SEARCH

Search Details

Nakahara Kenji

Research Faculty of Agriculture　Fundamental AgriScience Research　Agrobiology and BioresourcesLecturer
Institute for Academic InnovationLecturer

Researcher basic information

■ Degree
  • 博士（農学）
■ URL
researchmap URLホームページURL■ Various IDs
J-Global ID■ Research Keywords and Fields
Research Keyword
  • 応用分子細胞生物学
  • 植物病理学
Research Field
  • Environmental Science/Agriculture Science, Plant protection science
■ Educational Organization

Research activity information

■ Papers
  • Characterization of wheat virus Q from wheat in Hokkaido, Japan
    Satomi Adegawa; Asuka Seino; Kazuhiro Yaguchi; Kaito Toyoshima; Kento Mori; Kai Suto; Naoya Yamaguchi; Chihiro Souma; Takako Suzuki; Kenji S. Nakahara
    Journal of General Plant Pathology, Springer Science and Business Media LLC, 10 Oct. 2025, [Peer-reviewed], [Last author, Corresponding author], [International Magazine]
    English, Scientific journal
  • Two Stabiliser loci suppress Tam3 transposition without compromising transposase production in Antirrhinum
    Shasha Wang; Takako Uchiyama; Hiroyuki Kuwabara; Megumi Hirata; Ikumi Yuasa; Kenji Nakahara; Cathie Martin; Yuji Kishima
    Plant Physiology, Oxford University Press (OUP), 02 Sep. 2025, [Peer-reviewed]
    Scientific journal, Abstract

    Snapdragon (Antirrhinum majus) exhibits occasional genetic instabilities that manifest as variegations and morphological chimeras. Stabiliser (St) is a historical locus that stabilizes phenotypically unstable or mutable traits in Antirrhinum. Here, we characterized two St loci, the previously described Old Stabiliser (OSt) and New Stabiliser (NSt), that specifically suppress the transposition of the Class II DNA transposable element Tam3 in Antirrhinum. Both St loci harbor Tam3 derivatives with unique structures: OSt contains a pseudo-Tam3 copy whose 5'-terminal region has been rearranged compared to the cognate Tam3 element, and NSt consists of two intact Tam3 copies in a head-to-head orientation. Neither locus interferes with the production of the intact Tam3 transposase (TPase) or the nuclear import of TPase. Both OSt and NSt produce specific sRNAs from their 5'-terminal regions containing multiple TPase-binding motifs. We performed sRNA induction experiments by TRV-mediated virus-induced gene silencing targeting the 5’-terminal region of the Tam3 sequence, which suppressed Tam3 transposition in the ost/nst plants. These results suggest that specific sRNAs can repress Tam3 transposition by interacting with the TPase-binding motifs within the Tam3 element or with the TPase itself.
  • In-frame deletion mutant of eIF4E1 attenuates cucumber mosaic virus virulence by interfering with 2b function in tomato
    Sozib Ghos; Ayaka Kawakubo; Md; Shamim Akhter; Akinori Yoshimura; Miyuki Suto; Kami Murakami; Chiaki Konishi; Hiroki Atarashi; Kenji S. Nakahara
    Physiologia Plantarum, 177, e70369, 03 Jul. 2025, [Peer-reviewed], [Last author, Corresponding author]
    English, Scientific journal, 45296035
  • Induction of necrosis symptoms by potato virus X in AGO2-silenced tomato plants associates with reduced transcript accumulation of copper chaperon for superoxide dismutase gene
    Joon Kwon; Kento Mori; Tetsuo Maoka; Teruo Sano; Kenji S. Nakahara
    Virus Research, 348, 199436, Elsevier BV, Oct. 2024, [Peer-reviewed], [Last author, Corresponding author], [International Magazine]
    English, Scientific journal
  • Monitoring systemic infection by cucumber mosaic virus using a small fluorescent protein iLOV in plants
    Ayaka Kawakubo; Jean-Luc Gallois; Kenji S. Nakahara
    Journal of General Plant Pathology, 89, 47, 52, Springer Science and Business Media LLC, 12 Oct. 2022, [Peer-reviewed], [Last author, Corresponding author]
    Scientific journal
  • Aspartic protease inhibitor enhances resistance to potato virus Y and A in transgenic potato plants
    Zhila Osmani; Mohammad Sadegh Sabet; Kenji S. Nakahara
    BMC Plant Biology, 22, 241, Springer Science and Business Media LLC, 12 May 2022, [Peer-reviewed], [Last author]
    English, Scientific journal, Abstract

    Background

    Viruses are the major threat to commercial potato (Solanum tuberosum) production worldwide. Because viral genomes only encode a small number of proteins, all stages of viral infection rely on interactions between viral proteins and host factors. Previously, we presented a list of the most important candidate genes involved in potato plants’ defense response to viruses that are significantly activated in resistant cultivars. Isolated from this list, Aspartic Protease Inhibitor 5 (API5) is a critical host regulatory component of plant defense responses against pathogens. The purpose of this study is to determine the role of StAPI5 in defense of potato against potato virus Y and potato virus A, as well as its ability to confer virus resistance in a transgenic susceptible cultivar of potato (Desiree). Potato plants were transformed with Agrobacterium tumefaciens via a construct encoding the potato StAPI5 gene under the control of the Cauliflower mosaic virus (CaMV) 35S promoter.

    Results

    Transgenic plants overexpressing StAPI5 exhibited comparable virus resistance to non-transgenic control plants, indicating that StAPI5 functions in gene regulation during virus resistance. The endogenous StAPI5 and CaMV 35S promoter regions shared nine transcription factor binding sites. Additionally, the net photosynthetic rate, stomatal conductivity, and maximum photochemical efficiency of photosystem II were significantly higher in virus-infected transgenic plants than in wild-type plants.

    Conclusion

    Overall, these findings indicate that StAPI5 may be a viable candidate gene for engineering plant disease resistance to viruses that inhibit disease development.
  • First report of Citrus tristeza virus in Bangladesh
    Md. Shamim Akhter; Mohammad Monirul Hasan Tipu; Md. Siddiqur Rahman; Rummana Islam; Md. Iqbal Faruk; Md. Matiar Rahman; Kenji S. Nakahara
    Australasian Plant Disease Notes, 17, 1, 12, Springer Science and Business Media LLC, May 2022, [Peer-reviewed], [Corresponding author]
    Scientific journal
  • Resistance induction based on the understanding of molecular interactions between plant viruses and host plants
    Md. Shamim Akhter; Kenji S. Nakahara; Chikara Masuta
    Virology Journal, 18, 1, 176, Springer Science and Business Media LLC, Dec. 2021, [Peer-reviewed]
    English, Scientific journal, Abstract
    Background
    Viral diseases cause significant damage to crop yield and quality. While fungi- and bacteria-induced diseases can be controlled by pesticides, no effective approaches are available to control viruses with chemicals as they use the cellular functions of their host for their infection cycle. The conventional method of viral disease control is to use the inherent resistance of plants through breeding. However, the genetic sources of viral resistance are often limited. Recently, genome editing technology enabled the publication of multiple attempts to artificially induce new resistance types by manipulating host factors necessary for viral infection.



    Main body
    In this review, we first outline the two major (R gene-mediated and RNA silencing) viral resistance mechanisms in plants. We also explain the phenomenon of mutations of host factors to function as recessive resistance genes, taking the eIF4E genes as examples. We then focus on a new type of virus resistance that has been repeatedly reported recently due to the widespread use of genome editing technology in plants, facilitating the specific knockdown of host factors. Here, we show that (1) an in-frame mutation of host factors necessary to confer viral resistance, sometimes resulting in resistance to different viruses and that (2) certain host factors exhibit antiviral resistance and viral-supporting (proviral) properties.



    Conclusion
    A detailed understanding of the host factor functions would enable the development of strategies for the induction of a new type of viral resistance, taking into account the provision of a broad resistance spectrum and the suppression of the appearance of resistance-breaking strains.


  • Effect of aphid biology and morphology on plant virus transmission
    Wikum H Jayasinghe; Md Shamim Akhter; Kenji Nakahara; Midatharahally N Maruthi
    Pest Management Science, Wiley, 21 Sep. 2021, [Peer-reviewed]
    Scientific journal
  • Identification of a defense response gene involved in signaling pathways against PVA and PVY in potato
    Zhila Osmani; Mohammad Sadegh Sabet; Kenji S. Nakahara; Ali Mokhtassi-Bidgoli; Khabat Vahabi; Ahmad Moieni; Masoud Shams-Bakhsh
    GM Crops & Food, 12, 1, 86, 105, Informa UK Limited, 02 Jan. 2021, [Peer-reviewed]
    Scientific journal
  • Artificially edited alleles of the eukaryotic translation initiation factor 4E1 gene differentially reduce susceptibility to cucumber mosaic virus and potato virus Y in tomato
    Atarashi H; Jayasinghe W.H; Kwon J; Kim H; Taninaka Y; Igarashi M; Ito K; Yamada T; Masuta C; Nakahara K.S
    Frontiers in Microbiology, 11, 564310, Frontiers Media SA, Dec. 2020, [Peer-reviewed], [Last author, Corresponding author]
    English, Scientific journal, Eukaryotic translation initiation factors, including eIF4E, are susceptibility factors for viral infection in host plants. Mutation and double-stranded RNA-mediated silencing of tomato eIF4E genes can confer resistance to viruses, particularly members of the Potyvirus genus. Here, we artificially mutated the eIF4E1 gene on chromosome 3 of a commercial cultivar of tomato (Solanum lycopersicum L.) by using CRISPR/Cas9. We obtained three alleles, comprising two deletions of three and nine nucleotides (3DEL and 9DEL) and a single nucleotide insertion (1INS), near regions that encode amino acid residues important for binding to the mRNA 5' cap structure and to eIF4G. Plants homozygous for these alleles were termed 3DEL, 9DEL, and 1INS plants, respectively. In accordance with previous studies, inoculation tests with potato virus Y (PVY; type member of the genus Potyvirus) yielded a significant reduction in susceptibility to the N strain (PVYN), but not to the ordinary strain (PVYO), in 1INS plants. 9DEL among three artificial alleles had a deleterious effect on infection by cucumber mosaic virus (CMV, type member of the genus Cucumovirus). When CMV was mechanically inoculated into tomato plants and viral coat accumulation was measured in the non-inoculated upper leaves, the level of viral coat protein was significantly lower in the 9DEL plants than in the parental cultivar. Tissue blotting of microperforated inoculated leaves of the 9DEL plants revealed significantly fewer infection foci compared with those of the parental cultivar, suggesting that 9DEL negatively affects the initial steps of infection with CMV in a mechanically inoculated leaf. In laboratory tests, viral aphid transmission from an infected susceptible plant to 9DEL plants was reduced compared with the parental control. Although many pathogen resistance genes have been discovered in tomato and its wild relatives, no CMV resistance genes have been used in practice. RNA silencing of eIF4E expression has previously been reported to not affect susceptibility to CMV in tomato. Our findings suggest that artificial gene editing can introduce additional resistance to that achieved with mutagenesis breeding, and that edited eIF4E alleles confer an alternative way to manage CMV in tomato fields.
  • RNA silencing-related genes contribute to tolerance of infection with potato virus X and Y in a susceptible tomato plant
    Joon Kwon; Atsushi Kasai; Tetsuo Maoka; Chikara Masuta; Teruo Sano; Kenji S. Nakahara
    Virology Journal, 17, 1, 149, Springer Science and Business Media LLC, Oct. 2020, [Peer-reviewed], [Corresponding author]
    Scientific journal, Abstract

    Background
    In plants, the RNA silencing system functions as an antiviral defense mechanism following its induction with virus-derived double-stranded RNAs. This occurs through the action of RNA silencing components, including Dicer-like (DCL) nucleases, Argonaute (AGO) proteins, and RNA-dependent RNA polymerases (RDR). Plants encode multiple AGOs, DCLs, and RDRs. The functions of these components have been mainly examined in Arabidopsis thaliana and Nicotiana benthamiana. In this study, we investigated the roles of DCL2, DCL4, AGO2, AGO3 and RDR6 in tomato responses to viral infection. For this purpose, we used transgenic tomato plants (Solanum lycopersicum cv. Moneymaker), in which the expression of these genes were suppressed by double-stranded RNA-mediated RNA silencing.




    Methods
    We previously created multiple DCL (i.e., DCL2 and DCL4) (hpDCL2.4) and RDR6 (hpRDR6) knockdown transgenic tomato plants and here additionally did multiple AGO (i.e., AGO2 and AGO3) knockdown plants (hpAGO2.3), in which double-stranded RNAs cognate to these genes were expressed to induce RNA silencing to them. Potato virus X (PVX) and Y (PVY) were inoculated onto these transgenic tomato plants, and the reactions of these plants to the viruses were investigated. In addition to observation of symptoms, viral coat protein and genomic RNA were detected by western and northern blotting and reverse transcription-polymerase chain reaction (RT-PCR). Host mRNA levels were investigated by quantitative RT-PCR.




    Results
    Following inoculation with PVX, hpDCL2.4 plants developed a more severe systemic mosaic with leaf curling compared with the other inoculated plants. Systemic necrosis was also observed in hpAGO2.3 plants. Despite the difference in the severity of symptoms, the accumulation of PVX coat protein (CP) and genomic RNA in the uninoculated upper leaves was not obviously different among hpDCL2.4, hpRDR6, and hpAGO2.3 plants and the empty vector-transformed plants. Moneymaker tomato plants were asymptomatic after infection with PVY. However, hpDCL2.4 plants inoculated with PVY developed symptoms, including leaf curling. Consistently, PVY CP was detected in the uninoculated symptomatic upper leaves of hpDCL2.4 plants through western blotting. Of note, PVY CP was rarely detected in other asymptomatic transgenic or wild-type plants. However, PVY was detected in the uninoculated upper leaves of all the inoculated plants using reverse transcription-polymerase chain reactions. These findings indicated that PVY systemically infected asymptomatic Moneymaker tomato plants at a low level (i.e., no detection of CP via western blotting).




    Conclusion
    Our results indicate that the tomato cultivar Moneymaker is susceptible to PVX and shows mild mosaic symptoms, whereas it is tolerant and asymptomatic to systemic PVY infection with a low virus titer. In contrast, in hpDCL2.4 plants, PVX-induced symptoms became more severe and PVY infection caused symptoms. These results indicate that DCL2, DCL4, or both contribute to tolerance to infection with PVX and PVY. PVY CP and genomic RNA accumulated to a greater extent in DCL2.4-knockdown plants. Hence, the contribution of these DCLs to tolerance to infection with PVY is at least partly attributed to their roles in anti-viral RNA silencing, which controls the multiplication of PVY in tomato plants. The necrotic symptoms observed in the PVX-infected hpAGO2.3 plants suggest that AGO2, AGO3 or both are also distinctly involved in tolerance to infection with PVX.


  • First report of Botrytis porri causing Botrytis leaf blight on leek in Japan
    T. Misawa; R. Ueno; D. Kurose; K.S. Nakahara
    New Disease Reports, 41, 19, 19, British Society for Plant Pathology, 30 Mar. 2020, [Peer-reviewed]
    English, Scientific journal
  • Intracellular proliferation of clover yellow vein virus is unaffected by the recessive resistance gene cyv1 of Pisum sativum.
    Yosuke Taninaka; Kenji S Nakahara; Yuka Hagiwara-Komoda
    Microbiology and immunology, 64, 1, 76, 82, Jan. 2020, [Peer-reviewed], [International Magazine]
    English, Scientific journal, The pea cyv1 gene is a yet-to-be-identified recessive resistance gene that inhibits the infection of clover yellow vein virus (ClYVV). Previous studies confirmed that the cell-to-cell movement of ClYVV is inhibited in cyv1-carrying pea plants; however, the effect of cyv1 on viral replication remains unknown. In this study, we developed a new pea protoplast transfection method to investigate ClYVV propagation at the single-cell level. Using this method, we revealed that ClYVV accumulates to similar levels in both ClYVV-susceptible and cyv1-carrying pea protoplasts. Thus, the cyv1-mediated resistance would not suppress intracellular ClYVV replication.
  • Precise exchange of HC-Pro cistron between soybean mosaic virus and clover yellow vein virus: Impact on virus viability and host range specificity
    Wang, Y; Xu, W; Abe, J; Nakahara, K.S; Hajimorad, M.R
    Phytopathology, Sep. 2019, [Peer-reviewed]
    English, Scientific journal
  • CRISPR/Cas9-mediated editing of genes encoding rgs-CaM-like proteins in transgenic potato plants.
    Osmani, Z; Jin, S; Mikami, M; Endo, M; Atarashi, H; Fujino, K; Yamada, T; Nakahara, KS
    Methods in Molecular Biology, 2028, 153, 165, Jun. 2019, [Invited], [Corresponding author], [International Magazine]
    English, Scientific journal, A tobacco calmodulin-like protein, rgs-CaM, has been shown to interact with viruses in a variety of ways; it contributes to geminivirus infections but is also involved in primed immunity to the cucumber mosaic virus. Sequence similarity searches revealed several calmodulin-like proteins similar to rgs-CaM (rCML) in Arabidopsis and other Solanaceae plants, including potato (Solanum tuberosum). To analyze the functions of each rCML, mutations were introduced into potato rCMLs using the CRISPR/Cas9 system. Here, we describe our protocol of the CRISPR/Cas9-mediated targeted mutagenesis in stably transformed potato plants.
  • Recessive resistance governed by a major quantitative trait locus restricts clover yellow vein virus in mechanically but not graft-inoculated cultivated soybeans
    Abe J; Wang Y; Yamada T; Sato M; Ono T; Atsumi G; Abe J; Hajimorad MR; Nakahara KS
    Mol Plant-Microbe Interactions, 32, 8, 1026, 1037, American Phytopathological Society(APS), Mar. 2019, [Peer-reviewed], [Corresponding author]
    English, Scientific journal
  • rgs-CaM Detects and Counteracts Viral RNA Silencing Suppressors in Plant Immune Priming
    Eun Jin Jeon; Kazuki Tadamura; Taiki Murakami; Jun-ichi Inaba; Bo Min Kim; Masako Sato; Go Atsumi; Kazuyuki Kuchitsu; Chikara Masuta; Kenji S. Nakahara
    JOURNAL OF VIROLOGY, 91, 19, e00761-17, Oct. 2017, [Peer-reviewed], [Last author, Corresponding author]
    English, Scientific journal
  • Paired immunities that impose trade-offs on viruses secure durability of the immunities in plants
    Kenji Nakahara
    Seikagaku, 89, 3, 436, 440, Japanese Biochemical Society, 2017, [Invited], [Corresponding author]
    Japanese, Scientific journal
  • Microperforated leaf blotting on polyvinylidene difluoride and nylon membranes to analyze spatial distribution of endogenous and viral gene expression in plant leaves
    Taiki Murakami; Ryou Tayama; Kenji S. Nakahara
    JOURNAL OF GENERAL PLANT PATHOLOGY, 82, 5, 254, 260, Sep. 2016, [Peer-reviewed], [Corresponding author]
    English, Scientific journal
  • Trade-Offs for Viruses in Overcoming Innate Immunities in Plants
    Yuri Miyashita; Go Atsumi; Kenji S. Nakahara
    MOLECULAR PLANT-MICROBE INTERACTIONS, 29, 8, 595, 598, Aug. 2016, [Peer-reviewed], [Corresponding author]
    English, Scientific journal
  • P3N-PIPO, a Frameshift Product from the P3 Gene, Pleiotropically Determines the Virulence of Clover Yellow Vein Virus in both Resistant and Susceptible Peas
    Go Atsumi; Haruka Suzuki; Yuri Miyashita; Sun Hee Choi; Yusuke Hisa; Shunsuke Rihei; Ryoko Shimada; Eun Jin Jeon; Junya Abe; Kenji S. Nakahara; Ichiro Uyeda
    JOURNAL OF VIROLOGY, 90, 16, 7388, 7404, Aug. 2016, [Peer-reviewed], [Corresponding author]
    English, Scientific journal
  • Trade-Offs for Viruses in Overcoming Innate Immunities in Plants
    Yuri Miyashita; Go Atsumi; Kenji S. Nakahara
    Molecular plant-microbe interactions : MPMI, 2016, 1, 1, 4, 01 Jul. 2016
    English, Scientific journal
  • Truncated yet functional viral protein produced via RNA polymerase slippage implies underestimated coding capacity of RNA viruses
    Yuka Hagiwara-Komoda; Sun Hee Choi; Masanao Sato; Go Atsumi; Junya Abe; Junya Fukuda; Mie N. Honjo; Atsushi J. Nagano; Keisuke Komoda; Kenji S. Nakahara; Ichiro Uyeda; Satoshi Naito
    SCIENTIFIC REPORTS, 6, 21411, Feb. 2016, [Peer-reviewed], [Corresponding author]
    English, Scientific journal
  • Construction of infectious cDNA clones derived from the potyviruses Clover yellow vein virus and Bean yellow mosaic virus
    Kenji S. Nakahara; Kei Nishino; Ichiro Uyeda
    Methods in Molecular Biology, 1236, 219, 227, 2015, [Invited], [Corresponding author]
    English, Scientific journal
  • ウイルスに対する植物の自然免疫機構
    忠村一毅; 中原健二
    化学と生物, 52, 12, 805, 813, Dec. 2014, [Invited], [Corresponding author]
    Japanese, Research society
  • Interaction between viral RNA silencing suppressors and host factors in plant immunity
    Kenji S. Nakahara; Chikara Masuta
    CURRENT OPINION IN PLANT BIOLOGY, 20, 88, 95, Aug. 2014, [Peer-reviewed], [Invited], [Corresponding author]
    English, Scientific journal
  • P3N-PIPO of Clover yellow vein virus exacerbates symptoms in pea infected with White clover mosaic virus and is implicated in viral synergism
    Yusuke Hisa; Haruka Suzuki; Go Atsumi; Sun Hee Choi; Kenji S. Nakahara; Ichiro Uyeda
    VIROLOGY, 449, 200, 206, Jan. 2014, [Peer-reviewed], [Corresponding author]
    English, Scientific journal
  • Quantitative and qualitative involvement of P3N-PIPO in overcoming recessive resistance against Clover yellow vein virus in pea carrying the cyv1 gene
    Sun Hee Choi; Yuka Hagiwara-Komoda; Kenji S. Nakahara; Go Atsumi; Ryoko Shimada; Yusuke Hisa; Satoshi Naito; Ichiro Uyeda
    Journal of Virology, 87, 13, 7326, 7337, Jul. 2013, [Peer-reviewed], [Corresponding author]
    English, Scientific journal
  • Characterization of the recessive resistance gene cyv1 of Pisum sativum against Clover yellow vein virus
    Sun Hee Choi; Kenji S. Nakahara; Marcelo Andrade; Ichiro Uyeda
    JOURNAL OF GENERAL PLANT PATHOLOGY, 78, 4, 269, 276, Jul. 2012, [Peer-reviewed]
    English, Scientific journal
  • Tobacco calmodulin-like protein provides secondary defense by binding to and directing degradation of virus RNA silencing suppressors
    Kenji S. Nakahara; Chikara Masuta; Syouta Yamada; Hanako Shimura; Yukiko Kashihara; Tomoko S. Wada; Ayano Meguro; Kazunori Goto; Kazuki Tadamura; Kae Sueda; Toru Sekiguchi; Jun Shao; Noriko Itchoda; Takeshi Matsumura; Manabu Igarashi; Kimihito Ito; Richard W. Carthew; Ichiro Uyeda
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 109, 25, 10113, 10118, Jun. 2012, [Peer-reviewed], [Lead author, Corresponding author]
    English, Scientific journal
  • Heterologous expression of viral suppressors of RNA silencing complements virulence of the HC-Pro mutant of clover yellow vein virus in pea
    Go Atsumi; Kenji S. Nakahara; Tomoko Sugikawa Wada; Sun Hee Choi; Chikara Masuta; Ichiro Uyeda
    ARCHIVES OF VIROLOGY, 157, 6, 1019, 1028, Jun. 2012, [Peer-reviewed]
    English, Scientific journal
  • White clover mosaic virus-induced gene silencing in pea
    Yukari Ido; Kenji S. Nakahara; Ichiro Uyeda
    JOURNAL OF GENERAL PLANT PATHOLOGY, 78, 2, 127, 132, Mar. 2012, [Peer-reviewed], [Corresponding author]
    English, Scientific journal
  • Wound-induced rgs-CaM gets ready for counterresponse to an early stage of viral infection
    Kazuki Tadamura; Kenji S. Nakahara; Chikara Masuta; Ichiro Uyeda
    Plant Signaling and Behavior, 7, 12, 1548, 1551, 2012, [Peer-reviewed], [Corresponding author]
    English, Scientific journal
  • Sensitive PCR-based Detection of Apple Chlorotic Leaf Spot Virus Heterogenous in Apple Trees
    Kenji S. Nakahara; Kouji Yoshida; Kouichi Suzaki; Nobuyuki Yoshikawa; Tsutae Ito
    JARQ-JAPAN AGRICULTURAL RESEARCH QUARTERLY, 45, 4, 411, 421, Oct. 2011, [Peer-reviewed], [Lead author, Corresponding author]
    English, Scientific journal
  • Cargo sorting to lysosome-related organelles regulates siRNA-mediated gene silencing
    Dinari A. Harris; Kevin Kim; Kenji Nakahara; Constanza Vasquez-Doorman; Richard W. Carthew
    JOURNAL OF CELL BIOLOGY, 194, 1, 77, 87, Jul. 2011, [Peer-reviewed]
    English, Scientific journal
  • Screening and analysis of genes expressed upon infection of broad bean with Clover yellow vein virus causing lethal necrosis
    Kenji S. Nakahara; Hiroaki Kitazawa; Go Atsumi; Sun Hee Choi; Yuji Suzuki; Ichiro Uyeda
    VIROLOGY JOURNAL, 8, 355, Jul. 2011, [Peer-reviewed], [Lead author, Corresponding author]
    English, Scientific journal
  • Involvement of the P1 Cistron in Overcoming eIF4E-Mediated Recessive Resistance Against Clover yellow vein virus in Pea
    Kenji S. Nakahara; Ryoko Shimada; Sun-Hee Choi; Haruko Yamamoto; Jun Shao; Ichiro Uyeda
    MOLECULAR PLANT-MICROBE INTERACTIONS, 23, 11, 1460, 1469, Nov. 2010, [Peer-reviewed], [Lead author, Corresponding author]
    English, Scientific journal
  • Silencing by small RNAs is linked to endosomal trafficking
    Young Sik Lee; Sigal Pressman; Arlise P. Andress; Kevin Kim; Jamie L. White; Justin J. Cassidy; Xin Li; Kim Lubell; Do Hwan Lim; Ik Sang Cho; Kenji Nakahara; Jonathan B. Preall; Priya Bellare; Erik J. Sontheimer; Richard W. Carthew
    NATURE CELL BIOLOGY, 11, 9, 1150, U243, Sep. 2009, [Peer-reviewed]
    English, Scientific journal
  • The cyv-2 resistance to Clover yellow vein virus in pea is controlled by the eukaryotic initiation factor 4E
    Marcelo Andrade; Yosuke Abe; Kenji S. Nakahara; Ichiro Uyeda
    JOURNAL OF GENERAL PLANT PATHOLOGY, 75, 3, 241, 249, Jun. 2009, [Peer-reviewed]
    English, Scientific journal
  • Activation of the Salicylic Acid Signaling Pathway Enhances Clover yellow vein virus Virulence in Susceptible Pea Cultivars
    Go Atsumi; Uiko Kagaya; Hiroaki Kitazawa; Kenji Suto Nakahara; Ichiro Uyeda
    MOLECULAR PLANT-MICROBE INTERACTIONS, 22, 2, 166, 175, Feb. 2009, [Peer-reviewed]
    English, Scientific journal
  • Point mutations in helper component protease of clover yellow vein virus are associated with the attenuation of RNA-silencing suppression activity and symptom expression in broad bean
    M. L. M. Yambao; H. Yagihashi; H. Sekiguchi; T. Sekiguchi; T. Sasaki; M. Sato; G. Atsumi; Y. Tacahashi; K. S. Nakahara; I. Uyeda
    ARCHIVES OF VIROLOGY, 153, 1, 105, 115, Jan. 2008, [Peer-reviewed]
    English, Scientific journal
  • The RNAi pathway initiated by Dicer-2 in Drosophila
    K. Kim; Y. S. Lee; D. Harris; K. Nakahara; R. W. Carthew
    COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY, 71, 39, 44, 2006, [Peer-reviewed]
    English, International conference proceedings
  • Targets of microRNA regulation in the Drosophila oocyte proteorne
    K Nakahara; K Kim; C Sciulli; Dowd, SR; JS Minden; RW Carthew
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 102, 34, 12023, 12028, Aug. 2005, [Peer-reviewed], [Lead author]
    English, Scientific journal
  • Stem cell division is regulated by the microRNA pathway
    SD Hatfield; HR Shcherbata; KA Fischer; K Nakahara; RW Carthew; H Ruohola-Baker
    NATURE, 435, 7044, 974, 978, Jun. 2005, [Peer-reviewed]
    English, Scientific journal
  • Selective involvement of members of the eukaryotic initiation factor 4E family in the infection of Arabidopsis thaliana by potyviruses
    M Sato; K Nakahara; M Yoshii; M Ishikawa; Uyeda, I
    FEBS LETTERS, 579, 5, 1167, 1171, Feb. 2005, [Peer-reviewed]
    English, Scientific journal
  • Distinct roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA silencing pathways
    YS Lee; K Nakahara; JW Pham; K Kim; ZY He; EJ Sontheimer; RW Carthew
    CELL, 117, 1, 69, 81, Apr. 2004, [Peer-reviewed]
    English, Scientific journal
  • Expanding roles for miRNAs and siRNAs in cell regulation
    K Nakahara; RW Carthew
    CURRENT OPINION IN CELL BIOLOGY, 16, 2, 127, 133, Apr. 2004, [Peer-reviewed], [Lead author]
    English
  • The central and C-terminal domains of VPg of Clover yellow vein virus are important for VPg-HCPro and VPg-VPg interactions
    MLM Yambao; C Masuta; K Nakahara; Uyeda, I
    JOURNAL OF GENERAL VIROLOGY, 84, 10, 2861, 2869, Oct. 2003, [Peer-reviewed]
    English, Scientific journal
  • Multiple citrus viroids in citrus from Japan and their ability to produce exocortis-like symptoms in citron
    T Ito; H Ieki; K Ozaki; T Iwanami; K Nakahara; T Hataya; T Ito; M Isaka; T Kano
    PHYTOPATHOLOGY, 92, 5, 542, 547, May 2002, [Peer-reviewed]
    English, Scientific journal
  • Cloning and sequencing of endochitinase genes from Gliocladium virens and Trichoderma species
    Kenji Nakahara; Kouji Yoshida; Tsutae Ito; Kouich Suzaki; Akira Kudo
    Archives of Phytopathology and Plant Protection, 33, 6, 519, 527, 2001, [Peer-reviewed], [Lead author]
    English, Scientific journal
  • Comparisons of gene diagnostic methods for the practical diagnosis of chrysanthemum stunt viroid in chrysanthemum plants
    Tatsuji Hataya; Kenji Nakahara; Kazuyoshi Furuta; Eishiro Shikata
    Archives of Phytopathology and Plant Protection, 32, 179, 192, 1999, [Peer-reviewed]
    English, Scientific journal
  • A simple, rapid method of nucleic acid extraction without tissue homogenization for detecting viroids by hybridization and RT-PCR
    K Nakahara; T Hataya; Uyeda, I
    JOURNAL OF VIROLOGICAL METHODS, 77, 1, 47, 58, Jan. 1999, [Peer-reviewed], [Lead author]
    English, Scientific journal
  • Inosine 5 '-triphosphate can dramatically increase the yield of NASBA products targeting GC-rich and intramolecular base-paired viroid RNA
    K Nakahara; T Hataya; Uyeda, I
    NUCLEIC ACIDS RESEARCH, 26, 7, 1854, 1855, Apr. 1998, [Peer-reviewed], [Lead author]
    English, Scientific journal
  • A mixture of synthetic oligonucleotide probes labeled with biotin for the sensitive detection of potato spindle tuber viroid
    K Nakahara; T Hataya; Y Hayashi; T Sugimoto; Kimura, I; E Shikata
    JOURNAL OF VIROLOGICAL METHODS, 71, 2, 219, 227, Apr. 1998, [Peer-reviewed], [Lead author]
    English, Scientific journal
  • An improved procedure for extracting nucleic acids from citrus tissues for diagnosis of citrus viroids
    Kenji Nakaraha; Tatsuji Hataya; Ichiro Uyeda; Hiroyuki Ieki
    Annals of the Phytopathologicial Society of Japan, 64, 6, 532, 538, The Phytopathological Society of Japan, 1998, [Peer-reviewed], [Lead author]
    English, Scientific journal, When detecting viroids in citrus tissues, contaminating polysaccharides and phenolic compounds often make it difficult to consistently extract nucleic acids. To extract the nucleic acids consistently, the method was improved as follows: the contaminants were removed by differential 2-butoxyethanol precipitation instead of a combination of 2-methoxyethanol extraction and cetyltrimethylammonium bromide precipitation. In contrast to the conventional method, the modified one consistently extracted some citrus viroids, i.e., citrus exocortis viroid (CEVd), group I citrus viroid (CVd-I) and hop stunt viroid-citrus isolate, which is a variant of group II citrus viroid (CVd-II), after they were sufficiently subjected to sequential polyacrylamide gel electrophoresis (sPAGE) and dot blot hybridization using digoxigenin (DIG)-labeled cRNA probes. A viroid-like RNA presumed to be a group III citrus viroid (CVd-III) was detected in nucleic acids extracted from Japanese citrus samples by sPAGE. The cDNA fragments of the viroid-like RNA were cloned and sequenced. The nucleotide sequences of the cDNAs were completely identical to those reported previously for CVd-IIIa and CVd-IIIb abroad. CVd-III was detected from several other Japanese citrus samples using the DIG-labeled cRNA probe prepared from the cloned cDNA. Many of these samples were also co-infected with other citrus viroids.
  • Citrus viroid Ia is a derivative of citrus bent leaf viroid (CVd-Ib) by partial sequence duplications in the right terminal region
    T Hataya; K Nakahara; T Ohara; H Ieki; T Kano
    ARCHIVES OF VIROLOGY, 143, 5, 971, 980, 1998, [Peer-reviewed]
    English, Scientific journal
  • Reactions of Potato Cultivars in Japan to Potato Spindle Tuber Viroid and Its Gene Diagnosis
    NAKAHARA Kenji
    Ann. Rept. Plant Prot. North Japan, 48, 48, 69, 74, The Society of Plant Protection of North Japan, 1997, [Peer-reviewed], [Lead author]
    Japanese, Scientific journal, To investigate reactions of potato cultivars in Japan to potato spindle tuber viroid (PSTVd), PSTVd was inoculated to seven potato cultivars. All cultivars inoculated showed some symptoms, but the severity of symptoms on both leaves and tubers were different among the seven cultivars. The cultivars Hokkaikogane, Norin No.1 and Danshaku-imo developed relatively severer symptoms than the other cultivars Waseshiro, Konahubuki, Mayqueen and Astarte. PSTVd in these plant leaves and tubers were detected by dot blot hybridization using digoxigenin (DIG) labeled cDNA probe. PSTVd accumulation did not differ among cultivars significantly. The result indicated that all cultivars were susceptible to PSTVd and this hybridization method was applicable to diagnose PSTVd in potato leaves and tubers directly.
■ Other Activities and Achievements
■ Syllabus
  • 大学院共通授業科目（一般科目）：自然科学・応用科学, 2024年, 修士課程, 大学院共通科目
  • バイオテクノロジー学特論, 2024年, 修士課程, 農学院
  • バイオテクノロジー学特論演習, 2024年, 修士課程, 農学院
  • 農業植物科学特論, 2024年, 修士課程, 農学院
  • 農業植物科学特論演習, 2024年, 修士課程, 農学院
  • 生物学概論, 2024年, 学士課程, 農学部
  • 生物資源科学特講, 2024年, 学士課程, 農学部
  • 植物ウイルス病学, 2024年, 学士課程, 農学部
  • 生物学実習, 2024年, 学士課程, 農学部
  • 生物資源科学実験Ⅰ, 2024年, 学士課程, 農学部
■ Research Themes
  • Molecular basis for the development of virus resistance genetic resources through in-frame deletion editing of host genes
    Grants-in-Aid for Scientific Research
    01 Apr. 2022 - 31 Mar. 2025
    中原 健二; 薦田 優香
    インフレーム塩基欠失型ゲノム編集(iGE、3の倍数の欠失)アレルによる抵抗性それぞれについて以下のような結果が得られた。eIF4E1_9DELによるCMV抵抗性については、eIF4E1_9DELが2bのRNAサイレンシング抑制活性をどのように阻害しているのかメカニズムの解明を進めた。まず、オートファジーによる分解ターゲットとして知られる2bのオートファジーによる分解をeIF4E_9DELが結合することで促進している可能性について検討した。N. benthamiana葉へのアグロインフィルトレーションによりeIF4E_9DELと2bタンパク質を共発現して2bの蓄積量をオートファジー阻害剤用いて検証したが、2bのオートファジーによる分解が促進される証拠は得られなかった。一方で、eIF4E機能の条件欠損酵母を用いた検証で、eIF4E_9DELタンパク質が変異によりeIF4E本来の翻訳開始因子としての機能を失っていることが明らかとなり、eIF4E_9DELのCMV感染阻害効果は、eIF4E本来の機能とは別の働きであると結論することができた。
    BAM2のiGEアレルについては、12延期欠失に加えて、3塩基、6塩基、144塩基欠失のiGEアレルが得られており、それらのTYLCV 抵抗性について比較検証した結果、インフレーム欠損変異では最小の3塩基欠損で、他の欠損変異よりも発病の割合が低く、発病した個体においても、TYLCVのゲノムDNAの蓄積量が野生型に比べて低下しており、より強いTYLCV抵抗性が示された。また、トマト黄化えそウイルス(TSWV)抵抗性についても同様に比較検証を開始して、再現実験による検証が必要ではあるが、1回目の接種試験で、やはり3塩基欠損で発病個体数が少なく、病徴も弱いことからより強いTSWV抵抗性が示された。
    Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 23K23608
  • Molecular basis for developing antiviral genetic resources derived from host genes by genome editing with in-frame deletions
    Grants-in-Aid for Scientific Research
    01 Apr. 2022 - 31 Mar. 2025
    中原 健二; 薦田 優香
    Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 22H02343
  • ダイズ矮化ウイルス感染の分子機構と宿主因子の解明
    科学研究費助成事業
    01 Apr. 2020 - 31 Mar. 2023
    薦田 優香; 中原 健二
    本研究では、北日本のダイズ圃場に慢性的に発生するダイズ矮化病の原因とされる、ダイズ矮化ウイルス（Soybean dwarf virus, SbDV）の増殖機構の解明を目指している。ウイルス増殖機構の解明には、感染性cDNAクローンの作出と機械的接種法の確立が求められる。本年度は昨年度に引き続き、SbDVの機械的接種法確立のための条件検討を進めた。SbDVの感染性cDNAクローンからSbDV RNAを試験管内合成し、これを接種源とした。発芽後間もないダイズおよびエンドウの芽生えに、微細なワイヤ－を用いてRNAを接種したところ、いずれの宿主植物においても、50％以上の効率で感染させることに成功した。そこで本接種法を利用し、SbDVがコードする遺伝子の機能についてのさらなる解析を進めた。
    SbDVには2種類の外被タンパク質（CP、minor CP）が存在する。CP翻訳時に終止コドンのリードスルーが起こることで、CPのN末端側にリードスルードメイン（RTD）が付加された形のminor CPが合成される。これまでに、minor CPに存在するRTDの機能として、アブラムシの媒介に関与する可能性が示唆されている。一方、RTDの植物体内における機能は未知である。そこで本研究では、RTDがウイルスの細胞間移行に重要かどうかについて解析を行った。RTDの翻訳が起こらないようCPの終止コドン位置に複数の終止コドンを挿入したSbDV cDNAを構築した。この変異SbDVのゲノムRNAを、上記機械的接種法を用いてダイズおよびエンドウの芽生えに接種した。その結果、変異SbDVは野生型SbDVと同程度の全身感染効率を示した。すなわち、SbDVのRTDはウイルスの細胞間移行および長距離移行に必須ではないことが明らかとなった。
    日本学術振興会, 基盤研究(C), 酪農学園大学, 20K06056
  • Molecular mechanism of calmodulin-like proteins-mediated recognition and control of viral virulent factors
    Grants-in-Aid for Scientific Research
    08 Nov. 2019 - 31 Mar. 2022
    中原 健二; AKHTER MD. Shamim; AKHTER MD.
    受け入れ研究者は、これまでカルモジュリン様タンパク質(CML)とCMV 2bを含むウイルスのRNAサイレンシング抑制タンパク質との相互作用について研究してきた。CMLと2bの相互作用の背景メカニズムを解明するために、昨年までのDr. Akhter特別研究員と受け入れ研究者の研究で、葉緑体外包膜タンパク質がCMLと2b両方と結合することを酵母ツーハイブリッド法により見出した。また、CMV 2bがオートファジーのキー遺伝子であるATG8と結合することを酵母ツーハイブリッド法による試験で見出した。本年度は、これらの結合、すなわち、CMV 2bとCML、CMLと葉緑体外包膜タンパク質、葉緑体外包膜タンパク質とCMV 2b、およびCML 2bとATG8が植物細胞内でも結合するのかどうか、それらのタンパク質とルシフェラーゼの部分断片を融合させた融合タンパク質を一過発現し、結合の有無をルシフェラーゼ活性で測定するsplit luciferase相補解析により検証した。植物細胞での一過発現には、Nicotiana benthamiana葉でのアグロバクテリウムのインフィルトレーションにより一過発現系を用いた。その結果、split luciferase相補解析により植物細胞内においていずれの組み合わせでも結合していることが確かめられた。そこで、これらの結合が、それぞれのタンパク質の機能や役割にどのように関わるのか、検証を進め、葉緑体外包膜タンパク質の高発現下で、CMV 2bの蓄積量が低下して、RNAサイレンシング抑制活性が弱まることが分かった。CMV 2bは葉緑体外包膜タンパク質と結合するとATG8との結合を介したオートファジーによる分解が促進している可能性が考えられた。受け入れ研究者の以前の研究でCMLは2bと結合しオートファジーによる分解に導くことでウイルス防御に関わることを明らかにした。そしてDr. Akhter特別研究員との本研究により、このCMLを介したウイルス防御機構に葉緑体外包膜タンパク質が重要な貢献をしていることが示唆された。
    Japan Society for the Promotion of Science, Grant-in-Aid for JSPS Fellows, Hokkaido University, 19F19391
  • Analysis of potyvirus propagation mechanisms using in vitro experimental systems
    Grants-in-Aid for Scientific Research
    01 Apr. 2017 - 31 Mar. 2022
    Komoda Yuka
    This study aimed to develop an experimental system to analyze the multiplication process of potyviruses that cause damage to various agricultural crops. Using an infectious cDNA clone of clover yellow vein virus (ClYVV) and pea, a natural host of ClYVV, a mesophyll protoplast transfection method was newly developed. This experimental system made it possible to analyze some of the virus-plant interactions, such as the involvement of the recessive resistance genes of pea in virus multiplication at the single-cell level. Furthermore, we showed that this experimental system can be applied to virus propagation assays using not only potyviruses but also luteoviruses.
    Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), Rakuno Gakuen University, 17K07670
  • Analysis of defense responses based on innate immunity against viroid infection and mechanism to develop dwarfing and necrosis
    Grants-in-Aid for Scientific Research
    01 Apr. 2018 - 31 Mar. 2021
    Sano Teruo
    In tomato plants infected with the virulent strain of potato spindle tuber viroid (PSTVd), stress-responsive microRNAs (miR398 and miR398a-3p) were excessively induced, and the expression of SOD genes encoding reactive oxygen species (ROS) scavenging enzymes were down-regulated, which caused loss of normal ROS scavenging function resulted in excessive accumulation of ROS, leading to severe pathological symptoms accompanied by necrosis. The nucleotides at position 42 and 64 of the attenuated strain of PSTVd were key nucleotides for the attenuation, and in cooperation with the nucleotides 43, 310, and 311/312, contributed to its low and stable accumulation. The attenuated strain did not elicit an excessive defense responses, such as pathogenesis-related protein 1b1. A novel 2-oxoglutarate Fe (II)-dependent oxygenase gene associated with dwarfing and leaf malformation characteristic of viroid disease was identified in tomato plant (Solanum lycopersicum).
    Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hirosaki University, 18H02201
  • 全身獲得抵抗性の新しい概念の証明とそれを利用した抵抗性育種戦略の例証
    科学研究費補助金(基盤研究(B))
    Apr. 2016 - Mar. 2020
    中原 健二
    文部科学省, Principal investigator, Competitive research funding
  • Molecular mechanisms underlying a plant phase-change from facilitative to resistant responses against pathogens(Fostering Joint International Research)
    Grants-in-Aid for Scientific Research
    2017 - 2019
    Nakahara Kenji
    In order to identify calmodulin-like proteins (CML) that are involved in an antiviral defese via interaction with virus RNA silencing suppressors (RSS), we sought CMLs that have an affinity to RSS of cucumber mosaic virus, 2b. As a result, 6 CMLs including CML43 were found to bind to 2b. Then, we screened endogenous proteins that bind to CML43 and identified two proteins, which may be factors that mediate the CML associated antiviral defense.
    Japan Society for the Promotion of Science, Fund for the Promotion of Joint International Research (Fostering Joint International Research), Hokkaido University, 16KK0167
  • Development of in vitro assay systems using legumes and stone fruits for analyses of potyvirus replication
    Grants-in-Aid for Scientific Research
    01 Apr. 2014 - 31 Mar. 2018
    Komoda Yuka
    Potyviruses cause diseases in many important crops. To reveal the replication mechanism of potyviruses, we attempted to generate an in vitro potyviral translation-replication system using their natural host plants. The establishment of the natural host-derived in vitro system remains incomplete. We investigated the expression mechanism of pipo ORF discovered recently in potyviral genomes, by using conventional in vitro and in vivo assay systems. The pipo ORF exists in the -1 (or +2) reading frame relative to the viral polyprotein ORF, and is expressed as a fusion protein with the N-terminal half of P3 (P3N-PIPO). We revealed that the pipo ORF is expressed via transcriptional slippage during viral replication. We also found that, in addition to P3N-PIPO, another frameshift product, P3N-ALT, is also produced via transcriptional slippage.
    Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), 26450050
  • ウイルスの病原関連分子パターン認識を介した免疫誘導機構の解明
    科学研究費補助金(基盤研究(C))
    Apr. 2013 - Mar. 2016
    中原 健二
    文部科学省, Principal investigator, Competitive research funding
  • Functional analysis of host factors involved in antiviral defense networks in plants
    Grants-in-Aid for Scientific Research
    2008 - 2011
    NAKAHARA Kenji
    This study uncovered a novel mechanism of antiviral defense cooperatively acting with RNA silencing. In the defense, a calmodulin-like protein, rgs-CaM, binds to diverse viral RNA silencing suppressors and directs degradation of them by autophagy. Thus, the rgs-CaM-mediated defense seemed a host countermeasure against viral RNA silencing suppressors to reinforce antiviral RNA silencing in tobacco.
    Japan Society for the Promotion of Science, Grant-in-Aid for Young Scientists (A), Hokkaido University, Principal investigator, Competitive research funding, 20688002
  • Modification of plant gene expression by viral gene silencing suppressors
    Grants-in-Aid for Scientific Research
    2006 - 2010
    UYEDA Ichiro; MASUTA Chikara; NAKAHARA Kenji
    Plants defense against virus infection by Post Transcriptional Gene Silencing (PTGS). We analyzed how PTGS was involved in pathogenicity and obtained the following results. (1) Suppressor activity of viral HC-Pro gene is required for induction of necrotic symptoms. (2) rgs-CaM gene has affinity to many viral suppressor genes. Its interaction destabilizes bound proteins and consequently strengthen defense against virus infection by elevating PTGS activity. (3) Y satellite RNA of cucumber mosaic virus (Y-satRNA) induces bright yellow mosaic in tobacco instead of regular green mosaic. Molecular mechanism of how Y-satRNA induces yellow mosaic is elucidated.
    Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (S), Hokkaido University, 18108001
  • HC-Pro/rgsCaMによるRNAサイレンシング阻害の分子機構
    科学研究費補助金(若手研究(B))
    2005 - 2006
    中原健二
    昨年の研究で、キイロショウジョウバエのS2培養細胞ではCIYVVのHC-ProはRNAサイレンシング抑制能を示さなかったが、HC-Proと相互作用することが知られるタバコのrgsCaMはsiRNA形成後の過程でRNAサイレンシングを抑制することが分かった。次にこれらの遺伝子とともに外来遺伝子ルシフェラーゼの遺伝子がS2細胞のゲノムに組み込まれた安定発現細胞を作成してルシフェラーゼ活性を指標にその発現に対するrgsCaMとCIYVV HC-Proの影響を調べた。なぜなら、安定発現するルシフェラーゼはジーンサイレンシングによる部分的な発現抑制を受けることが知られているからである。その結果、rgsCaMだけでなくCIYVVのHC-Proもルシフェラーゼの発現を上昇させている可能性が示された。そしてこれらの培養細胞のゲノムに組み込まれたルシフェラーゼ遺伝子のコピー数、それから転写されたmRNAの蓄積量、翻訳されたルシフェラーゼタンパク質の蓄積量を相対的に比較したところ、rgsCaMとCIYVVのHC-Proは翻訳以降の過程で発現を促進していると思われた。シクロヘキシミドを培養液に加えて培養細胞の翻訳を止めて、経時的にルシフェラーゼ活性を調べることによりルシフェラーゼタンパク質の分解速度を細胞間で比較したところ、HC-Proの発現の有無で分解速度に差がなかったことから、少なくともCIY...
    文部科学省, 若手研究(B), 北海道大学, Principal investigator, Competitive research funding, 17780032
  • ウイルスに対する防御機構における二本鎖RNA特異的アデノシンデアミナーゼの役割
    科学研究費助成事業
    2001 - 2001
    中原 健二
    本研究は植物のウイルスに対する感染防御機構、RNAサイレンシングの分子機構の解明を目的とする。RNAサイレンシングは二本鎖RNAを形成した塩基配列を特異的に認識して分解することが分かっている。植物ウイルスの多くはRNAウイルスあり複製中間体として二本鎖RNAを形成するので理にかなった認識機構である。本認識機構に関わる宿主遺伝子の探索を目的として、二本鎖RNA特異的アデノシンデアミナーゼ(dsRAD)遺伝子のクローニングを試みた。この遺伝子は動物においてインターフェロン及びウイルス感染により発現が誘導されることが報告されているが、植物でまだ見つかっていない。
    そこで申請者はDNAデーターベース上で検索を行ったところ、dsRAD遺伝子をコードする可能性のある塩基配列をシロイヌナズナで見出し、部分的なcDNAを得た。さらに5'および3'RACE法により全長cDNAをクローニングして動物のdsRADと比較したところ、アデノシンデアミナーゼ活性部分はあるが、二本鎖RNAとの結合部分がないことが分かった。動物でも同様のアデノシンデアミナーゼが見つかっており、tRNAを修飾していることが報告されている。従って、本遺伝子はdsRADではなくtRNAに作用するアデノシンデアミナーゼでRNAサイレンシングにおける役割はないものと思われた。シロイヌナズナには他にdsRADの候補は見出せず、dsRADはRNAサイレンシングには関わっていないと思われた。
    これを支持する結果が、最近、動物のRNAサイレンシングであるRNAiに関する実験でZamoreらにより報告されている。そこで、植物ウイルスのRNAサイレンシング阻害遺伝子を用いてRNAサイレンシング阻害遺伝子を用いてRNAサイレンシングに関わる宿主遺伝子を探索することにした。
    本科学研究費補助金の継続を辞退し、渡米して本研究を鋭意進めているところである。
    日本学術振興会, 若手研究(B), 秋田県立大学, 13760040
  • ウイルスに対する植物の自然免疫機構
    Competitive research funding
■ Industrial Property Rights
  • リンゴクロロティックリーフスポットウイルスの検出方法及び検出用プライマー
    Patent right
    3459976
  • 新規なcDNAサブトラクション法
    Patent right
    3459977