As shown in the figure below, this field broadly covers the cytoplasmic genome and has three research target areas: (1) control technology, (2) genetic understanding, and (3) utilization and development.

A01　Control technology

Planned Research

A01-1：Development of cytoplasmic genome editing and gene transfer technologies

PI: Shin-ichi ARIMURA

The University of Tokyo
Lab HP　https://lpmg-u-tokyo-en.labby.jp/
Researchmap　https://researchmap.jp/Shin-ichiArimura/?lang=en
ORCID　https://orcid.org/0000-0002-9537-1626

Co-Investigators

Masahito HOSOKAWA, Waseda University
Hideki TAKANASHI, The University of Tokyo
Miki OKUNO, Kurume University
Fumiko ISHIZUNA, Tokyo Kasei Gakuin University

Masahito HOSOKAWA
Hideki TAKANASHI
Miki OKUNO
Fumiko ISHIZUNA

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To extend the technology of plant cytoplasmic genome editing, (1) apply the technology to various species other than plants as a joint research within and outside of our field. (2) To challenge stable gene transfer beyond the current limitations of genome editing (targeted truncation and base substitution) as a challenge to freely modify cytoplasmic genomes. In particular, we will transiently introduce DNA sequences encoding genome-editing enzymes (in collaboration with the Numata Group) and apply Gene drive to amplify and stabilize inserted genes to create stable foreign gene insertions in the mitochondrial genome of multicellular organisms, which has not been achieved in the world. In addition, to understand the genetic individuality and heterogeneity of mitochondria that exist in cells, we will (3) establish a single organelle genomics analysis technology and (4) elucidate plant mitochondrial heterogeneity using (3). While providing materials and technologies, including newly developed technologies, both within and outside the field, we aim to (5) simplify the technologies (kits and manuals) and spread the use of cytoplasmic genome modification technologies that can be used by anyone.

Publications　*corresponding author

  1. Nakazato I, Okuno M, Zhou C, Itoh T, Tsutsumi N, Takenaka M, Arimura SI* (2022) Targeted base editing in the mitochondrial genome of Arabidopsis thaliana. Proc Natl Acad Sci USA, 119: e2121177119.
  2. Nakazato I, Okuno M, Yamamoto H, Tamura Y, Itoh T, Shikanai T, Takanashi H, Tsutsumi N, Arimura SI* (2021). Targeted base editing in the plastid genome of Arabidopsis thaliana. Nat Plants, 7: 906–913.
  3. Arimura SI*, Ayabe H, Sugaya H, Okuno M, Tamura Y, Tsuruta Y, Watari Y, Yanase S, Yamauchi T, Itoh T, Toyoda A, Takanashi H, Tsutsumi N (2020) Targeted gene disruption of ATP synthases 6-1 and 6-2 in the mitochondrial genome of Arabidopsis thaliana by mitoTALENs. Plant J, 104: 1459–1471.
  4. Kazama T*, Okuno M, Watari Y, Yanase S, Koizuka C, Tsuruta Y, Sugaya H, Toyoda A, Itoh T, Tsutsumi N, Toriyama K, Koizuka N*, Arimura SI* (2019) Curing cytoplasmic male sterility via TALEN-mediated mitochondrial genome editing. Nat Plants, 5: 722–730.
  5. Arimura SI*, Fujimoto M, Doniwa Y, Kadoya N, Nakazono M, Sakamoto W, Tsutsumi N (2008) Arabidopsis ELONGATED MITOCHONDRIA1 is required for localization of DYNAMIN-RELATED PROTEIN3A to mitochondrial fission sites. Plant Cell, 20: 1555–1566.
  6. Arimura SI, Yamamoto J, Aida G, Nakazono M, Tsutsumi N* (2004) Frequent fusion and fission of plant mitochondria with unequal nucleoid distribution. Proc Natl Acad Sci USA, 101:7805–7808.
  7. Arimura SI, Tsutsumi N* (2002) A dynamin-like protein (ADL2b), rather than FtsZ, is involved in Arabidopsis mitochondrial division. Proc Natl Acad Sci USA, 99: 5727–5731.

A01-2：Introduction of nucleic acids and bioactive molecules into organelles

PI : Keiji NUMATA

Kyoto University
Lab HP　http://pixy.polym.kyoto-u.ac.jp/index_en.html
Researchmap　https://researchmap.jp/keijinumata_biopoly/?lang=en
ORCID　https://orcid.org/0000-0003-2199-7420

Co-Investigators

Yuma YAMADA, Hokkaido University
Simon LAW, RIKEN

Yuma YAMADA
Simon LAW

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It is difficult to modify cytoplasmic genomes, especially mitochondrial genomes of multicellular organisms, using existing technologies. In this study, we will develop and provide composite biotechnologies that enable selective gene transfer to genome-preserving organelles of diverse target organisms by using fused peptides with different functions, methods combining carbon nanotubes and peptides, and the nanocapsule MITO-Porter. Numata uses the “peptide method,” a DNA carrier fused with multiple functional peptides, and Yamada uses MITO-Porter, which has been successfully used for mitochondrial delivery of drugs in mammals, to introduce nucleic acids such as DNA into specific organelles within cells. Numata et al. have succeeded for the first time in the world in selectively introducing genes into plant mitochondria by utilizing a fusion peptide consisting of a yeast-derived mitochondrial transfer sequence and a polycation in addition to the cell-permeable peptide CPP. In this research, 1) introduction of the protein into organelles, and 2) development of technology to select modified organelles and cells will be carried out. By delivering the enzymes and coding sequences to the organelle in combination with the genome editing technology of the Arimura group, target-specific insertion of the introduced gene and disappearance of the wild-type genome will be attempted. In addition, we will provide delivery materials to various target organisms in this research area and consult with them about the modification, and establish a standard technology for cytoplasmic genome gene transfer.

Publications　*corresponding author

  1. Miyamoto T*, Tsuchiya K, Toyooka K, Goto Y, Tateishi A, Numata K* (2022) Relaxation of the plant cell wall barrier via zwitterionic liquid pretreatment for micelle complex-mediated DNA delivery to specific plant organelles. Angew Chem Int Ed, 61: e202204234.
  2. Law SSY, Liou G, Nagai Y, Gimenez Dejoz J, Tateishi A, Tsuchiya K, Kodama Y, Fujigaya T, Numata K* (2022) Polymer-coated carbon nanotube hybrids with functional peptides for gene delivery into plant mitochondria. Nat Commun, 13: 2417.
  3. Thagun C, Horii Y, Mori M, Fujita S, Ohtani M, Tsuchiya K, Kodama Y, Odahara M*, Numata K* (2022) Non-transgenic gene modulation via spray delivery of nucleic acid/peptide complexes into plant nuclei and chloroplasts. ACS Nano, 16: 3506–3521.
  4. Miyamoto T, Toyooka K, Chuah J, Odahara M, Higchi-Takeuchi M, Goto Y, Motoda Y, Kigawa T, Kodama Y, Numata K* (2022) Synthetic peptide–guided protein delivery in plants via a distinct endocytic route. JACS Au, 2: 223–233.
  5. Thagun C, Chuah J, Numata K* (2019) Targeted gene delivery into various plastids mediated by clustered cell-penetrating and chloroplast-targeting peptides. Adv Sci, 6: 1902064.

Publicly Offered Research

A01-3：Development and evaluation of plant mitochondria-specific epigenome editing tools

Kenji OSABE

Plant Epigenetics Unit, Okinawa Institute of Science and Technology
Lab HP　https://www.oist.jp/research/research-units/peu
Researchmap　https://researchmap.jp/kenjiosabe?lang=en
ORCID　https://orcid.org/0000-0002-5216-1055

B01　Genetic understanding

Planned Research

B01-1：Elucidation of the regulatory mechanism of gene expression in plant cytoplasmic genomes

PI : Mizuki TAKENAKA

Kyoto University
Lab HP　https://sites.google.com/view/shikanailab/%E3%83%9B%E3%83%BC%E3%83%A0
Researchmap　https://researchmap.jp/7000021262?lang=en
ORCID　https://orcid.org/0000-0002-3242-5092

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Gene expression in plant cytoplasmic genomes is regulated in response to organ, developmental, and environmental conditions. The regulatory mechanisms are far more complex than originally thought, and many nuclear-encoded proteins have been identified as regulators of transcription, RNA cleavage, RNA editing, splicing, translation, and RNA degradation. However, the integrated regulatory mechanisms by which they work in tandem remain largely unexplored. In this study, we aim to understand the dynamic regulatory mechanism of organelle gene expression in which multiple molecules work in tandem. To this end, it is necessary to elucidate the relationship between each regulatory factor and genomic DNA and RNA. In collaboration with the Arimura and Yamori groups, we will screen for gene regulatory mutants from random base substitution mutants in the cytoplasmic genome to elucidate the relationship between DNA and RNA sequences and the regulation of gene expression. (2) In collaboration with the Arimura group, introduce mutations near the transcription start site of organelle genes to conduct detailed promoter analysis, which has been technically difficult. (3) We will also convert each C-to-U RNA editing site to T at the DNA stage and analyze its effects in detail to clarify the biological significance of RNA editing as a regulatory mechanism of gene expression. (4) Information synchronization among mitochondria is essential for the regulatory expression of multiple mitochondrial genomic genes. In collaboration with the Arimura, Ishihara, and Nishimura groups, we will analyze the effects of mitochondrial fusion/division and abnormal formation/distribution of nucleoid bodies on the regulation of mitochondrial genomic gene expression. Based on the above analysis, we will contribute to this field by aiming to understand the regulatory mechanism of gene expression, which is one of the important pipes connecting the cytoplasmic genome and the life phenomena it plays a role in.

Publications　*corresponding author

  1. Takenaka M*, Takenaka S, Barthel T, Frink B, Haag S, Verbitskiy D, Oldenkott B, Schallenberg-Rüdinger M, Feiler CG, Weiss MS, Palm GJ, Weber G (2021) DYW domain structures imply an unusual regulation principle in plant organellar RNA editing catalysis. Nat Catal, 4: 510–522.
  2. Guillaumot D, Lopez-Obando M, Baudry K, Avon A, Rigaill G, Falcon de Longevialle A, Broche B, Takenaka M, Berthomé R, De Jaeger G, Delannoy E, Lurin C* (2017) Two interacting PPR proteins are major Arabidopsis editing factors in plastid and mitochondria. Proc Natl Acad Sci USA, 114: 8877–8882.
  3. Takenaka M*, Zehrmann A, Verbitskiy D, Kugelmann M, Härtel B, Brennicke A (2012) Multiple organellar RNA editing factor (MORF) family proteins are required for RNA editing in mitochondria and plastids of plants. Proc Natl Acad Sci USA, 109: 5104–5109.
  4. Zehrmann A, Verbitskiy D, van der Merwe JA, Brennicke A, Takenaka M* (2009) A DYW domaincontaining pentatricopeptide repeat protein is required for RNA editing at multiple sites in mitochondria of Arabidopsis thaliana. Plant Cell, 21: 558–567.
  5. Takenaka M*, Brennicke A (2009) Multiplex single-base extension typing to identify nuclear genes required for RNA editing in plant organelles. Nucl Acids Res, 37: e13.

B01-2：Maternal inheritance, dynamics, and quality control mechanisms of the mitochondrial genome

PI : Miyuki SATO

Gunma University
Lab HP　http://makukinou.showa.gunma-u.ac.jp/index.html
Researchmap　https://researchmap.jp/read0153917/?lang=en
ORCID　https://orcid.org/0000-0002-1944-4918

Co-Investigator

Tomotake KANKI, Kyushu University

Tomotake KANKI

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The mitochondrial genome has unique properties that differ from the nuclear genome, such as “maternal inheritance” and “heteroplasmy” (the ability to exist in a mixed state of intracellular mtDNA). Although these characteristics are fundamental concepts for understanding mitochondrial diseases for which no treatment has been established, their molecular basis and physiological significance are still unresolved issues in mitochondrial genome research. Using a variety of model systems (C. elegans, mouse, mammalian cultured cells, yeast), this research group aims to elucidate the following issues, starting from mitophagy, a mitochondrial quality control mechanism. (1) Elucidation of the molecular mechanism and physiological significance of maternal inheritance, focusing on paternal mitochondria-specific mitophagy, (2) Elucidation of the mechanism that generates differences between sperm and egg mitochondria during germ cell differentiation, (3) Elucidation of the mechanism that maintains heteroplasmy and analysis of its effect on mitochondrial function, (4) Understanding the mechanism of mitochondrial genome quality control by mitophagy and its application to mitochondrial disease model organisms. To accomplish these tasks, we will conduct mitochondrial genome editing in individual animals (nematode worms and mice) using the technology developed by the A01 group, construct an experimental system that can easily distinguish between male and female mitochondrial genomes, and construct mitochondrial disease model organisms to promote research. We will also contribute to the further development of cytoplasmic genome regulation methods by providing feedback to Group A01 on the findings of these mitochondrial genome-specific behaviors and inheritance patterns.

Publications　*corresponding author

  1. Sato M*, Sato K, Tomura K, Kosako H, Sato K* (2018) The autophagy receptor ALLO-1 and the IKKE-1 kinase control clearance of paternal mitochondria in Caenorhabditis elegans. Nat Cell Biol, 20: 81–91.
  2. Saegusa K, Sato M*, Morooka N, Hara T, Sato K* (2018) SFT-4/Surf4 control ER export of soluble cargo proteins and participate in ER exit site organization. J Cell Biol, 217: 2073–2085.
  3. Sakaguchi A, Sato M (co-first author), Sato K, Gengyo-Ando K, Yorimitsu T, Nakai J, Hara T, Sato K, Sato K* (2015) REI-1 is a guanine nucleotide exchange factor regulating RAB-11 localization and function in C. elegans embryos. Dev Cell, 35: 211–221.
  4. Sato M*, Konuma R, Sato K, Tomura K, Sato K* (2015) Fertilization-induced K63-linked ubiquitylation mediates clearance of maternal membrane proteins. Development, 141: 1324–1331.
  5. Sato M, Sato K* (2011) Degradation of paternal mitochondria by fertilization-triggered autophagy in C. elegans embryos. Science, 334: 1141–1144.

B01-3：Cytoplasmic genome/chloroplast nucleoid dynamics, repair, and maternal inheritance controlled by ChloroTALEN

PI : Yoshiki NISHIMURA

Waseda University
Lab HP　https://sites.google.com/view/shikanailab/research/nishimura-group/english-ver-nishimura-group
Researchmap　https://researchmap.jp/7000008742/?lang=en
ORCID　https://orcid.org/0000-0001-8686-9206

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Based on chloroplast transitional TALEN (chloroTALEN) and live imaging technology of chloroplast nucleoid, we will investigate the dynamics of chloroplast nucleolus-like bodies during the cytoplasmic genome repair process and the regulatory mechanism of maternal inheritance. (1) Spatiotemporal analysis of chloroplast DNA repair: Chloroplasts, the key component of photosynthesis in plants, have their own DNA (chloroplast DNA) and mechanisms for its replication and gene expression. Chloroplast DNA encodes a set of genes essential for photosynthesis and maintenance of chloroplast function, and its repair and stable inheritance are life-and-death matter for plants. In this study, with the cooperation of the Arimura group, We will develop and induce the expression of a chloroplast-transitional TALEN (chloroTALEN) that introduces a double strand break (DSB) in chloroplast DNA in the model green alga Chlamydomonas, and capture the behavior of chloroplast DNA repair factors in response to this by live imaging. We aim to understand the chloroplast DNA repair system spatiotemporally by such analysis. (2) Development of technology for artificial control of chloroplast maternal inheritance. In the green alga Chlamydomonas, chloroplast DNA is maternally inherited. We will try to artificially convert and control maternal inheritance to paternal inheritance by expressing chloroTALEN in zygotic females and fragmenting their chloroplast DNA. We will also select mutants in which paternal inheritance is promoted and identify the responsible gene, thereby approaching the molecular mechanism of maternal inheritance. (3) The techniques established in this research and the knowledge obtained will be applied to animals and land plants in cooperation with the Sato, Arimura, and Takenaka groups, aiming to understand cytoplasmic genome repair and maternal inheritance mechanisms in eukaryotes as a whole, thereby contributing to the advancement of this research field.

Publications　*corresponding author

  1. Takusagawa M, Kobayashi Y, Fukao Y, Hidaka K, Endo M, Sugiyama H, Hamaji T, Kato Y, Miyakawa I, Misumi O, Shikanai T, Nishimura Y* (2021) HBD1 protein with a tandem repeat of two HMG box domains is a DNA clip to organize chloroplast nucleoids in Chlamydomonas reinhardtii. Proc Natl Acad Sci USA, 118:1–8.
  2. Kamimura Y, Tanaka H, Kobayashi Y, Shikanai T, Nishimura Y* (2018) Chloroplast nucleoids as a transformable network revealed by live imaging with a micro fluidic device. Commun Biol, 1: 1–7.
  3. Kobayashi Y, Misumi O, Odahara M, Ishibashi K, Hirono M, Hidaka K, Endo M, Sugiyama H, Iwasaki H, Kuroiwa T, Shikanai T, Nishimura Y* (2017) Holliday junction resolvases mediate chloroplast nucleoid segregation. Science, 356: 631–634.
  4. Nishimura Y*, Shikanai T, Nakamura S, Kawai-Yamada M, Uchimiya H (2012) Gsp1 triggers the sexual developmental program including inheritance of chloroplast DNA and mitochondrial DNA in Chlamydomonas reinhardtii. Plant Cell, 24: 2401–2414.
  5. Nishimura Y*, Misumi O, Matsunaga S, Higashiyama T, Yokota A, Kuroiwa T (1999) The active digestion of uniparental chloroplast DNA in a single zygote of Chlamydomonas reinhardtii is revealed by using the optical tweezer. Proc Natl Acad Sci USA, 96: 12577–12582.

Publicly Offered Research

B01-4：Analysis of maternal chloroplast inheritance based on the discovery of sex-specific differences in chloroplast nucleoid structure

Yusuke KOBAYASHI

Faculty of Science, Ibaraki University
Researchmap　https://researchmap.jp/yusuke2828?lang=en

B01-5：Elucidating the role of microRNAs in establishing uniparental inheritance of chloroplasts

Tomohito YAMASAKI

Science and Technology Department, Natural Science Cluster, Kochi University
Lab HP　http://science.cc.kochi-u.ac.jp/?course=4091
Researchmap　https://researchmap.jp/ytomohito?lang=en
ORCID　https://orcid.org/0000-0001-9157-0209

B01-6：Manipulation of host mitochondria by infected microorganisms

Takumi KOSHIBA

Division of Biology, Faculty of Science, Fukuoka University
Lab HP　https://www.sci.fukuoka-u.ac.jp/lab/chem/koshiba/
Researchmap　https://researchmap.jp/read0124036/?lang=en
ORCID　https://orcid.org/0000-0001-8535-5043

B01-7：Physics of gene regulation of mitochondrial DNA by assembly of nucleoid

Tetsuya YAMAMOTO

Institute for Chemical Reaction Design and Discovery, Hokkaido Univeristy
Lab HP　https://www.icredd.hokudai.ac.jp/yamamoto-tetsuya
Researchmap　https://researchmap.jp/tetsuwis?lang=en
ORCID　https://orcid.org/0000-0002-6786-8299

B01-8：Exploring the dynamics of plastid transcriptional regulation by improved chromatin immunoprecipitation

Sho FUJII

Department of Biology, Faculty of Agricultrue and Life Science, Hirosaki Unvieristy
Lab HP　https://sites.google.com/view/shofujii-hirosaki-u/
Researchmap　https://researchmap.jp/shofujii?lang=en
ORCID　https://orcid.org/0000-0002-6260-5596

B02　Application and development

Planned Research

B02-1：Establishment of technology to enhance biological functions through mitochondrial intervention

PI : Naotada ISHIHARA

Osaka University
Lab HP　https://mitochondria.jp/englishpage
Researchmap　https://researchmap.jp/10325516/?lang=en
ORCID　https://orcid.org/0000-0002-6305-7149

Co-Investigator

Emi OGASAWARA, Osaka University

Emi OGASAWARA

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The elongated, branched mitochondria of cultured mammalian cells are active within the living cell, and their morphology is regulated by a balance between fusion and fission. However, the details of how mitochondrial internal DNA (mtDNA) is distributed and functionally expressed, and the coordinated regulation of the membrane and genome, are unknown. In this study, we aim to understand the molecular basis for the regulation of mitochondrial genome functions (e.g., respiratory chain complex formation, mtDNA stabilization, and cytoplasmic inheritance) from the perspective of multiple membrane mitochondrial dynamics. In our previous studies, we have found that respiratory chain formation is activated by nucleoid dispersion and that mitochondrial mitogenic factors and mtDNA binding and packaging factors are involved in its regulation. We aim to understand the molecular details by identifying the relevant factors and understanding the molecular mechanisms in detail. Mitochondrial dysfunction contributes not only to mitochondrial diseases but also to aging, diabetes, metabolic diseases, and neurodegenerative diseases. Based on the results of this research, we will construct mitochondrial activation technology in cells and individuals. Based on the molecular findings obtained in this area, we will seek collaboration with clinical research groups in related diseases. The molecular understanding gained from this research will also provide fundamental knowledge for innovations in cytoplasmic genome regulation technologies across species. The project will also contribute to the development of this field by providing broad support for the analysis of mitochondrial function, including respiratory measurements in mammals within the field.

Publications　*corresponding author

  1. Ishihara T, Ban-Ishihara R, Ota A, Ishihara N* (2022) Mitochondrial nucleoid trafficking regulated by the inner-membrane AAA-ATPase ATAD3A modulates respiratory complex formation. Proc Natl Acad Sci USA, 119: e2210730119.
  2. Hanada Y, Ishihara N*, Wang L, Otera H, Ishihara T, Koshiba T, Mihara K, Ogawa Y, Nomura M (2020) MAVS is energized by Mff which senses mitochondrial metabolism via AMPK for acute antiviral immunity. Nat Commun, 11: 5711.
  3. Ban T, Ishihara T, Kohno H, Saita S, Ichimura A, Maenaka K, Oka T, Mihara K, Ishihara N* (2017) Molecular basis of selective mitochondrial fusion by heterotypic action between OPA1 and cardiolipin. Nat Cell Biol, 19: 856–863.
  4. Ban-Ishihara R, Ishihara T, Sasaki N, Mihara K, Ishihara N* (2013) Dynamics of nucleoid structure regulated by mitochondrial fission contributes to cristae reformation and release of cytochrome c. Proc Natl Acad Sci USA, 110: 11863–11868.
  5. Ishihara N, Nomura M, Jofuku A, Kato H, Suzuki SO, Masuda K, Otera H, Nakanishi Y, Nonaka I, Goto Y, Taguchi N, Morinaga H, Maeda M, Takayanagi R, Yokota S, Mihara K* (2009) Mitochondrial fission factor Drp1 is essential for embryonic development and synapse formation in mice. Nat Cell Biol, 11: 958–966.

B02-2：Creation of high photosynthetically competent plants by cytoplasmic genome editing technology and elucidation of their Functions

PI : Wataru YAMORI

The University of Tokyo
Lab HP　https://park.itc.u-tokyo.ac.jp/yamori-lab/english-page.html
Researchmap　https://researchmap.jp/wataru.yamori/?lang=en
ORCID　https://orcid.org/0000-0001-7215-4736

Co-Investigators

Hiroshi FUKAYAMA, Kobe University
Hiroyoshi MATSUMURA, Ritsumeikan University

Hiroshi FUKAYAMA
Hiroyoshi MATSUMURA

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Efforts to improve crop photosynthesis are underway in Japan and abroad, but all of these studies have mainly focused on plant modification by introducing single or multiple foreign genes targeting the nuclear genome, and have not led to drastic photosynthetic improvement. It is well known that the major rate-limiting factor of photosynthesis is ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), whose catalytic site rbcL is encoded in the chloroplast genome. However, it has been difficult to improve the catalytic properties of Rubisco because its catalytic site rbcL is encoded in chloroplast genome. Therefore, this project group aims to enhance the photosynthetic ability based on Rubisco modification and elucidate its function by utilizing the “chloroplast genome editing technology” provided by the Arimura Group. Yamori will select photosynthetically competent Arabidopsis mutants from random mutant populations targeting the entire chloroplast genome or the Rubisco catalytic site rbcL gene to elucidate their functions. He also aims to elucidate the catalytic reaction mechanism of Rubisco and design highly catalytically active Rubisco by targeted single nucleotide replacement technology at sites predicted to affect Rubisco catalytic reaction. Fukayama will establish an experimental system in which rbcL is knocked out by chloroplast genome editing and a foreign rbcL with a chloroplast transit peptide is introduced into rice plants. Using this experimental system, we will introduce Rubisco rbcL, a C4 plant with high catalytic speed, and C4-optimized rice rbcL designed using the genetic algorithm GAOptimizer. Analysis of these transformants will be used to search for mutations in the rbcL gene that are effective in improving the Rubisco catalytic properties and to improve the Rubisco enzyme properties. Matsumura will conduct crystal structure analysis and cryo-EM analysis to fully elucidate the catalytic mechanism of the highly efficient and functional Rubisco produced by this project group. YAMORI will support the research on the functional analysis of the photosynthesis/respiration system using chlorophyll fluorescence and gas exchange measurements, and MATSUMURA will support the structure-function analysis of organelle proteins using “artificial binding proteins”.

Publications　*corresponding author

  1. Qu Y, Sakoda K, Fukayama H, Kondo E, Suzuki Y, Makino A, Terashima I, Yamori W* (2021) Overexpression of both Rubisco and Rubisco activase rescues rice photosynthesis and biomass under heat stress. Plant Cell Environ, 44: 2308–2320.
  2. Yamori W*, Kusumi K, Iba K, Terashima I (2020) Increased stomatal conductance induces rapid changes to photosynthetic rate in response to naturally fluctuating light conditions in rice. Plant Cell Environ, 43: 1230– 1240.
  3. Ushijima T, Hanada K, Gotoh E, Yamori W, Kodama Y, Tanaka H, Kusano M, Fukushima A, Tokizawa M, Yamamoto Y, Tada Y, Suzuki Y, Matsushita T* (2017) Light controls protein localization through phytochrome-mediated alternative promoter selection. Cell, 171: 1316–1325.
  4. Yamori W*, Shikanai T (2016) Physiological functions of cyclic electron transport around photosystem I in sustaining photosynthesis and plant growth. Annu Rev Plant Biol, 67: 81–106.
  5. Yamori W*, Hikosaka K, Way DA (2014) Temperature response of photosynthesis in C3, C4 and CAM plants: Temperature acclimation and temperature adaptation. Photosyn Res, 119: 101–117.

B02-3：Sexual regulation and symbiosis mechanisms by genome editing of the cytoplasmic symbiotic bacterium Wolbachia.

PI : Takashi KIUCHI

The University of Tokyo
Lab HP　https://sites.google.com/view/igblab-ut-aba/top
Researchmap　https://researchmap.jp/takashikiuchi/?lang=en
ORCID　https://orcid.org/0000-0003-3616-1650

Co-Investigator

Keisuke SHOJI, Tokyo University of Agriculture and Technology

Keisuke SHOJI

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Wolbachia, a symbiotic bacterium that infects about half of all arthropods, behaves like an organelle within the cell and is passed on to the next generation through maternal inheritance. In order to spread its infection within a host population, Wolbachia may manipulate the sex and reproduction of the host, for example, by specifically killing the male (male-killing). It is also known that Wolbachia-infected mosquitoes are less likely to transmit dengue virus and malaria. Insect control techniques using Wolbachia that apply these unique life phenomena have been devised, and some of them have already been implemented. However, extracellular culture or transplantation of Wolbachia is difficult, there is no genetic manipulation technology, and the execution factors and mechanisms of action of Wolbachia-specific life phenomena, such as symbiosis mechanisms (maternal inheritance, reduced pathogen vectors) and sexual control mechanisms, remain unresolved except in some cases. Therefore, in this research project, we will elucidate the function of the Wolbachia gene, which has not been elucidated so far, through the development of the world's first Wolbachia (Wol) TALEN. In the future, we aim to develop insect control technology using Wolbachia. To this end, the following three issues will be addressed. (1) We aim to develop WolTALEN by constructing a system to transport TALEN within Wolbachia through technical cooperation within this project. Targeting the male-killing factor Oscar (Nat Commun 2022), which we have identified, and using the functional reversion of Masc (Nature 2014), a protein responsible for malefication and gene dosage compensation, as an indicator, we can rapidly evaluate the effects of genome editing. (2) We aim to elucidate the sexual regulation and symbiotic mechanisms of Wolbachia, which are becoming organelles, and to develop applications using these mechanisms, as well as to discuss about their commonality and diversity by comparing them with mitochondria and chloroplasts, and contribute to the development of symbiotic organelle studies. We will play a bridging role to intracellular symbiotic bacteria research in this research area and provide WolTALEN within the area (publicly recruited groups) to create diversity and ripple effects in the research.

Publications　*corresponding author

  1. Kiuchi T*, Katsuma S (2022) Functional characterization of silkworm PIWI proteins by embryonic RNAi. Methods Mol Biol, 2360: 19–31.
  2. Katsuma S*, Hirota K, Matsuda-Imai N, Fukui T, Muro T, Nishino K, Kosako H, Shoji K, Takanashi H, Fujii T, Arimura S, Kiuchi T (2022) A Wolbachia factor for male killing in lepidopteran insects. Nat Commun, 13: 6764.
  3. Cortes-Silva N, Ulmer J, Kiuchi T, Hsieh E, Cornilleau G, Ladid I, Dingli F, Loew D, Katsuma S, Drinnenberg IA* (2020) CenH3-independent kinetochore assembly in Lepidoptera requires CCAN, including CENPT. Curr Biol, 30: 561–572.e10.
  4. Fukui T†, Kawamoto M†, Shoji K†, Kiuchi T†, Sugano S, Shimada T, Suzuki Y, Katsuma S* (2015) The endosymbiotic bacterium Wolbachia selectively kills male hosts by targeting the masculinizing gene. PLoS Pathogens, 11: e1005048. (†contributed equally to this work)
  5. Kiuchi T, Koga H, Kawamoto M, Shoji K, Sakai H, Arai Y, Ishihara G, Kawaoka S, Sugano S, Shimada T, Suzuki Y, Suzuki MG, Katsuma S* (2014) A single female-specific piRNA is the primary determiner of sex in the silkworm. Nature, 509: 633–636.

B02-4：Elucidation of the "male-killing" system hidden in mitochondria and its application to breeding

PI : Tomohiko KAZAMA

Kyushu University
Lab HP　https://www.agr.kyushu-u.ac.jp/lab/plantmb/English/index_En.html
Researchmap　https://researchmap.jp/read0143904/?lang=en
ORCID　https://orcid.org/0000-0003-3808-3991

Co-Investigator

Kinya TORIYAMA, Tohoku University

Kinya TORIYAMA

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Aiming to elucidate biological phenomena and improve important traits controlled by cytoplasmic genomes, we are challenging to elucidate a novel “male-killing” system that causes pollen development inhibition in the mitochondrial genome, and to establish a novel F1 breeding system by applying this system. Using precise mutagenesis (mitoTALECD) and gene disruption (mitoTALEN) of the mitochondrial genome, (1) we will clarify the functions of mitochondrial genes whose functions are still unknown (in collaboration with the Arimura Group). (2) We will also identify nuclear genes that may be involved in the regulation of the expression of these genes and elucidate their regulatory mechanisms (in collaboration with the Takenaka Group). Furthermore, by artificially controlling the combination of mitochondrial and nuclear genes identified in (1) and (2), (3) we will create new “male-killing” plants by genome editing. If these processes reveal the sequence conditions essential to function as a “male-killer” gene, the “male-killer” gene can be synthesized artificially. By introducing the synthesized artificial “male-killing” gene using organelle gene transfer technology (in collaboration with the Numata Group), it will be possible to create “male-killing” plants that do not rely on genetic resources. Furthermore, by applying the results to other plants, it will be possible to create “male-killing” plants in plants where “male-killing” has not yet been obtained, which is a trait with high commercial value, such as efficient F1 seed production. Thus, we show that it is possible to apply this Japanese-originated technology to organelle breeding. Furthermore, we will clarify the biological significance of “male-killing” in plants by comparing it with the male-killing mechanism in Wolbachia (in collaboration with the Kiuchi group).

Publications　*corresponding author

  1. Takatsuki A, Kazama T, Arimura SI, Toriyama K* (2022) TALEN-mediated depletion of the mitochondrial gene orf312 proves that it is a Tadukan-type cytoplasmic male sterility-causative gene in rice. Plant J, 110: 994–1004.
  2. Omukai S, Arimura SI, Toriyama K, Kazama T* (2021) Disruption of mitochondrial open reading frame 352 partially restores pollen development in cytoplasmic male sterile rice. Plant Physiol, 187: 236–246.
  3. Kazama T*, Okuno M, Watari Y, Yanase S, Koizuka C, Tsuruta Y, Sugaya H, Toyoda A, Itoh T, Tsutsumi N, Toriyama K, Koizuka N*, Arimura SI* (2019) Curing cytoplasmic male sterility via TALEN-mediated mitochondrial genome editing. Nat Plants, 5: 722–730.
  4. Kazama T*, Itabashi E, Fujii S, Nakamura T, Toriyama K (2016) Mitochondrial ORF79 levels determine pollen abortion in cytoplasmic male sterile rice. Plant J, 85, 707–716.
  5. Kazama T, Toriyama K* (2003) A pentatricopeptide repeat-containing gene that promotes the processing of aberrant atp6 RNA of cytoplasmic male-sterile rice. FEBS Lett, 544: 99–102.

Publicly Offered Research

B02-5：Physiological significance of chloroplast genomic gene regulation in stomatal guard cells

Juntaro NEGI

Department of Biology, Faculty of Sciences, Kyushu University
Lab HP　https://www.biology.kyushu-u.ac.jp/~plant/en/index.html
Researchmap　https://researchmap.jp/juntaro-negi?lang=en
ORCID　https://orcid.org/0000-0001-7457-1269

B02-6：Genetic engineering of Wolbachia for functional genomics and strain development

Manabu OTE

Department of Tropical Medicine, The Jikei University School of Medicine
Lab HP　http://jikei-tropmed.jp/
Researchmap　https://researchmap.jp/wolbachia?lang=en
ORCID　https://orcid.org/0000-0003-1480-0319

B02-7：iPS Cell-Based Modeling of mtDNA Mutation-Driven Neuropathology in Mitochondrial Disease

Naoki YAHATA

Department of Developmental Biology, Fujita Health University School of medicine
Lab HP　https://www.fujita-hu.ac.jp/graduate/medical/laboratories/developmental_neurobiology.html
Researchmap　https://researchmap.jp/ko60_KE01sh1?lang=en
ORCID　https://orcid.org/0000-0003-4125-4536

B02-8：Pioneering curative approaches for mitochondrial disorders through mtDNA editing

Haruna TANI

Institute of Development, Aging and Cancer, Tohoku University
Lab HP　https://www.modomics-medicine.com/
Researchmap　https://researchmap.jp/70930303?lang=en
ORCID　https://orcid.org/0009-0006-1277-8145

B02-9：Regulation of mtDNA mutation through activation of mitochondrial quality control

Shiori AKABANE

Kanagawa Cancer Center Research Institute, Cancer Biology Division
Lab HP　https://kcch.kanagawa-pho.jp/kccri/index_en.html
Researchmap　https://researchmap.jp/7000020098?lang=en
ORCID　https://orcid.org/0009-0009-9088-0606

B02-10：Regulation of organelle genome stability by chlorophagy and mitophagy in plants

Masanori IZUMI

CSRS, RIKEN
Lab HP　https://molecular-bioregulation.riken.jp/index_en.html
Researchmap　https://researchmap.jp/7000010004?lang=en
ORCID　https://orcid.org/0000-0001-5222-9163

B02-11：Organelle genome editing uncovers the mechanisms of drug resistance in malaria parasites

Makoto HIRAI

Department of Tropical Medicine and Parasitology, Faculty of Medicine, Juntendo University
Lab HP　https://www.juntendo.ac.jp/graduate/laboratory/labo/kiseityu/en-index.html
Researchmap　https://researchmap.jp/malariamut?lang=en
ORCID　https://orcid.org/0000-0002-5001-9653

B02-12：Genomic regulation of unculturable bacterial parasites in animal and plant cells

Kensaku MAEJIMA

Graduate School of Agricultural and Life Sciences, The University of Tokyo
Lab HP　https://webpark1802.sakura.ne.jp/planpath/en/index-en.html
Researchmap　https://researchmap.jp/k-maejima?lang=en
ORCID　https://orcid.org/0000-0003-1960-6232

B02-13：Mitochondrial DNA editing in malaria parasites: deciphering resistance mechanisms of Electron Transport Chain inhibitors

Takaya SAKURA

Institute of Tropical Medicine (NEKKEN), Nagasaki University
Lab HP　https://www.tm.nagasaki-u.ac.jp/molecdyna/
Researchmap　https://researchmap.jp/-2qE3_xt?lang=en
ORCID　https://orcid.org/0000-0002-8320-8485

Research Support

  • Shin-ichi ARIMURA／Genome editing
  • Keiji NUMATA／Gene delivery
  • Yuma YAMADA／Gene delivery
  • Masahito HOSOKAWA／Single organella analysis
  • Naotada ISHIHARA／Respiratory bioactivity measurement
  • Wataru YAMORI／Photosynthesis and respiration activity measurement
  • Keisuke SHOJI／Bioinformatic analysis
  • Hiroyoshi MATSUMURA／Protein structural analysis

Advisory Councils

Masaaki DEMURA／Nikkei Science, Inc.

Ralph Bock／Max Plank Institute

Pal Maliga／Waksman Institute

Wataru SAKAMOTO／Okayama University

Hideki SUMIMOTO／Kyushu University

Tetsuya HIGASHIYAMA／The University of Tokyo

Tetsuro MIMURA／Kyoto University of Advanced Science

Takashi YAMAMOTO／Hiroshima University

A01　Control technology

B01　Genetic understanding

B02　Application and development