研究者詳細 - 兵頭　究

論文 - 兵頭　究

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Evidence for the replication of a plant rhabdovirus in its arthropod mite vector. 国際誌

Hideki Kondo, Miki Fujita, Paul Telengech, Kazuyuki Maruyam, Kiwamu Hyodo, Aline Daniele Tassi, Ronald Ochoa, Ida Bagus Andika, Nobuhiro Suzuki

Virus research 351 199522 - 199522 2025年1月

記述言語：英語掲載種別：研究論文（学術雑誌）

Transmission of plant viruses that replicate in the insect vector is known as persistent-propagative manner. However, it remains unclear whether such virus-vector relationships also occur between plant viruses and other biological vectors such as arthropod mites. In this study, we investigated the possible replication of orchid fleck virus (OFV), a segmented plant rhabdovirus, within its mite vector (Brevipalpus californicus s.l.) using quantitative RT-qPCR, western blotting and next-generation sequencing. Time-course RT-qPCR and western blot analyses showed an increasing OFV accumulation pattern in mites after virus acquisition. Since OFV genome expression requires the transcription of polyadenylated mRNAs, polyadenylated RNA fractions extracted from the viruliferous mite samples and OFV-infected plant leaves were used for RNA-seq analysis. In the mite and plant datasets, a large number of sequence reads were aligned to genomic regions of OFV RNA1 and RNA2 corresponding to transcribed viral gene mRNAs. This includes the short polyadenylated transcripts originating from the leader and trailer regions at the ends of the viral genome, which are believed to play a crucial role in viral transcription/replication. In contrast, a low number of reads were mapped to the non-transcribed regions (gene junctions). These results strongly suggested that OFV gene expression occurs both in mites and plants. Additionally, deep sequencing revealed the accumulation of OFV-derived small RNAs in mites, although their size profiles differ from those found in plants. Taken together, our results indicated that OFV replicates within a mite vector and is targeted by the RNA-silencing mechanism.

DOI： 10.1016/j.virusres.2024.199522

SHORT AND CROOKED AWN, Encoding an Epigenetic Regulator EMF1, Promotes Barley Awn Development.

Koki Nakamura, Yuichi Kikuchi, Mizuho Shiraga, Toshihisa Kotake, Kiwamu Hyodo, Shin Taketa, Yoko Ikeda

Plant & cell physiology 2024年12月

記述言語：英語掲載種別：研究論文（学術雑誌）

The awn is a bristle-like extension from the lemma of grass spikelets. In barley, the predominant cultivars possess long awns that contribute to grain yield and quality through photosynthesis. Barley is a useful cereal crop to investigate the mechanism of awn development as various awn morphological mutants are available. Here, we identified the gene causative of the short and crooked awn (sca) mutant, which exhibits a short and curved awn phenotype. Intercrossing experiments revealed that the sca mutant induced in the Japanese cultivar (cv.) 'Akashinriki' is allelic to independently isolated moderately short-awn mutant breviaristatum-a (ari-a). Map-based cloning and sequencing revealed that SCA encodes the Polycomb group-associated protein EMBRYONIC FLOWER 1 (EMF1). We found that SCA affects awn development through the promotion of cell proliferation, elongation, and cell wall synthesis. RNA sequencing of cv. Bowman (BW) backcross-derived near-isogenic lines of sca and ari-a6 alleles showed that SCA is directly or indirectly involved in promoting the expression of genes related to awn development. Additionally, SCA represses various transcription factors essential for floral organ development and plant architecture, such as MADS-box and KNOX1 genes. Notably, the repression of the C-class MADS-box gene HvMADS58 by SCA in awns is associated with the accumulation of the repressive histone modification H3K27me3. These findings highlight the potential role of SCA-mediated gene regulation, including histone modification, as a novel pathway in barley awn development.

DOI： 10.1093/pcp/pcae150

Replication of single viruses across the kingdoms, Fungi, Plantae, and Animalia. 国際誌

Paul Telengech, Kiwamu Hyodo, Hiroaki Ichikawa, Ryusei Kuwata, Hideki Kondo, Nobuhiro Suzuki

Proceedings of the National Academy of Sciences of the United States of America 121 ( 25 ) e2318150121 2024年6月

記述言語：英語掲載種別：研究論文（学術雑誌）

It is extremely rare that a single virus crosses host barriers across multiple kingdoms. Based on phylogenetic and paleovirological analyses, it has previously been hypothesized that single members of the family Partitiviridae could cross multiple kingdoms. Partitiviridae accommodates members characterized by their simple bisegmented double-stranded RNA genome; asymptomatic infections of host organisms; the absence of an extracellular route for entry in nature; and collectively broad host range. Herein, we show the replicability of single fungal partitiviruses in three kingdoms of host organisms: Fungi, Plantae, and Animalia. Betapartitiviruses of the phytopathogenic fungusRosellinia necatrix could replicate in protoplasts of the carrot (Daucus carota), Nicotiana benthamiana and Nicotiana tabacum, in some cases reaching a level detectable by agarose gel electrophoresis. Moreover, betapartitiviruses showed more robust replication than the tested alphapartitiviruses. One of the fungal betapartitiviruses, RnPV18, could persistently and stably infect carrot plants regenerated from virion-transfected protoplasts. Both alpha- and betapartitiviruses, although with different host preference, could replicate in two insect cell lines derived from the fall armyworm Spodoptera frugiperda and the fruit fly Drosophila melanogaster. Our results indicate the replicability of single partitiviruses in members of three kingdoms and provide insights into virus adaptation, host jumping, and evolution.

DOI： 10.1073/pnas.2318150121

Discovery and Genome Characterization of a Closterovirus from Wheat Plants with Yellowing Leaf Symptoms in Japan

Hideki Kondo, Hitomi Sugahara, Miki Fujita, Kiwamu Hyodo, Ida Bagus Andika, Hiroshi Hisano, Nobuhiro Suzuki

Pathogens 12 ( 3 ) 358 - 358 2023年2月

掲載種別：研究論文（学術雑誌）出版者・発行元：MDPI AG

Many aphid-borne viruses are important pathogens that affect wheat crops worldwide. An aphid-transmitted closterovirus named wheat yellow leaf virus (WYLV) was found to have infected wheat plants in Japan in the 1970s; however, since then, its viral genome sequence and occurrence in the field have not been investigated. We observed yellowing leaves in the 2018/2019 winter wheat-growing season in an experimental field in Japan where WYLV was detected five decades ago. A virome analysis of those yellow leaf samples lead to the discovery of a closterovirus together with a luteovirus (barley yellow dwarf virus PAV variant IIIa). The complete genomic sequence of this closterovirus, named wheat closterovirus 1 isolate WL19a (WhCV1-WL19a), consisted of 15,452 nucleotides harboring nine open reading frames. Additionally, we identified another WhCV1 isolate, WL20, in a wheat sample from the winter wheat-growing season of 2019/2020. A transmission test indicated that WhCV1-WL20 was able to form typical filamentous particles and transmissible by oat bird-cherry aphid (Rhopalosiphum pad). Sequence and phylogenetic analyses showed that WhCV1 was distantly related to members of the genus Closterovirus (family Closteroviridae), suggesting that the virus represents a novel species in the genus. Furthermore, the characterization of WhCV1-WL19a-derived small RNAs using high-throughput sequencing revealed highly abundant 22-nt-class small RNAs potentially derived from the 3′-terminal end of the WhCV1 negative-strand genomic RNA, indicating that this terminal end of the WhCV1 genome is likely particularly targeted for the synthesis of viral small RNAs in wheat plants. Our results provide further knowledge on closterovirus diversity and pathogenicity and suggest that the impact of WhCV1 on wheat production warrants further investigations.

DOI： 10.3390/pathogens12030358

Identification and characterization of host factors involved in plant RNA virus replication

Kiwamu Hyodo

JOURNAL OF GENERAL PLANT PATHOLOGY 87 ( 6 ) 415 - 417 2021年11月

Identification of an RNA silencing suppressor encoded by a symptomless fungal hypovirus, Cryphonectria hypovirus 4

Annisa Aulia, Kiwamu Hyodo, Sakae Hisano, Hideki Kondo, Bradley I. Hillman, Nobuhiro Suzuki

Biology 10 ( 2 ) 1 - 16 2021年2月

掲載種別：研究論文（学術雑誌）

Previously, we have reported the ability of a symptomless hypovirus Cryphonectria hypovirus 4 (CHV4) of the chestnut blight fungus to facilitate stable infection by a co-infecting mycoreovirus 2 (MyRV2)—likely through the inhibitory effect of CHV4 on RNA silencing (Aulia et al., Virology, 2019). In this study, the N-terminal portion of the CHV4 polyprotein, termed p24, is identified as an autocatalytic protease capable of suppressing host antiviral RNA silencing. Using a bacterial expression system, CHV4 p24 is shown to cleave autocatalytically at the di-glycine peptide (Gly214-Gly215) of the polyprotein through its protease activity. Transgenic expression of CHV4 p24 in Cryphonectria parasitica suppresses the induction of one of the key genes of the antiviral RNA silencing, dicer-like 2, and stabilizes the infection of RNA silencing-susceptible virus MyRV2. This study shows functional similarity between CHV4 p24 and its homolog p29, encoded by the symptomatic prototype hypovirus CHV1.

DOI： 10.3390/biology10020100

Identification of a Novel Quinvirus in the Family Betaflexiviridae That Infects Winter Wheat. 国際誌

Hideki Kondo, Naoto Yoshida, Miki Fujita, Kazuyuki Maruyama, Kiwamu Hyodo, Hiroshi Hisano, Tetsuo Tamada, Ida Bagus Andika, Nobuhiro Suzuki

Frontiers in microbiology 12 715545 - 715545 2021年

記述言語：英語掲載種別：研究論文（学術雑誌）

Yellow mosaic disease in winter wheat is usually attributed to the infection by bymoviruses or furoviruses; however, there is still limited information on whether other viral agents are also associated with this disease. To investigate the wheat viromes associated with yellow mosaic disease, we carried out de novo RNA sequencing (RNA-seq) analyses of symptomatic and asymptomatic wheat-leaf samples obtained from a field in Hokkaido, Japan, in 2018 and 2019. The analyses revealed the infection by a novel betaflexivirus, which tentatively named wheat virus Q (WVQ), together with wheat yellow mosaic virus (WYMV, a bymovirus) and northern cereal mosaic virus (a cytorhabdovirus). Basic local alignment search tool (BLAST) analyses showed that the WVQ strains (of which there are at least three) were related to the members of the genus Foveavirus in the subfamily Quinvirinae (family Betaflexiviridae). In the phylogenetic tree, they form a clade distant from that of the foveaviruses, suggesting that WVQ is a member of a novel genus in the Quinvirinae. Laboratory tests confirmed that WVQ, like WYMV, is potentially transmitted through the soil to wheat plants. WVQ was also found to infect rye plants grown in the same field. Moreover, WVQ-derived small interfering RNAs accumulated in the infected wheat plants, indicating that WVQ infection induces antiviral RNA silencing responses. Given its common coexistence with WYMV, the impact of WVQ infection on yellow mosaic disease in the field warrants detailed investigation.

DOI： 10.3389/fmicb.2021.715545

Virome Analysis of Aphid Populations That Infest the Barley Field: The Discovery of Two Novel Groups of Nege/Kita-Like Viruses and Other Novel RNA Viruses. 査読国際誌

Hideki Kondo, Miki Fujita, Hiroshi Hisano, Kiwamu Hyodo, Ida Bagus Andika, Nobuhiro Suzuki

Frontiers in microbiology 11 509 - 509 2020年

記述言語：英語掲載種別：研究論文（学術雑誌）

Aphids (order Hemiptera) are important insect pests of crops and are also vectors of many plant viruses. However, little is known about aphid-infecting viruses, particularly their diversity and relationship to plant viruses. To investigate the aphid viromes, we performed deep sequencing analyses of the aphid transcriptomes from infested barley plants in a field in Japan. We discovered virus-like sequences related to nege/kita-, flavi-, tombus-, phenui-, mononega-, narna-, chryso-, partiti-, and luteoviruses. Using RT-PCR and sequence analyses, we determined almost complete sequences of seven nege/kitavirus-like virus genomes; one of which was a variant of the Wuhan house centipede virus (WHCV-1). The other six seem to belong to four novel viruses distantly related to Wuhan insect virus 9 (WhIV-9) or Hubei nege-like virus 4 (HVLV-4). We designated the four viruses as barley aphid RNA virus 1 to 4 (BARV-1 to -4). Moreover, some nege/kitavirus-like sequences were found by searches on the transcriptome shotgun assembly (TSA) libraries of arthropods and plants. Phylogenetic analyses showed that BARV-1 forms a clade with WHCV-1 and HVLV-4, whereas BARV-2 to -4 clustered with WhIV-9 and an aphid virus, Aphis glycines virus 3. Both virus groups (tentatively designated as Centivirus and Aphiglyvirus, respectively), together with arthropod virus-like TSAs, fill the phylogenetic gaps between the negeviruses and kitaviruses lineages. We also characterized the flavi/jingmen-like and tombus-like virus sequences as well as other RNA viruses, including six putative novel viruses, designated as barley aphid RNA viruses 5 to 10. Interestingly, we also discovered that some aphid-associated viruses, including nege/kita-like viruses, were present in different aphid species, raising a speculation that these viruses might be distributed across different aphid species with plants being the reservoirs. This study provides novel information on the diversity and spread of nege/kitavirus-related viruses and other RNA viruses that are associated with aphids.

DOI： 10.3389/fmicb.2020.00509

Hijacking of host cellular components as proviral factors by plant-infecting viruses

Kiwamu Hyodo, Tetsuro Okuno

Advances in Virus Research 37 - 86 2020年

Diverse Partitiviruses From the Phytopathogenic Fungus, Rosellinia necatrix. 国際誌

Paul Telengech, Sakae Hisano, Cyrus Mugambi, Kiwamu Hyodo, Juan Manuel Arjona-López, Carlos José López-Herrera, Satoko Kanematsu, Hideki Kondo, Nobuhiro Suzuki

Frontiers in microbiology 11 1064 - 1064 2020年

記述言語：英語掲載種別：研究論文（学術雑誌）

Partitiviruses (dsRNA viruses, family Partitiviridae) are ubiquitously detected in plants and fungi. Although previous surveys suggested their omnipresence in the white root rot fungus, Rosellinia necatrix, only a few of them have been molecularly and biologically characterized thus far. We report the characterization of a total of 20 partitiviruses from 16 R. necatrix strains belonging to 15 new species, for which "Rosellinia necatrix partitivirus 11-Rosellinia necatrix partitivirus 25" were proposed, and 5 previously reported species. The newly identified partitiviruses have been taxonomically placed in two genera, Alphapartitivirus, and Betapartitivirus. Some partitiviruses were transfected into reference strains of the natural host, R. necatrix, and an experimental host, Cryphonectria parasitica, using purified virions. A comparative analysis of resultant transfectants revealed interesting differences and similarities between the RNA accumulation and symptom induction patterns of R. necatrix and C. parasitica. Other interesting findings include the identification of a probable reassortment event and a quintuple partitivirus infection of a single fungal strain. These combined results provide a foundation for further studies aimed at elucidating mechanisms that underly the differences observed.

DOI： 10.3389/fmicb.2020.01064

Two novel fungal negative-strand RNA viruses related to mymonaviruses and phenuiviruses in the shiitake mushroom (Lentinula edodes). 査読

Lin YH, Fujita M, Chiba S, Hyodo K, Andika IB, Suzuki N, Kondo H

Virology 533 125 - 136 2019年7月

Hijacking a host scaffold protein, RACK1, for replication of a plant RNA virus. 査読

Hyodo K, Suzuki N, Okuno T

The New phytologist 221 ( 2 ) 935 - 945 2019年1月

The photosystem II repair cycle requires FtsH turnover through the EngA GTPase. 査読

Kato Y, Hyodo K, Sakamoto W

Plant physiology 178 ( 2 ) 596 - 611 2018年8月

Integration of danger peptide signals with herbivore-associated molecular pattern signaling amplifies anti-herbivore defense responses in rice. 査読国際誌

Tomonori Shinya, Shigetaka Yasuda, Kiwamu Hyodo, Rena Tani, Yuko Hojo, Yuka Fujiwara, Kei Hiruma, Takuma Ishizaki, Yasunari Fujita, Yusuke Saijo, Ivan Galis

The Plant journal : for cell and molecular biology 94 ( 4 ) 626 - 637 2018年5月

記述言語：英語掲載種別：研究論文（学術雑誌）

Plant defense against herbivores is modulated by herbivore-associated molecular patterns (HAMPs) from oral secretions (OS) and/or saliva of insects. Furthermore, feeding wounds initiate plant self-damage responses modulated by danger-associated molecular patterns (DAMPs) such as immune defense-promoting plant elicitor peptides (Peps). While temporal and spatial co-existence of both patterns during herbivory implies a possibility of their close interaction, the molecular mechanisms remain undetermined. Here we report that exogenous application of rice (Oryza sativa) peptides (OsPeps) can elicit multiple defense responses in rice cell cultures. Specific activation of OsPROPEP3 gene transcripts in rice leaves by wounding and OS treatments further suggests a possible involvement of the OsPep3 peptide in rice-herbivore interactions. Correspondingly, we found that simultaneous application of OsPep3 and Mythimna loreyi OS significantly amplifies an array of defense responses in rice cells, including mitogen-activated protein kinase activation, and generation of defense-related hormones and metabolites. The induction of OsPROPEP3/4 by OsPep3 points to a positive auto-feedback loop in OsPep signaling which may contribute to additional enhancement of defense signal(s). Finally, the overexpression of the OsPep receptor OsPEPR1 increases the sensitivity of rice plants not only to the cognate OsPeps but also to OS signals. Our findings collectively suggest that HAMP-DAMP signal integration provides a critical step in the amplification of defense signaling in plants.

DOI： 10.1111/tpj.13883

Dual function of a cis-acting RNA element that acts as a replication enhancer and a translation repressor in a plant positive-stranded RNA virus 査読

Kiwamu Hyodo, Hikari Nagai, Tetsuro Okuno

VIROLOGY 512 74 - 82 2017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：ACADEMIC PRESS INC ELSEVIER SCIENCE

The genome of red clover necrotic mosaic virus is divided into two positive-stranded RNA molecules of RNA1 and RNA2, which have no 5' cap structure and no 3' poly(A) tail. Previously, we showed that any mutations in the cis-acting RNA replication elements of RNA2 abolished its cap-independent translational activity, suggesting a strong link between RNA replication and translation. Here, we investigated the functions of the 5' untranslated region (UTR) of RNA2 and revealed that the basal stem-structure (5'BS) predicted in the 5' UTR is essential for robust RNA replication. Interestingly, RNA2 mutants with substitution or deletion in the right side of the 5'BS showed strong translational activity, despite their impaired replication competency. Furthermore, nucleotide sequences other than the 5'BS of the 5' UTR were essential to facilitate the replication-associated translation. Overall, these cis-acting RNA elements seem to coordinately regulate the balance between RNA replication and replication-associated translation.

DOI： 10.1016/j.virol.2017.09.008

Requirement for eukaryotic translation initiation factors in cap-independent translation differs between bipartite genomic RNAs of red clover necrotic mosaic virus 査読

Yuri Tajima, Hiro-oki Iwakawa, Kiwamu Hyodo, Masanori Kaido, Kazuyuki Mise, Tetsuro Okuno

VIROLOGY 509 152 - 158 2017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：ACADEMIC PRESS INC ELSEVIER SCIENCE

The bipartite genomic RNAs of red clover necrotic mosaic virus (RCNMV) lack a 5' cap and a 3' poly(A) tail. RNA1 encodes viral replication proteins, and RNA2 encodes a movement protein (MP). These proteins are translated in a cap-independent manner. We previously identified two cis-acting RNA elements that cooperatively recruit eukaryotic translation initiation factor (eIF) complex eIF4F or eIFiso4F to RNA1. Such cis-acting RNA elements and host factors have not been identified in RNA2. Here we found that translation of RNA1 was significantly compromised in Arabidopsis thaliana carrying eif4f mutation. RNA1 replicated efficiently in eifiso4fl mutants, suggesting vigorous translation of the replication proteins from RNA1 in the plants. In contrast, MP accumulation was decreased in eifiso4f1 mutants but not in eif4f mutants. Collectively, these results suggest that RCNMV uses different eIF complexes for translation of its bipartite genomic RNAs, which may contribute to fine-tuning viral gene expression during infection.

DOI： 10.1016/j.virol.2017.06.015

Roles of superoxide anion and hydrogen peroxide during replication of two unrelated plant RNA viruses in Nicotiana benthamiana 査読

Kiwamu Hyodo, Nobuhiro Suzuki, Kazuyuki Mise, Tetsuro Okuno

Plant Signaling and Behavior 12 ( 6 ) e1338223 2017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：Taylor and Francis Inc.

Reactive oxygen species (ROS), including superoxide anion (O2 −), hydrogen peroxide (H2O2), and hydroxyl radical, act as signaling molecules to transduce biotic and abiotic stimuli into stress adaptations in plants. A respiratory burst oxidase homolog B of Nicotiana benthamiana (NbRBOHB) is responsible for O2 − production to inhibit pathogen infection during plant innate immunity. RBOH-derived O2 − can be immediately converted into H2O2 by the action of superoxide dismutase. Interestingly, we recently showed that red clover necrotic mosaic virus (RCNMV), a plant positive-strand RNA [(+)RNA] virus, hijacks the host's ROS-generating machinery during infection. An RCNMV replication protein associates with NbRBOHB and triggers intracellular ROS bursts. These bursts are required for robust viral RNA replication. However, what types of ROS are required for viral replication is currently unknown. Here, we found that RCNMV replication was sensitive to an O2 − scavenger but insensitive to an H2O2 scavenger. Interestingly, replication of another plant (+)RNA virus, brome mosaic virus, was sensitive to both types of scavengers. These results indicate a virus-specific pattern requirement of O2 − and H2O2 for (+)RNA virus replication and suggest a conserved nature of the roles of ROS in (+)RNA virus replication.

DOI： 10.1080/15592324.2017.1338223

Harnessing host ROS-generating machinery for the robust genome replication of a plant RNA virus 査読

Kiwamu Hyodo, Kenji Hashimoto, Kazuyuki Kuchitsu, Nobuhiro Suzuki, Tetsuro Okuno

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 114 ( 7 ) E1282 - E1290 2017年2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：NATL ACAD SCIENCES

As sessile organisms, plants have to accommodate to rapid changes in their surrounding environment. Reactive oxygen species (ROS) act as signaling molecules to transduce biotic and abiotic stimuli into plant stress adaptations. It is established that a respiratory burst oxidase homolog B of Nicotiana benthamiana (NbRBOHB) produces ROS in response to microbe-associated molecular patterns to inhibit pathogen infection. Plant viruses are also known as causative agents of ROS induction in infected plants; however, the function of ROS in plant-virus interactions remains obscure. Here, we show that the replication of red clover necrotic mosaic virus (RCNMV), a plant positive-strand RNA [(+) RNA] virus, requires NbRBOHB-mediated ROS production. The RCNMV replication protein p27 plays a pivotal role in this process, redirecting the subcellular localization of NbRBOHB and a subgroup II calcium-dependent protein kinase of N. benthamiana (NbCDPKiso2) from the plasma membrane to the p27-containing intracellular aggregate structures. p27 also induces an intracellular ROS burst in an RBOH-dependent manner. NbCDPKiso2 was shown to be an activator of the p27-triggered ROS accumulations and to be required for RCNMV replication. Importantly, this RBOH-derived ROS is essential for robust viral RNA replication. The need for RBOHderived ROS was demonstrated for the replication of another (+) RNA virus, brome mosaic virus, suggesting that this characteristic is true for plant (+) RNA viruses. Collectively, our findings revealed a hitherto unknown viral strategy whereby the host ROS-generating machinery is diverted for robust viral RNA replication.

DOI： 10.1073/pnas.1610212114

Chloroplast genome sequences of seven strains of the bloom-forming raphidophyte Heterosigma akashiwo 査読

Sergio Seoane, Kiwamu Hyodo, Shoko Ueki

Genome Announcements 5 ( 41 ) 2017年

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

We report here the complete chloroplast genome sequences of seven strains of the bloom-forming raphidophyte Heterosigma akashiwo. These ~160-kb sequences contain 124 protein-, 6 rRNA-, and 34 tRNA-coding sequences. Notable sequence variations were observed among these seven sequenced and two previously characterized strains.

DOI： 10.1128/genomeA.01030-17

Structural Analysis of Long Single-Stranded RNA Molecules with Atomic Force Microscopy Imaging

Jamie L. Gilmore, Aiko Yoshida, Katashi Deguchi, Suguru Asai, Hideki Aizaki, Masahiro Kumeta, Kiwamu Hyodo, Tetsuro Okuno, Takaji Wakita, Kunio Takeyasu

3RD INTERNATIONAL MULTIDISCIPLINARY MICROSCOPY AND MICROANALYSIS CONGRESS (INTERM) 186 3 - 9 2017年

記述言語：英語掲載種別：研究論文（国際会議プロシーディングス）出版者・発行元：SPRINGER INTERNATIONAL PUBLISHING AG

Characterization of the structure of long RNA molecules (>1 kb) is usually a time-consuming and tedious process. In this study, we have developed an imaging procedure for obtaining images of the extended secondary structures of long RNA molecules combined with automated MATLAB-based data processing algorithms for identification of the domain architecture of the molecules in these images. These algorithms include a molecule autoselection procedure based on height and area thresholding, a morphological thinning procedure to generate skeletons of the molecule in order to analyze the branched structure of the molecules, and a procedure to generate local volume profiles along the main chain of the molecule for identification of domains and prediction of the number of nucleotides comprising each domain. The single-molecule nature of this technique also allows for the identification of varying conformations of the molecule and assessment of the conformational flexibility of the identified domain organization.

DOI： 10.1007/978-3-319-46601-9_1

Pathogenesis mediated by proviral host factors involved in translation and replication of plant positive-strand RNA viruses 査読

Kiwamu Hyodo, Tetsuro Okuno

CURRENT OPINION IN VIROLOGY 17 11 - 18 2016年4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：ELSEVIER SCI LTD

Viral pathogenesis comes from complex interactions between viruses and hosts. All the processes of viral infection, including translation of viral factors and replication of viral genomes, define viral pathogenesis; therefore, molecular insights into the mechanisms underlying viral replication strategies unambiguously pave the way for our comprehensive understanding of viral pathogenesis and disease outcome, as well as for developing new antiviral strategies against plant virus disease. Recent studies of plant positive-strand RNA [(+)RNA] viruses have advanced our understanding of co-opted host factors and their roles in viral translation and replication. It is becoming clear that plant (+)RNA viruses harness host factors involved in membrane trafficking and lipid metabolism to establish the viral replication complex (VRC). In this review, we aim to discuss the contribution of co-opted host factors in translation and genome replication of plant (+)RNA viruses mainly focusing on those involved in the biogenesis of the VRC, which may act as a central hub in almost all the processes of viral infection as well as viral pathogenesis.

DOI： 10.1016/j.coviro.2015.11.004

Phosphatidic Acid Produced by Phospholipase D Promotes RNA Replication of a Plant RNA Virus 査読

Kiwamu Hyodo, Takako Taniguchi, Yuki Manabe, Masanori Kaido, Kazuyuki Mise, Tatsuya Sugawara, Hisaaki Taniguchi, Tetsuro Okuno

PLOS PATHOGENS 11 ( 5 ) e1004909 2015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：PUBLIC LIBRARY SCIENCE

Eukaryotic positive-strand RNA [(+) RNA] viruses are intracellular obligate parasites replicate using the membrane-bound replicase complexes that contain multiple viral and host components. To replicate, (+) RNA viruses exploit host resources and modify host metabolism and membrane organization. Phospholipase D (PLD) is a phosphatidylcholine-and phosphatidylethanolamine-hydrolyzing enzyme that catalyzes the production of phosphatidic acid (PA), a lipid second messenger that modulates diverse intracellular signaling in various organisms. PA is normally present in small amounts (less than 1% of total phospholipids), but rapidly and transiently accumulates in lipid bilayers in response to different environmental cues such as biotic and abiotic stresses in plants. However, the precise functions of PLD and PA remain unknown. Here, we report the roles of PLD and PA in genomic RNA replication of a plant (+) RNA virus, Red clover necrotic mosaic virus (RCNMV). We found that RCNMV RNA replication complexes formed in Nicotiana benthamiana contained PLD alpha and PLD beta. Gene-silencing and pharmacological inhibition approaches showed that PLDs and PLDs-derived PA are required for viral RNA replication. Consistent with this, exogenous application of PA enhanced viral RNA replication in plant cells and plant-derived cell-free extracts. We also found that a viral auxiliary replication protein bound to PA in vitro, and that the amount of PA increased in RCNMV-infected plant leaves. Together, our findings suggest that RCNMV hijacks host PA-producing enzymes to replicate.

DOI： 10.1371/journal.ppat.1004909

GAPDH-A Recruits a Plant Virus Movement Protein to Cortical Virus Replication Complexes to Facilitate Viral Cell-to-Cell Movement 査読

Masanori Kaido, Kazutomo Abe, Akira Mine, Kiwamu Hyodo, Takako Taniguchi, Hisaaki Taniguchi, Kazuyuki Mise, Tetsuro Okuno

PLOS PATHOGENS 10 ( 11 ) e1004505 2014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：PUBLIC LIBRARY SCIENCE

The formation of virus movement protein (MP)-containing punctate structures on the cortical endoplasmic reticulum is required for efficient intercellular movement of Red clover necrotic mosaic virus (RCNMV), a bipartite positive-strand RNA plant virus. We found that these cortical punctate structures constitute a viral replication complex (VRC) in addition to the previously reported aggregate structures that formed adjacent to the nucleus. We identified host proteins that interacted with RCNMV MP in virus-infected Nicotiana benthamiana leaves using a tandem affinity purification method followed by mass spectrometry. One of these host proteins was glyceraldehyde 3-phosphate dehydrogenase-A (NbGAPDH-A), which is a component of the Calvin-Benson cycle in chloroplasts. Virus-induced gene silencing of NbGAPDH-A reduced RCNMV multiplication in the inoculated leaves, but not in the single cells, thereby suggesting that GAPDH-A plays a positive role in cell-to-cell movement of RCNMV. The fusion protein of NbGAPDH-A and green fluorescent protein localized exclusively to the chloroplasts. In the presence of RCNMV RNA1, however, the protein localized to the cortical VRC as well as the chloroplasts. Bimolecular fluorescence complementation assay and GST pulldown assay confirmed in vivo and in vitro interactions, respectively, between the MP and NbGAPDH-A. Furthermore, gene silencing of NbGAPDH-A inhibited MP localization to the cortical VRC. We discuss the possible roles of NbGAPDH-A in the RCNMV movement process.

DOI： 10.1371/journal.ppat.1004505

Host and viral RNA-binding proteins involved in membrane targeting, replication and intercellular movement of plant RNA virus genomes 査読

K. Hyodo, M. Kaido, T. Okuno

Frontiers in Plant Science 5 321 2014年7月

Traffic jam on the cellular secretory pathway generated by a replication protein from a plant RNA virus 査読

Kiwamu Hyodo, Masanori Kaido, Tetsuro Okuno

Plant Signaling and Behavior 9 2014年

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

Although positive-strand RNA [(+)RNA] viruses have a limited coding capacity, they can replicate efficiently in host cells because of their ability to use host-derived proteins, membranes, lipids, and metabolites, and to rewire cellular trafficking pathways. Previously, we showed that a plant RNA virus, the Red clover necrotic mosaic virus (RCNM V), hijacked Arf1 and Sar1, which are small GTPases that regulate the biogenesis of COPI and COPII vesicles, respectively, for viral RNA replication. These small GTPases are relocated from appropriate subcellular compartments to the viral RNA replication sites by p27 replication protein, which raises the possibility that RCNM V interferes with the cellular secretory pathway. Here, we examined this possibility by using green fluorescent protein-fused rice SCAM P1 and Arabidopsis LRR 84A as secretory pathway marker proteins and showed that p27 inhibited the trafficking of these proteins. RCNM Vmediated inhibition of the host secretion pathway and its possible impact on plant-virus interaction are discussed. © 2014 Landes Bioscience.

DOI： 10.4161/psb.28644

ADPribosylation factor 1 plays an essential role in the replication of a plant RNA virus 査読

Hyodo K, Mine A, Taniguchi T, Kaido M, Mise K, Taniguchi H, Okuno T

J Virol. 87 ( 1 ) 163 - 176 2013年1月

Differential roles of Hsp70 and Hsp90 in the assembly of the replicase complex of a positive-strand RNA plant virus 査読

Mine A, Hyodo K, Tajima Y, Kusumanegara K, Taniguchi T, Kaido M, Mise K, Taniguchi H, Okuno T

J Virol. 86 ( 22 ) 12091 - 12104 2012年11月

記述言語：英語出版者・発行元：American Society for Microbiology

Assembly of viral replicase complexes of eukaryotic positive-strand RNA viruses is a regulated process: multiple viral and host components must be assembled on intracellular membranes and ordered into quaternary complexes capable of synthesizing viral RNAs. However, the molecular mechanisms underlying this process are poorly understood. In this study, we used a model virus, Red clover necrotic mosaic virus (RCNMV), whose replicase complex can be detected readily as the 480-kDa functional protein complex. We found that host heat shock proteins Hsp70 and Hsp90 are required for RCNMV RNA replication and that they interact with p27, a virus-encoded component of the 480-kDa replicase complex, on the endoplasmic reticulum membrane. Using a cell-free viral translation/replication system in combination with specific inhibitors of Hsp70 and Hsp90, we found that inhibition of p27-Hsp70 interaction inhibits the formation of the 480-kDa complex but instead induces the accumulation of large complexes that are nonfunctional in viral RNA synthesis. In contrast, inhibition of p27-Hsp90 interaction did not induce such large complexes but rendered p27 incapable of binding to a specific viral RNA element, which is a critical step for the assembly of the 480-kDa replicase complex and viral RNA replication. Together, our results suggest that Hsp70 and Hsp90 regulate different steps in the assembly of the RCNMV replicase complex.

DOI： 10.1128/JVI.01659-12

Identification of domains in p27 auxiliary replicase protein essential for its association with the endoplasmic reticulum membranes in Red clover necrotic mosaic virus. 査読

Kusumanegara K, Mine A, Hyodo K, Kaido M, Mise K, Okuno T

Virology 433 ( 1 ) 131 - 141 2012年11月

Identification ofamino acids in auxiliary replicase protein p27 critical for its RNA-bindingactivity and the assembly of the replicase complex in Red clover necrotic mosaic virus 査読

Hyodo K, Mine A, Iwakawa HO, Kaido M, Mise K, Okuno T

Virology 413 ( 2 ) 300 - 309 2011年5月

Template recognition mechanisms by replicase proteins differ between bipartitepositive-strand genomic RNAs of a plant virus 査読

Iwakawa HO, Mine A, Hyodo K, An M, Kaido M, Mise K, Okuno T

J Virol. 85 ( 1 ) 497 - 509 2011年1月

記述言語：英語出版者・発行元：American Society for Microbiology

Recognition of RNA templates by viral replicase proteins is one of the key steps in the replication process of all RNA viruses. However, the mechanisms underlying this phenomenon, including primary RNA elements that are recognized by the viral replicase proteins, are not well understood. Here, we used aptamer pulldown assays with membrane fractionation and protein-RNA coimmunoprecipitation in a cell-free viral translation/replication system to investigate how viral replicase proteins recognize the bipartite genomic RNAs of the Red clover necrotic mosaic virus (RCNMV). RCNMV replicase proteins bound specifically to a Y-shaped RNA element (YRE) located in the 3' untranslated region (UTR) of RNA2, which also interacted with the 480-kDa replicase complexes that contain viral and host proteins. The replicase-YRE interaction recruited RNA2 to the membrane fraction. Conversely, RNA1 fragments failed to interact with the replicase proteins supplied in trans. The results of protein-RNA coimmunoprecipitation assays suggest that RNA1 interacts with the replicase proteins coupled with their translation. Thus, the initial template recognition mechanisms employed by the replicase differ between RCNMV bipartite genomic RNAs and RNA elements are primary determinants of the differential replication mechanism.

DOI： 10.1128/JVI.01754-10

Interactions between p27 and p88 replicase proteins of Red clover necrotic mosaic virus play an essential role in viral RNA replication and suppression of RNA silencing via the 480-kDaviral replicase complex assembly 査読

Mine A, Hyodo K, Takeda A, Kaido M, Mise K, Okuno T

Virology 407 ( 2 ) 213 - 224 2010年11月

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