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Masayuki Ishikawa - One of the best experts on this subject based on the ideXlab platform.
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structural basis for the recognition evasion arms race between Tomato Mosaic Virus and the resistance gene tm 1
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Kazuhiro Ishibashi, Masayuki Ishikawa, Tsuyoshi Inoue, Hiroyoshi Matsumura, Masahiko Kato, Chihoko Kobayashi, Yuichiro Kezuka, Takamasa Nonaka, Etsuko KatohAbstract:The Tomato Mosaic Virus (ToMV) resistance gene Tm-1 encodes a protein that shows no sequence homology to functionally characterized proteins. Tm-1 binds ToMV replication proteins and thereby inhibits replication complex formation. ToMV mutants that overcome this resistance have amino acid substitutions in the helicase domain of the replication proteins (ToMV-Hel). A small region of Tm-1 in the genome of the wild Tomato Solanum habrochaites has been under positive selection during its antagonistic coevolution with ToMV. Here we report crystal structures for the N-terminal inhibitory domains of Tm-1 and a natural Tm-1 variant with an I91-to-T substitution that has a greater ability to inhibit ToMV RNA replication and their complexes with ToMV-Hel. Each complex contains a Tm-1 dimer and two ToMV-Hel monomers with the interfaces between Tm-1 and ToMV-Hel bridged by ATP. Residues in ToMV-Hel and Tm-1 involved in antagonistic coevolution are found at the interface. The structural differences between ToMV-Hel in its free form and in complex with Tm-1 suggest that Tm-1 affects nucleoside triphosphatase activity of ToMV-Hel, and this effect was confirmed experimentally. Molecular dynamics simulations of complexes formed by Tm-1 with ToMV-Hel variants showed how the amino acid changes in ToMV-Hel impair the interaction with Tm-1 to overcome the resistance. With these findings, together with the biochemical properties of the interactions between ToMV-Hel and Tm-1 variants and effects of the mutations in the polymorphic residues of Tm-1, an atomic view of a step-by-step coevolutionary arms race between a plant resistance protein and a viral protein emerges.
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the resistance protein tm 1 inhibits formation of a Tomato Mosaic Virus replication protein host membrane protein complex
Journal of Virology, 2013Co-Authors: Kazuhiro Ishibashi, Masayuki IshikawaAbstract:ABSTRACT The Tm-1 gene of Tomato confers resistance to Tomato Mosaic Virus (ToMV). Tm-1 encodes a protein that binds ToMV replication proteins and inhibits the RNA-dependent RNA replication of ToMV. The replication proteins of resistance-breaking mutants of ToMV do not bind Tm-1, indicating that the binding is important for inhibition. In this study, we analyzed how Tm-1 inhibits ToMV RNA replication in a cell-free system using evacuolated tobacco protoplast extracts. In this system, ToMV RNA replication is catalyzed by replication proteins bound to membranes, and the RNA polymerase activity is unaffected by treatment with 0.5 M NaCl-containing buffer and remains associated with membranes. We show that in the presence of Tm-1, negative-strand RNA synthesis is inhibited; the replication proteins associate with membranes with binding that is sensitive to 0.5 M NaCl; the viral genomic RNA used as a translation template is not protected from nuclease digestion; and host membrane proteins TOM1, TOM2A, and ARL8 are not copurified with the membrane-bound 130K replication protein. Deletion of the polymerase read-through domain or of the 3′ untranslated region (UTR) of the genome did not prevent the formation of complexes between the 130K protein and the host membrane proteins, the 0.5 M NaCl-resistant binding of the replication proteins to membranes, and the protection of the genomic RNA from nucleases. These results indicate that Tm-1 binds ToMV replication proteins to inhibit key events in replication complex formation on membranes that precede negative-strand RNA synthesis.
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expression purification and functional characterization of an n terminal fragment of the Tomato Mosaic Virus resistance protein tm 1
Protein Expression and Purification, 2013Co-Authors: Masahiko Kato, Masayuki Ishikawa, Kazuhiro Ishibashi, Chihoko Kobayashi, Etsuko KatohAbstract:Abstract Tm-1, the protein product of Tm-1 , a semidominant resistance gene of Tomato, inhibits Tomato Mosaic Virus (ToMV) replication by binding to ToMV replication proteins. Previous studies suggested the importance of the Tm-1 N-terminal region for its inhibitory activity; however, it has not been determined if the N-terminal region is sufficient for inhibition. Furthermore, the three-dimensional structure of Tm-1 has not been determined. In this study, an N-terminal fragment of Tm-1 (residues 1–431) as a fusion protein containing an upstream maltose-binding protein was expressed in E. coli Rosetta (DE3) cells at 30 °C and then purified. The solubility of the fusion protein was greater when the cells were cultured at 30 °C than when cultured at lower or higher temperatures. The purified N-terminal Tm-1 fragment from which the maltose-binding protein tag had been removed has inhibitory activity against ToMV RNA replication.
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coevolution and hierarchical interactions of Tomato Mosaic Virus and the resistance gene tm 1
PLOS Pathogens, 2012Co-Authors: Kazuhiro Ishibashi, Tetsuo Meshi, Natsuki Mawatari, Shuhei Miyashita, Hirohisa Kishino, Masayuki IshikawaAbstract:During antagonistic coevolution between Viruses and their hosts, Viruses have a major advantage by evolving more rapidly. Nevertheless, Viruses and their hosts coexist and have coevolved, although the processes remain largely unknown. We previously identified Tm-1 that confers resistance to Tomato Mosaic Virus (ToMV), and revealed that it encodes a protein that binds ToMV replication proteins and inhibits RNA replication. Tm-1 was introgressed from a wild Tomato species Solanum habrochaites into the cultivated Tomato species Solanum lycopersicum. In this study, we analyzed Tm-1 alleles in S. habrochaites. Although most part of this gene was under purifying selection, a cluster of nonsynonymous substitutions in a small region important for inhibitory activity was identified, suggesting that the region is under positive selection. We then examined the resistance of S. habrochaites plants to ToMV. Approximately 60% of 149 individuals from 24 accessions were resistant to ToMV, while the others accumulated detectable levels of coat protein after inoculation. Unexpectedly, many S. habrochaites plants were observed in which even multiplication of the Tm-1-resistance-breaking ToMV mutant LT1 was inhibited. An amino acid change in the positively selected region of the Tm-1 protein was responsible for the inhibition of LT1 multiplication. This amino acid change allowed Tm-1 to bind LT1 replication proteins without losing the ability to bind replication proteins of wild-type ToMV. The antiviral spectra and biochemical properties suggest that Tm-1 has evolved by changing the strengths of its inhibitory activity rather than diversifying the recognition spectra. In the LT1-resistant S. habrochaites plants inoculated with LT1, mutant Viruses emerged whose multiplication was not inhibited by the Tm-1 allele that confers resistance to LT1. However, the resistance-breaking mutants were less competitive than the parental strains in the absence of Tm-1. Based on these results, we discuss possible coevolutionary processes of ToMV and Tm-1.
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crystal structure of the superfamily 1 helicase from Tomato Mosaic Virus
Journal of Virology, 2012Co-Authors: Masaki Nishikiori, Masayuki Ishikawa, Shigeru Sugiyama, Hongyu Xiang, Mayumi Niiyama, Kazuhiro Ishibashi, Tsuyoshi Inoue, Hiroyoshi Matsumura, Etsuko KatohAbstract:The genomes of the Tomato Mosaic Virus and many other plant and animal positive-strand RNA Viruses of agronomic and medical importance encode superfamily 1 helicases. Although helicases play important roles in viral replication, the crystal structures of viral superfamily 1 helicases have not been determined. Here, we report the crystal structure of a fragment (S666 to Q1116) of the replication protein from Tomato Mosaic Virus. The structure reveals a novel N-terminal domain tightly associated with a helicase core. The helicase core contains two RecA-like α/β domains without any of the accessory domain insertions that are found in other superfamily 1 helicases. The N-terminal domain contains a flexible loop, a long α-helix, and an antiparallel six-stranded β-sheet. On the basis of the structure, we constructed deletion mutants of the S666-to-Q1116 fragment and performed split-ubiquitin-based interaction assays in Saccharomyces cerevisiae with TOM1 and ARL8, host proteins that are essential for Tomato Mosaic Virus RNA replication. The results suggested that both TOM1 and ARL8 interact with the long α-helix in the N-terminal domain and that TOM1 also interacts with the helicase core. Prediction of secondary structures in other viral superfamily 1 helicases and comparison of those structures with the S666-to-Q1116 structure suggested that these helicases have a similar fold. Our results provide a structural basis of viral superfamily 1 helicases.
Hiroshi Nyunoya - One of the best experts on this subject based on the ideXlab platform.
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Plant Protein-Mediated Inhibition of Virus Cell-to-Cell Movement: Far-Western Screening and Biological Analysis of a Plant Protein Interacting with a Viral Movement Protein
Methods in Molecular Biology, 2019Co-Authors: Nobumitsu Sasaki, Yasuhiko Matsushita, Hiroshi NyunoyaAbstract:Cell-to-cell movement via plasmodesmata is a crucial step for plant RNA Viruses to determine their host ranges. Many Viruses including Tomato Mosaic Virus (ToMV) encode one or more movement proteins (MPs) that are indispensable for cell-to-cell movement. During movement processes, MPs are thought to interact directly with many plant proteins that may be involved in supporting or inhibiting cell-to-cell movement of Viruses. In order to understand the molecular mechanisms that regulate viral spread positively or negatively, it is important to discover such MP-interacting plant proteins and analyze their functions in viral cell-to-cell movement in efficient ways. In this chapter, we provide protocols of a radioisotope-based far-western screening strategy to construct a λ phage cDNA library from a nonhost Brassica campestris (syn. rapa) for ToMV and identify plant proteins that bind directly to the 32P-labeled probe of ToMV MP, and subsequently a biolistic bombardment method to examine whether a plant protein selected have a function as an inhibitory factor that can interfere with Virus cell-to-cell movement.
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altered subcellular localization of a tobacco membrane raft associated remorin protein by tobamoVirus infection and transient expression of viral replication and movement proteins
Frontiers in Plant Science, 2018Co-Authors: Nobumitsu Sasaki, Hiroshi Nyunoya, Eita TakashimaAbstract:Remorins are plant specific proteins found in plasma membrane microdomains (termed lipid or membrane rafts) and plasmodesmata. A potato remorin is reported to be involved in regulating negatively potexVirus movement and plasmodesmal permeability. In this study, we isolated cDNAs of tobacco remorins (NtREMs) and examined roles of an NtREM in infection by Tomato Mosaic Virus (ToMV). Subcellular localization analysis by using fluorescently tagged NtREM, ToMV, and viral replication and movement proteins indicated that Virus infection and transient expression of the viral proteins promoted the formation of NtREM aggregates by alteration of the subcellular distribution of NtREM, which was localized uniformly on the plasma membrane under normal conditions. NtREM aggregates were often observed to be associated closely with endoplasmic reticulum networks and also bodies of the 126K replication and movement proteins. The bimolecular fluorescence complementation assay indicated that NtREM might interact directly with the movement protein on the plasma membrane and around plasmodesmata. In addition, transient overexpression of NtREM facilitated ToMV cell-to-cell movement. Based on these results, we discuss possible roles of the tobacco remorin in tobamoVirus movement.
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Altered Subcellular Localization of a Tobacco Membrane Raft-Associated Remorin Protein by TobamoVirus Infection and Transient Expression of Viral Replication and Movement Proteins
Frontiers Media S.A., 2018Co-Authors: Nobumitsu Sasaki, Hiroshi Nyunoya, Eita TakashimaAbstract:Remorins are plant specific proteins found in plasma membrane microdomains (termed lipid or membrane rafts) and plasmodesmata. A potato remorin is reported to be involved in negatively regulating potexVirus movement and plasmodesmal permeability. In this study, we isolated cDNAs of tobacco remorins (NtREMs) and examined roles of an NtREM in infection by Tomato Mosaic Virus (ToMV). Subcellular localization analysis using fluorescently tagged NtREM, ToMV, and viral replication and movement proteins (MPs) indicated that Virus infection and transient expression of the viral proteins promoted the formation of NtREM aggregates by altering the subcellular distribution of NtREM, which was localized uniformly on the plasma membrane under normal conditions. NtREM aggregates were often observed associated closely with endoplasmic reticulum networks and bodies of the 126K replication and MPs. The bimolecular fluorescence complementation assay indicated that NtREM might interact directly with the MP on the plasma membrane and around plasmodesmata. In addition, transient overexpression of NtREM facilitated ToMV cell-to-cell movement. Based on these results, we discuss possible roles of the tobacco remorin in tobamoVirus movement
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Image_5_Altered Subcellular Localization of a Tobacco Membrane Raft-Associated Remorin Protein by TobamoVirus Infection and Transient Expression of Viral Replication and Movement Proteins.TIFF
2018Co-Authors: Nobumitsu Sasaki, Eita Takashima, Hiroshi NyunoyaAbstract:Remorins are plant specific proteins found in plasma membrane microdomains (termed lipid or membrane rafts) and plasmodesmata. A potato remorin is reported to be involved in negatively regulating potexVirus movement and plasmodesmal permeability. In this study, we isolated cDNAs of tobacco remorins (NtREMs) and examined roles of an NtREM in infection by Tomato Mosaic Virus (ToMV). Subcellular localization analysis using fluorescently tagged NtREM, ToMV, and viral replication and movement proteins (MPs) indicated that Virus infection and transient expression of the viral proteins promoted the formation of NtREM aggregates by altering the subcellular distribution of NtREM, which was localized uniformly on the plasma membrane under normal conditions. NtREM aggregates were often observed associated closely with endoplasmic reticulum networks and bodies of the 126K replication and MPs. The bimolecular fluorescence complementation assay indicated that NtREM might interact directly with the MP on the plasma membrane and around plasmodesmata. In addition, transient overexpression of NtREM facilitated ToMV cell-to-cell movement. Based on these results, we discuss possible roles of the tobacco remorin in tobamoVirus movement.
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The nonstructural protein pC6 of rice grassy stunt Virus trans-complements the cell-to-cell spread of a movement-defective Tomato Mosaic Virus
Archives of Virology, 2011Co-Authors: Akihiro Hiraguri, Nobumitsu Sasaki, Hiroshi Nyunoya, Osamu Netsu, Takumi Shimizu, Tamaki Uehara-ichiki, Toshihiro Omura, Takahide SasayaAbstract:The nonstructural protein pC6 encoded by rice grassy stunt Virus is thought to correspond functionally to the nonstructural protein pC4 of rice stripe Virus, which can support viral cell-to-cell movement. In a trans -complementation experiment with a movement-defective Tomato Mosaic Virus, pC6 and pC4 facilitated intercellular transport of the Virus. Transient expression of pC6, fused with green fluorescent protein, in epidermal cells was predominantly observed close to the cell wall as well as in a few punctate structures, presumably associated with plasmodesmata. These results suggest that pC6 has a role similar to that of pC4 in viral cell-to-cell movement.
Tetsuo Meshi - One of the best experts on this subject based on the ideXlab platform.
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coevolution and hierarchical interactions of Tomato Mosaic Virus and the resistance gene tm 1
PLOS Pathogens, 2012Co-Authors: Kazuhiro Ishibashi, Tetsuo Meshi, Natsuki Mawatari, Shuhei Miyashita, Hirohisa Kishino, Masayuki IshikawaAbstract:During antagonistic coevolution between Viruses and their hosts, Viruses have a major advantage by evolving more rapidly. Nevertheless, Viruses and their hosts coexist and have coevolved, although the processes remain largely unknown. We previously identified Tm-1 that confers resistance to Tomato Mosaic Virus (ToMV), and revealed that it encodes a protein that binds ToMV replication proteins and inhibits RNA replication. Tm-1 was introgressed from a wild Tomato species Solanum habrochaites into the cultivated Tomato species Solanum lycopersicum. In this study, we analyzed Tm-1 alleles in S. habrochaites. Although most part of this gene was under purifying selection, a cluster of nonsynonymous substitutions in a small region important for inhibitory activity was identified, suggesting that the region is under positive selection. We then examined the resistance of S. habrochaites plants to ToMV. Approximately 60% of 149 individuals from 24 accessions were resistant to ToMV, while the others accumulated detectable levels of coat protein after inoculation. Unexpectedly, many S. habrochaites plants were observed in which even multiplication of the Tm-1-resistance-breaking ToMV mutant LT1 was inhibited. An amino acid change in the positively selected region of the Tm-1 protein was responsible for the inhibition of LT1 multiplication. This amino acid change allowed Tm-1 to bind LT1 replication proteins without losing the ability to bind replication proteins of wild-type ToMV. The antiviral spectra and biochemical properties suggest that Tm-1 has evolved by changing the strengths of its inhibitory activity rather than diversifying the recognition spectra. In the LT1-resistant S. habrochaites plants inoculated with LT1, mutant Viruses emerged whose multiplication was not inhibited by the Tm-1 allele that confers resistance to LT1. However, the resistance-breaking mutants were less competitive than the parental strains in the absence of Tm-1. Based on these results, we discuss possible coevolutionary processes of ToMV and Tm-1.
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identification of an amino acid residue required for differential recognition of a viral movement protein by the Tomato Mosaic Virus resistance gene tm 22
Journal of Plant Physiology, 2011Co-Authors: Michie Kobayashi, Tetsuo Meshi, Katsuyuki Hirai, Ayako Yamamotokatou, Shinpei Katou, Yuko Ohashi, Ichiro MitsuharaAbstract:Abstract The Tm-2 gene of Tomato and its allelic gene, Tm-2 2 , confer resistance to Tomato Mosaic Virus (ToMV) and encode a member of the coiled-coil/nucleotide binding-ARC/leucine-rich repeat (LRR) protein class of plant resistance ( R ) genes. Despite exhibiting only four amino acid differences between the products of Tm-2 and Tm-2 2 , Tm-2 2 confers resistance to ToMV mutant B7, whereas Tm-2 is broken by ToMV-B7. An Agrobacterium -mediated transient expression system was used to study the mechanism of differential recognition of the movement proteins (MPs), an avirulence factor for ToMV resistance, of ToMV-B7 by Tm-2 and Tm-2 2 . Although resistance induced by Tm-2 and Tm-2 2 is not usually accompanied by hypersensitive response (HR), Tm-2 and Tm-2 2 induced HR-like cell death by co-expression with MP of a wild-type ToMV, a strain that causes resistance for these R genes, and Tm-2 2 but not Tm-2 induced cell death with B7-MP in this system. Site-directed amino acid mutagenesis revealed that Tyr-767 in the LRR of Tm-2 2 is required for the specific recognition of the B7-MP. These results suggest that the Tyr residue in LRR contributes to the recognition of B7-MP, and that Tm-2 and Tm-2 2 are involved in HR cell death.
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Inducible viral inoculation system with cultured plant cells facilitates a biochemical approach for Virus-induced RNA silencing
Archives of Virology, 2010Co-Authors: Atsushi Tamai, Tetsuo Meshi, Koji Dohi, Masasi Mori, Masayuki IshikawaAbstract:An inducible Virus infection system was demonstrated to be an efficient protein expression system for inducing synchronous Virus vector multiplication in suspension-cultured plant cells. A GFP-tagged Tomato Mosaic Virus (ToMV-GFP) derivative that has a defect in its 130 K protein, a silencing suppressor of ToMV, was synchronously infected to tobacco BY2 cultured cells using this system. In the infection-induced cells, viral RNA was degraded rapidly, and a cytosol extract prepared from the infected cells showed RNA degradation activity specific for ToMV- or GFP-related sequences. In lysate prepared from cells infected by ToMV-GFP carrying the wild-type 130 K protein, sequence-specific RNA degradation activity was suppressed, although siRNA derived from the Virus was generated. Furthermore, the 130 K protein interfered with 3′-end methylation of siRNA. The inducible Virus infection system may provide a method for biochemical analysis of antiviral RNA silencing and silencing suppression by ToMV.
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over expression of putative transcriptional coactivator kelp interferes with Tomato Mosaic Virus cell to cell movement
Molecular Plant Pathology, 2009Co-Authors: Nobumitsu Sasaki, Atsushi Tamai, Tetsuo Meshi, Yasuhiko Matsushita, Yuichiro Watanabe, Masakazu Deguchi, Takuya Ogata, Shoko Nagai, Shigeki Kawakami, Hiroshi NyunoyaAbstract:Tomato Mosaic Virus (ToMV) encodes a movement protein (MP) that is necessary for Virus cell-to-cell movement. We have demonstrated previously that KELP, a putative transcriptional coactivator of Arabidopsis thaliana, and its orthologue from Brassica campestris can bind to ToMV MP in vitro. In this study, we examined the effects of the transient over-expression of KELP on ToMV infection and the intracellular localization of MP in Nicotiana benthamiana, an experimental host of the Virus. In co-bombardment experiments, the over-expression of KELP inhibited Virus cell-to-cell movement. The N-terminal half of KELP (KELPdC), which had been shown to bind to MP, was sufficient for inhibition. Furthermore, the over-expression of KELP and KELPdC, both of which were co-localized with ToMV MP, led to a reduction in the plasmodesmal association of MP. In the absence of MP expression, KELP was localized in the nucleus and the cytoplasm by the localization signal in its N-terminal half. It was also shown that ToMV amplified normally in protoplasts prepared from leaf tissue that expressed KELP transiently. These results indicate that over-expressed KELP interacts with MP in vivo and exerts an inhibitory effect on MP function for Virus cell-to-cell movement, but not on Virus amplification in individual cells.
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overexpression of a host factor tom1 inhibits Tomato Mosaic Virus propagation and suppression of rna silencing
Virology, 2008Co-Authors: Yuka Hagiwarakomoda, Tetsuo Meshi, Atsuko Mochizuki, Masamichi Nishiguchi, Katsuyuki Hirai, Masayuki IshikawaAbstract:A plant integral membrane protein TOM1 is involved in the multiplication of Tomato Mosaic Virus (ToMV). TOM1 interacts with ToMV replication proteins and has been suggested to tether the replication proteins to the membranes where the viral RNA synthesis takes place. We have previously demonstrated that inactivation of TOM1 results in reduced ToMV multiplication. In the present study, we show that overexpression of TOM1 in tobacco also inhibits ToMV propagation. TOM1 overexpression led to a decreased accumulation of the soluble form of the replication proteins and interfered with the ability of the replication protein to suppress RNA silencing. The reduced accumulation of the soluble replication proteins was also observed in a silencing suppressor-defective ToMV mutant. Based on these results, we propose that RNA silencing suppression is executed by the soluble form of the replication proteins and that efficient ToMV multiplication requires balanced accumulation of the soluble and membrane-bound replication proteins.
Nobumitsu Sasaki - One of the best experts on this subject based on the ideXlab platform.
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Plant Protein-Mediated Inhibition of Virus Cell-to-Cell Movement: Far-Western Screening and Biological Analysis of a Plant Protein Interacting with a Viral Movement Protein
Methods in Molecular Biology, 2019Co-Authors: Nobumitsu Sasaki, Yasuhiko Matsushita, Hiroshi NyunoyaAbstract:Cell-to-cell movement via plasmodesmata is a crucial step for plant RNA Viruses to determine their host ranges. Many Viruses including Tomato Mosaic Virus (ToMV) encode one or more movement proteins (MPs) that are indispensable for cell-to-cell movement. During movement processes, MPs are thought to interact directly with many plant proteins that may be involved in supporting or inhibiting cell-to-cell movement of Viruses. In order to understand the molecular mechanisms that regulate viral spread positively or negatively, it is important to discover such MP-interacting plant proteins and analyze their functions in viral cell-to-cell movement in efficient ways. In this chapter, we provide protocols of a radioisotope-based far-western screening strategy to construct a λ phage cDNA library from a nonhost Brassica campestris (syn. rapa) for ToMV and identify plant proteins that bind directly to the 32P-labeled probe of ToMV MP, and subsequently a biolistic bombardment method to examine whether a plant protein selected have a function as an inhibitory factor that can interfere with Virus cell-to-cell movement.
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altered subcellular localization of a tobacco membrane raft associated remorin protein by tobamoVirus infection and transient expression of viral replication and movement proteins
Frontiers in Plant Science, 2018Co-Authors: Nobumitsu Sasaki, Hiroshi Nyunoya, Eita TakashimaAbstract:Remorins are plant specific proteins found in plasma membrane microdomains (termed lipid or membrane rafts) and plasmodesmata. A potato remorin is reported to be involved in regulating negatively potexVirus movement and plasmodesmal permeability. In this study, we isolated cDNAs of tobacco remorins (NtREMs) and examined roles of an NtREM in infection by Tomato Mosaic Virus (ToMV). Subcellular localization analysis by using fluorescently tagged NtREM, ToMV, and viral replication and movement proteins indicated that Virus infection and transient expression of the viral proteins promoted the formation of NtREM aggregates by alteration of the subcellular distribution of NtREM, which was localized uniformly on the plasma membrane under normal conditions. NtREM aggregates were often observed to be associated closely with endoplasmic reticulum networks and also bodies of the 126K replication and movement proteins. The bimolecular fluorescence complementation assay indicated that NtREM might interact directly with the movement protein on the plasma membrane and around plasmodesmata. In addition, transient overexpression of NtREM facilitated ToMV cell-to-cell movement. Based on these results, we discuss possible roles of the tobacco remorin in tobamoVirus movement.
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Altered Subcellular Localization of a Tobacco Membrane Raft-Associated Remorin Protein by TobamoVirus Infection and Transient Expression of Viral Replication and Movement Proteins
Frontiers Media S.A., 2018Co-Authors: Nobumitsu Sasaki, Hiroshi Nyunoya, Eita TakashimaAbstract:Remorins are plant specific proteins found in plasma membrane microdomains (termed lipid or membrane rafts) and plasmodesmata. A potato remorin is reported to be involved in negatively regulating potexVirus movement and plasmodesmal permeability. In this study, we isolated cDNAs of tobacco remorins (NtREMs) and examined roles of an NtREM in infection by Tomato Mosaic Virus (ToMV). Subcellular localization analysis using fluorescently tagged NtREM, ToMV, and viral replication and movement proteins (MPs) indicated that Virus infection and transient expression of the viral proteins promoted the formation of NtREM aggregates by altering the subcellular distribution of NtREM, which was localized uniformly on the plasma membrane under normal conditions. NtREM aggregates were often observed associated closely with endoplasmic reticulum networks and bodies of the 126K replication and MPs. The bimolecular fluorescence complementation assay indicated that NtREM might interact directly with the MP on the plasma membrane and around plasmodesmata. In addition, transient overexpression of NtREM facilitated ToMV cell-to-cell movement. Based on these results, we discuss possible roles of the tobacco remorin in tobamoVirus movement
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Image_5_Altered Subcellular Localization of a Tobacco Membrane Raft-Associated Remorin Protein by TobamoVirus Infection and Transient Expression of Viral Replication and Movement Proteins.TIFF
2018Co-Authors: Nobumitsu Sasaki, Eita Takashima, Hiroshi NyunoyaAbstract:Remorins are plant specific proteins found in plasma membrane microdomains (termed lipid or membrane rafts) and plasmodesmata. A potato remorin is reported to be involved in negatively regulating potexVirus movement and plasmodesmal permeability. In this study, we isolated cDNAs of tobacco remorins (NtREMs) and examined roles of an NtREM in infection by Tomato Mosaic Virus (ToMV). Subcellular localization analysis using fluorescently tagged NtREM, ToMV, and viral replication and movement proteins (MPs) indicated that Virus infection and transient expression of the viral proteins promoted the formation of NtREM aggregates by altering the subcellular distribution of NtREM, which was localized uniformly on the plasma membrane under normal conditions. NtREM aggregates were often observed associated closely with endoplasmic reticulum networks and bodies of the 126K replication and MPs. The bimolecular fluorescence complementation assay indicated that NtREM might interact directly with the MP on the plasma membrane and around plasmodesmata. In addition, transient overexpression of NtREM facilitated ToMV cell-to-cell movement. Based on these results, we discuss possible roles of the tobacco remorin in tobamoVirus movement.
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The nonstructural protein pC6 of rice grassy stunt Virus trans-complements the cell-to-cell spread of a movement-defective Tomato Mosaic Virus
Archives of Virology, 2011Co-Authors: Akihiro Hiraguri, Nobumitsu Sasaki, Hiroshi Nyunoya, Osamu Netsu, Takumi Shimizu, Tamaki Uehara-ichiki, Toshihiro Omura, Takahide SasayaAbstract:The nonstructural protein pC6 encoded by rice grassy stunt Virus is thought to correspond functionally to the nonstructural protein pC4 of rice stripe Virus, which can support viral cell-to-cell movement. In a trans -complementation experiment with a movement-defective Tomato Mosaic Virus, pC6 and pC4 facilitated intercellular transport of the Virus. Transient expression of pC6, fused with green fluorescent protein, in epidermal cells was predominantly observed close to the cell wall as well as in a few punctate structures, presumably associated with plasmodesmata. These results suggest that pC6 has a role similar to that of pC4 in viral cell-to-cell movement.
Kazuhiro Ishibashi - One of the best experts on this subject based on the ideXlab platform.
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structural basis for the recognition evasion arms race between Tomato Mosaic Virus and the resistance gene tm 1
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Kazuhiro Ishibashi, Masayuki Ishikawa, Tsuyoshi Inoue, Hiroyoshi Matsumura, Masahiko Kato, Chihoko Kobayashi, Yuichiro Kezuka, Takamasa Nonaka, Etsuko KatohAbstract:The Tomato Mosaic Virus (ToMV) resistance gene Tm-1 encodes a protein that shows no sequence homology to functionally characterized proteins. Tm-1 binds ToMV replication proteins and thereby inhibits replication complex formation. ToMV mutants that overcome this resistance have amino acid substitutions in the helicase domain of the replication proteins (ToMV-Hel). A small region of Tm-1 in the genome of the wild Tomato Solanum habrochaites has been under positive selection during its antagonistic coevolution with ToMV. Here we report crystal structures for the N-terminal inhibitory domains of Tm-1 and a natural Tm-1 variant with an I91-to-T substitution that has a greater ability to inhibit ToMV RNA replication and their complexes with ToMV-Hel. Each complex contains a Tm-1 dimer and two ToMV-Hel monomers with the interfaces between Tm-1 and ToMV-Hel bridged by ATP. Residues in ToMV-Hel and Tm-1 involved in antagonistic coevolution are found at the interface. The structural differences between ToMV-Hel in its free form and in complex with Tm-1 suggest that Tm-1 affects nucleoside triphosphatase activity of ToMV-Hel, and this effect was confirmed experimentally. Molecular dynamics simulations of complexes formed by Tm-1 with ToMV-Hel variants showed how the amino acid changes in ToMV-Hel impair the interaction with Tm-1 to overcome the resistance. With these findings, together with the biochemical properties of the interactions between ToMV-Hel and Tm-1 variants and effects of the mutations in the polymorphic residues of Tm-1, an atomic view of a step-by-step coevolutionary arms race between a plant resistance protein and a viral protein emerges.
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the resistance protein tm 1 inhibits formation of a Tomato Mosaic Virus replication protein host membrane protein complex
Journal of Virology, 2013Co-Authors: Kazuhiro Ishibashi, Masayuki IshikawaAbstract:ABSTRACT The Tm-1 gene of Tomato confers resistance to Tomato Mosaic Virus (ToMV). Tm-1 encodes a protein that binds ToMV replication proteins and inhibits the RNA-dependent RNA replication of ToMV. The replication proteins of resistance-breaking mutants of ToMV do not bind Tm-1, indicating that the binding is important for inhibition. In this study, we analyzed how Tm-1 inhibits ToMV RNA replication in a cell-free system using evacuolated tobacco protoplast extracts. In this system, ToMV RNA replication is catalyzed by replication proteins bound to membranes, and the RNA polymerase activity is unaffected by treatment with 0.5 M NaCl-containing buffer and remains associated with membranes. We show that in the presence of Tm-1, negative-strand RNA synthesis is inhibited; the replication proteins associate with membranes with binding that is sensitive to 0.5 M NaCl; the viral genomic RNA used as a translation template is not protected from nuclease digestion; and host membrane proteins TOM1, TOM2A, and ARL8 are not copurified with the membrane-bound 130K replication protein. Deletion of the polymerase read-through domain or of the 3′ untranslated region (UTR) of the genome did not prevent the formation of complexes between the 130K protein and the host membrane proteins, the 0.5 M NaCl-resistant binding of the replication proteins to membranes, and the protection of the genomic RNA from nucleases. These results indicate that Tm-1 binds ToMV replication proteins to inhibit key events in replication complex formation on membranes that precede negative-strand RNA synthesis.
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expression purification and functional characterization of an n terminal fragment of the Tomato Mosaic Virus resistance protein tm 1
Protein Expression and Purification, 2013Co-Authors: Masahiko Kato, Masayuki Ishikawa, Kazuhiro Ishibashi, Chihoko Kobayashi, Etsuko KatohAbstract:Abstract Tm-1, the protein product of Tm-1 , a semidominant resistance gene of Tomato, inhibits Tomato Mosaic Virus (ToMV) replication by binding to ToMV replication proteins. Previous studies suggested the importance of the Tm-1 N-terminal region for its inhibitory activity; however, it has not been determined if the N-terminal region is sufficient for inhibition. Furthermore, the three-dimensional structure of Tm-1 has not been determined. In this study, an N-terminal fragment of Tm-1 (residues 1–431) as a fusion protein containing an upstream maltose-binding protein was expressed in E. coli Rosetta (DE3) cells at 30 °C and then purified. The solubility of the fusion protein was greater when the cells were cultured at 30 °C than when cultured at lower or higher temperatures. The purified N-terminal Tm-1 fragment from which the maltose-binding protein tag had been removed has inhibitory activity against ToMV RNA replication.
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coevolution and hierarchical interactions of Tomato Mosaic Virus and the resistance gene tm 1
PLOS Pathogens, 2012Co-Authors: Kazuhiro Ishibashi, Tetsuo Meshi, Natsuki Mawatari, Shuhei Miyashita, Hirohisa Kishino, Masayuki IshikawaAbstract:During antagonistic coevolution between Viruses and their hosts, Viruses have a major advantage by evolving more rapidly. Nevertheless, Viruses and their hosts coexist and have coevolved, although the processes remain largely unknown. We previously identified Tm-1 that confers resistance to Tomato Mosaic Virus (ToMV), and revealed that it encodes a protein that binds ToMV replication proteins and inhibits RNA replication. Tm-1 was introgressed from a wild Tomato species Solanum habrochaites into the cultivated Tomato species Solanum lycopersicum. In this study, we analyzed Tm-1 alleles in S. habrochaites. Although most part of this gene was under purifying selection, a cluster of nonsynonymous substitutions in a small region important for inhibitory activity was identified, suggesting that the region is under positive selection. We then examined the resistance of S. habrochaites plants to ToMV. Approximately 60% of 149 individuals from 24 accessions were resistant to ToMV, while the others accumulated detectable levels of coat protein after inoculation. Unexpectedly, many S. habrochaites plants were observed in which even multiplication of the Tm-1-resistance-breaking ToMV mutant LT1 was inhibited. An amino acid change in the positively selected region of the Tm-1 protein was responsible for the inhibition of LT1 multiplication. This amino acid change allowed Tm-1 to bind LT1 replication proteins without losing the ability to bind replication proteins of wild-type ToMV. The antiviral spectra and biochemical properties suggest that Tm-1 has evolved by changing the strengths of its inhibitory activity rather than diversifying the recognition spectra. In the LT1-resistant S. habrochaites plants inoculated with LT1, mutant Viruses emerged whose multiplication was not inhibited by the Tm-1 allele that confers resistance to LT1. However, the resistance-breaking mutants were less competitive than the parental strains in the absence of Tm-1. Based on these results, we discuss possible coevolutionary processes of ToMV and Tm-1.
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crystal structure of the superfamily 1 helicase from Tomato Mosaic Virus
Journal of Virology, 2012Co-Authors: Masaki Nishikiori, Masayuki Ishikawa, Shigeru Sugiyama, Hongyu Xiang, Mayumi Niiyama, Kazuhiro Ishibashi, Tsuyoshi Inoue, Hiroyoshi Matsumura, Etsuko KatohAbstract:The genomes of the Tomato Mosaic Virus and many other plant and animal positive-strand RNA Viruses of agronomic and medical importance encode superfamily 1 helicases. Although helicases play important roles in viral replication, the crystal structures of viral superfamily 1 helicases have not been determined. Here, we report the crystal structure of a fragment (S666 to Q1116) of the replication protein from Tomato Mosaic Virus. The structure reveals a novel N-terminal domain tightly associated with a helicase core. The helicase core contains two RecA-like α/β domains without any of the accessory domain insertions that are found in other superfamily 1 helicases. The N-terminal domain contains a flexible loop, a long α-helix, and an antiparallel six-stranded β-sheet. On the basis of the structure, we constructed deletion mutants of the S666-to-Q1116 fragment and performed split-ubiquitin-based interaction assays in Saccharomyces cerevisiae with TOM1 and ARL8, host proteins that are essential for Tomato Mosaic Virus RNA replication. The results suggested that both TOM1 and ARL8 interact with the long α-helix in the N-terminal domain and that TOM1 also interacts with the helicase core. Prediction of secondary structures in other viral superfamily 1 helicases and comparison of those structures with the S666-to-Q1116 structure suggested that these helicases have a similar fold. Our results provide a structural basis of viral superfamily 1 helicases.