研究者詳細 - 石川　一也

論文 - 石川　一也

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Lytic Transglycosylase Deficiency Increases Susceptibility to β‐lactam Antibiotics But Reduces Susceptibility to Vancomycin in Escherichia coli 査読

Takahiko Kimura, Kazuya Ishikawa, Ryosuke Nakagawa, Kazuyuki Furuta, Chikara Kaito

Microbiology and Immunology 2025年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Wiley

ABSTRACT

In Staphylococcus aureus, a gram‐positive pathogen, vancomycin‐resistant strains become susceptible to β‐lactam antibiotics, referred to as the “seesaw effect.” However, in gram‐negative bacteria, the phenomenon is less clear. Here, we analyzed the gene‐knockout effects of eight lytic transglycosylases (slt, mltA, mltB, mltC, mltD, mltE, mltF, mltG) on antibiotic sensitivity in Escherichia coli. Knockout of both slt and mltG increased sensitivity to β‐lactam antibiotics and reduced sensitivity to vancomycin. The β‐lactam antibiotic sensitivity and vancomycin resistance of the slt‐knockout mutant were abolished by the introduction of the wild‐type slt gene but remained unchanged by the introduction of the mutant slt gene encoding an amino acid substitution variant of the transglycosylase catalytic centre. The double‐knockout strain for slt and mltB was more sensitive to ampicillin and more resistant to vancomycin than each single‐knockout strain. The double‐knockout strain for slt and mltG was more sensitive to ampicillin and more resistant to vancomycin than each single‐knockout strain. These results suggest that loss of lytic transglycosylase activity causes β‐lactam antibiotic sensitivity and vancomycin resistance in E. coli.

DOI： 10.1111/1348-0421.13227

Enterobacterial common antigen repeat-unit flippase WzxE is required for Escherichia coli growth under acidic conditions, low temperature, and high osmotic stress conditions 査読

Saki Yamaguchi, Kazuya Ishikawa, Kazuyuki Furuta, Chikara Kaito

Applied and Environmental Microbiology 91 ( 5 ) 2025年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：American Society for Microbiology

ABSTRACT

Colanic acid and enterobacterial common antigen (ECA) are cell-surface polysaccharides that are produced by many Escherichia coli isolates. Colanic acid is induced under acidic, low temperature, and high-salt conditions and is important for E. coli resistance to these stresses; however, the role of ECA in these stresses is less clear. Here, we observed that knockout of flippase wzxE , which translocates lipid-linked ECA repeat units from the cytoplasmic side of the inner membrane to the periplasmic side, resulted in the sensitivity of E. coli BW25113 to acidic conditions. The wzxE -knockout mutant showed reduced growth potential and viable counts in vegetable extracts with acidic environments, including cherry tomatoes, carrots, celery, lettuce, and spinach. A double-knockout strain of wzxE and wecF (glycosyltransferase that adds the third-and-final sugar of the lipid-linked ECA repeat unit) was not sensitive to acidic conditions, with similar results obtained for a double-knockout strain of wzxE and wcaJ (glycosyltransferase that initiates colanic acid lipid-linked repeat-unit biosynthesis). The wzxE -knockout mutant was sensitive to low temperatures or high-salt conditions, which induced colanic acid synthesis, and these sensitivities were abolished by the additional knockout of wcaJ . These results suggest that lipid-linked ECA repeat units confer E. coli susceptibility to acidic, low temperatures, and high-salt conditions in a colanic acid-dependent manner and that wzxE suppresses this negative effect.

IMPORTANCE

The role of the common enterobacterial antigen, a polysaccharide that is conserved throughout enterobacteria, in stress resistance is unclear. Our results suggest that lipid-linked enterobacterial common antigen repeat units (which are typically translocated across the inner membrane by the flippase WzxE) cause sensitivity of Escherichia coli to acidic, low-temperature, and high-salt conditions in a manner dependent on colanic acid. The wzxE -knockout mutant was sensitive to crude vegetable extracts, suggesting that the development of WzxE inhibitors could lead to novel food poisoning prevention agents. Considering previous findings that lipid-linked ECA repeat units are flipped by both WzxE and the flippase for colanic acid lipid-linked repeat-unit, the colanic acid-dependence of the wzxE -knockout phenotype proposes a model in which a large amount of colanic acid under stress conditions occupies the flippase for colanic acid lipid-linked repeat unit, leading to accumulation of lipid-linked ECA repeat units on the inner membrane.

DOI： 10.1128/aem.02595-24

Xenopus laevis as an infection model for human pathogenic bacteria 査読

Ayano Kuriu, Kazuya Ishikawa, Kohsuke Tsuchiya, Kazuyuki Furuta, Chikara Kaito

Infection and Immunity 2025年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：American Society for Microbiology

ABSTRACT

Animal infection models are essential for understanding bacterial pathogenicity and corresponding host immune responses. In this study, we investigated whether juvenile Xenopus laevis could be used as an infection model for human pathogenic bacteria. Xenopus frogs succumbed to intraperitoneal injection containing the human pathogenic bacteria Staphylococcus aureus , Pseudomonas aeruginosa , and Listeria monocytogenes . In contrast, non-pathogenic bacteria Bacillus subtilis and Escherichia coli did not induce mortality in Xenopus frogs. The administration of appropriate antibiotics suppressed mortality caused by S. aureus and P. aeruginosa . Strains lacking the agr locus, cvfA ( rny ) gene, or hemolysin genes in S. aureus , LIPI-1-deleted mutant of L. monocytogenes , which attenuate virulence within mammals, exhibited reduced virulence in Xenopus frogs compared with their respective wild-type counterparts. Bacterial distribution analysis revealed that S. aureus persisted in the blood, liver, heart, and muscles of Xenopus frogs until death. These results suggested that intraperitoneal injection of human pathogenic bacteria induces sepsis-like symptoms in Xenopus frogs, supporting their use as a valuable animal model for evaluating antimicrobial efficacy and identifying virulence genes in various human pathogenic bacteria.

DOI： 10.1128/iai.00126-25

RpoB H481Y Rifampicin Resistance Mutation‐Associated Oxidative Stress Sensitivity Reduces the Virulence of Staphylococcus aureus 査読

Tomonori Kano, Kazuya Ishikawa, Lina Imai, Kazuyuki Furuta, Chikara Kaito

Microbiology and Immunology 69 ( 2 ) 128 - 131 2024年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Wiley

ABSTRACT

In this study, we have established a Staphylococcus aureus RpoB H481Y mutant strain and demonstrated that it is sensitive to menadione or hydrogen peroxide‐induced oxidative stress and exhibits reduced virulence against a silkworm infection model. Furthermore, the reduced virulence of the RpoB H481Y mutant was abrogated in the presence of N‐acetylcysteine, a reactive oxygen species scavenger. These results suggest that oxidative stress sensitivity caused by the RpoB H481Y rifampicin resistance mutation attenuates the virulence of S. aureus.

DOI： 10.1111/1348-0421.13190

Overexpression of diglucosyldiacylglycerol synthase leads to daptomycin resistance in Bacillus subtilis 査読

Ryogo Yamamoto, Kazuya Ishikawa, Yusuke Miyoshi, Kazuyuki Furuta, Shin-Ichi Miyoshi, Chikara Kaito

Journal of Bacteriology 206 ( 10 ) e00307 - e00324 2024年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：American Society for Microbiology

ABSTRACT

The lipopeptide antibiotic daptomycin exhibits bactericidal activity against Gram-positive bacteria by forming a complex with phosphatidylglycerol (PG) and lipid II in the cell membrane, causing membrane perforation. With the emergence of daptomycin-resistant bacteria, understanding the mechanisms of bacterial resistance to daptomycin has gained great importance. In this study, we aimed to identify the genetic factors contributing to daptomycin resistance in Bacillus subtilis , a model Gram-positive bacterium. Our findings demonstrated that overexpression of ugtP , which encodes diglucosyldiacylglycerol synthase, induces daptomycin resistance in B. subtilis . Specifically, overexpression of ugtP resulted in increased levels of diglucosyldiacylglycerol (Glc ₂ DAG) and decreased levels of acidic phospholipids cardiolipin and PG, as well as the basic phospholipid lysylphosphatidylglycerol. However, ugtP overexpression did not alter the cell surface charge and the susceptibility to the cationic antimicrobial peptide nisin or the cationic surfactant hexadecyltrimethylammonium bromide. Furthermore, by serial passaging in the presence of daptomycin, we obtained daptomycin-resistant mutants carrying ugtP mutations. These mutants showed increased levels of Glc ₂ DAG and a >4-fold increase in the minimum inhibitory concentration of daptomycin. These results suggest that increased Glc ₂ DAG levels, driven by ugtP overexpression, modify the phospholipid composition and confer daptomycin resistance in B. subtilis without altering the cell surface charge of the bacteria.

IMPORTANCE

Daptomycin is one of the last-resort drugs for the treatment of methicillin-resistant Staphylococcus aureus infections, and the emergence of daptomycin-resistant bacteria has become a major concern. Understanding the mechanism of daptomycin resistance is important for establishing clinical countermeasures against daptomycin-resistant bacteria. In the present study, we found that overexpression of ugtP , which encodes diglucosyldiacylglycerol synthase, induces daptomycin resistance in B. subtilis , a model Gram-positive bacteria. The overexpression of UgtP increased diglucosyldiacylglycerol levels, resulting in altered phospholipid composition and daptomycin resistance. These findings are important for establishing clinical strategies against daptomycin-resistant bacteria, including their detection and management.

DOI： 10.1128/jb.00307-24

Overexpression of the flagellar motor protein MotB sensitizes Bacillus subtilis to aminoglycosides in a motility-independent manner 査読

Mio Uneme, Kazuya Ishikawa, Kazuyuki Furuta, Atsuko Yamashita, Chikara Kaito

PLOS ONE 19 ( 4 ) e0300634 - e0300634 2024年4月

掲載種別：研究論文（学術雑誌）出版者・発行元：Public Library of Science (PLoS)

The flagellar motor proteins, MotA and MotB, form a complex that rotates the flagella by utilizing the proton motive force (PMF) at the bacterial cell membrane. Although PMF affects the susceptibility to aminoglycosides, the effect of flagellar motor proteins on the susceptibility to aminoglycosides has not been investigated. Here, we found that MotB overexpression increased susceptibility to aminoglycosides, such as kanamycin and gentamicin, in Bacillus subtilis without affecting swimming motility. MotB overexpression did not affect susceptibility to ribosome-targeting antibiotics other than aminoglycosides, cell wall-targeting antibiotics, DNA synthesis-inhibiting antibiotics, or antibiotics inhibiting RNA synthesis. Meanwhile, MotB overexpression increased the susceptibility to aminoglycosides even in the motA-deletion mutant, which lacks swimming motility. Overexpression of the MotB mutant protein carrying an amino acid substitution at the proton-binding site (D24A) resulted in the loss of the enhanced aminoglycoside-sensitive phenotype. These results suggested that MotB overexpression sensitizes B. subtilis to aminoglycosides in a motility-independent manner. Notably, the aminoglycoside-sensitive phenotype induced by MotB requires the proton-binding site but not the MotA/MotB complex formation.

DOI： 10.1371/journal.pone.0300634

Bilirubin Distribution in Plants at the Subcellular and Tissue Levels 査読

Kazuya Ishikawa, Yutaka Kodama

Plant and Cell Physiology 65 ( 5 ) 762 - 769 2024年2月

担当区分：筆頭著者掲載種別：研究論文（学術雑誌）出版者・発行元：Oxford University Press (OUP)

Abstract

In heterotrophs, heme degradation produces bilirubin, a tetrapyrrole compound that has antioxidant activity. In plants, heme is degraded in plastids and is believed to be converted to phytochromobilin rather than bilirubin. Recently, we used the bilirubin-inducible fluorescent protein UnaG to reveal that plants produce bilirubin via a non-enzymatic reaction with NADPH. In the present study, we used an UnaG-based live imaging system to visualize bilirubin accumulation in Arabidopsis thaliana and Nicotiana benthamiana at the organelle and tissue levels. In chloroplasts, bilirubin preferentially accumulated in the stroma, and the stromal bilirubin level increased upon dark treatment. Investigation of intracellular bilirubin distribution in leaves and roots showed that it accumulated mostly in plastids, with low levels detected in the cytosol and other organelles, such as peroxisomes, mitochondria and the endoplasmic reticulum. A treatment that increased bilirubin production in chloroplasts decreased the bilirubin level in peroxisomes, implying that a bilirubin precursor is transported between the two organelles. At the cell and tissue levels, bilirubin showed substantial accumulation in the root elongation region but little or none in the root cap and guard cells. Intermediate bilirubin accumulation was observed in other shoot and root tissues, with lower levels in shoot tissues. Our data revealed the distribution of bilirubin in plants, which has implications for the transport and physiological function of tetrapyrroles.

DOI： 10.1093/pcp/pcae017

その他リンク： https://academic.oup.com/pcp/article-pdf/65/5/762/58008797/pcae017.pdf

Knockout of adenylosuccinate synthase purA increases susceptibility to colistin in Escherichia coli 査読

Tomonori Kano, Kazuya Ishikawa, Kazuyuki Furuta, Chikara Kaito

FEMS Microbiology Letters 371 fnae007 2024年2月

掲載種別：研究論文（学術雑誌）出版者・発行元：Oxford University Press (OUP)

Abstract

Colistin is a cationic cyclic antimicrobial peptide used as a last resort against multidrug-resistant gram-negative bacteria. To understand the factors involved in colistin susceptibility, we screened colistin-sensitive mutants from an E. coli gene-knockout library (Keio collection). The knockout of purA, whose product catalyzes the synthesis of adenylosuccinate from IMP in the de novo purine synthesis pathway, resulted in increased sensitivity to colistin. Adenylosuccinate is subsequently converted to AMP, which is phosphorylated to produce ADP, a substrate for ATP synthesis. The amount of ATP was lower in the purA-knockout mutant than that in the wild-type strain. ATP synthesis is coupled with proton transfer, and it contributes to the membrane potential. Using the membrane potential probe, 3,3′-diethyloxacarbocyanine iodide [DiOC2(3)], we found that the membrane was hyperpolarized in the purA-knockout mutant compared to that in the wild-type strain. Treatment with the proton uncoupler, carbonyl cyanide m-chlorophenyl hydrazone (CCCP), abolished the hyperpolarization and colistin sensitivity in the mutant. The purA-knockout mutant exhibited increased sensitivity to aminoglycosides, kanamycin, and gentamicin; their uptake requires a membrane potential. Therefore, the knockout of purA, an adenylosuccinate synthase, decreases ATP synthesis concurrently with membrane hyperpolarization, resulting in increased sensitivity to colistin.

DOI： 10.1093/femsle/fnae007

Short‐chain fatty acids stimulate dendrite elongation in dendritic cells by inhibiting histone deacetylase 査読

Takuho Inamoto, Kazuyuki Furuta, Cheng Han, Mio Uneme, Tomonori Kano, Kazuya Ishikawa, Chikara Kaito

The FEBS Journal 290 ( 24 ) 5794 - 5810 2023年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Wiley

Dendritic cells activate immune responses by presenting pathogen‐derived molecules. The dendrites of dendritic cells contribute to the incorporation of foreign antigens or presenting antigens to T cells. Short‐chain fatty acids (SCFAs), such as acetic, propionic, butyric and valeric acids, have many effects on immune responses by activating specific receptors or inhibiting a histone deacetylase (HDAC), although their effect on dendrite formation in dendritic cells is unknown. In the present study, we aimed to investigate the effect of SCFAs on dendrite elongation using a dendritic cell line (DC2.4 cells) and mouse bone marrow‐derived dendritic cells. We found that SCFAs induced dendrite elongation. The elongation was reduced by inhibitors of Src family kinase (SFK), phosphatidylinositol‐3 kinase (PI3K), Rho family GTPases (Cdc42, Rac1) or actin polymerization, indicating that SCFAs promote dendrite elongation by activating actin polymerization via the SFK/PI3K/Rho family GTPase signaling pathway. We showed that agonists for SCFA receptors GPR43 and GPR109a did not promote dendrite elongation. By contrast, HDAC inhibitors, including trichostatin A, promoted dendrite elongation in DC2.4 cells, and the promoting activity of trichostatin A was decreased by inhibiting the SFK/PI3K/Rho family GTPase signaling pathway or actin polymerization. Furthermore, DC2.4 cells treated with valeric acid showed enhanced uptake of soluble proteins, insoluble beads and Staphylococcus aureus. We also found that treatment with valeric acid enhanced major histocompatibility complex class II‐mediated antigen presentation in bone marrow‐derived dendritic cells. These results suggest that SCFAs promote dendrite elongation by inhibiting HDAC, stimulating the SFK/PI3K/Rho family pathway and activating actin polymerization, resulting in increased antigen uptake and presentation in dendritic cells.

DOI： 10.1111/febs.16945

Bilirubin is produced nonenzymatically in plants to maintain chloroplast redox status 査読国際誌

Kazuya Ishikawa, Xiaonan Xie, Yasuhide Osaki, Atsushi Miyawaki, Keiji Numata, Yutaka Kodama

Science Advances 9 ( 23 ) eadh478 2023年6月

担当区分：筆頭著者記述言語：英語掲載種別：研究論文（学術雑誌）

Bilirubin, a potent antioxidant, is a product of heme catabolism in heterotrophs. Heterotrophs mitigate oxidative stress resulting from free heme by catabolism into bilirubin via biliverdin. Although plants also convert heme to biliverdin, they are generally thought to be incapable of producing bilirubin because they lack biliverdin reductase, the enzyme responsible for bilirubin biosynthesis in heterotrophs. Here, we demonstrate that bilirubin is produced in plant chloroplasts. Live-cell imaging using the bilirubin-dependent fluorescent protein UnaG revealed that bilirubin accumulated in chloroplasts. In vitro, bilirubin was produced nonenzymatically through a reaction between biliverdin and reduced form of nicotinamide adenine dinucleotide phosphate at concentrations comparable to those in chloroplasts. In addition, increased bilirubin production led to lower reactive oxygen species levels in chloroplasts. Our data refute the generally accepted pathway of heme degradation in plants and suggest that bilirubin contributes to the maintenance of redox status in chloroplasts.

DOI： 10.1126/sciadv.adh4787

Binding of Tau-derived peptide-fused GFP to plant microtubules in Arabidopsis thaliana 査読

Hiroshi Inaba, Kazusato Oikawa, Kazuya Ishikawa, Yutaka Kodama, Kazunori Matsuura, Keiji Numata

PLOS ONE 18 ( 6 ) e0286421 2023年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Public Library of Science (PLoS)

Studies on how exogenous molecules modulate properties of plant microtubules, such as their stability, structure, and dynamics, are important for understanding and modulating microtubule functions in plants. We have developed a Tau-derived peptide (TP) that binds to microtubules and modulates their properties by binding of TP-conjugated molecules in vitro. However, there was no investigation of TPs on microtubules in planta. Here, we generated transgenic Arabidopsis thaliana plants stably expressing TP-fused superfolder GFP (sfGFP-TP) and explored the binding properties and effects of sfGFP-TP on plant microtubules. Our results indicate that the expressed sfGFP-TP binds to the plant microtubules without inhibiting plant growth. A transgenic line strongly expressing sfGFP-TP produced thick fibrous structures that were stable under conditions where microtubules normally depolymerize. This study generates a new tool for analyzing and modulating plant microtubules.

DOI： 10.1371/journal.pone.0286421

Knockout of ribosomal protein RpmJ leads to zinc resistance in Escherichia coli 査読

Riko Shirakawa, Kazuya Ishikawa, Kazuyuki Furuta, Chikara Kaito

PLOS ONE 18 ( 3 ) e0277162 2023年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Public Library of Science (PLoS)

Zinc is an essential metal for cells, but excess amounts are toxic. Other than by regulating the intracellular zinc concentration by zinc uptake or efflux, the mechanisms underlying bacterial resistance to excess zinc are unknown. In the present study, we searched for zinc-resistant mutant strains from the Keio collection, a gene knockout library of Escherichia coli, a model gram-negative bacteria. We found that knockout mutant of RpmJ (L36), a 50S ribosomal protein, exhibited zinc resistance. The rpmJ mutant was sensitive to protein synthesis inhibitors and had altered translation fidelity, indicating ribosomal dysfunction. In the rpmJ mutant, the intracellular zinc concentration was decreased under excess zinc conditions. Knockout of ZntA, a zinc efflux pump, abolished the zinc-resistant phenotype of the rpmJ mutant. RNA sequence analysis revealed that the rpmJ mutant exhibited altered gene expression of diverse functional categories, including translation, energy metabolism, and stress response. These findings suggest that knocking out RpmJ alters gene expression patterns and causes zinc resistance by lowering the intracellular zinc concentration. Knockouts of other ribosomal proteins, including RplA, RpmE, RpmI, and RpsT, also led to a zinc-resistant phenotype, suggesting that deletion of ribosomal proteins is closely related to zinc resistance.

DOI： 10.1371/journal.pone.0277162

Using the organelle glue technique to engineer the plant cell metabolome 査読

Kazuya Ishikawa, Makoto Kobayashi, Miyako Kusano, Keiji Numata, Yutaka Kodama

Plant Cell Reports 42 ( 3 ) 599 - 607 2023年1月

担当区分：筆頭著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Springer Science and Business Media LLC

DOI： 10.1007/s00299-023-02982-2

その他リンク： https://link.springer.com/article/10.1007/s00299-023-02982-2/fulltext.html

Knockout of ykcB , a Putative Glycosyltransferase, Leads to Reduced Susceptibility to Vancomycin in Bacillus subtilis 査読

Kazuya Ishikawa, Riko Shirakawa, Daiki Takano, Tomoki Kosaki, Kazuyuki Furuta, Chikara Kaito

Journal of Bacteriology 204 ( 12 ) 2022年12月

担当区分：筆頭著者掲載種別：研究論文（学術雑誌）出版者・発行元：American Society for Microbiology

Although vancomycin is effective against Gram-positive bacteria, vancomycin-resistant bacteria are a major public health concern. While the vancomycin-resistance mechanisms of clinically important bacteria such as Staphylococcus aureus , Enterococcus faecium , and Streptococcus pneumoniae are well studied, they remain unclear in other Gram-positive bacteria.

DOI： 10.1128/jb.00387-22

Live‐cell imaging of the chloroplast outer envelope membrane using fluorescent dyes 査読国際誌

Shintaro Ichikawa, Kazuya Ishikawa, Hitoshi Miyakawa, Yutaka Kodama

Plant Direct 6 ( 11 ) e462 2022年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Wiley

UNLABELLED: Chloroplasts are organelles composed of sub-organellar compartments-stroma, thylakoids, and starch granules-and are surrounded by outer and inner envelope membranes (OEM and IEM, respectively). The chloroplast OEM and IEM play key roles not only as a barrier separating the chloroplast components from the cytosol but also in the interchange of numerous metabolites and proteins between the chloroplast interior and the cytosol. Fluorescent protein markers for the chloroplast OEM have been widely used to visualize the outermost border of chloroplasts. However, the use of marker proteins requires an established cellular genetic transformation method, which limits the plant species in which marker proteins can be used. Moreover, the high accumulation of OEM marker proteins often elicits abnormal morphological phenotypes of the OEM. Because the OEM can currently only be visualized using exogenous marker proteins, the behaviors of the chloroplast and/or its OEM remain unknown in wild-type cells of various plant species. Here, we visualized the OEM using live-cell staining with the fluorescent dyes rhodamine B and Nile red in several plant species, including crops. We propose rhodamine B and Nile red as new tools for visualizing the chloroplast OEM in living plant cells that do not require genetic transformation. SIGNIFICANCE STATEMENT: We established a live-cell imaging method to visualize the chloroplast outer envelope membrane by staining living cells with fluorescent dyes. This method does not require genetic transformation and allows the observation of the chloroplast outer envelope membrane in various plant species.

DOI： 10.1002/pld3.462

その他リンク： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/pld3.462

Organellar Glue: A Molecular Tool to Artificially Control Chloroplast–Chloroplast Interactions 査読国際誌

Shintaro Ichikawa, Shota Kato, Yuta Fujii, Kazuya Ishikawa, Keiji Numata, Yutaka Kodama

ACS Synthetic Biology 11 ( 10 ) 3190 - 3197 2022年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：American Chemical Society (ACS)

Organelles can physically interact to facilitate various cellular processes such as metabolite exchange. Artificially regulating these interactions represents a promising approach for synthetic biology. Here, we artificially controlled chloroplast-chloroplast interactions in living plant cells with our organelle glue (ORGL) technique, which is based on reconstitution of a split fluorescent protein. We simultaneously targeted N-terminal and C-terminal fragments of a fluorescent protein to the chloroplast outer envelope membrane or cytosol, respectively, which induced chloroplast-chloroplast interactions. The cytosolic C-terminal fragment likely functions as a bridge between two N-terminal fragments, thereby bringing the chloroplasts in close proximity to interact. We modulated the frequency of chloroplast-chloroplast interactions by altering the ratio of N- and C-terminal fragments. We conclude that the ORGL technique can successfully control chloroplast-chloroplast interactions in plants, providing a proof of concept for the artificial regulation of organelle interactions in living cells.

DOI： 10.1021/acssynbio.2c00367

The endoplasmic reticulum membrane–bending protein RETICULON facilitates chloroplast relocation movement in Marchantia polymorpha 査読国際誌

Kazuya Ishikawa, Ryota Konno, Satoyuki Hirano, Yuta Fujii, Masayuki Fujiwara, Yoichiro Fukao, Yutaka Kodama

Plant Journal 111 ( 1 ) 205 - 216 2022年7月

担当区分：筆頭著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Wiley

Plant cells alter the intracellular positions of chloroplasts to ensure efficient photosynthesis, a process controlled by the blue light receptor phototropin. Chloroplasts migrate toward weak light (accumulation response) and move away from excess light (avoidance response). Chloroplasts are encircled by the endoplasmic reticulum (ER), which forms a complex network throughout the cytoplasm. To ensure rapid chloroplast relocation, the ER must alter its structure in conjunction with chloroplast relocation movement, but little is known about the underlying mechanism. Here, we searched for interactors of phototropin in the liverwort Marchantia polymorpha and identified a RETICULON (RTN) family protein; RTN proteins play central roles in ER tubule formation and ER network maintenance by stabilizing the curvature of ER membranes in eukaryotic cells. Marchantia polymorpha RTN1 (MpRTN1) is localized to ER tubules and the rims of ER sheets, which is consistent with the localization of RTNs in other plants and heterotrophs. The Mprtn1 mutant showed an increased ER tubule diameter, pointing to a role for MpRTN1 in ER membrane constriction. Furthermore, Mprtn1 showed a delayed chloroplast avoidance response but a normal chloroplast accumulation response. The live cell imaging of ER dynamics revealed that ER restructuring was impaired in Mprtn1 during the chloroplast avoidance response. These results suggest that during the chloroplast avoidance response, MpRTN1 restructures the ER network and facilitates chloroplast movement via an interaction with phototropin. Our findings provide evidence that plant cells respond to fluctuating environmental conditions by controlling the movements of multiple organelles in a synchronized manner.

DOI： 10.1111/tpj.15787

その他リンク： https://onlinelibrary.wiley.com/doi/full-xml/10.1111/tpj.15787

Arabidopsis group C Raf-like protein kinases negatively regulate abscisic acid signaling and are direct substrates of SnRK2 査読国際誌

Yoshiaki Kamiyama, Misaki Hirotani, Shinnosuke Ishikawa, Fuko Minegishi, Sotaro Katagiri, Conner J. Rogan, Fuminori Takahashi, Mika Nomoto, Kazuya Ishikawa, Yutaka Kodama, Yasuomi Tada, Daisuke Takezawa, Jeffrey C. Anderson, Scott C. Peck, Kazuo Shinozaki, Taishi Umezawa

Proceedings of the National Academy of Sciences 118 ( 30 ) e2100073118 - e2100073118 2021年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Proceedings of the National Academy of Sciences

The phytohormone abscisic acid (ABA) plays a major role in abiotic stress responses in plants, and subclass III SNF1-related protein kinase 2 (SnRK2) kinases mediate ABA signaling. In this study, we identified Raf36, a group C Raf-like protein kinase in <italic>Arabidopsis</italic>, as a protein that interacts with multiple SnRK2s. A series of reverse genetic and biochemical analyses revealed that 1) Raf36 negatively regulates ABA responses during postgermination growth, 2) the N terminus of Raf36 is directly phosphorylated by SnRK2s, and 3) Raf36 degradation is enhanced in response to ABA. In addition, Raf22, another C-type Raf-like kinase, functions partially redundantly with Raf36 to regulate ABA responses. A comparative phosphoproteomic analysis of ABA-induced responses of wild-type and <italic>raf22raf36-1</italic> plants identified proteins that are phosphorylated downstream of Raf36 and Raf22 in planta. Together, these results support a model in which Raf36/Raf22 function mainly under optimal conditions to suppress ABA responses, whereas in response to ABA, the SnRK2 module promotes Raf36 degradation as a means of alleviating Raf36-dependent inhibition and allowing for heightened ABA signaling to occur.

DOI： 10.1073/pnas.2100073118

その他リンク： https://syndication.highwire.org/content/doi/10.1073/pnas.2100073118

Mitochondrial movement during its association with chloroplasts in Arabidopsis thaliana 査読国際誌

Kazusato Oikawa, Takuto, Imai, Chonprakun Thagun, Kiminori Toyooka, Takeshi Yoshizumi, Kazuya Ishikawa, Yutaka Kodama, Keiji Numata

Communications Biology 4 ( 1 ) 1 - 13 2021年3月

記述言語：英語掲載種別：研究論文（学術雑誌）

Plant mitochondria move dynamically inside cells and this movement is classified into two types: directional movement, in which mitochondria travel long distances, and wiggling, in which mitochondria travel short distances. However, the underlying mechanisms and roles of both types of mitochondrial movement, especially wiggling, remain to be determined. Here, we used confocal laser-scanning microscopy to quantitatively characterize mitochondrial movement (rate and trajectory) in Arabidopsis thaliana mesophyll cells. Directional movement leading to long-distance migration occurred at high speed with a low angle-change rate, whereas wiggling leading to short-distance migration occurred at low speed with a high angle-change rate. The mean square displacement (MSD) analysis could separate these two movements. Directional movement was dependent on filamentous actin (F-actin), whereas mitochondrial wiggling was not, but slightly influenced by F-actin. In mesophyll cells, mitochondria could migrate by wiggling, and most of these mitochondria associated with chloroplasts. Thus, mitochondria migrate via F-actin-independent wiggling under the influence of F-actin during their association with chloroplasts in Arabidopsis.

DOI： 10.1038/s42003-021-01833-8

Apple latent spherical virus (ALSV)-induced gene silencing in a medicinal plant, Lithospermum erythrorhizon 査読国際誌

Yuki Izuishi, Natsumi Isaka, Hao Li, Kohei Nakanishi, Joji Kageyama, Kazuya Ishikawa, Tomoo Shimada, Chikara Masuta, Nobuyuki Yoshikawa, Hiroaki Kusano, Kazufumi Yazaki

Scientific Reports 10 ( 1 ) 13555 - 13555 2020年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Springer Science and Business Media LLC

Lithospermum erythrorhizon is a medicinal plant that produces shikonin, a red lipophilic naphthoquinone derivative that accumulates exclusively in roots. The biosynthetic steps required to complete the naphthalene ring of shikonin and its mechanism of secretion remain unclear. Multiple omics studies identified several candidate genes involved in shikonin production. The functions of these genes can be evaluated using virus-induced gene silencing (VIGS) systems, which have been shown advantageous in introducing iRNA genes into non-model plants. This study describes the development of a VIGS system using an apple latent spherical virus (ALSV) vector and a target gene, phytoene desaturase (LePDS1). Virus particles packaged in Nicotiana benthamiana were inoculated into L. erythrorhizon seedlings, yielding new leaves with albino phenotype but without disease symptoms. The levels of LePDS1 mRNAs were significantly lower in the albino plants than in mock control or escape plants. Virus-derived mRNA was detected in infected plants but not in escape and mock plants. Quantitative PCR and deep sequencing analysis indicated that transcription of another hypothetical PDS gene (LePDS2) also decreased in the defective leaves. Virus infection, however, had no effect on shikonin production. These results suggest that virus-based genetic transformation and the VIGS system silence target genes in soil-grown L. erythrorhizon.

DOI： 10.1038/s41598-020-70469-1

その他リンク： http://www.nature.com/articles/s41598-020-70469-1

Structural and functional relationships between plasmodesmata and plant endoplasmic reticulum–plasma membrane contact sites consisting of three synaptotagmins 査読国際誌

Kazuya Ishikawa, Kentaro Tamura, Yoichiro Fukao, Tomoo Shimada

New Phytologist 226 ( 3 ) 798 - 808 2020年5月

担当区分：筆頭著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Wiley

Synaptotagmin 1 (SYT1) has been recognised as a tethering factor of plant endoplasmic reticulum (ER)-plasma membrane (PM) contact sites (EPCSs) and partially localises to around plasmodesmata (PD). However, other components of EPCSs associated with SYT1 and functional links between the EPCSs and PD have not been identified. We explored interactors of SYT1 by immunoprecipitation and mass analysis. The dynamics, morphology and spatial arrangement of the ER in Arabidopsis mutants lacking the EPCS components were investigated using confocal microscopy and electron microscopy. PD permeability of EPCS mutants was assessed using a virus movement protein and free green fluorescent protein (GFP) as indicators. We identified two additional components of the EPCSs, SYT5 and SYT7, that interact with SYT1. The mutants of the three SYTs were defective in the anchoring of the ER to the PM. The ER near the PD entrance appeared to be weakly squeezed in the triple mutant compared with the wild-type. The triple mutant suppressed cell-to-cell movement of the virus movement protein, but not GFP diffusion. We revealed major additional components of EPCS associated with SYT1 and suggested that the EPCSs arranged around the PD squeeze the ER to regulate active transport via PD.

DOI： 10.1111/nph.16391

その他リンク： https://onlinelibrary.wiley.com/doi/full-xml/10.1111/nph.16391

Subcellular localisation of an endoplasmic reticulum-plasma membrane tethering factor, SYNAPTOTAGMIN 1, is affected by fluorescent protein fusion 招待査読

Kazuya Ishikawa, Kentaro Tamura, Tomoo Shimada

Plant Signaling & Behavior 13 ( 12 ) e1547577 - e1547577 2018年12月

Synaptotagmin-Associated Endoplasmic Reticulum-Plasma Membrane Contact Sites Are Localized to Immobile ER Tubules 査読国際誌

Kazuya Ishikawa, Kentaro Tamura, Haruko Ueda, Yoko Ito, Akihiko Nakano, Ikuko Hara-Nishimura, Tomoo Shimada

Plant Physiology 178 ( 2 ) 641 - 653 2018年10月

担当区分：筆頭著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：American Society of Plant Biologists (ASPB)

The plant endoplasmic reticulum (ER), which is morphologically divided into tubules and sheets, seems to flow continuously as a whole, but locally, mobile and immobile regions exist. In eukaryotes, the ER physically and functionally interacts with the plasma membrane (PM) at domains called ER-PM contact sites (EPCSs). Extended synaptotagmin family proteins are concentrated in the cortical ER to form one type of EPCS; however, it is unclear whether the localization of extended synaptotagmin corresponds to the EPCS and where in the cortical ER the EPCSs are formed. Here, we analyzed the spatiotemporal localization of SYNAPTOTAGMIN1 (SYT1), a synaptotagmin in Arabidopsis (Arabidopsis thaliana), to investigate the precise distribution of SYT1-associated EPCSs in the cortical ER. Three-dimensional imaging using superresolution confocal live imaging microscopy demonstrated that SYT1 was specifically localized to the ER-PM boundary. Time-lapse imaging revealed that SYT1 was distributed to immobile ER tubules, but not to mobile tubules. Moreover, SYT1 was frequently localized to the edges of ER sheets that were transformed into immobile ER tubules over time. A lower intracellular calcium ion concentration resulted in an increased EPCS area and disrupted the ER network. Finally, SYT1 deficiency caused a reduction of the immobile tubules and enlargement of the ER meshes. Taken together, our findings show that SYT1-associated EPCS are distributed to immobile tubules and play an important role in the formation of the tubular ER network. This provides important insight into the relationship between the function and dynamics/morphology of the cortical ER.

DOI： 10.1104/pp.18.00498

Dual targeting of a virus movement protein to ER and plasma membrane subdomains is essential for plasmodesmata localization 査読

Kazuya Ishikawa, Masayoshi Hashimoto, Akira Yusa, Hiroaki Koinuma, Yugo Kitazawa, Osamu Netsu, Yasuyuki Yamaji, Shigetou Namba

PLOS Pathogens 13 ( 6 ) e1006463 - e1006463 2017年6月

EXA1, a GYF domain protein, is responsible for loss-of-susceptibility to plantago asiatica mosaic virus in Arabidopsis thaliana 査読

Masayoshi Hashimoto, Yutaro Neriya, Takuya Keima, Nozomu Iwabuchi, Hiroaki Koinuma, Yuka Hagiwara-Komoda, Kazuya Ishikawa, Misako Himeno, Kensaku Maejima, Yasuyuki Yamaji, Shigetou Namba

The Plant Journal 88 ( 1 ) 120 - 131 2016年10月

Passive virus movements with organelle dynamics 招待査読

Kazuya Ishikawa, Masayoshi Hashimoto, Shigetou Namba

Oncotarget 6 ( 31 ) 30437 - 30438 2015年10月

Rapid detection of fig mosaic virus using reverse transcription loop-mediated isothermal amplification 査読

Kazuya Ishikawa, Kensaku Maejima, Osamu Netsu, Misato Fukuoka, Takamichi Nijo, Masayoshi Hashimoto, Daisuke Takata, Yasuyuki Yamaji, Shigetou Namba

Journal of General Plant Pathology 81 ( 5 ) 382 - 389 2015年9月

Cell Death Triggered by a Putative Amphipathic Helix of Radish mosaic virus Helicase Protein Is Tightly Correlated With Host Membrane Modification 査読

Masayoshi Hashimoto, Ken Komatsu, Ryo Iwai, Takuya Keima, Kensaku Maejima, Takuya Shiraishi, Kazuya Ishikawa, Tetsuya Yoshida, Yugo Kitazawa, Yukari Okano, Yasuyuki Yamaji, Shigetou Namba

Molecular Plant-Microbe Interactions 28 ( 6 ) 675 - 688 2015年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Scientific Societies

Systemic necrosis is one of the most severe symptoms caused by plant RNA viruses. Recently, systemic necrosis has been suggested to have similar features to a defense response referred to as the hypersensitive response (HR), a form of programmed cell death. In virus-infected plant cells, host intracellular membrane structures are changed dramatically for more efficient viral replication. However, little is known about whether this replication-associated membrane modification is the cause of the symptoms. In this study, we identified an amino-terminal amphipathic helix of the helicase encoded by Radish mosaic virus (RaMV) (genus Comovirus) as an elicitor of cell death in RaMV-infected plants. Cell death caused by the amphipathic helix had features similar to HR, such as SGT1-dependence. Mutational analyses and inhibitor assays using cerulenin demonstrated that the amphipathic helix–induced cell death was tightly correlated with dramatic alterations in endoplasmic reticulum (ER) membrane structures. Furthermore, the cell death–inducing activity of the amphipathic helix was conserved in Cowpea mosaic virus (genus Comovirus) and Tobacco ringspot virus (genus Nepovirus), both of which are classified in the family Secoviridae. Together, these results indicate that ER membrane modification associated with viral intracellular replication may be recognized to prime defense responses against plant viruses.

DOI： 10.1094/mpmi-01-15-0004-r

Nucleocapsid Protein from Fig Mosaic Virus Forms Cytoplasmic Agglomerates That Are Hauled by Endoplasmic Reticulum Streaming 査読

Kazuya Ishikawa, Chihiro Miura, Kensaku Maejima, Ken Komatsu, Masayoshi Hashimoto, Tatsuya Tomomitsu, Misato Fukuoka, Akira Yusa, Yasuyuki Yamaji, Shigetou Namba

Journal of Virology 89 ( 1 ) 480 - 491 2015年1月

担当区分：筆頭著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：American Society for Microbiology

<title>ABSTRACT</title>Although many studies have demonstrated intracellular movement of viral proteins or viral replication complexes, little is known about the mechanisms of their motility. In this study, we analyzed the localization and motility of the nucleocapsid protein (NP) of<italic>Fig mosaic virus</italic>(FMV), a negative-strand RNA virus belonging to the recently established genus<named-content content-type="genus-species">Emaravirus</named-content>. Electron microscopy of FMV-infected cells using immunogold labeling showed that NPs formed cytoplasmic agglomerates that were predominantly enveloped by the endoplasmic reticulum (ER) membrane, while nonenveloped NP agglomerates also localized along the ER. Likewise, transiently expressed NPs formed agglomerates, designated NP bodies (NBs), in close proximity to the ER, as was the case in FMV-infected cells. Subcellular fractionation and electron microscopic analyses of NP-expressing cells revealed that NBs localized in the cytoplasm. Furthermore, we found that NBs moved rapidly with the streaming of the ER in an actomyosin-dependent manner. Brefeldin A treatment at a high concentration to disturb the ER network configuration induced aberrant accumulation of NBs in the perinuclear region, indicating that the ER network configuration is related to NB localization. Dominant negative inhibition of the class XI myosins, XI-1, XI-2, and XI-K, affected both ER streaming and NB movement in a similar pattern. Taken together, these results showed that NBs localize in the cytoplasm but in close proximity to the ER membrane to form enveloped particles and that this causes passive movements of cytoplasmic NBs by ER streaming.

<bold>IMPORTANCE</bold>Intracellular trafficking is a primary and essential step for the cell-to-cell movement of viruses. To date, many studies have demonstrated the rapid intracellular movement of viral factors but have failed to provide evidence for the mechanism or biological significance of this motility. Here, we observed that agglomerates of nucleocapsid protein (NP) moved rapidly throughout the cell, and we performed live imaging and ultrastructural analysis to identify the mechanism of motility. We provide evidence that cytoplasmic protein agglomerates were passively dragged by actomyosin-mediated streaming of the endoplasmic reticulum (ER) in plant cells. In virus-infected cells, NP agglomerates were surrounded by the ER membranes, indicating that NP agglomerates form the basis of enveloped virus particles in close proximity to the ER. Our work provides a sophisticated model of macromolecular trafficking in plant cells and improves our understanding of the formation of enveloped particles of negative-strand RNA viruses.

DOI： 10.1128/jvi.02527-14

Complete nucleotide sequence and genome structure of a Japanese isolate of hibiscus latent Fort Pierce virus, a unique tobamovirus that contains an internal poly(A) region in its 3′ end 査読

Tetsuya Yoshida, Yugo Kitazawa, Ken Komatsu, Yutaro Neriya, Kazuya Ishikawa, Naoko Fujita, Masayoshi Hashimoto, Kensaku Maejima, Yasuyuki Yamaji, Shigetou Namba

Archives of Virology 159 ( 11 ) 3161 - 3165 2014年11月

Recognition of floral homeotic MADS domain transcription factors by a phytoplasmal effector, phyllogen, induces phyllody 査読

Kensaku Maejima, Ryo Iwai, Misako Himeno, Ken Komatsu, Yugo Kitazawa, Naoko Fujita, Kazuya Ishikawa, Misato Fukuoka, Nami Minato, Yasuyuki Yamaji, Kenro Oshima, Shigetou Namba

The Plant Journal 78 ( 4 ) 541 - 554 2014年5月

Development of an on-site plum pox virus detection kit based on immunochromatography 査読

Kensaku Maejima, Misako Himeno, Osamu Netsu, Kazuya Ishikawa, Tetsuya Yoshida, Naoko Fujita, Masayoshi Hashimoto, Ken Komatsu, Yasuyuki Yamaji, Shigetou Namba

Journal of General Plant Pathology 80 ( 2 ) 176 - 183 2014年3月

Fig mosaic emaravirus p4 protein is involved in cell-to-cell movement 査読

Kazuya Ishikawa, Kensaku Maejima, Ken Komatsu, Osamu Netsu, Takuya Keima, Takuya Shiraishi, Yukari Okano, Masayoshi Hashimoto, Yasuyuki Yamaji, Shigetou Namba

Journal of General Virology 94 ( 3 ) 682 - 686 2013年3月

Identification and characterization of two novel genomic RNA segments of fig mosaic virus, RNA5 and RNA6 査読

Kazuya Ishikawa, Kensaku Maejima, Ken Komatsu, Yugo Kitazawa, Masayoshi Hashimoto, Daisuke Takata, Yasuyuki Yamaji, Shigetou Namba

Journal of General Virology 93 ( 7 ) 1612 - 1619 2012年7月

Infection of capilloviruses requires subgenomic RNAs whose transcription is controlled by promoter-like sequences conserved among flexiviruses 査読

Ken Komatsu, Hisae Hirata, Takako Fukagawa, Yasuyuki Yamaji, Yukari Okano, Kazuya Ishikawa, Tatsushi Adachi, Kensaku Maejima, Masayoshi Hashimoto, Shigetou Namba

Virus Research 167 ( 1 ) 8 - 15 2012年7月

First report of fig mosaic virus infecting common fig (Ficus carica) in Japan 査読

Kazuya Ishikawa, Kensaku Maejima, Susumu Nagashima, Nobuo Sawamura, Yusuke Takinami, Ken Komatsu, Masayoshi Hashimoto, Yasuyuki Yamaji, Jun Yamamoto, Shigetou Namba

Journal of General Plant Pathology 78 ( 2 ) 136 - 139 2012年1月

Identification of three MAPKKKs forming a linear signaling pathway leading to programmed cell death in Nicotiana benthamiana 査読

Masayoshi Hashimoto, Ken Komatsu, Kensaku Maejima, Yukari Okano, Takuya Shiraishi, Kazuya Ishikawa, Yusuke Takinami, Yasuyuki Yamaji, Shigetou Namba

BMC Plant Biology 12 ( 1 ) 103 - 103 2012年

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