scholarly journals Targeted gene suppression through double-stranded RNA application using easy-to-use methods in Arabidopsis thaliana

2022 ◽  
Vol 65 (1) ◽  
Author(s):  
Minsu Park ◽  
Tae Young Um ◽  
Geupil Jang ◽  
Yang Do Choi ◽  
Chanseok Shin
Keyword(s):  
Arabidopsis Thaliana ◽  
Target Genes ◽  
Fluorescent Protein ◽  
Specific Interaction ◽  
Mrna Levels ◽  
Messenger Rnas ◽  
Small Interfering Rnas ◽  
Double Stranded Rna ◽  
Reverse Transcription Pcr ◽  
Green Fluorescent

AbstractRNA interference (RNAi) is an RNA-dependent gene silencing process that is regulated by the interaction between the RNA-induced silencing complex (RISC) and double-stranded RNA (dsRNA). Exogenous dsRNAs are imported directly into the cytoplasm, where they are cleaved by Dicer into short dsRNA fragments of 20–25 base pairs. These short dsRNA fragments, called small interfering RNAs (siRNAs) have sequence-specific interaction with target genes. The guide strand, onto which siRNAs are incorporated in the RISC interacts with the target mRNA sequence, thereby inducing cleavage and degradation of target messenger RNAs (mRNAs) by ribonucleases. Recent studies have shown that plant dsRNA treatments can induce RNAi. However, the dsRNA application methods and delivery systems involved have not been well examined. In this study, dsRNA was introduced to Arabidopsis thaliana by two methods: dipping and spray. We synthesized two dsRNAs designed to target mRNAs encoding enhanced green fluorescent protein (EGFP). After applying dsRNAs that target EGFP, we found an obvious reduction in GFP expression. This was determined using fluorescence microscopy and quantitative reverse transcription PCR to assess the mRNA levels of the auxin-sensitive reporter DR5-EGFP Arabidopsis thaliana. Our data revealed that applying target gene-specific exogenous dsRNAs can induce suppression of target genes of interest whether the dipping or spray method is used. This study therefore provides a foundation for understanding how to apply and deliver dsRNAs in plants.

scholarly journals The Evolutionary Significance of RNAi in the Fungal Kingdom

2020 ◽  
Vol 21 (24) ◽  
pp. 9348
Author(s):  
Carlos Lax ◽  
Ghizlane Tahiri ◽  
José Alberto Patiño-Medina ◽  
José T. Cánovas-Márquez ◽  
José A. Pérez-Ruiz ◽  
...  
Keyword(s):  
Target Genes ◽  
Regulation Of Gene Expression ◽  
Genome Integrity ◽  
Evolutionary Significance ◽  
Messenger Rnas ◽  
Small Interfering Rnas ◽  
Rnase Iii ◽  
Double Stranded Rna ◽  
Main Component ◽  
Fungal Kingdom

RNA interference (RNAi) was discovered at the end of last millennium, changing the way scientists understood regulation of gene expression. Within the following two decades, a variety of different RNAi mechanisms were found in eukaryotes, reflecting the evolutive diversity that RNAi entails. The essential silencing mechanism consists of an RNase III enzyme called Dicer that cleaves double-stranded RNA (dsRNA) generating small interfering RNAs (siRNAs), a hallmark of RNAi. These siRNAs are loaded into the RNA-induced silencing complex (RISC) triggering the cleavage of complementary messenger RNAs by the Argonaute protein, the main component of the complex. Consequently, the expression of target genes is silenced. This mechanism has been thoroughly studied in fungi due to their proximity to the animal phylum and the conservation of the RNAi mechanism from lower to higher eukaryotes. However, the role and even the presence of RNAi differ across the fungal kingdom, as it has evolved adapting to the particularities and needs of each species. Fungi have exploited RNAi to regulate a variety of cell activities as different as defense against exogenous and potentially harmful DNA, genome integrity, development, drug tolerance, or virulence. This pathway has offered versatility to fungi through evolution, favoring the enormous diversity this kingdom comprises.

scholarly journals Induction of Transgene Suppression in Plants via External Application of Synthetic dsRNA

2019 ◽  
Vol 20 (7) ◽  
pp. 1585 ◽  
Author(s):  
Alexandra Dubrovina ◽  
Olga Aleynova ◽  
Alexander Kalachev ◽  
Andrey Suprun ◽  
Zlata Ogneva ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Crop Improvement ◽  
Plant Genome ◽  
Mrna Levels ◽  
Small Interfering Rnas ◽  
External Application ◽  
Transcript Levels ◽  
Exogenous Application ◽  
Green Fluorescent

Recent investigations show that exogenously applied small interfering RNAs (siRNA) and long double-stranded RNA (dsRNA) precursors can be taken up and translocated in plants to induce RNA interference (RNAi) in the plant or in its fungal pathogen. The question of whether genes in the plant genome can undergo suppression as a result of exogenous RNA application on plant surface is almost unexplored. This study analyzed whether it is possible to influence transcript levels of transgenes, as more prone sequences to silencing, in Arabidopsis genome by direct exogenous application of target long dsRNAs. The data revealed that in vitro synthesized dsRNAs designed to target the gene coding regions of enhanced green fluorescent protein (EGFP) or neomycin phosphotransferase II (NPTII) suppressed their transcript levels in Arabidopsis. The fact that, simple exogenous application of polynucleotides can affect mRNA levels of plant transgenes, opens new opportunities for the development of new scientific techniques and crop improvement strategies.

scholarly journals Diverging Affinity of Tospovirus RNA Silencing Suppressor Proteins, NSs, for Various RNA Duplex Molecules

2010 ◽  
Vol 84 (21) ◽  
pp. 11542-11554 ◽  
Author(s):  
Esther Schnettler ◽  
Hans Hemmes ◽  
Rik Huismann ◽  
Rob Goldbach ◽  
Marcel Prins ◽  
...  
Keyword(s):  
Rna Silencing ◽  
Fluorescent Protein ◽  
Micro Rna ◽  
Impatiens Necrotic Spot Virus ◽  
Small Interfering Rnas ◽  
Double Stranded Rna ◽  
Cell Extracts ◽  
Green Fluorescent

ABSTRACT The tospovirus NSs protein was previously shown to suppress the antiviral RNA silencing mechanism in plants. Here the biochemical analysis of NSs proteins from different tospoviruses, using purified NSs or NSs containing cell extracts, is described. The results showed that all tospoviral NSs proteins analyzed exhibited affinity to small double-stranded RNA molecules, i.e., small interfering RNAs (siRNAs) and micro-RNA (miRNA)/miRNA* duplexes. Interestingly, the NSs proteins from tomato spotted wilt virus (TSWV), impatiens necrotic spot virus (INSV), and groundnut ringspot virus (GRSV) also showed affinity to long double-stranded RNA (dsRNA), whereas tomato yellow ring virus (TYRV) NSs did not. The TSWV NSs protein was shown to be capable of inhibiting Dicer-mediated cleavage of long dsRNA in vitro. In addition, it suppressed the accumulation of green fluorescent protein (GFP)-specific siRNAs during coinfiltration with an inverted-repeat-GFP RNA construct in Nicotiana benthamiana. In vivo interference of TSWV NSs in the miRNA pathway was shown by suppression of an enhanced GFP (eGFP) miRNA sensor construct. The ability to stabilize miRNA/miRNA* by different tospovirus NSs proteins in vivo was demonstrated by increased accumulation and detection of both miRNA171c and miRNA171c* in tospovirus-infected N. benthamiana. All together, these data suggest that tospoviruses interfere in the RNA silencing pathway by sequestering siRNA and miRNA/miRNA* molecules before they are uploaded into their respective RNA-induced silencing complexes. The observed affinity to long dsRNA for only a subset of the tospoviruses studied is discussed in light of evolutional divergence and their ancestral relation to the animal-infecting members of the Bunyaviridae.

scholarly journals Extracellular stimuli specifically regulate localized levels of individual neuronal mRNAs

2007 ◽  
Vol 178 (6) ◽  
pp. 965-980 ◽  
Author(s):  
Dianna E. Willis ◽  
Erna A. van Niekerk ◽  
Yukio Sasaki ◽  
Mariano Mesngon ◽  
Tanuja T. Merianda ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Mrna Levels ◽  
Messenger Rnas ◽  
Sensory Axons ◽  
Neurotrophin 3 ◽  
Extracellular Stimuli ◽  
Actin Mrna ◽  
Regulate Protein ◽  
Green Fluorescent ◽  
Localized Levels

Subcellular regulation of protein synthesis requires the correct localization of messenger RNAs (mRNAs) within the cell. In this study, we investigate whether the axonal localization of neuronal mRNAs is regulated by extracellular stimuli. By profiling axonal levels of 50 mRNAs detected in regenerating adult sensory axons, we show that neurotrophins can increase and decrease levels of axonal mRNAs. Neurotrophins (nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3) regulate axonal mRNA levels and use distinct downstream signals to localize individual mRNAs. However, myelin-associated glycoprotein and semaphorin 3A regulate axonal levels of different mRNAs and elicit the opposite effect on axonal mRNA levels from those observed with neurotrophins. The axonal mRNAs accumulate at or are depleted from points of ligand stimulation along the axons. The translation product of a chimeric green fluorescent protein–β-actin mRNA showed similar accumulation or depletion adjacent to stimuli that increase or decrease axonal levels of endogenous β-actin mRNA. Thus, extracellular ligands can regulate protein generation within subcellular regions by specifically altering the localized levels of particular mRNAs.

scholarly journals Rotavirus Virus-Like Particles as Surrogates in Environmental Persistence and Inactivation Studies

2004 ◽  
Vol 70 (7) ◽  
pp. 3904-3909 ◽  
Author(s):  
Santiago Caballero ◽  
F. Xavier Abad ◽  
Fabienne Loisy ◽  
Françoise S. Le Guyader ◽  
Jean Cohen ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Uv Light ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Virus Removal ◽  
Virus Like Particles ◽  
Reverse Transcription Pcr ◽  
Actual Field ◽  
Uv Dose ◽  
Green Fluorescent

ABSTRACT Virus-like particles (VLPs) with the full-length VP2 and VP6 rotavirus capsid proteins, produced in the baculovirus expression system, have been evaluated as surrogates of human rotavirus in different environmental scenarios. Green fluorescent protein-labeled VLPs (GFP-VLPs) and particles enclosing a heterologous RNA (pseudoviruses), whose stability may be monitored by flow cytometry and antigen capture reverse transcription-PCR, respectively, were used. After 1 month in seawater at 20°C, no significant differences were observed between the behaviors of GFP-VLPs and of infectious rotavirus, whereas pseudovirus particles showed a higher decay rate. In the presence of 1 mg of free chlorine (FC)/liter both tracers persisted longer in freshwater at 20°C than infectious viruses, whereas in the presence of 0.2 mg of FC/liter no differences were observed between tracers and infectious rotavirus at short contact times. However, from 30 min of contact with FC onward, the decay of infectious rotavirus was higher than that of recombinant particles. The predicted Ct value for a 90% reduction of GFP-VLPs or pseudoviruses induces a 99.99% inactivation of infectious rotavirus. Both tracers were more resistant to UV light irradiation than infectious rotavirus in fresh and marine water. The effect of UV exposure was more pronounced on pseudovirus than in GFP-VLPs. In all types of water, the UV dose to induce a 90% reduction of pseudovirus ensures a 99.99% inactivation of infectious rotavirus. Recombinant virus surrogates open new possibilities for the systematic validation of virus removal practices in actual field situations where pathogenic agents cannot be introduced.

scholarly journals Decreased Vancomycin Susceptibility in Staphylococcus aureus Caused by IS256Tempering of WalKR Expression

2013 ◽  
Vol 57 (7) ◽  
pp. 3240-3249 ◽  
Author(s):  
Christopher R. E. McEvoy ◽  
Brian Tsuji ◽  
Wei Gao ◽  
Torsten Seemann ◽  
Jessica L. Porter ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Fluorescent Protein ◽  
Site Directed Mutagenesis ◽  
Infection Model ◽  
Content Type ◽  
Reverse Transcription Pcr ◽  
Phenotype Analysis ◽  
Dosing Regimens ◽  
Mrsa Strains ◽  
Green Fluorescent

ABSTRACTVancomycin-intermediateStaphylococcus aureus(VISA) strains often arise by mutations in the essential two-component regulatorwalKR; however their impact onwalKRfunction has not been definitively established. Here, we investigated 10 MRSA strains recovered serially after exposure of vancomycin-susceptibleS. aureus(VSSA) JKD6009 to simulated human vancomycin dosing regimens (500 mg to 4,000 mg every 12 h) using a 10-day hollow fiber infection model. After continued exposure to the vancomycin regimens, two isolates displayed reduced susceptibility to both vancomycin and daptomycin, developing independent IS256insertions in thewalKR5′ untranslated region (5′ UTR). Quantitative reverse transcription-PCR (RT-PCR) revealed a 50% reduction inwalKRgene expression in the IS256mutants compared to the VSSA parent. Green fluorescent protein (GFP) reporter analysis, promoter mapping, and site-directed mutagenesis confirmed these findings and showed that the IS256insertions had replaced two SigA-likewalKRpromoters with weaker, hybrid promoters. Removal of IS256reverted the phenotype to VSSA, showing that reduced expression of WalKR did induce the VISA phenotype. Analysis of selected WalKR-regulated autolysins revealed upregulation ofssaAbut no change in expression ofsakandsceDin both IS256mutants. Whole-genome sequencing of the two mutants revealed an additional IS256insertion withinagrCfor one mutant, and we confirmed that this mutation abolishedagrfunction. These data provide the first substantial analysis ofwalKRpromoter function and show that prolonged vancomycin exposure can result in VISA through an IS256-mediated reduction inwalKRexpression; however, the mechanisms by which this occurs remain to be determined.

scholarly journals Colonization of Arabidopsis thaliana with Salmonella enterica and Enterohemorrhagic Escherichia coli O157:H7 and Competition by Enterobacter asburiae

2003 ◽  
Vol 69 (8) ◽  
pp. 4915-4926 ◽  
Author(s):  
Michael B. Cooley ◽  
William G. Miller ◽  
Robert E. Mandrell
Keyword(s):  
Escherichia Coli ◽  
Arabidopsis Thaliana ◽  
Salmonella Enterica ◽  
Fluorescent Protein ◽  
Enterohemorrhagic Escherichia Coli ◽  
Plant Responses ◽  
Human Pathogens ◽  
E Coli ◽  
Green Fluorescent ◽  
Enterobacter Asburiae

ABSTRACT Enteric pathogens, such as Salmonella enterica and Escherichia coli O157:H7, have been shown to contaminate fresh produce. Under appropriate conditions, these bacteria will grow on and invade the plant tissue. We have developed Arabidopsis thaliana (thale cress) as a model system with the intention of studying plant responses to human pathogens. Under sterile conditions and at 100% humidity, S. enterica serovar Newport and E. coli O157:H7 grew to 109 CFU g−1 on A. thaliana roots and to 2 × 107 CFU g−1 on shoots. Furthermore, root inoculation led to contamination of the entire plant, indicating that the pathogens are capable of moving on or within the plant in the absence of competition. Inoculation with green fluorescent protein-labeled S. enterica and E. coli O157:H7 showed invasion of the roots at lateral root junctions. Movement was eliminated and invasion decreased when nonmotile mutants of S. enterica were used. Survival of S. enterica serovar Newport and E. coli O157:H7 on soil-grown plants declined as the plants matured, but both pathogens were detectable for at least 21 days. Survival of the pathogen was reduced in unautoclaved soil and amended soil, suggesting competition from indigenous epiphytes from the soil. Enterobacter asburiae was isolated from soil-grown A. thaliana and shown to be effective at suppressing epiphytic growth of both pathogens under gnotobiotic conditions. Seed and chaff harvested from contaminated plants were occasionally contaminated. The rate of recovery of S. enterica and E. coli O157:H7 from seed varied from undetectable to 19% of the seed pools tested, depending on the method of inoculation. Seed contamination by these pathogens was undetectable in the presence of the competitor, Enterobacter asburiae. Sampling of 74 pools of chaff indicated a strong correlation between contamination of the chaff and seed (P = 0.025). This suggested that contamination of the seed occurred directly from contaminated chaff or by invasion of the flower or silique. However, contaminated seeds were not sanitized by extensive washing and chlorine treatment, indicating that some of the bacteria reside in a protected niche on the seed surface or under the seed coat.

scholarly journals The miR-26b-5p/KPNA2 Axis Is an Important Regulator of Burkitt Lymphoma Cell Growth

Cancers ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 1464
Author(s):  
Fubiao Niu ◽  
Marta Kazimierska ◽  
Ilja M. Nolte ◽  
Miente Martijn Terpstra ◽  
Debora de Jong ◽  
...  
Keyword(s):  
Cell Growth ◽  
Cell Lines ◽  
Burkitt Lymphoma ◽  
Target Genes ◽  
Fluorescent Protein ◽  
Luciferase Reporter ◽  
Argonaute 2 ◽  
A Genome ◽  
Green Fluorescent ◽  
Competition Assays

The expression of several microRNAs (miRNAs) is known to be changed in Burkitt lymphoma (BL), compared to its normal counterparts. Although for some miRNAs, a role in BL was demonstrated, for most of them, their function is unclear. In this study, we aimed to identify miRNAs that control BL cell growth. Two BL cell lines were infected with lentiviral pools containing either 58 miRNA inhibitors or 44 miRNA overexpression constructs. Eighteen constructs showed significant changes in abundance over time, indicating that they affected BL growth. The screening results were validated by individual green fluorescent protein (GFP) growth competition assays for fifteen of the eighteen constructs. For functional follow-up studies, we focused on miR-26b-5p, whose overexpression inhibited BL cell growth. Argonaute 2 RNA immunoprecipitation (Ago2-IP) in two BL cell lines revealed 47 potential target genes of miR-26b-5p. Overlapping the list of putative targets with genes showing a growth repression phenotype in a genome-wide CRISPR/Cas9 knockout screen, revealed eight genes. The top-5 candidates included EZH2, COPS2, KPNA2, MRPL15, and NOL12. EZH2 is a known target of miR-26b-5p, with oncogenic properties in BL. The relevance of the latter four targets was confirmed using sgRNAs targeting these genes in individual GFP growth competition assays. Luciferase reporter assay confirmed binding of miR-26b-5p to the predicted target site for KPNA2, but not to the other genes. In summary, we identified 18 miRNAs that affected BL cell growth in a loss- or gain-of-function screening. A tumor suppressor role was confirmed for miR-26b-5p, and this effect could at least in part be attributed to KPNA2, a known regulator of OCT4, c-jun, and MYC.

scholarly journals Non-Target Effects of Green Fluorescent Protein (GFP)-Derived Double-Stranded RNA (dsRNA-GFP) Used in Honey Bee RNA Interference (RNAi) Assays

Insects ◽  
2013 ◽  
Vol 4 (1) ◽  
pp. 90-103 ◽  
Author(s):  
Francis Nunes ◽  
Aline Aleixo ◽  
Angel Barchuk ◽  
Ana Bomtorin ◽  
Christina Grozinger ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Rna Interference ◽  
Honey Bee ◽  
Fluorescent Protein ◽  
Double Stranded Rna ◽  
Green Fluorescent ◽  
Target Effects

Relative Strengths and Regulation of Different Promoter-Associated Sequences for Various blaSHV Genes and Their Relationships to β-Lactam Resistance

2017 ◽  
Vol 27 (2) ◽  
pp. 91-101 ◽  
Author(s):  
Yao Zhai ◽  
Zhao Zhang ◽  
Zhanwei Wang ◽  
Yusheng Chen ◽  
Qi Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
Mrna Expression ◽  
Inhibitory Concentration ◽  
Fluorescent Protein ◽  
Mrna Levels ◽  
Associated Sequences ◽  
Regulation Function ◽  
Green Fluorescent ◽  
Mrna Expression Levels

<b><i>Aims:</i></b> This work investigated the relative strengths of different <i>bla</i><sub>SHV</sub> promoter-associated sequences and their regulation function in <i>bla</i><sub>SHV</sub> expression and β-lactam resistance. <b><i>Methods:</i></b> Recombinant plasmids with the promoter-associated sequences (P-W, P-S, P-IS, and P-WPD), <i>tac</i> promoter, and combined fragments of promoter and <i>bla</i><sub>SHV</sub> were separately constructed and transformed into <i>Escherichia coli</i> DH5α. The relative strengths of the promoters indicated by the intensities of green fluorescent protein and the mRNA expression levels of <i>bla</i><sub>SHV</sub> were compared. The minimum inhibitory concentration and extended spectrum β-lactamase phenotypes were evaluated. <b><i>Results:</i></b> The relative strengths were ranked as P-<i>tac</i> > P-WPD > P-IS > P-S > P-W. The mRNA expression and β-lactam resistance levels of the different promoter-associated sequence groups were generally consistent with the strength rank, but the extent of <i>gfp</i> and <i>bla</i><sub>SHV</sub> mRNA levels varied significantly in each group. The β-lactam resistance levels were inconsistent with the strength rank in certain <i>bla</i><sub>SHV</sub> groups. In relation to the different promoter-associated sequences,<i> bla</i><sub>SHV-ESBLs</sub> displayed significantly different change modes of β-lactam resistance compared with <i>bla</i><sub>SHV-non-ESBLs</sub>. <b><i>Conclusion:</i></b> The mRNA expression and β-lactam resistance of the <i>bla</i><sub>SHV</sub> showed consistencies and inconsistencies with the strengths of the promoter-associated sequences. The mechanisms accounting for these discrepancies need further investigation.

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