Barriers to Efficient Foliar Uptake of dsRNA and Molecular Barriers to dsRNA Activity in Plant Cells

Bennett, Michael J.; Deikman, Jill; Hendrix, Bill; Iandolino, Alberto

doi:10.3389/fpls.2020.00816

Cited by 48 publications

(62 citation statements)

References 28 publications

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“…Hence, the use of formulations and delivery carriers to prolong dsRNA persistence and improve efficacy are required. In recent reports, several companies have claimed the development of co-formulants to overcome the hurdles mentioned above [ 56 ]. To protect dsRNA in the gut, various nanoparticles and peptide-based formulations have shown the successful enhancement of RNAi efficacy [ 57 , 58 ].…”

Section: Discussionmentioning

confidence: 99%

Silencing of Double-Stranded Ribonuclease Improves Oral RNAi Efficacy in Southern Green Stinkbug Nezara viridula

Sharma

Taning

Smagghe

et al. 2021

Insects

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Variability in RNA-interference (RNAi) efficacy among different insect orders poses a big hurdle in the development of RNAi-based pest control strategies. The activity of double-stranded ribonucleases (dsRNases) in the digestive canal of insects can be one of the critical factors affecting oral RNAi efficacy. Here, the involvement of these dsRNases in the southern green stinkbug Nezaraviridula was investigated. First, the full sequence of the only dsRNase (NvdsRNase) in the transcriptome of N. viridula was obtained, followed by an oral feeding bioassay to evaluate the effect of NvdsRNase-silencing on oral RNAi efficacy. The NvdsRNase was first silenced in nymphs by NvdsRNase-dsRNA injections, followed by exposure to an artificial diet containing a lethal αCop-specific dsRNA. A significantly higher mortality was observed in the NvdsRNase-silenced nymphs when placed on the dsαCop-containing diet (65%) than in the dsGFP injected and dsαCop fed control (46.67%). Additionally, an ex vivo dsRNA degradation assay showed a higher stability of dsRNA in the saliva and midgut juice of NvdsRNase-silenced adults. These results provide evidence for the involvement of NvdsRNase in the reduction of oral RNAi efficacy in N. viridula. This information will be useful in further improving potential RNAi-based strategies to control this pest.

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Section: Discussionmentioning

confidence: 99%

Silencing of Double-Stranded Ribonuclease Improves Oral RNAi Efficacy in Southern Green Stinkbug Nezara viridula

Sharma

Taning

Smagghe

et al. 2021

Insects

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“…With further study, we learned that the systemic GFP silencing in 16C could be specifically initiated using 22nt dsRNA (7) (10) and that topical delivery of dsRNA targeting GFP in the 16C line initiated an amplification process that is characterized by production of transitive small RNAs both 5' and 3' of region targeted with dsRNA, especially when using a 22nt dsRNA (10).We adapted the topical dsRNA technology to several other dicot species targeting both endogenous genes and transgenes. However, we were unable to identify another genetic system in which we observed systemic gene silencing after topical dsRNA application (11,36). Further, the observation of abundant transitive small RNAs 5' of region targeted was not replicated in most transgenes and all endogenous gene targets that we were able to silence using a topical dsRNA technique.…”

Section: Discussionmentioning

confidence: 74%

“…We adapted the topical dsRNA technology to several other dicot species targeting both endogenous genes and transgenes. However, we were unable to identify another genetic system in which we observed systemic gene silencing after topical dsRNA application(11, 36). Further, the observation of abundant transitive small RNAs 5’ of region targeted was not replicated in most transgenes and all endogenous gene targets that we were able to silence using a topical dsRNA technique.…”

Section: Discussionmentioning

confidence: 90%

High transgene expression is associated with systemic GFP silencing in Nicotiana benthamiana

Hendrix

Hoffer

Sanders

et al. 2021

Preprint

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Gene silencing in plants using topical dsRNA is a new approach that has the potential to be a sustainable component of the agricultural production systems of the future. However, more research is needed to enable this technology as an economical and efficacious supplement to current crop protection practices. Systemic gene silencing is one key enabling aspect. The objective of this research was to better understand systemic transgene silencing in Nicotiana benthamiana. Previous reports details sequencing of the integration site of the Green Fluorescent Protein (GFP) transgene in the well-known N. benthamiana GFP16C event revealed inadvertent co-integration of part of a bacterial transposase. To determine the effect of this transgene configuration on systemic silencing, new GFP transgenic lines with or without the transposase sequences were produced. GFP expression levels in the 19 single-copy events and three hemizygous 16C lines produced for this study ranged from 50-72% of the homozygous 16C line. GFP expression was equivalent to 16C in a two-copy event. Local GFP silencing was observed in all transgenic and 16C hemizygous lines after topical application of delivery formulations with a GFP targeting dsRNA. The 16C-like systemic silencing phenotype was only observed in the two-copy line. The partial transposase had no impact on transgene expression level, local GFP silencing, small RNA abundance and distribution, or systemic GFP silencing in the transgenic lines. We conclude that high transgene expression level is a key enabler of systemic transgene silencing in N. benthamiana.

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“…Some authors have also reported the possibility to use SIGS to target endogenous genes in plants and downregulate their mRNA levels both locally and systemically [ 116 , 127 , 129 ]. To our knowledge, the data present in the literature are limited to the foliar application of dsRNA molecules to silence transgenes expressed in model plant species, like Arabidopsis and N. benthamiana ; however, these results open new scenarios for the use of SIGS also to target endogenous gene sequences, like susceptibility genes in grapevine and other crops, to enhance plant defense responses.…”

Section: Rnai: Host- or Spray-induced Gene Silencing Against Fungimentioning

confidence: 99%

Biotechnological Approaches: Gene Overexpression, Gene Silencing, and Genome Editing to Control Fungal and Oomycete Diseases in Grapevine

Capriotti

Baraldi

Mezzetti

et al. 2020

IJMS

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Downy mildew, powdery mildew, and grey mold are some of the phytopathological diseases causing economic losses in agricultural crops, including grapevine, worldwide. In the current scenario of increasing global warming, in which the massive use of agrochemicals should be limited, the management of fungal disease has become a challenge. The knowledge acquired on candidate resistant (R) genes having an active role in plant defense mechanisms has allowed numerous breeding programs to integrate these traits into selected cultivars, even though with some limits in the conservation of the proper qualitative characteristics of the original clones. Given their gene-specific mode of action, biotechnological techniques come to the aid of breeders, allowing them to generate simple and fast modifications in the host, without introducing other undesired genes. The availability of efficient gene transfer procedures in grapevine genotypes provide valid tools that support the application of new breeding techniques (NBTs). The expertise built up over the years has allowed the optimization of these techniques to overexpress genes that directly or indirectly limit fungal and oomycetes pathogens growth or silence plant susceptibility genes. Furthermore, the downregulation of pathogen genes which act as virulence effectors by exploiting the RNA interference mechanism, represents another biotechnological tool that increases plant defense. In this review, we summarize the most recent biotechnological strategies optimized and applied on Vitis species, aimed at reducing their susceptibility to the most harmful fungal and oomycetes diseases. The best strategy for combating pathogenic organisms is to exploit a holistic approach that fully integrates all these available tools.

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Barriers to Efficient Foliar Uptake of dsRNA and Molecular Barriers to dsRNA Activity in Plant Cells

Cited by 48 publications

References 28 publications

Silencing of Double-Stranded Ribonuclease Improves Oral RNAi Efficacy in Southern Green Stinkbug Nezara viridula

Silencing of Double-Stranded Ribonuclease Improves Oral RNAi Efficacy in Southern Green Stinkbug Nezara viridula

High transgene expression is associated with systemic GFP silencing in Nicotiana benthamiana

Biotechnological Approaches: Gene Overexpression, Gene Silencing, and Genome Editing to Control Fungal and Oomycete Diseases in Grapevine

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