Carbon dots for efficient siRNA delivery and gene silencing in plants

Abstract

The Initiation of RNA interference (RNAi) by topically applied double stranded RNA (dsRNA) has potential applications for plant functional genomics, crop improvement and crop protection. The primary obstacle for the development of this technology is efficient delivery of RNAi effectors. The plant cell wall is a particularly challenging barrier to the delivery of macromolecules. Many of the transfection agents that are commonly used with animal cells produce nanocomplexes that are significantly larger than the size exclusion limit of the plant cell wall. Utilizing a class of very small nanoparticles called carbon dots, a method of delivering siRNA into the model plant Nicotiana benthamiana and tomato is described. Low-pressure spray application of these formulations with a spreading surfactant resulted in strong silencing of GFP transgenes in both species. The delivery efficacy of carbon dot formulations was also demonstrated by silencing endogenous genes that encode two sub-units of magnesium chelatase, an enzyme necessary for chlorophyll synthesis. The strong visible phenotypes observed with the carbon dot facilitated delivery were validated by measuring significant reductions in the target gene transcript and/or protein levels. Methods for the delivery of RNAi effectors into plants, such as the carbon dot formulations described here, could become valuable tools for gene silencing in plants with practical applications in plant functional genomics and agriculture.

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

“…siRNA applied with a nuclease inhibitor or polybrene which binds dsRNA was still detectable at 24 h. Both sets of experimental results strongly suggest that nucleases act as a barrier to efficient dsRNA delivery. This assertion is further supported by recent publications demonstrating that agents such as carbon dots, single-walled carbon nanotubes, and clay nanosheets enhance nuclease stability and delivery efficiency of applied nucleic acids in plants (Mitter et al, 2017;Demirer et al, 2019;Schwartz et al, 2019).…”

“…The applied siRNA was extracted from the samples and analyzed by anionic exchange HPLC. Results indicate that siRNA applied without a nuclease inhibitor or polybrene was completely degraded by 6 h. siRNA applied with nuclease inhibitors or polybrene was still detectable at 24 h. efficiency in plant cells (Bennett et al, 2017;Demirer et al, 2019;Schwartz et al, 2019).…”

“…Systemic Silencing Using Topical dsRNA Has Only Been Documented for the GFP Transgene in N. benthamiana Systemic RNAi has been observed for the GFP transgene in N. benthamiana line 16C (Ruiz et al, 1998) using topical RNA with dsRNAs that are 124 bp or 22 bp, but not 21 bp (Dalakouras et al, 2016;Schwartz et al, 2019;Hendrix et al, 2020; unpublished Bayer Crop Science research). The ability of 22 bp siRNAs to cause systemic silencing is likely related to their ability to induce production of secondary sRNAs (Chen et al, 2010;Cuperus et al, 2010;Dalakouras et al, 2020;Hendrix et al, 2020).…”

High-Pressure-Sprayed Double Stranded RNA Does Not Induce RNA Interference of a Reporter Gene

“…Different classes of adjuvants, including cationic nanoparticles, surfactants, clay nanosheets, peptide-based agents, and carbon dots have been used to boast plant defense against pests by facilitating the delivery of exogenous dsRNAs through the cell wall and subsequently cell membrane (Jiang et al, 2014;Mitter et al, 2017a;Schwartz et al, 2019;Worrall et al, 2019;Zheng et al, 2019). Indeed, it has been shown that these adjuvants improved plant defense against virus infection by increasing the uptake of dsRNA into plant cells and by protecting the dsRNAs from early degradation (Unnamalai et al, 2004;Mitter et al, 2017b).…”

RNA Interference Strategies for Future Management of Plant Pathogenic Fungi: Prospects and Challenges

“…Interestingly, this formulation also seems to facilitate uptake and systemic dissemination within the sprayed host plant [67]. The use of a class of very small nanoparticles, called carbon dots, for the delivery of siRNA to the Nicotiana benthamiana and tomato plants, has also been reported [87]. In addition, liposome-based delivery method has been applied in insects, fungi, and nematodes [88,89,90] with success in altering gene expression and/or mortality.…”