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Use of cell cultures in vitro to assess the uptake of long dsRNA in plant cells

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Abstract
While RNAi approaches based on transgenic plants overexpressing double-stranded RNAs (dsRNAs) have yielded a number of genetically modified plants with interesting new traits, several problems associated with the use of genetically modified organisms caused a shift in interest towards exogenous application of the RNA molecules. A major challenge in the environmental RNAi approach is the delivery of the dsRNA molecules into cells. The presence of a cell wall provides a challenging barrier for the internalization of dsRNAs into plant cells. While small interfering RNAs (siRNA) have previously been demonstrated to be internalized in plant cells, the ability of long dsRNAs to cross the cell wall is under discussion. In this study, confocal fluorescence microscopy revealed the successful internalization of a Cy5-labeled long dsRNA (dsGFP, 500 bp) in Arabidopsis thaliana PSB-D cell cultures. However, a similar approach was not successful in Nicotiana tabacum BY-2 cell cultures. This discrepancy between the two cell cultures suggests a pivoting role of the cell wall in the uptake of dsRNAs. The use of carrier molecules complexed with dsRNA can overcome this internalization barrier, thus facilitating cellular dsRNA uptake. Complexation of the dsRNA with a cell-penetrating peptide (CPP)-based carrier was shown to accelerate its internalization in Arabidopsis cells compared to uptake of the free dsRNA. These results confirm the potential of dsRNAs to cross the plant cell wall of particular cells and the beneficial effect of carriers to improve dsRNA delivery in cells.
Keywords
PENETRATING PEPTIDES, VIRUS-RESISTANCE, GENE, RNA, EXPRESSION, INTERFERENCE, DELIVERY, WALLS, RNAi, Arabidopsis cells, BY-2, dsRNA delivery, Peptide, dsRNA carrier, Cellular uptake

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MLA
De Schutter, Kristof, et al. “Use of Cell Cultures in Vitro to Assess the Uptake of Long DsRNA in Plant Cells.” IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY-PLANT, vol. 58, no. 4, 2022, pp. 511–20, doi:10.1007/s11627-022-10260-1.
APA
De Schutter, K., Verbeke, I., Kontogiannatos, D., Dubruel, P., Swevers, L., Van Damme, E., & Smagghe, G. (2022). Use of cell cultures in vitro to assess the uptake of long dsRNA in plant cells. IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY-PLANT, 58(4), 511–520. https://doi.org/10.1007/s11627-022-10260-1
Chicago author-date
De Schutter, Kristof, Isabel Verbeke, Dimitrios Kontogiannatos, Peter Dubruel, Luc Swevers, Els Van Damme, and Guy Smagghe. 2022. “Use of Cell Cultures in Vitro to Assess the Uptake of Long DsRNA in Plant Cells.” IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY-PLANT 58 (4): 511–20. https://doi.org/10.1007/s11627-022-10260-1.
Chicago author-date (all authors)
De Schutter, Kristof, Isabel Verbeke, Dimitrios Kontogiannatos, Peter Dubruel, Luc Swevers, Els Van Damme, and Guy Smagghe. 2022. “Use of Cell Cultures in Vitro to Assess the Uptake of Long DsRNA in Plant Cells.” IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY-PLANT 58 (4): 511–520. doi:10.1007/s11627-022-10260-1.
Vancouver
1.
De Schutter K, Verbeke I, Kontogiannatos D, Dubruel P, Swevers L, Van Damme E, et al. Use of cell cultures in vitro to assess the uptake of long dsRNA in plant cells. IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY-PLANT. 2022;58(4):511–20.
IEEE
[1]
K. De Schutter et al., “Use of cell cultures in vitro to assess the uptake of long dsRNA in plant cells,” IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY-PLANT, vol. 58, no. 4, pp. 511–520, 2022.
@article{8758435,
  abstract     = {{While RNAi approaches based on transgenic plants overexpressing double-stranded RNAs (dsRNAs) have yielded a number of genetically modified plants with interesting new traits, several problems associated with the use of genetically modified organisms caused a shift in interest towards exogenous application of the RNA molecules. A major challenge in the environmental RNAi approach is the delivery of the dsRNA molecules into cells. The presence of a cell wall provides a challenging barrier for the internalization of dsRNAs into plant cells. While small interfering RNAs (siRNA) have previously been demonstrated to be internalized in plant cells, the ability of long dsRNAs to cross the cell wall is under discussion. In this study, confocal fluorescence microscopy revealed the successful internalization of a Cy5-labeled long dsRNA (dsGFP, 500 bp) in Arabidopsis thaliana PSB-D cell cultures. However, a similar approach was not successful in Nicotiana tabacum BY-2 cell cultures. This discrepancy between the two cell cultures suggests a pivoting role of the cell wall in the uptake of dsRNAs. The use of carrier molecules complexed with dsRNA can overcome this internalization barrier, thus facilitating cellular dsRNA uptake. Complexation of the dsRNA with a cell-penetrating peptide (CPP)-based carrier was shown to accelerate its internalization in Arabidopsis cells compared to uptake of the free dsRNA. These results confirm the potential of dsRNAs to cross the plant cell wall of particular cells and the beneficial effect of carriers to improve dsRNA delivery in cells.}},
  author       = {{De Schutter, Kristof and Verbeke, Isabel and Kontogiannatos, Dimitrios and Dubruel, Peter and Swevers, Luc and Van Damme, Els and Smagghe, Guy}},
  issn         = {{1054-5476}},
  journal      = {{IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY-PLANT}},
  keywords     = {{PENETRATING PEPTIDES,VIRUS-RESISTANCE,GENE,RNA,EXPRESSION,INTERFERENCE,DELIVERY,WALLS,RNAi,Arabidopsis cells,BY-2,dsRNA delivery,Peptide,dsRNA carrier,Cellular uptake}},
  language     = {{eng}},
  number       = {{4}},
  pages        = {{511--520}},
  title        = {{Use of cell cultures in vitro to assess the uptake of long dsRNA in plant cells}},
  url          = {{http://doi.org/10.1007/s11627-022-10260-1}},
  volume       = {{58}},
  year         = {{2022}},
}

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