scholarly journals MdWRKY46-Enhanced Apple Resistance to Botryosphaeria dothidea by Activating the Expression of MdPBS3.1 in the Salicylic Acid Signaling Pathway

2019 ◽  
Vol 32 (10) ◽  
pp. 1391-1401 ◽  
Author(s):  
Xian-Yan Zhao ◽  
Chen-Hui Qi ◽  
Han Jiang ◽  
Ming-Shuang Zhong ◽  
Qiang Zhao ◽  
...  
Keyword(s):  
Salicylic Acid ◽  
Disease Resistance ◽  
Signaling Pathway ◽  
Transcriptional Activation ◽  
Viral Vector ◽  
Botryosphaeria Dothidea ◽  
Mobility Shift ◽  
Electrophoretic Mobility Shift Assays ◽  
Transgenic Apple ◽  
Wrky Proteins

Salicylic acid (SA) is closely related to disease resistance of plants. WRKY transcription factors have been linked to the growth and development of plants, especially under stress conditions. However, the regulatory mechanism of WRKY proteins involved in SA production and disease resistance in apple is not clear. In this study, MdPBS3.1 responded to Botryosphaeria dothidea and enhanced resistance to B. dothidea. Electrophoretic mobility shift assays, yeast one-hybrid assays, and chromatin immunoprecipitation and quantitative PCR demonstrated that MdWRKY46 can directly bind to a W-box motif in the promoter of MdPBS3.1. Glucuronidase transactivation and luciferase analysis further showed that MdWRKY46 can activate the expression of MdPBS3.1. Finally, B. dothidea inoculation in transgenic apple calli and fruits revealed that MdWRKY46 improved resistance to B. dothidea by the transcriptional activation of MdPBS3.1. Viral vector-based transformation assays indicated that MdWRKY46 elevates SA content and transcription of SA-related genes, including MdPR1, MdPR5, and MdNPR1 in an MdPBS3.1-dependent way. These findings provide new insights into how MdWRKY46 regulates plant resistance to B. dothidea through the SA signaling pathway.

scholarly journals NbWRKY40 Positively Regulates the Response of Nicotiana benthamiana to Tomato Mosaic Virus via Salicylic Acid Signaling

2021 ◽  
Vol 11 ◽  
Author(s):  
Yaoyao Jiang ◽  
Weiran Zheng ◽  
Jing Li ◽  
Peng Liu ◽  
Kaili Zhong ◽  
...  
Keyword(s):  
Salicylic Acid ◽  
Mosaic Virus ◽  
Transcriptional Activation ◽  
Nicotiana Benthamiana ◽  
Yeast Cells ◽  
Tomato Mosaic Virus ◽  
Plant Responses ◽  
Mobility Shift ◽  
Localization Analysis ◽  
Electrophoretic Mobility Shift Assays

WRKY transcription factors play important roles in plants, including responses to stress; however, our understanding of the function of WRKY genes in plant responses to viral infection remains limited. In this study, we investigate the role of NbWRKY40 in Nicotiana benthamiana resistance to tomato mosaic virus (ToMV). NbWRKY40 is significantly downregulated by ToMV infection, and subcellular localization analysis indicates that NbWRKY40 is targeted to the nucleus. In addition, NbWRKY40 activates W-box-dependent transcription in plants and shows transcriptional activation in yeast cells. Overexpressing NbWRKY40 (OEWRKY40) inhibits ToMV infection, whereas NbWRKY40 silencing confers susceptibility. The level of salicylic acid (SA) is significantly higher in OEWRKY40 plants compared with that of wild-type plants. In addition, transcript levels of the SA-biosynthesis gene (ICS1) and SA-signaling genes (PR1b and PR2) are dramatically higher in OEWRKY40 plants than in the control but lower in NbWRKY40-silenced plants than in the control. Furthermore, electrophoretic mobility shift assays show that NbWRKY40 can bind the W-box element of ICS1. Callose staining reveals that the plasmodesmata is decreased in OEWRKY40 plants but increased in NbWRKY40-silenced plants. Exogenous application of SA also reduces viral accumulation in NbWRKY40-silenced plants infected with ToMV. RT-qPCR indicates that NbWRKY40 does not affect the replication of ToMV in protoplasts. Collectively, our findings suggest that NbWRKY40 likely regulates anti-ToMV resistance by regulating the expression of SA, resulting in the deposition of callose at the neck of plasmodesmata, which inhibits viral movement.

scholarly journals ATF4-Dependent Oxidative Induction of the DNA Repair Enzyme Ape1 Counteracts Arsenite Cytotoxicity and Suppresses Arsenite-Mediated Mutagenesis

2007 ◽  
Vol 27 (24) ◽  
pp. 8834-8847 ◽  
Author(s):  
Hua Fung ◽  
Pingfang Liu ◽  
Bruce Demple
Keyword(s):  
Dna Repair ◽  
Transcriptional Activation ◽  
Activity Levels ◽  
Cellular Mechanisms ◽  
Mobility Shift ◽  
Repair Enzyme ◽  
Base Excision ◽  
Transcription Suppression ◽  
Tk6 Cells ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT Arsenite is a human carcinogen causing skin, bladder, and lung tumors, but the cellular mechanisms underlying these effects remain unclear. We investigated expression of the essential base excision DNA repair enzyme apurinic endonuclease 1 (Ape1) in response to sodium arsenite. In mouse 10T½ fibroblasts, Ape1 induction in response to arsenite occurred about equally at the mRNA, protein, and enzyme activity levels. Analysis of the APE1 promoter region revealed an AP-1/CREB binding site essential for arsenite-induced transcriptional activation in both mouse and human cells. Electrophoretic mobility shift assays indicated that an ATF4/c-Jun heterodimer was the responsible transcription factor. RNA interference targeting c-Jun or ATF4 eliminated arsenite-induced APE1 transcription. Suppression of Ape1 or ATF4 sensitized both mouse fibroblasts (10T½) and human lymphoblastoid cells (TK6) to arsenite cytotoxicity. Expression of Ape1 from a transgene did not efficiently restore arsenite resistance in ATF4-depleted cells but did offset initial accumulation of abasic DNA damage following arsenite treatment. Mutagenesis by arsenite (at the TK and HPRT loci in TK6 cells) was observed only for ATF4-depleted cells, which was strongly offset by Ape1 expression from a transgene. Therefore, the ATF4-mediated up-regulation of Ape1 and other genes plays a key role against arsenite-mediated toxicity and mutagenesis.

scholarly journals Transcriptional Activation of Multiple Operons Involved inpara-Nitrophenol Degradation by Pseudomonas sp. Strain WBC-3

2014 ◽  
Vol 81 (1) ◽  
pp. 220-230 ◽  
Author(s):  
Wen-Mao Zhang ◽  
Jun-Jie Zhang ◽  
Xuan Jiang ◽  
Hongjun Chao ◽  
Ning-Yi Zhou
Keyword(s):  
Transcriptional Activation ◽  
Transcriptional Regulator ◽  
Pseudomonas Sp ◽  
Mobility Shift ◽  
Content Type ◽  
High Affinity ◽  
Dnase I Footprinting ◽  
Nitrophenol Degradation ◽  
Transcriptional Start Sites ◽  
Electrophoretic Mobility Shift Assays

ABSTRACTPseudomonassp. strain WBC-3 utilizespara-nitrophenol (PNP) as a sole carbon and energy source. The genes involved in PNP degradation are organized in the following three operons:pnpA,pnpB, andpnpCDEFG. How the expression of the genes is regulated is unknown. In this study, an LysR-type transcriptional regulator (LTTR) is identified to activate the expression of the genes in response to the specific inducer PNP. While the LTTR coding genepnpRwas found to be not physically linked to any of the three catabolic operons, it was shown to be essential for the growth of strain WBC-3 on PNP. Furthermore, PnpR positively regulated its own expression, which is different from the function of classical LTTRs. A regulatory binding site (RBS) with a 17-bp imperfect palindromic sequence (GTT-N11-AAC) was identified in allpnpA,pnpB,pnpC, andpnpRpromoters. Through electrophoretic mobility shift assays and mutagenic analyses, this motif was proven to be necessary for PnpR binding. This consensus motif is centered at positions approximately −55 bp relative to the four transcriptional start sites (TSSs). RBS integrity was required for both high-affinity PnpR binding and transcriptional activation ofpnpA,pnpB, andpnpR. However, this integrity was essential only for high-affinity PnpR binding to the promoter ofpnpCDEFGand not for its activation. Intriguingly, unlike other LTTRs studied, no changes in lengths of the PnpR binding regions of thepnpAandpnpBpromoters were observed after the addition of the inducer PNP in DNase I footprinting.

Regulation of the Bacillus subtilis mannitol utilization genes: promoter structure and transcriptional activation by the wild-type regulator (MtlR) and its mutants

Microbiology ◽  
2014 ◽  
Vol 160 (1) ◽  
pp. 91-101 ◽  
Author(s):  
Kambiz Morabbi Heravi ◽  
Josef Altenbuchner
Keyword(s):  
Bacillus Subtilis ◽  
Transcriptional Activation ◽  
Transcription Initiation ◽  
Phosphotransferase System ◽  
Mobility Shift ◽  
Dnase I Footprinting ◽  
Proposed Model ◽  
Rna Polymerase Holoenzyme ◽  
Electrophoretic Mobility Shift Assays

Expression of mannitol utilization genes in Bacillus subtilis is directed by P mtlA , the promoter of the mtlAFD operon, and P mtlR , the promoter of the MtlR activator. MtlR contains phosphoenolpyruvate-dependent phosphotransferase system (PTS) regulation domains, called PRDs. The activity of PRD-containing MtlR is mainly regulated by the phosphorylation/dephosphorylation of its PRDII and EIIBGat-like domains. Replacing histidine 342 and cysteine 419 residues, which are the targets of phosphorylation in these two domains, by aspartate and alanine provided MtlR-H342D C419A, which permanently activates P mtlA in vivo. In the mtlR-H342D C419A mutant, P mtlA was active, even when the mtlAFD operon was deleted from the genome. The mtlR-H342D C419A allele was expressed in an Escherichia coli strain lacking enzyme I of the PTS. Electrophoretic mobility shift assays using purified MtlR-H342D C419A showed an interaction between the MtlR double-mutant and the Cy5-labelled P mtlA and P mtlR DNA fragments. These investigations indicate that the activated MtlR functions regardless of the presence of the mannitol-specific transporter (MtlA). This is in contrast to the proposed model in which the sequestration of MtlR by the MtlA transporter is necessary for the activity of MtlR. Additionally, DNase I footprinting, construction of P mtlA -P licB hybrid promoters, as well as increasing the distance between the MtlR operator and the −35 box of P mtlA revealed that the activated MtlR molecules and RNA polymerase holoenzyme likely form a class II type activation complex at P mtlA and P mtlR during transcription initiation.

scholarly journals C/EBPα activates the transcription of triacylglycerol hydrolase in 3T3-L1 adipocytes

2005 ◽  
Vol 388 (3) ◽  
pp. 959-966 ◽  
Author(s):  
Enhui WEI ◽  
Richard LEHNER ◽  
Dennis E. VANCE
Keyword(s):  
Transcriptional Activation ◽  
Promoter Region ◽  
Ectopic Expression ◽  
3T3 Cells ◽  
Mobility Shift ◽  
Protein Levels ◽  
Triacylglycerol Hydrolase ◽  
Enhancer Binding Protein ◽  
Nih 3T3 ◽  
Electrophoretic Mobility Shift Assays

TGH (triacylglycerol hydrolase) catalyses the lipolysis of intracellular stored triacylglycerol. To explore the mechanisms that regulate TGH expression in adipose tissue, we studied the expression of TGH during the differentiation of 3T3-L1 adipocytes. TGH mRNA and protein levels increased dramatically in 3T3-L1 adipocytes compared with pre-adipocytes. Electrophoretic mobility shift assays demonstrated enhanced binding of nuclear proteins of adipocytes to the distal murine TGH promoter region (−542/−371 bp), yielding one adipocyte-specific migrating complex. Competitive and supershift assays demonstrated that the distal TGH promoter fragment bound C/EBPα (CCAAT/enhancer-binding protein α). Transient transfections of different mutant TGH promoter–luciferase constructs into 3T3-L1 adipocytes and competitive electromobility shift assays showed that the C/EBP-binding elements at positions −470/−459 bp and −404/−390 bp are important for transcriptional activation. Co-transfection with C/EBPα cDNA and TGH promoter constructs in 3T3-L1 pre-adipocytes demonstrated that C/EBPα increased TGH promoter activity. Ectopic expression of C/EBPα in NIH 3T3 cells activated TGH mRNA expression without causing differentiation into adipocytes. These experiments directly link increased TGH expression in adipocytes to transcriptional regulation by C/EBPα. This is the first evidence that C/EBPα participates directly in the regulation of an enzyme associated with lipolysis.

scholarly journals Genomic organization, chromosomal mapping and promoter analysis of the mouse selenocysteine tRNA gene transcription-activating factor (mStaf) gene

2000 ◽  
Vol 346 (1) ◽  
pp. 45-51 ◽  
Author(s):  
Kazushige ADACHI ◽  
Masato KATSUYAMA ◽  
Shigeaki SONG ◽  
Takami OKA
Keyword(s):  
Transcriptional Activation ◽  
Trna Gene ◽  
Zinc Finger Protein ◽  
Regulatory Elements ◽  
Chromosomal Mapping ◽  
Proximal Promoter ◽  
Mobility Shift ◽  
Responsive Element ◽  
Specificity Protein ◽  
Electrophoretic Mobility Shift Assays

mStaf is a zinc-finger protein that activates the transcription of the mouse selenocysteine tRNA gene. The mStaf gene is approx. 35 kb long and split into 16 exons. All exon-intron junction sequences conform to the GT/AG rule. The transcription start site is located 83 bp upstream of the initiation codon. Chromosomal mapping localized the gene to mouse chromosome 7, region E3-F1. Sequence analysis of the proximal promoter region revealed several potential regulatory elements; these include the recognition elements of Sp1, Nkx, CP2, E2A, SIF (SIS-inducible factor), TFII-I and cAMP-responsive element (CRE), but no TATA sequences. Transfection experiments demonstrated that the 5ʹ-flanking region (-1894 to +37) of the mStaf gene drives transcription in mouse NMuMG cells and that a construct containing a fragment from -387 to +37 showed the highest transcriptional activity. Deletion and mutation experiments suggested that four Sp1 sites played an important role for the basal promoter activity. Furthermore, electrophoretic mobility-shift assays demonstrated that Sp3 but not other Sp (specificity protein) family members binds to three of the Sp1 sites. Our present study suggests that Sp3 is involved in the basal transcriptional activation of the mStaf gene.

Transcriptional regulation of rat Na+/H+exchanger isoform-2 (NHE-2) gene by Sp1 transcription factor

2001 ◽  
Vol 280 (5) ◽  
pp. C1168-C1175 ◽  
Author(s):  
Liqun Bai ◽  
James F. Collins ◽  
Hua Xu ◽  
Fayez K. Ghishan
Keyword(s):  
Transcriptional Regulation ◽  
Transcriptional Activation ◽  
Collecting Duct ◽  
Gene Promoter ◽  
Minimal Promoter ◽  
Mobility Shift ◽  
Sp1 Transcription Factor ◽  
Medullary Collecting Duct ◽  
High Level ◽  
Electrophoretic Mobility Shift Assays

The rat Na+/H+ exchanger isoform-2 ( NHE-2) gene promoter lacks a TATA box and is very GC rich. A minimal promoter extending from bp −36 to +116 directs high-level expression of NHE-2 in mouse inner medullary collecting duct (mIMCD-3) cells. Four Sp1 consensus elements were found in this region. The introduction of mutations within these Sp1 consensus elements and DNA footprinting revealed that only two of them were utilized and are critical for basal transcriptional activation in mIMCD-3 cells. The use of Sp1, Sp3, and Sp4 antisera in electrophoretic mobility shift assays demonstrated that Sp1, Sp3, and Sp4 bound to this minimal promoter. We further analyzed the transcriptional regulation of NHE-2 by members of the Sp1 multigene family. In Drosophila SL2 cells, which lack endogenous Sp1, the minimal promoter cannot drive transcription. Introduction of Sp1 activated transcription over 100-fold, suggesting that Sp1 is critical for transcriptional regulation. However, neither Sp3 nor Sp4 was able to activate transcription in these cells. Furthermore, in mIMCD-3 cells, Sp1-mediated transcriptional activation was repressed by expression of Sp3 and Sp4. These data suggest that Sp1 is critical for the basal promoter function of rat NHE-2 and that Sp3 and Sp4 may repress transcriptional activation by competing with Sp1 for binding to core cis-elements.

scholarly journals ZjSEP3 modulates flowering time by regulating the LHY promoter

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Weilin Gao ◽  
Liman Zhang ◽  
Jiurui Wang ◽  
Zhiguo Liu ◽  
Yao Zhang ◽  
...  
Keyword(s):  
Transgenic Plants ◽  
Flowering Time ◽  
Plant Species ◽  
Transcriptional Activation ◽  
Chlorophyll Synthesis ◽  
Early Flowering ◽  
Mobility Shift ◽  
Expression Of Genes ◽  
Electrophoretic Mobility Shift Assays ◽  
Early Flowering Phenotype

Abstract Background SEPALLATA3 (SEP3), which is conserved across various plant species, plays essential and various roles in flower and fruit development. However, the regulatory network of the role of SEP3 in flowering time at the molecular level remained unclear. Results Here, we investigated that SEP3 in Ziziphus jujuba Mill. (ZjSEP3) was expressed in four floral organs and exhibited strong transcriptional activation activity. ZjSEP3 transgenic Arabidopsis showed an early-flowering phenotype and altered the expression of some genes related to flowering. Among them, the expression of LATE ELONGATED HYPOCOTYL (AtLHY), the key gene of circadian rhythms, was significantly suppressed. Yeast one-hybrid (Y1H) and electrophoretic mobility shift assays (EMSAs) further verified that ZjSEP3 inhibited the transcription of AtLHY by binding to the CArG-boxes in its promoter. Moreover, ZjSEP3 also could bind to the ZjLHY promoter and the conserved binding regions of ZjSEP3 were found in the LHY promoter of various plant species. The ectopic regulatory pathway of ZjSEP3-AtLHY was further supported by the ability of 35S::AtLHY to rescue the early-flowering phenotype in ZjSEP3 transgenic plants. In ZjSEP3 transgenic plants, total chlorophyll content and the expression of genes involved in chlorophyll synthesis increased during vegetative stages, which should contribute to its early flowering and relate to the regulatory of AtLHY. Conclusion Overall, ZjSEP3-AtLHY pathway represents a novel regulatory mechanism that is involved in the regulation of flowering time.

scholarly journals The transcriptional repressor gene Mad3 is a novel target for regulation by E2F1

2003 ◽  
Vol 370 (1) ◽  
pp. 307-313 ◽  
Author(s):  
Elizabeth J. FOX ◽  
Stephanie C. WRIGHT
Keyword(s):  
Cell Cycle ◽  
Transcriptional Activation ◽  
S Phase ◽  
Mobility Shift ◽  
Binding Factor ◽  
Flanking Region ◽  
Novel Target ◽  
Electrophoretic Mobility Shift Assays

Mad family proteins are transcriptional repressors that antagonize the activity of the c-Myc proto-oncogene product. Mad3 is expressed specifically during the S-phase of the cell cycle in both proliferating and differentiating cells, suggesting that its biological function is probably linked to processes that occur during this period. To determine the mechanisms that regulate the cell-cycle-specific transcription of Mad3, we used reporter gene assays in stably transfected fibroblasts. We show that the activation of Mad3 at the G1—S boundary is mediated by a single E2F (E2 promoter binding factor)-binding site within the 5′-flanking region of the gene. Mutation of this element eliminated transcriptional activation at S-phase, suggesting that the positively acting E2F proteins play a role in Mad3 regulation. Using electrophoretic mobility-shift assays and chromatin immunoprecipitation, we show that E2F1 binds to the Mad3 5′-flanking region both in vitro and in vivo. We thus identify Mad3 as a novel transcriptional target of E2F1.

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