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 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 NbALD1 mediates resistance to turnip mosaic virus by regulating the accumulation of salicylic acid and the ethylene pathway in Nicotiana benthamiana

2019 ◽  
Vol 20 (7) ◽  
pp. 990-1004 ◽  
Author(s):  
Shu Wang ◽  
Kelei Han ◽  
Jiejun Peng ◽  
Jinping Zhao ◽  
Liangliang Jiang ◽  
...  
Keyword(s):  
Salicylic Acid ◽  
Mosaic Virus ◽  
Nicotiana Benthamiana ◽  
Turnip Mosaic Virus

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 Salicylic Acid and Jasmonic Acid Are Essential for Systemic Resistance Against Tobacco mosaic virus in Nicotiana benthamiana

2014 ◽  
Vol 27 (6) ◽  
pp. 567-577 ◽  
Author(s):  
Feng Zhu ◽  
De-Hui Xi ◽  
Shu Yuan ◽  
Fei Xu ◽  
Da-Wei Zhang ◽  
...  
Keyword(s):  
Salicylic Acid ◽  
Jasmonic Acid ◽  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Nicotiana Benthamiana ◽  
Foliar Application ◽  
Mass Spectrometry Analysis ◽  
Systemic Resistance ◽  
Long Distance ◽  
Resistance Response

Systemic resistance is induced by pathogens and confers protection against a broad range of pathogens. Recent studies have indicated that salicylic acid (SA) derivative methyl salicylate (MeSA) serves as a long-distance phloem-mobile systemic resistance signal in tobacco, Arabidopsis, and potato. However, other experiments indicate that jasmonic acid (JA) is a critical mobile signal. Here, we present evidence suggesting both MeSA and methyl jasmonate (MeJA) are essential for systemic resistance against Tobacco mosaic virus (TMV), possibly acting as the initiating signals for systemic resistance. Foliar application of JA followed by SA triggered the strongest systemic resistance against TMV. Furthermore, we use a virus-induced gene-silencing–based genetics approach to investigate the function of JA and SA biosynthesis or signaling genes in systemic response against TMV infection. Silencing of SA or JA biosynthetic and signaling genes in Nicotiana benthamiana plants increased susceptibility to TMV. Genetic experiments also proved the irreplaceable roles of MeSA and MeJA in systemic resistance response. Systemic resistance was compromised when SA methyl transferase or JA carboxyl methyltransferase, which are required for MeSA and MeJA formation, respectively, were silenced. Moreover, high-performance liquid chromatography–mass spectrometry analysis indicated that JA and MeJA accumulated in phloem exudates of leaves at early stages and SA and MeSA accumulated at later stages, after TMV infection. Our data also indicated that JA and MeJA could regulate MeSA and SA production. Taken together, our results demonstrate that (Me)JA and (Me)SA are required for systemic resistance response against TMV.

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.

scholarly journals Genome-Wide Identification Reveals That Nicotiana benthamiana Hypersensitive Response (HR)-Like Lesion Inducing Protein 4 (NbHRLI4) Mediates Cell Death and Salicylic Acid-Dependent Defense Responses to Turnip Mosaic Virus

2021 ◽  
Vol 12 ◽  
Author(s):  
Xinyang Wu ◽  
Yuchao Lai ◽  
Shaofei Rao ◽  
Lanqing Lv ◽  
Mengfei Ji ◽  
...  
Keyword(s):  
Cell Death ◽  
Salicylic Acid ◽  
Mosaic Virus ◽  
Hypersensitive Response ◽  
Nicotiana Benthamiana ◽  
Defense Responses ◽  
Turnip Mosaic Virus ◽  
Genome Wide ◽  
Transient Overexpression ◽  
Basal Immunity

Hypersensitive response (HR)-like cell death is an important mechanism that mediates the plant response to pathogens. In our previous study, we reported that NbHIR3s regulate HR-like cell death and basal immunity. However, the host genes involved in HR have rarely been studied. Here, we used transcriptome sequencing to identify Niben101Scf02063g02012.1, an HR-like lesion inducing protein (HRLI) in Nicotiana benthamiana that was significantly reduced by turnip mosaic virus (TuMV). HRLIs are uncharacterized proteins which may regulate the HR process. We identified all six HRLIs in N. benthamiana and functionally analyzed Niben101Scf02063g02012.1, named NbHRLI4, in response to TuMV. Silencing of NbHRLI4 increased TuMV accumulation, while overexpression of NbHRLI4 conferred resistance to TuMV. Transient overexpression of NbHRLI4 caused cell death with an increase in the expression of salicylic acid (SA) pathway genes but led to less cell death level and weaker immunity in plants expressing NahG. Thus, we have characterized NbHRLI4 as an inducer of cell death and an antiviral regulator of TuMV infection in a SA-mediated manner.

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