scholarly journals p24G1 Encoded by Grapevine Leafroll-Associated Virus 1 Suppresses RNA Silencing and Elicits Hypersensitive Response-Like Necrosis in Nicotiana Species

Viruses ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 1111
Author(s):  
Chen-Wei Zhang ◽  
Qing Liu ◽  
Qi Zeng ◽  
Wen-Ting Huang ◽  
Qi Wang ◽  
...  
Keyword(s):  
Hypersensitive Response ◽  
Rna Silencing ◽  
Molecular Mechanisms ◽  
Potato Virus X ◽  
Mobility Shift ◽  
Nicotiana Species ◽  
P24 Protein ◽  
Systemic Rna ◽  
Interfering Rna ◽  
Electrophoretic Mobility Shift Assays

Grapevine leafroll-associated virus 1 (GLRaV-1) is a major pathogen associated with grapevine leafroll disease. However, the molecular mechanisms underlying GLRaV-1 interactions with plant cells are unclear. Using Agrobacterium infiltration-mediated RNA-silencing assays, we demonstrated that GLRaV-1 p24 protein (p24G1) acts as an RNA-silencing suppressor (RSS), inhibiting local and systemic RNA silencing. Electrophoretic mobility shift assays showed that p24G1 binds double-stranded 21-nucleotide small interfering RNA (siRNA), and that siRNA binding is required but not sufficient for its RSS activity. p24G1 localizes in the nucleus and can self-interact through its amino acid 10 to 210 region. Dimerization is needed for p24G1 interaction with importin α1 before moving to the nucleus, but is not required for its siRNA binding and RSS activity. Expression of p24G1 from a binary pGD vector or potato virus X-based vector elicited a strong hypersensitive response in Nicotiana species, indicating that p24G1 may be a factor in pathogenesis. Furthermore, p24G1 function in pathogenesis required its RSS activity, dimerization and nuclear localization. In addition, the region of amino acids 122–139 played a crucial role in the nuclear import, siRNA binding, silencing suppression and pathogenic activity of p24G1. These results contribute to our understanding of the molecular mechanisms underlying GLRaV-1 infection.

scholarly journals Identification of an RNA silencing suppressor encoded by a mastrevirus

2014 ◽  
Vol 95 (9) ◽  
pp. 2082-2088 ◽  
Author(s):  
Yaqin Wang ◽  
Mingqing Dang ◽  
Huwei Hou ◽  
Yuzhen Mei ◽  
Yajuan Qian ◽  
...  
Keyword(s):  
Rna Silencing ◽  
Silencing Suppressor ◽  
Mobility Shift ◽  
Rep Protein ◽  
Rna Silencing Suppressor ◽  
Gfp Reporter ◽  
Gfp Reporter Gene ◽  
Systemic Rna ◽  
Gel Mobility Shift ◽  
Interfering Rna

Wheat dwarf virus (WDV) is a DNA virus belonging to the genus Mastrevirus of the family Geminiviridae. In this study, we report that the Rep protein encoded by WDV is a RNA silencing supressor as determined by co-infiltration assays using transgenic Nicotiana benthamiana line 16c carrying the GFP reporter gene. The Rep protein was shown to inhibit both local and systemic RNA silencing of the GFP gene as well as the spread of systemic GFP RNA silencing signals. Gel mobility shift assays showed that the Rep protein binds 21 nt and 24 nt small interfering RNA (siRNA) duplexes and single-stranded (ss)-siRNA. To our knowledge, this is the first identification of an RNA silencing suppressor encoded by mastreviruses. Furthermore, deletion mutagenesis indicates that both the N- and C-terminal regions of the Rep protein are not critical for silencing suppression and self-interaction, but the N terminus of Rep is necessary for its pathogenicity.

scholarly journals Inducible Systemic RNA Silencing in Caenorhabditis elegans

2003 ◽  
Vol 14 (7) ◽  
pp. 2972-2983 ◽  
Author(s):  
Lisa Timmons ◽  
Hiroaki Tabara ◽  
Craig C. Mello ◽  
Andrew Z. Fire
Keyword(s):  
Rna Interference ◽  
Caenorhabditis Elegans ◽  
Rna Silencing ◽  
Molecular Mechanisms ◽  
Normal Animal ◽  
Cell Types ◽  
Animal Cells ◽  
Tissue Specific ◽  
C Elegans ◽  
Systemic Rna

Introduction of double-stranded RNA (dsRNA) can elicit a gene-specific RNA interference response in a variety of organisms and cell types. In many cases, this response has a systemic character in that silencing of gene expression is observed in cells distal from the site of dsRNA delivery. The molecular mechanisms underlying the mobile nature of RNA silencing are unknown. For example, although cellular entry of dsRNA is possible, cellular exit of dsRNA from normal animal cells has not been directly observed. We provide evidence that transgenic strains of Caenorhabditis elegans transcribing dsRNA from a tissue-specific promoter do not exhibit comprehensive systemic RNA interference phenotypes. In these same animals, modifications of environmental conditions can result in more robust systemic RNA silencing. Additionally, we find that genetic mutations can influence the systemic character of RNA silencing in C. elegans and can separate mechanisms underlying systemic RNA silencing into tissue-specific components. These data suggest that trafficking of RNA silencing signals in C. elegans is regulated by specific physiological and genetic factors.

scholarly journals Upregulation of a KN1 homolog by transposon insertion promotes leafy head development in lettuce

2020 ◽  
Vol 117 (52) ◽  
pp. 33668-33678
Author(s):  
Changchun Yu ◽  
Chenghuan Yan ◽  
Yuling Liu ◽  
Yali Liu ◽  
Yue Jia ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Vegetable Crops ◽  
Mobility Shift ◽  
Unique Type ◽  
Chip Sequencing ◽  
Head Development ◽  
Enhanced Expression ◽  
Trait Locus ◽  
Leafy Head ◽  
Electrophoretic Mobility Shift Assays

Leafy head is a unique type of plant architecture found in some vegetable crops, with leaves bending inward to form a compact head. The genetic and molecular mechanisms underlying leafy head in vegetables remain poorly understood. We genetically fine-mapped and cloned a major quantitative trait locus controlling heading in lettuce. The candidate gene (LsKN1) is a homolog of knotted 1 (KN1) from Zea mays. Complementation and CRISPR/Cas9 knockout experiments confirmed the role of LsKN1 in heading. In heading lettuce, there is a CACTA-like transposon inserted into the first exon of LsKN1 (LsKN1▽). The transposon sequences act as a promoter rather than an enhancer and drive high expression of LsKN1▽. The enhanced expression of LsKN1▽ is necessary but not sufficient for heading in lettuce. Data from ChIP-sequencing, electrophoretic mobility shift assays, and dual luciferase assays indicate that the LsKN1▽ protein binds the promoter of LsAS1 and down-regulates its expression to alter leaf dorsoventrality. This study provides insight into plant leaf development and will be useful for studies on heading in other vegetable crops.

scholarly journals Multiple Functions of Rice Dwarf Phytoreovirus Pns10 in Suppressing Systemic RNA Silencing

2010 ◽  
Vol 84 (24) ◽  
pp. 12914-12923 ◽  
Author(s):  
Bo Ren ◽  
Yuanyuan Guo ◽  
Feng Gao ◽  
Peng Zhou ◽  
Feng Wu ◽  
...  
Keyword(s):  
Nicotiana Benthamiana ◽  
Rna Silencing ◽  
Rna Stability ◽  
Antiviral Defense ◽  
Double Stranded Rna ◽  
Systemic Spread ◽  
Dsrna Viruses ◽  
Systemic Rna ◽  
Interfering Rna ◽  
Diverse Groups

ABSTRACT RNA silencing is a potent mechanism of antiviral defense response in plants and other organisms. For counterdefense, viruses have evolved a variety of suppressors of RNA silencing (VSRs) that can inhibit distinct steps of a silencing pathway. We previously identified Pns10 encoded by Rice dwarf phytoreovirus (RDV) as a VSR, the first of its kind from double-stranded RNA (dsRNA) viruses. In this study we investigated the mechanisms of Pns10 function in suppressing systemic RNA silencing in the widely used Nicotiana benthamiana model plant. We report that Pns10 suppresses local and systemic RNA silencing triggered by sense mRNA, enhances viral replication and/or viral RNA stability in inoculated leaves, accelerates the systemic spread of viral infection, and enables viral invasion of shoot apices. Mechanistically, Pns10 interferes with the perception of silencing signals in recipient tissues, binds double-stranded small interfering RNA (siRNAs) with two-nucleotide 3′ overhangs, and causes the downregulated expression of RDR6. These results significantly deepen our mechanistic understanding of the VSR functions encoded by a dsRNA virus and contribute additional evidence that binding siRNAs and interfering with RDR6 expression are broad mechanisms of VSR functions encoded by diverse groups of viruses.

Functional consensus for mammalian osmotic response elements

1999 ◽  
Vol 276 (3) ◽  
pp. C667-C673 ◽  
Author(s):  
Joan D. Ferraris ◽  
Chester K. Williams ◽  
Akihiko Ohtaka ◽  
Arlyn García-Pérez
Keyword(s):  
Molecular Mechanisms ◽  
Full Range ◽  
Renal Medulla ◽  
Binding Activity ◽  
Organic Osmolytes ◽  
Mobility Shift ◽  
Osmotic Response ◽  
Eukaryotic Gene ◽  
Electrophoretic Mobility Shift Assays ◽  
Response Region

The molecular mechanisms underlying adaptation to hyperosmotic stress through the accumulation of organic osmolytes are largely unknown. Yet, among organisms, this is an almost universal phenomenon. In mammals, the cells of the renal medulla are uniquely exposed to high and variable salt concentrations; in response, renal cells accumulate the osmolyte sorbitol through increased transcription of the aldose reductase (AR) gene. In cloning the rabbit AR gene, we found the first evidence of an osmotic response region in a eukaryotic gene. More recently, we functionally defined a minimal essential osmotic response element (ORE) having the sequence CGGAAAATCAC(C) (bp −1105 to −1094). In the present study, we systematically replaced each base with every other possible nucleotide and tested the resulting sequences individually in reporter gene constructs. Additionally, we categorized hyperosmotic response by electrophoretic mobility shift assays of a 17-bp sequence (−1108 to −1092) containing the native ORE as a probe against which the test constructs would compete for binding. In this manner, binding activity was assessed for the full range of osmotic responses obtained. Thus we have arrived at a functional consensus for the mammalian ORE, NGGAAAWDHMC(N). This finding should accelerate the discovery of genes previously unrecognized as being osmotically regulated.

scholarly journals Synergistic binding of bHLH transcription factors to the promoter of the maize NADP-ME gene used in C4 photosynthesis is based on an ancient code found in the ancestral C3 state

2018 ◽  
Author(s):  
Ana Rita Borba ◽  
Tânia S. Serra ◽  
Alicja Górska ◽  
Paulo Gouveia ◽  
André M. Cordeiro ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Regulatory Networks ◽  
Molecular Mechanisms ◽  
Mobility Shift ◽  
Helix Loop Helix ◽  
Specific Accumulation ◽  
Cell Type Specific ◽  
Bhlh Transcription Factors ◽  
Electrophoretic Mobility Shift Assays ◽  
Element Pair

AbstractC4 photosynthesis has evolved repeatedly from the ancestral C3 state to generate a carbon concentrating mechanism that increases photosynthetic efficiency. This specialised form of photosynthesis is particularly common in the PACMAD clade of grasses, and is used by many of the world’s most productive crops. The C4 cycle is accomplished through cell-type specific accumulation of enzymes but cis-elements and transcription factors controlling C4 photosynthesis remain largely unknown. Using the NADP-Malic Enzyme (NADP-ME) gene as a model we aimed to better understand molecular mechanisms associated with the evolution of C4 photosynthesis. Two basic Helix-Loop-Helix (bHLH) transcription factors, ZmbHLH128 and ZmbHLH129, were shown to bind the C4NADP-ME promoter from maize. These proteins form heterodimers and ZmbHLH129 impairs trans-activation by ZmbHLH128. Electrophoretic mobility shift assays indicate that a pair of cis-elements separated by a seven base pair spacer synergistically bind either ZmbHLH128 or ZmbHLH129. This pair of cis-elements is found in both C3 and C4 species of the PACMAD clade. Our analysis is consistent with this cis-element pair originating from a single motif present in the ancestral C3 state. We conclude that C4 photosynthesis has co-opted an ancient C3 regulatory code built on G-box recognition by bHLH to regulate the NADP-ME gene. More broadly, our findings also contribute to the understanding of gene regulatory networks controlling C4 photosynthesis.

scholarly journals The Myb domain of LUX ARRHYTHMO in complex with DNA: expression, purification and crystallization

2016 ◽  
Vol 72 (5) ◽  
pp. 356-361 ◽  
Author(s):  
Catarina S. Silva ◽  
Xuelei Lai ◽  
Max Nanao ◽  
Chloe Zubieta
Keyword(s):  
Dna Binding ◽  
Molecular Mechanisms ◽  
Binding Activity ◽  
Mobility Shift ◽  
Data Set ◽  
Dna Binding Activity ◽  
Protein Dna Interactions ◽  
Reported Data ◽  
Electrophoretic Mobility Shift Assays ◽  
Myb Domain

LUX ARRHYTHMO (LUX) is a Myb-domain transcription factor that plays an important role in regulating the circadian clock.Luxmutations cause severe clock defects and arrhythmia in constant light and dark. In order to examine the molecular mechanisms underlying the function of LUX, the DNA-binding Myb domain was cloned, expressed and purified. The DNA-binding activity of the Myb domain was confirmed using electrophoretic mobility shift assays (EMSAs), demonstrating that the LUX Myb domain is able to bind to DNA with nanomolar affinity. In order to investigate the specificity determinants of protein–DNA interactions, the protein was co-crystallized with a 10-mer cognate DNA. Initial crystallization results for the selenomethionine-derivatized protein and data-set collection statistics are reported. Data collection was performed using theMeshAndCollectworkflow available at the ESRF.

Antiparasitic Treatment of Patients with P. falciparum Malaria Reduces the Ability of Patient Serum to Induce Tissue Factor by Decreasing NF-κB Activation

1995 ◽  
Vol 73 (01) ◽  
pp. 039-048 ◽  
Author(s):  
A Bierhaus ◽  
Ch J Hemmer ◽  
N Mackman ◽  
R Kutob ◽  
R Ziegler ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Falciparum Malaria ◽  
De Novo ◽  
Neutralizing Antibody ◽  
Mobility Shift ◽  
Before And After ◽  
Tf Gene ◽  
Electrophoretic Mobility Shift Assays ◽  
Antiparasitic Treatment ◽  
Stimulation Of

SummarySerum from patients with P. falciparum malaria at day 1 (pretherapy) induces tissue factor (TF) in cultured endothelial cells. TF induction depends on de novo transcription as shown in Nuclear Run On assays. Electrophoretic mobility shift assays demonstrated binding of AP-1 and NF- κB/Rel proteins to their recognition sites in the TF promotor. After therapy (day 28), stimulation of TF antigen by patient serum is reduced by 70%. When serum obtained before and after therapy was compared, a decrease of NF-κB activation was evident. Activation of NF-κB-like proteins was in part dependent on TNFα in patient serum, since a TNFα neutralizing antibody reduced induction of TF transcription and translation and induction of NF-κB-like proteins. Induction of TF activity was suppressed by pDTC, an inhibitor of NF-κB activation. When different promotor constructs of the TF gene were tested, induction was dependent upon the presence of the intact NF-κB-like binding site in the TF promotor. A mutant with deleted NF-κB, but intact AP-1 sites was not inducible. Mutation of the AP-1 sites did not prevent induction, but reduced inducibility by pretherapy serum. Therefore, NF-κB/Rel proteins are responsible for induction of TF transcription by pretherapy serum, but AP-1 is needed for highest inducibility. The effect of antiparasitic therapy on the induction of TF by serum from patients with complicated P. falciparum malaria is dependent on a therapy-mediated loss of activation of NF-κB-like proteins in post-treatment patient serum.

scholarly journals Copy number-dependent DNA methylation of the Pyricularia oryzae MAGGY retrotransposon is triggered by DNA damage

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Ba Van Vu ◽  
Quyet Nguyen ◽  
Yuki Kondo-Takeoka ◽  
Toshiki Murata ◽  
Naoki Kadotani ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Copy Number ◽  
Rna Silencing ◽  
Molecular Mechanisms ◽  
De Novo ◽  
Pyricularia Oryzae ◽  
Transcriptional Gene Silencing ◽  
Physical Interaction ◽  
Uncharacterized Protein ◽  
Form Complex

AbstractTransposable elements are common targets for transcriptional and post-transcriptional gene silencing in eukaryotic genomes. However, the molecular mechanisms responsible for sensing such repeated sequences in the genome remain largely unknown. Here, we show that machinery of homologous recombination (HR) and RNA silencing play cooperative roles in copy number-dependent de novo DNA methylation of the retrotransposon MAGGY in the fungusPyricularia oryzae. Genetic and physical interaction studies revealed thatRecAdomain-containing proteins, includingP. oryzaehomologs ofRad51, Rad55, andRad57, together with an uncharacterized protein, Ddnm1, form complex(es) and mediate either the overall level or the copy number-dependence of de novo MAGGY DNA methylation, likely in conjunction with DNA repair. Interestingly,P. oryzaemutants of specific RNA silencing components (MoDCL1andMoAGO2)were impaired in copy number-dependence of MAGGY methylation. Co-immunoprecipitation of MoAGO2 and HR components suggested a physical interaction between the HR and RNA silencing machinery in the process.

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