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N-glycan maturation and plant development
Wahyu Indra Fanata,Kyoung Hwan Lee,Bo Hwa Son,Jae Yong Yoo,Rikno Harmoko,Ki Seong Ko,Nirmal Kumar Ramasamy,Kyung Hwa Kim,Hyun Suk Jung,Jae-Yean Kim,Sang Yeol Lee,Kyun Oh Lee 한국당과학회 2012 한국당과학회 학술대회 Vol.2012 No.1
Plant produce complex N-glycans with ß1,2-xylose and core α1,3-fucose residues linked to the conserved core oligosaccharide. Production of heterologous glycoprotein in plants for therapeutic purposes is limited by the presence of plant specific oligosaccharide residues that are considered to arouse immunogenic response in the recipient body. Therefore, efforts for developing humanized N-glycosylation system in plants are required for the large scale production of safe therapeutic glycoproteins. In this regard, we isolated a rice mutant lacking complex N-glycans. However, the mutant showed severe developmental defects such as retarded shoot and root development, failure in tiller formation, and finally resulted in early developmental lethality. It is also shown that the mutant contains low amount of cellulose and high content of lignin. Interestingly, callus induced from the mutant seeds was maintainable and proliferate similarly compared with that of WT despite the size were relatively smaller.
Isolation and characterization of AtXBP1, a functional homolog of XBP1/Hac1, in Arabidopsis
Wahyu Indra Fanata,Jeong Chan Moon,Joo Mi Jeon,Bo Hwa Son,In Jung Jung,Jae Yong Yoo,Jae Ho Cha,Je Hein Kim,Rikno Harmoko,Ki Seong Ko,Sang Yeol Lee,Kyun Oh Lee 한국당과학회 2009 한국당과학회 학술대회 Vol.2009 No.1
A number of environmental and physiological stimuli, such as perturbation in calcium homeostasis, shift of endoplasmic reticulum (ER) redox potential, altered glycosylation and elevated protein synthesis lead to accumulation of unfolded or misfolded proteins in the ER lumen, and subsequently impose stress to ER. Such condition activates a set of signaling pathway termed the unfolded protein response (UPR). To date, a large number of components for UPR signaling pathway has been isolated and characterized in yeast or mammals. Plants also show a quite similar response compared to the UPR in yeast and mammals when they are exposure to the stimuli. However, the plant UPR and its signaling pathway are rarely studied. In this report, we present our research regarding to the characterization of a UPR signaling component AtXBP1 in Arabidopsis. AtXBP1 is alternatively spliced generating two isoforms; long isoform (AtXBP1L) and short isoform (AtXBP1S). Overexpression of AtXBP1S has shown to up regulate the expression of ER stress responsive genes. Futhermore, expression of the luciferase gene, driven by BiP3 promoter, under the ER stress condition has been significantly decreased in the atxbp1 mutant. Electrophoretic mobility shift assay revealed that only AtXBP1S is able to interact with putative XBP1 binding cis-elements in AtBiP2 and AtBiP3 promoters. These results support our hypothesis that the AtXBP1 is an important regulatory component in the UPR signaling in Arabidopsis. [Supported by BK21 program]
Poster Session : Characterization of DJ-1 Like gene in arabidopsis
( Wahyu Indra Duwi Fanata ),( Min Hee Jung ),( Joon Yung Cha ),( Netty Ermawati ),( Ying Shi Liang ),( Mukhamad Su`udi ),( Sun Ping ),( Sun Shin Cha ),( Kon Ho Lee ),( Dae Young Son ) 한국생화학분자생물학회 (구 한국생화학회) 2006 생화학분자생물학회 춘계학술발표논문집 Vol.2006 No.-
N-Acetylglucosaminyltransferase I (GnTI) is Developmentally essential in Rice (Oryza sativa)
Wahyu Indra Fanata,Jeong Chan Moon,Joo Mi Jeon,Bo Hwa Son,In Jung Jung,Jae Yong Yoo,Jae Ho Cha,Je Hein Kim,Rikno Harmoko,Ki Seong Ko,Sang Yeol Lee,Kyun Oh Lee 한국당과학회 2009 한국당과학회 학술대회 Vol.2009 No.1
N-acetylglucosaminyltransferase I (GnTI) is an enzyme that controls the formation of hybrid and complex type N-glycans. Plant produce complex N-glycans with β1,2-xylose and core α1,3-fucose residues linked to the conserved core oligosaccharide. Production of heterologous glycoprotein in plant for therapeutic purposes is limited by the presence of plant specific oligosaccharide residues that are considered to arouse immunogenic response in the recipient body. Therefore, efforts for developing humanized N-glycosylation system in plants are required for the large scale production of safe therapeutic glycoproteins. In this regard, we isolated a rice mutant lacking GnTI activity. Biochemical analyses showed that rice gnt1 mutant predominantly produced high mannose type glycans and β1,2-xylose and core α1,3-fucose were absent on the endogenous glycoproteins. However, the rice gnt1 mutant showed severe developmental defects such as retarded shoot and root development, failure in tiller formation, and finally resulted in early developmental lethality. Interestingly, callus induced from gnt1 seeds was maintainable and proliferation rate of gnt1 callus was similar with that of WT. However, gnt1 calli were relatively smaller than that of WT. [Supported by EB-NCRC & BK21 program]
Plant made pharmaceuticals (PMP) as a convenient, safe and economical alternative to the shortfall
Wahyu Indra Fanata,In Jung Jung,Bo Hwa Son,Jae Yong Yoo,Je Hein Kim,Rikno Harmoko,Ki Seong Ko,Sang Yeol Lee,Kyun Oh Lee 한국당과학회 2010 한국당과학회 학술대회 Vol.2010 No.1
Since the completion of the human genome project, development of new therapeutic and diagnostic proteins have been exponentially increased, but widespread use of these molecules has been hampered by production bottlenecks such as low yields, poor and inconsistent product quality and a shortage of production capacity. Especially, we are facing a growing demand for protein diagnostics and therapeutics, but lack the capacity to meet those demands using established facilities. Over the last decade, plants have emerged as a convenient, safe and economical alternative to mainstream expression systems which are based on the large-scale culture of microbes or animal cells, or transgenic animals. The production of plant-made pharmaceuticals and technical proteins is known as Molecular Farming. The objective is to harness the power of agriculture to cultivate and harvest plants or plant cells producing recombinant therapeutics, diagnostics, industrial enzymes and green chemicals. Molecular Farming has the potential to provide virtually unlimited quantities of recombinant antibodies, vaccines, blood substitutes, growth factors, cytokines and enzymes for use as diagnostic and therapeutic tools in health care, the life sciences and the chemical industry. The overall aim of our research is to develop new plant molecular farming systems which are economic, efficient, stable and safe. To accomplish the purpose, we are performing specific research objectives in regards to plant molecular farming.
Callus Induction and Regeneration from Anther Cultures of Indonesian Indica Black Rice Cultivar
Anisa Maharani,Wahyu Indra Duwi Fanata,Faida Nur Laeli,김경민,Tri Handoyo 한국작물학회 2020 Journal of crop science and biotechnology Vol.23 No.1
The assembly of superior varieties and collection of rice germplasm involves the process of selecting and storing elders that have superior genotypic properties and phenotypes. The anther culture techniques on indica black rice cultivar have a high difficulty factor to get plants, because of the low regeneration ability at the plant formation phase from the anther callus. This study aimed to investigate the influence of the cold-pretreatment time on anther, the combination of plant growth regulators (PGR’s) concentrations, and putrescine concentrations in media for the increase callus induction and plant regeneration of indica black rice. The optimization of the cold pre-treatment time was important to obtain the high-frequency callus induction, which showed that anther at the 4°C for 8 days formed the high callus induction (20%). To accelerate the callus induction, the application of 20 µM putrescine in the MS medium could produce more friable embryogenic callus for 24 days with 27% of callus formation. Generally, the optimal medium for the high frequency of callus induction contained 2 mgL-1 NAA+0.5 mgL-1 Kinetin+20 µM putrescine. Especially indica black rice cultivars, the best media to get a high plant regeneration frequency were N6 media containing the combination of 2 mgL-1 IAA and 2,5 mgL-1 Kinetin. The total callus regenerated to plantlet about 12.5%. The study of the callus induction and in-vitro plant regeneration medium for indica black rice were still important to develop to get the best result for other cultivars.
RNA-Dependent RNA Polymerase 6 Is Required for Efficient hpRNA-Induced Gene Silencing in Plants
RIKNOHARMOKO,이균오,Wahyu Indra Duwi Fanata,유재용,고기성,임영길,엠다나짐우단,Tri Agus Siswoyo,이승식,김둘이,이상열 한국분자세포생물학회 2013 Molecules and cells Vol.35 No.3
In plants, transgenes with inverted repeats are used to in-duce efficient RNA silencing, which is also frequently induced by highly transcribed sense transgenes. RNA silencing induced by sense transgenes is dependent on RNA-dependent RNA polymerase 6 (RDR6), which con-verts single-stranded (ss) RNA into double-stranded (ds) RNA. By contrast, it has been proposed that RNA silencing induced by self-complementary hairpin RNA (hpRNA) does not require RDR6, because the hpRNA can directly fold back on itself to form dsRNA. However, it is unclear whether RDR6 plays a role in hpRNA-induced RNA silencing by amplifying dsRNA to spread RNA silencing within the plant. To address the efficiency of hpRNA-induced RNA silencing in the presence or absence of RDR6, Wild type (WT, Col-0) and rdr6-11 Arabidopsis thaliana lines expressing green fluorescent protein (GFP) were generated and transformed with a GFP-RNA interference (RNAi) construct. Whereas most GFP-RNAi-transformed WT lines exhibited almost complete silencing of GFP expression in the T1 generation, various levels of GFP expression remained among the GFP-RNAi-transformed rdr6-11 lines. Homozygous expression of GFP-RNAi in the T3 generation was not sufficient to induce complete GFP silencing in several rdr6-11 lines. Our results indicate that RDR6 is required for efficient hpRNA-induced RNA silencing in plants.
RNA-dependent RNA polymerase 6 is required for efficient hpRNA-induced gene silencing in plants
Harmoko, Rikno,Fanata, Wahyu Indra Duwi,Yoo, Jae Yong,Ko, Ki Seong,Rim, Yeong Gil,Uddin, Mohammad Nazim,Siswoyo, Tri Agus,Lee, Seung Sik,Kim, Dool Yi,Lee, Sang Yeol,Lee, Kyun Oh Springer-Verlag 2013 Molecules and cells Vol.35 No.3
Loss of ALG3 function (alg3) Leads to Enhanced ER Quality Control (ERQC) in Arabidopsis thaliana
Bo Hwa Son,Wahyu Indra Fanata,Jae Yong Yoo,Rikno Harmoko,Ki Seong Ko,Nirmal Kumar Ramasamy,Kyung Hwa Kim,Thiyagarajan Thulasinathan,Sang Yeol Lee,Kyun Oh Lee 한국당과학회 2012 한국당과학회 학술대회 Vol.2012 No.1
In plants, glycoproteins have been implicated in a wide variety of cellular processes including production of cell walls, pollination, pathogen defence and cell-to-cell communication. In addition, they have attracted considerable interest from the medical field as the main cause of food and pollen allergies. N-glycosylation is a complex process that encompasses the biosynthesis and modification of sugar moieties in the endoplasmic reticulum (ER) and Golgi. The core oligosaccharide Glc3Man9GlcNAc2 is assembled by a series of membrane-bound glycosyltransferases as the lipid carrier dolichylpyrophosphate-linked glycan in the endoplasmic reticulum (ER). The first step of this assembly pathway on the ER luminal side is mediated by ALG3 (asparagine-linked glycosylation 3), which is a highly conserved reaction among eukaryotic cells. In Arabidopsis ALG3 mutant (alg3), an immature lipid-linked oligosaccharide structure, M5ER, was synthesized and efficiently processed into complex-type glycans. Although no high-mannose-type glycoproteins are detected in alg3 plants, these plants do not show a growth phenotype under normal growth conditions. However, the glycosylation abnormalities result in activation of marker genes diagnostic of the unfolded protein response and alg3 mutant showed a stress sensitive phenotype. These results indicate that ALG3 is a critical factor for correct N-glycosylation of proteins and is involved in the ER stress response.