• The Plant Pathology Journal
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• v.25 no.4
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• pp.333-343
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• 2009

Here, we show that an endophytic bacterial strain, Enterobacter asburiae B1 exhibits the ability to elicit ISR in cucumber, tobacco and Arabidopsis thaliana. This indicates that strain B1 has a widespread ability to elicit ISR on various host plants. In this study, E. asburiae strain B1 did not show antifungal activity against tested major fungal pathogens, Colletotrichum orbiculare, Botrytis cinerea, Phytophthora capsici, Rhizoctonia solani, and Fusarium oxysporum. Moreover, the siderophore production by E. asburiae strain B1 was observed under in vitro condition. In greenhouse experiments, the root treatment of strain B1 significantly reduced disease severity of cucumber anthracnose caused by fungal pathogen C. orbiculare compared to nontreated control plants. By root treatment of strain B1 more than 50% disease control against anthracnose on cucumber was observed in all greenhouse experiments. Simultaneously, under the greenhouse condition, the soil drench of strain B1 and a chemical inducer benzothiadiazole (BTH) to tobacco plants induced GUS activity which is linked with activation of PR promoter gene. Furthermore, in Arabidopsis thaliana plants the soil drench of strain B1 induced the defense gene expression of PR1 and PDF1.2 related to salicylic acid and jasmonic acid/ethylene signaling pathways, respectively. In this study, for the main focus on root colonization by strain B1 associated with defense responses, bacterial cells of strain B1 was tagged with the gfp gene encoding the green fluorescent protein in order to determine the colonization pattern of strain B1 in cucumber. The gfp-tagged B1 cells were found on root surface and internal colonization in root, stem, and leaf. In addition to this, the scanning electron microscopy observation showed that E. asburiae strain B1 was able to colonized cucumber root surface.

• Journal of Microbiology and Biotechnology
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• v.25 no.7
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• pp.988-998
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• 2015

The filamentous fungus Aspergillus oryzae is a well-known expression host used to express homologous and heterologous proteins in a number of industrial applications. To facilitate higher yields of proteins of interest, we constructed the pAsOP vector to express heterologous proteins in A. oryzae. pAsOP carries a selectable marker, pyrG, derived from Aspergillus nidulans, and a strong promoter and a terminator of the amyB gene derived from A. oryzae. pAsOP transformed A. oryzae efficiently via the PEG-CaCl₂-mediated transformation method. As proof of concept, green fluorescent protein (GFP) was successfully expressed in A. oryzae transformed by pAsOP-GFP. Additionally, we identified a novel fungal α-amylase (PcAmy) gene from Penicillium sp. and cloned the gene into the vector. After transformation by pAsOPPcAmy, the α-amylase PcAmy from Penicillium sp. was successfully expressed in a heterologous host system for the first time. The α-amylase activity in the A. oryzae transformant was increased by 62.3% compared with the untransformed A. oryzae control. The PcAmy protein produced in the system had an optimum pH of 5.0 and optimum temperature of 30^oC. As a cold-adapted enzyme, PcAmy shows potential value in industrial applications because of its high catalytic activity at low temperature. Furthermore, the expression vector reported in this study provides promising utility for further scientific research and biotechnological applications.

• Journal of Microbiology and Biotechnology
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• v.20 no.2
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• pp.238-244
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• 2010

Rhizobia are well-known for their ability to infect and nodulate legume roots, forming a nitrogen-fixing symbiosis of agricultural importance. In addition, recent studies have shown that rhizobia can colonize roots and aerial plant tissues of rice as a model plant of the Graminaceae family. Here we show that rhizobia can invade tobacco, a model plant belonging to the Solanaceae family. Inoculation of seedling. roots with five GFP-tagged rhizobial species followed by microscopy and viable plating analyses indicated their colonization of the surface and interior of the whole vegetative plant. Blockage of ascending epiphytic migration by coating the hypocotyls with Vaseline showed that the endophytic rhizobia can exit the leaf interior through stomata and colonize the external phyllosphere habitat. These studies indicate rhizobia can colonize both below- and above-ground tissues of tobacco using a dynamic invasion process that involves both epiphytic and endophytic lifestyles.

• Korean Journal of Microbiology
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• v.51 no.4
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• pp.435-439
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• 2015

THO/TREX complex plays an important role in transcriptional elongation, mRNA processing, nuclear RNA export, and genome stability. A fission yeast, Schizosaccharomyces pombe, SPBC577.04 gene encoding the ortholog of THOC5, a component of THO/TREX complex, was identified and characterized. The S. pombe thoc5 (spthoc5) is not essential for both growth and mRNA export, but deletion of the spthoc5 gene caused growth defect and slight accumulation of $poly(A)^+$ RNA in the nucleus. And the functional spThoc5-GFP protein is localized mainly in the nucleus. Co-immunoprecipitation analysis showed that the Hpr1(THOC1) protein, an evolutionally well-conserved component of THO/TREX complex, interacted with spThoc5 as well as Tho2(THOC2), another subunit of THO complex. These results suggest that S. pombe Thoc5 as a component of THO/TREX complex is also involved in mRNA export from the nucleus.

• BMB Reports
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• v.53 no.10
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• pp.521-526
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• 2020

Endogenous retroviruses (ERVs) are retrotransposons present in various metazoan genomes and have been implicated in metazoan evolution as well as in nematodes and humans. The long terminal repeat (LTR) retrotransposons contain several regulatory sequences including promoters and enhancers that regulate endogenous gene expression and thereby control organismal development and response to environmental change. ERVs including the LTR retrotransposons constitute 8% of the human genome and less than 0.6% of the Caenorhabditis elegans (C. elegans) genome, a nematode genetic model system. To investigate the evolutionarily conserved mechanism behind the transcriptional activity of retrotransposons, we generated a transgenic worm model driving green fluorescent protein (GFP) expression using Human endogenous retroviruses (HERV)-K LTR as a promoter. The promoter activity of HERV-K LTR was robust and fluorescence was observed in various tissues throughout the developmental process. Interestingly, persistent GFP expression was specifically detected in the adult vulva muscle. Using deletion constructs, we found that the region from positions 675 to 868 containing the TATA box was necessary for promoter activity driving gene expression in the vulva. Interestingly, we found that the promoter activity of the LTR was dependent on che-1 transcription factor, a sensory neuron driver, and lin-15b, a negative regulator of RNAi and germline gene expression. These results suggest evolutionary conservation of the LTR retrotransposon activity in transcriptional regulation as well as the possibility of che-1 function in non-neuronal tissues.

• Journal of Plant Biotechnology
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• v.36 no.1
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• pp.68-74
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• 2009

We have previously demonstrated that overexpression and characterization of Brassica rapa type-l metallothionein gene (BrMT1) in Arabidopsis which showed enhanced resistance to cadmium and ROS. Here, we present the consistent study of our previous report about BrMTs. BrMT3 expressing DTY167 cells showed resistance to Zn and Pb as well as Cd. Thus, we have developed the BrMT3 overexpression Arabidopsis to enhance capacity for metal stresses. Successful expression and localization were achieved using the rubisco transit peptides of RbcS-BrMT3-GFP protein, which was confirmed by western blot analysis with the GFP antibody and green fluorescence signal from the chloroplast. BrMT3 overexpression Arabidopsis plants exhibited a higher resistance to cadmium compared to control plants. This result indicates that BrMT3 would be applicable to the development of plants with enhanced resistance against heavy metal stresses.

• Molecules and Cells
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• v.40 no.11
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• pp.828-836
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• 2017

Eukaryotic cells consist of a complex network of thousands of proteins present in different organelles where organelle-specific cellular processes occur. Identification of the subcellular localization of a protein is important for understanding its potential biochemical functions. In the post-genomic era, localization of unknown proteins is achieved using multiple tools including a fluorescent-tagged protein approach. Several fluorescent-tagged protein organelle markers have been introduced into dicot plants, but its use is still limited in monocot plants. Here, we generated a set of multicolored organelle markers (fluorescent-tagged proteins) based on well-established targeting sequences. We used a series of pGWBs binary vectors to ameliorate localization and co-localization experiments using monocot plants. We constructed different fluorescent-tagged markers to visualize rice cell organelles, i.e., nucleus, plastids, mitochondria, peroxisomes, golgi body, endoplasmic reticulum, plasma membrane, and tonoplast, with four different fluorescent proteins (FPs) (G3GFP, mRFP, YFP, and CFP). Visualization of FP-tagged markers in their respective compartments has been reported for dicot and monocot plants. The comparative localization of the nucleus marker with a nucleus localizing sequence, and the similar, characteristic morphology of mCherry-tagged Arabidopsis organelle markers and our generated organelle markers in onion cells, provide further evidence for the correct subcellular localization of the Oryza sativa (rice) organelle marker. The set of eight different rice organelle markers with four different FPs provides a valuable resource for determining the subcellular localization of newly identified proteins, conducting co-localization assays, and generating stable transgenic localization in monocot plants.

• The Plant Pathology Journal
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• v.26 no.3
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• pp.238-244
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• 2010

Spatial and temporal distribution of Bacillus licheniformis N1 was investigated over time on the leaves, petioles and crowns of the strawberry plants. Bacterial population on the strawberry plants was quantified over time by selective plating. Bacterial population of N1 containing a plasmid pWH43G carrying green fluorescent protein (GFP) declined relatively faster on the plant surface as compared to the Strain N1 itself. However, this result was found to be enough to utilize the strain to visualize bacterial colonization on the plant surface. When B. licheniformis N1 was treated together with Silwet L-77 at 0.03%, the bacterial population on plant surface persisted for up to 7 days. B. licheniformis N1 (pWH43G) containing Silwet L-77 was applied on the strawberry plants and the GFP expressing bacteria were visualized by confocal laser scanning microscopy. Bacterial persistence was also investigated in a growth chamber and in a plastic house after N1 bioformulation treatment on the strawberry plant. The Strain N1 colonized three different tissues well and persisted over 3 to 5 days on the strawberry plants. They formed bacterial aggregates on plant surfaces for at least 3 days, resulting in a biofilm to resist fluctuating plant surface environment. However, the bacterial persistence dramatically declined after 7 days in all tested tissues in a plastic house. This study suggest that B. licheniformis N1 colonizes the strawberry plant surface and persists for a long time in a controlled growth chamber, while it can not persist over 7 days on the plant surface in a plastic house.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.12
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• pp.6211-6216
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• 2012

Objective: To investigate the effects of gambogic acid (GA) on the growth of human malignant glioma cells. Methods: U251MG and U87MG human glioma cell lines were treated with GA and growth and proliferation were investigated by MTT and colony formation assays. Cell apoptosis was analyzed by annexin V FITC/PI flow cytometry, mitochondrial membrane potential assays and DAPI nuclear staining. Monodansylcadaverine (MDC) staining and GFP-LC3 localisation were used to detect autophagy. Western blotting was used to investigate the molecular changes that occurred in the course of GA treatment. Results: GA treatment significantly suppressed cell proliferation and colony formation, induced apoptosis in U251 and U87MG glioblastoma cells in a time- and dose-dependent manner. GA treatment also lead to the accumulation of monodansylcadaverine (MDC) in autophagic vacuoles, upregulated expressions of Atg5, Beclin 1 and LC3-II, and the increase of punctate fluorescent signals in glioblastoma cells pre-transfected with GFP-tagged LC3 plasmid. After the combination treatment of autophagy inhitors and GA, GA mediated growth inhibition and apoptotic cell death was further potentiated. Conclusion: Our results suggested that autophagic responses play roles as a self-protective mechanism in GA-treated glioblastoma cells, and autophagy inhibition could be a novel adjunctive strategy for enhancing chemotherapeutic effect of GA as an anti-malignant glioma agent.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.7
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• pp.4095-4099
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• 2013

Oxidative stress induces apoptosis in many cellular systems including glioblastoma cells, with caspase-8 activation was regarded as a major contribution to $H_2O_2$-induced cell death. This study focused on the role of the autophagic protein p62 in $H_2O_2$-induced apoptosis in U87MG cells. Oxidative stress was applied with $H_2O_2$, and cell apoptosis and viability were measured with use of caspase inhibitors or autophagic mediators or siRNA p62, GFP-p62 and GFP-p62-UBA (del) transfection. We found that $H_2O_2$-induced U87MG cell death was correlated with caspase-8. To understand the role of p62 in MG132-induced cell death, the levels of p62/SQSTM1 or autophagy in U87MG cells were modulated with biochemical or genetic methods. The results showed that the over-expression of wild type p62/SQSTM1 significantly reduced $H_2O_2$ induced cell death, but knockdown of p62 aggravated the process. In addition, inhibition of autophagy promoted p62 and active caspase-8 increasing $H_2O_2$-induced apoptosis while induction of autophagy manifested the opposite effect. We further demonstrated that the function of p62/SQSTM1 required its C-terminus UBA domain to attenuate $H_2O_2$ cytotoxity by inhibition of caspase-8 activity. Our results indicated that p62/SQSTM1 was a potential contributor to mediate caspase-8 activation by autophagy in oxidative stress process.