• Korean Journal of Agricultural Science
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• v.44 no.2
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• pp.145-180
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• 2017

Alternanthera mosaic virus (AltMV) is a member of the genus Potexvirus which has been known for less than twenty years, and has been detected in Australasia, Europe, North and South America, and Asia. The natural host range to date includes species in at least twenty-four taxonomically diverse plant families, with species in at least four other families known to be infected experimentally. AltMV has been shown to differ from Potato virus X (PVX), the type member of the genus Potexvirus, in a number of ways, including the subcellular localization of the Triple Gene Block 3 (TGB3) protein and apparent absence of interactions between TGB3 and TGB2. Differences between AltMV variants have allowed identification of viral determinants of pathogenicity, and identification of residues involved in interactions with host proteins. Infectious clones of AltMV differing significantly in symptom severity and efficiency of RNA silencing suppression have been produced, suitable either for high level protein expression (with efficient RNA silencing suppression) or for Virus-Induced Gene Silencing (VIGS; with weaker RNA silencing suppression), demonstrating a range of utility not available with most other plant viral vectors. The difference in silencing suppression efficiency was shown to be due to a single amino acid residue substitution in TGB1, and to differences in subcellular localization of TGB1 to the nucleus and nucleolus. The current state of knowledge of AltMV biology, including host range, strain differentiation, host interactions, and utility as a plant viral vector for both protein expression and VIGS are summarized.

• Journal of Plant Biotechnology
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• v.34 no.4
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• pp.307-312
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• 2007

Chlamydomonas reinhardtii was transformed with the coding sequence of the Tombus virus gene p19 to determine whether the gene functions as an RNAi suppressor in C. reinhardtii. Transformants were confirmed to have 1 to several copies of p19 gene in their chromosomes. When an RNAi strain of C. reinhardtii generated by transforming the inverted repeat (IR) sequence homologous to the 3'UTR region of the MAA7 gene was re-transformed with the gene p19, MAA7 transcript levels of transformants fluctuated and proliferation of trans-formants on the medium containing 5-FI was suppressed. Overall results suggest that p19-mediated silencing suppression works at a low level in C. reinhardtii because of difference in codon usage resulting in weak P19 expression unless p19-mediated silencing suppression in C. reinhardtii works in a different manner from higher plants.

• BMB Reports
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• v.41 no.5
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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.

• Horticultural Science & Technology
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• v.35 no.3
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• pp.323-332
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• 2017

Homology-specific transcriptional and post-transcriptional silencing, an intrinsic mechanism of gene regulation in most eukaryotes, can be induced by anti-sense, co-suppression, or hairpin-based double-stranded RNA. Hairpin-based RNA interference (RNAi) has been applied to analyze gene function and genetically modify crops. However, RNAi vector construction usually requires high-cost cloning steps and large amounts of time, or involves methods that are protected by intellectual property rights. We describe a more effective method for generating intron-spliced RNAi constructs. To produce intron-spliced hairpin RNA, an RNAi cassette was ligated with the first intron and splicing sequences of the Brassica rapa ssp. pekinensis histone deacetylase 1 gene. This method requires a single ligation of the PCR-amplified target gene to SpeI-NcoI and SacI-BglII enzyme sites to create a gene-specific silencing construct. We named the resulting binary vector system pKHi and verified its functionality by constructing a vector to silence DIHYDROFLAVONOL 4-REDUCTASE (DFR), transforming it into tobacco plants, and confirming DFR gene-silencing via PCR, RT-qPCR, and analysis of the accumulation of small interfering RNAs. Reduction of anthocyanin biosynthesis was also confirmed by analyzing flower color of the transgenic tobacco plants. This study demonstrates that small interfering RNAs generated through the pKHi vector system can efficiently silence target genes and could be used in developing genetically modified crops.

• Molecules and Cells
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• v.38 no.11
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• pp.959-965
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• 2015

Inducible and reversible gene silencing in desired types of cells is instrumental for deciphering gene functions using cultured cells or in vivo models. However, efficient conditional gene knockdown systems remain to be established. Here, we report the generation of an inducible expression system for short hairpin RNA (shRNA) targeted to PTEN, a well-documented dual-specificity phosphatase involved in tumor suppression and ontogenesis. Upon induction by doxycycline (DOX), the reverse tetracycline transcriptional activator (rtTA) switched on the concomitant expression of GFP and a miR-30 precursor, the subsequent processing of which released the embedded PTEN-targeted shRNA. The efficacy and reversibility of PTEN knockdown by this construct was validated in normal and neoplastic cells, in which PTEN deficiency resulted in accelerated cell proliferation, suppressed apoptosis, and increased invasiveness. Transgenic mice harboring the conditional shRNA-expression cassette were obtained; GFP expression and concurrent PTEN silencing were observed upon ectopic expression of rtTA and induction with Dox. Therefore, this study provides novel tools for the precise dissection of PTEN functions and the generation of PTEN loss of function models in specific subsets of cells during carcinogenesis and ontogenesis.

• The Korean Journal of Physiology and Pharmacology
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• v.22 no.2
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• pp.127-134
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• 2018

Myofibrillogenesis regulator-1 (MR-1) is a novel protein involved in cellular proliferation, migration, inflammatory reaction and signal transduction. However, little information is available on the relationship between MR-1 expression and the progression of atherosclerosis. Here we report atheroprotective effects of silencing MR-1 in a model of Ang II-accelerated atherosclerosis, characterized by suppression focal adhesion kinase (FAK) and nuclear factor kappaB ($NF-{\kappa}B$) signaling pathway, and atherosclerotic lesion macrophage content. In this model, administration of the siRNA-MR-1 substantially attenuated Ang II-accelerated atherosclerosis with stabilization of atherosclerotic plaques and inhibited FAK, Akt, mammalian target of rapamycin (mTOR) and NF-kB activation, which was associated with suppression of inflammatory factor and atherogenic gene expression in the artery. In vitro studies demonstrated similar changes in Ang II-treated vascular smooth muscle cells (VSMCs) and macrophages: siRNA-MR-1 inhibited the expression levels of proinflammatory factor. These studies uncover crucial proinflammatory mechanisms of Ang II and highlight actions of silencing MR-1 to inhibit Ang II signaling, which is atheroprotective.

• Biotechnology and Bioprocess Engineering:BBE
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• v.11 no.3
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• pp.273-276
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• 2006

RNA interference (RNAi), a process of sequence-specific gene suppression, has been known as a natural gene regulatory mechanism in a wide range of lower organisms. Recently, we have reported that a transfection of human U6 promoter (hU6) driven hairpin small-interference RNA (siRNA) plasmid specifically knocks down the target gene by post-transcriptional gene silencing in mammalian cells. Here we report that transfection of polymerase chain reaction (PCR) products, containing human U6 promoter with hairpin siRNA, knocks down the target gene expression in human teratocarcinoma NT-2/D1 cells. Moreover, we showed 3' end termination sequence, 5 Ts, is not critical elements for knocking down in PCR-based siRNA system. Therefore, the PCR-based siRNA system is a promising tool not only for the screening but also to temporally regulate gene expression in the human progenitor cells.

• Genomics & Informatics
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• v.13 no.2
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• pp.26-30
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• 2015

nc886 (=vtRNA2-1, pre-miR-886, or CBL3) is a newly identified non-coding RNA (ncRNA) that represses the activity of protein kinase R (PKR). nc886 is transcribed by RNA polymerase III (Pol III) and is intriguingly the first case of a Pol III gene whose expression is silenced by CpG DNA hypermethylation in several types of cancer. PKR is a sensor protein that recognizes evading viruses and induces apoptosis to eliminate infected cells. Like viral infection, nc886 silencing activates PKR and induces apoptosis. Thus, the significance of the nc886:PKR pathway in cancer is to sense and eliminate pre-malignant cells, which is analogous to PKR's role in cellular innate immunity. Beyond this tumor sensing role, nc886 plays a putative tumor suppressor role as supported by experimental evidence. Collectively, nc886 provides a novel example how epigenetic silencing of a ncRNA contributes to tumorigenesis by controlling the activity of its protein ligand.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.76.1-76
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• 2003

The coat protein (CP) of Turnip crinkle virus (TCV) is organized into 3 distinct domains, R domain (RNA-binding) connected by an arm, 5 domain and P domain. We have previously shown that the CP of TCV strongly suppresses RNA silencing, and have mapped N-terminal R domain of which is also the elicitor of resistance response in the Arabidopsis ecotype Di-17 carrying the HRT resistance gene. In order to map the region in the TCV CP that is responsible for silencing suppression, a series of CP mutants were constructed, transformed into Agrobacterium, coinfiltrated either with HC-Pro (the helper component proteinase of tobacco etch potyvirus) known as a suppressor of PTGS or GFP constructs into leaves of Nicotiana benthmiana expressing GFP transgenically. In the presence of HC-Pro, all CP mutants were well protected, accumulating mutant CP mRNAs and their proteins even 5 days post-infiltration (DPI). In the presence of GFP, some mutant constructs which showed the accumulation of CP mutants and GFP mRNAs at early stage but eventually degraded at 5 DPI. Only a mutant which carrying 4 amino acid deletion of R domain was tolerable to maintain suppressing activity, suggesting that the suppressing activity is not directly related with the eliciting activity. A transient assay also revealed that the mutants synthesized their proteins, suggesting that a full length of CP sequences and its intact structure are required to stabilize CP, which suppresses the RNA silencing.

• Journal of Plant Biotechnology
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• v.7 no.2
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• pp.87-95
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• 2005

The tomato ripening associated membrane protein (TRAMP) (Fray et al., 1994) is a member of the major intrinsic protein (MIP) family, defined as channels facilitating the passage of water and small solutes through membranes. During normal fruit ripening the TRAMP mRNA levels were increased whereas the expression levels of TRAMP in low ethylene ACO1-sense suppressed lines, Nr and rin fruits, were lower than at the breaker stage of wild type fruit. TRAMP mRNA is inhibited by $LaCl_3$, which is an inhibitor of $Ca^{2+}$-stimulated responses, treatment but drought condition did not affect TRAMP expression. The levels of TRAMP mRNA transcripts were substantially higher in the dark treated seedlings and fruits. These suggest that TRAMP function as a water channel may be doubted because of several reasons; no water content was changed during ripening in wild type, antisense and overexpression lines, TRAMP expression under light condition was lower than dark condition and TRAMP expression was not changed in drought condition. Co-suppression plant, 3588 was one of sense suppression lines, which contain CaMV 35S promoter and sense pNY507 cDNA, produced small antisense RNA, approximately 21-25 nucleotides in length, mediated post-transcriptional gene silencing. Therefore, TRAMP expression was inhibited by small antisense and multiple copies might induce gene silencing without any production of double strand RNA. Total seven selected volatile productions, isobutylthiazole, 6-methyl-5-hepten-2-one, hexanal, hexenal methylbutanal, hexenol, and methylbutanol, were highly reduced in sense line whereas total volatile production was increased in TRAMP antisense line. These results suggested TRAMP might change volatile related compounds.