• Journal of Plant Biotechnology
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• 제6권1호
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• pp.9-13
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• 2004

Transient uidA expression was used to optimize parameters required for biolistic transformation of suspension cells of Easter lily, Lilium longiflourm. Maximum uidA expression occurred following bombardment with gold particles as compared to tungsten. A 3hr pre-treatment of suspension cells with 0.125M osmoticum resulted in a 1.5X increase in uidA expression. A helium pressure of 1550 psi combined with a particle travelling distance of 6cm resulted in maximum uidA expression as compared to either 1100, 1200, or 1800 psi. Transient transformation resulted in up to 493 uidA expressing cells/Petri plate. For stable transformation suspension cells of Lilium longiflorum, were co-bombarded with plasmid DNA containing cucumber mosaic virus (CMV) replicase under the rice actin (Act1) promoter and either the bar or PAT genes under the cauliflower mosaic virus (CaMV 355) promoter. Ten regenerated plants contained the transgene as analyzed by PCR, and two of the ten plants were confirmed to contain the transgene by Southern hybridization. The two transgenic plants were independent transformants, one containing the bar gene and the other both the CMV replicase and bar genes. Plants were sprayed at the rosette stage and found to be resistant to 1000 mg/L of phosphinothricin (Trade name-Ignite) indicating expression of the bar gene throughout the leaves when bar was under control of the CaMV 35S promoter.

• The Plant Pathology Journal
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• 제37권2호
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• pp.182-193
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• 2021

The transcription factor SHE1 was identified as an interacting partner with the cucumber mosaic virus (CMV) 1a protein in the yeast two-hybrid system, by a pull-down assay, and via bimolecular fluorescent complementation. Using fluorescent-tagged proteins and confocal microscopy, the CMV 1a protein itself was found distributed predominantly between the nucleus and the tonoplast membrane, although it was also found in speckles in the cytoplasm. The SHE1 protein was localized in the nucleus, but in the presence of the CMV 1a protein was partitioned between the nucleus and the tonoplast membrane. SHE1 expression was induced by infection of tobacco with four tested viruses: CMV, tobacco mosaic virus, potato virus X and potato virus Y. Transgenic tobacco expressing the CMV 1a protein showed constitutive expression of SHE1, indicating that the CMV 1a protein may be responsible for its induction. However, previously, such plants also were shown to have less resistance to local and systemic movement of tobacco mosaic virus (TMV) expressing the green fluorescent protein, suggesting that the CMV 1a protein may act to prevent the function of the SHE1 protein. SHE1 is a member of the AP2/ERF class of transcription factors and is conserved in sequence in several Nicotiana species, although two clades of SHE1 could be discerned, including both different Nicotiana species and cultivars of tobacco, varying by the presence of particular insertions or deletions.

• 한국식물병리학회:학술대회논문집
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• 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
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• pp.65.2-65
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• 2003

To protect the plant against several soil-borne pathogens, we are currently constructing disease-resistant transgenic root stock for the growth of cucurbitaceae vegetable plants, watermelon and gourd. We made a watermelon cDNA library from Cladosporium cucumerinum-Infected leaves for substractive hybriazation and differential screening. We isolated the several pathogen inducible cDNA clones, such as caffeoyl-CoA-methyltransferase, LAA induced protein, receptor-like kinase homolog, hydroxyproline-rich glycoprotein, catalase, calmodulin binding protein, mitochondrial ATPase beta subunit, methyl tRNA synthetase and WRKY transcription factors. We previously obtained CaMADS in pepper and galactinol synthase ( CsGolS) in cucumber that were confirmed to be related with disease-resistance. CaMADS and CsGolS2 were transformed into the inbred line 'GO701-2' gourd, the inbred line '6-2-2' watermelon and the Kong-dye watermelon by Agrobacterium tumerfaciens LBA4404. Plant growth regulators (zeatin, BAP and IAA) were used for shoot regeneration and root induction for optimal condition. Putative transgenic plants were selected in medium containing 100mg/L kanamycin and integration of the CaMADS and CsGO/S2 into the genomic DNA were demonstrated by the PCR analysis. We isolated major soil-borne pathogens, such as Monosporascus cannonballus, Didymella bryoniae, Cladosporium cuvumerinum from the cultivation area of watermelon or root stock, and successfully established artificial inoculation method for each pathogen. This work was supported by a grant from BioGreen 21 program, Rural Development Administration, Republic of Korea.

• Journal of Plant Biotechnology
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• 제29권1호
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• pp.7-13
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• 2002

Superoxide dismutase (SOD)를 과실에서 고발현시킨 형질전환 토마토 (서광과 꼬꼬)를 개발하였다. 카사바 배양세포에서 분리한 CuZnSOD (mSOD1)를 과실에 우세적으로 발현하는 ascorbate oxidase promoter (ASOp)를 이용하여 ASOp :: mSOD1/pBI101 벡터를 제작한 후 Agrobacterium 매개로 자엽 절편체를 형질전환하였다. Kanamycin 저항성 식물체를 기관발생 경로로 재분화시킨 후 Southern 분석으로 형질전환을 확인하였다. 서광과 꼬꼬 토마토의 형질전환체와 대조구 식물체의 과실을 성숙 단계별로 분류하여 단백질 함량과 SOD 비활성도 (units/mg protein)를 측정한 결과, 단백질 함량은 열매가 익은 단계로 갈수록 점점 감소하여 완전히 익은 단계에서 가장 낮았다. SOD 비활성도는 형질전환 토마토의 열매의 모든 단계에서 대조구보다 높았으며 완전히 성숙한 과실에서 가장 높았다. 성숙한 형질전환 서광과 꼬꼬 과실에서 SOD 비활성도는 비형질전환의 것보다 각각 약 1.6배와 약 2.2배 높았다. SOD isoenzyme gel 분석에서 도입한 mSOD1로 추정되는 CuZnSOD 밴드가 형질전환체에서 과실 성숙에 따라 강하게 발현되었다. 이상의 결과로서 ASO promoter에 의해 SOD 유전자가 토마토 과실에 특이적으로 발현됨이 확인되었다.

• 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.

• Journal of Microbiology and Biotechnology
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• 제17권2호
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• pp.280-286
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• 2007

We evaluated a commercial biopreparation of plant growth-promoting rhizobacteria (PGPR) strains Bacillus subtilis GB03 and B. amyloliquefaciens IN937a formulated with the carrier chitosan (Bio Yield) for its capacity to elicit growth promotion and induced systemic resistance against infection by Cucumber Mosaic Virus (CMV) and Pseudomonas syringae pv. tomato DC3000 in Arabidopsis thaliana. The biopreparation promoted plant growth of Arabidopsis hormonal mutants, which included auxin, gibberellic acid, ethylene, jasmonate, salicylic acid, and brassinosteroid insensitive lines as well as each wild-type. The biopreparation protected plants against CMV based on disease severity in wild-type plants. However, virus titre was not lower in control plants and those treated with biopreparation, suggesting that the biopreparation induced tolerance rather than resistance against CMV. Interestingly, the biopreparation induced resistance against CMV in NahG plants, as evidenced by both reduced disease severity and virus titer. The biopreparation also elicited induced resistance against P. syringae pv. tomato in the wild-type but not in NahG transgenic plants, which degrade endogenous salicylic acid, indicating the involvement of salicylic acid signaling. Our results indicate that some PGPR strains can elicit plant growth promotion by mechanisms that are different from known hormonal signaling pathways. In addition, the mechanism for elicitation of induced resistance by PGPR may be pathogen-dependent. Collectively, the two-Bacilli strain mixture can be utilized as a biological inoculant for both protection of plant against bacterial and viral pathogens and enhancement of plant growth.

• 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.

• 식물병연구
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• 제8권2호
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• pp.78-83
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• 2002

Tobacco diseases have not been recorded until 1900s in Korea, where tobacco plants were introduced at early 1700s. Practical researches on the disease have been conducted since mid 1960s. Major ten tobacco diseases were mosaic caused by tobacco mosaic virus·potato virus Y·cucumber mosaic virus, bacterial wilt, hollow stalk, wild fire caused by angular leaf spot strain, black shank, brown spot, powdery mildew and fusarium wilt. But their annual occurrences were varied according to changes of tobacco varieties and their cultivating practices. As no useful chemicals, several biological tactics have been developed to control the viral or bacterial diseases that give significant economic damages on sustainable crop yield, but not practicable to field farming condition yet. Transgenic tobacco plants containing foreign disease resistant genes have been developed by current bio-technology, but not released to farmers yet. Though some disease-resistant tobacco varieties have been developed by the conventional breeding technology and currently used by farmers, their disease controlling efficacy have been diminished by occurrence of the new strain or race. Future research on tobacco diseases has been focused on technical development to produce high quality tobacco with less production cost, which leads Korean tobacco industry to keep its competence against foreign industry and decreasing overall market.

• The Plant Pathology Journal
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• 제27권4호
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• pp.310-314
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• 2011

Nonstructural protein 3 (NS3) encoded by RNA3 of Rice stripe virus (RSV), known to be a suppressor of gene silencing, was cloned and sequenced. The cloned NS3 gene is composed of 636 nucleotides encoding 211 deduced amino acids, and showed a high degree of similarity with the equivalent genes isolated from Korea, Japan and China. The NS3 gene promoted the enhancement of transient gene expression and suppressed transgene co-silencing. In the transient GFP expression via agroinfiltration, GFP expression was dramatically enhanced in terms of both protein yield and expression period in the presence of NS3. The highest accumulation of GFP protein reached to 6.8% of total soluble proteins, which corresponded to a two-fold increase compared to that obtained in the absence of NS3. In addition, NS3 significantly suppressed the initiation of GFP co-silencing induced by the additive GFP infiltration in GFP-transgenic Nicotiana benthamiana. The NS3 gene was also found to be a stronger suppressor than Cucumber mosaic virus 2b. These observations are believed to be derived from the strong suppressive effect of NS3 on gene silencing, and indicate that NS3 could be used as an effective enhancer for the rapid production of foreign proteins in plants.

• The Plant Pathology Journal
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• 제29권2호
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• pp.182-192
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• 2013

Numerous root-associated bacteria (rhizobacteria) are known to elicit induced systemic resistance (ISR) in plants. Bacterial cell-density-dependent quorum sensing (QS) is thought to be important for ISR. Here, we investigated the role of QS in the ISR elicited by the rhizobacterium, Serratia marcescens strain 90-166, in tobacco. Since S. marcescens 90-166 produces at least three QS signals, QS-mediated ISR in strain 90-166 has been difficult to understand. Therefore, we investigated the ISR capacity of two transgenic tobacco (Nicotiana tabacum) plants that contained either bacterial acylhomoserine lactone-producing (AHL) or -degrading (AiiA) genes in conjunction with S. marcescens 90-166 to induce resistance against bacterial and viral pathogens. Root application of S. marcescens 90-166 increased ISR to the bacterial pathogens, Pectobacterium carotovorum subsp. carotovorum and Pseudomonas syringae pv. tabaci, in AHL plants and decreased ISR in AiiA plants. In contrast, ISR to Cucumber mosaic virus was reduced in AHL plants treated with S. marcescens 90-166 but enhanced in AiiA plants. Taken together, these data indicate that QS-dependent ISR is elicited by S. marcescens 90-166 in a pathogen-dependent manner. This study provides insight into QS-dependent ISR in tobacco elicited by S. marcescens 90-166.