scholarly journals Characterization of a DCL2-Insensitive Tomato Bushy Stunt Virus Isolate Infecting Arabidopsis thaliana

Viruses ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 1121
Author(s):  
Marco Incarbone ◽  
Hélene Scheer ◽  
Jean-Michel Hily ◽  
Lauriane Kuhn ◽  
Mathieu Erhardt ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Arabidopsis Thaliana ◽  
High Efficiency ◽  
Wide Spectrum ◽  
Model Organism ◽  
Plant Viruses ◽  
Tomato Bushy Stunt Virus ◽  
Defensive Reaction ◽  
Knock Out ◽  
Replication Sites

Tomato bushy stunt virus (TBSV), the type member of the genus Tombusvirus in the family Tombusviridae is one of the best studied plant viruses. The TBSV natural and experimental host range covers a wide spectrum of plants including agricultural crops, ornamentals, vegetables and Nicotiana benthamiana. However, Arabidopsis thaliana, the well-established model organism in plant biology, genetics and plant–microbe interactions is absent from the list of known TBSV host plant species. Most of our recent knowledge of the virus life cycle has emanated from studies in Saccharomyces cerevisiae, a surrogate host for TBSV that lacks crucial plant antiviral mechanisms such as RNA interference (RNAi). Here, we identified and characterized a TBSV isolate able to infect Arabidopsis with high efficiency. We demonstrated by confocal and 3D electron microscopy that in Arabidopsis TBSV-BS3Ng replicates in association with clustered peroxisomes in which numerous spherules are induced. A dsRNA-centered immunoprecipitation analysis allowed the identification of TBSV-associated host components including DRB2 and DRB4, which perfectly localized to replication sites, and NFD2 that accumulated in larger viral factories in which peroxisomes cluster. By challenging knock-out mutants for key RNAi factors, we showed that TBSV-BS3Ng undergoes a non-canonical RNAi defensive reaction. In fact, unlike other RNA viruses described, no 22nt TBSV-derived small RNA are detected in the absence of DCL4, indicating that this virus is DCL2-insensitive. The new Arabidopsis-TBSV-BS3Ng pathosystem should provide a valuable new model for dissecting plant–virus interactions in complement to Saccharomyces cerevisiae.

scholarly journals The MyLo CRISPR-Cas9 Toolkit: A Markerless Yeast Localization and Overexpression CRISPR-Cas9 Toolkit

2021 ◽  
Author(s):  
Bjorn DM Bean ◽  
Malcolm Whiteway ◽  
Vincent JJ Martin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
High Efficiency ◽  
Model Organism ◽  
Basic Research ◽  
Expression Vectors ◽  
Selectable Markers ◽  
Guide Rna ◽  
One Step ◽  
Selection For ◽  
The One

The genetic tractability of the yeast Saccharomyces cerevisiae has made it a key model organism for basic research and a target for metabolic engineering. To streamline the introduction of tagged genes and compartmental markers with powerful CRISPR-Cas9-based genome editing tools we constructed a Markerless Yeast Localization and Overexpression (MyLO) CRISPR-Cas9 Toolkit with three components: (i) a set of optimized S. pyogenes Cas9-guide RNA (gRNA) expression vectors with five selectable markers and the option to either pre-clone or co-transform the gRNAs; (ii) vectors for the one-step construction of integration cassettes expressing an untagged or GFP/RFP/HA-tagged gene of interest at one of three levels, supporting localization and overexpression studies; and (iii) integration cassettes containing moderately expressed GFP- or RFP-tagged compartmental markers for colocalization experiments. These components allow rapid, high efficiency genomic integrations and modifications with only transient selection for the Cas9 vector, resulting in markerless transformations. Thus, the MyLO toolkit packages CRISPR-Cas9 technology into a flexible, optimized bundle to simplify yeast research

scholarly journals In Vivo Production of RNA Aptamers and Nanoparticles: Problems and Prospects

Molecules ◽  
2021 ◽  
Vol 26 (5) ◽  
pp. 1422
Author(s):  
Ousama Al Shanaa ◽  
Andrey Rumyantsev ◽  
Elena Sambuk ◽  
Marina Padkina
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Large Scale ◽  
Potential Model ◽  
Model Organism ◽  
Rna Aptamers ◽  
Early Stages ◽  
Near Future

RNA aptamers are becoming increasingly attractive due to their superior properties. This review discusses the early stages of aptamer research, the main developments in this area, and the latest technologies being developed. The review also highlights the advantages of RNA aptamers in comparison to antibodies, considering the great potential of RNA aptamers and their applications in the near future. In addition, it is shown how RNA aptamers can form endless 3-D structures, giving rise to various structural and functional possibilities. Special attention is paid to the Mango, Spinach and Broccoli fluorescent RNA aptamers, and the advantages of split RNA aptamers are discussed. The review focuses on the importance of creating a platform for the synthesis of RNA nanoparticles in vivo and examines yeast, namely Saccharomyces cerevisiae, as a potential model organism for the production of RNA nanoparticles on a large scale.

scholarly journals Non-Ionizing Millimeter Waves Non-Thermal Radiation of Saccharomyces cerevisiae—Insights and Interactions

2021 ◽  
Vol 11 (14) ◽  
pp. 6635
Author(s):  
Ayan Barbora ◽  
Shailendra Rajput ◽  
Konstantin Komoshvili ◽  
Jacob Levitan ◽  
Asher Yahalom ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Division ◽  
Millimeter Waves ◽  
Model Organism ◽  
Horn Antenna ◽  
Colony Growth ◽  
Genetic Alterations ◽  
Power Delivery ◽  
Repair Gene ◽  
Safety Standards

Non-ionizing millimeter-waves (MMW) interact with cells in a variety of ways. Here the inhibited cell division effect was investigated using 85–105 GHz MMW irradiation within the International Commission on Non-Ionizing Radiation Protection (ICNIRP) non-thermal 20 mW/cm2 safety standards. Irradiation using a power density of about 1.0 mW/cm2 SAR over 5–6 h on 50 cells/μL samples of Saccharomyces cerevisiae model organism resulted in 62% growth rate reduction compared to the control (sham). The effect was specific for 85–105 GHz range and was energy- and cell density-dependent. Irradiation of wild type and Δrad52 (DNA damage repair gene) deleted cells presented no differences of colony growth profiles indicating non-thermal MMW treatment does not cause permanent genetic alterations. Dose versus response relations studied using a standard horn antenna (~1.0 mW/cm2) and compared to that of a compact waveguide (17.17 mW/cm2) for increased power delivery resulted in complete termination of cell division via non-thermal processes supported by temperature rise measurements. We have shown that non-thermal MMW radiation has potential for future use in treatment of yeast related diseases and other targeted biomedical outcomes.

scholarly journals Arabidopsis thaliana G3BP Ortholog Rescues Mammalian Stress Granule Phenotype across Kingdoms

2021 ◽  
Vol 22 (12) ◽  
pp. 6287
Author(s):  
Hendrik Reuper ◽  
Benjamin Götte ◽  
Lucy Williams ◽  
Timothy J. C. Tan ◽  
Gerald M. McInerney ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Fusion Proteins ◽  
Biological Function ◽  
Protein Complexes ◽  
Protein Family ◽  
Knock Out ◽  
Marker Proteins ◽  
Interaction Partners ◽  
Functional Analyses ◽  
Rna Protein Complexes

Stress granules (SGs) are dynamic RNA–protein complexes localized in the cytoplasm that rapidly form under stress conditions and disperse when normal conditions are restored. The formation of SGs depends on the Ras-GAP SH3 domain-binding protein (G3BP). Formations, interactions and functions of plant and human SGs are strikingly similar, suggesting a conserved mechanism. However, functional analyses of plant G3BPs are missing. Thus, members of the Arabidopsis thaliana G3BP (AtG3BP) protein family were investigated in a complementation assay in a human G3BP knock-out cell line. It was shown that two out of seven AtG3BPs were able to complement the function of their human homolog. GFP-AtG3BP fusion proteins co-localized with human SG marker proteins Caprin-1 and eIF4G1 and restored SG formation in G3BP double KO cells. Interaction between AtG3BP-1 and -7 and known human G3BP interaction partners such as Caprin-1 and USP10 was also demonstrated by co-immunoprecipitation. In addition, an RG/RGG domain exchange from Arabidopsis G3BP into the human G3BP background showed the ability for complementation. In summary, our results support a conserved mechanism of SG function over the kingdoms, which will help to further elucidate the biological function of the Arabidopsis G3BP protein family.

scholarly journals Ca2+-Dependent Protein Kinase 6 Enhances KAT2 Shaker Channel Activity in Arabidopsis thaliana

2021 ◽  
Vol 22 (4) ◽  
pp. 1596
Author(s):  
Elsa Ronzier ◽  
Claire Corratgé-Faillie ◽  
Frédéric Sanchez ◽  
Christian Brière ◽  
Tou Cheu Xiong
Keyword(s):  
Arabidopsis Thaliana ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Physical Interaction ◽  
Fluorescence Lifetime Imaging ◽  
Dependent Manner ◽  
In Planta ◽  
Knock Out ◽  
Calcium Dependent ◽  
Dependent Protein

Post-translational regulations of Shaker-like voltage-gated K+ channels were reported to be essential for rapid responses to environmental stresses in plants. In particular, it has been shown that calcium-dependent protein kinases (CPKs) regulate Shaker channels in plants. Here, the focus was on KAT2, a Shaker channel cloned in the model plant Arabidopsis thaliana, where is it expressed namely in the vascular tissues of leaves. After co-expression of KAT2 with AtCPK6 in Xenopuslaevis oocytes, voltage-clamp recordings demonstrated that AtCPK6 stimulates the activity of KAT2 in a calcium-dependent manner. A physical interaction between these two proteins has also been shown by Förster resonance energy transfer by fluorescence lifetime imaging (FRET-FLIM). Peptide array assays support that AtCPK6 phosphorylates KAT2 at several positions, also in a calcium-dependent manner. Finally, K+ fluorescence imaging in planta suggests that K+ distribution is impaired in kat2 knock-out mutant leaves. We propose that the AtCPK6/KAT2 couple plays a role in the homeostasis of K+ distribution in leaves.

scholarly journals Genetic analysis of a Piezo-like protein suppressing systemic movement of plant viruses in Arabidopsis thaliana

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Zhen Zhang ◽  
Xin Tong ◽  
Song-Yu Liu ◽  
Long-Xiang Chai ◽  
Fei-Fan Zhu ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Genetic Analysis ◽  
Plant Viruses ◽  
Systemic Movement

scholarly journals Saccharomyces cerevisiae (Baker’s Yeast) as an Interfering RNA Expression and Delivery System

2019 ◽  
Vol 20 (9) ◽  
pp. 942-952 ◽  
Author(s):  
Molly Duman-Scheel
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Interference ◽  
Genetic Model ◽  
Model Organism ◽  
Rna Expression ◽  
Commercial Development ◽  
Rna Integrity ◽  
Yeast Cultures ◽  
Ideal System ◽  
Interfering Rna

The broad application of RNA interference for disease prevention is dependent upon the production of dsRNA in an economically feasible, scalable, and sustainable fashion, as well as the identification of safe and effective methods for RNA delivery. Current research has sparked interest in the use of Saccharomyces cerevisiae for these applications. This review examines the potential for commercial development of yeast interfering RNA expression and delivery systems. S. cerevisiae is a genetic model organism that lacks a functional RNA interference system, which may make it an ideal system for expression and accumulation of high levels of recombinant interfering RNA. Moreover, recent studies in a variety of eukaryotic species suggest that this microbe may be an excellent and safe system for interfering RNA delivery. Key areas for further research and development include optimization of interfering RNA expression in S. cerevisiae, industrial-sized scaling of recombinant yeast cultures in which interfering RNA molecules are expressed, the development of methods for largescale drying of yeast that preserve interfering RNA integrity, and identification of encapsulating agents that promote yeast stability in various environmental conditions. The genetic tractability of S. cerevisiae and a long history of using this microbe in both the food and pharmaceutical industry will facilitate further development of this promising new technology, which has many potential applications of medical importance.

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