• Korean Journal of Agricultural Science
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• v.42 no.1
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• pp.7-13
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• 2015

Ninety genes randomly selected from tobacco whitefly (Bemisia tabaci) cDNA library was studied for selecting target gene in order to control of tobacco whitefly using TRV-VIGS vector (tobacco rattle virus-virus induced gene silencing vector) with RNAi. First of all, the occurrence of B. tabaci adult according to agro-infiltration of TRV was no significant difference. And that of TRV inserted tobacco whitefly cDNA showed a significant difference in each sample. P CV and N CV sample were more than 80% could be confirmed in 5 samples, for example, wh11, wh36, wh46, wh50 and wh71. Lastly, the occurrence of nymph and egg also showed a significant difference in each sample. That could be confirmed in 11 samples, for example, wh01, wh09, wh10, wh15, wh16, wh23, wh24, wh48, wh64 and wh66. In case of wh46, wh50 and wh71 sample could be confirmed that occurrence of B. tabaci adult was many, but occurrence of B. tabaci nymph and egg was a little. So sample showed a physioecological good effect to control of whitefly need to be investigated variation of gene expression in whitefly body using qRT-PCR through individual test.

• The Plant Pathology Journal
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• v.32 no.4
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• pp.371-376
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• 2016

Grapevine Algerian latent virus (GALV) is a member of the genus Tombusvirus in the Tombusviridae and infects not only woody perennial grapevine plant but also herbaceous Nicotiana benthamiana plant. In this study, we developed GALV-based gene expression and virus-induced gene silencing (VIGS) vectors in N. benthamiana. The GALV coat protein deletion vector, pGMG, was applied to express the reporter gene, green fluorescence protein (GFP), but the expression of GFP was not detected due to the necrotic cell death on the infiltrated leaves. The p19 silencing suppressor of GALV was engineered to inactivate its expression and GFP was successfully expressed with unrelated silencing suppressor, HC-Pro, from soybean mosaic virus. The pGMG vector was used to knock down magnesium chelatase (ChlH) gene in N. benthamaina and the silencing phenotype was clearly observed on systemic leaves. Altogether, the GALV-derived vector is expected to be an attractive tool for useful gene expression and VIGS vectors in grapevine as well as N. benthamiana.

• Molecules and Cells
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• v.26 no.3
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• pp.270-277
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• 2008

This study addresses the physiological functions of the Ran-binding protein homolog NbRanBP1 in Nicotiana benthamiana. Virus-induced gene silencing (VIGS) of NbRanBP1 caused stunted growth, leaf yellowing, and abnormal leaf morphology. The NbRanBP1 gene was constitutively expressed in diverse tissues and an NbRanBP1:GFP fusion protein was primarily localized to the nuclear rim and the cytosol. BiFC analysis revealed in vivo interaction between NbRanBP1 and NbRan1 in the nuclear envelope and the cytosol. Depletion of NbRanBP1 or NbRan1 reduced nuclear accumulation of a NbBTF3:GFP marker protein. In the later stages of development, NbRanBP1 VIGS plants showed stress responses such as reduced mitochondrial membrane potential, excessive production of reactive oxygen species, and induction of defense-related genes. The molecular role of RanBP1 in plants is discussed in comparison with RanBP1 function in yeast and mammals.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.77.1-77
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• 2003

We have isolated a full-length cDNA clone, CaCDPK4 encoding a typical calcium-dependent protein kinase (CDPK) from hot pepper cDNA library. Genomic southern blot analysis showed that it belongs to a multigene family, but represents a single copy gone in hot pepper genome. RNA expression pattern of this gene revealed that it is induced by infiltration of Xanthomonas axonopodis pv. glycines Bra into hot pepper leaves but not by water deficit stress. However, high salt treatment of NaCl (0.4 M) solution to hot pepper plants strongly induced CaCDPK4 gene. In addition, this gene is weakly responsive to the exogenous application of salicylic acid or ethephon. Biochemical study of the GST-CaCDPK4 recominant protein showed that it autophosphorylates in vitro and the presence of EGTA, a calcium chelater, eliminates the kinase activity of the recombinant protein. As a way to identify the in vivo function of CaCDPK4 in plants, VIGS (Virus-Induced Gene Silencing) was employed. Agrobacterium-mediated TRV silencing construct containing the kinase and calmodulin domain of CaCDPK4 resulted in cell death of Nicotiana benthamiana plants. A highly homologous H benthamiana CDPK gene, NbCDPK5, to CaCDPK4 was cloned from N. benthamiana cDNA library. VIGS of NbCDPK5 also resulted in cell death. The molecular characterization of this cell death phenotype is being under investigation.

• Molecules and Cells
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• v.38 no.10
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• pp.866-875
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• 2015

COPI vesicles are essential to the retrograde transport of proteins in the early secretory pathway. The COPI coatomer complex consists of seven subunits, termed ${\alpha}-$, ${\beta}-$, ${\beta}^{\prime}-$, ${\gamma}-$, ${\delta}-$, ${\varepsilon}-$, and ${\zeta}$-COP, in yeast and mammals. Plant genomes have homologs of these subunits, but the essentiality of their cellular functions has hampered the functional characterization of the subunit genes in plants. Here we have employed virus-induced gene silencing (VIGS) and dexamethasone (DEX)-inducible RNAi of the COPI subunit genes to study the in vivo functions of the COPI coatomer complex in plants. The ${\beta}^{\prime}-$, ${\gamma}-$, and ${\delta}$-COP subunits localized to the Golgi as GFP-fusion proteins and interacted with each other in the Golgi. Silencing of ${\beta}^{\prime}-$, ${\gamma}-$, and ${\delta}$-COP by VIGS resulted in growth arrest and acute plant death in Nicotiana benthamiana, with the affected leaf cells exhibiting morphological markers of programmed cell death. Depletion of the COPI subunits resulted in disruption of the Golgi structure and accumulation of autolysosome-like structures in earlier stages of gene silencing. In tobacco BY-2 cells, DEX-inducible RNAi of ${\beta}^{\prime}$-COP caused aberrant cell plate formation during cytokinesis. Collectively, these results suggest that COPI vesicles are essential to plant growth and survival by maintaining the Golgi apparatus and modulating cell plate formation.

• The Plant Pathology Journal
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• v.26 no.4
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• pp.402-408
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• 2010

Plant matrix metalloproteases (MMPs) are a family of apoplastic metalloproteases closely related to human matrilysins. Up-regulation of Nicotiana benthamiana matrix metalloprotease 1 (NMMP1) expression by treatment with pathogens, ethephon and aging indicates that the gene is related to plant defense and the aging process through ethylene signaling. NMMP1 expression was higher than in normal growth leaves following infection with an incompatible pathogen Pseudomonas syringae pv. tomato T1 or a compatible pathogen P. syringae pv. tabaci and in aged leaves. Transient overexpression of NMMP1 in N. benthamiana leaves lowered the growth of P. syringae pv. tabaci. However, NMMP1-silenced leaves showed increased growth of P. syringae pv. tabaci. These data strongly suggest that NMMP1 in N. benthamiana is a defense related gene, which is positively regulated by ethylene.

• The Plant Pathology Journal
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• v.28 no.1
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• pp.81-86
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• 2012

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.

• The Plant Pathology Journal
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• v.28 no.1
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• pp.40-48
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• 2012

$Potato$ $virus$ $X$ (PVX) replication is precisely regulated by regulatory viral sequences and by viral and/or host proteins. In a previous study, we identified a 54-kDa cellular tobacco protein that bound to a region within the first 46 nucleotides (nt) of the 5' non-translated region (NTR) of the viral genome. Optimal binding was dependent upon the presence of an ACCA sequence at nt 10-13. To identify host factors that bind to 5' NTR elements including AC-rich sequences as well as stemloop 1 (SL1), we used northwestern blotting and matrixassisted laser desorption/ionization time-of-flight mass spectrometry for peptide mass fingerprinting. We screened several host factors that might affect PVX replication and selected a candidate protein, $Nicotiana$ $tabacum$ WRKY transcription factor 1 (NtWRKY1). We used a $Tobacco$ $rattle$ $virus$ (TRV)-based virus-induced gene silencing (VIGS) system to investigate the role of NtWRKY1 in PVX replication. Silencing of $WRKY1$ in $Nicotiana$ $benthamiana$ caused lethal apical necrosis and allowed an increase in PVX RNA accumulation. This result could reflect the balancing of PVX accumulation in a systemic $N.$ $benthamiana$ host to maintain PVX survival and still produce a suitable appearance of mosaic and mottle symptoms. Our results suggest that PVX may recruit the WRKY transcription factor, which binds to the 5' NTR of viral genomic RNA and acts as a key regulator of viral infection.

• Molecules and Cells
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• v.28 no.1
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• pp.57-65
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• 2009

CDC48 is a member of the AAA ATPase superfamily. Yeast CDC48 and its mammalian homolog p97 are implicated in diverse cellular processes, including mitosis, membrane fusion, and ubiquitin-dependent protein degradation. However, the cellular functions of plant CDC48 proteins are largely unknown. In the present study, we performed virus-induced gene silencing (VIGS) screening and found that silencing of a gene encoding a tobacco CDC48 homolog, NgCDC48, resulted in severe abnormalities in leaf and shoot development in tobacco. Furthermore, transgenic tobacco plants (35S:anti-NgCDC48), in which the NgCDC48 gene was suppressed using the antisense RNA method, exhibited severely aberrant development of both vegetative and reproductive organs, resulting in arrested shoot and leaf growth and sterile flowers. Approximately 57-83% of 35S:anti-NgCDC48 plants failed to develop mature organs and died at early stage of development. Scanning electron microscopy showed that both adaxial and abaxial epidermal pavement cells in antisense transgenic leaves were significantly smaller and more numerous than those in wild type leaves. These results indicate that NgCDC48 is critically involved in cell growth and development of tobacco plants. An in vivo targeting experiment revealed that NgCDC48 resides in the endoplasmic reticulum (ER) in tobacco protoplasts. We consider the tantalizing possibility that CDC48-mediated degradation of an as-yet unidentified protein(s) in the ER might be a critical step for cell growth and expansion in tobacco leaves.

• Korean Journal of Agricultural Science
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• v.42 no.2
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• pp.81-86
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• 2015

Three genes selected from cDNA library of tobacco whitefly, Bemisia tabaci, were checked whether these genes expressed in plant or not, and confirmed the change of gene expression using qRT-PCR in the tobacco whitefly. First of all, three genes were inserted in Tobacco rattle virus (TRV) RNA2 vector using Sac I and Xho I restriction enzymes, and conducted agro-infiltration in tobacco plants (Nicotiana benthamianana). And then, it was confirmed that TRV RNA2 vector and genes inserted in TRV RNA2 vector were expressed in plant. So, after feeding the tobacco whitefly the plants inoculated the genes and induced RNAi of the genes, we plan to confirm the RNAi in the whitefly and investigate the changes of gene expression through the qRT-PCR.