Breakthrough Technologies

Carbon Dots for Efficient Small Interfering RNA Deliveryand Gene Silencing in Plants[OPEN]

Steven H. Schwartz,1,2,3 Bill Hendrix,4 Paul Hoffer,5 Rick A. Sanders, and Wei Zheng6

Bayer Crop Science, Woodland, California 95695

ORCID IDs: 0000-0002-5982-8553 (S.H.S.); 0000-0001-8799-1254 (B.H.); 0000-0003-3763-8510 (P.H.); 0000-0002-1402-1764 (R.A.S.);0000-0002-6881-3572 (W.Z).

The initiation of RNA interference (RNAi) by topically applied small interfering RNA has potential applications for plantfunctional genomics, crop improvement and crop protection, but the primary obstacle for the development of this technology isthe efficient delivery of RNAi effectors into the cell. The plant cell wall is a particularly challenging barrier for the delivery ofmacromolecules because many of the transfection agents that are commonly used with animal cells produce nanocomplexes thatare significantly larger than the size exclusion limit of the cell wall. Here, we illustrate the use of a class of very smallnanoparticles, called carbon dots, for delivering small interfering RNA into the model plants Nicotiana benthamiana and tomato(Solanum lycopersicum). Low-pressure spray application of these formulations with a spreading surfactant resulted in strongsilencing of GFP transgenes in both species. The delivery efficacy of carbon dot formulations was also demonstrated by thesilencing of endogenous genes that encode two subunits of magnesium chelatase, an enzyme necessary for chlorophyll synthesis.The strong visible phenotypes observed with the carbon dot-facilitated delivery were validated by measuring significantreductions in the target gene transcript and/or protein levels. Methods for the delivery of RNAi effectors into plants, such asthe carbon dot formulations described here, could become valuable tools for gene silencing in plants with practical applicationsin plant functional genomics and agriculture.

RNA interference (RNAi) comprises interrelated path-ways that mediate transcriptional gene silencing bymethylation of genomic sequences, posttranscriptionalgene silencing by cleavage of targeted RNA sequences,or translational repression by binding to targetedtranscripts (Matzke and Matzke, 2004; Frizzi andHuang, 2010). In plants, these pathways have a rolein resistance to pathogens (Rosa et al., 2018) and arealso required for normal development (Liu et al.,2017). The use of RNAi to silence specific genes hasbecome a valuable tool for plant functional genomics

(Eamens et al., 2008; McGinnis, 2010; Kumar and Salar,2017). Several agricultural applications of RNAi havebeen developed, including improvements in the nutri-tional composition of crops (Huang et al., 2004; Mroczkaet al., 2010; Chawla et al., 2012) and resistance to variousplant pathogens that can significantly reduce crop yieldand quality (Rosa et al., 2018). RNAi technology has alsobeen used to provide resistance to insect pests (Baumet al., 2007).The first uses of gene silencing in plant biology in-

volved stable transformation with antisense or cosup-pression constructs. With a better understanding of theRNAi pathway, more efficient methods were devel-oped that utilize double-stranded RNA (dsRNA) hair-pins (Smith et al., 2000) and artificial microRNAs(Schwab et al., 2006). Another significant advancement inRNAi technology has been the development of methodsfor transient gene silencing, such as virus-inducedgene silencing (VIGS; Burch-Smith et al., 2004; Watsonet al., 2005; Becker and Lange, 2010) andAgrobacteriumtumefaciens-mediated infiltration (Johansen andCarrington,2001). These transient silencing systems allow for the rapidtesting of gene function. However, both VIGS and A.tumefaciens-mediated infiltration require preparation ofconstructs for the expression of the RNAi effector andcontainment of the pathogen-infected plants. The efficientdelivery of topically applied RNAi effectors would be an-other valuable tool for plant functional genomics and mayhave some practical applications in agriculture.Because of the potential for therapeutic applications,

the delivery of RNAi effectors has been extensivelystudied in animal systems. Many transfection agents

1Author for contact: steven.h.schwartz@outlook.com.2Senior author.3Present address: InnerPlant, 1450 Drew Circle, Suite 150, Davis,

CA 95618.4Present address: Bayer Crop Science, 890 Embarcadero Drive,

West Sacramento, CA 95605.5Present address: California Governor’s Office of Emergency Ser-

vices, Radiological Preparedness Unit, 3650 Schriever Avenue,Mather, CA 95655.

6Present address: Hangzhou Huadi Group Co., Ltd., Hangzhou310012, China.

The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Steven H. Schwartz (steven.h.schwartz@outlook.com).

S.H.S., B.H., and W.Z. conceived of the experiments; B.H., P.H.,R.A.S., S.H.S., and W.Z. performed the experiments; S.H.S. wrote theoriginal manuscript; and all authors read the manuscript and assistedin the editing.

[OPEN]Articles can be viewed without a subscription.www.plantphysiol.org/cgi/doi/10.1104/pp.20.00733

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that enhance delivery into animal cells have been de-scribed (Kozielski et al., 2013). Common classes of thesetransfection agents include lipid nanoparticles, cationicpolymers, cell-penetrating peptides, and inorganicnanoparticles. The nanocomplexes that are formed bythe interaction of these transfection agents with nucleicacids provide some protection from nucleases and fa-cilitate cellular uptake by endocytosis or membranefusion. There have been reports describing the useof transfection agents for delivery into plant cells(Unnamalai et al., 2004; Cheon et al., 2009; Eggenbergeret al., 2011; Lakshmanan et al., 2013; Numata et al.,2014; Ziemienowicz et al., 2015; Cheon et al., 2017;Kimura et al., 2017; Miyamoto et al., 2019). The effi-ciency of many transfection agents, however, may belimited by delivery barriers that are unique to plants(Fig. 1). The plant cell wall is a particularly challengingbarrier for the delivery of RNA or other macromole-cules. The dense polysaccharide matrix of the cell wallhas a size exclusion limit that is between 3 and 10 nmin diameter (Carpita et al., 1979; Baron-Epel et al.,1988; Carpita and Gibeaut, 1993). Many of the nano-complexes used to transfect nucleic acids have a size inthe range of 100 to 200 nm, which is 10- to 20-fold largerthan the size exclusion of the plant cell wall. There havebeen several reports describing the use of smallernanostructures, such as single-walled carbon nano-tubes (Golestanipour et al., 2018; Demirer et al., 2019;Kwak et al., 2019) and DNA nanoparticles (Zhang et al.,2019), for the delivery of nucleic acids into plant cells.

Delivery of small interfering RNA (siRNA) and tran-sient silencing of a GFP transgene have been achievedby infiltration of formulations into leaf tissues with aneedleless syringe (Zhang et al., 2019). The use of claynanosheets to stabilize longer dsRNA and provideprotection from virus infection in plants has also beenreported (Mitter et al., 2017).

There are additional classes of nanomaterials thatmay be well suited for plant delivery. Quantum dots,for example, are small nanoparticles that have showngood efficacy in the transfection of animal cells(Yezhelyev et al., 2008). A significant disadvantage toquantum dots is that they are most often made fromheavy metals. In recent years, carbon dots have re-ceived a considerable amount of attention as a “green”alternative to quantum dots (Reckmeier et al., 2016; Yaoet al., 2019). Much of the interest in carbon dots hasevolved around their optical properties, which havepractical applications for bioimaging, photocatalysis,photovoltaic cells, and light-emitting diodes. The up-take of carbon dots in plants has been studied (Li et al.,2016, 2018; Qian et al., 2018), but their use for deliveryof nucleic acids into plant cells has not yet been repor-ted. There are reports describing the use of carbon dotsfor transfection of plasmids, long dsRNA, and siRNAinto animal cells (Liu et al., 2012; Wang et al., 2014; Daset al., 2015; Pierrat et al., 2015). With a high ratio ofcarbon dots to nucleic acids, relatively small nano-complexes can be formed. Using carbon dots with anaverage size of 19.7 nm, a nanocomplex with plasmid

Figure 1. Strategy for delivery of siRNA into plants. The first barrier for delivery is the cuticle, a water-impermeable layer thatcovers all the above-ground parts of the plant. Stomates, the pores that allow for gas exchange, can be a point of entry for for-mulations. With a spreading surfactant, formulations can flow across the leaf surface and “flood” stomates for delivery to themesophyll cells (Schonherr and Bukovac, 1972). Once in the leaf apoplast, nanocomplexes containing the siRNA cargo need todiffuse through the cell wall to reach the plasma membrane. The relatively small size-exclusion limit of the cell wall (,10 nm)would likely restrict the movement of larger nanocomplexes. Smaller nanocomplexes can reach the plasma membrane andfacilitate cellular uptake by endocytosis. Escape from the endomembrane vesicles can be achieved by a phenomenon called the“proton-sponge” effect, which causes osmotic swelling and lysis of the vesicles (Behr, 1997). Some portion of the siRNA may bereleased from the nanoparticles by changes in pH between cellular compartments or by the competitive binding of naturallyoccurring biomolecules.

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DNA was only marginally larger at 20.6 nm when themass ratio of carbon dots to DNA was 32 (Pierrat et al.,2015). Wang et al. showed that carbon dots with anaverage size of ;3.5 nm formed nanocomplexes withsiRNA that were ;5 nm in size. Because of their smallsize, we hypothesized that carbon dots might be usefulfor the delivery of siRNA through the plant cell walland subsequent barriers.

RESULTS

Preparation and Purification of Carbon Dots

Carbon dots can be synthesized by a “top-down” ap-proach, which involves the decomposition of structuredcarbon precursors such as graphene. Alternatively, the“bottom-up” approach begins with the carbonization ofsimple precursors, such as organic acids, sugars, or aminoacids (Yao et al., 2019). The bottom-up methods for pro-ducing carbon dots usually involve a hydrothermal reac-tion or pyrolysis of the carbon precursor to producenanoparticles with sizes that are typically between1 and 10 nm. Surface functionalization/passivation

of the carbon dots can increase the colloidal stabilityand allow for the binding of various ligands. Forexample, functionalization with amines producesparticles with a positive charge that can bind with thenegative charges of the phosphate backbone in nucleicacids. Polyethyleneimine (PEI) is a commonly usedcationic polymer for the functionalization of carbondots. Citrate-derived carbon dots functionalized withbranched PEIs (bPEIs) have been used to deliver plas-mid DNA and siRNA to animal cells (Liu et al., 2012;Pierrat et al., 2015).Functionalization of carbon dots with PEI is often

done in a “one-pot” synthesis where carbonization ofthe precursor and functionalization occur at the sametime. This is possible because aqueous solutions of PEIare relatively thermostable. There are several reportsdescribing the synthesis of carbon dots directly fromPEI or other amines with the assistance of H2O2 (Zhouet al., 2015) or by performing the reactions in chloro-form (van Dam et al., 2017). In those examples, PEIserves as both the carbon source for the core and thenitrogen source for surface passivation. In the workdescribed here, carbon dots were produced directlyfrom bPEIs of various Mr by heating solutions in amixture of chloroform and methanol to 155°C for arelatively short time. The bPEI-derived carbon dots had

Figure 2. Purification and size characterization of carbon dots. A,Carbon dots were produced from differentMr PEIs and purified by size-exclusion chromatography. The elution of the carbon dot preparationswas monitored by their absorption at 360 nm. B, The hydrodynamicdiameter of carbon dots in the peak fractions was determined by dy-namic light scattering.

Figure 3. GFP silencing observed with carbon dots produced fromdifferent-Mr PEIs. The silencing efficacy of fractions corresponding tothe primary peaks from each of the different-Mr PEI precursors wastested for activity with a 22-mer siRNA targetingGFP at a concentrationof 12 ng mL21. At 5 d after spray application, GFP fluorescence wasvisualized under a strong blue light. The appearance of red chlorophyllfluorescence under the strong blue lights indicates GFP silencing.Representative leaf images for each of the carbon dot preparations areshown. Scale bar5 5 cm. The percent silencing area given below eachimage was measured digitally from four leaves for each treatment.Fraction 6 from the 10-kD bPEI preparation, which eluted after theprimary peak, was included in the analysis because it displayed bettersilencing activity than the fraction corresponding to the primary peak.

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an absorption maximum of 363 nm and produced ablue fluorescencewith an emissionmaximumat 460 nm(Supplemental Fig. S1), characteristic of nitrogen-dopedcarbon dots.

The carbon dot preparations likely contained aheterogeneous mixture of precursors, by-products,and carbon dots of different sizes and physical prop-erties. Therefore, the preparations were fractionatedby size-exclusion chromatography (Fig. 2A), and thesize distributions within the fractions was determinedby dynamic light scattering (Fig. 2B). Based on the sizeexclusion chromatography and dynamic light scatteringmeasurements, the size distribution of the carbon dotsassociated well with the Mr of the PEI precursors. Thesmallest PEIs used in this study, with averageMr valuesof 1,200 and 1,800 D, produced the smallest carbon dots.The carbon dots produced from 5-, 10-, or 25-kD bPEIswere progressively larger.

Gel retardation assays were used to demonstrate thebinding of dsRNA to carbon dots and enhanced resis-tance of the bound dsRNA to nuclease degradation(Supplemental Fig. S2). The Escherichia coli RNase IIIand a 124 bp dsRNA were used for these in vitro pro-tection assays because this enzyme has a very high levelof activity with longer dsRNA. In assays that containedthe dsRNA alone, significant degradation was ob-served after 1 min and the dsRNA was mostly de-graded by 15 min. When dsRNA was formulated withthe carbon dots, there was little apparent degradationafter a 60-min incubationwith RNase III. This enhancedresistance to nucleases could be significant for efficacyin plants, which contain high levels of RNase activity inthe extracellular apoplast (Sangaev et al., 2011).

Carbon Dot Formulations Are Efficacious in Silencing GFPin the 16C Line of Nicotiana benthamiana

The GFP-expressing 16C line of N. benthamiana is anoften-used model for the study of RNAi (Ruiz et al.,1998; Dalakouras et al., 2016; Bally et al., 2018). Inwild-type plants, chlorophyll in the leaves displays astrong red fluorescence under UV or strong blue lights.In the GFP transgenic lines, this red fluorescence is

masked by the green fluorescence. The silencing ofGFP isthen easily detected by the unmasking of the red chlo-rophyllfluorescence. For the silencing experiments in thisstudy, 22-mer siRNAs were used. Several lines of re-search have demonstrated that the initiation of silencingwith 22-mer siRNAs may result in more secondarysiRNAproduction,which can enhanceRNAi phenotypes(Mlotshwa et al., 2008;Manavella et al., 2012; Dalakouraset al., 2016; Taochy et al., 2017). The sequences of theGFP-targeting 22-mer and other siRNAs used in this study areshown in Supplemental Table S1.

True leaves 3 and 4 from 17-d-old plants (SupplementalFig. S3) were treated by an adaxial spray of formula-tions with 0.4% (v/v) BREAK-THRU S 279, a nonionicspreading surfactant. The surfactant allows entry ofthe formulation into plants by stomatal flooding (Fig. 1),which is a commonly used method for delivery of agro-chemicals into leaf tissue (Schönherr and Bukovac, 1972).A typical concentration of siRNA used was 12 ngmL21 with an estimated application volume of 3.8 mL offormulation per square centimeter of leaf. These appli-cation parameters gave an approximate siRNA use rateof 45 ng siRNA per square centimeter of leaf. In theinitial screen for efficacy, carbon dots produced fromPEIs of different Mr were tested (Fig. 3). With thelowest-Mr PEI precursors (Mr 5 1,200 and 1,800 D),there was little or no visible GFP silencing. A limitedamount of the silencing was observed for carbon dotsderived from the largest PEI tested (Mr 5 25 kD).Much higher levels of silencing were observed withthe intermediate-sized carbon dots that were pro-duced from the 5- or 10-kD bPEIs.

Application of the 22-mer siRNA with the surfactantalone did not result in any detectable silencing. Becauseunmodified PEIs are one of the most often used trans-fection agents for delivery to animal cells, the unmod-ified 5-kD bPEI was also tested for delivery andsilencing efficacy with the 16C line (Supplemental Fig.S4). With these buffer conditions and the BREAK-THRU S 279 surfactant at 0.4% (v/v), the unmodified5 kD bPEI was not an effective transfection agentfor delivery to intact plant cells.

In the initial screen, some differences were observedin the delivery efficacy of fractions from the same

Table 1. Percent silenced area with different fractions of CD-5K

Different fractions of the CD-5K preparation were formulated with the GFP-targeting 22-mer siRNA at aconcentration of 8 ng mL21. The mean percent silenced area relative to the entire leaf area was calculateddigitally with four replicates per treatment. Means were separated using a Student’s t test. The fractions notconnected by a letter are statistically different (a 5 0.05). Images used for the analysis are included inSupplemental Fig. S5.

Fraction Connected Letters Silenced Area SE

%Fraction 5 A — — 43.6 3.81Fraction 6 A — — 36.2 6.13Fraction 7 A B — 35.6 7.33Fraction 8 A B — 28.5 5.59Fraction 9 — B — 20.4 2.36Fraction 10 — — C 4.9 1.14

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preparation. Fraction 6 from the 10-kD preparation, forexample, displayedmuch higher silencing efficacy thanfraction 4 from the same preparation (Fig. 3). As part ofthe optimization process, additional fractions from the5-kD bPEI preparations (CD-5K) were tested for ef-ficacy in silencing GFP in the 16C line. The carbondots from these different fractions ranged in size be-tween 2.7 and 3.9 nm (Supplemental Table S2). Basedon image analysis of the silenced area, fraction 5 fromthe CD-5K preparation was the most efficacious(Table 1; Supplemental Fig. S5). The later fractions,which contain smaller carbon dots, were less effica-cious in silencing GFP.For molecular validation of gene silencing, the CD-

5K fraction 5 was used. The application of a carbon dotformulation with a nontargeting siRNA did not impactthe typical green fluorescence of the 16C line under bluelights. Plants that were sprayed with a formulationcontaining a GFP-targeting 22-mer siRNA displayed astrong red fluorescence that covered most of the leaf(Fig. 4A). The level of GFP transcript reduction, mea-sured by reverse transcription quantitative PCR (RT-qPCR), was 84% (Fig. 4B). Immunoblot analysisshowed a similar reduction in GFP protein levels at 87%(Fig. 4, C–E). This level of GFP silencing was enough toinitiate the systemic spread of silencing. By 12 d aftertreatment, GFP silencing in newly emerging leaves be-came apparent (Supplemental Fig. S6).

Carbon Dot Formulations Are Efficacious in SilencingEndogenous Genes in N. benthamiana

The silencing of endogenous genes was tested bytargeting the genes encoding the H and I subunits ofmagnesium chelatase (CHLH and CHLI), an enzymenecessary for chlorophyll synthesis. True leaves 3 and 4from 17-d-old N. benthamiana plants were sprayed withcarbon dot formulations containing a nontargeting siRNA(Fig. 5A), a CHLI-targeting siRNA (Fig. 5B), or a CHLH-targeting siRNA (Fig. 5C). Leaves that were sprayed withformulations targeting either theH or I subunits displayedspots andpatches of bleaching that is indicative of reducedchlorophyll accumulation, whereas leaves that weresprayedwith the nontargeting siRNAdisplayed no visiblephenotype. Over several experiments, the bleaching phe-notype was strongest on the younger leaf 4 and with theformulation targeting the CHLH gene. Analysis of CHLHtranscript levels by RT-qPCR showed a 79% reduction inthe phenotypic tissues at 5 d after treatment (Fig. 5D). Thebleaching phenotype persisted for the duration of the ex-periments on the application leaves, which was up to 20 dafter treatment (Supplemental Fig. S7).The CHLH silencing phenotype was used to evaluate

the stability of carbon dot formulations. A single batchof formulation with the CHLH-targeting 22-mer siRNAwas sprayed on plants 1 d, 1 week, or 2 weeks afterpreparation. The plants were imaged 5 d after treatment(Supplemental Fig. S8). With the spray application at1 week, there did not appear to be a significant loss in

the efficacy of the carbon dot formulation; indicatingthat the siRNA is mostly intact, and that the formula-tion has good colloidal stability. The formulation thatwas stored for 2weeks at room temperature displayed a

Figure 4. Molecular validation of GFP silencing in the 16C line of N.benthamiana. Leaves 3 and 4 were sprayed with carbon dot formula-tions containing a nontargeting siRNA or a 22-mer siRNA targeting theGFP transgene. The final concentration of siRNAs in the formulationswas 12 ng mL21. All data came from leaf tissue collected 5 d aftertreatment. A, Phenotypic silencing of GFP was monitored by fluores-cence under blue lights. Scale bar 5 5 cm. B, Silencing of GFP wasvalidated by RT-qPCR analysis of transcript levels (P 5 0.00048). C,Immunoblot analysis of GFP protein. D, Coomassie-stained imagedemonstrating equal loading. E, Quantification of the band intensity inC with ImageJ. The reduction in GFP protein was statistically significantat P5 6.51E205. All error bars represent the mean6 SE and a Student’st test (two-tailed) was used for all statistical comparisons.

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bleaching phenotype, but there did appear to be somereduction in the phenotype.

Carbon Dot Formulations Are Efficacious for siRNADelivery and Silencing in Tomato

The efficacy of carbon dot formulations for siRNAdelivery and silencing in tomato (Solanum lycopersicum)was testedwith a line expressing enhancedGFP (eGFP).Plants that were treatedwith a formulation containing anontargeting siRNA displayed a strong green fluores-cence (Fig. 6A).With formulations containing the eGFP-targeting siRNA, silencing was apparent as spots andpatches at the lower siRNA concentrations (2 and 4 ngmL21). At an siRNA concentration of 8 ng mL21, thesilencing phenotype covered most of the leaf. Immu-noblot analysis of whole leaf extracts showed an88% reduction in GFP protein levels in tomato leavesthat were treated with the formulation containing8 ng mL21 of the GFP-targeting siRNA (Fig. 6, B–D).

The stomatal flooding application method with thespreading surfactant works well with fully expandedleaves. In younger leaves, spray application of carbondot formulations results in strong silencing toward thetip with limited silencing at the base (Fig. 7). In basip-etal plants, such as tomato, functional stomates developfirst at the leaf tip. The limited silencing at the base ofthe young leaves is likely due to reduced floodingwhere stomates have not yet fully developed. With thelimitations in stomatal flooding, other methods may bemore amenable for silencing genes in very young tis-sues. Delivery methods for DNA or RNA that rely onthe physical disruption of barriers have also been used

(Shang et al., 2007; Dalakouras et al., 2016). Thesephysical delivery methods appear to be most effectivewith young tissues and could be complementary to thecarbon dot-mediated delivery described here.

An Alternative Method to Produce Carbon Dots

Given the high level of activity observedwith CD-5K,a method was developed to produce carbon dots fromGlc with 5 kD bPEI for functionalization in a “one-pot”synthesis. These carbon dots, which were produced at alower temperature and in an aqueous solution, showedhigh levels of activity for the silencing of GFP in tomato(Fig. 8A) and of GFP and CHLH in N. benthamiana(Fig. 8, B and C). With the lower temperature and theaqueous solvent used for production of the Glc carbondots, methods could be developed that would requireless specialized lab equipment.

DISCUSSION

Transient gene silencing methods, such as VIGS andA. tumefaciens-mediated infiltration, have been impor-tant tools for studying the function of plant genes. Thedevelopment of delivery methods for topically appliedsiRNA would be another valuable tool for plant func-tional genomics. In addition to its simplicity, a topicalRNAi delivery method would not require the contain-ment of treated plants, making this method bettersuited for field studies. As discussed in the introduc-tion, the reagents that are most often used to transfectnucleic acids into animal cells form nanocomplexes that

Figure 5. Silencing of the magnesium chelataseH or I subunits (CHLH or CHLI) inN. benthamiana. Leaves 3 and 4 from 17-d-oldN. benthamiana plants were sprayed with siRNAs formulated with the purified CD-5K fraction 5. The final concentration ofsiRNAs in the formulations was 12 ng mL21. Representative plants for each treatment are shown at 4 d after application. Theapplication sites, leaves 3 and 4, are indicated by white arrows. A, Leaves that were sprayed with formulations containing anontargeting siRNA. Scale bar5 5 cm. B, Leaves sprayed with a 22-mer siRNA targeting CHLI. C, Leaves sprayed with a 22-mersiRNA targeting CHLH. D, Transcript analysis of CHLH. At 5 d after treatment, leaf 4 from four plants was sampled for RT-qPCRanalysis. Error bars represent the mean6 SE. A Student’s t test (two-tailed) was used to compare transcript levels. The reduction inCHLH transcript was statistically significant at P 5 3.48E206

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are too large to efficiently diffuse through the plant cellwall. Based on their small size, we hypothesized thatcarbon dots might readily pass through the cell walland provide an effective method for the delivery ofsiRNA into plant cells. Like other types of cationictransfection agents, amine-functionalized carbon dotscan bind to the negatively charged polyphosphatebackbone of nucleic acids. This binding provides pro-tection from nucleases, and the shielding of negativecharges can significantly enhance cellular uptake ofnucleic acids (Kozielski et al., 2013)Utilizing a microwave synthesizer, carbon dots were

produced directly from bPEIs in a solution of chloro-form and methanol. To make the technology more ac-cessible to other labs, an alternative method ofproducing carbon dots from Glc and bPEIs in anaqueous solution and at a lower temperature was alsodeveloped. Carbon dots were prepared from severalbPEIs of different Mr to yield a distribution of particlesizes that associated well with the size of the bPEIprecursor. Purification of these preparations was animportant step in optimizing the formulations—anycomponents of the preparation that can bind RNAbut are less efficacious for delivery will likely reducethe silencing activity by sequestering the siRNA. Byscreening fractions from the different preparations forsilencing activity, highly efficacious carbon dot for-mulations were identified.The size of the carbon dots appears to be an impor-

tant variable in determining their efficacy. The carbondots produced from the 25-kD bPEI precursors, with anaverage hydrodynamic diameter of 8.7 nm, had littlesilencing activity; this could be due to the size-exclusionlimit of the cell wall. Somewhat unexpectedly, thesmallest carbon dots that were prepared from thelower-Mr bPEIs also displayed limited silencing activ-ity. The highest level of silencing was achieved withintermediate-sized carbon dots produced from the 5- or10-kD bPEI precursors, with a hydrodynamic diameternear 3.9 nm. A small size may be a prerequisite for ef-ficient delivery through the plant cell wall, but it doesnot appear to be the only factor affecting delivery andsilencing efficiency of formulations. Subsequent bar-riers to delivery, such as endocytosis through theplasma membrane, escape from the endomembranesystem, and release of siRNA, will also impact the si-lencing efficiency. Carbon dot characteristics that areoptimal for passage through the cell wall may be

Figure 6. GFP silencing in fully expanded tomato leaves. True leaves 3and 4 received an abaxial spray of carbon dot formulations containing anontargeting siRNA or a 22-mer siRNA targeting eGFP. Final concen-trations of siRNAs are shown. A, eGFP fluorescence was imaged at 5 dafter treatment. Scale bar 5 5 cm. B, Immunoblot analysis of eGFPprotein levels from whole-leaf extracts at 5 d after treatment. C,Coomassie-stained gel demonstrating equal loading. D, Quantificationof the eGFP band intensity using ImageJ. Error bars represent the mean6 SE. A Student’s t test (two-tailed) was used to compare band intensi-ties. The reduction in eGFP protein was statistically significant atP 5 4.1E204

Figure 7. GFP silencing in younger tomato leaves. The fluorescenceimage of tomato leaves 5 d after treatmentwith a carbon dot formulationcontaining the eGFP-targeting siRNA at 8 ng mL21. Scale bar 5 5 cm.

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suboptimal for these later barriers. Further characteri-zation of the carbon dots and studies aimed at interro-gating delivery through the different barriers couldallow for further improvements in silencing efficiency.

With a low-pressure spray application, the optimizedcarbon dot formulations showed good efficacy in thedelivery of siRNA toN. benthamiana and tomato leaves.The targeting of a GFP transgene resulted in an almostcomplete loss of green fluorescence in both species. Thisgene-specific silencing was validated by measuring a.80% reduction in GFP transcript and protein levels.The efficacy of the carbon dot formulations was alsodemonstrated by targeting the H and I subunits ofmagnesium chelatase, which resulted in a visiblebleaching phenotype and a significant reduction in thetarget gene transcript levels.

There have been several recent reports describing theuse of nanomaterials for plant delivery and the silenc-ing of transgenes (Mitter et al., 2017; Zhang et al., 2019),but a visible whole-leaf phenotype has not yet beenreported. Leaf infiltration of a 21-mer siRNA linkedwith DNA-nanostructures resulted in silencing of aGFP transgene in N. benthamiana at 2 d post infiltration,but GFP protein levels recovered by 7 d post infiltration(Zhang et al., 2019). This transient silencing may be dueto the use of a 21-mer siRNA, which produces lesssecondary siRNA than 22-mer siRNA. Clay nanosheetsmay enhance the stability of dsRNA but did not en-hance the silencing of a b-glucuronidase reporter genein Arabidopsis (Arabidopsis thaliana) relative to the

dsRNA alone (Mitter et al., 2017). The carbon dot-facilitated delivery method for 22-mer siRNAs that isdescribed here may have some advantages in that astrong, persistent, and visible phenotype can be ach-ieved with a low-pressure spray application. The si-lencing of genes encoding theCHLH and CHLI subunitsis also the first report of the silencing of endogenous plantgenes with a nanomaterial-based delivery method. Thesimple spray application method and the strong silencingphenotypes achieved could make the current method auseful tool for plant functional genomics. Carbon dotformulations have recently been used to study therelationship between transgene expression levels,the production of secondary siRNA, and the systemicspread of silencing (B. Hendrix, P. Hoffer, R.A. Sanders,S.H. Schwartz, W. Zheng, B. Eads, D. Taylor, J. Deikman,unpublished data). Further improvements of nano-material deliverymethods, such as carbon dots, couldalso have practical agricultural applications.

MATERIALS AND METHODS

Plants and Growth Conditions

The plants used in this study included the 16C GFP expression line of Ni-cotiana benthamiana (Voinnet and Baulcombe, 1997) and a tomato (Solanumlycopersicum) line with constitutive expression of an enhanced GFP (eGFP;Seminis Vegetable). Constitutive expression of eGFP in tomato was accom-plished with the TraitMaker technology developed by Mendel Biotechnology(Ratcliffe et al., 2014). The HP375 line of tomato was transformed with a con-struct that contained a 35s promoter driving the expression of a LEXA DNAbinding domain fused to a GAL4 activation domain. Another transgenic linewas generated in the same tomato background with the eGFP transgene and anupstream LexA operator (opLEXA) sequence. The two lines were crossed and aline that was homozygous for both insertions was selected in a subsequentgeneration. Transactivation of the opLexA::eGFP by the LEXA/GAL4 fusionprotein resulted in constitutive expression of the eGFP reporter gene.

Plants were grown in 2.5-inch pots that were irrigated by an ebb-and-flowsystemwith Peters professional 20-20-20 fertilizer. Plants were kept in a growthchamber maintained at 25°C with a light intensity of 150 mmol m22 s21 and aday length of 16 h.

Preparation and Purification of Carbon Dots

bPEIs with an averageMr of 1,200 D, 1,800 D, and 10 kDwere obtained fromPolysciences. The 25-kD bPEI was obtained from Sigma-Aldrich. The 5-kDbPEI, Lupasol G100, was supplied by BASF as a 50% (w/w) solution—beforecarbon dot preparation, the water was removed by lyophilization. Carbon dotswere produced by a solvothermal reaction of the bPEIs in a solution of chlo-roform and methanol (4:1). Using a microwave synthesizer (Monowave 50,Anton Paar), 375 mg of bPEI in 5 mL of the solvent was heated to 155°C with aramp time of 5 min and then held at 155°C for an additional 7 min. The reactionproducts were dried under nitrogen and then resuspended in water. Any re-sidual chloroform was separated from the aqueous solution of carbon dots bycentrifugation at 7,500g for 5 min. The pH of the carbon dot solution was ad-justed to 8.0 with 4N HCl, and the final volume was adjusted to 5 mL withwater. For visible and fluorescence spectra, an aliquot was diluted in water(1:16). The absorption spectra were measured with standard 1-cm cuvettes inthe cuvette port of a SpectraMaxM2microplate reader (Molecular Devices). Thefluorescence spectra from the crude preparations and the fractions were mea-sured in 96-well plates.

An alternative method was developed to produce highly efficacious carbondots fromGlc and 5-kD bPEI in an aqueous solution and at lower temperatures.For the PEI-functionalized Glc carbon dots, a 5-mL solution containing 37.5 mgmL21 Glc and 75 mg mL21 5-kD bPEI was adjusted to pH 8.0 with 4N HCl anddegassed under vacuum. The solution was then heated in a microwave syn-thesizer to 100°C over 3 min and then held at 100°C for an additional 5 min. The

Figure 8. Efficacy of 5K bPEI functionalized Glc-carbon dots. FPLCfraction 5 from theGlc Lupasol G100 preparationwas used for testing ofsilencing efficacy. A, GFP silencing in tomato. B, GFP silencing in the16C line ofN. benthamiana. C,CHLH silencing inN. benthamianawiththe application leaves indicated by arrows. Scale bars 5 5 cm.

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visible spectra, fluorescence spectra, and FPLC chromatogram of the Glc carbondots were similar to those of carbon dots produced directly from the 5-kD PEI.

FPLC Fractionation of Carbon Dot Preparations

Carbondot preparationswere purified by size-exclusion chromatography.A2.5 3 20 cm Econo-column (Bio-Rad) filled with Sephadex G-50 superfine (GEHealthcare) was run on a Bio-Rad BioLogic Duo Flow FPLC system equippedwith a QuadTec detector. The column was eluted at 2 mL min21 with 50 mM

NaCl. Elution of the carbon dots was monitored at 360 nm. Five-milliliterfractions were collected starting after 30 mL.

Formulation of Carbon Dots

The final concentration of siRNA in the formulations was 12 ng mL21 or less.At higher concentrations of siRNA, aggregation can occur and a decrease in thesilencing efficacy of formulations was observed. A similar observation wasmade with carbon dot formulations used to deliver plasmid DNA to animalcells (Pierrat et al., 2015). Pierrat et al. (2015) also observed that the size of thenanocomplexes decreased when the ratio of carbon dots to plasmid was high.The optimal concentration of carbon dots for gene silencing was determinedempirically. A carbon dots:siRNA mass ratio of 40:50 worked well. The siRNAand the carbon dots were added separately to two tubes containing 10 mM MESbuffer pH 5.7 and 20 mM glycerol. The two tubes were then combined withvortex mixing. Prior to spray application, the formulations were left at roomtemperature for at least 1 h. Longer incubations at room temperature (up to1 week) did not significantly decrease the efficacy of the formulations(Supplemental Fig. S8).

Spray Application

The spreading surfactant BREAK-THRU S279 was added to a final con-centration of 0.4% (v/v)within 1 h of spraying. The spray applicationwasmadewith an Iwata HPM1 airbrush at;12 pounds per square inch andwith the fluidadjustment knob set to 1.5. With a slight depression of the airbrush trigger, avery light coat of the formulation was applied to the adaxial side of N. ben-thamiana leaves. The vertical growth habit of tomato allowed spray applicationsto the abaxial side of leaves, which allowed for betterflooding and consequentlyimproved gene silencing. The efficient stomatal flooding of the formulationswas apparent by a subtle change in the shade of green. With this light sprayapplication, there was no apparent damage to the leaves.

GFP Imaging and Analysis

The leaves were photographed using a custom-built imaging stationequipped with a Canon EOS 70D camera with an EFS 18-55 mmmacro 0.25-m/0.8-ft lens and a high-intensity blue LED light source (SL3500-D) equipped witha 460-nm filter (Photon System Instruments). Images were acquired using theCanon EOS Utility 2 software with tethered image acquisition. For GFP imag-ing, 58-mm Tiffen Green No. 11 and Yellow No. 12 filters were used in com-bination. An ISO speed (sensitivity) of 800, a 0.25-s exposure time, and an F-stopof 4.5 were typical camera settings for GFP image acquisition. The GFP-silencing phenotype first became apparent at 2 d after treatment but becamemore intense at later time points. Image analysis and molecular validation ofGFP silencing were performed on leaves harvested at 5 d after treatment.

The images were processed using ImageJ (https://imagej.nih.gov/ij/index.html). Briefly, the program operator utilized the threshold color panel tohighlight a border around each leaf. A border image was overlaid onto the leafimage, and the pixel number within the leaf border was quantified by thesoftware to give the total leaf area. A similar process was used to highlight aborder around the region displaying the red fluorescence that is demonstrativeof a GFP-silencing phenotype. The GFP-silenced areas were calculated by di-viding the phenotypic area by the total leaf area. A one-way ANOVAwas usedto compare the percent silenced area for the different formulations that weretested.

Validation of Gene Silencing

For RT-qPCR analysis, total RNA was extracted from phenotypic leaftissue collected 5 d after treatment using Trizol reagent (Invitrogen)according to the manufacturer’s protocol. The RNAwas dissolved in water,

and the concentration was measured using the Quant-iT RNA assay kit(Invitrogen). The total RNA samples were diluted to 5 ng mL21, and 50 ng oftotal RNA was used to synthesize random-primed, first-strand comple-mentary DNA using the High-Capacity Reverse Transcription kit (AppliedBiosystems). The reverse transcription products were used as a template forqPCR. The qPCR reaction mixtures consisted of 2 mL complementary DNA,3 mL of a primer-probe mix (0.5 mM of each primer and 0.25 mM probe finalconcentration), and 5 mL Taqman Universal PCR Master Mix (Applied Bi-osystems). The sequences for the primer-probe sets are provided inSupplemental Table S3. The reactions were performed using an Applied Biosys-tems 7900HT Fast Real-Time PCR System with 40 cycles of two-step cycling at95°C for 15 s and 60°C for 60 s. Target gene expressionwas expressed relative to areference gene, Protein Phosphatase2a (Nbv6.1trP16930; http://benthgenome.qut.edu.au/). Expression values were calculated using the comparative cyclethreshold (Ct) method: 2-(Ct Target – Ct Reference). A Student’s t test (two-tailed) wasused to compare the transcript levels between formulations containing the non-targeting siRNA and the targeting siRNA.

For immunoblot analysis of N. benthamiana or tomato, whole leaves wereharvested at 5 d after treatment, frozen, and then ground to a fine powder.Approximately 200 mg of ground tissue was homogenized in 300 mL of ice-coldbuffer containing 20mMTris-Cl (pH 8.0), 150mMNaCl, 0.1% (v/v) Triton X-100,and a protease inhibitor cocktail. The insoluble debris was removed bycentrifugation. The protein concentration was quantified by Bradfordassays, and 10 mg of total protein for each sample was run on a 12.5% Cri-terion Tris-HCl protein gel (3450014, Bio-Rad). Following electrophoresis,the proteins were transferred onto a polyvinylidene difluoride membraneand then blocked overnight with 5% skim milk in TBS plus 0.1% (v/v)Tween 20 (TBST). For GFP detection, a horseradish peroxidase-conjugatedantibody directed against the full-length GFP protein (sc-8334, Santa CruzBiotechnology) was diluted 1:1,000 in TBST and 5% (v/v) skim milk andincubated with the blot for 1 h. The blot was then washed four times withTBST for 10 min. Pierce ECL plus western substrate (32132, Thermo ScientificPierce Protein Biology) was used for chemiluminescent detection of the GFPprotein. ImageJ software was used to quantify band intensity for eachsample. A Student’s t test (two-tailed) was used to compare the GFP proteinlevels between formulations containing the nontargeting siRNA and theGFP-targeting siRNA.

Accession Numbers

Accession numbers for the genes tested can be found in the legend ofSupplemental Table S1.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Spectroscopic characterization of carbon dotsproduced from the 5-kD bPEI.

Supplemental Figure S2. Binding and nuclease protection of dsRNA incarbon dot formulations.

Supplemental Figure S3. N. benthamiana at the time of treatment, 17 d aftersowing.

Supplemental Figure S4. Silencing assays with unmodified 5-kD bPEI.

Supplemental Figure S5. The efficacy of CD-5K fractions from size-exclusion chromatography.

Supplemental Figure S6. Systemic silencing of GFP is initiated in the 16Cline with carbon dot formulations.

Supplemental Figure S7. Persistence of the bleaching phenotype caused byCHLH silencing in N. benthamiana.

Supplemental Figure S8. Colloidal stability and efficacy of a carbon dotformulation with the 22-mer siRNA targeting CHLH.

Supplemental Table S1. siRNA sequences used in this study.

Supplemental Table S2. Dynamic light scattering measurements of thefractionated carbon dots from the CD-5K preparation prior to formula-tion with siRNA.

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Supplemental Table S3. Oligonucleotide and probe sequences for RT-qPCR analysis of transcript levels.

ACKNOWLEDGMENTS

We thank Norma Medina, Brenda Reed, and Kaylene Yandell for theirassistance in growing and maintaining the plants used in this study.

Received June 12, 2020; accepted July 13, 2020; published August 6, 2020.

LITERATURE CITED

Bally J, Jung H, Mortimer C, Naim F, Philips JG, Hellens R, Bombarely A,Goodin MM, Waterhouse PM (2018) The rise and rise of Nicotianabenthamiana: A plant for all reasons. Annu Rev Phytopathol 56: 405–426

Baron-Epel O, Gharyal PK, Schindler M (1988) Pectins as mediators ofwall porosity in soybean cells. Planta 175: 389–395

Baum JA, Bogaert T, Clinton W, Heck GR, Feldmann P, Ilagan O,Johnson S, Plaetinck G, Munyikwa T, Pleau M, et al (2007) Control ofcoleopteran insect pests through RNA interference. Nat Biotechnol 25:1322–1326

Becker A, Lange M (2010) VIGS—genomics goes functional. Trends PlantSci 15: 1–4

Behr J-P (1997) The proton sponge: A trick to enter cells the viruses did notexploit. Chimia (Aarau) 51: 34–36

Burch-Smith TM, Anderson JC, Martin GB, Dinesh-Kumar SP (2004)Applications and advantages of virus-induced gene silencing for genefunction studies in plants. Plant J 39: 734–746

Carpita N, Sabularse D, Montezinos D, Delmer DP (1979) Determinationof the pore size of cell walls of living plant cells. Science 205: 1144–1147

Carpita NC, Gibeaut DM (1993) Structural models of primary cell walls inflowering plants: Consistency of molecular structure with the physicalproperties of the walls during growth. Plant J 3: 1–30

Chawla R, Shakya R, Rommens CM (2012) Tuber-specific silencing ofasparagine synthetase-1 reduces the acrylamide-forming potential ofpotatoes grown in the field without affecting tuber shape and yield.Plant Biotechnol J 10: 913–924

Cheon S-H, Kim Z-H, Choi H-Y, Kang S-H, Nam H-J, Kim J-Y, Kim D-I(2017) Effective delivery of siRNA to transgenic rice cells for enhancedtransfection using PEI-based polyplexes. Biotechnol Bioproc Eng 22:577–585

Cheon SH, Lee KH, Kwon JY, Choi SH, Song MN, Kim DI (2009) En-hanced delivery of siRNA complexes by sonoporation in transgenic ricecell suspension cultures. J Microbiol Biotechnol 19: 781–786

Dalakouras A, Wassenegger M, McMillan JN, Cardoza V, Maegele I,Dadami E, Runne M, Krczal G, Wassenegger M (2016) Induction ofsilencing in plants by high-pressure spraying of in vitro-synthesizedsmall RNAs. Front Plant Sci 7: 1327

Das S, Debnath N, Cui Y, Unrine J, Palli SR (2015) Chitosan, carbonquantum dot, and silica nanoparticle mediated dsRNA delivery for genesilencing in Aedes aegypti: A comparative analysis. ACS Appl MaterInterfaces 7: 19530–19535

Demirer GS, Zhang H, Matos JL, Goh NS, Cunningham FJ, Sung Y,Chang R, Aditham AJ, Chio L, Cho MJ, et al (2019) High aspect rationanomaterials enable delivery of functional genetic material withoutDNA integration in mature plants. Nat Nanotechnol 14: 456–464

Eamens A, Wang M-B, Smith NA, Waterhouse PM (2008) RNA silencingin plants: Yesterday, today, and tomorrow. Plant Physiol 147: 456–468

Eggenberger K, Mink C, Wadhwani P, Ulrich AS, Nick P (2011) Using thepeptide BP100 as a cell-penetrating tool for the chemical engineering ofactin filaments within living plant cells. ChemBioChem 12: 132–137

Frizzi A, Huang S (2010) Tapping RNA silencing pathways for plant bio-technology. Plant Biotechnol J 8: 655–677

Golestanipour A, Nikkhah M, Aalami A, Hosseinkhani S (2018) Genedelivery to tobacco root cells with single-walled carbon nanotubes andcell-penetrating fusogenic peptides. Mol Biotechnol 60: 863–878

Huang S, Adams WR, Zhou Q, Malloy KP, Voyles DA, Anthony J, KrizAL, Luethy MH (2004) Improving nutritional quality of maize proteinsby expressing sense and antisense zein genes. J Agric Food Chem 52:1958–1964

Johansen LK, Carrington JC (2001) Silencing on the spot. Induction andsuppression of RNA silencing in the Agrobacterium-mediated transientexpression system. Plant Physiol 126: 930–938

Kimura S, Kawano T, Iwasaki T (2017) Short polyhistidine peptides pen-etrate effectively into Nicotiana tabacum-cultured cells and Saccharomycescerevisiae cells. Biosci Biotechnol Biochem 81: 112–118

Kozielski KL, Tzeng SY, Green JJ (2013) Bioengineered nanoparticles forsiRNA delivery. Wiley Interdiscip Rev Nanomed Nanobiotechnol 5:449–468

Kumar S, Salar RK (2017) Control of gene expression by RNAi: A revo-lution in functional genomics. In SK Gahlawat, RK Salar, P Siwach, JSDuhan, S Kumar, and P Kaur, eds, Plant Biotechnology: Recent Ad-vancements and Developments. Springer Singapore, Singapore, pp17–57

Kwak SY, Lew TTS, Sweeney CJ, Koman VB, Wong MH, Bohmert-TatarevK, Snell KD, Seo JS, Chua NH, Strano MS (2019) Chloroplast-selectivegene delivery and expression in planta using chitosan-complexed single-walled carbon nanotube carriers. Nat Nanotechnol 14: 447–455

Lakshmanan M, Kodama Y, Yoshizumi T, Sudesh K, Numata K (2013)Rapid and efficient gene delivery into plant cells using designed peptidecarriers. Biomacromolecules 14: 10–16

Li H, Huang J, Lu F, Liu Y, Song Y, Sun Y, Zhong J, Huang H, Wang Y, LiS, et al (2018) Impacts of carbon dots on rice plants: Boosting the growthand improving the disease resistance. ACS Appl Bio Mater 1: 663–672

Li W, Zheng Y, Zhang H, Liu Z, Su W, Chen S, Liu Y, Zhuang J, Lei B(2016) Phytotoxicity, uptake, and translocation of fluorescent carbondots in mung bean plants. ACS Appl Mater Interfaces 8: 19939–19945

Liu C, Zhang P, Zhai X, Tian F, Li W, Yang J, Liu Y, Wang H, Wang W, Liu W(2012) Nano-carrier for gene delivery and bioimaging based on carbon dotswith PEI-passivation enhanced fluorescence. Biomaterials 33: 3604–3613

Liu W-W, Meng J, Cui J, Luan Y-S (2017) Characterization and function ofmicroRNA*s in plants. Front Plant Sci 8: 2200–2201

Manavella PA, Koenig D, Weigel D (2012) Plant secondary siRNA pro-duction determined by microRNA-duplex structure. Proc Natl Acad SciUSA 109: 2461–2466

Matzke MA, Matzke AJM (2004) Planting the seeds of a new paradigm.PLoS Biol 2: E133

McGinnis KM (2010) RNAi for functional genomics in plants. Brief FunctGenomics 9: 111–117

Mitter N, Worrall EA, Robinson KE, Li P, Jain RG, Taochy C, Fletcher SJ,Carroll BJ, Lu GQ, Xu ZP (2017) Clay nanosheets for topical delivery ofRNAi for sustained protection against plant viruses. Nat Plants 3: 16207

Miyamoto T, Tsuchiya K, Numata K (2019) Block copolymer/plasmidDNA micelles postmodified with functional peptides via thiol-maleimide conjugation for efficient gene delivery into plants. Biomac-romolecules 20: 653–661

Mlotshwa S, Pruss GJ, Peragine A, Endres MW, Li J, Chen X, Poethig RS,Bowman LH, Vance V (2008) DICER-LIKE2 plays a primary role intransitive silencing of transgenes in Arabidopsis. PLoS One 3: e1755

Mroczka A, Roberts PD, Fillatti JJ, Wiggins BE, Ulmasov T, Voelker T(2010) An intron sense suppression construct targeting soybean FAD2-1 requires a double-stranded RNA-producing inverted repeat T-DNAinsert. Plant Physiol 153: 882–891

Numata K, Ohtani M, Yoshizumi T, Demura T, Kodama Y (2014) Localgene silencing in plants via synthetic dsRNA and carrier peptide. PlantBiotechnol J 12: 1027–1034

Pierrat P, Wang R, Kereselidze D, Lux M, Didier P, Kichler A, Pons F,Lebeau L (2015) Efficient in vitro and in vivo pulmonary delivery ofnucleic acid by carbon dot-based nanocarriers. Biomaterials 51: 290–302

Qian K, Guo H, Chen G, Ma C, Xing B (2018) Distribution of different surfacemodified carbon dots in pumpkin seedlings. Sci Rep 8: 7991–7999

Ratcliffe OJ, Heard J, Kumimoto RW, Repetti PP, Reuber LT, Creelman R,Hempel FD, Gutterson NL, Canales R, inventors (January 21, 2014).Plants with improved water deficit and cold tolerance. United StatesPatent Application No. 11/981,813.

Reckmeier CJ, Schneider J, Susha AS, Rogach AL (2016) Luminescentcolloidal carbon dots: Optical properties and effects of doping [Invited].Opt Express 24: A312–A340

Rosa C, Kuo YW, Wuriyanghan H, Falk BW (2018) RNA interferencemechanisms and applications in plant pathology. Annu Rev Phytopa-thol 56: 581–610

Ruiz MT, Voinnet O, Baulcombe DC (1998) Initiation and maintenance ofvirus-induced gene silencing. Plant Cell 10: 937–946

656 Plant Physiol. Vol. 184, 2020

Schwartz et al.

https://plantphysiol.orgDownloaded on May 9, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

Sangaev SS, Kochetov AV, Ibragimova SS, Levenko BA, Shumny VK(2011) Physiological role of extracellular ribonucleases of higher plants.Russ J Genet 1: 44–50

Schönherr J, Bukovac MJ (1972) Penetration of stomata by liquids: De-pendence on surface tension, wettability, and stomatal morphology.Plant Physiol 49: 813–819

Schwab R, Ossowski S, Riester M, Warthmann N, Weigel D (2006) Highlyspecific gene silencing by artificial microRNAs in Arabidopsis. Plant Cell18: 1121–1133

Shang Y, Schwinn KE, Bennett MJ, Hunter DA, Waugh TL, PathiranaNN, Brummell DA, Jameson PE, Davies KM (2007) Methods for tran-sient assay of gene function in floral tissues. Plant Methods 3: 1–12

Smith NA, Singh SP, Wang MB, Stoutjesdijk PA, Green AG, WaterhousePM (2000) Total silencing by intron-spliced hairpin RNAs. Nature 407:319–320

Taochy C, Gursanscky NR, Cao J, Fletcher SJ, Dressel U, Mitter N, TuckerMR, Koltunow AMG, Bowman JL, Vaucheret H, et al (2017) A geneticscreen for impaired systemic RNAi highlights the crucial role of DICER-LIKE2. Plant Physiol 175: 1424–1437

Unnamalai N, Kang BG, Lee WS (2004) Cationic oligopeptide-mediateddelivery of dsRNA for post-transcriptional gene silencing in plant cells.FEBS Lett 566: 307–310

van Dam B, Nie H, Ju B, Marino E, Paulusse JMJ, Schall P, Li M,Dohnalová K (2017) Excitation-dependent photoluminescence fromsingle-carbon dots. Small 13: 1702098

Voinnet O, Baulcombe DC (1997) Systemic signalling in gene silencing.Nature 389: 553

Wang Q, Zhang C, Shen G, Liu H, Fu H, Cui D (2014) Fluorescent carbondots as an efficient siRNA nanocarrier for its interference therapy ingastric cancer cells. J Nanobiotechnology 12: 58

Watson JM, Fusaro AF, Wang M, Waterhouse PM (2005) RNA silencingplatforms in plants. FEBS Lett 579: 5982–5987

Yao B, Huang H, Liu Y, Kang Z (2019) Carbon dots: A small conundrum.Trends Chem 1: 235–246

Yezhelyev MV, Qi L, O’Regan RM, Nie S, Gao X (2008) Proton-spongecoated quantum dots for siRNA delivery and intracellular imaging.J Am Chem Soc 130: 9006–9012

Zhang H, Demirer GS, Zhang H, Ye T, Goh NS, Aditham AJ, CunninghamFJ, Fan C, Landry MP (2019) DNA nanostructures coordinate gene si-lencing in mature plants. Proc Natl Acad Sci USA 116: 7543–7548

Zhou X, Pan Y, Xu J, Wang A, Wu S, Shen J (2015) The carbonization ofpolyethyleneimine: Facile fabrication of N-doped graphene oxide andgraphene quantum dots. RSC Advances 5: 105855–105861

Ziemienowicz A, Pepper J, Eudes F (2015) Applications of CPPs in genomemodulation of plants. Methods Mol Biol 1324: 417–434

Plant Physiol. Vol. 184, 2020 657

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