Intron-assisted, viroid-based production of insecticidal circular double-stranded RNA in <i>Escherichia coli</i>

Ortolá, Beltrán; Cordero, Teresa; Daròs, José‐Antonio

doi:10.1080/15476286.2021.1872962

Cited by 16 publications

(21 citation statements)

References 52 publications

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“…In conclusion, here we have presented the use of minimal circular replicating RNAs able to systemic spread combined with the production of a tailored sRNA for targeted silencing. This is a conceptual novelty that further expands the biotechnological applications of viroids in general, and ELVd in particular (41, 53).…”

Section: Discussionmentioning

confidence: 98%

Minimal RNA replicons for targeted gene silencing based on an asymptomatic viroid

Marquez‐Molins

Urrutia-Perez

Hernandez-Azurdia

et al. 2022

Preprint

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Gene silencing for functional studies in plants has been largely facilitated by manipulating viral genomes with inserts from host genes to trigger virus induced gene silencing (VIGS) against the corresponding mRNAs. However, viral genomes encode multiple proteins and disrupt plant homeostasis by interfering with endogenous cell mechanisms. To circumvent this issue, we have developed a silencing method based on the minimal autonomously-infectious nucleic acids currently known: viroids. In particular, Eggplant latent viroid (ELVd), an asymptomatic viroid, was manipulated with insertions between 21 to 42 nucleotides and our results show that larger insertions are tolerated but secondary structure is critical for their stability. Additionally, these ELVd constructs are able of local and systemic spread and can silence a target gene in eggplant. Inspired by the design of artificial microRNAs, we have developed a standardized procedure to generate stable insertions into the ELVd genome capable of silencing the desired target gene. Analogously to VIGS, we have termed our approach Viroid Induced Gene Silencing (VdIGS) and demonstrate that is a promising tool for dissecting gene functions in eggplant. Overall, this represents the use of minimal circular replicating RNAs able to spread systemically combined with the production of a tailored sRNA for targeted silencing.

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Section: Discussionmentioning

confidence: 98%

Minimal RNA replicons for targeted gene silencing based on an asymptomatic viroid

Marquez‐Molins

Urrutia-Perez

Hernandez-Azurdia

et al. 2022

Preprint

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“…Nevertheless, we believe that a self-splicing intron, such as that used in our study, must be universally applied because it only requires a guanosine nucleoside for processing (Zaug and Cech 1986). In this way, the use of this self-splicing intron allows the correct formation of the hairpin motif-not only in plants but also in species belonging to other domains (Ortolá et al 2021). This technique can even be applied in vitro if more complex experimental strategies require it.…”

Section: Discussionmentioning

confidence: 98%

A Virus-Induced Gene-Silencing Vector Based on a Mild Isolate of Watermelon Mosaic Virus for Reverse Genetic Analyses in Cucurbits

Houhou

Aragonés

Butković

et al. 2021

Preprint

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As a response to viral infections, host plants trigger an RNA-mediated gene silencing defense, to which viruses respond with the expression of viral-encoded RNA silencing suppressors. If virus clones are manipulated to include sequences homologous to host endogenous genes, these are also targeted by the plant RNA silencing machinery. This so-called virus-induced gene silencing (VIGS) has become a powerful technique for reverse genetic analyses in plants, as an alternative to labor-intensive genome transformation. We show that a mild isolate of Watermelon mosaic virus (WMV, genus Potyvirus) can be used as a VIGS vector for reverse genetic analyses in melon. Recombinant WMV clones —in which fragments of the melon Phytoene desaturase (PDS) mRNA were inserted in sense, antisense, and hairpin modalities— induced a distinctive phenotype and significant silencing of the endogenous gene. While the foreign fragments in sense and antisense orientations were stable in the viral progeny, the hairpin was quickly lost. Nevertheless, the hairpin construct triggered a maintained silencing effect comparable to those of the sense and antisense constructs. The suitability of WMV as a VIGS vector was further confirmed targeting melon Magnesium chelatase subunit I (CHLI). These results also support that, although potyviruses express a strong silencing suppressor that usually precludes VIGS, mild isolates of this kind of viruses can be used as VIGS vectors. Finally, to facilitate the use of this new tool by cucurbit geneticists, we describe plasmid pGWMV-VIGS that allows easy cloning fragments of the genes of interest in a single Gibson assembly reaction.

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“…One approach is to produce RNAs that have a stable and compact three-dimensional structure that is resistant to ribonucleases in vivo, and to carry the target RNA segment on this scaffold. Such scaffolds for the production of recombinant RNA molecules of interest include tRNA [46][47][48], 5S rRNA [49,50], tRNA/pre-miRNA structures [51,52], and viroidderived RNA structures [53,54]. The concept of using the structures of tRNA and 5S rRNA as scaffolds is based on the rationale that these structures will be recognized as endogenous molecules by the RNA metabolizing machinery in the producing bacteria, resulting in suppression of rapid degradation of the additional RNA segment of interest.…”

Section: Escherichia Colimentioning

confidence: 99%

RNA Interference-Based Pesticides and Antiviral Agents: Microbial Overproduction Systems for Double-Stranded RNA for Applications in Agriculture and Aquaculture

Hashiro

Yasueda

2022

Applied Sciences

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RNA interference (RNAi)-based pesticides are pest control agents that use RNAi mechanisms as the basis of their action. They are regarded as environmentally friendly and are a promising alternative to conventional chemical pesticides. The effective substance in RNAi-based pesticides is double-stranded RNA (dsRNA) designed to match the nucleotide sequence of a target essential gene of the pest of concern. When taken up by the pest, this exerts an RNAi effect and inhibits some vital biochemical/biological process in the pest. dsRNA products are also expected to be applied for the control of viral diseases in aquaculture by RNAi, especially in shrimp farming. A critical issue in the practical application of RNAi agents is that production of the dsRNA must be low-cost. Here, we review recent methods for microbial production of dsRNAs using representative microorganisms (Escherichia coli, Pseudomonas syringae, Corynebacterium glutamicum, Chlamydomonas reinhardtii, and others) as host strains. The characteristics of each dsRNA production system are discussed.

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Intron-assisted, viroid-based production of insecticidal circular double-stranded RNA in Escherichia coli

Abstract: Daròs (2021): Intronassisted, viroid-based production of insecticidal circular double-stranded RNA in Escherichiacoli, RNA Biology,

Cited by 16 publications

References 52 publications

Minimal RNA replicons for targeted gene silencing based on an asymptomatic viroid

Minimal RNA replicons for targeted gene silencing based on an asymptomatic viroid

A Virus-Induced Gene-Silencing Vector Based on a Mild Isolate of Watermelon Mosaic Virus for Reverse Genetic Analyses in Cucurbits

RNA Interference-Based Pesticides and Antiviral Agents: Microbial Overproduction Systems for Double-Stranded RNA for Applications in Agriculture and Aquaculture

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