• The Plant Pathology Journal
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• 제20권3호
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• pp.206-211
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• 2004

Transgenic Nicotiana benthamiana plants harboring coat protein (CP) gene of Zucchini green mottle mosaic virus (ZGMMV) were generated for virus-resistant screening and complementation analysis of related viruses for environmental safety assessment (SA) of living modified organism (LMO) purposes. Transformation of leaf disc of N.benthamiana was performed by using Agrobacterium-mediated method and the pZGC-PPGA748 containing the ZGMMV CP and NPTII genes. Two kinds of transgenic homozygous groups, virus-resistant and virus-susceptible N.benthamiana lines, were obtained by screening of challenging homologous virus for Tl generations. These two pathologically different lines can be useful for host-virus interactions and LMO environmental SA.

• The Plant Pathology Journal
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• 제19권3호
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• pp.159-161
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• 2003

The coat protein (CP) gene of Apple mosaic virus (ApMV), a member of the genus Ilarvirus, was selected for the design of virus-specific primers for amplification and molecular detection of the virus in cultivated apple. A combined assay of reverse transcription and polymerase chain reaction (RT-PCR) was performed with a single pair of ApMV-specific primers and crude nucleic acid extracts from virus-infected apple for rapid detection of the virus. The PCR product was verified by restriction mapping analysis and by sequence determination. The lowest concentration of template viral RNA required for detection was 100 fg. This indicates that the RT-PCR for detection of the virus is a 10$^3$times more sensitive, reproducible and time-saving method than the enzyme-linked immunosorbent assay. The specificity of the primers was verified using other unrelated viral RNAs. No PCR product was observed when Cucumber mosaic virus (Cucumovirus) or a crude extract of healthy apple was used as a template in RT-PCR with the same primers. The PCR product (669 bp) of the CP gene of the virus was cloned into the plasmid vector and result-ant recombinant (pAPCP1) was selected for molecule of apple transformation to breed virus-resistant transgenic apple plants as the next step. This method can be useful for early stage screening of in vitro plantlet and genetic resources of resistant cultivar of apple plants.

• The Plant Pathology Journal
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• 제18권6호
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• pp.317-322
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• 2002

An isolate of Odontoglossum ringspot virus (ORSV) was identified from Cymbidium var. 'Grace Kelly' showing ringspot symptom on the floral and leaf parts, and was denoted as cymbidium ringspot isolate (ORSV-CR). In ultrathin sections of leaf tissue from diseased Cymbidium plants, clusters of virus particles were observed in the vacuole and cytoplasm. In the Western blot hybridization, the virus strongly reacted with ORSV-specific antiserum indistinguishable from ORSV, suggesting that the vims is serologically identical with ORSV. ORSV-CR sap was inoculated onto 20 species belonging to 12 genera. Systemic infection occurred in Cymbidium sp., Nicotiana benthamiana and N. clevelandii, the host of which was found to be different from that of ORSV-Cy, the Korean strain of ORSV. The analysis of coat protein (CP) gene showed that ORSV-CR was highly homologous to the known isolates of ORSV, with over 95.6% identity in amino acid level. Phylogenetic tree analysis of CP showed that ORSV-CR was clustered with the known ORSV isolates, suggesting that ORSV is a very stable tobamovirus.

• The Plant Pathology Journal
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• 제22권3호
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• pp.260-264
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• 2006

Talaromyces flavus mediates the transmission of Apple stem grooving virus (ASGV) to several host plants. The ASGV-F carried by T.flavus was partially purified from the fungus. Based on sequence analysis and homology searches, this is closely related to other ASGV strains isolated from host plants. The partially purified viral coat protein (CP) was separated on a 12% SDS-polyacrylamide gel and analyzed by Western blotting with an ASGV anti-serum. A single band at 28 kDa reacted with the ASGV anti-serum. The deduced amino acid sequence of the ORF-l showed conserved domains, including an NTP-binding helicase motif, GFAGSGKT. The amino acid sequences of the helicase and CP showed strong homology to other ASGV strains (98%). All ASGV isolated from plants and fungi had salt bridges composed of the CP and the GFAGSGKT motif of the helicase, which are commonly conserved in plant viruses. These results suggest that ASGV-F is one of ASGV strains isolated from T.flavus based on sequence similarity as well as the serological analysis of CP.

• The Plant Pathology Journal
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• 제18권3호
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• pp.130-137
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• 2002

Potato virus Y (PVY) is one of the most important viruses in many field crops in Korea. In this study, 31 PVY isolates were isolated from infected potato (Solanum tuberosum), tobacco (Nicotiana tabacum), pea (Pisum sativum), and weeds (Veronica persica, Lamium amplexicause and Capsella bursa-pastoris) showing different mosaic symptoms in Jeonbuk, Chungnam, Gangwon, and Gyeongbuk areas in Korea. The 640 nucleotide region containing the C-terminal portion of coat protein (CP) gene and 3'non-translated region (NTR) was amplified by reverse transcription-polymerase chain reaction (RT-PCR) using PVY-specific oligonucleotide primers. Sequence analyses of the amplified DNA fragments showed that the C-terminal portion of CP gene was not significantly different from that of previously reported PVY strains from potato (PVY-OK and -T) and tobacco (PVY-VN) in Korea. Homologies of the deduced CP amino acid sequences were 93.3-99.0% to corresponding regions of the other PVY strains including PV $Y^{N}$, PV $Y^{o}$ , PV $Y^{OK}$ , PV $Y^{T}$ , and PV $Y^{VN}$ . In contrast the sequences located at the 3'-NTR showed more diverse sequence homologies (76.4-99.7%). These results indicate that the C-terminal portion of the CP gene was relatively conserved while sequences at the 3'NTR were more diverse and variable over the host species and the regions where they were isolated.e isolated.

• The Plant Pathology Journal
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• 제16권4호
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• pp.231-235
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• 2000

The 3,000 nucleotides of 3'-terminal region of the genomic RNA of a new isolate of carlavirus from a Korean native lily (Lilum lancitoium) was cloned and its nucleotide sequences were determined. The coat protein (CP) gene of the virus showed 72.0% to 72.8% nucleotide sequence identities and 86.9% to 88.0% amino acid sequence identities with those of the four strains (two Korean, one Dutch, and one Japanese isolates) of lily symptomless virus (LSV). Interestingly, different amino acid sequences between the new isolate and LSV strains were located at the N-terminal region of the CP. Pairwise amino acid sequence comparison of the CP gene revealed sequence identities of 22.0% to 71.1% between the virus and other 9 carlavirus species. The 25 kDa and 12 kDa proteins genes of the virus share 30.7% to 76.3% and 31.1% to 85.8% amino acid sequence identities, respectively, with those of 8 other carlaviruses. The 16 kDa protein gene of the virus shares 16.7% to 72.9% amino acid sequence identities with that of 9 other carlaviruses. These data indicate that the virus, designated as lily latent virus (LiLV), is a distinct of the Carlavirus genus and distinguished from the known strains of LSV.

• The Plant Pathology Journal
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• 제23권4호
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• pp.281-286
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• 2007

Interactions between viral proteins and host proteins are essential for virus replication. Especially, translation of viral genes completely depends on the host machinery. In potyviruses, interactions of genome-linked viral protein (VPg) with host translation factors including eIF4E, eIF(iso)4E, and poly(A)-binding protein (PABP) has previously been characterized. In this study, we investigated interactions between Soybean mosaic virus (SMV) viral proteins and host translation factors by yeast two-hybrid system. SMV VPg interacted with eIF4E, eIF(iso)4E, and PABP in yeast two-hybrid system, while SMV helper component proteinase (HC-pro) interacted with neither of those proteins. The interaction between SMV NIb and PABP was also detected. These results are consistent with those reported previously in other potyviruses. Interestingly, we found reproducible and specific interactions between SMV coat protein (CP) and PABP. Deletion analysis showed that the region of CP comprising amino acids 116 to 206 and the region of PABP comprising amino acids 520 to 580 are involved in CP/PABP interactions. Soybean library screening with SMV NIb by yeast two-hybrid assay also identified several soybean proteins including chlorophyll a/b binding preprotein, photo-system I-N subunit, ribulose 1,5-biphosphate carboxylase, ST-LSI protein, translation initiation factor 1, TIR-NBS type R protein, RNA binding protein, ubiquitin, and LRR protein kinase. Altogether, these results suggest that potyviral replicase may comprise a multi-protein complex with PABP, CP, and other host factors.

• Applied Biological Chemistry
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• 제38권1호
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• pp.49-54
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• 1995

한국 마늘에 감염된 바이러스의 종류와 병 발생 메카니즘을 구명하기 위하여, 마늘 바이러스 cDNA clone들을 분리하였다. 24개 cDNA clone들의 부분적인 염기 서열을 결정하였고, 이 중 poly(A) tail을 가진 5개 clone들의 염기 서열을 결정하였다. 이를 이미 알려진 다른 식물 바이러스와 비교했을 때, clone V9은 일차구조가 carlavirus와 유사성을 보이므로 GLV cDNA clone으로 여겨진다. Northern blot 결과로부터 GLV genome의 크기는 8.5 knt이고, poly(A) tail을 가지고 있다는 것을 알 수 있었다. clone V9의 3' 말단부분에는 바이러스 복제과정에서 cis-acting element로 작용한다고 여겨지는 hexanucleotide motif(5'-ACCUAA)가 존재한다. 또한 carlavirus의 껍질 단백질 subgenomic RNA의 5' 말단에 보존되어 있는 5'-TTAGGT도 나타난다. 이들은 모두 carlavirus의 특징들이다. 껍질 단백질 유전자를 pRSET-A 발현 벡터에 재조합하고, E. coli BL21에서 발현시켰다. 발현된 껍질 단백질을 $Ni^{2+}$ NTA affinity chromatography에 의해 정제하였다. 껍질 단백질을 토끼에 주사하여 항체를 만든 후, immunoblot을 한 결과 GLV 껍질 단백질에 해당하는 24 kDa polypeptide가 인지되었다. 또한 다양한 마늘 품종에 대해서 immunoblot을 한 결과, GLV 껍질 단백질의 크기와 GLV의 감염정도가 마늘 품종에 따라서 차이가 있다는 것을 알 수 있었다.

• 한국식품저장유통학회지
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• 제22권6호
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• pp.901-907
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• 2015

CMV 바이러스 저항성 H15 고추 도입유전자의 발현산물인 CMV-CP 단백질의 80개씩의 아미노산 비교에서 35% 보다 큰 상동성을 갖거나 8개 이상의 일련된 아미노산 염기 서열이 일치하는 알레르겐은 존재하지 않는 것이 확인되었으며, 따라서 CMV-CP 단백질 서열은 기지의 알레르겐과 구조적 및 면역학적으로 연관된 유사성이 없음을 확인하였다. 형질전환에 따른 특성 변화를 비교하기 위해 서로 다른 지역에서 재배된 CMV 바이러스 저항성 H15 고추 및 그 모본의 발현 단백질 항원 농도와 그 분포를 비교한 결과 발현된 단백질의 양상과 양은 재배 연도 및 환경적 영향에 따른 차이가 발견되었지만, 동일한 연도에 같은 장소에서 재배된 유전자변형 고추와 그 모본 사이에서의 차이는 발견되지 않아 형질전환으로 인한 특성변화는 없는 것으로 확인 되었다. 환자 혈청을 이용해 immunoblotting법과 ELISA법으로 확인한 항원-항체 반응성 비교에서도 CMV 바이러스 저항성 H15 고추와 그 모본 P2377사이에 차이가 없는 것이 확인되었다. 이러한 결과를 종합할 때, 유전자변형 고추 H15는 그 모본과 비교하여 형질전환으로 인한 알레르기성의 변화는 없는 것으로 판단된다.

• The Plant Pathology Journal
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• 제21권2호
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• pp.167-171
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• 2005

We isolated the Cucumber green mottle mosaic virus(CGMMV) particles from watermelon leaves and designated as CGMMV-HY1 as a watermelon isolate and attempted to characterize the pathogenic isolate responsible for such an epidemic in watermelon and also to monitor dominant viral isolates in greenhouse. The watermelon plants infected with CGMMV generally showed mottle mosaic, mosaic, growth stunting, necrosis and deformed fruit. The reactions of indicator plants to CGMMV-HY1 were the local lesions on Nicotiana tabacum cv. White Burley, Nicotiana tabacum cv. Samsun, and Chenopodium amaranticola, and the mosaic symptoms only on Cucumis sativus, but the CGMMV-HY1 did not infect Nicotiana sylvesytis, Datura stramonium, Chenopodium quinoa, and Petunia hybrida. Purified virus particles were rod-shaped and about 300 nm long. The coat protein (CP) of purified CGMMV-HY1 was single band with molecular weight of about 16.5 kDa which was confirmed by western blot analysis probed with monoclonal antibody of CGMMV-HY1. The genomic and subgenomic RNAs of 6.4 kb and 0.75 kb were revealed by the electrophoresis on 1.2% formaldehydedenatured agarose gel. Viral and complementary CGMMV-specific primer sets were designed for spanning the genome using previously reported CGMMV sequences. A 464bp of CP gene of CGMMV-HY1 was amplified by RT-PCR and cloned into PGEM-T easy vector. The nucleotide sequence of CP gene of CGMMV-HY1 shared 98%, 99%, and 100% identities with that of CGMMV strains W, KOM, and KW respectively. Based on these results, we identified CGMMV-HY1 as a CGMMV isolate of watermelon, a member of Tobamovirus.