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Emi Ota,Chikara Masuta,Minoru Takeshita 한국식물병리학회 2023 Plant Pathology Journal Vol.39 No.6
A defective RNA3 (D3Yα) of strain Y of cucumber mosaic virus (CMV-Y) was examined on host-specific maintenance, experimental conditions, and a viral factor required for its generation in plants. D3Yα was stably maintained in cucumber but not in tomato plants for 28 days post inoculation (dpi). D3Yα was generated in Nicotiana tabacum or N. benthamiana after prolonged infection in the second and the third passages, but not in plants of N. benthamiana grown at low temperature at 28 dpi or infected with CMV-Y mutant that had the 2b gene deleted. Collectively, we suggest that generation and retention of D3Yα depends on potential host plants and experimental conditions, and that the 2b protein has a role for facilitation of generation of D3Yα.
김보민,Jun-ichi Inaba,Chikara Masuta 한국원예학회 2011 Horticulture, Environment, and Biotechnology Vol.52 No.2
The Arabidopsis gene AINTEGUMENTA (At-ANT) functions in cell proliferation and organ growth. The ANT protein has two copies of the AP2 domains, R1 and R2. Recently, a partial cDNA sequence of the At-ANT homolog in Antirrhinum majus (Am-ANT) was reported (Delgado-Benarroch et al., 2009). Here, we used virus-induced gene silencing (VIGS) to analyze the function of the reported Am-ANT. We then determined the open reading frame (ORF) of Am-ANT and its predicted amino acid sequence. We induced VIGS using the Cucumber mosaic virus vector (CMV-A1) that contained partial sequence of Am-ANT (A1:ANT) and suppressed the level of Am-ANT mRNA and noted for any phenotypic changes. The function of Am-ANT was very similar to that of At-ANT. The A1:ANT-infected Antirrhinum plants had smaller floral organs and leaves, even though cell sizes were unchanged in flowers and larger in leaves. The CMV-based VIGS showed that the isolated Am-ANT gene was indeed functional in cell proliferation and organ growth as observed for At-ANT. In conclusion, we found that the CMV vector had the advantage of systemically infecting A. majus without severe symptoms for functional analysis of A. majus genes.
Furuta, Kazuyoshi,Nagashima, Saki,Inukai, Tsuyoshi,Masuta, Chikara The Korean Society of Plant Pathology 2017 Plant Pathology Journal Vol.33 No.1
One of the major problems in strawberry production is difficulty in diagnosis of anthracnose caused by Colletotrichum acutatum or Glomerella cingulata in latent infection stage. We here developed a diagnostic tool for the latent infection consisting of initial culturing of fungi, DNA extraction, synthesis of PCR-amplified probes and microtube hybridization (MTH) using a macroarray. The initial culturing step is convenient to lure the fungi out of the plant tissues, and to extract PCR-inhibitor-free DNA directly from fungal hyphae. For specific detection of the fungi, PCR primers were designed to amplify the fungal MAT1-2 gene. The subsequent MTH step using the PCR products as probes can replace the laborious electrophoresis step providing us sequence information and high-throughput screening. Using this method, we have conducted a survey for a few thousands nursery plants every year for three consecutive years, and finally succeeded in eliminating latent infection in the third year of challenge.
홍진성,리선주,김은지,김태성,류기현,Chikara Masuta,이긍표 한국식물병리학회 2012 Plant Pathology Journal Vol.28 No.1
We developed a reassortant RNA virus vector derived from Cucumber mosaic virus (CMV), which has advantages of very wide host range and can efficiently induce gene silencing in a few model plants. Certain CMV isolates, however, show limited host ranges presumably because they naturally co-evolved with their own hosts. We used a reassortant comprised of two strains of CMV,Y-CMV and Gn-CMV, to broaden the host range and to develop a virus vector for virus-induced gene silencing (VIGS). Gn-CMV could infect chili pepper and tomato more efficiently than Y-CMV. Gn-CMV RNA1, 3 and Y-CMV RNA2-A1 vector were newly reconstructed,and the transcript mixture of RNA1 and 3 genomes of Gn-CMV and RNA2 genome of Y-CMV RNA2 containing portions of the endogenous phytoene desaturase (PDS) gene (CMV2A1::PDSs) was inoculated onto chili pepper (cv. Chung-yang), tomato (cvs. Bloody butcher,Tigerella, Silvery fir tree, and Czech bush) and Nicotiana benthamiana. All the tested plants infected by the reassortant CMV vector showed typical photo-bleaching phenotypes and reduced expression levels of PDS mRNA. These results suggest that the reassortant CMV vector would be a useful tool for the rapid induction of the RNA silencing of endogenous genes in chili pepper and tomato plants.
Kazuyoshi Furuta,Shusuke Kawakubo,Jun Sasaki,Chikara Masuta 한국식물병리학회 2024 Plant Pathology Journal Vol.40 No.1
Garlic can be infected by a variety of viruses, but mixed infections with leek yellow stripe virus, onion yellow dwarf virus, and allexiviruses are the most damaging, so an easy, inexpensive on-site method to simultaneously detect at least these three viruses with a certain degree of accuracy is needed to produce virus-free plants. The most common laboratory method for diagnosis is multiplex reverse transcription polymerase chain reaction (RT-PCR). However, allexiviruses are highly diverse even within the same species, making it difficult to design universal PCR primers for all garlic-growing regions in the world. To solve this problem, we developed an inexpensive on-site detection system for the three garlic viruses that uses a commercial mobile PCR device and a compact electrophoresis system with a blue light. In this system, virus-specific bands generated by electrophoresis can be identified by eye in real time because the PCR products are labeled with a fluorescent dye, FITC. Because the electrophoresis step might eventually be replaced with a lateral flow assay (LFA), we also demonstrated that a uniplex LFA can be used for virus detection; however, multiplexing and a significant cost reduction are needed before it can be used for on-site detection.
Kazuyoshi Furuta,Saki Nagashima,Tsuyoshi Inukai,Chikara Masuta 한국식물병리학회 2017 Plant Pathology Journal Vol.33 No.1
One of the major problems in strawberry productionis difficulty in diagnosis of anthracnose caused by Colletotrichumacutatum or Glomerella cingulata in latentinfection stage. We here developed a diagnostic toolfor the latent infection consisting of initial culturingof fungi, DNA extraction, synthesis of PCR-amplifiedprobes and microtube hybridization (MTH) using amacroarray. The initial culturing step is convenient tolure the fungi out of the plant tissues, and to extractPCR-inhibitor-free DNA directly from fungal hyphae. For specific detection of the fungi, PCR primers weredesigned to amplify the fungal MAT1-2 gene. The subsequentMTH step using the PCR products as probescan replace the laborious electrophoresis step providingus sequence information and high-throughputscreening. Using this method, we have conducted asurvey for a few thousands nursery plants every yearfor three consecutive years, and finally succeeded ineliminating latent infection in the third year of challenge.
Yamaguchi, Naoya,Seshimo, Yuko,Yoshimoto, Eri,Ahn, Hong Il,Ryu, Ki Hyun,Choi, Jang Kyung,Masuta, Chikara Microbiology Society 2005 The Journal of general virology Vol.86 No.8
<P>Five isolates of Cucumber mosaic virus (CMV) from Lilium sp. (lily), which were isolated from specimens in Japan, Korea and Taiwan, were unable to support satellite RNA (satRNA) accumulation. In order to map the CMV sequences that are involved in satRNA support, HL-CMV (Japanese lily isolate), Y-CMV (ordinary strain) and Y-satellite RNA (Y-sat) were used as the source material. The pseudorecombinants between Y-CMV and HL-CMV revealed that RNA1 was essential for satRNA replication in lily. The results of chimeric constructs and various mutations showed that two amino acid residues (at positions 876 and 891) in the 1a protein were the determinants for the inability of HL-CMV to support a satRNA. Specifically, Thr at position 876 had a more pronounced effect than Met at position 891. Specific changes in RNA sequence were also detected in the 3' terminus of Y-sat and these particular alterations allowed it to be supported by HL-CMV. It is believed that, through evolution, the adaptation of CMV to lily resulted in the introduction of amino acid changes in the 1a protein, changes that coincidentally affected the ability of lily CMV to support satRNAs.</P>
Hong, Jin-Sung,Rhee, Sun-Ju,Kim, Eun-Ji,Kim, Tae-Sung,Ryu, Ki-Hyun,Masuta, Chikara,Lee, Gung-Pyo The Korean Society of Plant Pathology 2012 Plant Pathology Journal Vol.28 No.1
We developed a reassortant RNA virus vector derived from $Cucumber$ $mosaic$ $virus$ (CMV), which has advantages of very wide host range and can efficiently induce gene silencing in a few model plants. Certain CMV isolates, however, show limited host ranges presumably because they naturally co-evolved with their own hosts. We used a reassortant comprised of two strains of CMV, Y-CMV and Gn-CMV, to broaden the host range and to develop a virus vector for virus-induced gene silencing (VIGS). Gn-CMV could infect chili pepper and tomato more efficiently than Y-CMV. Gn-CMV RNA1, 3 and Y-CMV RNA2-A1 vector were newly reconstructed, and the transcript mixture of RNA1 and 3 genomes of Gn-CMV and RNA2 genome of Y-CMV RNA2 containing portions of the endogenous phytoene desaturase (PDS) gene (CMV2A1::PDSs) was inoculated onto chili pepper (cv. Chung-yang), tomato (cvs. Bloody butcher, Tigerella, Silvery fir tree, and Czech bush) and $Nicotiana$ $benthamiana$. All the tested plants infected by the reassortant CMV vector showed typical photo-bleaching phenotypes and reduced expression levels of $PDS$ mRNA. These results suggest that the reassortant CMV vector would be a useful tool for the rapid induction of the RNA silencing of endogenous genes in chili pepper and tomato plants.