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

Schutter, Kristof De; Verbeke, Isabel; Kontogiannatos, Dimitriοs; Dubruel, Peter; Swevers, Luc; Damme, Els J. M. Van; Smagghe, Guy

doi:10.1007/s11627-022-10260-1

Cited by 4 publications

(3 citation statements)

References 49 publications

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“…In the current study, we leveraged the electrostatic bonding between the negatively charged phosphate groups of dsRNA and the positively charged lysine and histidine residues (at pI 9.74 and 7.59, respectively) in the KH tail added to the VgP to form dsRNA-VgP nanoparticles that could be used as a delivery tool. Upon mixing of the KH-tailed peptide with dsRNA, retardation of dsRNA in the agarose gel was observed (Figures 4A, B), with the retardation being positively correlated with increasing amounts of the interacting KH-peptide; this retardation could be attributed to the formation of larger nanoparticles, in line with previously published studies (Unnamalai et al, 2004;Mo et al, 2012;De Schutter et al, 2022).…”

Section: Discussionsupporting

confidence: 89%

A crustacean vitellogenin-derived peptide as an oocyte-specific delivery vehicle for gene silencing

et al. 2023

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Gene silencing by dsRNA is well documented in crustaceans, but RNA interference (RNAi) in developing oocytes is yet to be achieved. The main obstacle to RNAi in the oocytes of oviparous animals derives from their protective layers, including the cytosolic membrane, the vitelline envelope, and a layer of follicular cells. These layers form a barrier preventing the entry of large nonspecific molecules, such as double-stranded RNA (dsRNA). This article describes a sophisticated tool – designated OSDel [oocyte-specific delivery] – for the delivery of dsRNA for gene silencing in the developing oocyte. The methodology exploits the process of receptor-mediated endocytosis (RME) taking place in the crustacean female for internalizing vitellogenin (Vg) (the precursor of the major yolk protein, vitellin) into oocytes. In this process, the extracellular domain of the Vg receptor (VgR) interacts with a distinct amino acid sequence of Vg and internalizes it to form yolk droplets. Here, we tested the premise that a distinct 24 amino-acid peptide derived from Macrobrachium rosenbergii Vg (designated VgP) would interact with VgR to form particles of a size suitable for piggy-backing dsRNA into oocytes via RME. We found that fluorescently labeled VgP had a micromolar affinity for the ligand-binding domain (LBD) of the VgR and could indeed be delivered to and internalized in oocytes. As model system to illustrate the applicability of the OSDel, we injected vitellogenic females with dsRNA encoding the eye development gene PAX6 piggybacked on VgP. Proof that the dsRNA had been successfully internalized into the developing oocytes and had silenced the PAX6 gene was derived from impaired eye development in 87% of the embryos. The ability to manipulate embryos by simple injection into vitellogenic crustacean females may prove to be a powerful high throughput tool for functional genomics investigation in crustacean embryos and for silencing genes relevant to crustacean aquaculture and biotechnology.

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Section: Discussionsupporting

confidence: 89%

A crustacean vitellogenin-derived peptide as an oocyte-specific delivery vehicle for gene silencing

et al. 2023

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“…[13] NPs have also emerged as promising carriers for dsRNA, as they improve its stability and enhance RNAi efficiency. [14][15][16][17][18][19][20][21][22][23][24] For example, cationic lipid NPs have been applied to deliver dsRNA and protect plants against fungal diseases, thereby increasing dsRNA stability. [21] RNA NPs, on the other hand, can be constructed using structural motifs from bottom up, and they can selfassemble into specific structures with strong stability.…”

Section: Introductionmentioning

confidence: 99%

Development and application of an RNA nanostructure to induce transient RNAi in difficult transgenic plants

Zhao,

Liu,

Liu

et al. 2024

Biotechnology Journal

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The development of RNA interference (RNAi) is crucial for studying plant gene function. Its use, is limited to a few plants with well‐established transgenic techniques. Spray‐induced gene silencing (SIGS) introduces exogenous double‐stranded RNA (dsRNA) into plants by spraying, injection, or irrigation, triggering the RNAi pathway to instantly silence target genes. As is a transient RNAi technology that does not rely on transgenic methods, SIGS has significant potential for studying gene function in plants lacking advanced transgenic technology. In this study, to enhance their stability and delivery efficiency, siRNAs were used as structural motifs to construct RNA nanoparticles (NPs) of four shapes: triangle, square, pentagon, and hexagon. These NPs, when synthesized by Escherichia coli, showed that triangular and square shapes accumulated more efficiently than pentagon and hexagon shapes. Bioassays revealed that RNA squares had the highest RNAi efficiency, followed by RNA triangles, with GFP‐dsRNA showing the lowest efficiency at 4 and 7 days post‐spray. We further explored the use of RNA squares in inducing transient RNAi in plants that are difficult to transform genetically. The results indicated that Panax notoginseng‐derived MYB2 (PnMYB2) and Camellia oleifera‐derived GUT (CoGUT) were significantly suppressed in P. notoginseng and C. oleifera, respectively, following the application of PnMYB2‐ and CoGUT‐specific RNA squares. These findings suggest that RNA squares are highly effective in SIGS and can be utilized for gene function research in plants.

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“…A aplicação foliar de biopesticidas à base de dsRNA pode ser uma estratégia viável para controlar pragas com hábitos minadores, sugadores ou insetos de solo que atacam as raízes , uma vez que evidências indicam a capacidade de penetração foliar e distribuição do dsRNA pelas diferentes partes das plantas, com resultados maiores na presença adjuvantes De SCHUTTER et al, 2022;. Os mecanismos de penetração e transporte de dsRNA nas plantas ainda não estão totalmente esclarecidos, no entanto, acredita-se que o dsRNA seja preferencialmente absorvido pelas folhas via estômatos e inicialmente difundido via apoplasto (HOANG et al, 2022).…”

Section: Controle De Pragas Por Rnaiunclassified

Aplicação de dsRNA sintético ou associado a nanocarreadores de zeína para controle de Tuta absoluta (Meyrick, 1917) por RNAi

Rodrigues

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

Cited by 4 publications

References 49 publications

A crustacean vitellogenin-derived peptide as an oocyte-specific delivery vehicle for gene silencing

A crustacean vitellogenin-derived peptide as an oocyte-specific delivery vehicle for gene silencing

Development and application of an RNA nanostructure to induce transient RNAi in difficult transgenic plants

Aplicação de dsRNA sintético ou associado a nanocarreadores de zeína para controle de Tuta absoluta (Meyrick, 1917) por RNAi

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