2020年12月, 茨城大学学長学術表彰 奨励賞, 茨城大学

2019年09月, 奨励賞, 日本植物形態学会
小林 優介

2019年02月, 井上研究奨励賞, 井上科学振興財団
小林 優介

2018年09月, 若手奨励賞, 日本植物学会
小林 優介

2017年09月, 平瀬賞, 日本植物形態学会
小林 優介

HBD1 protein with a tandem repeat of two HMG-box domains is a DNA clip to organize chloroplast nucleoids in Chlamydomonas reinhardtii
Mari Takusagawa; Yusuke Kobayashi; Yoichiro Fukao; Kumi Hidaka; Masayuki Endo; Hiroshi Sugiyama; Takashi Hamaji; Yoshinobu Kato; Isamu Miyakawa; Osami Misumi; Toshiharu Shikanai; Yoshiki Nishimura, Compaction of bulky DNA is a universal issue for all DNA-based life forms. Chloroplast nucleoids (chloroplast DNA–protein complexes) are critical for chloroplast DNA maintenance and transcription, thereby supporting photosynthesis, but their detailed structure remains enigmatic. Our proteomic analysis of chloroplast nucleoids of the green alga Chlamydomonas reinhardtii identified a protein (HBD1) with a tandem repeat of two DNA-binding high mobility group box (HMG-box) domains, which is structurally similar to major mitochondrial nucleoid proteins transcription factor A, mitochondrial (TFAM), and ARS binding factor 2 protein (Abf2p). Disruption of the HBD1 gene by CRISPR-Cas9–mediated genome editing resulted in the scattering of chloroplast nucleoids. This phenotype was complemented when intact HBD1 was reintroduced, whereas a truncated HBD1 with a single HMG-box domain failed to complement the phenotype. Furthermore, ectopic expression of HBD1 in the mitochondria of yeast Δabf2 mutant successfully complemented the defects, suggesting functional similarity between HBD1 and Abf2p. Furthermore, in vitro assays of HBD1, including the electrophoretic mobility shift assay and DNA origami/atomic force microscopy, showed that HBD1 is capable of introducing U-turns and cross-strand bridges, indicating that proteins with two HMG-box domains would function as DNA clips to compact DNA in both chloroplast and mitochondrial nucleoids., Proceedings of the National Academy of Sciences
Proceedings of the National Academy of Sciences, 2021年05月18日, ［査読有り］

Holliday Junction Resolvase MOC1 Maintains Plastid and Mitochondrial Genome Integrity in Algae and Bryophytes
Yusuke Kobayashi; Masaki Odahara; Yasuhiko Sekine; Takashi Hamaji; Sumire Fujiwara; Yoshiki Nishimura; Shin-ya Miyagishima, When DNA double-strand breaks occur, four-stranded DNA structures called Holliday junctions (HJs) form during homologous recombination. Because HJs connect homologous DNA by a covalent link, resolution of HJ is crucial to terminate homologous recombination and segregate the pair of DNA molecules faithfully. We recently identified Monokaryotic Chloroplast1 (MOC1) as a plastid DNA HJ resolvase in algae and plants. Although Cruciform cutting endonuclease1 (CCE1) was identified as a mitochondrial DNA HJ resolvase in yeasts, homologs or other mitochondrial HJ resolvases have not been identified in other eukaryotes. Here, we demonstrate that MOC1 depletion in the green alga Chlamydomonas reinhardtii and the moss Physcomitrella patens induced ectopic recombination between short dispersed repeats in ptDNA. In addition, MOC1 depletion disorganized thylakoid membranes in plastids. In some land plant lineages, such as the moss P. patens, a liverwort and a fern, MOC1 dually targeted to plastids and mitochondria. Moreover, mitochondrial targeting of MOC1 was also predicted in charophyte algae and some land plant species. Besides causing instability of plastid DNA, MOC1 depletion in P. patens induced short dispersed repeat-mediated ectopic recombination in mitochondrial DNA and disorganized cristae in mitochondria. Similar phenotypes in plastids and mitochondria were previously observed in mutants of plastid-targeted (RECA2) and mitochondrion-targeted (RECA1) recombinases, respectively. These results suggest that MOC1 functions in the double-strand break repair in which a recombinase generates HJs and MOC1 resolves HJs in mitochondria of some lineages of algae and plants as well as in plastids in algae and plants., Oxford University Press (OUP)
Plant Physiology, 2020年12月, ［査読有り］

Dynamic Motion of Chloroplast Nucleoids Captured by the Microfluidic System　　　　　　　　　　　　　　　
Kamimura Y; Kobayashi Y; Nishimura Y
Cyotologia, 2020年09月, ［査読有り］

Molecular structure and evolution of chloroplast nucleoids
Yusuke Kobayashi, The Japanese Society of Plant Morphology
PLANT MORPHOLOGY, 2020年, ［査読有り］, ［招待有り］

Evolutionary Changes in DnaA-Dependent Chromosomal Replication in Cyanobacteria.
Ryudo Ohbayashi; Shunsuke Hirooka; Ryo Onuma; Yu Kanesaki; Yuu Hirose; Yusuke Kobayashi; Takayuki Fujiwara; Chikara Furusawa; Shin-Ya Miyagishima, Replication of the circular bacterial chromosome is initiated at a unique origin (oriC) in a DnaA-dependent manner in which replication proceeds bidirectionally from oriC to ter. The nucleotide compositions of most bacteria differ between the leading and lagging DNA strands. Thus, the chromosomal DNA sequence typically exhibits an asymmetric GC skew profile. Further, free-living bacteria without genomes encoding dnaA were unknown. Thus, a DnaA-oriC-dependent replication initiation mechanism may be essential for most bacteria. However, most cyanobacterial genomes exhibit irregular GC skew profiles. We previously found that the Synechococcus elongatus chromosome, which exhibits a regular GC skew profile, is replicated in a DnaA-oriC-dependent manner, whereas chromosomes of Synechocystis sp. PCC 6803 and Nostoc sp. PCC 7120, which exhibit an irregular GC skew profile, are replicated from multiple origins in a DnaA-independent manner. Here we investigate the variation in the mechanisms of cyanobacterial chromosome replication. We found that the genomes of certain free-living species do not encode dnaA and such species, including Cyanobacterium aponinum PCC 10605 and Geminocystis sp. NIES-3708, replicate their chromosomes from multiple origins. Synechococcus sp. PCC 7002, which is phylogenetically closely related to dnaA-lacking free-living species as well as to dnaA-encoding but DnaA-oriC-independent Synechocystis sp. PCC 6803, possesses dnaA. In Synechococcus sp. PCC 7002, dnaA was not essential and its chromosomes were replicated from a unique origin in a DnaA-oriC independent manner. Our results also suggest that loss of DnaA-oriC-dependency independently occurred multiple times during cyanobacterial evolution and raises a possibility that the loss of dnaA or loss of DnaA-oriC dependency correlated with an increase in ploidy level.
Frontiers in microbiology, 2020年, ［査読有り］

Responses of unicellular predators to cope with the phototoxicity of photosynthetic prey　　　　　　　　　　　　　　　
Uzuka A; Kobayashi Y; Onuma R; Hirooka S; Kanesaki Y; Yoshikawa H; Fujiwara T; Miyagishima SY; These authors contributed equally to this work
Nature communications, 2019年12月, ［査読有り］

Chloroplast nucleoids as a transformable network revealed by live imaging with a microfluidic device　　　　　　　　　　　　　　　
Kamimura Y; Tanaka H; Kobayashi Y; Shikanai T; Nishimura Y
Communications Biology, 2018年05月, ［査読有り］

Identification of Holliday junction resolvases crucial for the chloroplast nucleoid morphology and segregation
Yusuke Kobayashi; Osami Misumi; Yoshiki Nishimura, The Japanese Society of Plant Morphology
PLANT MORPHOLOGY, 2018年

Finding holliday junction resolvases: A crucial factor for chloroplast nucleoid segregation
Yusuke Kobayashi; Osami Misumi; Yoshiki Nishimura, Japan Mendel Society
Cytologia, 2017年12月01日, ［査読有り］, ［招待有り］

Holliday junction resolvases mediate chloroplast nucleoid segregation
Yusuke Kobayashi; Osami Misumi; Masaki Odahara; Kota Ishibashi; Masafumi Hirono; Kumi Hidaka; Masayuki Endo; Hiroshi Sugiyama; Hiroshi Iwasaki; Tsuneyoshi Kuroiwa; Toshiharu Shikanai; Yoshiki Nishimura, Holliday junctions, four-stranded DNA structures formed during homologous recombination, are disentangled by resolvases that have been found in prokaryotes and eukaryotes but not in plant organelles. Here, we identify monokaryotic chloroplast 1 (MOC1) as a Holliday junction resolvase in chloroplasts by analyzing a green alga Chlamydomonas reinhardtii mutant defective in chloroplast nucleoid (DNA-protein complex) segregation. MOC1 is structurally similar to a bacterial Holliday junction resolvase, resistance to ultraviolet (Ruv) C, and genetically conserved among green plants. Reduced or no expression of MOC1 in Arabidopsis thaliana leads to growth defects and aberrant chloroplast nucleoid segregation. In vitro biochemical analysis and high-speed atomic force microscopic analysis revealed that A. thaliana MOC 1 (AtMOC1) binds and cleaves the core of Holliday junctions symmetrically. MOC1 may mediate chloroplast nucleoid segregation in green plants by resolving Holliday junctions., AMER ASSOC ADVANCEMENT SCIENCE
SCIENCE, 2017年05月, ［査読有り］

Dynamic Interplay between Nucleoid Segregation and Genome Integrity in Chlamydomonas Chloroplasts
Masaki Odahara; Yusuke Kobayashi; Toshiharu Shikanai; Yoshiki Nishimura, The chloroplast (cp) genome is organized as nucleoids that are dispersed throughout the cp stroma. Previously, a cp homolog of bacterial recombinase RecA (cpRECA) was shown to be involved in the maintenance of cp genome integrity by repairing damaged chloroplast DNA and by suppressing aberrant recombination between short dispersed repeats in the moss Physcomitrella patens. Here, overexpression and knockdown analysis of cpRECA in the green alga Chlamydomonas reinhardtii revealed that cpRECA was involved in cp nucleoid dynamics as well as having a role in maintaining cp genome integrity. Overexpression of cpRECA tagged with yellow fluorescent protein or hemagglutinin resulted in the formation of giant filamentous structures that colocalized exclusively to chloroplast DNA and cpRECA localized to cp nucleoids in a heterogenous manner. Knockdown of cpRECA led to a significant reduction in cp nucleoid number that was accompanied by nucleoid enlargement. This phenotype resembled those of gyrase inhibitor-treated cells and monokaryotic chloroplast mutant cells and suggested that cpRECA was involved in organizing cp nucleoid dynamics. The cp genome also was destabilized by induced recombination between short dispersed repeats in cpRECA-knockdown cells and gyrase inhibitor-treated cells. Taken together, these results suggest that cpRECA and gyrase are both involved in nucleoid dynamics and the maintenance of genome integrity and that the mechanisms underlying these processes may be intimately related in C. reinhardtii cps., AMER SOC PLANT BIOLOGISTS
PLANT PHYSIOLOGY, 2016年12月, ［査読有り］

C-Terminal Region of Sulfite Reductase Is Important to Localize to Chloroplast Nucleoids in Land Plants
Yusuke Kobayashi; Takuto Otani; Kota Ishibashi; Toshiharu Shikanai; Yoshiki Nishimura, Chloroplast (cp) DNA is compacted into cpDNA-protein complexes, called cp nucleoids. An abundant and extensively studied component of cp nucleoids is the bifunctional protein sulfite reductase (SiR). The preconceived role of SiR as the core cp nucleoid protein, however, is becoming less likely because of the recent findings that SiRs do not associate with cp nucleoids in some plant species, such as Zea mays and Arabidopsis thaliana. To address this discrepancy, we have performed a detailed phylogenetic analysis of SiRs, which shows that cp nucleoid-type SiRs share conserved C-terminally encoded peptides (CEPs). The CEPs are likely to form a bacterial ribbon helix helix DNA-binding motif, implying a potential role in attaching SiRs onto cp nucleoids. A proof-of-concept experiment was conducted by fusing the nonnucleoid-type SiR from A. thaliana (AtSiR) with the CEP from the cp nucleoid-type SiR of Phaseolus yulgaris. The addition of the CEP drastically altered the intra-cp localization of AtSiR to cp nucleoids. Our analysis supports the possible functions of CEPs in determining the localization of SiRs to cp nucleoids and illuminates a possible evolutionary scenario for SiR as a cp nucleoid protein., OXFORD UNIV PRESS
GENOME BIOLOGY AND EVOLUTION, 2016年05月, ［査読有り］

Eukaryotic Components Remodeled Chloroplast Nucleoid Organization during the Green Plant Evolution
Yusuke Kobayashi; Mari Takusagawa; Naomi Harada; Yoichiro Fukao; Shohei Yamaoka; Takayuki Kohchi; Koichi Hori; Hiroyuki Ohta; Toshiharu Shikanai; Yoshiki Nishimura, Chloroplast (cp) DNA is thought to originate from the ancestral endosymbiont genome and is compacted to form nucleoprotein complexes, cp nucleoids. The structure of cp nucleoids is ubiquitously observed in diverse plants from unicellular algae to flowering plants and is believed to be a multifunctional platform for various processes, including cpDNA replication, repair/recombination, transcription, and inheritance. Despite its fundamental functions, the protein composition for cp nucleoids in flowering plants was suggested to be divergent from those of bacteria and algae, but the evolutionary process remains elusive. In this research, we aimed to reveal the evolutionary history of cp nucleoid organization by analyzing the key organisms representing the three evolutionary stages of eukaryotic phototrophs: the chlorophyte alga Chlamydomonas reinhardtii, the charophyte alga Klebsormidium flaccidum, and the most basal land plant Marchantia polymorpha. To clarify the core cp nucleoid proteins in C. reinhardtii, we performed an LC-MS/MS analysis using highly purified cp nucleoid fractions and identified a novel SAP domain-containing protein with a eukaryotic origin as a constitutive core component. Then, homologous genes for cp nucleoid proteins were searched for in C. reinhardtii, K. flaccidum, and M. polymorpha using the genome databases, and their intracellular localizations and DNA binding activities were investigated by cell biological/biochemical analyses. Based on these results, we propose a model that recurrent modification of cp nucleoid organization by eukaryotic factors originally related to chromatin organization might have been the driving force for the diversification of cp nucleoids since the early stage of green plant evolution., OXFORD UNIV PRESS
GENOME BIOLOGY AND EVOLUTION, 2016年01月, ［査読有り］

Development of a Heat-Shock Inducible Gene Expression System in the Red Alga Cyanidioschyzon merolae
Nobuko Sumiya; Takayuki Fujiwara; Yusuke Kobayashi; Osami Misumi; Shin-ya Miyagishima, The cell of the unicellular red alga Cyanidioschyzon merolae contains a single chloroplast and mitochondrion, the division of which is tightly synchronized by a light/dark cycle. The genome content is extremely simple, with a low level of genetic redundancy, in photosynthetic eukaryotes. In addition, transient transformation and stable transformation by homologous recombination have been reported. However, for molecular genetic analyses of phenomena that are essential for cellular growth and survival, inducible gene expression/suppression systems are needed. Here, we report the development of a heat-shock inducible gene expression system in C. merolae. CMJ101C, encoding a small heat shock protein, is transcribed only when cells are exposed to an elevated temperature. Using a superfolder GFP as a reporter protein, the 200-bp upstream region of CMJ101C orf was determined to be the optimal promoter for heat-shock induction. The optimal temperature to induce expression is 50 degrees C, at which C. merolae cells are able to proliferate. At least a 30-min heat shock is required for the expression of a protein of interest and a 60-min heat shock yields the maximum level of protein expression. After the heat shock, the mRNA level decreases rapidly. As an example of the system, the expression of a dominant negative form of chloroplast division DRP5B protein, which has a mutation in the GTPase domain, was induced. Expression of the dominant negative DRP5B resulted in the appearance of aberrant-shaped cells in which two daughter chloroplasts and the cells are still connected by a small DRP5B positive tube-like structure. This result suggests that the dominant negative DRP5B inhibited the final scission of the chloroplast division site, but not the earlier stages of division site constriction. It is also suggested that cell cycle progression is not arrested by the impairment of chloroplast division at the final stage., PUBLIC LIBRARY SCIENCE
PLOS ONE, 2014年10月, ［査読有り］

Algae Sense Exact Temperatures: Small Heat Shock Proteins Are Expressed at the Survival Threshold Temperature in Cyanidioschyzon merolae and Chlamydomonas reinhardtii
Yusuke Kobayashi; Naomi Harada; Yoshiki Nishimura; Takafumi Saito; Mami Nakamura; Takayuki Fujiwara; Tsuneyoshi Kuroiwa; Osami Misumi, The primitive red alga Cyanidioschyzon merolae inhabits acidic hot springs and shows robust resistance to heat shock treatments up to 63 A degrees C. Microarray analysis was performed to identify the key genes underlying the high temperature tolerance of this organism. Among the upregulated genes that were identified, we focused on two small heat shock proteins (sHSPs) that belong to a unique class of HSP families. These two genes are located side by side in an inverted repeat orientation on the same chromosome and share a promoter. These two genes were simultaneously and rapidly upregulated in response to heat shock treatment (> 1,000-fold more than the control). Interestingly, upregulation appeared to be triggered not by a difference in temperatures, but rather by the absolute temperature. Similar sHSP structural genes have been reported in the green alga Chlamydomonas reinhardtii, but the threshold temperature for the expression of these sHSP-encoding genes in Ch. reinhardtii was different from the threshold temperature for the expression of the sHSP genes from Cy. merolae. These results indicate the possible importance of an absolute temperature sensing system in the evolution and tolerance of high-temperature conditions among unicellular microalgae., OXFORD UNIV PRESS
GENOME BIOLOGY AND EVOLUTION, 2014年10月, ［査読有り］

シアノバクテリアの進化実験で迫る葉緑体相同組換え機構の多様性
小林優介; 大林龍胆; 宮城島進也
Plant Morphology, 2019年

葉緑体核様体の進化と構造のダイナミクス
小林 優介; 三角 修己; 西村 芳樹
化学と生物, 2018年09月, ［招待有り］

葉緑体ゲノムの分配はHollidayジャンクション解離酵素MOC1により保障される
小林 優介; 三角 修己; 西村 芳樹
ライフサイエンス 新着論文レビュー, 2017年05月, ［招待有り］

葉緑体核様体コア因子の多様性と進化
小林優介; 田草川真理; 田草川真理; 原田尚実; 深尾陽一朗; 深尾陽一朗; 山岡尚平; 河内孝之; 堀孝一; 太田啓之; 太田啓之; 鹿内利治; 西村芳樹
日本植物学会大会研究発表記録, 2015年

葉緑体DNAの遺伝機構　　　　　　　　　　　　　　　
日本遺伝学会第96回大会＠高知工科大学, 2024年09月, ［招待有り］

Hollidayジャンクション切断酵素MOC1は色素体とミトコンドリアDNAの安定性に寄与する　　　　　　　　　　　　　　　
Yusuke Kobayashi
2020年度日本分子生物学会ワークショップ, 2020年12月

葉緑体DNA遺伝に必須な Holliday ジャンクション切断機構の発見　　　　　　　　　　　　　　　
小林 優介
第20回植物オルガネラワークショップ, 2018年03月, ［招待有り］

Hollidayジャンクション解離酵素MOC1は葉緑体核様体の形態・分配を保障する　　　　　　　　　　　　　　　
小林 優介
新光合成＆光合成若手の会ジョイント若手ワークショップ, 2017年08月, ［招待有り］

日本植物生理学会

日本植物形態学会

日本植物学会

光合成を基盤とした細胞内共生進化の初期における宿主細胞の進化過程の解明
基盤研究(A)
国立遺伝学研究所
2020年04月01日 - 2024年03月31日

母性遺伝を保障する葉緑体DNAの「組換え抑制」と「選択的複製」の解析
若手研究
茨城大学
2020年04月01日 - 2023年03月31日

ミドリゾウリムシを用いた宿主・共生体の代謝・概日時計を介した双方向制御機構の解析
特別研究員奨励費
2018年04月25日 - 2021年03月31日

ミドリゾウリムシにおける共生藻獲得に伴う遺伝子発現・代謝動態とその意義の解明
研究活動スタート支援
国立遺伝学研究所
2017年08月25日 - 2019年03月31日

オルガネラゲノム核様体の構造様式：その蛋白質構成、ダイナミズムに迫る
特別研究員奨励費
京都大学
2014年04月25日 - 2017年03月31日