Carbon Dots for Efficient Small Interfering RNA Delivery and Gene Silencing in Plants

Schwartz, Steven H.; Hendrix, Bill; Hoffer, Paul; Sanders, Rick; Zheng, Wei

doi:10.1104/pp.20.00733

Cited by 138 publications

(127 citation statements)

References 50 publications

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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: 73%

“…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: 88%

“…Short interfering RNAs 22nt in length were chemically synthesized and used to target the GFP and magnesium chelatase subunit H ( CHL-H ) genes in the 16C line (sequences, Fig 2S). Carbon dot formulations were used to topically deliver these dsRNAs or a scrambled control sequence to 16C seedlings (11). Application leaves from the plants were removed and photographed 6 days after dsRNA application (Fig 2).…”

Section: Resultsmentioning

confidence: 99%

“…Topical dsRNA delivery was performed using carbon dots produced in-house as described (11). Briefly, the chemically synthesized dsRNAs were complexed to carbon dots overnight at room temperature in a solution containing 40 mM glycerol, 10 mM MES pH 5.7 and 12 μg/ml dsRNA.…”

Section: Methodsmentioning

confidence: 99%

“…The RISC functions as a specific endonuclease that cleaves target transcripts identified by base pairing chemistry. Recently, methods have been developed to silence plant genes using topically delivered dsRNAs (7)(8)(9)(10)(11). The most efficacious versions of these methods deliver 21-24nt dsRNAs that initiate silencing without initial dicer processing to produce efficacious gene silencing effectors.…”

Section: Introductionmentioning

confidence: 99%

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High transgene expression is associated with systemicGFPsilencing inNicotiana 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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Section: Discussionmentioning

confidence: 73%

Section: Discussionmentioning

confidence: 88%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

High transgene expression is associated with systemicGFPsilencing inNicotiana benthamiana

Hendrix

Hoffer

Sanders

et al. 2021

Preprint

Self Cite

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Interactions of Nanoparticles with Plants

Roy

2023

Bioinspired and Green Synthesis of Nanostructures

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Functionalized Carbon Dot‐Delivered RNA Nano Fungicides as Superior Tools to Control Phytophthora Pathogens through Plant RdRP1 Mediated Spray‐Induced Gene Silencing

Wang

Zhang

et al. 2023

Adv Funct Materials

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Spray-induced gene silencing (SIGS) represents an attractive avenue for plant protection, but limited uptake efficiency of double-stranded RNA (dsRNA) has restricted its application against the notorious oomycetes, Phytophthora. To control Phytophthora, dsRNAs targeting two essential genes in Phytophthora are screened for their ability to partially decrease Phytophthora capsici (P. capsici) infection and fecundity. To further facilitate SIGS for Phytophthora control, functionalized carbon dots (CDs) are complexed with the screened dsRNAs (dsRNA-CDs) through electrostatic interaction. dsRNA-CDs significantly enhanced the control efficacy of dsRNA against Phytophthora infestans, Phytophthora sojae, and both the wild-type and fungicide-resistant P. capsici. The synergism is based on enhancing dsRNA stability and internalization. Dual treatment with dsRNA-CDs and the corresponding fungicide reduced the amount of fungicide required to achieve the same protective effect by 90%. Plant RdRP1 is the main effector in processing various lengths of small RNAs to induce translation inhibition in Phytophthora. Notably, here the first application of a nano-delivery system to improve the effect of SIGS against Phytophthora pathogens is reported. Moreover, the elucidation of how CD facilitates dsRNA internalization in recipient cell and the molecular mechanism of SIGS can benefit the application of dsRNA-CDs in other plant-pathogen pathosystems through improving dsRNA bioactivity and reducing chemical fungicides application.

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Carbon Dots for Efficient Small Interfering RNA Delivery and Gene Silencing in Plants

Abstract: The development of a formulation for siRNA delivery enables posttranscriptional gene silencing in plants with a simple spray application.

Cited by 138 publications

References 50 publications

High transgene expression is associated with systemicGFPsilencing inNicotiana benthamiana

High transgene expression is associated with systemicGFPsilencing inNicotiana benthamiana

Interactions of Nanoparticles with Plants

Functionalized Carbon Dot‐Delivered RNA Nano Fungicides as Superior Tools to Control Phytophthora Pathogens through Plant RdRP1 Mediated Spray‐Induced Gene Silencing

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