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, [Last author, Corresponding author]
Scientific journal

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, 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,

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.




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.




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 (PVY^N), but not to the ordinary strain (PVY^O), 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,


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.





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.





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).





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, AMER SOC MICROBIOLOGY, Oct. 2017, [Peer-reviewed], [Corresponding author]
English, Scientific journal, Primary infection of a plant with a pathogen that causes high accumulation of salicylic acid in the plant typically via a hypersensitive response confers enhanced resistance against secondary infection with a broad spectrum of pathogens, including viruses. This phenomenon is called systemic acquired resistance (SAR), which is a plant priming for adaption to repeated biotic stress. However, the molecular mechanisms of SAR-mediated enhanced inhibition, especially of virus infection, remain unclear. Here, we show that SAR against cucumber mosaic virus (CMV) in tobacco plants (Nicotiana tabacum) involves a calmodulin-like protein, rgs-CaM. We previously reported the antiviral function of rgs-CaM, which binds to and directs degradation of viral RNA silencing suppressors (RSSs), including CMV 2b, via autophagy. We found that rgs-CaM-mediated immunity is ineffective against CMV infection in normally growing tobacco plants but is activated as a result of SAR induction via salicylic acid signaling. We then analyzed the effect of overexpression of rgs-CaM on salicylic acid signaling. Overexpressed and ectopically expressed rgs-CaM induced defense reactions, including cell death, generation of reactive oxygen species, and salicylic acid signaling. Further analysis using a combination of the salicylic acid analogue benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) and the Ca2+ ionophore A23187 revealed that rgs-CaM functions as an immune receptor that induces salicylic acid signaling by simultaneously perceiving both viral RSS and Ca2+ influx as infection cues, implying its autoactivation. Thus, secondary infection of SAR-induced tobacco plants with CMV seems to be effectively inhibited through 2b recognition and degradation by rgs-CaM, leading to reinforcement of antiviral RNA silencing and other salicylic acid-mediated antiviral responses.
IMPORTANCE Even without an acquired immune system like that in vertebrates, plants show enhanced whole-plant resistance against secondary infection with pathogens; this so-called systemic acquired resistance (SAR) has been known for more than half a century and continues to be extensively studied. SAR-induced plants strongly and rapidly express a number of antibiotics and pathogenesis-related proteins targeted against secondary infection, which can account for enhanced resistance against bacterial and fungal pathogens but are not thought to control viral infection. This study showed that enhanced resistance against cucumber mosaic virus is caused by a tobacco calmodulin-like protein, rgs-CaM, which detects and counteracts the major viral virulence factor (RNA silencing suppressor) after SAR induction. rgs-CaM-mediated SAR illustrates the growth versus defense trade-off in plants, as it targets the major virulence factor only under specific biotic stress conditions, thus avoiding the cost of constitutive activation while reducing the damage from virus infection.

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, SPRINGER JAPAN KK, Sep. 2016, [Peer-reviewed], [Corresponding author]
English, Scientific journal, Leaf blotting to detect proteins and investigate their spatial distribution in leaves has so far mainly been used to detect viral coat proteins that accumulate abundantly in infected leaves, but rarely to detect endogenous plant proteins. We improved the method for detecting endogenous proteins. We found that microperforating leaves with bundled pins before blotting, then pressing leaves with a rolling pin onto polyvinylidene difluoride (PVDF) membranes enabled even blotting of sap. This microperforated leaf blotting (mPLB) was also suitable for use with nylon membranes to detect leaf RNA. The mPLB revealed that accumulation of two endogenous proteins, calmodulin-like rgs-CaM and actin, was respectively positively and negatively associated with that of viral coat protein in tobacco leaves infected with cucumber mosaic virus (CMV). When a tobacco plant primed with benzothiadiazole was inoculated with CMV, mPLB showed that the infection was restricted to some areas of the leaf and that in these areas the mRNA encoding tobacco pathogenesis-related protein 1, an indicator of salicylic acid-mediated immune responses, was induced. These results demonstrate the effectiveness of mPLB for investigating the spatial distribution of endogenous and viral gene expression in leaves.

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, AMER PHYTOPATHOLOGICAL SOC, Aug. 2016, [Peer-reviewed], [Corresponding author]
English, Scientific journal, Plants recognize viral infection via an immune receptor, i.e., nucleotide-binding site (NB)-leucine-rich repeat (LRR) proteins. Another immune receptor, receptor-like kinase proteins, which share an LRR domain with NB-LRRs, perceive conserved molecules of pathogens called pathogen-or microbe associated molecular patterns, but NB-LRRs generally perceive particular viral proteins. As viruses can evolve more rapidly than the host immune system, how do plant immune systems, which rely on the perception of proteins, remain effective? Viral adaptive evolution may be controlled by penalties that result from mutations in viral proteins that are perceived by NB-LRRs. Our recent studies in pea (Pisum sativum) suggest a penalty of increased susceptibility to another immune system. When a viral protein mutates to evade one immune system, the virus with the mutated protein becomes more susceptible to another. Such antagonistic pleiotropy of a viral protein by two independent plant immune systems may have precedents. Plants may rely on pairs of immune systems to constrain adaptive evolution by viruses and thereby maintain durable antiviral immunity.

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, AMER SOC MICROBIOLOGY, Aug. 2016, [Peer-reviewed], [Corresponding author]
English, Scientific journal, Peas carrying the cyv1 recessive resistance gene are resistant to clover yellow vein virus (ClYVV) isolates No. 30 (Cl-No. 30) and 90-1 (Cl-90-1) but can be infected by a derivative of Cl-90-1 (Cl-90-1 Br2). The main determinant for the breaking of cyv1 resistance by Cl-90-1 Br2 is P3N-PIPO produced from the P3 gene via transcriptional slippage, and the higher level of P3N-PIPO produced by Cl-90-1 Br2 than by Cl-No. 30 contributes to the breaking of resistance. Here we show that P3N-PIPO is also a major virulence determinant in susceptible peas that possess another resistance gene, Cyn1, which does not inhibit systemic infection with ClYVV but causes hypersensitive reaction-like lethal systemic cell death. We previously assumed that the susceptible pea cultivar PI 226564 has a weak allele of Cyn1. Cl-No. 30 did not induce cell death, but Cl-90-1 Br2 killed the plants. Our results suggest that P3N-PIPO is recognized by Cyn1 and induces cell death. Unexpectedly, heterologously strongly expressed P3N-PIPO of Cl-No. 30 appears to be recognized by Cyn1 in PI 226564. The level of P3N-PIPO accumulation from the P3 gene of Cl-No. 30 was significantly lower than that of Cl-90-1 Br2 in a Nicotiana benthamiana transient assay. Therefore, Cyn1-mediated cell death also appears to be determined by the level of P3N-PIPO. The more efficiently a ClYVV isolate broke cyv1 resistance, the more it induced cell death systemically (resulting in a loss of the environment for virus accumulation) in susceptible peas carrying Cyn1, suggesting that antagonistic pleiotropy of P3N-PIPO controls the resistance breaking of ClYVV.
IMPORTANCE
Control of plant viral disease has relied on the use of resistant cultivars; however, emerging mutant viruses have broken many types of resistance. Recently, we revealed that Cl-90-1 Br2 breaks the recessive resistance conferred by cyv1, mainly by accumulating a higher level of P3N-PIPO than that of the nonbreaking isolate Cl-No. 30. Here we show that a susceptible pea line recognized the increased amount of P3N-PIPO produced by Cl-90-1 Br2 and activated the salicylic acid-mediated defense pathway, inducing lethal systemic cell death. We found a gradation of virulence among ClYVV isolates in a cyv1-carrying pea line and two susceptible pea lines. This study suggests a trade-off between breaking of recessive resistance (cyv1) and host viability; the latter is presumably regulated by the dominant Cyn1 gene, which may impose evolutionary constraints upon P3N-PIPO for overcoming resistance. We propose a working model of the host strategy to sustain the durability of resistance and control fast-evolving viruses.

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, Plants recognize viral infection via an immune receptor, i.e., nucleotide-binding site (NB)-leucine-rich repeat (LRR) proteins. Another immune receptor, receptor-like kinase proteins, which share an LRR domain with NB-LRRs, perceive conserved molecules of pathogens called pathogen- or microbe-associated molecular patterns, but NB-LRRs generally perceive particular viral proteins. As viruses can evolve more rapidly than the host immune system, how do plant immune systems, which rely on the perception of proteins, remain effective? Viral adaptive evolution may be controlled by penalties that result from mutations in viral proteins that are perceived by NB-LRRs. Our recent studies in pea (Pisum sativum) suggest a penalty of increased susceptibility to another immune system. When a viral protein mutates to evade one immune system, the virus with the mutated protein becomes more susceptible to another. Such antagonistic pleiotropy of a viral protein by two independent plant immune systems may have precedents. Plants may rely on pairs of immune systems to constrain adaptive evolution by viruses and thereby maintain durable antiviral immunity. [Formula: see text] Copyright © 2016 The Author(s). This is an open access article distributed under the CC BY-NC 4.0 International license .

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, NATURE PUBLISHING GROUP, Feb. 2016, [Peer-reviewed], [Corresponding author]
English, Scientific journal, RNA viruses use various strategies to condense their genetic information into small genomes. Potyviruses not only use the polyprotein strategy, but also embed an open reading frame, pipo, in the P3 cistron in the -1 reading frame. PIPO is expressed as a fusion protein with the N-terminal half of P3 (P3N-PIPO) via transcriptional slippage of viral RNA-dependent RNA polymerase (RdRp). We herein show that clover yellow vein virus (ClYVV) produces a previously unidentified factor, P3N-ALT, in the +1 reading frame via transcriptional slippage at a conserved G(1-2)A(6-7) motif, as is the case for P3N-PIPO. The translation of P3N-ALT terminates soon, and it is considered to be a C-terminal truncated form of P3. In planta experiments indicate that P3N-ALT functions in cell-to-cell movement along with P3N-PIPO. Hence, all three reading frames are used to produce functional proteins. Deep sequencing of ClYVV RNA from infected plants endorses the slippage by viral RdRp. Our findings unveil a virus strategy that optimizes the coding capacity.

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, CURRENT BIOLOGY LTD, Aug. 2014, [Peer-reviewed], [Invited], [Corresponding author]
English, Scientific journal, To elucidate events in the molecular arms race between the host and pathogen in evaluating plant immunity, a zigzag model is useful for uncovering aspects common to different host pathogen interactions. By analogy of the steps in virus host interactions with the steps in the standard zigzag model outlined in recent papers, we may regard RNA silencing as pattern-triggered immunity (PTI) against viruses, RNA silencing suppressors (RSSs) as effectors to overcome host RNA silencing and resistance gene (R-gene)-mediated defense as effector-triggered immunity (ETI) recognizing RSSs as avirulence proteins. However, because the standard zigzag model does not fully apply to some unique aspects in the interactions between a plant host and virus, we here defined a model especially designed for viruses. Although we simplified the phenomena involved in the virus host interactions in the model, certain specific interactive steps can be explained by integrating additional host factors into the model. These host factors are thought to play an important role in maintaining the efficacy of the various steps in the main pathway of defense against viruses in this model for virus plant interactions. For example, we propose candidates that may interact with viral RSSs to induce the resistance response.

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, ACADEMIC PRESS INC ELSEVIER SCIENCE, Jan. 2014, [Peer-reviewed], [Corresponding author]
English, Scientific journal, Mixed infection of pea (Pisum sativum) with Clover yellow vein virus (ClYVV) and White clover mosaic virus (WClMV) led to more severe disease symptoms (a phenomenon called viral synergism). Similar to the mixed ClYVV/WClMV infection, a WClMV-based vector encoding P3N-PIPO of ClYVV exacerbated the disease symptoms. Infection with the WClMV vector encoding ClYVV HC-Pro (a suppressor of RNA silencing involved in potyviral synergisms), also resulted in more severe symptoms, although to a lesser extent than infection with the vector encoding P3N-PIPO. Viral genomic RNA accumulated soon after inoculation (at 2 and 4 days) at higher levels in leaves inoculated with WClMV encoding HC-Pro but at lower levels in leaves inoculated with WClMV encoding P3N-PIPO than in peas infected with WClMV encoding GFP. Our results suggest that ClYVV P3N-PIPO is involved in the synergism between ClYVV and WClMV during pea infection through an unknown mechanism different from suppression of RNA silencing. (C) 2013 Elsevier Inc. All rights reserved.

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, In pea carrying cyv1, a recessive gene for resistance to Clover yellow vein virus (ClYVV), ClYVV isolate Cl-no30 was restricted to the initially infected cells, whereas isolate 90-1 Br2 overcame this resistance. We mapped the region responsible for breaking of cyv1-mediated resistance by examining infection of cyv1 pea with chimeric viruses constructed from parts of Cl-no30 and 90-1 Br2. The breaking of resistance was attributed to the P3 cistron, which is known to produce two proteins: P3, from the main open reading frame (ORF), and P3N-PIPO, which has the N-terminal part of P3 fused to amino acids encoded by a small open reading frame (ORF) called PIPO in the +2 reading frame. We introduced point mutations that were synonymous with respect to the P3 protein but nonsynonymous with respect to the P3N-PIPO protein, and vice versa, into the chimeric viruses. Infection of plants with these mutant viruses revealed that both P3 and P3N-PIPO were involved in overcoming cyv1-mediated resistance. Moreover, P3N-PIPO quantitatively affected the virulence of Cl-no30 in cyv1 pea. Additional expression in trans of the P3NPIPO derived from Cl-no30, using White clover mosaic virus as a vector, enabled Cl-no30 to move to systemic leaves in cyv1 pea. Susceptible pea plants infected with chimeric ClYVV possessing the P3 cistron of 90-1 Br2, and which were therefore virulent toward cyv1 pea, accumulated more P3N-PIPO than did those infected with Cl-no30, suggesting that the higher level of P3NPIPO in infected cells contributed to the breaking of resistance by 90-1 Br2. This is the first report showing that P3N-PIPO is a virulence determinant in plants resistant to a potyvirus. © 2013, American Society for Microbiology.

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, SPRINGER TOKYO, Jul. 2012, [Peer-reviewed]
English, Scientific journal, Two recessive resistance genes against Clover yellow vein virus (ClYVV), cyv1 and cyv2, have been previously reported. We recently screened resistant peas from a separate set of pea lines and classified them into two groups according to their distinct modes of resistance. We later revealed that one group carries cyv2, encoding eukaryotic translation initiation factor 4E (eIF4E), in linkage group (LG) VI. We explored the possibility that the resistance gene, tentatively designated non-cyv2, that confers resistance to the other group, was actually cyv1. We found that PI 236493, which carries cyv1, had restricted cell-to-cell movement of ClYVV similar to that in non-cyv2 peas including PI 429853. PI 429853 was crossed with susceptible line PI 250438. Mapping of F-2 progeny revealed that non-cyv2 was 4 cM from the simple sequence repeat marker AB40, whose loci are close to cyv1, mo, and sbm-2 mapped in LG II, which mediates resistance to other potyviruses. Moreover, PI 429853 crossed with PI 236493 produced F-1 progeny resistant to ClYVV, raising the possibility that non-cyv2 is allelic to cyv1. Because mo was previously mapped with eIF(iso)4E in LG II, we examined the possibility that non-cyv2, cyv1, and mo encoded eIF(iso)4E. However, there was no difference in the nucleotide sequence of the eIF(iso)4E-coding region between susceptible and resistant pea lines. The eIF(iso)4E gene was equivalently expressed in both PI 429853 and PI 250438 before and after ClYVV infection. Our results suggest that these resistance genes are unlikely to encode eIF(iso)4E on LG II.

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, NATL ACAD SCIENCES, Jun. 2012, [Peer-reviewed], [Lead author, Corresponding author]
English, Scientific journal, RNA silencing (RNAi) induced by virus-derived double-stranded RNA (dsRNA), which is in a sense regarded as a pathogen-associated molecular pattern (PAMP) of viruses, is a general plant defense mechanism. To counteract this defense, plant viruses express RNA silencing suppressors (RSSs), many of which bind to dsRNA and attenuate RNAi. We showed that the tobacco calmodulin-like protein, rgs-CaM, counterattacked viral RSSs by binding to their dsRNA-binding domains and sequestering them from inhibiting RNAi. Autophagy-like protein degradation seemed to operate to degrade RSSs with the sacrifice of rgs-CaM. These RSSs could thus be regarded as secondary viral PAMPs. This study uncovered a unique defense system in which an rgs-CaM-mediated countermeasure against viral RSSs enhanced host antiviral RNAi in tobacco.

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, SPRINGER WIEN, Jun. 2012, [Peer-reviewed]
English, Scientific journal, Many plant viruses encode suppressors of RNA silencing, including the helper component-proteinase (HC-Pro) of potyviruses. Our previous studies showed that a D-to-Y mutation at amino acid position 193 in HC-Pro (HC-Pro-D193Y) drastically attenuated the virulence of clover yellow vein virus (ClYVV) in legume plants. Furthermore, RNA-silencing suppression (RSS) activity of HC-Pro-D193Y was significantly reduced in Nicotiana benthamiana. Here, we examine the effect of expression of heterologous suppressors of RNA silencing, i.e., tomato bushy stunt virus p19, cucumber mosaic virus 2b, and their mutants, on the virulence of the ClYVV point mutant with D193Y (Cl-D193Y) in pea. P19 and 2b fully and partially complemented Cl-D193Y multiplication and virulence, including lethal systemic HR in pea, respectively, but the P19 and 2b mutants with defects in their RSS activity did not. Our findings strongly suggest that the D193Y mutation exclusively affects RSS activity of HC-Pro and that RSS activity is necessary for ClYVV multiplication and virulence in pea.

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, SPRINGER JAPAN KK, Mar. 2012, [Peer-reviewed], [Corresponding author]
English, Scientific journal, Double-stranded RNAs formed in secondary structures and replicative intermediates of viral genomes are thought to strongly elicit RNA silencing. This phenomenon is known as virus-induced gene silencing (VIGS). VIGS is a powerful tool for modifying gene expression in host plants. We constructed a virus vector based on White clover mosaic virus (WC1MV) and demonstrated VIGS of phytoene desaturase (PDS) in pea. Photobleaching of tissues, caused by VIGS of PDS, was observed in restricted areas of upper leaves and stems. We confirmed that the PDS mRNA and subgenomic RNAs of WC1MV were reduced in the photobleached tissues.

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, Plants and animals can recognize the invasion of pathogens through their perception of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRR s). Plant PRR s identified have been exclusively receptor-like kinases/ proteins (RLK/Ps), and no RLK/P that can detect viruses has been identified to date. RNA silencing (RNAinterference, RNAi) is regarded as an antiviral basal immunity because the majority of plant viruses has RNA as their genomes and encode RNA silencing suppressor (RSS) proteins to counterattack antiviral RNAi. Many RSSs were reported to bind to double-stranded RNA s (dsRNA s), which are regarded as viral PAMPs. We have recently identified a tobacco calmodulin (CaM)-like protein, rgs-CaM, as a PRR that binds to diverse viral RSSs through its affinity for the dsRNA-binding domains. Because rgs-CaM seems to target RSSs for autophagic degradation with self-sacrifice, the expression level of rgs-CaM is important for antiviral activity. Here, we found that the rgs-CaM expression was induced immediately (within 1 h) after wounding at a wound site on tobacco leaves. Since the invasion of plant viruses is usually associated with wounding, and several hours are required for viruses to replicate to a detectable level in invaded cells, the wound-induced expression of rgs-CaM seems to be linked to its antiviral function, which should be ready before the virus establishes infection. CaMs and CaM-like proteins usually transduce calcium signals through their binding to endogenous targets. Therefore, rgs- CaM is a unique CaM-like protein in terms of binding to exogenous targets and functioning as an antiviral PRR. © 2012 Landes Bioscience.

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, JAPAN INT RESEARCH CENTER AGRICULTURAL SCIENCES, Oct. 2011, [Peer-reviewed], [Lead author, Corresponding author]
English, Scientific journal, This paper presents a useful process for the detection of Apple chlorotic leaf spot virus (ACLSV) in apple trees. The 3'-terminal 1.8-kb genomic cDNAs of 15 ACLSV isolates, of which 11 induce a decline (of varying severity) in the condition of inoculated Malus prunifolia var. ringo rootstock and four do not, were amplified by reverse transcription (RT) coupled with polymerase chain reaction (PCR). Single-strand conformation polymorphism analysis of the cDNAs revealed that most of the isolates inducing the decline were composed of at least two to four sequence variants per apple tree, whereas isolates inducing no decline were occupied with a major type sequence. Direct sequencing of the cDNAs showed heterogeneity in the nucleotide sequence of the analyzed region; most of the variation in these positions appeared to specify the same amino acids upon translation of the 50K protein and capsid protein (CP). To detect all isolates, degenerate primers were designed with consideration of the sequence varieties of the viral genomes. RT-nested PCR and its improved methods have a 10(4)-fold higher sensitivity than conventional RT-PCR, and consistently amplify ACLSV genomic cDNA in RNA extracts from apple leaf and bark during growing and dormant seasons, respectively.

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, ROCKEFELLER UNIV PRESS, Jul. 2011, [Peer-reviewed]
English, Scientific journal, M ammals lacking BLOC-3 have impaired formation of melanosomes, a type of lysosome-related organelle (LRO), and, in earlier work, we found that a subunit of the BLOC-3 complex inhibits loading of Argonaute (Ago) proteins with small ribonucleic acids (RNAs) in Drosophila melanogaster cells. Small RNAs such as small interfering RNAs (siRNAs) direct Ago proteins to repress the stability of messenger RNA transcripts. In this paper, we show that BLOC-3 is required for biogenesis of Drosophila LROs called pigment granules. Other complexes that sort cargo to pigment LROs also negatively regulate siRNA activity. However, regulation is not obligately linked to biogenesis of LROs but instead to specific cargo-sorting processes. Negative regulation is also not linked to sorting into all LROs but only a specific class of pigment LRO. Thus, regulation of siRNA activity is tied to sorting of specific types of cargo to particular LROs.

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, BIOMED CENTRAL LTD, Jul. 2011, [Peer-reviewed], [Lead author, Corresponding author]
English, Scientific journal, Clover yellow vein virus (ClYVV) causes lethal systemic necrosis in legumes, including broad bean (Vicia faba) and pea (Pisum sativum). To identify host genes involved in necrotic symptom expression after ClYVV infection, we screened cDNA fragments in which expression was changed in advance of necrotic symptom expression in broad bean (V. faba cv. Wase) using the differential display technique and secondarily with Northern blot analysis. Expression changes were confirmed in 20 genes, and the six that exhibited the most change were analyzed further. These six genes included a gene that encodes a putative nitrate-induced NOI protein (VfNOI), and another was homologous to an Arabidopsis gene that encodes a glycine-and proline-rich protein GPRP (VfGPRP). We recently reported that necrotic symptom development in ClYVV-infected pea is associated with expression of salicylic acid (SA)-dependent pathogenesis-related (PR) proteins and requires SA-dependent host responses. Interestingly, VfNOI and VfGPRP expression was correlated with that of the putative SA-dependent PR proteins in ClYVV-infected broad bean. However, broad bean infected with a recombinant ClYVV expressing the VfGPRP protein showed weaker symptoms and less viral multiplication than that infected with ClYVV expressing the GFP protein. These results imply that VfGPRP plays a role in defense against ClYVV rather than in necrotic symptom expression.

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, AMER PHYTOPATHOLOGICAL SOC, Nov. 2010, [Peer-reviewed], [Lead author, Corresponding author]
English, Scientific journal, Two recessive genes (cyv1 and cyv2) are known to confer resistance against Clover yellow vein virus (ClYVV) in pea. cyv2 has recently been revealed to encode eukaryotic translation initiation factor 4E (eIF4E) and is the same allele as sbm1 and whiz against other potyviruses. Although mechanical inoculation with crude sap is rarely able to cause infection of a cyv2 pea, biolistic inoculation of the infectious ClYVV cDNA clone does. At the infection foci, the breaking virus frequently emerges, resulting in systemic infection. Here, a derived cleaved-amplified polymorphic sequence analysis showed that the breakings were associated with a single nonsynonymous mutation on the ClYVV genome, corresponding to an amino-acid substitution at position 24 (isoleucine to valine) on the PI cistron. ClYVV with the point mutation was able to break the resistance. This is a first report demonstrating that PI is involved in eIF4E-mediated recessive resistance.

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, NATURE PUBLISHING GROUP, Sep. 2009, [Peer-reviewed]
English, Scientific journal, Small RNAs direct RNA-induced silencing complexes (RISCs) to regulate stability and translation of mRNAs(1,2). RISCs associated with target mRNAs often accumulate in discrete cytoplasmic foci known as GW-bodies(3). However, RISC proteins can associate with membrane compartments such as the Golgi and endoplasmic reticuium(4). Here, we show that GW-bodies are associated with late endosomes (multivesicular bodies, MVBs). Blocking the maturation of MVBs into lysosomes by loss of the tethering factor HPS4 (ref. 5) enhances short interfering RNA (siRNA)- and micro RNA (miRNA)-mediated silencing in Drosophila melanogaster and humans. It also triggers over-accumulation of GW-bodies. Blocking MVB formation by ESCRT (endosomal sorting complex required for transport)6 depletion results in impaired miRNA silencing and loss of GW-bodies. These results indicate that active RISCs are physically and functionally coupled to MVBs. We further show that MVBs promote the competence of RISCs in loading small RNAs. We suggest that the recycling of RISCs is promoted by MVBs, resulting in RISCs more effectively engaging with small RNA effectors and possibly target RNAs. It may provide a means to enhance the dynamics of RNA silencing in the cytoplasm.

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, SPRINGER TOKYO, Jun. 2009, [Peer-reviewed]
English, Scientific journal, The same mutant allele of eukaryotic initiation factor 4E (eIF4E) that confers resistance to Pea seed-borne mosaic virus (sbm-1) and the white lupine strain of Bean yellow mosaic virus (wlv) also confers resistance to Clover yellow vein virus (ClYVV) in pea. The eIF4E genes from several pea lines were isolated and sequenced. Analysis of the eIF4E amino acid sequences from several resistant lines revealed that some lines, including PI 378159, have the same sequence as reported for sbm-1 and wlv. When eIF4E from a susceptible pea line was expressed from a ClYVV vector after mechanical inoculation of resistant PI 378159, the virus caused systemic infection, similar to its effects in susceptible line PI 250438. The resistance to ClYVV in line PI 378159 was characterized through a cross with PI 193835, which reportedly carries cyv-2. Mechanical inoculation of the F1 progeny with ClYVV resulted in no infection, indicating that the resistance gene in PI 378159 is identical to cyv-2 in PI 193835. Furthermore, particle bombardment of pea line PI 193835 with infectious cDNA of ClYVV (pClYVV/C3-S65T) resulted in the same resistance mode as that described for PI 378159. These results demonstrate that the resistance to ClYVV conferred by cyv-2 is mediated by eIF4E and that cyv-2 is identical to sbm-1 and wlv.

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, AMER PHYTOPATHOLOGICAL SOC, Feb. 2009, [Peer-reviewed]
English, Scientific journal, The wild-type strain (Cl-WT) of Clover yellow vein virus (ClYVV) systemically induces cell death in pea cv. Plant introduction (PI) 118501 but not in PI 226564. A single incompletely dominant gene, Cyn1, controls systemic cell death in PI 118501. Here, we show that activation of the salicylic acid (SA) signaling pathway enhances ClYVV virulence in susceptible pea cultivars. The kinetics of virus accumulation was not significantly different between PI 118501 (Cyn1) and PI 226564 (cyn1); however, the SA-responsive chitinase gene (SA-CHI) and the hypersensitive response (HR)-related gene homologous to tobacco HSR203J were induced only in PI 118501 (Cyn1). Two mutant viruses with mutations in P1/HCPro, which is an RNA-silencing suppressor, reduced the ability to induce cell death and SA-CHI expression. The application of SA and of its analog benzo (1,2,3) thiadiazole-7-carbothioic acid S-methyl ester (BTH) partially complemented the reduced virulence of mutant viruses. These results suggest that high activation of the SA signaling pathway is required for ClYVV virulence. Interestingly, BTH could enhance Cl-WT symptoms in PI 226564 (cyn1). However, it could not enhance symptoms induced by White clover mosaic virus and Bean yellow mosaic virus. Our report suggests that the SA signaling pathway has opposing functions in compatible interactions, depending on the virus-host combination.

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, SPRINGER WIEN, Jan. 2008, [Peer-reviewed]
English, Scientific journal, Helper component protease (HC-Pro) is a potyvirus-encoded multifunctional protein and a major determinant of symptom expression in a susceptible plant. Here, we show the involvement of clover yellow vein virus (ClYVV) HC-Pro in necrotic symptom expression in broad bean (Vicia faba cv. Wase). In this host, lethal necrosis was induced by ClYVV no. 30, from which a spontaneous, mosaic-inducing mutant (MM) was obtained. Mapping with chimeric viruses between ClYVV no. 30 and MM attributed the symptom attenuation to two mutations at the HC-Pro positions 27 (threonine to isoleucine) and 193 (aspartic acid to tyrosine). Although neither mutant with the single amino acid substitution at position 27 or 193 (ClYVV/T27I or D193Y) induced the lethal necrosis, ClYVV/T27I still retained the ability to induce necrotic symptoms, but ClYVV/D193Y scarcely did so. The virus accumulation of ClYVV/D193Y was also lower than that of ClYVV no. 30. The mutations, T27I and D193Y, are located in a putative zinc finger domain and in one (N-terminal) of the two RNA binding domains, respectively, of HC-Pro. RNA-silencing suppression (RSS) activity of P1/HC-Pro in Nicotiana benthamiana was weakened by both mutations. Our results suggest a correlation between viral virulence and RSS function and the importance of the two domains in HC-Pro.

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, COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT, 2006, [Peer-reviewed]
English, International conference proceedings, Injection or expression of double-stranded RNA (dsRNA) in Drosophila serves as a trigger that causes cells to specifically cleave homologous mRNA transcripts. Our approach is to identify essential components of the RNA interference (RNAi) mechanism by isolating and characterizing mutations that cause the RNAi response to be abnormal. These studies have thus far led to the identification of seven genetic loci that encode proteins acting at various steps in the RNAi process. We have molecularly identified several of these proteins. Two are members of the Dicer family. Dicer-1 and Dicer-2 are required for short interfering RNA (siRNA)-directed mRNA cleavage by facilitating distinct steps in the assemble of the RNA-induced silencing complex (RISC). AGO2 is a RISC component that both carries out transcript cleavage and facilitates RISC maturation. Other factors appear to function as regulators of RISC assembly rather than as core factors for RNAi.

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, NATL ACAD SCIENCES, Aug. 2005, [Peer-reviewed], [Lead author]
English, Scientific journal, MicroRNAs (miRNAs) are a class of small RNAs that silence gene expression. In animal cells, miRNAs bind to the 3 ' untranslated regions of specific mRNAs and inhibit their translation. Although some targets of a handful of miRNAs are known, the number and identities of mRNA targets in the genome are uncertain, as are the developmental functions of miRNA regulation. To identify the global range of miRNA-regulated genes during oocyte maturation of Drosophila, we compared the proteome from wild-type oocytes with the proteome from oocytes lacking the dicer-1 gene, which is essential for biogenesis of miRNAs. Most identified proteins appeared to be subject to translation inhibition. Their transcripts contained putative binding sites in the 3 ' untranslated region for a subset of miRNAs, based on computer modeling. The fraction of genes subject to direct and indirect repression by miRNAs during oocyte maturation appears to be small (4%), and the genes tend to share a common functional relationship in protein biogenesis and turnover. The preponderance of genes that control global protein abundance suggests this process is under tight control by miRNAs at the onset of fertilization.

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, NATURE PUBLISHING GROUP, Jun. 2005, [Peer-reviewed]
English, Scientific journal, One of the key characteristics of stem cells is their capacity to divide for long periods of time in an environment where most of the cells are quiescent. Therefore, a critical question in stem cell biology is how stem cells escape cell division stop signals. Here, we report the necessity of the microRNA (miRNA) pathway(1-4) for proper control of germline stem cell (GSC) division in Drosophila melanogaster. Analysis of GSCs mutant for dicer-1 (dcr-1), the double-stranded RNaseIII essential for miRNA biogenesis, revealed a marked reduction in the rate of germline cyst production. These dcr-1 mutant GSCs exhibit normal identity but are defective in cell cycle control. On the basis of cell cycle markers and genetic interactions, we conclude that dcr-1 mutant GSCs are delayed in the G1 to S transition, which is dependent on the cyclin-dependent kinase inhibitor Dacapo, suggesting that miRNAs are required for stem cells to bypass the normal G1/S checkpoint. Hence, the miRNA pathway might be part of a mechanism that makes stem cells insensitive to environmental signals that normally stop the cell cycle at the G1/S transition.

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, ELSEVIER SCIENCE BV, Feb. 2005, [Peer-reviewed]
English, Scientific journal, Arabidopsis thaliana plants with mutations in the genes encoding eukaryotic initiation factor (eIF4E) or isoform of eIF4E (eIF(iso)4E) were tested for susceptibility to Clover yellow vein virus (ClYVV), a member of the genus Potyvirus. ClYVV accumulated in both inoculated and upper uninoculated leaves of mutant plants lacking eIF(iso)4E, but not in mutant plants lacking eIF4E. In contrast, Turnip mosaic virus (TuMV), another member of the genus Potyvirus, multiplied in mutant plants lacking eIF4E but not in mutant plants lacking eIF(iso)4E. These results suggest the selective involvement of members of the eIF4E family in infection by potyviruses. (C) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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, CELL PRESS, Apr. 2004, [Peer-reviewed]
English, Scientific journal, The RNase III enzyme Dicer processes RNA into siRNAs and miRNAs, which direct a RNA-induced silencing complex (RISC) to cleave mRNA or block its translation (RNAi). We have characterized mutations in the Drosophila dicer-1 and dicer-2 genes. Mutation in dicer-1 blocks processing of miRNA precursors, whereas dicer-2 mutants are defective for processing siRNA precursors. It has been recently found that Drosophila Dicer-1 and Dicer-2 are also components of siRNA-dependent RISC (siRISC). We find that Dicer-1 and Dicer-2 are required for siRNA-directed mRNA cleavage, though the RNase III activity of Dicer-2 is not required. Dicer-1 and Dicer-2 facilitate distinct steps in the assembly of siRISC. However, Dicer-1 but not Dicer-2 is essential for miRISC-directed translation repression. Thus, siRISCs and miRISCs are different with respect to Dicers in Drosophila.

Expanding roles for miRNAs and siRNAs in cell regulation
K Nakahara, RW Carthew
CURRENT OPINION IN CELL BIOLOGY, 16, 2, 127, 133, CURRENT BIOLOGY LTD, Apr. 2004, [Peer-reviewed], [Lead author]
English, The role of small RNAs as key regulators of mRNA turnover and translation has been well established. Recent advances indicate that the small RNAs termed microRNAs play important roles in cell proliferation, apoptosis and differentiation. Moreover, the microRNA mechanism is an efficient means to regulate production of a diverse range of proteins. As new microRNAs and their mRNA targets rapidly emerge, it is becoming apparent that RNA-based regulation of mRNAs may rival ubiquitination as a mechanism to control protein levels.

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, SOC GENERAL MICROBIOLOGY, Oct. 2003, [Peer-reviewed]
English, Scientific journal, Interactions between the major proteins of Clover yellow vein virus (ClYVV) were investigated using a GAL4 transcription activator-based yeast two-hybrid system (YTHS). Self-interactions manifested by VPg and HCPro and an interaction between Nib and NlaPro were observed in ClYVV. In addition, a strong HCPro-VPg interaction was detected by both YTHS and by in vitro far-Western blot analysis in ClYVV. A potyvirus HCPro-VPg interaction has not been reported previously. Using YTHS, domains in ClYVV for the VPg self-interaction and the HCPro-VPg interaction were mapped. The VPg C-terminal region (38 amino acids) was important for the VPg-VPg interaction and the central 19 amino acids were needed for the HCPro-VPg interaction.

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, AMER PHYTOPATHOLOGICAL SOC, May 2002, [Peer-reviewed]
English, Scientific journal, Sequential polyacrylamide gel electrophoresis analyses showed many viroid-like RNAs in samples collected from citrus trees in Japan. Reverse transcription polymerase chain reaction and sequencing analyses of the amplified fragments verified that they were derived from variants of six citrus viroids, Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd) including CVd-I-LSS (a distinct variant of CBLVd), Hop stunt viroid, Citrus viroid III, Citrus viroid IV, and Citrus viroid OS. The samples induced symptoms with variable severity in Arizona 861-S1 'Etrog' citrons (Citrus medica L.) likely due to the varying accumulation patterns produced by the different viroids. Some of the symptoms caused by the samples harboring the citrus viroids other than CEVd were as severe as those caused by CEVd. Some source citrus trees showing the severe bark scaling characteristic of exocortis disease in trifoliate orange (Poncirus trifoliata (L.) Raf.) rootstocks contained only citrus viroids other than CEVd in complex. This indicates that certain exocortis-like diseases in Japan were caused by some combination of citrus viroids not including CEVd.

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, ELSEVIER SCIENCE BV, Jan. 1999, [Peer-reviewed], [Lead author]
English, Scientific journal, A simple, rapid method of nucleic acid extraction on a microcentrifuge tube scale for detecting viroids is presented. Five distinct citrus viroids (CVds), chrysanthemum stunt viroid (CSVd), hop stunt viroid (HSVd), hop latent viroid (HLVd) and potato spindle tuber viroid (PSTVd) were detected in their natural host plants by hybridization using cRNA probes and reverse transcription-polymerase chain reaction (RT-PCR). Nucleic acids (NA) were liberated from tissues by incubation in a buffer containing potassium ethyl xanthogenate (PEX) without tissue homogenization, and then precipitated with ethanol (NA-PEX). All the viroids except CVd-IV could be detected clearly in NA-PEX by hybridization. HSVd, HLVd and PSTVd could also be detected in NA-PEX by RT-PCR. Although CVds and CSVd could not be detected in NA-PEX by RT-PCR, they were detected after further purification: differential precipitation with 2-butoxyethanol and HCl treatment followed by ethanol-precipitation. In addition, PCR in the presence of tetramethylammonium chloride specifically amplified the cDNA of all five distinct CVds under the same temperature and cycle conditions. Since all the viroids could be detected in NA liberated by PEX, the amount of NA extracted by the method described here is sufficient for detecting viroids, enabling the processing of a large number of samples. (C) 1999 Elsevier Science B.V. All rights reserved.

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, OXFORD UNIV PRESS, Apr. 1998, [Peer-reviewed], [Lead author]
English, Scientific journal, Nucleic acid sequence-based amplification (NASBA) according to the standard protocol failed to amplify cRNA of viroids, probably because of their CC-rich and intramolecular base-paired structure. However, NASBA in the presence of inosine 5'-triphosphate successfully amplified the cRNAs to viroids in total nucleic acid extracts from citrus plants. As sequence specificity of the cRNA to viroids was confirmed by northern analysis, the amplification and fidelity of cRNAs are sufficient for the sensitive and specific detection of viroids.

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, ELSEVIER SCIENCE BV, Apr. 1998, [Peer-reviewed], [Lead author]
English, Scientific journal, Five kinds of synthetic oligonucleotide probes labeled with biotin (BIO) were designed for the detection of potato spindle tuber viroid (PSTVd), and their sensitivities were compared with that of a digoxigenin (DIG)- or BIO-labeled cDNA probe. Although each oligonucleotide probe alone was less sensitive than the DIG-cDNA probe, sensitivity was increased by using a mixture of two or all of the five oligonucleotide probes. The sensitivity of a PSmix1-5 probe, which was a mixture of five oligonucleotides, was the same as that of a DIG-labeled cDNA probe, which can detect 7.8 pg of purified PSTVd and PSTVd in nucleic acids, equivalent to extracts from 20 mu g of infected potato leaf and 310 mu g of infected potato tuber. Using the PSmix1-5 probe, PSTVd in all leaves and tubers of seven potato cultivars could be detected without any background. Moreover, with the PSmix1-5 probe, the hybridization time could be shortened to 2 h without any decrease in sensitivity, whereas the sensitivity of the cDNA probes clearly decreased when the hybridization time was shortened. Hybridization using a mixture of several oligonucleotide probes may be applicable to the gene diagnosis of other viroids and viruses. (C) 1998 Elsevier Science B.V. All rights reserved.

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, SPRINGER-VERLAG WIEN, 1998, [Peer-reviewed]
English, Scientific journal, Nucleotide sequences of group I citrus viroids Ia (CVd-Ia) and citrus bent leaf viroid (CBLVd, formerly designated CVd-Ib) isolated from citrus plants in Japan, the Philippines and China have been determined. Citrus samples in Japan and the Philippines contained CVd-Ia, which consists of 328 nucleotides(nt). Although 10 nt longer than the type CBLVd-225A in Israel they share 94% identity in overall nucleotide sequence. The Philippines sample also contained a 329-nt long CVd-Ia sequence variant, in which one base insertion and three substitutions were observed. A citrus in China contained CBLVd, which consists of 318 nt and shares 98% identity to CBLVd-225A. CVd-Ia was clearly separated from CBLVd by two 5-nt insertions located in upper (5'-ACCUG-3') and the lower (5'-CUUCU-3') strand of the right terminal region (which is also designated T2 domain) in rodlike secondary structure. Since both of the additional 5-nt sequences are similar to the adjacent sequences (5'-AGUUG-3' and 5'-CUUCU-3'), we hypothesize that CVd-Ia is a derivative of CBLVd caused by partial sequence duplications and substitutions taking place in the right terminal region.

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.

ダイズとツルマメ間の組み換え自殖系統を用いたダイズのクローバ葉脈黄化ウイルスに対する非宿主抵抗性遺伝子のファインマッピング
阿部純也, 山田哲也, 阿部純, 中原健二, 日本植物病理学会大会プログラム・講演要旨予稿集, 2016, 134, 03 Mar. 2016
Japanese

クローバ葉脈黄化ウイルスがコードするP3N‐PIPOおよび新規タンパク質の発現機構
薦田(萩原)優香, CHOI S. H, 佐藤昌直, 厚見剛, 阿部純也, 中原健二, 上田一郎, 内藤哲, 日本植物病理学会大会プログラム・講演要旨予稿集, 2015, 149, 10 Mar. 2015
Japanese

ダイズとツルマメ間の組み換え自殖系統を用いたクローバ葉脈黄化ウイルスの全身感染抵抗性に関与するQTL解析
阿部純也, 山田哲也, 阿部純, 中原健二, 日本植物病理学会大会プログラム・講演要旨予稿集, 2015, 150, 10 Mar. 2015
Japanese

ダイズおよびツルマメに対するクローバ葉脈黄化ウイルスの病原性の比較
阿部純也, 山田哲也, 阿部純, HAJIMORAD M.Reza, 中原健二, 日本植物病理学会大会プログラム・講演要旨予稿集, 2014, 151, 10 May 2014
Japanese

Silencing by small RNAs is linked to endosomal trafficking (vol 11, pg 1150, 2009)
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, 12, 1495, 1495, Dec. 2009
NATURE PUBLISHING GROUP, English, Others

(261) Construction of Infectious cDNA Clone to CS Strain of Bean yellow mosaic virus(Abstracts of the Papers Presented at the Annual Meeting of the Society)
Nishino K., Nakahara K., Uyeda I., Annals of the Phytopathological Society of Japan, 74, 3, 236, 236, 25 Aug. 2008
The Phytopathological Society of Japan (PSJ), Japanese

(234) Functional Analysis of the Novel XYPPX Repeat Gene Associated with Virus Infection(Abstracts of the Papers Presented at the Annual Meeting of the Society)
Atsumi G., Nakahara K. S., Masuta C., Uyeda I., Annals of the Phytopathological Society of Japan, 74, 3, 229, 229, 25 Aug. 2008
The Phytopathological Society of Japan (PSJ), Japanese

(287) Identification of a Virus Factor Involved in the Recessive Resistance to Clover yellow vein virus in Pea(Abstracts of the Papers Presented at the Annual Meeting of the Society)
Shimada R., Andrade Marcelo, Nakahara K., Uyeda I., Annals of the Phytopathological Society of Japan, 74, 3, 243, 243, 25 Aug. 2008
The Phytopathological Society of Japan (PSJ), Japanese

(288) White clover mosaic virus Induced RNA Silencing(Abstracts of the Papers Presented at the Annual Meeting of the Society)
Nakahara K., Ido Y., Atsumi G., Uyeda I., Annals of the Phytopathological Society of Japan, 74, 3, 243, 244, 25 Aug. 2008
The Phytopathological Society of Japan (PSJ), Japanese

(17)Construction of a cDNA Infectious Clone of White clover mosaic virus-RC and Analyses of its Subgenomic RNA Developing a Viral Vector(Abstracts Presented at the Meeting of the Hokkaido Division,Abstracts of Papers Presented at the Division Meetings of the Phytopathological Society of Japan, 2007)
Ido Y., Atsumi G., Nakahara K., Uyeda I., Annals of the Phytopathological Society of Japan, 74, 1, 83, 83, 20 Feb. 2008
The Phytopathological Society of Japan (PSJ), Japanese

(243) Relationship between Clover yellow vein virus-induced Systemic Cell Death and RNA Silencing Suppressor Activity in Pea(Abstracts of the Papers Presented at the Annual Meeting of the Society)
Atsumi G., Wada T., Yambao M.L.M., Nakahara K.S., Masuta C., Uyeda I., Annals of the Phytopathological Society of Japan, 73, 3, 240, 240, 25 Aug. 2007
The Phytopathological Society of Japan (PSJ), Japanese

P1 protein of Clover yellow vein virus affects RNA silencing suppressor activity of helper component protease (HC-Pro)(Abstracts Presented at the Meeting of the Hokkaido Division,Abstracts of Papers Presented at the Division Meetings of the Phytopathological Society of Japan, 2006)
Sekiguchi T., Yambao M. L. M., Atsumi G., Nakahara K., Uyeda I., Annals of the Phytopathological Society of Japan, 73, 1, 77, 77, 25 Feb. 2007
The Phytopathological Society of Japan (PSJ), Japanese

Complete Nucleotide Sequence of Bean yellow mosaic virus CS(Abstracts Presented at the Meeting of the Hokkaido Division,Abstracts of Papers Presented at the Division Meetings of the Phytopathological Society of Japan, 2006)
Nishino K., Nakahara K., Uyeda I., Annals of the Phytopathological Society of Japan, 73, 1, 75, 76, 25 Feb. 2007
The Phytopathological Society of Japan (PSJ), Japanese

(343) Characterization of Clover yellow vein virus (ClYVV) Mutant That Breaks the Resistance in Pea Line W6-15452(Abstract of the Paper Presented at the 2006 Annual Meeting in Sapporo)
Andrade M., Abe Y., Nakahara K., Uyeda I., Annals of the Phytopathological Society of Japan, 72, 4, 293, 293, 25 Nov. 2006
The Phytopathological Society of Japan (PSJ), English

(358) Mutation in P1/HCPro Gene of Clover yellow vein virus Reduces Ability to Activate SA-mediated Defence Responce(Abstract of the Paper Presented at the 2006 Annual Meeting in Sapporo)
Atsumi G., Nakahara K., Uyeda I., Annals of the Phytopathological Society of Japan, 72, 4, 297, 297, 25 Nov. 2006
The Phytopathological Society of Japan (PSJ), Japanese

(312) Suppression of Post-transcriptional Gene Silencing by Clover yellow vein virus P1/HC-Pro and Tobacco rgsCaM in Drosophila melanogaster S2 Cells(Abstract of the Paper Presented at the 2006 Annual Meeting in Sapporo)
Nakahara K., Ueda I., Annals of the Phytopathological Society of Japan, 72, 4, 285, 285, 25 Nov. 2006
The Phytopathological Society of Japan (PSJ), Japanese

Salicylic Acid is Involved in Clover yellow vein virus (C1YVV) Virulence in Pea (Abstracts Presented at the Meeting of the Hokkaido Division,Abstracts of Papers Presented at the Division Meetings of the Phytopathological Society of Japan 2005)
Atsumi G., Nakahara K., Uyeda I., Annals of the Phytopathological Society of Japan, 72, 1, 84, 85, 25 Feb. 2006
The Phytopathological Society of Japan (PSJ), Japanese

(320) Involvement of the Eukaryotic Initiation Factor 4E in the Resistance in Pea to Clover Yellow Vein Virus(Abstracts of the Papers Presented at the 2005 Annual Meeting in Shizuoka)
Abe Y., Nakahara K., Andrade M., Uyeda I., Annals of the Phytopathological Society of Japan, 71, 3, 271, 271, 25 Aug. 2005
The Phytopathological Society of Japan (PSJ), Japanese

(8) Analysis of the Mutation for the Host Adaptation of Citrus exocortis viroid(Abstracts Presented at the Meeting of the Hokkaido Division, Sapporo, October 22, 2004, Abstracts of Papers Presented at the Division Meetings of the Phytopathological Society of Japan, 2003)
Kawamura T., Yonemoto T., Nakahara K., Hataya T., Annals of the Phytopathological Society of Japan, 71, 1, 83, 83, 25 Feb. 2005
The Phytopathological Society of Japan (PSJ), Japanese

Analysis of Nucleotide Sequences Cloned from CMV-infected Tomato by NASBA-based RNA Subtraction
Nakahara K., Yoshida K., Kitagawa Y., Annals of the Phytopathological Society of Japan, 66, 2, 151, 151, 25 Aug. 2000
The Phytopathological Society of Japan (PSJ), Japanese

Apple Fruit Crinkle Viroid (AFCVd) Causes a Graft-transmissible Blister Bark on Apple cv. Nero 26
Ito T., Suzaki K., Nakahara K., Machita I., Matsunaka K., Yoshida K., Annals of the Phytopathological Society of Japan, 65, 3, 394, 394, 25 Jun. 1999
The Phytopathological Society of Japan (PSJ), Japanese

Genomic Diversity of ACLSV Isolates Contained in Apple Trees : (2)
Nakahara K., Ito T., Yoshida K., Suzaki K., Annals of the Phytopathological Society of Japan, 65, 3, 380, 381, 25 Jun. 1999
The Phytopathological Society of Japan (PSJ), Japanese

(1) Genomic Diversity of ACLSV Isolates Contained in Apple Trees (Abstracts Presented at the Meeting of the Tohoku Division, 1998)
Nakahara K., Ito T., Yoshida K., Suzaki K., Annals of the Phytopathological Society of Japan, 64, 6, 605, 605, 25 Dec. 1998
The Phytopathological Society of Japan (PSJ), Japanese

(2) Detection of Citrus Exocortis Viroid Using Nucleic Acid Sequence-based Amplification(Abstracts of the Papers Presented at the Annual Meeting of the Society)
NAKAHARA K., HATAYA T., UYEDA I., Annals of the Phytopathological Society of Japan, 63, 3, 193, 193, 25 Jun. 1997
The Phytopathological Society of Japan (PSJ), Japanese

Analysis of Rice Dwarf Phytoreovirus Genome Segments Involved in Insect Transmission
TANJI Y., ANDO Y., KIMURA I., HATAYA T., NAKAHARA K., UYEDA I., Annals of the Phytopathological Society of Japan, 62, 6, 649, 649, 25 Dec. 1996
The Phytopathological Society of Japan (PSJ), Japanese

Detection and Nucleotide Sequence of Group IV Citrus Viroid
NAKAHARA K., HATAYA T., SHIWAKU K., UETANI Y., Annals of the Phytopathological Society of Japan, 62, 6, 649, 649, 25 Dec. 1996
The Phytopathological Society of Japan (PSJ), Japanese

Rapid and Simple Procedure for Extraction of Nucleic Acids from Citrus Tissues and Its Application to Gene Diagnosis of Citrus Viroids
NAKAHARA K., HATAYA T., IEKI H., Annals of the Phytopathological Society of Japan, 62, 3, 322, 322, 25 Jun. 1996
The Phytopathological Society of Japan (PSJ), Japanese

An Improved Procedure for Extraction of Nucleic Acids from Citrus Tissues and Its Application to Gene Diagnosis of Citrus Viroids
NAKAHARA K., HATAYA T., UYEDA I., Annals of the Phytopathological Society of Japan, 61, 6, 647, 647, 25 Dec. 1995
The Phytopathological Society of Japan (PSJ), Japanese

Detection of Potato Spindle Tuber Viroid (PSTVd) by Using Mixed Synthetic Oligonucleotide Probes Labeled with Biotin
NAKAHARA K., HATAYA T., HAYASHI Y., SUGIMOTO T., KIMURA I., SHIKATA E., Annals of the Phytopathological Society of Japan, 61, 3, 279, 279, 25 Jun. 1995
The Phytopathological Society of Japan (PSJ), Japanese

Detection and Nucleotide Sequence of Group I Citrus Viroid
HATAYA T., NAKAHARA K., OHARA T., IEKI H., KANO T., SHIKATA E., KIMURA I., Annals of the Phytopathological Society of Japan, 61, 3, 279, 279, 25 Jun. 1995
The Phytopathological Society of Japan (PSJ), Japanese

Reactions of Japanese Potato Cultivars to Potato Spindle Tuber Viroid and Its Gene Diagnosis
NAKAHARA K, HATAYA T, HAYASHI Y, SUGIMOTO T, KIMURA I, SHIKATA E, Annals of the Phytopathological Society of Japan, 60, 6, 793, 793, 25 Dec. 1994
The Phytopathological Society of Japan (PSJ), Japanese

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