Methods for the Cost-Effective Production of Bacteria-Derived Double-Stranded RNA for in vitro Knockdown Studies

Verdonckt, Thomas-Wolf; Broeck, Jozef Vanden

doi:10.3389/fphys.2022.836106

Cited by 17 publications

(29 citation statements)

References 48 publications

(69 reference statements)

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“…Moreover, the application of living dsRNA-producing bacteria for gene silencing has been used in nematodes [ 160 ], insects [ 225 ], crustaceans [ 226 ], mammalian cells [ 227 ], and filamentous fungi [ 215 ]. However, in order to avoid bacterial release into the environment, it is necessary to purify the RNA, which can make the process more expensive and limit the amount of dsRNAs produced [ 26 , 228 , 229 , 230 , 231 , 232 ].…”

Section: Rna Interference (Rnai): the Targeted Management For Plant D...mentioning

confidence: 99%

New Insights on the Integrated Management of Plant Diseases by RNA Strategies: Mycoviruses and RNA Interference

Bocos-Asenjo

Niño‐Sánchez

Ginésy

2022

IJMS

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RNA-based strategies for plant disease management offer an attractive alternative to agrochemicals that negatively impact human and ecosystem health and lead to pathogen resistance. There has been recent interest in using mycoviruses for fungal disease control after it was discovered that some cause hypovirulence in fungal pathogens, which refers to a decline in the ability of a pathogen to cause disease. Cryphonectria parasitica, the causal agent of chestnut blight, has set an ideal model of management through the release of hypovirulent strains. However, mycovirus-based management of plant diseases is still restricted by limited approaches to search for viruses causing hypovirulence and the lack of protocols allowing effective and systemic virus infection in pathogens. RNA interference (RNAi), the eukaryotic cell system that recognizes RNA sequences and specifically degrades them, represents a promising. RNA-based disease management method. The natural occurrence of cross-kingdom RNAi provides a basis for host-induced gene silencing, while the ability of most pathogens to uptake exogenous small RNAs enables the use of spray-induced gene silencing techniques. This review describes the mechanisms behind and the potential of two RNA-based strategies, mycoviruses and RNAi, for plant disease management. Successful applications are discussed, as well as the research gaps and limitations that remain to be addressed.

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Section: Rna Interference (Rnai): the Targeted Management For Plant D...mentioning

confidence: 99%

New Insights on the Integrated Management of Plant Diseases by RNA Strategies: Mycoviruses and RNA Interference

Bocos-Asenjo

Niño‐Sánchez

Ginésy

2022

IJMS

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“…Production of a large amount of dsRNA is required for silencing the expression of a gene in vivo. The bacterial system is the most common between in vivo dsRNA expressing alternatives [136]. This production involves the efficient release of the dsRNA from the cells without affecting RNA integrity during the extraction process [136].…”

Section: Dsrna Production Costsmentioning

confidence: 99%

“…The bacterial system is the most common between in vivo dsRNA expressing alternatives [136]. This production involves the efficient release of the dsRNA from the cells without affecting RNA integrity during the extraction process [136]. In experimental settings three main approaches are used to produce dsRNA: chemical synthesis of NTPs, in vitro synthesis through RNA-dependent RNA polymerases and fermentation through microorganisms.…”

Section: Dsrna Production Costsmentioning

confidence: 99%

“…The delivery of dsRNA using bacteria has many advantages in comparison with plant-mediated dsRNA delivery, other microorganisms, or in vitro synthesized dsRNA [35]. E. coli HT115 (DE3) were first used as a heterologous dsRNA production system by [147] and become since then a useful tool for functional studies in invertebrate physiology [131,136,148,149]. Still, other kind of bacteria, like Bacillus thuringiensis (Bt) [134,135,150], B. subtilis [151,152], symbiotic bacteria [153][154][155][156][157], Pseudomonas syringae, Corynebacterium glutamicum, and Chlamydomonas reinhardtii are effective for biopesticide production strains that have been widely used for control of several pests.…”

Section: Production Of Dsrna In Recombinant Microorganismsmentioning

confidence: 99%

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Current Scenario of Exogenously Induced RNAi for Lepidopteran Agricultural Pest Control: From dsRNA Design to Topical Application

Lucena-Leandro¹,

Abreu²,

Vidal³

et al. 2022

Preprint

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Global crop yields are estimated to be reduced by 30–40% per year on account of plant pests and pathogens. Agricultural insect pests raise concerns about constraining global food security and climate changes contributing to the rise of infestation. The current management relies on plant breeding, associated or not with transgenes and chemical pesticides. Both approaches face serious technology obsolescence on the field due to resistance breakdown or development of insecticide resistance. The need for new Modes of Action (MoA) approaches in managing crop health grows each year, driven by market demands to reduce economic losses and phytosanitary requirements to meet the consumer perception. Disabling pest genes by sequence-specific expression silencing is considered a promising tool in the development of environment and health respectful biopesticides. The specificity conferred by long dsRNA-base solutions give support to minimizing effects on off-targeted genes in the insect pest genome and the target gene in non-target organisms (NTOs). In this review, we summarize the current status of gene silencing by RNA interference (RNAi) for agricultural control. More specifically, we focus on the engineering, development and application of gene silencing to control Lepidoptera by the employment of non-transforming dsRNA technologies. Despite some delivery and stability drawbacks of topical applications, we reviewed works showing convincing proof-of-concept results that point to imminent innovative solutions. Considerations about the regulamentation of the ongoing research on dsRNA-based pesticides to produce commercialized products for exogenous application are discussed. Academic and industry initiatives reveal a worthy effort to accomplish controlling Lepidoptera pests with this new mode of action to provide more sustainable and reliable technologies to field management. New data on genomics of this taxon encourage the increment of a customized target genes portfolio. As a case of study, we illustrate how dsRNA and associated methodologies could be applied to control an important Lepidopteran coffee pest.

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“…(Goodfellow et al The application of dsRNA insecticides via direct spraying of the microbial fermentation material (expressing the dsRNA) has potential drawbacks related to crop spraying itself as well as challenges and costs of the formulation. Although this effectively triggers knockdown of some insects' target genes including, hemocoel of orthopteran insects (Verdonckt & Vanden Broeck, 2022), it is less effective in insects such as Helicoverpa pests due to immune responses to the presence of bacteria in the hemolymph (Li et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

A novel sustainable platform for scaled manufacturing of double-stranded RNA biopesticides

Nwokeoji

Chou³

et al. 2022

Preprint

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RNA interference (RNAi) represents one of the most conserved pathways evolved by eukaryotic cells for regulating gene expression. RNAi utilises non-translatable double-stranded RNA (dsRNA) molecules to sequester or degrade mRNA molecules gene. In RNAi, specifically-designed exogenous dsRNA delivered to the cell can silence a target gene, a phenomenon that has been exploited in many functional studies and explored in biopesticide applications. The search for safe and sustainable crop pest management options drives the need to offset the effect of inorganic pesticides on biodiversity. The prospect of replacing inorganic pesticides with dsRNA crop spray is gaining popularity, enhanced by its high-target specificity and low-environmental impact. However, for dsRNA to reach the pesticide market, it must be produced cost-effectively and sustainably. In this paper, we develop a high-yield expression media that generates up to 15-fold dsRNA yield compared to existing expression media utilising 1mM IPTG. We also optimise a low-cost purification method that generates high-quality and purified dsRNA. The developed method circumvents the need for hazardous chemical reagents often found in commercial kits or commercial nucleases to eliminate contaminating DNA or single-stranded RNA (ssRNA) species. We also demonstrate that the production platform is scalable, generating 6.29 mg dsRNA from 259 mg wet E. coli cell pellet. The results also provide structural insights into the heterogeneous dsRNA species within the microbial-derived dsRNA pool. Finally, we also show that the purified 'naked' dsRNA, without prior formulation, can induce insect toxicity under field conditions. This study provides a novel, complete, low-cost process dsRNA platform with potential for application in industrial dsRNA production.

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Methods for the Cost-Effective Production of Bacteria-Derived Double-Stranded RNA for in vitro Knockdown Studies

Cited by 17 publications

References 48 publications

New Insights on the Integrated Management of Plant Diseases by RNA Strategies: Mycoviruses and RNA Interference

New Insights on the Integrated Management of Plant Diseases by RNA Strategies: Mycoviruses and RNA Interference

Current Scenario of Exogenously Induced RNAi for Lepidopteran Agricultural Pest Control: From dsRNA Design to Topical Application

A novel sustainable platform for scaled manufacturing of double-stranded RNA biopesticides

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