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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.

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Acknowledgements

The PSB-D and BY-2 cells were obtained from the Plant Systems Biology department of the VIB-UGent (Belgium). The pPTD-DRBD vector was a gift from Steven Dowdy (Addgene plasmid # 35622).

Funding

This research was funded by the Research Foundation-Flanders (Belgium) and the Special Research Fund of Ghent University (Belgium).

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Authors and Affiliations

  1. Laboratory of Agrozoology, Department of Plants and Crops, Faculty of Bioscience Engineering, Ghent University, 9000, Ghent, Belgium

    Kristof De Schutter & Guy Smagghe

  2. Department of Biotechnology, Faculty of Bioscience Engineering, Ghent University, 9000, Ghent, Belgium

    Isabel Verbeke & Els J. M. Van Damme

  3. Insect Molecular Genetics and Biotechnology, Institute of Biosciences & Applications, NCSR “Demokritos”, Aghia Paraskevi, Athens, Greece

    Dimitriοs Kontogiannatos & Luc Swevers

  4. Polymer Chemistry and Biomaterials Group, Department of Organic and Macromolecular Chemistry, Center of Macromolecular Chemistry (CMAC), Faculty of Sciences, Ghent University, Ghent, Belgium

    Peter Dubruel

  5. Center for Advanced Light Microscopy, Ghent University, Ghent, Belgium

    Els J. M. Van Damme

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  1. Kristof De Schutter
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  2. Isabel Verbeke
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  3. Dimitriοs Kontogiannatos
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  4. Peter Dubruel
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  5. Luc Swevers
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  6. Els J. M. Van Damme
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  7. Guy Smagghe
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Contributions

KDS, DK, LS, EJMVD, and GS conceived and designed research. KDS and IV conducted experiments. KDS analyzed data. KDS, EJMVD, and GS wrote and revised the manuscript. All authors have read and approved the final manuscript.

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Correspondence to Guy Smagghe.

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De Schutter, K., Verbeke, I., Kontogiannatos, D. et al. Use of cell cultures in vitro to assess the uptake of long dsRNA in plant cells. In Vitro Cell.Dev.Biol.-Plant 58, 511–520 (2022). https://doi.org/10.1007/s11627-022-10260-1

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