Biomedical subjects

Kyung-Hee Paek

Publications and source records attributed to Kyung-Hee Paek.

At least 19 recordsLinked to original sources

PubMed2006

Functional study of hot pepper 26S proteasome subunit RPN7 induced by Tobacco mosaic virus from nuclear proteome analysis.

Two-dimensional gel electrophoresis (2-DE) was applied for the screening of Tobacco mosaic virus (TMV)-induced hot pepper (Capsicum annuum cv. Bugang) nuclear proteins. From differentially expressed protein spots, we acquired the matched peptide mass fingerprint (PMF) data, analyzed by MALDI-TOF MS, from the non-redundant hot pepper EST protein FASTA database using the VEMS 2.0 software. Among six identified nuclear proteins, the hot pepper 26S proteasome subunit RPN7 (CaRPN7) was subjected to further study. The level of CaRPN7 mRNA was specifically increased during incompatible TMV-P(0) interaction, but not during compatible TMV-P(1.2) interaction. When CaRPN7::GFP fusion protein was targeted in onion cells, the nuclei had been broken into pieces. In the hot pepper leaves, cell death was exacerbated and genomic DNA laddering was induced by Agrobacterium-mediated transient overexpression of CaPRN7. Thus, this report presents that the TMV-induced CaRPN7 may be involved in programmed cell death (PCD) in the hot pepper plant.

Amino Acid Sequence↗

PubMed2006

Tobacco Tsip1, a DnaJ-type Zn finger protein, is recruited to and potentiates Tsi1-mediated transcriptional activation.

Tobacco stress-induced1 (Tsi1) is an ethylene-responsive-element binding protein/APETALA2-type transcription factor that plays an important role in both biotic and abiotic stress signaling pathways. We show that Tsi1-interacting protein1 (Tsip1), a DnaJ-type Zn finger protein, interacts with Tsi1 in vitro and in yeast (Saccharomyces cerevisiae). The transcript level of Tsip1 in tobacco (Nicotiana tabacum) increased upon treatment with salicylic acid (SA), ethylene, gibberellic acid, NaCl, and virus challenge. Tsip1 appeared to be physically associated with the chloroplast surface but dissociated from it after SA treatment. Tsip1 colocalized and coimmunoprecipitated with Tsi1 in plant cells following SA treatment. Tsip1 expression increased Tsi1-mediated transcription and was able to functionally compensate for loss of the Tsi1 transcriptional activation domain through a direct interaction with Tsi1. Transgenic plants simultaneously coexpressing Tsi1 and Tsip1 displayed stronger pathogen resistance and salt tolerance than did transgenic plants expressing either Tsi1 or Tsip1 alone. Concurrent with this, the expression of a subset of stress-related genes was induced in a cooperative manner in Tsi1/Tsip1 transgenic plants. These results together implied that Tsi1 recruits Tsip1 to the promoters of stress-related genes to potentiate Tsi1-mediated transcriptional activation.

Amino Acid Sequence↗

PubMed2006

An Arabidopsis homologue of human seven-in-absentia-interacting protein is involved in pathogen resistance.

Human seven-in-absentia (SIAH)-interacting protein (SIP) is a component of the E3 ligase complex targeting beta-catenin for destruction. Arabidopsis has one SIP protein (AtSIP) with 32% amino acid sequence identity to SIP. To investigate the functions of AtSIP, we isolated an atsip knockout mutant, and generated transgenic plants overexpressing AtSIP. The growth rates and morphologies of the atsip and transgenic plants were indistinguishable from those of wild type. However, atsip plants were more susceptible to Pseudomonas syringae infection, and the transgenic plants overexpressing AtSIP were more resistant. Consistent with this, RNA blot analysis showed that the AtSIP gene is strongly induced by wounding and hydrogen peroxide treatment. In addition, when plants were infected with P. syringae, AtSIP was transiently induced prior to PR-1 induction. These observations show that Arabidopsis AtSIP plays a role in resistance to pathogenic infection.

Arabidopsis↗

PubMed2006

In vivo binding of hot pepper bZIP transcription factor CabZIP1 to the G-box region of pathogenesis-related protein 1 promoter.

We find that salicylic acid and ethephon treatment in hot pepper increases the expression of a putative basic/leucine zipper (bZIP) transcription factor gene, CabZIP1. CabZIP1 mRNA is expressed ubiquitously in various organs. The green fluorescent protein-fused transcription factor, CabZIP1::GFP, can be specifically localized to the nucleus, an action that is consistent with the presence of a nuclear localization signal in its protein sequence. Transient overexpression of the CabZIP1 transcription factor results in an increase in PR-1 transcripts level in Nicotiana benthamiana leaves. Using chromatin immunoprecipitation, we demonstrate that CabZIP1 binds to the G-box elements in native promoter of the hot pepper pathogenesis-related protein 1 (CaPR-1) gene in vivo. Taken together, our results suggest that CabZIP1 plays a role as a transcriptional regulator of the CaPR-1 gene.

Amino Acid Sequence↗

PubMed2006

Suppression of CaCYP1, a novel cytochrome P450 gene, compromises the basal pathogen defense response of pepper plants.

A putative cytochrome P450 gene from chili pepper, Capsicum annuum L. Bukang cytochrome P450 (CaCYP1), was identified using cDNA microarray analysis of gene expression following induction of the leaf hypersensitive response by inoculation of pepper plants with the non-host pathogen Xanthomonas axonopodis pv. glycines 8ra. The full-length cDNA of CaCYP1 encoded a protein of 514 amino acid residues, which contained a putative hydrophobic membrane anchoring domain in the N-terminal region, and a heme-binding motif in the C-terminal region. Analysis of the deduced amino acid sequence of CaCYP1 revealed that it has high homology to Arabidopsis CYP89A5, the function of which is unknown. Expression of CaCYP1 was preferentially increased in pepper plants in response to non-host pathogen inoculation and also during the host resistance response. CaCYP1 expression also increased following treatment with salicylic acid and abscisic acid, while treatment with ethylene had a mild effect. Using a virus-induced gene silencing-based reverse genetics approach, we demonstrated that suppression of CaCYP1 results in enhanced susceptibility to bacterial pathogens. Interestingly, gene silencing of CaCYP1 in pepper plants resulted in the reduced expression of the defense-related genes CaLTP1, CaSIG4, and Cadhn. Our results indicated that CaCYP1, a novel cytochrome P450 in pepper plants, may play a role in plant defense response pathways that involve salicylic acid and abscisic acid signaling pathways.

Amino Acids↗

PubMed2006

Arabidopsis tonoplast proteins TIP1 and TIP2 interact with the cucumber mosaic virus 1a replication protein.

The cucumber mosaic virus (CMV) replication complex has previously been shown to associate with cellular membranes. However, it remains unknown whether any host factors participate in this process. In this study, five groups of Arabidopsis tonoplast intrinsic protein (TIP) genes were isolated and the proteins they encoded were evaluated with regard to their interactions with CMV proteins. TIP1 and TIP2 were found to interact with the CMV 1a protein in the Sos recruitment system, whereas no interactions with the other three TIP subgroups were observed in this assay. The interaction of CMV 1a with the TIP1 and TIP2 proteins was confirmed via co-immunoprecipitation assays. Additionally, CMV 1a co-localized with TIP1 and TIP2 in transfected Arabidopsis protoplasts. The findings of this study suggest that members of two TIP subfamilies might affect CMV replication via interaction with CMV 1a in the tonoplasts.

Aquaporins↗

PubMed2005

Induction of a cytosolic pyruvate kinase 1 gene during the resistance response to Tobacco mosaic virus in Capsicum annuum.

Hot pepper (Capsicum annuum L. cv. Bugang) plants exhibit a hypersensitive response (HR) upon infection by Tobacco mosaic virus (TMV) pathotype P(0). Previously, to elucidate molecular mechanism that underlies this resistance, hot pepper cv. Bugang leaves were inoculated with TMV-P(0) and genes specifically up-regulated during the HR were isolated by microarray analysis. One of the clones, Capsicum annuum cytosolic pyruvate kinase 1 (CaPK(c)1) gene was increased specifically in the incompatible interaction with TMV-P(0). The expression of CaPK(c)1 gene was also triggered not only by various hormones such as salicylic acid (SA), ethylene, and methyl jasmonate (MeJA), but also NaCl and wounding. These results suggest that CaPK(c)1 responds to several defense-related abiotic stresses in addition to TMV infection.

Amino Acid Sequence↗

PubMed2005

A hot pepper gene encoding WRKY transcription factor is induced during hypersensitive response to Tobacco mosaic virus and Xanthomonas campestris.

Plant WRKY transcription factors were previously implicated in the alteration of gene expression in response to various pathogens. The WRKY proteins constitute a large family of plant transcription factors, whose precise functions have yet to be elucidated. Using a domain-specific differential display procedure, we isolated a WRKY gene, which is rapidly induced during an incompatible interaction between hot pepper and Tobacco mosaic virus (TMV) or Xanthomonas campestris pv . vesicatoria (Xcv). The full-length cDNA of CaWRKY-a (Capsicum annuum WRKY-a) encodes a putative polypeptide of 546 amino acids, containing two WRKY domains with a zinc finger motif. The expression of CaWRKY-a could be rapidly induced by not only chemical elicitor such as salicylic acid (SA) or ethephon but also wounding treatments. The nuclear localization of CaWRKY-a was determined in transient expression system using tobacco BY-2 cells by polyethylene glycol (PEG)-mediated transformation experiment. With oligonucleotide molecules containing the putative W-box sequences as a probe, we confirmed that CaWRKY-a protein had W-box-binding activity. These results suggest that CaWRKY-a might be involved as a transcription factor in plant defense-related signal transduction pathways.

Amino Acid Sequence↗

PubMed2005

Novel protein kinase interacts with the Cucumber mosaic virus 1a methyltransferase domain.

The Cucumber mosaic virus (CMV)-encoded 1a protein has been implicated to play a role in replication of the viral genome along with 2a and one or more host factors. To identify the host cell factors interacting with CMV 1a, we used the yeast two-hybrid system using tobacco cDNA library. One of the cDNA clones encoded a protein homologous to the Arabidopsis putative protein kinase and was designated as Tcoi2 (Tobacco CMV 1a interacting protein 2). Tcoi2 specifically interacted with methyltransferase (MT) domain of CMV 1a protein in yeast cell. In vitro analyses using recombinant proteins showed that Tcoi2 also specifically interacted with CMV 1a MT domain. Tcoi2 did not have autophosphorylation activity but phosphorylated CMV 1a MT domain. Analysis of the subcellular localization of the Tcoi2 fused to GFP demonstrated that it is targeted to the endoplasmic reticulum. These results suggest Tcoi2 as a novel host factor that is capable of interacting and phosphorylating MT domain of CMV 1a protein.

Amino Acid Sequence↗

PubMed2005

BnNHL18A shows a localization change by stress-inducing chemical treatments.

The two genes, named BnNHL18A and BnNHL18B, showing sequence homology with Arabidopsis NDR1/HIN1-like (NHL) genes, were isolated from cDNA library prepared with oilseed rape (Brassica napus) seedlings treated with NaCl. The transcript level of BnNHL18A was increased by sodium chloride, ethephon, hydrogen peroxide, methyl jasmonate, or salicylic acid treatment. The coding regions of BnNHL18A and BnNHL18B contain a sarcolipin (SLN)-like sequence. Analysis of the localization of smGFP fusion proteins showed that BnNHL18A is mainly localized to endoplasmic reticulum (ER). This result suggests that the SLN-like sequence plays a role in retaining proteins in ER membrane in plants. In response to NaCl, hydrogen peroxide, ethephon, and salicylic acid treatments, the protein localization of BnNHL18A was changed. Our findings suggest a common function of BnNHL18A in biotic and abiotic stresses, and demonstrate the presence of the shared mechanism of protein translocalization between the responses to plant pathogen and to osmotic stress.

Acetates↗

PubMed2005

CaAlaAT1 catalyzes the alanine: 2-oxoglutarate aminotransferase reaction during the resistance response against Tobacco mosaic virus in hot pepper.

Hot pepper (Capsicum annuum L. cv. Bugang) plants exhibit a hypersensitive response (HR) upon infection by Tobacco mosaic virus (TMV) pathotype P0. To elucidate molecular mechanism that underlies this resistance, hot pepper cv. Bugang leaves were inoculated with TMV-P0 and genes specifically up-regulated during the HR were isolated by differential screening. One of the clones, CaAlaAT1 encoding a putative alanine aminotransferase (EC 2.6.1.2) exhibited organ-specific expression pattern and the transcript accumulated abundantly in red (ripe) fruit tissues. CaAlaAT1 transcript was also induced in older leaves during senescence. The expression of CaAlaAT1 gene was increased in the incompatible interaction with TMV-P0 but was not in the compatible interaction with TMV-P1.2. When a strain of Xanthomonas campestris pv. vesicatoria (Xcv) carrying an AvrBs2 gene was infiltrated into the leaves of a pepper cv. ECW 20R carrying Bs2 resistance gene, a marked induction and maintenance of CaAlaAT1 gene expression was observed. The expression of CaAlaAT1 gene was triggered by salicylic acid (SA) and ethylene but not by methyl jasmonate (MeJA). CaAlaAT1 seemed to be localized mostly at the cytosol from the polyethylene glycol (PEG)-mediated transformation experiment. CaAlaAT1 seemed to catalyze alanine: 2-oxoglutarate aminotransferase (AKT) reaction, which was a main activity among the four activities in vitro, during the resistance response against TMV in hot pepper. These results suggest that CaAlaAT1, a protein known to be involved in metabolic reactions, might be one of the components in the plant's defense signal pathway against pathogens.

Alanine Transaminase↗

PubMed2005

Internalization and trafficking mechanisms of coxsackievirus B3 in HeLa cells.

Coxsackievirus B3 (CVB3) is nonenveloped and has a single-stranded positive-sense RNA genome. CVB3 induces myocarditis and ultimately dilated cardiomyopathy. Although there are mounting evidences of an interaction between CVB3 particles and the cellular receptors, coxsackievirus and adenovirus receptor (CAR) and decay-accelerating factor (DAF), very little is known about the mechanisms of internalization and trafficking. In the present study, we used the CVB3 H3 strain, which is CAR-dependent but DAF-independent Woodruff variant and found that during entry, CVB3 particles were colocalized in clathrin, after interacting primarily with CAR, which was not recycled to the plasma membrane. We also found that CVB3 internalization was dependent on the function of dynamin, a large GTPase that has an essential role in endocytosis. Heat-shock cognate protein, Hsc70, which acts as a chaperone in the release of coat proteins from clathrin-coated vesicles (CCV), played a role in CVB3 trafficking processes. Moreover, endosomal acidification was crucial for CVB3 endocytosis. Finally, CVB3 was colocalized in early endosome autoantigen 1 (EEA1) molecules, which are involved in endosome-endosome tethering and fusion. In conclusion, these data together indicate that CVB3 uses clathrin-mediated endocytosis and is transcytosed to early endosomes.

Base Sequence↗

PubMed2005

In vitro and in planta interaction evidence between Nicotiana tabacum thaumatin-like protein 1 (TLP1) and cucumber mosaic virus proteins.

Using a yeast two-hybrid system, we identified a plant cellular factor that interacts with the proteins of the Cucumber mosaic virus (CMV). Initially 14 candidate genes were isolated from Nicotiana tabacum, using a full-length CMV 1a gene as bait. Among the candidate genes, two were encoding thaumatin-like proteins (TLP), and were designated as Nicotiana tabacum thaumatin-like protein 1 (NtTLP1). Consistent with this observation, recombinant GST-NtTLP1 protein, which was expressed and purified in E. coli, bound tightly to CMV 1a in vitro. In planta interaction was also verified via co-immunoprecipitation. Additionally, NtTLP1 specifically interacted with the CMV movement-related proteins, movement protein and coat protein, in yeast. Real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the expression of NtTLP1 increased as the result of CMV inoculation.

Arabidopsis Proteins↗

PubMed2004

Molecular characterization of pepper germin-like protein as the novel PR-16 family of pathogenesis-related proteins isolated during the resistance response to viral and bacterial infection.

To understand the molecular defense mechanism controlling the hypersensitive response (HR) better, we examined the hot pepper plant (Capsicum annuum L. cv. Bugang), which exhibits an HR in response to infection by Tobacco mosaic virus pathotype P0 (TMV-P0). A full-length cDNA clone was isolated by differential screening of a cDNA library that was constructed with mRNA extracted from hot pepper leaves during the resistance response to TMV-P0. The predicted amino acid sequence of the cDNA clone exhibited a high sequence similarity to germin-like protein (GLP). The CaGLP1 (Capsicum annuum GLP1) cDNA contains a single open reading frame of 660 bp encoding 220 amino acid residues. Upon inoculation with TMV or Xanthomonas, CaGLP1 transcripts were specifically accumulated in the incompatible interaction but not in the compatible interaction. In plants treated with salicylic acid (SA) or ethephon, which are signal molecules in the defense-related signal transduction pathway, CaGLP1 transcripts were accumulated rapidly. As far as we know, this is the first report that plant GLPs can be specifically induced during a defense response against viral infection. These data suggest that in the hot pepper plant CaGLP1 may be involved in the defense response to viral pathogens, and thus be classified as a new family of PR proteins, 'PR-16'.

Amino Acid Sequence↗

PubMed2004

Capsicum annuum tobacco mosaic virus-induced clone 1 expression perturbation alters the plant's response to ethylene and interferes with the redox homeostasis.

Capsicum annuum tobacco mosaic virus (TMV)-induced clone 1 (CaTin1) gene was expressed early during incompatible interaction of hot pepper (Caspsicum annuum) plants with TMV and Xanthomonas campestris. RNA-blot analysis showed that CaTin1 gene was expressed only in roots in untreated plants and induced mainly in leaf in response to ethylene, NaCl, and methyl viologen but not by salicylic acid and methyl jasmonate. The ethylene dependence of CaTin1 induction upon TMV inoculation was demonstrated by the decrease of CaTin1 expression in response to several inhibitors of ethylene biosynthesis or its action. Transgenic tobacco (Nicotiana tabacum) plants expressing CaTin1 gene in sense- or antisense-orientation showed interesting characteristics such as the accelerated growth and the enhanced resistance to biotic as well as abiotic stresses. Such characteristics appear to be caused by the elevated level of ethylene and H2O2. Moreover, in transgenic plants expressing antisense CaTin1 gene, the expression of some pathogenesis-related genes was enhanced constitutively, which may be mainly due to the increased ethylene level. The promoter of CaTin1 has four GCC-boxes, two AT-rich regions, and an elicitor-inducible W-box. The induction of the promoter activity by ethylene depends on GCC-boxes and by TMV on W-box. Taken together, we propose that the CaTin1 up-regulation or down-regulation interferes with the redox balance of plants leading to the altered response to ethylene and biotic as well as abiotic stresses.

Acetates↗

PubMed2004

Pathogenesis-related protein 10 isolated from hot pepper functions as a ribonuclease in an antiviral pathway.

A hot pepper (Capsicum annuum) cDNA clone encoding pathogenesis-related protein 10 (CaPR-10) was isolated by differential screening of a cDNA library prepared from pepper leaves inoculated with tobacco mosaic virus pathotype (TMV-P0). CaPR-10 transcripts were induced in the incompatible interaction with TMV-P0 or Xanthomonas campestris pv. vesicatoria (Xcv) but not induced in the compatible interaction. Characterization of enzymatic properties of CaPR-10 indicated that the recombinant protein exhibits a ribonucleolytic activity against TMV RNA, as well as against pepper total RNA, and shows its putative antiviral activity in several conditions. The CaPR-10 protein existed at very low level in leaf tissue but was dramatically induced as soon as plants were inoculated with TMV-P0, and this was correlated with the increase of its ribonucleolytic activity. Immunoblot analysis and pull-down assays using proteins extracted from pepper leaves showed that TMV-P0 inoculation led to the phosphorylation of CaPR-10, a modification that should affect its capacity for RNase function. We present data that the induction and subsequent phosphorylation of CaPR-10 increased its ribonucleolytic activity to cleave invading viral RNAs, and this activity should be important to its antiviral pathway during viral attack in vivo.

Amino Acid Sequence↗

PubMed2004

Ornithine decarboxylase gene (CaODC1) is specifically induced during TMV-mediated but salicylate-independent resistant response in hot pepper.

A gene encoding putative ornithine decarboxylase (ODC) has been isolated by differential screening of a cDNA library from the resistant hot pepper (Capsicum annuum L.) inoculated with avirulent tobacco mosaic virus (TMV) pathotype P0. In hot pepper plants, transcripts of the CaODC1 (C. annuum ODC1) gene started to accumulate at 24 h post-inoculation of TMV-P0 and the signal was spread systemically. The transcript level of CaODC1 was increased rapidly in a hot pepper resistant to Xanthomonas campestris pv. vesicatoria (Xcv) but not in a susceptible hot pepper after inoculation. These results suggest possible role(s) for CaODC1 in plant defense against a broad range of pathogens including viruses and bacteria.

Amino Acid Sequence↗

PubMed2003

Activation of the programmed cell death pathway by inhibition of proteasome function in plants.

Proteasomes constitute the major machinery to degrade or process proteins by ATP/ubiquitin-mediated proteolysis. Recent findings suggest a pivotal role of the ubiquitin/proteasome pathway in the regulation of apoptosis in animal cells. Here we show that virus-induced gene silencing of two different subunits of the 26 S proteasome, the alpha 6 subunit of the 20 S proteasome and RPN9 subunit of 19 S regulatory complex, both activated the programmed cell death (PCD) program, accompanied by reduced proteasome activity and accumulation of polyubiquitinated proteins. These results demonstrate that disruption of proteasome function leads to PCD in plant cells. The affected cells showed morphological markers of PCD, including nuclear condensation and DNA fragmentation, accompanied by the 10-fold higher production of reactive oxygen species and increased ion leakage for 3-fold. Similar to apoptosis in animal system, mitochondrial membrane potential was decreased, cytochrome c released from mitochondria to cytosol, and caspase 9- and caspase 3-like proteolytic activities detected in the cells. Interestingly, this proteasome-mediated PCD stimulated the expression of only a subset of transcripts that are highly induced during pathogen-mediated hypersensitive response cell death, indicating that the two PCD pathways are differentially regulated. Taken together, these results provide the first direct evidence that proteasomes play a role in the regulatory program of PCD in plants. Controlled inhibition of proteasome activities may be involved in developmentally or environmentally activated plant cell death programs.

Apoptosis↗