorsten Thiergart, Brigitte Pickel, Sjoerd Gremmen, Anna Piasecka, Mariola Piślewska-Bednarek, Ryohei Thomas Nakano, Youssef Belkhadir, Paweł Bednarek, Stéphane Hacquard
    Proceedings of the National Academy of Sciences of the United States of America 118 49 2021年12月07日 
    In nature, roots of healthy plants are colonized by multikingdom microbial communities that include bacteria, fungi, and oomycetes. A key question is how plants control the assembly of these diverse microbes in roots to maintain host-microbe homeostasis and health. Using microbiota reconstitution experiments with a set of immunocompromised Arabidopsis thaliana mutants and a multikingdom synthetic microbial community (SynCom) representative of the natural A. thaliana root microbiota, we observed that microbiota-mediated plant growth promotion was abolished in most of the tested immunocompromised mutants. Notably, more than 40% of between-genotype variation in these microbiota-induced growth differences was explained by fungal but not bacterial or oomycete load in roots. Extensive fungal overgrowth in roots and altered plant growth was evident at both vegetative and reproductive stages for a mutant impaired in the production of tryptophan-derived, specialized metabolites (cyp79b2/b3). Microbiota manipulation experiments with single- and multikingdom microbial SynComs further demonstrated that 1) the presence of fungi in the multikingdom SynCom was the direct cause of the dysbiotic phenotype in the cyp79b2/b3 mutant and 2) bacterial commensals and host tryptophan metabolism are both necessary to control fungal load, thereby promoting A. thaliana growth and survival. Our results indicate that protective activities of bacterial root commensals are as critical as the host tryptophan metabolic pathway in preventing fungal dysbiosis in the A. thaliana root endosphere.
  • Ryosuke Sugiyama, Rui Li, Ayuko Kuwahara, Ryo Nakabayashi, Naoyuki Sotta, Tetsuya Mori, Takehiro Ito, Naoko Ohkama-Ohtsu, Toru Fujiwara, Kazuki Saito, Ryohei Thomas Nakano, Paweł Bednarek, Masami Yokota Hirai
    Proceedings of the National Academy of Sciences of the United States of America 118 22 2021年06月01日 
    Specialized (secondary) metabolic pathways in plants have long been considered one-way routes of leading primary metabolite precursors to bioactive end products. Conversely, endogenous degradation of such "end" products in plant tissues has been observed following environmental stimuli, including nutrition stress. Therefore, it is of general interest whether specialized metabolites can be reintegrated into primary metabolism to recover the invested resources, especially in the case of nitrogen- or sulfur-rich compounds. Here, we demonstrate that endogenous glucosinolates (GLs), a class of sulfur-rich plant metabolites, are exploited as a sulfur source by the reallocation of sulfur atoms to primary metabolites such as cysteine in Arabidopsis thaliana Tracer experiments using 34S- or deuterium-labeled GLs depicted the catabolic processing of GL breakdown products in which sulfur is mobilized from the thioglucoside group in GL molecules, potentially accompanied by the release of the sulfate group. Moreover, we reveal that beta-glucosidases BGLU28 and BGLU30 are the major myrosinases that initiate sulfur reallocation by hydrolyzing particular GL species, conferring sulfur deficiency tolerance in A. thaliana, especially during early development. The results delineate the physiological function of GL as a sulfur reservoir, in addition to their well-known functions as defense chemicals. Overall, our findings demonstrate the bidirectional interaction between primary and specialized metabolism, which enhances our understanding of the underlying metabolic mechanisms via which plants adapt to their environments.
  • Tomohisa Shimasaki, Sachiko Masuda, Ruben Garrido-Oter, Takashi Kawasaki, Yuichi Aoki, Arisa Shibata, Wataru Suda, Ken Shirasu, Kazufumi Yazaki, Ryohei Thomas Nakano, Akifumi Sugiyama
    mBio 12 3 e0084621  2021年05月28日 
    Plant roots constitute the primary interface between plants and soilborne microorganisms and harbor microbial communities called the root microbiota. Recent studies have demonstrated a significant contribution of plant specialized metabolites (PSMs) to the assembly of root microbiota. However, the mechanistic and evolutionary details underlying the PSM-mediated microbiota assembly and its contribution to host specificity remain elusive. Here, we show that the bacterial genus Arthrobacter is predominant specifically in the tobacco endosphere and that its enrichment in the tobacco endosphere is partially mediated by a combination of two unrelated classes of tobacco-specific PSMs, santhopine and nicotine. We isolated and sequenced Arthrobacter strains from tobacco roots as well as soils treated with these PSMs and identified genomic features, including but not limited to genes for santhopine and nicotine catabolism, that are associated with the ability to colonize tobacco roots. Phylogenomic and comparative analyses suggest that these genes were gained in multiple independent acquisition events, each of which was possibly triggered by adaptation to particular soil environments. Taken together, our findings illustrate a cooperative role of a combination of PSMs in mediating plant species-specific root bacterial microbiota assembly and suggest that the observed interaction between tobacco and Arthrobacter may be a consequence of an ecological fitting process. IMPORTANCE Host secondary metabolites have a crucial effect on the taxonomic composition of its associated microbiota. It is estimated that a single plant species produces hundreds of secondary metabolites; however, whether different classes of metabolites have distinctive or common roles in the microbiota assembly remains unclear. Here, we show that two unrelated classes of secondary metabolites in tobacco play a cooperative role in the formation of tobacco-specific compositions of the root bacterial microbiota, which has been established as a consequence of independent evolutionary events in plants and bacteria triggered by different ecological effects. Our findings illustrate mechanistic and evolutionary aspects of the microbiota assembly that are mediated by an arsenal of plant secondary metabolites.
  • Jana Hucklenbroich, Tamara Gigolashvili, Anna Koprivova, Philipp Spohr, Mahnaz Nezamiv, Chegini, Gunnar W. Klau, Stanislav Kopriva, Ryohei Thomas Nakano
    2021年05月25日
  • 登達也, 中野亮平
    アグリバイオ 5 1 2021年
  • Lisa K Mahdi, Menghang Huang, Xiaoxiao Zhang, Ryohei Thomas Nakano, Leïla Brulé Kopp, Isabel M L Saur, Florence Jacob, Viera Kovacova, Dmitry Lapin, Jane E Parker, James M Murphy, Kay Hofmann, Paul Schulze-Lefert, Jijie Chai, Takaki Maekawa
    Cell host & microbe 28 6 813 - 824 2020年12月09日 
    HeLo domain-containing mixed lineage kinase domain-like protein (MLKL), a pseudokinase, mediates necroptotic cell death in animals. Here, we report the discovery of a conserved protein family across seed plants that structurally resembles vertebrate MLKL. The Arabidopsis genome encodes three MLKLs (AtMLKLs) with overlapping functions in disease resistance mediated by Toll-interleukin 1-receptor domain intracellular immune receptors (TNLs). The HeLo domain of AtMLKLs confers cell death activity but is dispensable for immunity. Cryo-EM structures reveal a tetrameric configuration, in which the HeLo domain is buried, suggestive of an auto-repressed complex. The mobility of AtMLKL1 along microtubules is reduced by chitin, a fungal immunity-triggering molecule. An AtMLKL1 phosphomimetic variant exhibiting reduced mobility enhances immunity. Coupled with the predicted presence of HeLo domains in plant helper NLRs, our data reveal the importance of HeLo domain proteins for TNL-dependent immunity and argue for a cell death-independent immune mechanism mediated by MLKLs.
  • Ryohei Thomas Nakano
    2020年09月26日 
    AbstractPlant specialized metabolites (PSMs) influence the taxonomic compositions of the root-associated microbiota; however, the underlying molecular mechanisms and evolutionary trajectories remain elusive. Here, we show that the bacterial genus Arthrobacter is predominant in the tobacco endosphere, and that its enrichment is mediated by a combination of two tobacco-specific PSMs, santhopine and nicotine The isolation and whole genome sequencing of a representative set of Arthrobacter strains identified independent genomic features, including but not limited to genes for santhopine and nicotine catabolism, which are associated with the colonization competence of tobacco roots. Taken together, these data suggest that plant species-specific root microbiota assembly is mediated by bacterial catabolism of a cocktail of PSMs synthesized by the host plant.
  • Araki KS, Nagano AJ, Nakano RT, Kitazume T, Yamaguchi K, Hara-Nishimura I, Shigenobu S, Kudoh H
    Scientific reports 10 1 13291 - 13291 2020年08月 [査読有り][通常論文]
     
    © 2020, The Author(s). The rhizome is a plant organ that develops from a shoot apical meristem but penetrates into belowground environments. To characterize the gene expression profile of rhizomes, we compared the rhizome transcriptome with those of the leaves, shoots and roots of a rhizomatous Brassicaceae plant, Cardamine leucantha. Overall, rhizome transcriptomes were characterized by the absence of genes that show rhizome-specific expression and expression profiles intermediate between those of shoots and roots. Our results suggest that both endogenous developmental factors and external environmental factors are important for controlling the rhizome transcriptome. Genes that showed relatively high expression in the rhizome compared to shoots and roots included those related to belowground defense, control of reactive oxygen species and cell elongation under dark conditions. A comparison of transcriptomes further allowed us to identify the presence of an ER body, a defense-related belowground organelle, in epidermal cells of the C. leucantha rhizome, which is the first report of ER bodies in rhizome tissue.
  • Nakano RT, Ishihama N, Wang Y, Takagi J, Uemura T, Schulze-Lefert P, Nakagami H
    Methods in molecular biology (Clifton, N.J.) 2139 79 - 88 2020年01月 [査読有り][通常論文]
     
    Proteins in the extracellular space (apoplast) play a crucial role at the interface between plant cells and their proximal environment. Consequently, it is not surprising that plants actively control the apoplastic proteomic profile in response to biotic and abiotic cues. Comparative quantitative proteomics of plant apoplastic fluids is therefore of general interest in plant physiology. We here describe an efficient method to isolate apoplastic fluids from Arabidopsis thaliana leaves inoculated with a nonadapted powdery mildew pathogen.
  • Tomohiro Uemura, Ryohei Thomas Nakano, Junpei Takagi, Yiming Wang, Katharina Kramer, Iris Finkemeier, Hirofumi Nakagami, Kenichi Tsuda, Takashi Ueda, Paul Schulze-Lefert, Akihiko Nakano
    Plant Physiology 179 2 519 - 532 2019年02月 [査読有り][通常論文]
     
    Spatiotemporal coordination of protein trafficking among organelles is essential for eukaryotic cells. The post-Golgi interface, including the trans-Golgi network (TGN), is a pivotal hub for multiple trafficking pathways. The Golgi-released independent TGN (GI-TGN) is a compartment described only in plant cells, and its cellular and physiological roles remain elusive. In Arabidopsis (Arabidopsis thaliana), the SYNTAXIN OF PLANTS (SYP) 4 group Qa-SNARE (soluble N-ethylmaleimide) membrane fusion proteins are shared components of TGN and GI-TGN and regulate secretory and vacuolar transport. Here we reveal that GI-TGNs mediate the transport of the R-SNARE VESICLE-ASSOCIATED MEMBRANE PROTEIN (VAMP) 721 to the plasma membrane. In interactions with a nonadapted powdery mildew pathogen, the SYP4 group of SNAREs is required for the dynamic relocation of VAMP721 to plant-fungus contact sites via GI-TGNs, thereby facilitating complex formation with its cognate SNARE partner PENETRATION1 to restrict pathogen entry. Furthermore, quantitative proteomic analysis of leaf apoplastic fluid revealed constitutive and pathogen-inducible secretion of cell wall-modification enzymes in a SYP4- and VAMP721-dependent manner. Hence, the GI-TGN acts as a transit compartment between the Golgi apparatus and the plasma membrane. We propose a model in which the GA-TGN matures into the GI-TGN and then into secretory vesicles by increasing the abundance of VAMP721-dependent secretory pathway components.
  • Ruben Garrido-Oter, Ryohei Thomas Nakano, Nina Dombrowski, Ka-Wai Ma, Alice C. McHardy, Paul Schulze-Lefert
    Cell Host & Microbe 24 1 155 - 167.e5 2018年07月 [査読有り][通常論文]
     
    Rhizobia are a paraphyletic group of soil-borne bacteria that induce nodule organogenesis in legume roots and fix atmospheric nitrogen for plant growth. In non-leguminous plants, species from the Rhizobiales order define a core lineage of the plant microbiota, suggesting additional functional interactions with plant hosts. In this work, genome analyses of 1,314 Rhizobiales isolates along with amplicon studies of the root microbiota reveal the evolutionary history of nitrogen-fixing symbiosis in this bacterial order. Key symbiosis genes were acquired multiple times, and the most recent common ancestor could colonize roots of a broad host range. In addition, root growth promotion is a characteristic trait of Rhizobiales in Arabidopsis thaliana, whereas interference with plant immunity constitutes a separate, strain-specific phenotype of root commensal Alphaproteobacteria. Additional studies with a tripartite gnotobiotic plant system reveal that these traits operate in a modular fashion and thus might be relevant to microbial homeostasis in healthy roots.
  • Mariola Piślewska-Bednarek, Ryohei Thomas Nakano, Kei Hiruma, Marta Pastorczyk, Andrea Sanchez-Vallet, Suthitar Singkaravanit-Ogawa, Danuta Ciesiołka, Yoshitaka Takano, Antonio Molina, Paul Schulze-Lefert, Paweł Bednarek
    Plant Physiology 176 1 538 - 551 2018年01月 [査読有り][通常論文]
     
    Glutathione (GSH) and indole glucosinolates (IGs) exert key functions in the immune system of the model plant Arabidopsis (Arabidopsis thaliana). Appropriate GSH levels are important for execution of both pre- and postinvasive disease resistance mechanisms to invasive pathogens, whereas an intact PENETRATION2 (PEN2)-pathway for IG metabolism is essential for preinvasive resistance in this species. Earlier indirect evidence suggested that the latter pathway involves conjugation of GSH with unstable products of IG metabolism and further processing of the resulting adducts to biologically active molecules. Here we describe the identification of Glutathione-S-Transferase class-tau member 13 (GSTU13) as an indispensable component of the PEN2 immune pathway for IG metabolism. gstu13 mutant plants are defective in the pathogen-triggered biosynthesis of end products of the PEN2 pathway, including 4-O-β-D-glucosyl-indol-3-yl formamide, indole-3-ylmethyl amine, and raphanusamic acid. In line with this metabolic defect, lack of functional GSTU13 results in enhanced disease susceptibility toward several fungal pathogens including Erysiphe pisi, Colletotrichum gloeosporioides, and Plectosphaerella cucumerina. Seedlings of gstu13 plants fail also to deposit the (1,3)-β-glucan cell wall polymer, callose, after recognition of the bacterial flg22 epitope. We show that GSTU13 mediates specifically the role of GSH in IG metabolism without noticeable impact on other immune functions of this tripeptide. We postulate that GSTU13 connects GSH with the pathogen-triggered PEN2 pathway for IG metabolism to deliver metabolites that may have numerous functions in the innate immune system of Arabidopsis.
  • Ryohei Thomas Nakano
    The Plant Journal 89 2 204 - 220 2016年09月 [査読有り][通常論文]
     
    The endoplasmic reticulum body (ER body) is an organelle derived from the ER that occurs in only three families of the order Brassicales and is suggested to be involved in plant defense. ER bodies in Arabidopsis thaliana contain large amounts of b-glucosidases, but the physiological functions of ER bodies and these enzymes remain largely unclear. Here we show that PYK10, the most abundant b-glucosidase in A. thaliana root ER bodies, hydrolyzes indole glucosinolates (IGs) in addition to the previously reported in vitro substrate scopolin. We found a striking co-expression between ER body-related genes (including PYK10), glucosinolate biosynthetic genes and the genes for so-called specifier proteins affecting the terminal products of myrosinase-mediated glucosinolate metabolism, indicating that these systems have been integrated into a common transcriptional network. Consistent with this, comparative metabolite profiling utilizing a number of A. thaliana relatives within Brassicaceae identified a clear phylogenetic co-occurrence between ER bodies and IGs, but not between ER bodies and scopolin. Collectively, our findings suggest a functional link between ER bodies and glucosinolate metabolism in planta. In addition, in silico three-dimensional modeling, combined with phylogenomic analysis, suggests that PYK10 represents a clade of 16 myrosinases that arose independently from the other well-documented class of six thioglucoside glucohydrolases. These findings provide deeper insights into how glucosinolates are metabolized in cruciferous plants and reveal variation of the myrosinase-glucosinolate system within individual plants.
  • Ryohei Thomas Nakano
    CELL 165 2 464 - 474 2016年04月 [査読有り][通常論文]
     
    A staggering diversity of endophytic fungi associate with healthy plants in nature, but it is usually unclear whether these represent stochastic encounters or provide host fitness benefits. Although most characterized species of the fungal genus Colletotrichum are destructive pathogens, we show here that C. tofieldiae (Ct) is an endemic endophyte in natural Arabidopsis thaliana populations in central Spain. Colonization by Ct initiates in roots but can also spread systemically into shoots. Ct transfers the macronutrient phosphorus to shoots, promotes plant growth, and increases fertility only under phosphorus-deficient conditions, a nutrient status that might have facilitated the transition from pathogenic to beneficial lifestyles. The host's phosphate starvation response (PSR) system controls Ct root colonization and is needed for plant growth promotion (PGP). PGP also requires PEN2-dependent indole glucosinolate metabolism, a component of innate immune responses, indicating a functional link between innate immunity and the PSR system during beneficial interactions with Ct.
  • Nakano, Ryohei T., Yamada, Kenji, Bednarek, Paweł, Nishimura, Mikio, Hara-Nishimura, Ikuko
    Frontiers in Plant Science 5 73 - 73 2014年 [査読有り][通常論文]
     
    The endoplasmic reticulum (ER) forms highly organized network structures composed of tubules and cisternae. Many plant species develop additional ER-derived structures, most of which are specific for certain groups of species. In particular, a rod-shaped structure designated as the ER body is produced by plants of the Brassicales order, which includes Arabidopsis thaliana. Genetic analyses and characterization of A. thaliana mutants possessing a disorganized ER morphology or lacking ER bodies have provided insights into the highly organized mechanisms responsible for the formation of these unique ER structures. The accumulation of proteins specific for the ER body within the ER plays an important role in the formation of ER bodies. However, a mutant that exhibits morphological defects of both the ER and ER bodies has not been identified. This suggests that plants in the Brassicales order have evolved novel mechanisms for the development of this unique organelle, which are distinct from those used to maintain generic ER structures. In A. thaliana, ER bodies are ubiquitous in seedlings and roots, but rare in rosette leaves. Wounding of rosette leaves induces de novo formation of ER bodies, suggesting that these structures are associated with resistance against pathogens and/or herbivores. ER bodies accumulate a large amount of beta-glucosidases, which can produce substances that potentially protect against invading pests. Biochemical studies have determined that the enzymatic activities of these beta-glucosidases are enhanced during cell collapse. These results suggest that ER bodies are involved in plant immunity, although there is no direct evidence of this. In this review, we provide recent perspectives of ER and ER body formation in A. thaliana, and discuss clues for the functions of ER bodies. We highlight defense strategies against biotic stress that are unique for the Brassicales order, and discuss how ER structures could contribute to these strategies.
  • Nakano, Ryohei Thomas, Matsushima, Ryo, Nagano, Atsushi J., Fukao, Yoichiro, Fujiwara, Masayuki, Kondo, Maki, Nishimura, Mikio, Hara-Nishimura, Ikuko
    PLoS ONE 7 11 e49103  2012年 [査読有り][通常論文]
     
    The endoplasmic reticulum (ER) has a unique, network-like morphology. The ER structures are composed of tubules, cisternae, and three-way junctions. This morphology is highly conserved among eukaryotes, but the molecular mechanism that maintains ER morphology has not yet been elucidated. In addition, certain Brassicaceae plants develop a unique ER-derived organelle called the ER body. This organelle accumulates large amounts of PYK10, a beta-glucosidase, but its physiological functions are still obscure. We aimed to identify a novel factor required for maintaining the morphology of the ER, including ER bodies, and employed a forward-genetic approach using transgenic Arabidopsis thaliana (GFP-h) with fluorescently-labeled ER. We isolated and investigated a mutant (designated endoplasmic reticulum morphology3, ermo3) with huge aggregates and abnormal punctate structures of ER. ERMO3 encodes a GDSL-lipase/esterase family protein, also known as MVP1. Here, we showed that, although ERMO3/MVP1/GOLD36 was expressed ubiquitously, the morphological defects of ermo3 were specifically seen in a certain type of cells where ER bodies developed. Coimmunoprecipitation analysis combined with mass spectrometry revealed that ERMO3/MVP1/GOLD36 interacts with the PYK10 complex, a huge protein complex that is thought to be important for ER body-related defense systems. We also found that the depletion of transcription factor NAI1, a master regulator for ER body formation, suppressed the formation of ER-aggregates in ermo3 cells, suggesting that NAI1 expression plays an important role in the abnormal aggregation of ER. Our results suggest that ERMO3/MVP1/GOLD36 is required for preventing ER and other organelles from abnormal aggregation and for maintaining proper ER morphology in a coordinated manner with NAI1.
  • 中野 亮平, 松島 良, 上田 晴子, 林 八寿子, 田村 謙太郎, 嶋田 知生, 西村 いくこ
    日本植物生理学会年会およびシンポジウム 講演要旨集 2009 0 35 - 35 日本植物生理学会 2009年 [査読有り][通常論文]
     
    小胞体はチューブ状構造とシート状構造が three-way junctionと呼ばれる三叉の接続を介して複雑に組み合わさったネットワーク状の構造をとっている．この形態の形成・維持に寄与する因子を同定するため，小胞体の形態に異常を示す変異体を3系統単離し，endoplasmic reticulum morphology（ermo）と命名した．ermo1とermo2では，小胞体に由来する1 μm前後の球状構造体が数多く観察され，ermo2ではそれらが細胞内の一カ所に凝集していた．一方ermo3では球状の構造体は観察されず，小胞体が一カ所に凝集していた．これらの変異体では異常な構造・凝集体の他に正常なネットワークも観察された．マッピングの結果，ermo1とermo2は小胞体ーゴルジ体間の輸送においてそれぞれCOPI小胞とCOPII小胞の出芽に関わると考えられている遺伝子の変異であることがわかった．しかし，これらの変異体において細胞内輸送に明らかな異常は観察できていない．このことから，ERMO1およびERMO2は小胞体の形態維持に関わる新奇機能をもっている，あるいは変異体における輸送異常は検出できないレベルだが，小胞体ーゴルジ体間の輸送が小胞体形態に重要であることが考えられた．また，ERMO2およびERMO3はオルガネラを細胞内に分布させるのに必要であることが示された．
  • Nagano, Atsushi J., Maekawa, Akinori, Nakano, Ryohei Thomas, Miyahara, Mado, Higaki, Takumi, Kutsuna, Natsumaro, Hasezawa, Seiichiro, Hara-Nishimura, Ikuko
    Plant and Cell Physiology 50 12 2015 - 2022 2009年 [査読有り][通常論文]
     
    Although fluorescence microscopy screening has proven useful in the identification of genes involved in plant organelle biogenesis and integrity, the quantitative and statistical study of the geometric phenotype is highly limited. This situation could generate unconscious bias in the understanding and presentation of a mutant phenotype. Therefore, we have developed an automated quantification system for green fluorescent protein (GFP) images, which enabled us to easily obtain quantitative data on ER bodies (an endoplasmic reticulum-derived organelle). We isolated an ER body morphology mutant of Arabidopsis thaliana, leb-1 (long ER body). The leb-1 mutant had significantly fewer and larger ER bodies than the wild-type. An amino acid substitution of Cys29 with tyrosine (C29Y) on PYK10, a major component protein of ER bodies, was found in leb-1. Non-reducing SDSPAGE revealed that the electrophoretic mobility of PYK10 in the leb-1 mutant was clearly different from that in the wild type. This difference suggests that the C29Y amino acid substitution caused a tertiary structural change of the PYK10 protein. While the bglu21-1 and pyk10-1 single mutations slightly affected the number and morphology of the ER bodies, a bglu21-1 pyk10-1 double mutant had fewer and larger ER bodies than the wild type. The quantitative ER body phenotypes of leb-1 were similar to those of bglu21-1 pyk10-1 and bglu21-1 leb-1, suggesting that the leb-1 mutation allele acts dominantly to the BGLU21 wild-type allele. The leb-1 type PYK10 protein, which has an abnormal structure, may competitively inhibit interactions between the wild-type BGLU21/PYK10 protein and an unknown partner.
  • Nakano, Ryohei Thomas, Matsushima, Ryo, Ueda, Haruko, Tamura, Kentaro, Shimada, Tomoo, Li, Lixin, Hayashi, Yasuko, Kondo, Maki, Nishimura, Mikio, Hara-Nishimura, Ikuko
    THE PLANT CELL 21 11 3672 - 3685 2009年 [査読有り][通常論文]
     
    The endoplasmic reticulum (ER) is composed of tubules, sheets, and three-way junctions, resulting in a highly conserved polygonal network in all eukaryotes. The molecular mechanisms responsible for the organization of these structures are obscure. To identify novel factors responsible for ER morphology, we employed a forward genetic approach using a transgenic Arabidopsis thaliana plant (GFP-h) with fluorescently labeled ER. We isolated two mutants with defects in ER morphology and designated them endoplasmic reticulum morphology1 (ermo1) and ermo2. The cells of both mutants developed a number of ER-derived spherical bodies, similar to 1 mu m in diameter, in addition to the typical polygonal network of ER. The spherical bodies were distributed throughout the ermo1 cells, while they formed a large aggregate in ermo2 cells. We identified the responsible gene for ermo1 to be GNOM-LIKE1 (GNL1) and the gene for ermo2 to be SEC24a. Homologs of both GNL1 and SEC24a are involved in membrane trafficking between the ER and Golgi in yeast and animal cells. Our findings, however, suggest that GNL1/ERMO1 and SEC24a/ERMO2 have a novel function in ER morphology in higher plants.

講演・口頭発表等

その他活動・業績

共同研究・競争的資金等の研究課題

  • 日本学術振興会：科学研究費助成事業 基盤研究(B)
    研究期間 : 2015年04月 -2018年03月 
    代表者 : 植村 知博, 中野 亮平
     
    植物のトランスゴルジ網が担う病原菌応答の研究をおこない、シロイヌナズナに対する非宿主うどんこ病菌によってGI-TGNを介して分泌経路が活性化することを発見した。また、細胞壁修飾関連酵素がこの経路によって分泌されることを明らかにした。塩ストレス応答におけるTGN動態の観察をおこない、塩ストレス応答によってTGNが小さくなっていることを発見した。この過程を詳細に観察するために、共焦点レーザー顕微鏡による塩処理開始から6時間の長時間観察システムを構築した。
  • 高等植物の小胞体の形態形成・維持機構の解明
    日本学術振興会：科学研究費助成事業 特別研究員奨励費
    研究期間 : 2009年 -2011年 
    代表者 : 中野 亮平
     
    小胞体は複雑なネットワーク状の構造をとりダイナミックに運動している.また,オイルボディなど様々な構造体の形成の場となっている.アブラナ科などに特異的に観察されるER bodyもその1つである.このような構造がどのように形成され維持されているか解明するために,小胞体の異常な凝集体を生じるシロイヌナズナ変異体ermoの解析を行ってきた.昨年度までの解析でGDSL-lipase/esterase familyに属するタンパク質をコードする遺伝子に変異を同定し,今年度さらに詳細な解析を行った. ERMO3-HAを発現する植物体を作出し,HA抗体を用いた共免疫沈降実験および質量解析を行った結果ERMO3はPYK10複合体と呼ばれる巨大複合体を構成することがわかった.この複合体は植食者や菌などにより細胞が破砕された際にのみ生じると考えられている.発現解析の結果この遺伝子は植物体全体でユビキタスに発現していることがわかった.しかしながら,ERMO3の発現しているはずのロゼット葉の多くの細胞では小胞体の凝集は観察されなかった.ロゼット葉において少数みられた,小胞体の凝集を示す細胞ではER bodyが常に観察され,小胞体の形態異常とER bodyとお強い相関が示唆された.ER bodyを形成できなくなるnai1変異体との二重変異体では小胞体は正常な形態をとっていたことから,転写因子NAl1の下流で発現するなんらかの因子がermo3変異体細胞内で小胞体の凝集を引き起こしていることが明らかとなった.生化学的な相互作用解析および遺伝学的な解析から,ERMO3はERbodyを形成する細胞(すなわちNAl1を発現する細胞)において小胞体の凝集を抑圧するように働いており,その作用機序にはPYK10複合体あるいはその構成因子が関与していることが示唆された.

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