Shigeo S. Sugano, Keishi Osakabe and Yuriko Osakabe : Crop Breeding Using CRISPR/Cas9, in Crop Improvement Through Microbial Biotechnology, Elsevier, NY, USA, Mar. 2018.
13.
Yuriko Osakabe and Keishi Osakabe : Genome editing to improve abiotic stress responses in plants. in Gene Editing in Plants, Progress in Molecular Biology and Translational Science, ELSEVIER, Jul. 2017.
Yuriko Osakabe and Keishi Osakabe : Genome editing in higher plants. Targeted genome editing using site-specific nucleases, Springer, Jan. 2015.
(要約)
Genome editing with engineered nucleases (``GEEN'') has been developed as one of the effective genetic engineering methods using `molecular scissors', or artificially engineered nucleases to digest the DNAs at the desired locations in the genome of various organisms including plant species. The DNA binding domains, Zinc finger (ZF) proteins, have firstly been used for the plant genome editing tool as the designed ZF nucleases (ZFNs), and now TAL-effectors (TALE) and the RNA-DNA recognition system, CRISPR/Cas, instead of protein-DNA recognition are used for the targeted gene modifications in not only model plants but also crop species as powerful genome editing tools. The key for genome editing is the target gene-specific double-stranded DNA break (DSB) by using the designed endonucleases, in which the site-directed mutagenesis via nonhomologous end joining (NHEJ) repair pathway and/or gene targeting via homologous recombination (HR), can efficiently be occurred at the specific sites in genome. This chapter provides the overview of the recent advances in genome editing technologies to understand the more insights into current plant molecular biology and breeding technology.
Yuriko Osakabe, Keishi Osakabe and Kazuo Shinozaki : Plant environmental stress responses for survival and biomass enhancement Climate change and abiotic stress tolerance, Wiley-VCH, Germany, 2013.
19.
Keishi Osakabe, Masaki Endo and Seiichi Toki : Site-directed mutagenesis in higher plants., The Smiling Hippo, Oct. 2012.
(要約)
Double-strand DNA breaks (DSBs) are the most threatening type of DNA damages in living cells. For the maintenance of genome integrity and cell survival, it is critical that cells repair such breaks accurately and faithfully. Cells possess several DNA repair pathways to deal with DSBs. Genetic and biochemical studies have provided considerable data on the genes and their encoded proteins involved in these repair systems and their functions. The completion of genome sequencing of Arabidopsis and several major crops has greatly facilitated the search and study of genes involved in DNA repair. Several Arabidopsis mutants that are deficient in homologous recombination (HR, Box 1) and non-homologous end joining (NHEJ, Box 1) have been identified
Yuriko Osakabe and Keishi Osakabe : Abiotic stress responses in plants, Nova Science Publishers. USA, NY, 2012.
22.
Yuriko Osakabe and Keishi Osakabe : Plant abiotic stress responses and nutrients, Nova Science Publishers. USA, NY, 2012.
23.
Yuriko Osakabe, Shinya Kajita and Keishi Osakabe : Abiotic stress responses in woody plants; molecular perspective in engineering woody plant tolerance to abiotic stress and enhance biomass, NY, 2012.
24.
Keishi Osakabe, Masaki Endo, Kiyomi Abe and Seiichi Toki : Chapter 19 Homologous recombination in higher plants., Springer, Jun. 2010.
(要約)
Homologous recombination (HR) is one of two major repair systems for double-stranded DNA breaks (DSBs). A deficiency in either repair pathway is highly toxic to eukaryotes. HR also forms the basis of the mechanism of programmed genetic recombination, such as meiotic recombination, thus producing genetic variation. Indeed, breeding programs depend heavily on the crossover events of meiotic HR. Genetic and biochemical studies have provided evidence on the proteins involved in HR and their functions in higher plants. These include several HR repair-gene-deficient Arabidopsis mutants showing hypersensitivity to DSB-inducing agents. Unlike vertebrates, however, with a few exceptions these mutants do not show embryonic lethality or severe growth defects. This could represent a distinct advantage of plant systems for the study of HR. In this chapter, recent advances in the HR mechanism in eukaryotes are discussed, including signal transduction in higher plants in relation to HR repair.
25.
Keishi Osakabe, Susumu Miura, Yasuo Matsumoto, Mikiko Yamakado, Shinya Kajita, Shinya Kawai, Yoshihiro Katayama, Kunio Hata and Noriyuki Morohoshi : Agrobacterium-mediated transformation of poplar and using antisense RNA method to inhibit lignin biosynthesis., Uni Publisher, Dec. 1992.
(要約)
We have cloned and characterized several cDNAs for peroxidases from hybrid aspen, Populus kitakamiensis. To analyze the function of these peroxidases, an efficient method was established for transformation of the poplar hybrid Populus kitakamiensis (Populus sieboldii × Populus gradidentata) using a binary disarmed strain of Agrobacterium tumefaciens LBA4404 and Ti-binary vectors. We altered the expressin of a peroxidase isozyme by introducing an identified genomic gene for a peroxidase (prxA1) under the control of the CaMV35S promoter. Transgenic poplars obtained by introducing the chimeric peroxidase gene (CaMV35S promoter-prxA1) were shown to have an increase in total peroxidase activity that was accounted for by the specific overproduction of the peroxidase isozyme (PrxA1). From this study, the anionic peroxidase isozyme encoded by the identified genomic gene, prxA1, was demonstrated to be the anionic peroxidase isozyme with a pI of 4.4 among various isozymes of poplar peroxidase. On the basis of this assignment, we characterized the tissue-specific and UV-light-inducible regulation of expression of this isozyme.
学術論文(審査論文):
1.
Takatoshi Kiba, Kahori Mizutani, Aimi Nakahara, Yumiko Takebayashi, Mikiko Kojima, Tokunori Hobo, Yuriko Osakabe, Keishi Osakabe and Hitoshi SakakibaraHitoshi Sakakibara : The trans-zeatin-type side-chain modification of cytokinins controls rice growth., Plant Physiology, Vol.192, No.3, 2457-2474, 2023.
(要約)
Cytokinins (CKs), a class of phytohormones with vital roles in growth and development, occur naturally with various side-chain structures, including N6-(Δ2-isopentenyl)adenine-, cis-zeatin- and trans-zeatin (tZ)-types. Recent studies in the model dicot plant Arabidopsis (Arabidopsis thaliana) have demonstrated that tZ-type CKs are biosynthesized via cytochrome P450 monooxygenase (P450) CYP735A and have a specific function in shoot growth promotion. Although the function of some of these CKs has been demonstrated in a few dicotyledonous plant species, the importance of these variations and their biosynthetic mechanism and function in monocots and in plants with distinctive side-chain profiles other than Arabidopsis, such as rice (Oryza sativa), remain elusive. In this study, we characterized CYP735A3 and CYP735A4 to investigate the role of tZ-type CKs in rice. Complementation test of the Arabidopsis CYP735A-deficient mutant and CK profiling of loss-of-function rice mutant cyp735a3 cyp735a4 demonstrated that CYP735A3 and CYP735A4 encode P450s required for tZ-type side-chain modification in rice. CYP735As are expressed in both roots and shoots. The cyp735a3 cyp735a4 mutants exhibited growth retardation concomitant with reduction in CK activity in both roots and shoots, indicating that tZ-type CKs function in growth promotion of both organs. Expression analysis revealed that tZ-type CK biosynthesis is negatively regulated by auxin, abscisic acid, and CK and positively by dual nitrogen nutrient signals, namely glutamine-related and nitrate-specific signals. These results suggest that tZ-type CKs control the growth of both roots and shoots in response to internal and environmental cues in rice.
Kohji Yamada, Toya Yamamoto, Kanon Uwasa, Keishi Osakabe and Yoshitaka Takano : The establishment of multiple knockout mutants of Colletotrichum orbiculare by CRISPR-Cas9 and Cre-loxP systems., Fungal genetics and biology : FG & B, 2023.
(要約)
Colletotrichum orbiculare is employed as a model fungus to analyze molecular aspects of plant-fungus interactions. Although gene disruption via homologous recombination (HR) was established for C. orbiculare, this approach is laborious due to its low efficiency. Here we developed methods to generate multiple knockout mutants of C. orbiculare efficiently. We first found that CRISPR-Cas9 system massively promoted gene-targeting efficiency. By transiently introducing a CRISPR-Cas9 vector, more than 90% of obtained transformants were knockout mutants. Furthermore, we optimized a self-excision Cre-loxP marker recycling system for C. orbiculare because a limited availability of desired selective markers hampers sequential gene disruption. In this system, the integrated selective marker is removable from the genome via Cre recombinase driven by a xylose-inducible promoter, enabling the reuse of the same selective marker for the next transformation. Using our CRISPR-Cas9 and Cre-loxP systems, we attempted to identify functional sugar transporters involved in fungal virulence. Multiple disruptions of putative quinate transporter genes restricted fungal growth on media containing quinate as a sole carbon source, confirming their functionality as quinate transporters. However, our analyses showed that quinate acquisition was dispensable for infection to host plants. In addition, we successfully built mutations of 17 cellobiose transporter genes in a strain. From the data of knockout mutants that we established in this study, we inferred that repetitive rounds of gene disruption using CRISPR-Cas9 and Cre-loxP systems do not cause adverse effects on fungal virulence and growth. Therefore, these systems will be powerful tools to perform a systematic loss-of-function approach for C. orbiculare.
Bioengineering approaches to modify lignin content and structure in plant cell walls have shown promise for facilitating biochemical conversions of lignocellulosic biomass into valuable chemicals. Despite numerous research efforts, however, the effect of altered lignin chemistry on the supramolecular assembly of lignocellulose and consequently its deconstruction in lignin-modified transgenic and mutant plants is not fully understood. In this study, we aimed to close this gap by analyzing lignin-modified rice (Oryza sativa L.) mutants deficient in 5-HYDROXYCONIFERALDEHYDE O-METHYLTRANSFERASE (CAldOMT) and CINNAMYL ALCOHOL DEHYDROGENASE (CAD). A set of rice mutants harboring knockout mutations in either or both OsCAldOMT1 and OsCAD2 was generated in part by genome editing and subjected to comparative cell wall chemical and supramolecular structure analyses. In line with the proposed functions of CAldOMT and CAD in grass lignin biosynthesis, OsCAldOMT1-deficient mutant lines produced altered lignins depleted of syringyl and tricin units and incorporating noncanonical 5-hydroxyguaiacyl units, whereas OsCAD2-deficient mutant lines produced lignins incorporating noncanonical hydroxycinnamaldehyde-derived units. All tested OsCAldOMT1- and OsCAD2-deficient mutants, especially OsCAldOMT1-deficient lines, displayed enhanced cell wall saccharification efficiency. Solid-state nuclear magnetic resonance (NMR) and X-ray diffraction analyses of rice cell walls revealed that both OsCAldOMT1- and OsCAD2 deficiencies contributed to the disruptions of the cellulose crystalline network. Further, OsCAldOMT1 deficiency contributed to the increase of the cellulose molecular mobility more prominently than OsCAD2 deficiency, resulting in apparently more loosened lignocellulose molecular assembly. Such alterations in cell wall chemical and supramolecular structures may in part account for the variations of saccharification performance of the OsCAldOMT1- and OsCAD2-deficient rice mutants.
Tatpong Boontawon, Takehito Nakazawa, Chikako Inoue, 刑部 敬史, Moriyuki Kawauchi, Masahiro Sakamoto, Yoichi Honda : Efficient genome editing with CRISPR/Cas9 in Pleurotus ostreatus, AMB Express, Vol.11, 2021年.
(要約)
Pleurotus ostreatus is one of the most commercially produced edible mushrooms worldwide. Improved cultivated strains with more useful traits have been obtained using classical breeding, which is laborious and time-consuming. Here, we attempted efficient gene mutagenesis using plasmid-based CRISPR/Cas9 as the first step for non-genetically modified (non-GM) P. ostreatus generation. Plasmids harboring expression cassettes of Cas9 and different single guide RNAs targeting fcy1 and pyrG were individually transferred into fungal protoplasts of the PC9 strain, which generated some strains exhibiting resistance to 5-fluorocytosine and 5-fluoroorotic acid, respectively. Genomic PCR followed by sequencing revealed small insertions/deletions or insertion of a fragment from the plasmid at the target site in some of the drug-resistant strains. The results demonstrated efficient CRISPR/Cas9-assisted genome editing in P. ostreatus, which could contribute to the molecular breeding of non-GM cultivated strains in the future. Furthermore, a mutation in fcy1 via homology-directed repair using this CRISPR/Cas9 system was also efficiently introduced, which could be applied not only for precise gene disruption, but also for insertions leading to heterologous gene expression in this fungus.
Masafumi Omori, Hisayo Yamane, Keishi Osakabe, Yuriko Osakabe and Ryutaro Tao : Targeted mutagenesis of CENTRORADIALIS using CRISPR/Cas9 system through the improvement of genetic transformation efficiency of tetraploid highbush blueberry., The Journal of Horticultural Science & Biotechnology, 2020.
T. Wakabayashi, M. Hamana, A. Mori, R. Akiyama, K. Ueno, Keishi Osakabe, Yuriko Osakabe, H. Suzuki, H. Takikawa, M. Mizutani and Y. Sugimoto : Direct conversion of carlactonoic acid to orobanchol by cytochrome P450 CYP722C in strigolactone biosynthesis., Science Advances, Vol.5, No.12, eaax9067, 2019.
(要約)
Strigolactones (SLs) are carotenoid-derived phytohormones and rhizosphere signaling molecules for arbuscular mycorrhizal fungi and root parasitic weeds. Why and how plants produce diverse SLs are unknown. Here, cytochrome P450 CYP722C is identified as a key enzyme that catalyzes the reaction of BC-ring closure leading to orobanchol, the most prevalent canonical SL. The direct conversion of carlactonoic acid to orobanchol without passing through 4-deoxyorobanchol is catalyzed by the recombinant enzyme. By knocking out the gene in tomato plants, orobanchol was undetectable in the root exudates, whereas the architecture of the knockout and wild-type plants was comparable. These findings add to our understanding of the function of the diverse SLs in plants and suggest the potential of these compounds to generate crops with greater resistance to infection by noxious root parasitic weeds.
R. Akiyama, H.J. Lee, M. Nakayasu, Keishi Osakabe, Yuriko Osakabe, N. Umemoto, K. Saito, T. Muranaka, Y. Sugimoto and M. Mizutani : Characterization of steroid 5α-reductase involved in α-tomatine biosynthesis in tomatoes., Plant Biotechnology, Vol.36, No.4, 253-263, 2019.
H Suzuki, EO Fukushima, Y. Shimizu, H. Seki, Y Fujisawa, M. Ishimoto, Keishi Osakabe, Yuriko Osakabe and T Muranaka : Lotus japonicus triterpenoid profile and characterization of the CYP716A51 and LjCYP93E1 genes involved in their biosynthesis in planta., Plant & Cell Physiology, Vol.60, No.11, 2496-2509, 2019.
(要約)
Lotus japonicus is an important model legume plant in several fields of research, such as secondary (specialized) metabolism and symbiotic nodulation. This plant accumulates triterpenoids; however, less information regarding its composition, content and biosynthesis is available compared with Medicago truncatula and Glycine max. In this study, we analyzed the triterpenoid content and composition of L. japonicus. Lotus japonicus accumulated C-28-oxidized triterpenoids (ursolic, betulinic and oleanolic acids) and soyasapogenols (soyasapogenol B, A and E) in a tissue-dependent manner. We identified an oxidosqualene cyclase (OSC) and two cytochrome P450 enzymes (P450s) involved in triterpenoid biosynthesis using a yeast heterologous expression system. OSC9 was the first enzyme derived from L. japonicus that showed α-amyrin (a precursor of ursolic acid)-producing activity. CYP716A51 showed triterpenoid C-28 oxidation activity. LjCYP93E1 converted β-amyrin into 24-hydroxy-β-amyrin, a metabolic intermediate of soyasapogenols. The involvement of the identified genes in triterpenoid biosynthesis in L. japonicus plants was evaluated by quantitative real-time PCR analysis. Furthermore, gene loss-of-function analysis of CYP716A51 and LjCYP93E1 was conducted. The cyp716a51-mutant L. japonicus hairy roots generated by the genome-editing technique produced no C-28 oxidized triterpenoids. Likewise, the complete abolition of soyasapogenols and soyasaponin I was observed in mutant plants harboring Lotus retrotransposon 1 (LORE1) in LjCYP93E1. These results indicate that the activities of these P450 enzymes are essential for triterpenoid biosynthesis in L. japonicus. This study increases our understanding of triterpenoid biosynthesis in leguminous plants and provides information that will facilitate further studies of the physiological functions of triterpenoids using L. japonicus.
E. Toda, N. Koiso, A. Takebayashi, M. Ichikawa, T. Kiba, Keishi Osakabe, Yuriko Osakabe, H. Sakakibara, N. Kato and T. Okamoto : An efficient DNA- and selectable-marker-free genome-editing system using zygotes in rice. https://doi.org/10.1038/s41477-019-0386-z, Nature Plants, Vol.5, No.4, 363-368, 2019.
(要約)
Technology involving the targeted mutagenesis of plants using programmable nucleases has been developing rapidly and has enormous potential in next-generation plant breeding. Notably, the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease (Cas9) (CRISPR-Cas9) system has paved the way for the development of rapid and cost-effective procedures to create new mutant populations in plants. Although genome-edited plants from multiple species have been produced successfully using a method in which a Cas9-guide RNA (gRNA) expression cassette and selectable marker are integrated into the genomic DNA by Agrobacterium tumefaciens-mediated transformation or particle bombardment, CRISPR-Cas9 integration increases the chance of off-target modifications, and foreign DNA sequences cause legislative concerns about genetically modified organisms. Therefore, DNA-free genome editing has been developed, involving the delivery of preassembled Cas9-gRNA ribonucleoproteins (RNPs) into protoplasts derived from somatic tissues by polyethylene glycol-calcium (PEG-Ca)-mediated transfection in tobacco, Arabidopsis, lettuce, rice, Petunia, grapevine, apple and potato, or into embryo cells by biolistic bombardment in maize and wheat. However, the isolation and culture of protoplasts is not feasible in most plant species and the frequency of obtaining genome-edited plants through biolistic bombardment is relatively low. Here, we report a genome-editing system via direct delivery of Cas9-gRNA RNPs into plant zygotes. Cas9-gRNA RNPs were transfected into rice zygotes produced by in vitro fertilization of isolated gametes and the zygotes were cultured into mature plants in the absence of selection agents, resulting in the regeneration of rice plants with targeted mutations in around 14-64% of plants. This efficient plant-genome-editing system has enormous potential for the improvement of rice as well as other important crop species.
Sugano S. Shigeo, Nishihama Ryuichi, Shirakawa Makoto, Takagi Junpei, Matsuda Yoriko, Ishida Sakiko, Shimada Tomoo, Hara-Nishimura Ikuko, Keishi Osakabe and Kohchi Takayuki : Efficient CRISPR/Cas9-based genome editing and its application to conditional genetic analysis in Marchantia polymorpha. doi: 10.1371/journal.pone.0205117, PLoS ONE, Vol.13, No.10, e0205117, 2018.
(要約)
Marchantia polymorpha is one of the model species of basal land plants. Although CRISPR/Cas9-based genome editing has already been demonstrated for this plant, the efficiency was too low to apply to functional analysis. In this study, we show the establishment of CRISPR/Cas9 genome editing vectors with high efficiency for both construction and genome editing. Codon optimization of Cas9 to Arabidopsis achieved over 70% genome editing efficiency at two loci tested. Systematic assessment revealed that guide sequences of 17 nt or shorter dramatically decreased this efficiency. We also demonstrated that a combinatorial use of this system and a floxed complementation construct enabled conditional analysis of a nearly essential gene. This study reports that simple, rapid, and efficient genome editing is feasible with the series of developed vectors.
Hashimoto Ryosuke, Ueta Risa, Abe Chihiro, Yuriko Osakabe and Keishi Osakabe : Efficient multiplex genome editing induces precise, and self-ligated type mutations in tomato plants, Frontiers in Plant Science, Vol.9, 916, 2018.
(要約)
Several expression systems for multiple guide RNA (gRNAs) have been developed in the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) system to induce multiple-gene modifications in plants. Here, we evaluated mutation efficiencies in the tomato genome using multiplex CRISPR/Cas9 vectors consisting of various expression promoters with multiple gRNA expression combinations. In transgenic tomato calli induced with these vectors, mutation patterns varied depending on the promoters used to express . By using the tomato α (α) promoter to drive , occurrence of various types of mutations with high efficiency was detected in the tomato genome. Furthermore, sequence analysis showed that the majority of mutations using the multiplex system with the α promoter corresponded to specific mutation pattern of deletions produced by self-ligation at two target sites of CRISPR/Cas9 with low mosaic mutations. These results suggest that optimizing the expression promoter used in CRISPR/Cas9-mediated mutation improves multiplex genome editing, and could be used effectively to disrupt functional domains precisely in the tomato genome.
Nakayasu Masaru, Akiyama Ryota, Lee Jae Hyoung, Keishi Osakabe, Yuriko Osakabe, Watanabe Bunta, Sugimoto Yukihiro, Umemoto Naoyuki, Saito Kazuki, Muranaka Toshiya and Mizutani Masaharu : Generation of -solanine-free hairy roots of potato by CRISPR/Cas9 mediated genome editing of the St16DOX gene, Plant Physiology and Biochemistry : PPB, Vol.131, 70-77, 2018.
(要約)
Potato (Solanum tuberosum) is a major food crop, while the most tissues of potato accumulates steroidal glycoalkaloids (SGAs) -solanine and -chaconine. Since SGAs confer a bitter taste on human and show the toxicity against various organisms, reducing the SGA content in the tubers is requisite for potato breeding. However, generation of SGA-free potato has not been achieved yet, although silencing of several SGA biosynthetic genes led a decrease in SGAs. Here, we show that the knockout of St16DOX encoding a steroid 16-hydroxylase in SGA biosynthesis causes the complete abolition of the SGA accumulation in potato hairy roots. Nine candidate guide RNA (gRNA) target sequences were selected from St16DOX by in silico analysis, and the two or three gRNAs were introduced into a CRISPR/Cas9 vector designated as pMgP237-2A-GFP that can express multiplex gRNAs based on the pre-tRNA processing system. To establish rapid screening of the candidate gRNAs that can efficiently mutate the St16DOX gene, we used a potato hairy root culture system for the introduction of the pMgP237 vectors. Among the transgenic hairy roots, two independent lines showed no detectable SGAs but accumulated the glycosides of 22,26-dihydroxycholesterol, which is the substrate of St16DOX. Analysis of the two lines with sequencing exhibited the mutated sequences of St16DOX with no wild-type sequences. Thus, generation of SGA-free hairy roots of tetraploid potato was achieved by the combination of the hairy root culture and the pMgP237-2A-GFP vector. This experimental system is useful to evaluate the efficacy of candidate gRNA target sequences in the short-term.
Sigeo Sugano, Hiroko Suzuki, Eisuke Shimokita, Hirofumi Chiba, Sumihare Noji, Yuriko Osakabe and Keishi Osakabe : Genome editing in the mushroom-forming basidiomycetes, Coprinopsis cinerea, optimized by high-throughput transformation system., Scientific Reports, Vol.7, 2017.
(要約)
Mushroom-forming basidiomycetes produce a wide range of metabolites and have great value not only as food but also as an important global natural resource. Here, we demonstrate CRISPR/Cas9-based genome editing in the model species Coprinopsis cinerea. Using a high-throughput reporter assay with cryopreserved protoplasts, we identified a novel promoter, CcDED1 pro , with seven times stronger activity in this assay than the conventional promoter GPD2. To develop highly efficient genome editing using CRISPR/Cas9 in C. cinerea, we used the CcDED1 pro to express Cas9 and a U6-snRNA promoter from C. cinerea to express gRNA. Finally, CRISPR/Cas9-mediated GFP mutagenesis was performed in a stable GFP expression line. Individual genome-edited lines were isolated, and loss of GFP function was detected in hyphae and fruiting body primordia. This novel method of high-throughput CRISPR/Cas9-based genome editing using cryopreserved protoplasts should be a powerful tool in the study of edible mushrooms.
Parthenocarpy in horticultural crop plants is an important trait with agricultural value for various industrial purposes as well as direct eating quality. Here, we demonstrate a breeding strategy to generate parthenocarpic tomato plants using the CRISPR/Cas9 system. We optimized the CRISPR/Cas9 system to introduce somatic mutations effectively into SlIAA9-a key gene controlling parthenocarpy-with mutation rates of up to 100% in the T0 generation. Furthermore, analysis of off-target mutations using deep sequencing indicated that our customized gRNAs induced no additional mutations in the host genome. Regenerated mutants exhibited morphological changes in leaf shape and seedless fruit-a characteristic of parthenocarpic tomato. And the segregated next generation (T1) also showed a severe phenotype associated with the homozygous mutated genome. The system developed here could be applied to produce parthenocarpic tomato in a wide variety of cultivars, as well as other major horticultural crops, using this precise and rapid breeding technique.
Kohji Murase, Shuji Shigenobu, Sota Fujii, Kazuki Ueda, Takanori Murata, Ai Sakamoto, Yuko Wada, Katsushi Yamaguchi, Yuriko Osakabe, Keishi Osakabe, Akira Kanno, Yukio Ozaki and Seiji Takayama : MYB transcription factor gene involved in sex determination in Asparagus officinalis, Genes to Cells, Vol.22, No.1, 115-123, 2017.
(要約)
Dioecy is a plant mating system in which individuals of a species are either male or female. Although many flowering plants evolved independently from hermaphroditism to dioecy, the molecular mechanism underlying this transition remains largely unknown. Sex determination in the dioecious plant Asparagus officinalis is controlled by X and Y chromosomes; the male and female karyotypes are XY and XX, respectively. Transcriptome analysis of A. officinalis buds showed that a MYB-like gene, Male Specific Expression 1 (MSE1), is specifically expressed in males. MSE1 exhibits tight linkage with the Y chromosome, specific expression in early anther development and loss of function on the X chromosome. Knockout of the MSE1 orthologue in Arabidopsis induces male sterility. Thus, MSE1 acts in sex determination in A. officinalis.
Fujimoto Satoru, Sugano S. Shigeo, Kuwata Keiko, Keishi Osakabe and Matsunaga Sachihiro : Visualization of specific repetitive genomic sequences with fluorescent TALEs in Arabidopsis thaliana., Journal of Experimental Botany, Vol.67, No.21, 6101-6110, 2016.
(要約)
Live imaging of the dynamics of nuclear organization provides the opportunity to uncover the mechanisms responsible for four-dimensional genome architecture. Here, we describe the use of fluorescent protein (FP) fusions of transcription activator-like effectors (TALEs) to visualize endogenous genomic sequences in Arabidopsis thaliana. The ability to engineer sequence-specific TALEs permits the investigation of precise genomic sequences. We could detect TALE-FP signals associated with centromeric, telomeric, and rDNA repeats and the signal distribution was consistent with that observed by fluorescent in situ hybridization. TALE-FPs are advantageous because they permit the observation of intact tissues. We used our TALE-FP method to investigate the nuclei of several multicellular plant tissues including roots, hypocotyls, leaves, and flowers. Because TALE-FPs permit live-cell imaging, we successfully observed the temporal dynamics of centromeres and telomeres in plant organs. Fusing TALEs to multimeric FPs enhanced the signal intensity when observing telomeres. We found that the mobility of telomeres was different in sub-nuclear regions. Transgenic plants stably expressing TALE-FPs will provide new insights into chromatin organization and dynamics in multicellular organisms.
T. Nomura, T. Sakurai, Yuriko Osakabe, Keishi Osakabe and H. Sakakibara : Efficient and heritable targeted mutagenesis in mosses using the CRISPR/Cas9 system, Plant & Cell Physiology, Vol.57, No.12, 2600-2610, 2016.
(要約)
Targeted genome modification by RNA-guided nucleases derived from the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9) system has seen rapid development in many organisms, including several plant species. In the present study, we succeeded in introducing the CRISPR/Cas9 system into the non-model organism Scopelophila cataractae, a moss that exhibits heavy metal tolerance, and the model organism Physcomitrella patens Utilizing the process by which moss plants regenerate from protoplasts, we conducted targeted mutagenesis by expression of single-chain guide RNA (sgRNA) and Cas9 in protoplasts. Using this method, the acquisition rate of strains exhibiting phenotypic changes associated with the target genes was approximately 45-69%, and strains with phenotypic changes exhibited various insertion and deletion mutations. In addition, we report that our method is capable of multiplex targeted mutagenesis (two independent genes) and also permits the efficient introduction of large deletions (∼3 kbp). These results demonstrate that the CRISPR/Cas9 system can be used to accelerate investigations of bryology and land plant evolution.
Chikako Nishitani, Narumi Hirai, Sadao Komori, Masato Wada, Kazuma Okada, Keishi Osakabe, Toshiya Yamamoto and Yuriko Osakabe : Efficient Genome Editing in Apple Using a CRISPR/Cas9 system., Scientific Reports, Vol.6, 31481, 2016.
(要約)
Genome editing is a powerful technique for genome modification in molecular research and crop breeding, and has the great advantage of imparting novel desired traits to genetic resources. However, the genome editing of fruit tree plantlets remains to be established. In this study, we describe induction of a targeted gene mutation in the endogenous apple phytoene desaturase (PDS) gene using the CRISPR/Cas9 system. Four guide RNAs (gRNAs) were designed and stably transformed with Cas9 separately in apple. Clear and partial albino phenotypes were observed in 31.8% of regenerated plantlets for one gRNA, and bi-allelic mutations in apple PDS were confirmed by DNA sequencing. In addition, an 18-bp gRNA also induced a targeted mutation. These CRIPSR/Cas9 induced-mutations in the apple genome suggest activation of the NHEJ pathway, but with some involvement also of the HR pathway. Our results demonstrate that genome editing can be practically applied to modify the apple genome.
Yuriko Osakabe, Takahito Watanabe, SS Sugano, R Ueta, R Ishihara, K Shinozaki and Keishi Osakabe : Optimization of CRISPR/Cas9 genome editing to modify abiotic stress responses in plants., Scientific Reports, Vol.6, 26685, 2016.
(要約)
Genome editing using the CRISPR/Cas9 system can be used to modify plant genomes, however, improvements in specificity and applicability are still needed in order for the editing technique to be useful in various plant species. Here, using genome editing mediated by a truncated gRNA (tru-gRNA)/Cas9 combination, we generated new alleles for OST2, a proton pump in Arabidopsis, with no off-target effects. By following expression of Cas9 and the tru-gRNAs, newly generated mutations in CRIPSR/Cas9 transgenic plants were detected with high average mutation rates of up to 32.8% and no off-target effects using constitutive promoter. Reducing nuclear localization signals in Cas9 decreased the mutation rate. In contrast, tru-gRNA Cas9 cassettes driven by meristematic- and reproductive-tissue-specific promoters increased the heritable mutation rate in Arabidopsis, showing that high expression in the germ line can produce bi-allelic mutations. Finally, the new mutant alleles obtained for OST2 exhibited altered stomatal closing in response to environmental conditions. These results suggest further applications in molecular breeding to improve plant function using optimized plant CRISPR/Cas9 systems.
Ayako Nishizawa-Yokoi, Tomas Cermak, Tomoki Hoshino, Kazuhiko Sugimoto, Hiroaki Saika, Akiko Mori, Keishi Osakabe, Masao Hamada, Yuichi Katayose, Colby Starker, Daniel F. Voytas and Seiichi Toki : A Defect in DNA Ligase4 Enhances the Frequency of TALEN-Mediated Targeted Mutagenesis in Rice., Plant Physiology, Vol.170, No.2, 653-666, 2015.
(要約)
We have established methods for site-directed mutagenesis via transcription activator-like effector nucleases (TALENs) in the endogenous rice (Oryza sativa) waxy gene and demonstrated stable inheritance of TALEN-induced somatic mutations to the progeny. To analyze the role of classical nonhomologous end joining (cNHEJ) and alternative nonhomologous end joining (altNHEJ) pathways in TALEN-induced mutagenesis in plant cells, we investigated whether a lack of DNA Ligase4 (Lig4) affects the kinetics of TALEN-induced double-strand break repair in rice cells. Deep-sequencing analysis revealed that the frequency of all types of mutations, namely deletion, insertion, combination of insertion with deletion, and substitution, in lig4 null mutant calli was higher than that in a lig4 heterozygous mutant or the wild type. In addition, the ratio of large deletions (greater than 10 bp) and deletions repaired by microhomology-mediated end joining (MMEJ) to total deletion mutations in lig4 null mutant calli was higher than that in the lig4 heterozygous mutant or wild type. Furthermore, almost all insertions (2 bp or greater) were shown to be processed via copy and paste of one or more regions around the TALENs cleavage site and rejoined via MMEJ regardless of genetic background. Taken together, our findings indicate that the dysfunction of cNHEJ leads to a shift in the repair pathway from cNHEJ to altNHEJ or synthesis-dependent strand annealing.
Ayako Nishizawa-Yokoi, Satoko Nonaka, Keishi Osakabe, Hiroaki Saika and Seiichi Toki : A universal positive-negative selection system for gene targeting in plants combining an antibiotic resistance gene and its antisense RNA., Plant Physiology, Vol.169, No.1, 362-370, 2015.
(要約)
Gene targeting (GT) is a useful technology for accurate genome engineering in plants. A reproducible approach based on a positive-negative selection system using hygromycin-resistance and the diphtheria toxin A subunit gene (DT-A) as positive and negative selection markers, respectively, is now available. However, to date this selection system has been applied exclusively in rice. To establish a universally applicable positive-negative GT system in plants, we designed a selection system using a combination of nptII and an antisense nptII construct. The concomitant transcription of both sense and antisense nptII suppresses significantly the level of expression of the sense nptII gene, and transgenic calli and plants become sensitive to the antibiotic G418. In addition, we were able to utilize the sense nptII gene as a positive selection marker and the antisense nptII construct as a negative selection marker for knock-out of the endogenous rice genes Waxy and Glb33 via GT. The approach developed here could be applied as a universal selection system for the enrichment of GT cells in several plant species.
Yuriko Osakabe and Keishi Osakabe : Genome editing with engineered nucleases in plants., Plant & Cell Physiology, Vol.56, No.3, 389-400, 2015.
(要約)
Numerous examples of successful 'genome editing' now exist. Genome editing uses engineered nucleases as powerful tools to target specific DNA sequences to edit genes precisely in the genomes of both model and crop plants, as well as a variety of other organisms. The DNA-binding domains of zinc finger (ZF) proteins were the first to be used as genome editing tools, in the form of designed ZF nucleases (ZFNs). More recently, transcription activator-like effector nucleases (TALENs), as well as the clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) system, which utilizes RNA-DNA interactions, have proved useful. A key step in genome editing is the generation of a double-stranded DNA break that is specific to the target gene. This is achieved by custom-designed endonucleases, which enable site-directed mutagenesis via a non-homologous end-joining (NHEJ) repair pathway and/or gene targeting via homologous recombination (HR) to occur efficiently at specific sites in the genome. This review provides an overview of recent advances in genome editing technologies in plants, and discusses how these can provide insights into current plant molecular biology research and molecular breeding technology.
Hiroaki Saika, Akiko Mori, Masaki Endo, Keishi Osakabe and Seiichi Toki : Rapid evaluation of the frequency of gene targeting in rice via a convenient positive-negative selection method, Plant Biotechnology, Vol.32, No.2, 169-173, 2015.
(要約)
Although gene targeting (GT) is a useful technology for precise mutagenesis of target sequences, its frequency is quite low. Establishing experimental procedures using a model system will enable us to improve this frequency and apply to GT as a universal system. Here, we propose a convenient system with which to evaluate the frequency of site-directed mutagenesis via GT using a positive-negative selection method. We constructed a GT vector harboring a partial rice acetolactate synthase gene with mutations conferring bispyribac sodium (BS) tolerance and a gene conferring blasticidin-S tolerance as a positive selection marker. In addition, diphtheria toxin A subunit gene was used as a negative selection marker to enrich GT cells. We regenerated GT candidate plants successfully at a frequency of 2.1 putative GT events/gram Agrobacterium-infected callus following dual selection on BS and blasticidin-S. Moreover, molecular analyses confirmed that GT events occurred in >80% of regenerated plants. Existing GT methods using positive-negative selection require that true putative GT events be verified by molecular analysis because of the growth of large numbers of cells in which partial GT vectors containing positive selection marker cassettes, but lacking the negative selection marker, have inserted at random loci. In contrast, the present method with dual selection on both BS and blasticidin-S allows direct enrichment of GT cells at high frequency without the need for further extensive molecular screening.
Yuriko Osakabe, Keishi Osakabe, Kazuo Shinozaki and Lam-Son P. Tran : Response of plants to water stress., Frontiers in Plant Science, Vol.5, 86, 2014.
(要約)
Water stress adversely impacts many aspects of the physiology of plants, especially photosynthetic capacity. If the stress is prolonged, plant growth, and productivity are severely diminished. Plants have evolved complex physiological and biochemical adaptations to adjust and adapt to a variety of environmental stresses. The molecular and physiological mechanisms associated with water-stress tolerance and water-use efficiency have been extensively studied. The systems that regulate plant adaptation to water stress through a sophisticated regulatory network are the subject of the current review. Molecular mechanisms that plants use to increase stress tolerance, maintain appropriate hormone homeostasis and responses and prevent excess light damage, are also discussed. An understanding of how these systems are regulated and ameliorate the impact of water stress on plant productivity will provide the information needed to improve plant stress tolerance using biotechnology, while maintaining the yield and quality of crops.
Keishi Osakabe, Ayako Nishizawa-Yokoi, Namie Ohtsuki, Yuriko Osakabe and Seiichi Toki : A mutated cytosine deaminase gene, codA (D314A), as an efficient negative selection marker for gene targeting in rice., Plant & Cell Physiology, Vol.55, No.3, 658-665, 2013.
(要約)
Gene targeting (GT) is a powerful tool manipulating a gene of interest in a given genome specifically and precisely. To achieve efficient GT in higher plants, both positive and negative selection markers are required. In particular, a strong negative selection system is needed for enrichment of cells to eliminate those cells in which random integration of the introduced DNA has occurred in GT experiments. Currently, non-conditional negative selection marker genes are used for GT experiments in rice plants, and no conditional negative selection system is available. In this study, we describe the development of an efficient conditional negative selection system in rice plants using Escherichia coli cytosine deaminase (codA). We found that a mutant codA gene, codA(D314A), acts more efficiently than the wild-type codA for negative selection in rice plants. The codA(D314A) marker was further used as a negative selection marker for GT experiments in rice. Our conditional negative selection system effectively eliminated the cells in which random integration event(s) occurred; the enrichment factor was approximately 100-fold. This enrichment factor was similar to that found when Corynebacterium diphtheriae toxin fragment A was used. Our results suggest the codA(D314A) marker gene as a promising negative selection marker for GT of rice.
Yoshinao Hara, Ryusuke Yokoyama, Keishi Osakabe, Seiichi Toki and Kazuhiko Nishitani : Function of xyloglucan endotransglucosylase/hydrolases in rice., Annals of Botany, Vol.114, No.6, 1309-1318, 2013.
(要約)
Although xyloglucans are ubiquitous in land plants, they are less abundant in Poales species than in eudicotyledons. Poales cell walls contain higher levels of -1,3/1,4 mixed-linked glucans and arabinoxylans than xyloglucans. Despite the relatively low level of xyloglucans in Poales, the xyloglucan endotransglucosylase/hydrolase (XTH) gene family in rice (Oryza sativa) is comparable in size to that of the eudicotyledon Arabidopsis thaliana. This raises the question of whether xyloglucan is a substrate for rice XTH gene products, whose enzyme activity remains largely uncharacterized. This study focused on OsXTH19 (which belongs to Group IIIA of the XTH family and is specifically expressed in growing tissues of rice shoots), and two other XTHs, OsXTH11 (Group I/II) and OsXTH20 (Group IIIA), for reference, and measurements were made of the enzymatic activities of three recombinant rice XTHs, i.e. OsXTH11, OsXTH20 and OsXTH19. All three OsXTH gene products have xyloglucan endohydrolase (XEH, EC 3·2·1·151) activity, and OsXTH11 has both XEH and xyloglucan endotransglycosylase (XET, EC 2·4·1207) activities. However, these proteins had neither hydrolase nor transglucosylase activity when glucuronoarabinoxylan or mixed-linkage glucan was used as the substrate. These results are consistent with histological observations demonstrating that pOsXTH19::GUS is expressed specifically in the vicinity of tissues where xyloglucan immunoreactivity is present. Transgenic rice lines over-expressing OsXTH19 (harbouring a Cauliflower Mosaic Virus 35S promoter::OsXTH19 cDNA construct) or with suppressed OsXTH19 expression (harbouring a pOsXTH19 RNAi construct) did not show dramatic phenotypic changes, suggesting functional redundancy and collaboration among XTH family members, as was observed in A. thaliana. OsXTH20 and OsXTH19 act as hydrolases exclusively on xyloglucan, while OsXTH11 exhibits both hydrolase and XET activities exclusively on xyloglucans. Phenotypic analysis of transgenic lines with altered expression of OsXTH19 suggests that OsXTH19 and related XTH(s) play redundant roles in rice growth.
Ayako Nishizawa-Yokoi, Masaki Endo, Keishi Osakabe, Hiroaki Saika and Seiichi Toki : Precise marker excision system using an animal-derived piggyBac transposon in plants., The Plant Journal : for Cell and Molecular Biology, Vol.77, No.3, 454-463, 2013.
(要約)
Accurate and effective positive marker excision is indispensable for the introduction of desired mutations into the plant genome via gene targeting (GT) using a positive/negative counter selection system. In mammals, the moth-derived piggyBac transposon system has been exploited successfully to eliminate a selectable marker from a GT locus without leaving a footprint. Here, we present evidence that the piggyBac transposon also functions in plant cells. To demonstrate the use of the piggyBac transposon for effective marker excision in plants, we designed a transposition assay system that allows the piggyBac transposition to be visualized as emerald luciferase (Eluc) luminescence in rice cells. The Eluc signal derived from piggyBac excision was observed in hyperactive piggyBac transposase-expressing rice calli. Polymerase chain reaction, Southern blot analyses and sequencing revealed the efficient and precise transposition of piggyBac in these calli. Furthermore, we have demonstrated the excision of a selection marker from a reporter locus in T0 plants without concomitant re-integration of the transposon and at a high frequency (44.0% of excision events), even in the absence of negative selection.
Yong-Ik Kwon, Kiyomi Abe, Masaki Endo, Keishi Osakabe, Namie Ohtsuki, Ayako Nishizawa-Yokoi, Akemi Tagiri, Hiroaki Saika and Seiichi Toki : DNA replication arrest leads to enhanced homologous recombination and cell death in meristems of rice OsRecQl4 mutants., BMC Plant Biology, Vol.13, 62, 2013.
(要約)
Mammalian BLM helicase is involved in DNA replication, DNA repair and homologous recombination (HR). These DNA transactions are associated tightly with cell division and are important for maintaining genome stability. However, unlike in mammals, cell division in higher plants is restricted mainly to the meristem, thus genome maintenance at the meristem is critical. The counterpart of BLM in Arabidopsis (AtRecQ4A) has been identified and its role in HR and in the response to DNA damage has been confirmed. However, the function of AtRecQ4A in the meristem during replication stress has not yet been well elucidated. We isolated the BLM counterpart gene OsRecQl4 from rice and analyzed its function using a reverse genetics approach. Osrecql4 mutant plants showed hypersensitivity to DNA damaging agents and enhanced frequency of HR compared to wild-type (WT) plants. We further analyzed the effect of aphidicolin--an inhibitor of S-phase progression via its inhibitory effect on DNA polymerases--on genome stability in the root meristem in osrecql4 mutant plants and corresponding WT plants. The following effects were observed upon aphidicolin treatment: a) comet assay showed induction of DNA double-strand breaks (DSBs) in mutant plants, b) TUNEL assay showed enhanced DNA breaks at the root meristem in mutant plants, c) a recombination reporter showed enhanced HR frequency in mutant calli, d) propidium iodide (PI) staining of root tips revealed an increased incidence of cell death in the meristem of mutant plants. These results demonstrate that the aphidicolin-sensitive phenotype of osrecql4 mutants was in part due to induced DSBs and cell death, and that OsRecQl4 plays an important role as a caretaker, maintaining genome stability during DNA replication stress in the rice meristem.
(キーワード)
Cell Death / DNA Breaks, Double-Stranded / DNA Replication / Homologous Recombination / Meristem / Mutation / Oryza sativa / Plant Proteins / RecQ Helicases / S Phase Cell Cycle Checkpoints
Yong-Ik Kwon, Kiyomi Abe, Keishi Osakabe, Masaki Endo, Ayako Nishizawa-Yokoi, Hiroaki Saika, Hiroaki Shimada and Seiichi Toki : Overexpression of OsRecQl4 and/or OsExo1 enhances DSB-induced homologous recombination in rice., Plant & Cell Physiology, Vol.53, No.12, 2142-2152, 2012.
(要約)
During homologous recombination (HR)-mediated DNA double-strand break (DSB) repair in eukaryotes, an initial step is the creation of a 3'-single-stranded DNA (ssDNA) overhang via resection of a 5' end. Rad51 polymerizes on this ssDNA to search for a homologous sequence, and the gapped sequence is then repaired using an undamaged homologous DNA strand as template. Recent studies in eukaryotes indicate that resection of the DSB site is promoted by the cooperative action of RecQ helicase family proteins: Bloom helicase (BLM) in mammals or Sgs1 in yeast, and exonuclease 1 (Exo1). However, the role of RecQ helicase and exonuclease during the 5'-resection process of HR in plant cells has not yet been defined. Here, we demonstrate that overexpression of rice proteins OsRecQl4 (BLM counterpart) and/or OsExo1 (Exo1 homolog) can enhance DSB processing, as evaluated by recombination substrate reporter lines in rice. These results could be applied to construct an efficient gene targeting system in rice.
Ayako Nishizawa-Yokoi, Satoko Nonaka, Hiroaki Saika, Yong-Ik Kwon, Keishi Osakabe and Seiichi Toki : Suppression of Ku70/80 or Lig4 leads to decreased stable transformation and enhanced homologous recombination in rice., The New Phytologist, Vol.196, No.4, 1048-1059, 2012.
(要約)
Evidence for the involvement of the nonhomologous end joining (NHEJ) pathway in Agrobacterium-mediated transferred DNA (T-DNA) integration into the genome of the model plant Arabidopsis remains inconclusive. Having established a rapid and highly efficient Agrobacterium-mediated transformation system in rice (Oryza sativa) using scutellum-derived calli, we examined here the involvement of the NHEJ pathway in Agrobacterium-mediated stable transformation in rice. Rice calli from OsKu70, OsKu80 and OsLig4 knockdown (KD) plants were infected with Agrobacterium harboring a sensitive emerald luciferase (LUC) reporter construct to evaluate stable expression and a green fluorescent protein (GFP) construct to monitor transient expression of T-DNA. Transient expression was not suppressed, but stable expression was reduced significantly, in KD plants. Furthermore, KD-Ku70 and KD-Lig4 calli exhibited an increase in the frequency of homologous recombination (HR) compared with control calli. In addition, suppression of OsKu70, OsKu80 and OsLig4 induced the expression of HR-related genes on treatment with DNA-damaging agents. Our findings suggest strongly that NHEJ is involved in Agrobacterium-mediated stable transformation in rice, and that there is a competitive and complementary relationship between the NHEJ and HR pathways for DNA double-strand break repair in rice.
(キーワード)
Agrobacterium / DNA Breaks, Double-Stranded / DNA End-Joining Repair / DNA Helicases / DNA Ligases / DNA Repair / DNA, Bacterial / DNA-Binding Proteins / Gene Expression Regulation, Plant / Gene Knockdown Techniques / Green Fluorescent Proteins / Homologous Recombination / Oryza sativa / Plant Proteins / Plants, Genetically Modified / Transformation, Genetic
Hiroaki Saika, Satoko Nonaka, Keishi Osakabe and Seiichi Toki : Sequential monitoring of transgene expression following Agrobacterium-mediated transformation of rice., Plant & Cell Physiology, Vol.53, No.11, 1974-1983, 2012.
(要約)
Although Agrobacterium-mediated transformation technology is now used widely in rice, many varieties of indica-type rice are still recalcitrant to Agrobacterium-mediated transformation. It was reported recently that T-DNA integration into the rice genome could be the limiting step in this method. Here, we attempted to establish an efficient sequential monitoring system for stable transformation events by visualizing stable transgene expression using a non-destructive and highly sensitive visible marker. Our results demonstrate that click beetle luciferase (ELuc) is an excellent marker allowing the observation of transformed cells in rice callus, exhibiting a sensitivity >30-fold higher than that of firefly luciferase. Since we have previously shown that green fluorescent protein (GFP) is a useful visual marker with which to follow transient and/or stable expression of transgenes in rice, we constructed an enhancer trap vector using both the gfbsd2 (GFP fused to the N-terminus of blasticidin S deaminase) and eluc genes. In this vector, the eluc gene is under the control of the Cauliflower mosaic virus 35S minimal promoter, while the gfbsd2 gene is under the control of the full-length rice elongation factor gene promoter. Observation of transformed callus under a dissecting microscope demonstrated that the level of ELuc luminescence reflected exclusively stable transgene expression, and that both transient and stable expression could be monitored by the level of GFP fluorescence. Moreover, we show that our system enables sequential quantification of transgene expression via differential measurement of ELuc luminescence and GFP fluorescence.
Yuriko Osakabe, Akiyoshi Kawaoka, Nobuyuki Nishikubo and Keishi Osakabe : Responses to environmental stresses in woody plants: key to survive and longevity., Journal of Plant Research, Vol.125, No.1, 1-10, 2011.
(要約)
Environmental stresses have adverse effects on plant growth and productivity, and are predicted to become more severe and widespread in decades to come. Especially, prolonged and repeated severe stresses affecting growth and development would bring down long-lasting effects in woody plants as a result of its long-term growth period. To counteract these effects, trees have evolved specific mechanisms for acclimation and tolerance to environmental stresses. Plant growth and development are regulated by the integration of many environmental and endogenous signals including plant hormones. Acclimation of land plants to environmental stresses is controlled by molecular cascades, also involving cross-talk with other stresses and plant hormone signaling mechanisms. This review focuses on recent studies on molecular mechanisms of abiotic stress responses in woody plants, functions of plant hormones in wood formation, and the interconnection of cell wall biosynthesis and the mechanisms shown above. Understanding of these mechanisms in depth should shed light on the factors for improvement of woody plants to overcome severe environmental stress conditions.
Yuriko Osakabe, Shinya Kajita and Keishi Osakabe : Genetic engineering of woody plants: current and future targets in a stressful environment., Physiologia Plantarum, Vol.142, No.2, 105-117, 2011.
(要約)
Abiotic stress is a major factor in limiting plant growth and productivity. Environmental degradation, such as drought and salinity stresses, will become more severe and widespread in the world. To overcome severe environmental stress, plant biotechnologies, such as genetic engineering in woody plants, need to be implemented. The adaptation of plants to environmental stress is controlled by cascades of molecular networks including cross-talk with other stress signaling mechanisms. The present review focuses on recent studies concerning genetic engineering in woody plants for the improvement of the abiotic stress responses. Furthermore, it highlights the recent advances in the understanding of molecular responses to stress. The review also summarizes the basis of a molecular mechanism for cell wall biosynthesis and the plant hormone responses to regulate tree growth and biomass in woody plants. This would facilitate better understanding of the control programs of biomass production under stressful conditions.
Keishi Osakabe, Yuriko Osakabe and Seiichi Toki : Site-directed mutagenesis in Arabidopsis using custom-designed zinc finger nucleases., Proceedings of the National Academy of Sciences of the United States of America, Vol.107, No.26, 12034-12039, 2010.
(要約)
Site-directed mutagenesis in higher plants remains a significant technical challenge for basic research and molecular breeding. Here, we demonstrate targeted-gene inactivation for an endogenous gene in Arabidopsis using zinc finger nucleases (ZFNs). Engineered ZFNs for a stress-response regulator, the ABA-INSENSITIVE4 (ABI4) gene, cleaved their recognition sequences specifically in vitro, and ZFN genes driven by a heat-shock promoter were introduced into the Arabidopsis genome. After heat-shock induction, gene mutations with deletion and substitution in the ABI4 gene generated via ZFN-mediated cleavage were observed in somatic cells at frequencies as high as 3%. The homozygote mutant line zfn_abi4-1-1 for ABI4 exhibited the expected mutant phenotypes, i.e., ABA and glucose insensitivity. In addition, ZFN-mediated mutagenesis was applied to the DNA repair-deficient mutant plant, atku80. We found that lack of AtKu80, which plays a role in end-protection of dsDNA breaks, increased error-prone rejoining frequency by 2.6-fold, with increased end-degradation. These data demonstrate that an approach using ZFNs can be used for the efficient production of mutant plants for precision reverse genetics.
(キーワード)
Amino Acid Sequence / Arabidopsis / Arabidopsis Proteins / Base Sequence / DNA Breaks, Double-Stranded / DNA Helicases / DNA Repair / DNA, Plant / Deoxyribonucleases / Genes, Plant / Molecular Sequence Data / Mutagenesis, Site-Directed / Mutation / Plants, Genetically Modified / Promoter Regions, Genetic / Protein Engineering / Recombinant Proteins / Transcription Factors
Yuriko Osakabe, Shinji Mizuno, Hidenori Tanaka, Kyonoshin Maruyama, Keishi Osakabe, Daisuke Todaka, Yasunari Fujita, Masatomo Kobayashi, Kazuo Shinozaki and Kazuko Yamaguchi-Shinozaki : Overproduction of the membrane-bound receptor-like protein kinase 1, RPK1, enhances abiotic stress tolerance in Arabidopsis., The Journal of Biological Chemistry, Vol.285, No.12, 9190-9201, 2010.
(要約)
RPK1 (receptor-like protein kinase 1) localizes to the plasma membrane and functions as a regulator of abscisic acid (ABA) signaling in Arabidopsis. In our current study, we investigated the effect of RPK1 disruption and overproduction upon plant responses to drought stress. Transgenic Arabidopsis overexpressing the RPK1 protein showed increased ABA sensitivity in their root growth and stomatal closure and also displayed less transpirational water loss. In contrast, a mutant lacking RPK1 function, rpk1-1, was found to be resistant to ABA during these processes and showed increased water loss. RPK1 overproduction in these transgenic plants thus increased their tolerance to drought stress. We performed microarray analysis of RPK1 transgenic plants and observed enhanced expression of several stress-responsive genes, such as Cor15a, Cor15b, and rd29A, in addition to H(2)O(2)-responsive genes. Consistently, the expression levels of ABA/stress-responsive genes in rpk1-1 had decreased compared with wild type. The results suggest that the overproduction of RPK1 enhances both the ABA and drought stress signaling pathways. Furthermore, the leaves of the rpk1-1 plants exhibit higher sensitivity to oxidative stress upon ABA-pretreatment, whereas transgenic plants overproducing RPK1 manifest increased tolerance to this stress. Our current data suggest therefore that RPK1 overproduction controls reactive oxygen species homeostasis and enhances both water and oxidative stress tolerance in Arabidopsis.
Yuriko Osakabe, Keishi Osakabe and Vincent L. Chiang : Isolation of 4-coumarate Co-A ligase gene promoter from loblolly pine (Pinus taeda) and characterization of tissue-specific activity in transgenic tobacco., Plant Physiology and Biochemistry : PPB, Vol.47, No.11-12, 1031-1036, 2009.
(要約)
We characterized promoter activity of a phenylpropanoid biosynthetic gene encoding 4-coumarate Co-A ligase (4CL), Pta4Clalpha, from Pinus taeda. Histochemical- and quantitative assays of GUS expression in the vascular tissue were performed using transgenic tobacco plants expressing promoter-GUS reporters. Deletion analysis of the Pta4Clalpha promoter showed that the region -524 to -252, which has two AC elements, controls the high expression levels in ray-parenchyma cells of older tobacco stems. High activity level of the promoter domain of Pta4CLalpha was also detected in the xylem cells under bending stress. DNA-protein complexes were detected in the reactions of the Pta4CLalpha promoter fragments with the nuclear proteins of xylem of P. taeda. The AC elements in the Pta4CLalpha promoter appeared to have individual roles during xylem development that are activated in a coordinated manner in response to stress in transgenic tobacco.
Yuriko Osakabe, Keishi Osakabe and Vincent L. Chiang : Characterization of the tissue-specific expression of phenylalanine ammonia-lyase gene promoter from loblolly pine (Pinus taeda) in Nicotiana tabacum., Plant Cell Reports, Vol.28, No.9, 1309-1317, 2009.
(要約)
We isolated the 5' flanking region of a gene for phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) from Pinus taeda, PtaPAL. To investigate the tissue-specific expression of the PtaPAL promoter, histochemical assay of GUS activity was performed using the transgenic tobacco expressing the PtaPAL promoter-GUS. The region of -897 to -420 in PtaPAL promoter showed high activities in the secondary xylem and response to bending stress. To characterize the cis-regulatory functions of the promoters for enzymes in phenylpropanoid biosynthesis, we examined the activity of chimeric promoters of PtaPAL and a 4-coumarate CoA ligase, Pta4CL alpha. The chimeric promoter showed similar activity as the Pta4CL alpha promoter. Electrophoretic mobility shift assays implicated -897 to -674 of PtaPAL promoter containing cis-elements of the expression in xylem of Pinus taeda. The results suggested that AC elements of PtaPAL have multiple functions in the expression under the various developmental stages and stress conditions in the transgenic tobacco.
Kiyomi Abe, Keishi Osakabe, Yuichi Ishikawa, Akemi Tagiri, Hiroaki Yamanouchi, Toshio Takyuu, Terutaka Yoshioka, Takuya Ito, Masatomo Kobayashi, Kazuo Shinozaki, Hiroaki Ichikawa and Seiichi Toki : Inefficient double-strand DNA break repair is associated with increased fasciation in Arabidopsis BRCA2 mutants., Journal of Experimental Botany, Vol.60, No.9, 2751-2761, 2009.
(要約)
BRCA2 is a breast tumour susceptibility factor with functions in maintaining genome stability through ensuring efficient double-strand DNA break (DSB) repair via homologous recombination. Although best known in vertebrates, fungi, and higher plants also possess BRCA2-like genes. To investigate the role of Arabidopsis BRCA2 genes in DNA repair in somatic cells, transposon insertion mutants of the AtBRCA2a and AtBRCA2b genes were identified and characterized. atbrca2a-1 and atbrca2b-1 mutant plants showed hypersensitivity to genotoxic stresses compared to wild-type plants. An atbrca2a-1/atbrca2b-1 double mutant showed an additive increase in sensitivity to genotoxic stresses compared to each single mutant. In addition, it was found that atbrca2 mutant plants displayed fasciation and abnormal phyllotaxy phenotypes with low incidence, and that the ratio of plants exhibiting these phenotypes is increased by gamma-irradiation. Interestingly, these phenotypes were also induced by gamma-irradiation in wild-type plants. Moreover, it was found that shoot apical meristems of the atbrca2a-1/atbrca2b-1 double mutant show altered cell cycle progression. These data suggest that inefficient DSB repair in the atbrca2a-1/atbrca2b-1 mutant leads to disorganization of the programmed cell cycle of apical meristems.
(キーワード)
Arabidopsis / Arabidopsis Proteins / BRCA2 Protein / Cell Cycle / DNA Breaks, Double-Stranded / DNA Damage / DNA Repair / Meristem / Mutation
Masaki Endo, Keishi Osakabe, Kazuko Ono, Hirokazu Handa, Tsutomu Shimizu and Seiichi Toki : Molecular breeding of a novel herbicide-tolerant rice by gene targeting., The Plant Journal : for Cell and Molecular Biology, Vol.52, No.1, 157-166, 2007.
(要約)
We have previously reported the production of a rice cell line tolerant to the acetolactate synthase (ALS)-inhibiting herbicide bispyribac (BS), and demonstrated that the BS-tolerant phenotype was due to a double mutation in the rice ALS gene. We further indicated that while changing either of the two amino acids (W548 L or S627I) individually resulted in a BS-tolerant phenotype, conversion of both amino acids simultaneously conferred increased tolerance to BS. As the BS-tolerant cell line had lost the ability to regenerate during two years of tissue culture selection, we attempted to introduce these two point mutations into the rice ALS gene via gene targeting (GT). Using our highly efficient Agrobacterium-mediated transformation system in rice, we were able to regenerate 66 independent GT rice plants from 1500 calli. Furthermore, two-thirds of these plants harbored the two point mutations exclusively, without any insertion of foreign DNA such as border sequences of T-DNA. The GT plants obtained in the present study are therefore equivalent to non-GM herbicide-tolerant rice plants generated by conventional breeding approaches that depend on spontaneous mutations. Surprisingly, GT rice homozygous for the modified ALS locus showed hyper-tolerance to BS when compared to BS-tolerant plants produced by a conventional transgenic system; ALS enzymatic activity in plants homozygous for the mutated ALS gene was inhibited only by extremely high concentrations of BS. These results indicate that our GT method has successfully created novel herbicide-tolerant rice plants that are superior to those produced by conventional mutation breeding protocols or transgenic technology.
Shinji Mizuno, Yuriko Osakabe, Kyonoshin Maruyama, Takuya Ito, Keishi Osakabe, Takahide Sato, Kazuo Shinozaki and Kazuko Yamaguchi-Shinozaki : Receptor-like protein kinase 2 (RPK 2) is a novel factor controlling anther development in Arabidopsis thaliana., The Plant Journal : for Cell and Molecular Biology, Vol.50, No.5, 751-766, 2007.
(要約)
Receptor-like kinases (RLK) comprise a large gene family within the Arabidopsis genome and play important roles in plant growth and development as well as in hormone and stress responses. Here we report that a leucine-rich repeat receptor-like kinase (LRR-RLK), RECEPTOR-LIKE PROTEIN KINASE2 (RPK2), is a key regulator of anther development in Arabidopsis. Two RPK2 T-DNA insertional mutants (rpk2-1 and rpk2-2) displayed enhanced shoot growth and male sterility due to defects in anther dehiscence and pollen maturation. The rpk2 anthers only developed three cell layers surrounding the male gametophyte: the middle layer was not differentiated from inner secondary parietal cells. Pollen mother cells in rpk2 anthers could undergo meiosis, but subsequent differentiation of microspores was inhibited by tapetum hypertrophy, with most resulting pollen grains exhibiting highly aggregated morphologies. The presence of tetrads and microspores in individual anthers was observed during microspore formation, indicating that the developmental homeostasis of rpk2 anther locules was disrupted. Anther locules were finally crushed without stomium breakage, a phenomenon that was possibly caused by inadequate thickening and lignification of the endothecium. Microarray analyses revealed that many genes encoding metabolic enzymes, including those involved in cell wall metabolism and lignin biosynthesis, were downregulated throughout anther development in rpk2 mutants. RPK2 mRNA was abundant in the tapetum of wild-type anthers during microspore maturation. These results suggest that RPK2 controls tapetal cell fate by triggering subsequent tapetum degradation, and that mutating RPK2 impairs normal pollen maturation and anther dehiscence due to disruption of key metabolic pathways.
Naozumi Mimida, Hiroko Kitamoto, Keishi Osakabe, Marina Nakashima, Yuji Ito, Wolf-Dietrich Heyer, Seiichi Toki and Hiroaki Ichikawa : Two alternatively spliced transcripts generated from OsMUS81, a rice homolog of yeast MUS81, are up-regulated by DNA-damaging treatments., Plant & Cell Physiology, Vol.48, No.4, 648-654, 2007.
(要約)
OsMUS81, a rice homolog of the yeast MUS81 endonuclease gene, produced two alternative transcripts, OsMUS81alpha and OsMUS81beta. OsMus81alpha contained a Helix-hairpin-Helix (HhH) motif at the N- and C-termini, and a conserved XPF-like motif in the center, while the OsMus81beta isoform lacked the second HhH motif by alternative splicing of a cryptic intron generating a truncated protein. The two transcripts were induced after DNA-damaging treatments such as high intensity light, UV-C and gamma-radiation. The yeast two-hybrid assay detected a strong interaction between OsMus81 and OsRad54 recombinational repair proteins. These findings suggest that OsMus81 functions in maintaining genome integrity through homologous recombination.
Keishi Osakabe, Kiyomi Abe, Toji Yoshioka, Yuriko Osakabe, Setsuko Todoriki, Hiroaki Ichikawa, Barbara Hohn and Seiichi Toki : Isolation and characterization of the RAD54 gene from Arabidopsis thaliana., The Plant Journal : for Cell and Molecular Biology, Vol.48, No.6, 827-842, 2006.
(要約)
Homologous recombination (HR) is an essential process in maintaining genome integrity and variability. In eukaryotes, the Rad52 epistasis group proteins are involved in meiotic recombination and/or HR repair. One member of this group, Rad54, belongs to the SWI2/SNF2 family of DNA-stimulated ATPases. Recent studies indicate that Rad54 has important functions in HR, both as a chromatin remodelling factor and as a mediator of the Rad51 nucleoprotein filament. Despite the importance of Rad54 in HR, no study of Rad54 from plants has yet been performed. Here, we cloned the full-length AtRAD54 cDNA sequence; an open reading frame of 910 amino acids encodes a protein with a predicted molecular mass of 101.9 kDa. Western blotting analysis showed that the AtRad54 protein was indeed expressed as a protein of approximately 110 kDa in Arabidopsis. The predicted protein sequence of AtRAD54 contains seven helicase domains, which are conserved in all other Rad54s. Yeast two-hybrid analysis revealed an interaction between Arabidopsis Rad51 and Rad54. AtRAD54 transcripts were found in all tissues examined, with the highest levels of expression in flower buds. Expression of AtRAD54 was induced by gamma-irradiation. A T-DNA insertion mutant of AtRAD54 devoid of full-length AtRAD54 expression was viable and fertile; however, it showed increased sensitivity to gamma-irradiation and the cross-linking reagent cisplatin. In addition, the efficiency of somatic HR in the mutant plants was reduced relative to that in wild-type plants. Our findings point to an important role for Rad54 in HR repair in higher plants.
(キーワード)
Amino Acid Sequence / Arabidopsis / Arabidopsis Proteins / Base Sequence / Blotting, Western / Cloning, Molecular / DNA Damage / DNA Helicases / DNA Repair / DNA, Complementary / DNA, Plant / DNA-Binding Proteins / Gamma Rays / Gene Expression Regulation, Plant / Genes, Plant / Green Fluorescent Proteins / Molecular Sequence Data / Promoter Regions, Genetic / Rad51 Recombinase / Recombination, Genetic
Masaki Endo, Yuichi Ishikawa, Keishi Osakabe, Shigeki Nakayama, Hidetaka Kaya, Takashi Araki, Kei-ichi Shibahara, Kiyomi Abe, Hiroaki Ichikawa, Lisa Valentine, Barbara Hohn and Seiichi Toki : Increased frequency of homologous recombination and T-DNA integration in Arabidopsis CAF-1 mutants., The EMBO Journal, Vol.25, No.23, 5579-5590, 2006.
(要約)
Chromatin assembly factor 1 (CAF-1) is involved in nucleo some assembly following DNA replication and nucleotide excision repair. In Arabidopsis thaliana, the three CAF-1 subunits are encoded by FAS1, FAS2 and, most likely, MSI1, respectively. In this study, we asked whether genomic stability is altered in fas1 and fas2 mutants that are lacking CAF-1 activity. Depletion of either subunit increased the frequency of somatic homologous recombination (HR) in planta approximately 40-fold. The frequency of transferred DNA (T-DNA) integration was also elevated. A delay in loading histones onto newly replicated or repaired DNA might make these DNA stretches more accessible, both to repair enzymes and to foreign DNA. Furthermore, fas mutants exhibited increased levels of DNA double-strand breaks, a G2-phase retardation that accelerates endoreduplication, and elevated levels of mRNAs coding for proteins involved in HR-all factors that could also contribute to upregulation of HR frequency in fas mutants.
Yuriko Osakabe, Kazuya NANTO, Hiroko KITAMURA, Keishi Osakabe, Shinya KAWAI, Noriyuki MOROHOSHI and Yoshihiro KATAYAMA : Immunological detection and cellular localization of the phenylalanine ammonia-lyase of a hybrid aspen, Plant Biotechnology, Vol.23, No.4, 399-404, 2006.
(要約)
The cellular-localization pattern of promoter activities of two phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) genes, palg1 and palg2b, of a hybrid aspen, Populus kitakamiensis, was determined using the GUS reporter system. The strong activities of palg2b promoter were detected in lignified tissues such as xylem and phloem fiber cells of the aspen stem, suggesting the specific function of the PALg2b in lignin biosynthesis. Immunoelectron microscopy and sub-cellular fractionation of xylem cells showed that the PAL activity was detected in both the plastid and cytosol of the xylem ray-parenchyma cells and the cytosol in the developing xylem cells of the aspen mature stem. Our results of the biochemical characterization of xylem PAL protein suggested that the PAL localization was varied during cell differentiation of the aspen xylem to function in lignin biosynthesis.
Masaki Endo, Keishi Osakabe, Hiroaki Ichikawa and Seiichi Toki : Molecular characterization of true and ectopic gene targeting events at the acetolactate synthase gene in Arabidopsis., Plant & Cell Physiology, Vol.47, No.3, 372-379, 2006.
(要約)
Precise modification of plant genomes via gene targeting (GT) is important for the study of gene function in vivo. A reliable GT system using the protoporphyrinogen oxidase (PPO) gene in Arabidopsis was reported 4 years ago; however, there are no subsequent successful reports of GT in Arabidopsis. A previous study showed ectopic gene targeting (EGT) of the endogenous gene in two-thirds of GT plants, which was an obstacle to efficient true gene targeting (TGT). The endogenous acetolactate synthase (ALS) gene is involved in the biosynthesis of branched chain amino acids in plants and is the site of action of several herbicides. To confirm the generality of the GT system in Arabidopsis, and to characterize the EGT event in plants in detail, we converted ALS from a herbicide (imazapyr)-susceptible to a -resistant form by GT. We obtained two imazapyr-resistant plants following GT. One of the targeting events was TGT while the other was EGT. After detailed Southern blotting, PCR and nucleotide sequence analysis of the EGT plant, we determined the genomic position and structure of the ectopically targeted site. Based on our findings, we discuss the possible mechanisms of EGT in plants.
Keishi Osakabe, Masaki Endo, Kiyoshi Kawai, Yaeko Nishizawa, Kazuko Ono, Kiyomi Abe, Yuichi Ishikawa, Hidemitsu Nakamura, Hiroaki Ichikawa, Shigeo Nishimura, Tsutomu Shimizu and Seiichi Toki : The Mutant Form of Acetolactate Synthase Genomic DNA from Rice is an Efficient Selectable Marker for Genetic Transformation, Molecular Breeding, Vol.16, No.4, 313-320, 2005.
(要約)
The proper use of a marker gene in a transformation process is critical for the production of transgenic plants. However, consumer concerns and regulatory requirements raise an objection to the presence of exogenous DNA in transgenic plants, especially antibiotic-resistant genes and promoters derived from viruses. One approach to overcome this problem is the elimination of marker genes from the plant genome by using several site-specific recombination systems. We propose an alternative method to solve this problem using a marker gene exclusively derived from the host plant DNA. We cloned a genomic DNA fragment containing regulatory and coding sequences of acetolactate synthase (ALS) gene from rice, and mutagenized the ALS gene into a herbicide-resistant form. After transfer of this construct to the rice genome, transgenic plants were efficiently selected with a herbicide, bispyribac-sodium salt, which inhibits the activity of wild type ALS. We also analyzed the regulatory feature of the rice ALS gene promoter with the gusA reporter gene and revealed that GUS expression was observed constitutively in aerial parts of rice seedlings and root tips. The marker system consisted exclusively of host plant DNA and enabled efficient selection in a monocot crop plant, rice. The selection system can potentially be applied to generate transgenic plants of other crop species and can be expected to be publicly acceptable.
Kiyomi Abe, Keishi Osakabe, Shigeki Nakayama, Masaki Endo, Akemi Tagiri, Setsuko Todoriki, Hiroaki Ichikawa and Seiichi Toki : Arabidopsis RAD51C gene is important for homologous recombination in meiosis and mitosis., Plant Physiology, Vol.139, No.2, 896-908, 2005.
(要約)
Rad51 is a homolog of the bacterial RecA recombinase, and a key factor in homologous recombination in eukaryotes. Rad51 paralogs have been identified from yeast to vertebrates. Rad51 paralogs are thought to play an important role in the assembly or stabilization of Rad51 that promotes homologous pairing and strand exchange reactions. We previously characterized two RAD51 paralogous genes in Arabidopsis (Arabidopsis thaliana) named AtRAD51C and AtXRCC3, which are homologs of human RAD51C and XRCC3, respectively, and described the interaction of their products in a yeast two-hybrid system. Recent studies showed the involvement of AtXrcc3 in DNA repair and functional role in meiosis. To determine the role of RAD51C in meiotic and mitotic recombination in higher plants, we characterized a T-DNA insertion mutant of AtRAD51C. Although the atrad51C mutant grew normally during vegetative developmental stage, the mutant produced aborted siliques, and their anthers did not contain mature pollen grains. Crossing of the mutant with wild-type plants showed defective male and female gametogeneses as evidenced by lack of seed production. Furthermore, meiosis was severely disturbed in the mutant. The atrad51C mutant also showed increased sensitivity to gamma-irradiation and cisplatin, which are known to induce double-strand DNA breaks. The efficiency of homologous recombination in somatic cells in the mutant was markedly reduced relative to that in wild-type plants.
Keishi Osakabe, Kiyomi Abe, Hiroaki Yamanouchi, Toshio Takyuu, Terutaka Yoshioka, Yuji Ito, Tomohiko Kato, Satoshi Tabata, Shunsuke Kurei, Yasushi Yoshioka, Yasunori Machida, Motoaki Seki, Masatomo Kobayashi, Kazuo Shinozaki, Hiroaki Ichikawa and Seiichi Toki : Arabidopsis Rad51B is important for double-strand DNA breaks repair in somatic cells., Plant Molecular Biology, Vol.57, No.6, 819-833, 2005.
(要約)
Rad51 paralogs belong to the Rad52 epistasis group of proteins and are involved in homologous recombination (HR), especially the assembly and stabilization of Rad51, which is a homolog of RecA in eukaryotes. We previously cloned and characterized two RAD51 paralogous genes in Arabidopsis, named AtRAD51C and AtXRCC3, which are considered the counterparts of human RAD51C and XRCC3, respectively. Here we describe the identification of RAD51B homologue in Arabidopsis, AtRAD51B. We found a higher expression of AtRAD51B in flower buds and roots. Expression of AtRAD51B was induced by genotoxic stresses such as ionizing irradiation and treatment with a cross-linking reagent, cisplatin. Yeast two-hybrid analysis showed that AtRad51B interacted with AtRad51C. We also found and characterized T-DNA insertion mutant lines. The mutant lines were devoid of AtRAD51B expression, viable and fertile. The mutants were moderately sensitive to gamma-ray and hypersensitive to cisplatin. Our results suggest that AtRAD51B gene product is involved in the repair of double-strand DNA breaks (DSBs) via HR.
(キーワード)
Amino Acid Sequence / Arabidopsis / Arabidopsis Proteins / Base Sequence / Blotting, Northern / Cisplatin / Cloning, Molecular / DNA / DNA Damage / DNA Repair / DNA, Bacterial / DNA, Complementary / Gamma Rays / Gene Expression Profiling / Gene Expression Regulation, Plant / Molecular Sequence Data / Mutagenesis, Insertional / Mutation / Phylogeny / Plants, Genetically Modified / Protein Isoforms / Sequence Alignment / Sequence Analysis, DNA / Sequence Homology, Amino Acid / Two-Hybrid System Techniques
Yuichi ISHIKAWA, Masaki ENDO, Kiyomi ABE, Keishi Osakabe, Nobuyoshi NAKAJIMA, Hikaru SAJI, Yuji ITO, Hiroaki ICHIKAWA, Toshiaki KAMEYA and Seiichi TOKI : Isolation of Four RAD23 Genes from Arabidopsis thaliana and Detection of Alternative Splicing Variants, Plant Biotechnology, Vol.21, No.1, 65-71, 2004.
(要約)
DNA damage recognition during nucleotide excision repair (NER) involves the homologous heterodimers Rad4:Rad23 in budding yeast and XPC:hHR23B in human. We report here the characteristics of four Arabidopsis homologues of RAD23 gene, named AtRAD23-1 to-4. AtRAD23-1, -3 and -4 expressed two alternatively spliced transcripts, long ones (AtRAD23-1, -3 and -4) and short ones (AtRAD23-1, -3 and -4). The predicted amino acid sequences of these genes possessed four conserved domains of Rad23 family; the ubiquitin-like domain, ubiquitin-associated domain I, XPC-binding domain and ubiquitin-associated domainII. AtRad23-3 and-4 lacked the C-terminus ubiquitin-like domain and the C-terminus XPC-binding domain, respectively, suggesting that these alternatively spliced variants may modulate functional AtRad23 proteins. Phylogenetic analysis showed that plant RAD23 genes could be divided into two classes and that Arabidopsis RAD23 genes were recently duplicated. AtRAD23-1-4 transcripts were detected in various tissues, with the highest expression level in flower buds.
Keishi Osakabe, Toji Yoshioka, Hiroaki Ichikawa and Seiichi Toki : Molecular cloning and characterization of RAD51-like genes from Arabidopsis thaliana., Plant Molecular Biology, Vol.50, No.1, 71-81, 2002.
(要約)
Homologous recombination is an essential process for the maintenance and variability of the genome. In eukaryotes, the Rad52 epistasis group proteins serve the main role for meiotic recombination and/or homologous recombinational repair. Rad51-like proteins, such as Rad55 and Rad57 in yeast, play a role in assembly or stabilization of multimeric Rad51 that promotes homologous pairing and strand exchange reactions. We cloned two RAD51-like genes named AtXRCC3 and AtRAD51C from Arabidopsis thaliana. Both AtXRCC3 and AtRAD51C expressed two alternatively spliced transcripts, and AtRAD51C produced two different sizes of isoforms, a long (AtRAD51Calpha) and a short one (AtRAD51Cbeta). The predicted protein sequences of these genes showed characteristic features of the RecA/Rad51 family; especially the amino acids around the ATP-binding motifs were well conserved. The transcripts of AtXRCC3 and AtRAD51C were detected in various tissues, with the highest level of expression in flower buds. Expression of both genes was induced by gamma-ray irradiation. The results of yeast two-hybrid assays suggested that Arabidopsis Rad51 family proteins form a complex, which could participate in meiotic recombination and/or homologous recombinational repair.
(キーワード)
Alternative Splicing / Amino Acid Sequence / Arabidopsis / Arabidopsis Proteins / Base Sequence / Cloning, Molecular / DNA, Complementary / DNA-Binding Proteins / Dose-Response Relationship, Radiation / Gene Expression Regulation, Plant / Molecular Sequence Data / Multigene Family / Phylogeny / Protein Binding / Rad51 Recombinase / Saccharomyces cerevisiae / Saccharomyces cerevisiae Proteins / Sequence Alignment / Sequence Analysis, DNA / Sequence Homology, Amino Acid / Two-Hybrid System Techniques
(文献検索サイトへのリンク)
● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 12139010
Keishi Osakabe, C C. Tsao, L Li, J L. Popko, T Umezawa, D T. Carraway, R H. Smeltzer, C P. Joshi and V L. Chiang : Coniferyl aldehyde 5-hydroxylation and methylation direct syringyl lignin biosynthesis in angiosperms., Proceedings of the National Academy of Sciences of the United States of America, Vol.96, No.16, 8955-8960, 1999.
(要約)
A central question in lignin biosynthesis is how guaiacyl intermediates are hydroxylated and methylated to the syringyl monolignol in angiosperms. To address this question, we cloned cDNAs encoding a cytochrome P450 monooxygenase (LsM88) and a caffeate O-methyltransferase (COMT) from sweetgum (Liquidambar styraciflua) xylem. Mass spectrometry-based functional analysis of LsM88 in yeast identified it as coniferyl aldehyde 5-hydroxylase (CAld5H). COMT expressed in Escherichia coli methylated 5-hydroxyconiferyl aldehyde to sinapyl aldehyde. Together, CAld5H and COMT converted coniferyl aldehyde to sinapyl aldehyde, suggesting a CAld5H/COMT-mediated pathway from guaiacyl to syringyl monolignol biosynthesis via coniferyl aldehyde that contrasts with the generally accepted route to sinapate via ferulate. Although the CAld5H/COMT enzyme system can mediate the biosynthesis of syringyl monolignol intermediates through either route, k(cat)/K(m) of CAld5H for coniferyl aldehyde was approximately 140 times greater than that for ferulate. More significantly, when coniferyl aldehyde and ferulate were present together, coniferyl aldehyde was a noncompetitive inhibitor (K(i) = 0.59 microM) of ferulate 5-hydroxylation, thereby eliminating the entire reaction sequence from ferulate to sinapate. In contrast, ferulate had no effect on coniferyl aldehyde 5-hydroxylation. 5-Hydroxylation also could not be detected for feruloyl-CoA or coniferyl alcohol. Therefore, in the presence of coniferyl aldehyde, ferulate 5-hydroxylation does not occur, and the syringyl monolignol can be synthesized only from coniferyl aldehyde. Endogenous coniferyl, 5-hydroxyconiferyl, and sinapyl aldehydes were detected, consistent with in vivo operation of the CAld5H/COMT pathway from coniferyl to sinapyl aldehydes via 5-hydroxyconiferyl aldehyde for syringyl monolignol biosynthesis.
(キーワード)
Angiosperms / Cloning, Molecular / Cytochrome P-450 Enzyme System / DNA, Complementary / Hydroxylation / Kinetics / Lignin / Methylation / Methyltransferases / Mixed Function Oxygenases / Molecular Sequence Data / Plant Proteins / Recombinant Proteins / Trees
(文献検索サイトへのリンク)
● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 10430877
W J. Hu, A Kawaoka, C J. Tsai, J Lung, Keishi Osakabe, H Ebinuma and V L. Chiang : Compartmentalized expression of two structurally and functionally distinct 4-coumarate:CoA ligase genes in aspen (Populus tremuloides)., Proceedings of the National Academy of Sciences of the United States of America, Vol.95, No.9, 5407-5412, 1998.
(要約)
4-Coumarate:CoA ligases (4CLs, EC 6.2.1.12) are a group of enzymes necessary for maintaining a continuous metabolic flux for the biosynthesis of plant phenylpropanoids, such as lignin and flavonoids, that are essential to the survival of plants. So far, various biochemical and molecular studies of plant 4CLs seem to suggest that 4CL isoforms in plants are functionally indistinguishable in mediating the biosynthesis of these phenolics. However, we have discovered two functionally and structurally distinct 4CL genes, Pt4CL1 and Pt4CL2 (63% protein sequence identity), that are differentially expressed in aspen (Populus tremuloides). The Escherichia coli-expressed and purified Pt4CL1 and Pt4CL2 proteins exhibited highly divergent substrate preference as well as specificity that reveal the association of Pt4CL1 with the biosynthesis of guaiacyl-syringyl lignin and the involvement of Pt4CL2 with other phenylpropanoid formation. Northern hybridization analysis demonstrated that Pt4CL1 mRNA is specifically expressed in lignifying xylem tissues and Pt4CL2 mRNA is specifically expressed in epidermal layers in the stem and the leaf, consistent with the promoter activities of Pt4CL1 and Pt4CL2 genes based on the heterologous promoter-beta-glucouronidase fusion analysis. Thus, the expression of Pt4CL1 and Pt4CL2 genes is compartmentalized to regulate the differential formation of phenylpropanoids that confer different physiological functions in aspen; Pt4CL1 is devoted to lignin biosynthesis in developing xylem tissues, whereas Pt4CL2 is involved in the biosynthesis of other phenolics, such as flavonoids, in epidermal cells.
L Li, J L. Popko, X H. Zhang, Keishi Osakabe, C J. Tsai, C P. Joshi and V L. Chiang : A novel multifunctional O-methyltransferase implicated in a dual methylation pathway associated with lignin biosynthesis in loblolly pine., Proceedings of the National Academy of Sciences of the United States of America, Vol.94, No.10, 5461-5466, 1997.
(要約)
S-adenosyl-L-methionine (SAM)-dependent O-methyltransferases (OMTs) catalyze the methylation of hydroxycinnamic acid derivatives for the synthesis of methylated plant polyphenolics, including lignin. The distinction in the extent of methylation of lignins in angiosperms and gymnosperms, mediated by substrate-specific OMTs, represents one of the fundamental differences in lignin biosynthesis between these two classes of plants. In angiosperms, two types of structurally and functionally distinct lignin pathway OMTs, caffeic acid 3-O-methyltransferases (CAOMTs) and caffeoyl CoA 3-O-methyltransferases (CCoAOMTs), have been reported and extensively studied. However, little is known about lignin pathway OMTs in gymnosperms. We report here the first cloning of a loblolly pine (Pinus taeda) xylem cDNA encoding a multifunctional enzyme, SAM:hydroxycinnamic Acids/hydroxycinnamoyl CoA Esters OMT (AEOMT). The deduced protein sequence of AEOMT is partially similar to, but clearly distinguishable from, that of CAOMTs and does not exhibit any significant similarity with CCoAOMT protein sequences. However, functionally, yeast-expressed AEOMT enzyme catalyzed the methylation of CAOMT substrates, caffeic and 5-hydroxyferulic acids, as well as CCoAOMT substrates, caffeoyl CoA and 5-hydroxyferuloyl CoA esters, with similar specific activities and was completely inactive with substrates associated with flavonoid synthesis. The lignin-related substrates were also efficiently methylated in crude extracts of loblolly pine secondary xylem. Our results support the notion that, in the context of amino acid sequence and biochemical function, AEOMT represents a novel SAM-dependent OMT, with both CAOMT and CCoAOMT activities and thus the potential to mediate a dual methylation pathway in lignin biosynthesis in loblolly pine xylem.
Yuriko Osakabe, Keishi Osakabe, Shinya Kawai, Yoshihiro Katayama and Noriyuki Morohoshi : Characterization of the structure and determination of mRNA levels of the phenylalanine ammonia-lyase gene family from Populus kitakamiensis., Plant Molecular Biology, Vol.28, 1133-1141, 1995.
(要約)
We isolated two new PAL genes, palg2b and palg4, from Populus kitakamiensis, palg2a and palg2b are clustered and palg2b encodes a polypeptide of 710 amino acids. The nucleotide sequence in the coding region of palg2b was 94.6% identical to that of palg2a. The promoter regions of palg1, palg2a and palg2b have several elements conserved among many phenylpropanoid biosynthetic genes. We measured the mRNA levels of the four PAL genes by S1 mapping using total RNA from stem tissues developing secondary xylem. Results showed that the transcript level of palg2b was higher than that of the other PAL genes.
Keishi Osakabe, H Koyama, S Kawai, Y Katayama and N Morohoshi : Molecular cloning of two tandemly arranged peroxidase genes from Populus kitakamiensis and their differential regulation in the stem., Plant Molecular Biology, Vol.28, No.4, 677-689, 1995.
(要約)
A genomic library was prepared from Populus kitakamiensis and screened with the cDNA for an anionic peroxidase from P. kitakamiensis. One genomic clone was isolated that contained two tandemly oriented genes for anionic peroxidases, prxA3a and prxA4a. Both genes consisted of four exons and three introns; the introns had consensus nucleotides, namely, GT and AG, at their 5' and 3' ends, respectively. The prxA3a and prxA4a genes encoded 347 and 343 amino acid residues, respectively, including putative signal sequences at the amino-termini. Putative promoters and polyadenylation signals were found in the flanking regions of both genes. The sequence of the coding region of prxA3a was completely identical to that of the cDNA clone pA3, whereas the sequence of the coding region of prxA4a was only 73% identical to that of the cDNA clone pA3. Northern blot analysis showed that the patterns of expression of the mRNAs that corresponded to prxA3a and prxA4a differed in stems of P. kitakamiensis.
Keishi Osakabe, Hirokazu Koyama, Shinya Kawai, Yoshihiro Katayama and Noriyuki Morohoshi : Molecular cloning and the nucleotide sequences of two novel cDNAs that encode anionic peroxidases of Populus kitakamiensis., Plant Science, Vol.103, No.2, 167-175, 1994.
(要約)
We have cloned and characterized two novel cDNAs for peroxidases from a cDNA library constructed with mRNAs from the young stem tissue of hybrid aspen, Populus kitakamiensis. Comparison of the deduced amino acid sequences of the two cDNAs, designated pA2 and pA3, with those encoded by other known genes for peroxidases revealed several already identified regions of homology (the regions of the two His residues, the distal and proximal His residues that are bound to protoheme). Southern blot analyses indicate that pA2 and pA3 correspond to two or three genes in each class of isoenzymes. The respective transcripts of the pA2 and pA3 groups were detected by use of gene class-specific probes and it was revealed that transcripts of both groups were expressed in the lignifying tissue of P. kitakamiensis.
67.
Shinya Kajita, Keishi Osakabe, Yoshihiro Katayama, Shinya Kawai, Yasuo Matsumoto, Kunio Hata and Noriyuki Morohoshi : Agrobacterium-mediated transformation of poplar using a disarmed binary vector and the over expression of a specific member of a family of poplar peroxidase genes in transgenic poplar cell., Plant Science, Vol.103, No.2, 231-239, 1994.
(要約)
An efficient method was established for transformation of the poplar hybrid Populus kitakamiensis (Populus sieboldii × Populus gradidentata) using a binary disarmed strain of Agrobacterium tumefaciens LBA4404 and Ti-binary vectors. The frequency of transformation of poplar leaf segments reached as high as 60%. In transgenic poplar plants, the gene for -glucuronidase (gus) was expressed at high levels under the control of the cauliflower mosaic virus 35S (CaMV35S) promoter. Poplars possess a number of peroxidase isozymes whose pattern of expression is tissue-specific, developmentally regulated and influenced by environmental factors. We altered the expressin of a peroxidase isozyme by introducing an identified genomic gene for a peroxidase (prxA1) under the control of the CaMV35S promoter. Transgenic poplars obtained by introducing the chimeric peroxidase gene (CaMV35S promoter-prxA1) were shown to have an increase in total peroxidase activity that was accounted for by the specific overproduction of the peroxidase isozyme (PrxA1). From this study, the anionic peroxidase isozyme encoded by the identified genomic gene, prxA1, was demonstrated to be the anionic peroxidase isozyme with a pI of 4.4 among various isozymes of poplar peroxidase. On the basis of this assignment, we characterized the tissue-specific and UV-light-inducible regulation of expression of this isozyme.
Naoki Wada, Keishi Osakabe and Yuriko Osakabe : Expanding the plant genome editing toolbox with recently developed CRISPR-Cas systems, Plant Physiology, Vol.188, No.4, 1825-1837, Jan. 2022.
(要約)
Since its first appearance, CRISPR-Cas9 has been developed extensively as a programmable genome-editing tool, opening a new era in plant genome engineering. However, CRISPR-Cas9 still has some drawbacks, such as limitations of the protospacer-adjacent motif (PAM) sequence, target specificity, and the large size of the cas9 gene. To combat invading bacterial phages and plasmid DNAs, bacteria and archaea have diverse and unexplored CRISPR-Cas systems, which have the potential to be developed as a useful genome editing tools. Recently, discovery and characterization of additional CRISPR-Cas systems have been reported. Among them, several CRISPR-Cas systems have been applied successfully to plant and human genome editing. For example, several groups have achieved genome editing using CRISPR-Cas type I-D and type I-E systems, which had never been applied for genome editing previously. In addition to higher specificity and recognition of different PAM sequences, recently developed CRISPR-Cas systems often provide unique characteristics that differ from well-known Cas proteins such as Cas9 and Cas12a. For example, type I CRISPR-Cas10 induces small indels and bi-directional long-range deletions ranging up to 7.2 kb in tomatoes (Solanum lycopersicum L.). Type IV CRISPR-Cas13 targets RNA, not double-strand DNA, enabling highly specific knockdown of target genes. In this article, we review the development of CRISPR-Cas systems, focusing especially on their application to plant genome engineering. Recent CRISPR-Cas tools are helping expand our plant genome engineering toolbox.
Naoki Wada, Ueta Risa, Yuriko Osakabe and Keishi Osakabe : Precision genome editing in plants: state-of-the-art in CRISPR/Cas9-based genome engineering, BMC Plant Biology, Vol.20, No.1, 234, May 2020.
(要約)
Traditionally, generation of new plants with improved or desirable features has relied on laborious and time-consuming breeding techniques. Genome-editing technologies have led to a new era of genome engineering, enabling an effective, precise, and rapid engineering of the plant genomes. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) has emerged as a new genome-editing tool, extensively applied in various organisms, including plants. The use of CRISPR/Cas9 allows generating transgene-free genome-edited plants ("null segregants") in a short period of time. In this review, we provide a critical overview of the recent advances in CRISPR/Cas9 derived technologies for inducing mutations at target sites in the genome and controlling the expression of target genes. We highlight the major breakthroughs in applying CRISPR/Cas9 to plant engineering, and challenges toward the production of null segregants. We also provide an update on the efforts of engineering Cas9 proteins, newly discovered Cas9 variants, and novel CRISPR/Cas systems for use in plants. The application of CRISPR/Cas9 and related technologies in plant engineering will not only facilitate molecular breeding of crop plants but also accelerate progress in basic research.
Yuriko Osakabe and Keishi Osakabe : Genome editing in higher plants., Targeted Genome Editing Using Engineered Nucleases: ZFNs, TALENs, and the CRISPR/Cas9 System, Oct. 2014.
Yuriko Osakabe, Nobuyuki Nishikubo and Keishi Osakabe : Phenylalanine ammonia-lyase in woody plants: a key switch of carbon accumulation in biomass., Japanese Journal of Plant Science, Vol.1, No.103-108, 103-108, 2007.
Kurihara Satoshi, Naoki Wada, Murakami Emi, Marui Kazuya, Yuriko Osakabe and Keishi Osakabe : Establishment of episomal vector-based CRISPR-Cas type I-D system for efficient genome editing in human cells, Genome Engineering:CRISPR Frontiers, Cold Spring Harbor Laboratory, NY, USA, Aug. 2024.
2.
Naoki Wada, Murakami Emi, Marui Kazuya, Yuriko Osakabe and Keishi Osakabe : Efficient gene knockout using CRISPR-Cas type I-D combined with TriFC system in diploid human cells, Genome Engineering:CRISPR Frontiers, Cold Spring Harbor Laboratory, NY, USA, Aug. 2024.
3.
Naoki Wada, Murakami Emi, Marui Kazuya, Yuriko Osakabe and Keishi Osakabe : Development of a highly efficient genome editing tool using Type I-D CRISPR-Cas, Keystone Symposia on Precision Genome Engineering, Jan. 2024.
4.
M Omori, H Yamane, Keishi Osakabe, Y Osakabe and R Tao : Transient expression assay to evaluate the utility of endogenous promoters for the efficient CRISPR/Cas9-mediated genome editing in tetraploid blueberry, The International Horticultural Congress, Angers, France, Aug. 2022.
5.
Miyamoto Takuji, Takada Rie, Tobimatsu Yuki, Suzuki Shiro, Masaomi Yamamura, Keishi Osakabe, Yuriko Osakabe, Sakamoto Masahiro and Umezawa Toshiaki : Knockout of OsWRKY36 and OsWRKY102 boosts lignification with altering culm morphology of rice, 60th Annual Meeting of the Phytochemical Society of North America, July 25-30, 2021 (Kelowna, Canada,on-line), Jul. 2021.
6.
Miyaji Tomoko, Tagami Shoya, Sakaguchi Kohei, Shimada Kanari, Nakashima Eiko, Fujii Syuki, Shinohara Keiko, Harada Yoko, Keishi Osakabe and Yuriko Osakabe : Genome editing of the model strawberry Fragaria vesca using plant-optimized CRISPR/Cas9 system, Frontiers in Genome Engineering 2019, Kobe Convention Center, Nov. 25-27, 2019, Nov. 2019.
7.
Keishi Osakabe, Naoki Wada, Marui Kazuya, Murakami Emi, Ueta Risa, Hashimoto Ryosuke, Hara Chihiro, Miyaji Tomoko and Yuriko Osakabe : Genome editing in plants by using a novel genome editing tool TiD, Frontiers in Genome Engineering 2019, Kobe Convention Center, Nov. 25-27, 2019, Nov. 2019.
8.
Yuriko Osakabe, Kira Nozomu, Ueta Risa, Sakamoto Hideki, Takahito Watanabe, Takayanagi Eiko, Hara Chihiro, Hashimoto Ryosuke, Kohji Yamada and Keishi Osakabe : Development of in planta-regeneration system for plant genome editing, Frontiers in Genome Engineering 2019, Kobe Convention Center, Nov. 25-27, 2019, Nov. 2019.
9.
Naoki Wada, Murakami Emi, Hashimoto Ryosuke, Yuriko Osakabe and Keishi Osakabe : DEVELOPMENT OF A NOVEL GENOME EDITING TOOL, TID SYSTEM, FOR MAMMALIAN GENOME ENGINEERING, Frontiers in Genome Engineering 2019, Kobe Convention Center, Nov. 25-27, 2019, Nov. 2019.
10.
Keishi Osakabe : Current and future of genome engineering in agricultural products, Bioengineering of lignocellulose for clean energy production: perspectives and opportunities, Kyoto Univ, Kyoto, Feb. 2019.
11.
Keishi Osakabe : Plant genome editing (invited lecture), International Workshop of Plant Cell Wall Study, South China Agricultral Univ., China, Oct. 2018.
12.
Okamoto Takashi, Toda Erika, Koiso Narumi, Takebayashi Arika, Ichikawa Masako, Kiba Takatoshi, Keishi Osakabe, Yuriko Osakabe, Sakakibara Hitoshi and Kato Norio : Genome editing in rice by direct delivery of preassembled CRISPR-Cas9 vectors or ribonucleoproteins into zygotes, International Association for Plant Biotechnology (IAPB) CONGRESS, Dublin, Ireland, Aug. 2018.
13.
Shimada Kanari, Iuchi Satoshi, Iuchi Atsuko, Kohji Yamada, Keishi Osakabe and Yuriko Osakabe : IDENTIFICATION OF AN ARABIDOPSIS MUTANT WITH ALTERED ROOT HAIR FORMATION, International Conference on Arabidopsis Research 2018 (ICAR2018), Turku Finland, Jul. 2018.
14.
Keishi Osakabe : Plant genome editing (Invited lecture), International Symposium on Forest and Tree Molecular Biology and Biotechnology (FTMB2018), Harbin, China, Jul. 2018.
15.
Chikako Nishitani, Narumi Hirai, Sadao Komori, Masato Wada, Kazuma Okada, Keishi Osakabe, Toshiya Yamamoto and Yuriko Osakabe : CRISPR/Cas9-mediated genome editing in apple, International Symposium on Forest and Tree Molecular Biology and Biotechnology (FTMB2018), Harbin, China, Jul. 2018.
16.
Risa Ueta, Abe Chihiro, Hashimoto Ryosuke, Yuriko Osakabe and Keishi Osakabe : High-efficient genome editing using CRISPR/Cas9 targeting functional genes in tomato, Taiwan-Japan Plant Biology 2017, Taipei, Taiwan, Nov 3-6, 2017, Nov. 2017.
17.
Keishi Osakabe : Current and future of genome editing in agricultural products, VICEA (Vienna International Science Conferences and Events Association) Plant Genome editing & Genome engineering, Wien, Jul. 2017.
18.
Risa Ueta, Chihiro Abe, Ryosuke Ishihara, Takahito Watanabe, Sigeo Sugano, Yuriko Osakabe and Keishi Osakabe : Site-directed mutagenesis of the tomato IAA9 gene by using the CRISPR/Cas9 system, Latest Advances in Plant Development and Environmental Response 2016, CSH - Asia Meetings, Nov. 2016.
Yuriko Osakabe, Risa Ueta, Sigeo Sugano, Takahito Watanabe, Kazuo Shinozaki and Keishi Osakabe : Genetic Engineering of Abiotic Stress Response in Plants, 3rd Conference of Cereal Biotechnology and Breeding, Nov. 2015.
21.
Keishi Osakabe : Development of genome engineering for non-model and crop plants (Invited lecture), International Symposium on RNAi and Genome editing methods, Tokushima, Mar. 2013.
22.
Yuriko Osakabe and Keishi Osakabe : Site-directed genome engineering of higher plants by genome editing tools; ZFN, International Symposium on RNAi and Genome editing methods, Tokushima, Mar. 2013.
和田 直樹, 村上 愛美, 刑部 祐里子, 刑部 敬史 : Development of a highly sensitive guide RNA evaluation system using Nano Luciferase, The 12th International Symposium Exploring the Global Sustainability, Kindai University, 201985, 2019年8月.