scholarly journals The transcription factor OpWRKY2 positively regulates the biosynthesis of the anticancer drug camptothecin in Ophiorrhiza pumila

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Xiaolong Hao ◽  
Chenhong Xie ◽  
Qingyan Ruan ◽  
Xichen Zhang ◽  
Chao Wu ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Anticancer Activity ◽  
Time Course ◽  
Indole Alkaloid ◽  
Fold Increase ◽  
Wrky Transcription Factor ◽  
Mobility Shift ◽  
Pathway Gene ◽  
Low Concentrations ◽  
Encoding Gene

AbstractThe limited bioavailability of plant-derived natural products with anticancer activity poses major challenges to the pharmaceutical industry. An example of this is camptothecin, a monoterpene indole alkaloid with potent anticancer activity that is extracted at very low concentrations from woody plants. Recently, camptothecin biosynthesis has been shown to become biotechnologically amenable in hairy-root systems of the natural producer Ophiorrhiza pumila. Here, time-course expression and metabolite analyses were performed to identify novel transcriptional regulators of camptothecin biosynthesis in O. pumila. It is shown here that camptothecin production increased over cultivation time and that the expression pattern of the WRKY transcription factor encoding gene OpWRKY2 is closely correlated with camptothecin accumulation. Overexpression of OpWRKY2 led to a more than three-fold increase in camptothecin levels. Accordingly, silencing of OpWRKY2 correlated with decreased camptothecin levels in the plant. Further detailed molecular characterization by electrophoretic mobility shift, yeast one-hybrid and dual-luciferase assays showed that OpWRKY2 directly binds and activates the central camptothecin pathway gene OpTDC. Taken together, the results of this study demonstrate that OpWRKY2 acts as a direct positive regulator of camptothecin biosynthesis. As such, a feasible strategy for the over-accumulation of camptothecin in a biotechnologically amenable system is presented.

scholarly journals AaWRKY17, a positive regulator of artemisinin biosynthesis, is involved in resistance to Pseudomonas syringae in Artemisia annua

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Tiantian Chen ◽  
Yongpeng Li ◽  
Lihui Xie ◽  
Xiaolong Hao ◽  
Hang Liu ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Pseudomonas Syringae ◽  
Artemisia Annua ◽  
Wrky Transcription Factor ◽  
Marker Genes ◽  
Mobility Shift ◽  
Positive Regulator ◽  
Pathway Gene ◽  
Transgenic Breeding ◽  
Artemisinin Biosynthesis

AbstractArtemisia annua, a traditional Chinese medicinal plant, remains the only plant source for artemisinin production, yet few genes have been identified to be involved in both the response to biotic stresses, such as pathogens, and artemisinin biosynthesis. Here, we isolated and identified the WRKY transcription factor (TF) AaWRKY17, which could significantly increase the artemisinin content and resistance to Pseudomonas syringae in A. annua. Yeast one-hybrid (Y1H), dual-luciferase (dual-LUC), and electrophoretic mobility shift assay (EMSA) results showed that AaWRKY17 directly bound to the W-box motifs in the promoter region of the artemisinin biosynthetic pathway gene amorpha-4,11-diene synthase (ADS) and promoted its expression. Real-time quantitative PCR (RT-qPCR) analysis revealed that the transcript levels of two defense marker genes, Pathogenesis-Related 5 (PR5) and NDR1/HIN1-LIKE 10 (NHL10), were greatly increased in AaWRKY17-overexpressing transgenic A. annua plants. Additionally, overexpression of AaWRKY17 in A. annua resulted in decreased susceptibility to P. syringae. These results indicated that AaWRKY17 acted as a positive regulator in response to P. syringae infection. Together, our findings demonstrated that the novel WRKY transcription factor AaWRKY17 could potentially be used in transgenic breeding to improve the content of artemisinin and pathogen tolerance in A. annua.

scholarly journals Altered Regulation of 15-Acetyldeoxynivalenol Production in Fusarium graminearum

2000 ◽  
Vol 66 (5) ◽  
pp. 2062-2065 ◽  
Author(s):  
Lifeng Chen ◽  
Susan P. McCormick ◽  
Thomas M. Hohn
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Reporter Gene ◽  
Fusarium Graminearum ◽  
Large Scale ◽  
Fold Increase ◽  
Gene Clusters ◽  
Gus Expression ◽  
Plasmid Integration ◽  
Pathway Gene

ABSTRACT Most Fusarium graminearum isolates produce low or undetectable levels of trichothecenes in liquid shake cultures, making it difficult to perform biochemical studies of trichothecene biosynthesis. To develop strains with higher levels of trichothecene production under liquid shake conditions we transformed F. graminearum with both a reporter gene containing a homologous trichothecene pathway gene promoter (TRI5) and a gene encoding a heterologous trichothecene pathway transcription factor (TRI6). The TRI5 and TRI6 genes are part of the trichothecene pathway gene clusters of both Fusarium sporotrichioides and F. graminearum. These genes encode trichodiene synthase (encoded by TRI5), the first enzyme in the trichothecene pathway, and a transcription factor (encoded by TRI6) required for pathway gene expression. Transformation of F. graminearum with plasmids containing either an F. graminearum TRI5 promoter fragment (FGTRI5P ) or FGTRI5P coupled with the β-d-glucuronidase (GUS) reporter gene resulted in the identification of several transformants capable of producing 45 to 200 mg of 15-acetyldeoxynivalenol (15-ADON)/liter in liquid shake culture after 7 days. Increased 15-ADON production was only observed in transformants where plasmid integration occurred through the FGTRI5P sequence and was not accompanied by increased GUS expression. 15-ADON production was further increased in liquid culture up to 1,200 mg/liter following introduction of the F. sporotrichioides TRI6 gene (FSTRI16) into F. graminearum. The effects of FSTRI6 on 15-ADON production also depended on plasmid integration via homologous recombination of the FGTRI5P fragment and resulted in a 100-fold increase in GUS expression. High-level production of 15-ADON in liquid shake cultures provides a convenient method for large-scale trichothecene preparation. The results suggest that targeting transformation vector integration toFGTRI5P alters pathway gene expression and are consistent with the proposed conservation of TRI6 function betweenFusarium species.

Laboratory selection at different temperatures modifies heat-shock transcription factor (HSF) activation in Drosophila melanogaster

2001 ◽  
Vol 204 (2) ◽  
pp. 315-323 ◽  
Author(s):  
D.N. Lerman ◽  
M.E. Feder
Keyword(s):  
Transcription Factor ◽  
Drosophila Melanogaster ◽  
Heat Shock ◽  
Time Course ◽  
Stress Protein ◽  
Laboratory Culture ◽  
Heat Shock Transcription Factor ◽  
Mobility Shift ◽  
Laboratory Selection ◽  
Mobility Shift Assay

The magnitude and time course of activation of the heat-shock transcription factor (HSF) differ among Drosophila melanogaster lines evolving at 18 degrees C, 25 degrees C or 28 degrees C for more than 20 years. At lower heat-shock temperatures (27–35 degrees C), flies from the 18 degrees C population had higher levels of activated HSF (as detected by an electrophoretic mobility shift assay) than those reared at 25 degrees C and 28 degrees C. At higher temperatures (36 and 37 degrees C), however, the 28 degrees C flies had the highest levels of HSF. These differences persisted after one generation of acclimation at 25 degrees C, suggesting that phenotypic plasticity was limited. In addition, larvae from the 28 degrees C lines activated HSF less rapidly after a 35 degrees C heat shock than those from the 18 degrees C and 25 degrees C populations. These results are similar but not identical to previously reported differences in expression of Hsp70 (the major heat-inducible stress protein in Drosophila melanogaster) among the experimental lines. We conclude that HSF activation evolves rapidly during laboratory culture at diverse temperatures and could play an important role in the evolution of the heat-shock response.

scholarly journals Activation of heat-shock transcription factor 1 by hypertonic shock in 3T3 cells

1996 ◽  
Vol 319 (2) ◽  
pp. 601-606 ◽  
Author(s):  
Roberta ALFIERI ◽  
Pier Giorgio PETRONINI ◽  
Simona URBANI ◽  
Angelo F BORGHETTI
Keyword(s):  
Transcription Factor ◽  
Heat Shock ◽  
Time Course ◽  
Binding Activity ◽  
Heat Shock Transcription Factor ◽  
3T3 Cells ◽  
Mobility Shift ◽  
Heat Shock Element ◽  
Competition Analysis ◽  
Hypertonic Shock

The exposure of 3T3 cells to a medium made hypertonic by the addition of NaCl induced activation of a heat-shock transcription factor (HSF). This activation, as monitored by gel-mobility-shift assays, occurred within 10 min of hypertonic shock and was dose-dependent in relation to the osmotic strength of the medium up to 0.7 osM. Competition analysis indicated that the effect of hypertonic shock on HSF binding activity was specific. The magnitude of the heat-shock element (HSE)-HSF binding induced by incubating the cells in a 0.7 osM medium was comparable in intensity and time course with that induced by a 44 °C heat shock. Following removal of the stressors, the decrease in HSF-HSE binding was more rapid in hypertonicity-shocked than in heat-shocked cells. Treatment of the cells with cycloheximide did not inhibit HSF-HSE binding, indicating that the activation was independent of new protein synthesis. By using a specifically directed polyclonal serum, HSF1 was identified as the transcription factor involved in the hypertonicity-induced activation. HSF was also activated when a membrane-impermeable osmolyte such as sucrose was used to increase the osmolarity of the medium. However, no HSF-HSE binding was observed after addition of glycerol (a freely membrane-permeable osmolyte) in excess. There was a temporal relationship between the hypertonicity-induced volume decrease, the increase in the intracellular K+ concentration and the induction of HSF-HSE binding. In contrast, an increase in the intracellular Na+ concentration was not required to induce HSF-HSE binding. However, unlike the heat-shock response, the activation of HSF by hypertonic shock did not lead to elongation of the RNA transcript of heat-shock protein 70.

scholarly journals Transcription Factor OpWRKY3 Is Involved in the Development and Biosynthesis of Camptothecin and Its Precursors in Ophiorrhiza pumila Hairy Roots

2019 ◽  
Vol 20 (16) ◽  
pp. 3996 ◽  
Author(s):  
Can Wang ◽  
Chao Wu ◽  
Yao Wang ◽  
Chenhong Xie ◽  
Min Shi ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Hairy Roots ◽  
Indole Alkaloid ◽  
Wrky Transcription Factor ◽  
Significant Feature ◽  
Total Production ◽  
Phylogenetic Tree Analysis ◽  
Reading Frame ◽  
Group Iii ◽  
Localization Analysis

The plant Ophiorrhiza pumila produces camptothecin (CPT), a kind of terpene indole alkaloid (TIAs) that has been widely used in treatment of cancer. Tryptophan-arginine-lysine-tyrosine (WRKY) transcription factors have been reported to play important roles in plant metabolism and development. In this study, a novel WRKY transcription factor named OpWRKY3 was isolated from O. pumila, with full-length open reading frame (ORF) of 1128 bp, encoding 375 amino acids. Phylogenetic tree analysis revealed that OpWRKY3 shared the highest homology with VvWRKY30, and it is a significant feature belonging to group III. OpWRKY3 was responsive to various treatments, including gibberellin (GA3), methyl jasmonate (MJ), acetylsalicylic acid (ASA), salicylic acid (SA), and abscisic acid (ABA). Besides, OpWRKY3 is expressed predominantly in stems. Subcellular localization analysis showed that OpWRKY3 localized in the nucleus. The biomass of OpWRKY3-SRDX transgenic hairy roots (S line) was visibly suppressed, while there were slight changes between overexpression of the OpWRKY3 line (OE line) and the control. In addition, the concentration and total production of camptothecin precursors including loganin and secologanin were significantly changed in both OE and S lines while total production of CPT was significantly changed in most transgenic lines. Thus, the present work revealed that OpWRKY3 may act as a regulator in the growth and development of O. pumila, and in production of camptothecin and its precursors.

scholarly journals Regulation of the CgPdr1 Transcription Factor from the Pathogen Candida glabrata

2010 ◽  
Vol 10 (2) ◽  
pp. 187-197 ◽  
Author(s):  
Sanjoy Paul ◽  
Jennifer A. Schmidt ◽  
W. Scott Moye-Rowley
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Abc Transporter ◽  
Candida Glabrata ◽  
Minor Effect ◽  
Deletion Mapping ◽  
Mobility Shift ◽  
Content Type ◽  
Zinc Cluster ◽  
Encoding Gene

ABSTRACTCandida glabratais an opportunistic human pathogen that is increasingly associated with candidemia, owing in part to the intrinsic and acquired high tolerance the organism exhibits for the important clinical antifungal drug fluconazole. This elevated fluconazole resistance often develops through gain-of-function mutations in the zinc cluster-containing transcriptional regulatorC. glabrataPdr1 (CgPdr1). CgPdr1 induces the expression of an ATP-binding cassette (ABC) transporter-encoding gene, CgCDR1.Saccharomyces cerevisiaehas two CgPdr1 homologues called ScPdr1 and ScPdr3. These factors control the expression of an ABC transporter-encoding gene called ScPDR5, which encodes a homologue of CgCDR1. Loss of the mitochondrial genome (ρ0cell) or overexpression of the mitochondrial enzyme ScPsd1 induces ScPDR5expression in a strictly ScPdr3-dependent fashion. ScPdr3 requires the presence of a transcriptional Mediator subunit called Gal11 (Med15) to fully induce ScPDR5transcription in response to ρ0signaling. ScPdr1 does not respond to either ρ0signals or ScPsd1 overproduction. In this study, we employed transcriptional fusions between CgPdr1 target promoters, like CgCDR1, to demonstrate that CgPdr1 stimulates gene expression via binding to elements called pleiotropic drug response elements (PDREs). Deletion mapping and electrophoretic mobility shift assays demonstrated that a single PDRE in the CgCDR1promoter was capable of supporting ρ0-induced gene expression. Removal of one of the two ScGal11 homologues fromC. glabratacaused a major defect in drug-induced expression of CgCDR1but had a quantitatively minor effect on ρ0-stimulated transcription. These data demonstrate that CgPdr1 appears to combine features of ScPdr1 and ScPdr3 to produce a transcription factor with chimeric regulatory properties.

scholarly journals Association of Serum Interleukin-6 Concentration with a Functional IL6 −6331T>C Polymorphism

2008 ◽  
Vol 54 (5) ◽  
pp. 841-850 ◽  
Author(s):  
Andrew J P Smith ◽  
Francesco D’Aiuto ◽  
Jutta Palmen ◽  
Jackie A Cooper ◽  
Jane Samuel ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Interleukin 6 ◽  
Artery Bypass ◽  
Fold Increase ◽  
Mobility Shift ◽  
Single Nucleotide ◽  
Functional Variants ◽  
Inflammatory State ◽  
Reporter Assays ◽  
Electrophoretic Mobility Shift Assays

Abstract Background: Interleukin-6 (IL-6) concentrations vary substantially among individuals. This study aimed to identify novel genetic markers to explain these differences. Methods: We sequenced a region 6-kb upstream of the IL6 [interleukin 6 (interferon, beta 2)] transcription start site in a search for functional variants and detected 3 common variants: −6331T>C, −6101A>T, and −5617/−5616C/A>T/G. IL6 −6331T>C (C allele frequency, 0.20; 95% confidence interval, 0.16–0.24) showed strong negative linkage disequilibrium with −174G>C (D′ = −0.97) and was studied further in 309 individuals who underwent coronary artery bypass grafting. Results: Patients with the TT genotype had higher IL-6 concentrations 6 h after surgery than those with the CC genotype (mean, 199.4 ng/L vs 114.9 ng/L; P = 0.02). A similar association was seen in a cohort of 173 patients who underwent intensive periodontal therapy: Individuals with the CC genotype had significantly lower IL-6 concentrations 24 h after therapy than TT patients (mean, 0.78 ng/L vs 5.00 ng/L; P < 0.0001). A similar trend was observed in 203 healthy individuals from northern Europe (1.29 ng/L for the TT genotype vs 0.89 ng/L for the CC genotype; P = 0.07). Reporter assays that used a sequence flanking the −6331 single-nucleotide polymorphism spliced upstream to the IL-6 minimal promoter driving luciferase gene expression demonstrated a 1.3-fold increase in promoter activity (P < 0.01) for constructs containing −6331T. Electrophoretic mobility shift assays revealed enhanced binding of transcription factor Oct-1 to the T allele. Conclusions: IL6 −6331T is associated with increased IL-6 concentrations in an acute inflammatory state via a mechanism involving binding of the Oct-1 transcription factor. This finding may help resolve conflicting studies based on the IL6 −174G>C variant.

An IRP-like protein from Plasmodium falciparum binds to a mammalian iron-responsive element

Blood ◽  
2001 ◽  
Vol 98 (8) ◽  
pp. 2555-2562 ◽  
Author(s):  
Mark Loyevsky ◽  
Timothy LaVaute ◽  
Charles R. Allerson ◽  
Robert Stearman ◽  
Olakunle O. Kassim ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Regulatory Protein ◽  
Protein S ◽  
Fold Increase ◽  
Binding Activity ◽  
Mobility Shift ◽  
Iron Responsive Element ◽  
Element Binding Protein ◽  
Responsive Element ◽  
Electrophoretic Mobility Shift Assays

Abstract This study cloned and sequenced the complementary DNA (cDNA) encoding of a putative malarial iron responsive element-binding protein (PfIRPa) and confirmed its identity to the previously identified iron-regulatory protein (IRP)–like cDNA from Plasmodium falciparum. Sequence alignment showed that the plasmodial sequence has 47% identity with human IRP1. Hemoglobin-free lysates obtained from erythrocyte-stage P falciparum contain a protein that binds a consensus mammalian iron-responsive element (IRE), indicating that a protein(s) with iron-regulatory activity was present in the lysates. IRE-binding activity was found to be iron regulated in the electrophoretic mobility shift assays. Western blot analysis showed a 2-fold increase in the level of PfIRPa in the desferrioxamine-treated cultures versus control or iron-supplemented cells. Malarial IRP was detected by anti-PfIRPa antibody in the IRE-protein complex fromP falciparum lysates. Immunofluorescence studies confirmed the presence of PfIRPa in the infected red blood cells. These findings demonstrate that erythrocyte P falciparum contains an iron-regulated IRP that binds a mammalian consensus IRE sequence, raising the possibility that the malaria parasite expresses transcripts that contain IREs and are iron-dependently regulated.

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