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Development and evaluation of a SYBR Green-based RT-qPCR assay with a specific primer set for tomato seed testing against tomato brown rugose fruit virus

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JOURNAL OF GENERAL PLANT PATHOLOGY 2025年第3期91卷 160-170页

作者： Ota, Emi Shinosaka, Hibiki ishibashi, kazuhiro Takeyama, Sawana Matsuyama, Momoko Tomitaka, Yasuhiro Matsushita, Yosuke Osaki, Kohei Kubota, Kenji NARO Inst Plant Protect Div Core Technol Pest Control Res 2-1-18 Kannondai Tsukuba Ibaraki 3058666 Japan NARO Ctr Seeds & Seedlings Dept DUS Test & Seed Inspect 2-2 Fujimoto Tsukuba Ibaraki 3050852 Japan NARO Inst Agrobiol Sci Div Plant Mol Regulat Res 2-1-2 Kannondai Tsukuba Ibaraki 3058602 Japan NARO Inst Plant Protect Div Crop Pest Control Res 2-1-18 Kannondai Tsukuba Ibaraki 3058666 Japan

Since its first occurrence in Israel and Jordan in 2014 and 2015, the tomato brown rugose fruit virus (ToBRFV) has become one of the most concerning pathogens affecting tomatoes and other crops worldwide. Its rapid spread is believed to result from the international trade of contaminated seeds and its seed transmissibility, underscoring the critical importance of seed health testing for ToBRFV to prevent further dissemination of the virus. To this end, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) protocols employing TaqMan probe chemistry have been widely adopted. However, the development of RT-qPCR protocols for ToBRFV seed testing using SYBR Green chemistry remains limited. The SYBR Green method offers the advantage of distinguishing ToBRFV from other tobamoviruses through melt curve analysis. In this study, we developed a SYBR Green-based RT-qPCR detection method using newly designed primer sets, which demonstrated high specificity for ToBRFV and sufficient sensitivity. While this protocol requires further optimization and validation for application in routine seed testing, it establishes a foundational approach for SYBR Green-based RT-qPCR seed testing. Additionally, this study raises an important question regarding the relationship between RT-qPCR results in seed tests and the likelihood of virus contamination or transmission via seeds.

关键词： ToBRFV Tobamoviruses SYBR green RT-qPCR Melt curve Tomato

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Studies on the Tm-1 gene of tomato and host specificity of tobamoviruses

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JOURNAL OF GENERAL PLANT PATHOLOGY 2010年第6期76卷 417-418页

作者： ishibashi, kazuhiro Natl Inst Agrobiol Sci Div Plant Sci Tsukuba Ibaraki 3058602 Japan

An abstract of the paper 'Studies on the Tm-1 Gene of Tomato and Host Specificity of Tobamoviruses,' by Young Scientist Award winner kazuhiro ishibashi discussed at the 2010 Annual Meeting of the Phytopatholog... 详细信息

关键词： TOBAMOVIRUSES ABSTRACTS

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Mechanisms of tomato mosaic virus RNA replication and its inhibition by the host resistance factor Tm-1

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CURRENT OPINION IN VIROLOGY 2014年 9卷 8-13页

作者： ishibashi, kazuhiro Ishikawa, Masayuki Natl Inst Agrobiol Sci Div Plant Sci Plant Microbe Interact Res Unit Tsukuba Ibaraki 3058602 Japan

In the plant immune system, sensor proteins encoded by dominant resistance genes activate a defense response upon pathogen infection. The tomato mosaic virus (ToMV) resistance gene Tm-1 is exceptional in that it inhibits ToMV multiplication without inducing a defense response. Several lines of evidence had suggested that Tm-1 encodes a direct inhibitor of ToMV RNA replication. The Tm-1 gene product was identified by purification of an inhibitor protein using a cell-free translation and replication system for ToMV RNA. Further analyses using the system showed that Tm-1 bound ToMV replication proteins, and that the Tm-1-bound ToMV replication proteins retained the ability to bind membranes, while Tm-1 inhibited replication complex formation on the membranes.

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Replication of Tobamovirus RNA

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ANNUAL REVIEW OF PHYTOPATHOLOGY, VOL 54 2016年第1期54卷 55-78页

作者： ishibashi, kazuhiro Ishikawa, Masayuki Natl Agr & Food Res Org NARO Plant & Microbial Res Unit Div Plant & Microbial Sci Inst Agrobiol Sci Tsukuba Ibaraki 3058602 Japan

Tobacco mosaic virus and other tobamoviruses have served as models for studying the mechanisms of viral RNA replication. In tobamoviruses, genomic RNA replication occurs via several steps: (a) synthesis of viral replication proteins by translation of the genomic RNA;(b) translation-coupled binding of the replication proteins to a 5'-terminal region of the genomic RNA;(c) recruitment of the genomic RNA by replication proteins onto membranes and formation of a complex with host proteins TOM1 and ARL8;(d) synthesis of complementary (negative-strand) RNA in the complex;and (e) synthesis of progeny genomic RNA. This article reviews current knowledge on tobamovirus RNA replication, particularly regarding how the genomic RNA is specifically selected as a replication template and how the replication proteins are activated. We also focus on the roles of the replication proteins in evading or suppressing host defense systems.

关键词： Tobacco mosaic virus Tomato mosaic virus replication protein host factor virus resistance

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A Split Staphylococcus aureus Cas9 as a Compact Genome-Editing Tool in Plants

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PLANT AND CELL PHYSIOLOGY 2017年第4期58卷 643-649页

作者： Kaya, Hidetaka ishibashi, kazuhiro Toki, Seiichi Natl Agr & Food Res Org Inst Agrobiol Sci Plant Genome Engn Res Unit 2-1-2 Kannondai Tsukuba Ibaraki 3058602 Japan Natl Agr & Food Res Org Inst Agrobiol Sci Plant & Microbial Res Unit 2-1-2 Kannondai Tsukuba Ibaraki 3058602 Japan Yokohama City Univ Grad Sch Nanobiosci 22-2 Seto Yokohama Kanagawa 2360027 Japan Yokohama City Univ Kihara Inst Biol Res 22-2 Seto Yokohama Kanagawa 2360027 Japan

Split-protein methods-where a protein is split into two inactive fragments that must re-assemble to form an active protein-can be used to regulate the activity of a given protein and reduce the size of gene transcription units. Here, we show that a Staphylococcus aureus Cas9 (SaCas9) can be split, and that split-SaCas9 expressed from Agrobacterium can induce targeted mutagenesis in Nicotiana benthamiana. Since SaCas9 is smaller than the more commonly used Cas9 derived from Streptococcus pyogenes, the split-SaCas9 provides the smallest tool yet for clustered regularly interspaced short pal-indromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) plant genome editing. Both sets of split-SaCas9 (_430N/431C and _739N/740C) exhibited genome-editing activity, and the activity of split-SaCas9_739N/740C was almost the same as that of full-length SaCas9. This result indicates that split-SaCas9_739N/740C is suitable for use in targeted mutagenesis. We also show that the split-SaCas9 fragment expressed from Tomato mosaic virus could induce targeted mutagenesis together with another fragment expressed from Agrobacterium, suggesting that a split-SaCas9 system using a plant virus vector is a promising tool for integration-free plant genome editing. Split-SaCas9 has the potential to regulate CRISPR/Cas9-mediated genome editing activity in plant cells both temporally and spatially.

关键词： CRISPR/Cas9 Split-SaCas9 Staphylococcus aureus Tomato mosaic virus

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Potato Virus X Vector-Mediated DNA-Free Genome Editing in Plants

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PLANT AND CELL PHYSIOLOGY 2020年第11期61卷 1946-1953页

作者： Ariga, Hirotaka Toki, Seiichi ishibashi, kazuhiro Natl Agr & Food Res Org Div Plant & Microbial Sci Inst Agrobiol Sci Plant & Microbial Res Unit Tsukuba Ibaraki 3058602 Japan Natl Agr & Food Res Org Div Appl Genet Plant Genome Engn Res Unit Inst Agrobiol Sci Tsukuba Ibaraki 3058602 Japan Yokohama City Univ Grad Sch Nanobiosci Yokohama Kanagawa 2360027 Japan Yokohama City Univ Kihara Inst Biol Res Yokohama Kanagawa 2360027 Japan Natl Agr & Food Res Org Genet Resources Ctr Plant Divers Res Team Tsukuba Ibaraki 3058602 Japan

Genome editing technology is important for plant science and crop breeding. Genome-edited plants prepared using general CRISPR-Cas9 methods usually contain foreign DNA, which is problematic for the production of genome-edited transgene-free plants for vegetative propagation or highly heterozygous hybrid cultivars. Here, we describe a method for highly efficient targeted mutagenesis in Nicotiana benthamiana through the expression of Cas9 and single-guide (sg)RNA using a potato virus X (PVX) vector. Following Agrobacterium-mediated introduction of virus vector cDNA, >60% of shoots regenerated without antibiotic selection carried targeted mutations, while similar to 18% of shoots contained T-DNA. The PVX vector was also used to express a base editor consisting of modified Cas9 fused with cytidine deaminase to introduce targeted nucleotide substitution in regenerated shoots. We also report exogenous DNA-free genome editing by mechanical inoculation of virions comprising the PVX vector expressing Cas9. This simple and efficient virus vector-mediated delivery of CRISPR-Cas9 could facilitate transgene-free gene editing in plants.

关键词： CRISPR-Cas9 Nicotiana benthamiana Plant genome editing RNA virus Virus vector

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An inhibitory interaction between viral and cellular proteins underlies the resistance of tomato to nonadapted tobamoviruses

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PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 2009年第21期106卷 8778-8783页

作者： ishibashi, kazuhiro Naito, Satoshi Meshi, Tetsuo Ishikawa, Masayuki Natl Inst Agrobiol Sci Div Plant Sci Tsukuba Ibaraki 3058602 Japan Hokkaido Univ Grad Sch Life Sci Sapporo Hokkaido 0608589 Japan

Any individual virus can infect only a limited range of hosts, and most plant species are "nonhosts" to a given virus;i.e., all members of the species are insusceptible to the virus. In nonhost plants, the factors that control virus resistance are not genetically tractable, and how the host range of a virus is determined remains poorly understood. Tomato (Solanum lycopersicum) is a nonhost species for Tobacco mild green mosaic virus (TMGMV) and Pepper mild mottle virus (PMMoV), members of the genus Tobamovirus. Previously, we identified Tm-1, a resistance gene of tomato to another tobamovirus, Tomato mosaic virus (ToMV), and found that Tm-1 binds to ToMV replication proteins to inhibit RNA replication. Tm-1 is derived from a wild tomato species, S. habrochaites, and ToMV-susceptible tomato cultivars have the allelic gene tm-1. The tm-1 protein can neither bind to ToMV replication proteins nor inhibit ToMV multiplication. Here, we show that transgenic tobacco plants expressing tm-1 exhibit resistance to TMGMV and PMMoV. The tm-1 protein bound to the replication proteins of TMGMV and PMMoV and inhibited their RNA replication in vitro. In one of the tm-1-expressing tobacco plants, a tm-1-insensitive TMGMV mutant emerged. In tomato protoplasts, this mutant TMGMV multiplied as efficiently as ToMV. However, in tomato plants, the mutant TMGMV multiplied with lower efficiency compared to ToMV and caused systemic necrosis. These results suggest that an inhibitory interaction between the replication proteins and tm-1 underlies a multilayered resistance mechanism to TMGMV in tomato.

关键词： Pepper mild mottle virus Tm-1 Tobacco mild green mosaic virus Tomato mosaic virus host range

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Heritable Tissue-Culture-Free Gene Editing in Nicotiana benthamiana through Viral Delivery of SpCas9 and sgRNA

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PLANT AND CELL PHYSIOLOGY 2024年第11期65卷 1743-1750页

作者： Yoshida, Tetsuya Ishikawa, Masayuki Toki, Seiichi ishibashi, kazuhiro Natl Agr & Food Res Org Inst Agrobiol Sci 2-1-2 Kannondai Tsukuba Ibaraki 3058518 Japan Ryukoku Univ Fac Agr 1-5 YokotaniSeta Oe Cho Otsu Shiga 5202194 Japan Yokohama City Univ Grad Sch Nanobiosci 22-2 SetoKanazawa Ku Yokohama Kanagawa 2360027 Japan Yokohama City Univ Kihara Inst Biol Res 641-12 Maioka ChoTotsuka Ku Yokohama Kanagawa 2440813 Japan

Conventional plant gene editing requires laborious tissue-culture-mediated transformation, which restricts the range of applicable plant species. In this study, we developed a heritable and tissue-culture-free gene editing method in Nicotiana benthamiana using tobacco ringspot virus (TRSV) as a vector for in planta delivery of Cas9 and single-guide RNA (sgRNA) to shoot apical meristems. Agrobacterium-mediated inoculation of the TRSV vector induced systemic and heritable gene editing in Nicotiana benthamiana PHYTOENE DESATURASE. Transient downregulation of RNA silencing enhanced gene editing efficiency, resulting in an order of magnitude increase (0.8-13.2%) in the frequency of transgenerational gene editing. While the TRSV system had a preference for certain sgRNA sequences, co-inoculation of a TRSV vector carrying only Cas9 and a tobacco rattle virus vector carrying sgRNA successfully introduced systemic mutations with all five tested sgRNAs. Extensively gene-edited lateral shoots occasionally grew from plants inoculated with the virus vectors, the transgenerational gene editing frequency of which ranged up to 100%. This virus-mediated heritable gene editing method makes plant gene editing easy, requiring only the inoculation of non-transgenic plants with a virus vector(s) to obtain gene-edited individuals.

关键词： Gene editing Nepovirus Plant virus Virus vector

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Tobamoviruses: old and new threats to tomato cultivation

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JOURNAL OF GENERAL PLANT PATHOLOGY 2023年第6期89卷 305-321页

作者： ishibashi, kazuhiro Kubota, Kenji Kano, Akihito Ishikawa, Masayuki NARO Inst Agrobiol Sci Div Plant Mol Regulat Res 2-1-2 Kannondai Tsukuba Ibaraki 3058518 Japan NARO Inst Plant Protect Div Core Technol Pest Control Res 2-1-18 Kannondai Tsukuba Ibaraki 3058666 Japan Takii & Co Ltd Plant Breeding & Expt Stn Konan Shiga 5203231 Japan

Mosaic diseases caused by tobamoviruses have posed significant threats to tomato production. In this review, we overview studies of tomato mosaic diseases published over the past century, which have led to several important discoveries in plant virology, such as the application of attenuated strains. A resistance breeding program established in the 1970s successfully controlled tomato mosaic virus for over 40 years;however, newly emerging tobamoviruses are posing serious challenges in current tomato production. We introduce recent biotechnological attempts to engineer tobamovirus-resistant tomato plants, which offer promising technologies for eradicating the current outbreak.

关键词： Tobamovirus Tomato mosaic virus Tobacco mosaic virus Tomato brown rugose fruit virus Tomato mottle mosaic virus

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Host membrane proteins involved in the replication of tobamovirus RNA

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CURRENT OPINION IN VIROLOGY 2012年第6期2卷 699-704页

作者： ishibashi, kazuhiro Miyashita, Shuhei Katoh, Etsuko Ishikawa, Masayuki Natl Inst Agrobiol Sci Div Plant Sci Tsukuba Ibaraki 3058602 Japan Japan Sci & Technol Agcy JST Precursory Res Embryon Sci & Technol PRESTO Kawaguchi Saitama 3320012 Japan Natl Inst Agrobiol Sci Agrogen Res Ctr Tsukuba Ibaraki 3058602 Japan

Eukaryotic positive-strand RNA viruses replicate their genomes in membrane-bound replication complexes composed of viral replication proteins and negative-strand RNA templates. These replication proteins are programmed to exhibit RNA polymerase and other replication-related activities only in replication complexes to avoid inducing double-stranded RNA-mediated host defenses. Host membrane components (e.g. proteins and lipids) should play important roles in the activation of replication proteins. Two host membrane proteins are components of the replication complex and activate the replication proteins of tobamoviruses. Interaction analyses using deletion mutants constructed based on structural information suggest a conformational change in replication proteins during the formation of a protein complex with RNA 5'-capping activity.

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