• Journal of Plant Biotechnology
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• 제7권3호
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• pp.143-148
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• 2005

The coat protein mediated resistance to potato virus Y is assessed here in transgenic potato plants (Solanum tuberosum L., cv Claustar). Therefore, the corresponding cDNA from tunisian isolate of the virus was cloned into Agrobacterium tumefaciens binary vector. The transgenic lines were subsequently analysed for the presence and expression of the transgene. The CP cDNA copy number was determined for kanamycin resistant plants. Three selected transgenic lines and their S1 progeny resulting from tuber germination showed a high protection level against the virus. These data appear to support the hypothesis that the virus resistance is mediated by the translated viral coat protein expressed in transgenic potato lines.

• Plant Biotechnology Reports
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• 제2권1호
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• pp.75-85
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• 2008

Prune dwarf virus (PDV) is an Ilarvirus systemically infecting almond trees and other Prunus species and spreading through pollen, among other means. We have studied strategies based on coat protein (cp) gene to block PDV replication in host plant cells. A Portuguese isolate of PDV was obtained from infected almond leaves and used to produce the cDNA of the cp gene. Various constructs were prepared based on this sequence, aiming for the transgenic expression of the original or modified PDV coat protein (cpPDVSense and cpPDVMutated) or for the expression of cpPDV RNA (cpPDVAntisense and cpPDVwithout start codon). All constructs were tested in a PDV host model, Nicotiana benthamiana, and extensive molecular characterization and controlled infections were performed on transformants and their progenies. Transgenic plants expressing the coat protein RNA were able to block the proliferation of a PDV isolate sharing only 91% homology with the isolate used for cpPDV cloning, as evaluated by DAS-ELISA on newly developed leaves. With cp expression, the blockage of PDV proliferation in newly developed leaves was only achieved with the construct cpPDV Mutated, where the coat protein has a substitution in the 14th aa residue, with arginine replaced by alanine. This result points to a possible role of the mutated amino acid in the virus ability to replicate and proliferate. This work reveals the possibility of achieving protection against PDV through either coat protein RNA or mutated cp sequence.

• BMB Reports
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• 제40권3호
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• pp.404-411
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• 2007

Transformation of cantaloupes with the coat protein (cp) gene of papaya ringspot virus type W (PRSV-W), Thai isolate, was used to introduce virus resistance. Binary vectors containing either the full length coat protein coding region under control of the 35S CaMV promoter(pSA1175), or the inverted-repeat of a coat protein coding region (pSA1304), were constructed and used for Agrobacteriummediated transformation of cotyledonary explants of the cantaloupe cultivar Sun Lady. Four independent transgenic lines were obtained using pSA1304 and one using pSA1175. Integration of the PRSV-W cp gene into the genome of these transgenic lines was verified by PCR amplification, GUS assays and Southern blot hybridization. In vitro inoculation of these lines with PRSV-W revealed that whereas the line containing pSA1175 remained sensitive, the four lines containing pSA1304 were resistant. The presence of small RNA species, presumably siRNA, corresponding to regions of the viral cp gene in transgenic lines resistant to PRSV-W supports the involvement of post-transcriptional gene silencing in the establishment of resistance.

• 한국연초학회지
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• 제19권2호
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• pp.117-123
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• 1997

Viral coat protein (CP) encoding cDNA with artificial start and stop codons was synthesized by reverse-transcriptase polymerase chain reaction (RT-PCR) from the Korean isolate of potato virus Y-vein nectrosis strain (pVY-VN). To make PVY CP cDNA to untranslatable form, three stop codons were inserted near the start codon by "megaprimer-PCR" method. The untranslatable CP cDNA was subcloned to plant expression vector and transferred to N. tabacum cv. NC82 by Agrobacterium-mediated transformation. Highly resistant plants to PVY infection were screened, based on symptom development after mechanical virus inoculation. By genomic PCR and Southern blot analysis, one or more copies of the untranslatable CP gene were found in all transformants. From northern blot analysis, highly resistant transgenic lines had very low level of CP transcript but susceptible lines had high level, suggesting resistance to PVY infection should be related to RNA-mediated mechanism.mechanism.

• 한국동물학회:학술대회논문집
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• 한국동물학회 1997년도 한국생물과학협회 학술발표대회
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• pp.289.1-289
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• 1997

No Abstract, See Full Text

• 한국식물병리학회:학술대회논문집
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• 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
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• pp.75.1-75
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• 2003

Barely yellow mosaic(BaYMV) and barely mild mosaic (BaMMV) bymoviruses are both transmitted by the soil-inhabiting fungus Polymyxa gramnis, and are responsible for economic losses in barley crops in Asia and Europe. Because chemical control of the vector is ineffective, the losses can only be prevented by growing resistant barley cultivars. The objective of this study is to produce resistant barley plants by transformation with viral coat protein(cp) genes. Resistance tests of T1 plants transformed with the BaYMV CP gene showed that at least four independent lines had clear resistance to BaYMV but two other lines were highly susceptible with severe symptoms. The CP gene was detected in all resistant T1 plants by genomic PCR. Most of T2 progenies derived from the resistant T1 lines also showed resistance. In contrast, only one out of 21 independent T2 lines transformed with the BAMMV CP gene tested showed clear resistance to BaMMV, and others were very susceptible. Further analyses of resistance and CP gene expression are in progress.

• The Plant Pathology Journal
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• 제19권1호
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• pp.19-23
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• 2003

Turnip crinkle vims (TCV) inoculation onto resistant Arabidopsis ecotype Dijon（Di-17) leads to a hypersensitive response (HR) on the inoculated leaves. A dominant gene, HRT, which confers an HR to TCV, has been cloned from Di-17 plants by map-based cloning. HRT is a LZ-NBS-LRR class resistance gene and it belongs to a small gene family that includes RPP8, which confers resistance to Peronospora parasitica Emco5. Outside of the LRR region, HRT and RPP8 proteins share 98% amino acid identity while their LRR regions are less conserved (87% identity). HRT-transformed Arabidopsis plants developed an HR but generally remained susceptible to TCV due to a dominant RRT allele, which is not compatible with resistance. However, several transgenic plants that over-expressed HRT much higher than Di-l7 showed micro-HR or no HR when inoculated with TCV and were resistant to infection. Both the HR and resistance are dependent on salicylic acid but independent of NPRI, ethylene, or jasmonic acid. Arabidopsis plants containing both TCV coat protein gene and HRT developed massive necrosis and death in seedlings, indicating that the TCV coat protein is an avirulence factor detected by the HRT.

• The Plant Pathology Journal
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• 제19권3호
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• pp.162-165
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• 2003

Transformation of Fuji apple (Malus domestica 'Fuji') was performed using Agrobacterium tumefaciens harboring a coat protein (CP) gene of Cucumber mosaic virus (CMV). A plasmid DNA containing the virus CP and NPT II genes was introduced into the loaves of apple by th e Agrobacterium - mediated transformation procedure. Regenerated transformants of the apple were obtained by kanamycin resistance conferred by the introduced NPT II gene. PCR analysis showed that 3 out of 20 putatively selected R0 plant lines contain the CMV-CP gene. Nine putative transgenic lines out of 20 lines were investigated with the PCR analysis; 5 regenerants produced a 450 bp DNA band and 3 regenerants showed a 671 bp DNA band for the NPT II and CMV-CP genes, respectively. Southern hybyidization results demonstrate the successful integration of the CMV-CP gene into the genome of the apple. This is the first report on the generation of useful vius resistance source of transgenic apple for molecular breeding program.

• The Plant Pathology Journal
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• 제22권4호
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• pp.353-359
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• 2006

Transgenic Nicotiana benthamiana plants harboring the coat protein(CP) gene of Zucchini green mottle mosaic virus(ZGMMV) were chosen as a model host for the environmental risk assessment of genetically modified plants with virus resistance. This study was focused on whether new virus type may arise during serial inoculation of one point CP mutant of ZGMMV on the transgenic plants. In vitro transcripts derived from the non-functional CP mutant were inoculated onto the virus-tolerant and -susceptible transgenic N. benthamiana plants. Any notable viral symptoms that could arise on the inoculated transgenic host plants were not detected, even though the inoculation experiment was repeated a total of ten times. This result suggests that potential risk associated with the CP-expressiing transgenic plants may not be significant. However, cautions must be taken as it does not guarantee environmental safety of these CP-mediated virus-resistant plants, considering the limited number of the transgenic plants tested in this study. Further study at a larger scale is needed to evaluate the environmental risk that might be associated with the CP-mediated virus resistant plant.

• BMB Reports
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• 제41권5호
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• pp.376-381
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• 2008

The discovery of RNA silencing inhibition by virus encoded suppressors or low temperature leads to concerns about the stability of transgenic resistance. RNA-dependent RNA polymerase (RdRp) has been previously characterized to be essential for transgene-mediated RNA silencing. Here we showed that low temperature led to the inhibition of RNA silencing, the loss of viral resistance and the reduced expression of host RdRp homolog (NtRdRP1) in transgenic T4 progeny with untranslatable potato virus Y coat protein (PVY-CP) gene. Moreover, RNA silencing and the associated resistance were differently inhibited by potato virus X (PVX) and tobacco mosaic virus (TMV) infections. The increased expression of NtRdRP1 in both PVX and TMV infected plants indicated its general role in response to viral pathogens. Collectively, we propose that biotic and abiotic stress factors affect RNA silencing-mediated resistance in transgenic tobacco plants and that their effects target different steps of RNA silencing.