A Protein Interaction Map of Soybean Mosaic Virus Strain G7H Based on the Yeast Two-Hybrid System

김국형,Sung-Hwan Kang,Won-Seok Lim 한국분자세포생물학회 2004 Molecules and cells Vol.18 No.1

A protein interaction map of Soybean mosaic virus (SMV) strain G7H was generated by the yeast twohybrid system. Clones encoding the genes P1, HC-Pro, P3, 6K1, CI, 6K2, VPg, NIa, NIb, and CP were fused downstream of the GAL4 binding domain (GAL4-BD) and of the GAL4 activation domain (GAL4-AD). The GAL4-BD and GAL4-AD fusion derivatives of each gene were co-transformed into yeast and transformants in which interaction took place were identified on selective media. Interacting fusion proteins were extracted from the yeast cells, run on SDS-PAGE gels and finally checked by Western blotting with GAL4 polyclonal antibodies. Strong interactions were detected between the pairs CP/CP, HC-Pro/HC-Pro, NIa/NIa, and CP/HC-Pro. Relatively weak but significant interaction was detected between VPg and NIa. Although not all of the protein-protein interactions previously reported in other potyviruses were detected, the interactions revealed here were, in general, similar to those reported previously.

Destruction of Cucumber Green Mottle Mosaic Virus by Heat Treatment and Rapid Detection of Virus Inactivation by RT-PCR

김국형,김상민,Sang-Hyun Nam,Jung-Myung Lee,Kyu-Ock Yim 한국분자세포생물학회 2003 Molecules and cells Vol.16 No.3

Nucleotide Sequences of Two Korean Isolates of Cucumber Green Mottle Mosaic Virus

김국형,김상민,이정명,Kyu-Ock Yim,Man-Ho Oh,Jin-Woo Park 한국분자세포생물학회 2003 Molecules and cells Vol.16 No.3

Highly Specific Detection of Five Exotic Quarantine Plant Viruses using RT-PCR

최호성,김국형,조원경,유지숙,이종승 한국식물병리학회 2013 Plant Pathology Journal Vol.29 No.1

To detect five plant viruses (Beet black scorch virus, Beet necrotic yellow vein virus, Eggplant mottled dwarf virus,Pelargonium zonate spot virus, and Rice yellow mottle virus) for quarantine purposes, we designed 15 RT-PCR primer sets. Primer design was based on the nucleotide sequence of the coat protein gene, which is highly conserved within species. All but one primer set successfully amplified the targets, and gradient PCRs indicated that the optimal temperature for the 14 useful primer sets was 51.9 oC. Some primer sets worked well regardless of annealing temperature while others required a very specific annealing temperature. A primer specificity test using plant total RNAs and cDNAs of other plant virusinfected samples demonstrated that the designed primer sets were highly specific and generated reproducible results. The newly developed RT-PCR primer sets would be useful for quarantine inspections aimed at preventing the entry of exotic plant viruses into Korea.

RT-PCR Detection of Five Quarantine Plant RNA Viruses Belonging to Potyand Tospoviruses

이종승,김국형,최홍수,조원경 한국식물병리학회 2011 Plant Pathology Journal Vol.27 No.3

In order to detect quarantine plant viruses, we developed reverse transcription-polymerase chain reaction (RT-PCR) primer pairs for five single-stranded (ss)plant RNA viruses that are not currently reported in Korea but could be potential harmful plant viral pathogens. Three viruses such as Chilli veinal mottle virus (ChiVMV), Colombian datura virus (CDV), and Tobacco etch virus (TEV) belong to the genus Potyvirus while Chrysanthemum stem necrosis virus (CSNV) and Iris yellow spot virus (IYSV) are members of the genus Tospovirus. To design RT-PCR primers, we used reported gene sequences corresponding to the capsid protein and polyprotein for ChiVMV, CDV, and TEV while using nucleocapsid protein regions for CSNV and IYSV. At least two different primer pairs were designed for each virus. Fifteen out of 16 primer pairs were successfully applied in detection of individual quarantine virus with high specificity and efficiency. Taken together, this study provides a rapid and useful protocol for detection of five quarantine viruses.

Complete Genome Sequences of the Genomic RNA of Soybean mosaic virus Stranins G7H and G5

임원석,김국형,김유호 한국식물병리학회 2003 Plant Pathology Journal Vol.19 No.3

The complete nucleotide sequences of the genomic RNAs of Soybean mosaic virus strains G5 (SMV-G5) and G7H (SMV-G7H) were determined and compared with sequences of other SMV strains. Each viral RNA was determined to be 9588 nucleotides in length excluding the poly (A) tail and contained an open reading frame to encode a polyprotein subsequently processed into up to ten proteins by proteolytic cleavage. Comparison of the amino acid sequences with those of other SMV strains showed high percentage of amino acid sequence homology with the same genome organization. The nucleotide and the deduced amino acid sequences between SMV-G5 and SMV-G7H were greater than 99% identity. When compared with those of other SMV strains in a phylogenetic analysis of the nucleotide and deduced amino acid sequences, they formed a distinct virus clade showing over 97% amino acid identity, but were more distantly related to the other potyvirus (44.1- 69.6% identity). Interestingly, SMV G7H strain caused a severe mosaic or necrosis symptom in soybean cultivars including Jinpum-1, Jinpum-2, and Sodam, whereas, no symptom was observed in SMV-G5 inoculation. Complete nucleotide sequences of these strains will give clues for determining symptom determinant(s) in future research.

Agroinfiltration-based Potato Virus X Replicons to Dissect the Requirements of Viral Infection

박상호,김국형 한국식물병리학회 2006 Plant Pathology Journal Vol.22 No.4

performed in in vitro transcription system using the bacteriophage T7 promoter. We constructed an efficient T-DNA based binary vector, pSNU1, and modified vectors carrying PVX replicons. The suitability of the construct to transiently express PVX RNA using Agrobacterium tumefaciens was tested by analysis of infectivity in plants. The expressed PVX RNA was infectous and systemically spread in three plant species including Nicotiana benthamiana, N. tabacum cv. Xanthi-nc, and Capsicum annuum cv. Chilsungcho. The PVX full length construct, pSPVXp31, was caused severe mosaic symptoms on N. benthamiana, severe necrotic lesions on C. annuum while milder symptoms and delayed mosaic symptoms were appeared on the systemic leaves on N. tabaccum. RT-PCR analysis confirmed the presence of PVX RNAs on both inoculated and systemic leaves in all three plant species tested. Our results indicated that PVX replicons were efficiently expressed PVX RNA in at least three tested species. Further investigation will be needed to elucidate the mechanism of PVX replication, translation, movement and assembly/disassembly processes.

Development of RT-PCR Based Method for Detecting Five Non-reported Quarantine Plant Viruses Infecting the Family Cucurbitaceae or Solanaceae

이종승,김국형,조원경,이수헌,최홍수 한국식물병리학회 2011 Plant Pathology Journal Vol.27 No.1

For quarantine purpose, we selected five plant RNA viruses including Cucumber vein yellowing virus (CVYV),Cucurbit yellow stunting disorder virus (CYSDV), Potato aucuba mosaic virus (PAMV), Potato yellow dwarf virus (PYDV), and Tomato chlorosis virus (ToCV), which are not reported in Korea and cause serious economic losses to the family Cucurbitaceae or Solanaceae. To detect those viruses, we employed RT-PCR technique with specific oligonucleotide primer pairs and tested their detection efficiency for each virus. To design RT-PCR primers,coat protein was used for CVYV, CYSDV, and ToCV whereas RNA polymerase and nucleocapsid regions were used for PAMV and PYDV, respectively. The development of an RT-PCR based method proved a useful tool for rapid detection and identification of quarantine virus infections.

Cis-acting Elements in the 3' Region of Potato virus X are Required for HostProtein Binding

권선정,김국형,Cynthia Hemenway 한국식물병리학회 2006 Plant Pathology Journal Vol.22 No.2

The 3' region of Potato virus X (PVX) has the 74 nt 3'- nontranslated region (NTR) that is conserved among all potexviruses and contains several cis-acting elements for minus-strand and plus-strand RNA accumulation. Three stem-loop structures (SL1-SL3), especially formation of SL3 and U-rich sequence of SL2, and near upstream elements in the 3' NTR were previously demonstrated as important cis-acting elements. To investigate the binding of these cis-acting elements within 3' end with host protein, we used the electrophoretic mobility shift assays (EMSA) and UV-cross linking analysis. The EMSA with cellular extracts from tobacco and RNA transcripts corresponding to the 150 nt of the 3' end of PVX RNA showed that the 3' end of PVX formed complexes with cellular proteins. The specificity of protein binding was confirmed through competition assay by using with 50-fold excess of specific and non-specific probes. We also conducted EMSA with RNAs containing various mutants on those cis-acting elements (Δ10, SL3B, SL2A and Δ21; J Mol Biol 326, 701-720) required for efficient PVX RNA accumulation. These analyses supported that these cisacting elements are required for interaction with host protein(s). UV-cross linking analysis revealed that at least three major host proteins of about 28, 32, and 42 kDa in mass bound to these cis-elements. These results indicate that cis-acting elements from 3' end which are important for minus and plus-strand RNA accumulation are also required for host protein binding.

식물 바이러스 증식에 관여하는 기주 요인의 중요성

박미리,김국형 한국식물병리학회 2005 식물병연구 Vol.11 No.2

All viruses have few genes relative to their hosts. Viruses, thus, utilize many host factors for efficient viral replication in host cell. Virus-host interactions are crucial determinations of host range, replication, and pathology. Host factors participate in most steps of positive-strand RNA virus infection, including entry, viral gene expression, virion assembly, and release. Recent data show that host factors play important roles in assembling the viral RNA replication complex, selecting and recruiting viral RNA replication templates, activating the viral complex for RNA synthesis, and the other steps. These virus-host interactions may contribute to the host specificity and/or pathology. Positive-strand RNA viruses encompass over two-thirds of all virus genera and include numerous pathogens. This review focuses on the importance of host factors involved in positivestrand plant RNA virus genome replication. 기주 식물체 내에서 식물바이러스의 증식과 이동 여부는 바이러스 게놈과 기주 간의 상호작용에 의해 결정된다. 바이러스는 기주 내에서 바이러스가 증식하고 이동하기 위해서는 기주의 요소들을 이용해야 하며, 이러한 기주 요소들은 바이러스의 기주내 침입(entry), 바이러스 유전자의 발현, 그리고 바이러스 입자형성(virion assembly) 등 모든 과정에서 직접적으로 관여를 하거나, 또는 기주 단백질 발현과 저항성을 조절하여 바이러스 증식에 간접적으로 관여를 한다. 기주 요소들과 상호작용을 통해서 바이러스 증식에 관여함으로써, 기주 특이성 및 바이러스의 병 발생에 관여를 할 것으로 보고 있다.