Inhibitory activity of carbon quantum dots against Phytophthora infestans and fungal plant pathogens and their effect on dsRNA-induced gene silencing

Abstract Carbon quantum dots (CQDs) have many potential applications due to their cell-penetrating ability, biocompatibility and tunable properties. Among a variety of characteristics, the inhibition of bacteria by CQDs is often reported. However, the effect on other microorganisms, such as plant pathogenic fungi and oomycetes, is poorly studied. Here we monitored the growth of the oomycete plant pathogen Phytophthora infestans in the presence of CQDs, as well as of another three fungal plant pathogens, namely Botrytis cinerea, Alternaria alternata and Fusarium oxysporum. Moreover, the ability of CQDs to improve gene silencing caused by exogenous dsRNA in P. infestans was studied, and the toxicity of CQDs to human keratinocytes was evaluated. Our results indicate significant inhibitory activity of CQDs against P. infestans at relatively low concentrations. In a species-specific manner and to a lesser extent, the growth of the three fungal plant pathogens was also affected. We also found that the treatment of P. infestans with naked dsRNA in vitro did not trigger gene silencing. However, the mixture of CQDs with dsRNA increased RNAi efficiency, by causing a significant reduction of the transcript levels of the target gene in developing sporangia. Finally, no cytotoxicity of the CQDs, in the concentrations active against the plant pathogens, was found.


Introduction
Carbon quantum dots (CQDs) are nanoparticles with sizes below 10 nm that gathered attention in the last years because of their excellent photoluminescent properties, low toxicity and good biocompatibility [1,2]. They have many potential applications, for example, in bioimaging [3], as biosensors [4] and as drug delivery carriers [5,6] and their properties are dependent on the precursor, the synthesis method and the surface functionalization [7][8][9][10].
The CQDs are synthesized by pyrolysis, microwave heating or hydrothermal reactions from a variety of organic, inorganic and natural carbon sources, such as graphite [11], plant materials [2,12], food and agricultural wastes [8,13,14]. In general, CQDs are considered low toxic to humans and mammals; however, cytotoxicity may occur depending on the concentration and the surface functionalization [15].
Recently, a number of studies reported antimicrobial activity of CQDs, mainly against pathogenic bacteria, and consider their use as alternatives of the conventional antibiotics [16,17]. Despite the well-documented antibacterial activity of the CQDs, very little is known about their effect on other microorganisms, such as fungi and oomycetes. A few studies report inhibitory activity of CQDs against plant pathogenic fungi and investigate their possible use as fungicides [18,19]; however, the mechanism, the magnitude and the spectrum of this activity are poorly studied.
Among a variety of applications, the CQDs have been successfully used to enhance the delivery of nucleic acids in different organisms, often aiming at increasing the efficiency of RNA interference (RNAi) [20][21][22][23]. Double-stranded RNA (dsRNA) coated with CQDs entered the leaves of cucumber plants in higher quantities, compared to dsRNA alone [24]. Increase in the gene silencing and mortality in mosquitos were achieved by dsRNA/CQDs complexes, with the highest efficiency among three different types of nanoparticles [25].
Constant efforts in potato production are made to control the wide-spread plant pathogen Phytophthora infestans [26]. It has the potential to develop rapidly and to be disastrous under favorable conditions, and its management relies heavily on the use of chemicals [27]. The ecological impact of fungicide usage on soils and waters, as well as the remarkable ability of P. infestans to evade control strategies, including host resistance and fungicides, implies the need to search for alternative approaches [27][28][29][30]. Such an approach includes silencing of essential genes by the application of dsRNA [31]. A crucial factor for the success of this strategy is the sensitivity of the target organism to RNAi [32]. It has been demonstrated that the uptake of dsRNA by P. infestans is limited, and thus the efficiency of the gene silencing [33].
The present study aims to explore the antifungal activity of CQDs against P. infestans and three other important, common plant pathogenic fungi, namely Botrytis cinerea, Fusarium oxysporum and Alternaria alternata; to assess the ability of CQDs to enhance the dsRNA-induced gene silencing in P. infestans and to evaluate the CQDs cytotoxicity.

Carbon quantum dots (CQDs)
Research-grade carbon quantum dots (CQDs) were purchased from Sigma-Aldrich (Product no. 900414; St. Louis, MO, USA) as ≥ 0.2% nanoparticles dispersion in water. According to the product specification, the quantum efficiency of the CQDs is ≥ 5% and the emission spectra from 450 to 550 nm.

Isolates and culturing of P. infestans and fungi
Phytophthora infestans CBS 120920 was obtained from Westerdijk Fungal Biodiversity Institute (Utrecht, Netherlands) and maintained on V8 agar medium (16 g/L agar, 3 g/L CaCO 3 , 100 mL/L Campbell's V8 juice) and rye agar medium (60 g/L ray, 15 g/L agar, 20 g/L sucrose and 0.05 g/L of β-sitosterol) at 20 °C in dark. B. cinerea, F. oxysporum and A. alternata were obtained from our culture collection and maintained on potato dextrose agar (PDA) medium at 25 °C.

Effect on mycelial growth of P. infestans
To assess the effect of CQDs on the mycelium of P. infestans the radial growth on solid medium was measured. In 12 mL V8 agar medium after autoclaving and short cooling (to approx. 60 °C) an appropriate amount of CQDs were added to receive the following concentrations: 1.25, 2.5, 5, 10, 20 and 40 µg/mL. V8 agar medium without the addition of CQDs was used as a control. The medium was poured in 90 mm-diameter plastic petri dishes and left to solidify. Next, a 5 mm-diameter agar plug with mycelium from the growing margin of a 7-day-old P. infestans culture was placed at the middle and incubated for 9 days at 20 °C in the dark. The diameter was measured in three directions and the mean was calculated. The experiment was performed in triplicate. Data were analyzed with one-way analysis of variance (ANOVA) followed by Tukey's Honest Significant Difference (Tukey's HSD) test for multiple comparison, in the environment of R studio, using aov() and TukeyHSD() functions, respectively.

Effect on sporangia/spores germination and development
The change in the optical density of the liquid medium was used to quantify the effect of CQDs on the germination and development of sporangia of P. infestans. The experiment was performed in 384-well high content screening microplates with Glass Bottom (Corning, New York, NY, USA). Sporangia were harvested from 10-day-old P. infestans rye agar cultures, by adding 3 mL of distilled water and vigorous, up and down pipetting. They were precipitated by centrifugation (5 min at 2500g) and adjusted to a concentration of about 3.6 × 10 5 sporangia/mL using a hemocytometer. Next, the sporangia suspension was placed at 4 °C for 2 h to induce zoospores release. In one well were added 10 µL of sporangia suspension, 5 µL of potato dextrose broth and 5 µL of CQDs suspended in water, in an appropriate amount, to reach the final concentrations of 10, 20, 40, 80 and 160 μg/ mL. The same amount of distilled water was added instead of CQDs suspension in the control treatment. The plate was incubated at 20 °C in darkness and the absorbance at 450 nm was measured using a CLARIOstar Plus microplate reader (BMG Labtech, Ortenberg, Germany) after 24, 48 and 72 h. To follow the sporangia development, microscopic observations were performed on an ImageXpress Pico automated cell imaging system (Molecular Devices, San Jose, CA, USA). The average of four replicates was calculated to measure the effect of each CQDs concentration. Data were analyzed with ANOVA followed by Tukey's Honest Significant Difference test for multiple comparisons.
A similar technique was used to assess the effect of CQDs on spores germination and development of B. cinerea, F. oxysporum and A. alternata. Spore suspensions were prepared by adding 5 mL distilled water into sporulating PDA cultures (about two weeks old) and scraping the surface with a spreader. The suspensions were filtered through cheesecloth to clean the mycelium debris, and the spores were counted using a hemocytometer. The concentrations were adjusted as follows: B. cinerea − 4 × 10 5 spores/ mL, F. oxysporum − 9 × 10 5 spores/mL and A. alternata − 1.8 × 10 5 spores/mL. The experiment was performed in a 96-well plate, in a total volume of 75 µL per well containing: 40 µL spore suspension, 29 µL potato dextrose broth and 6 µL water suspension of CQDs in appropriate amount to make final concentrations of 10, 20, 40, 80 and 160 μg/mL. The absorbance at 450 nm was measured using CLARIOstar Plus microplate reader (BMG Labtech, Ortenberg, Germany) after 24 and 48 h. Four replicates were performed and the data were analyzed using the same procedures as explained above.

dsRNA synthesis
RNA was extracted from P. infestans mycelium using an RNeasy Plant Mini Kit (Qiagen, Venlo, the Netherlands), treated with recombinant DNase I (TaKaRa, Tokyo, Japan) and purified applying the RNA purification protocol of the RNeasy Plant Mini Kit (Qiagen, Venlo, the Netherlands). Copy DNA (cDNA) was synthesized with reverse transcriptase ReadyScript cDNA Synthesis Mix (Sigma-Aldrich, Saint Louis, MO, USA). The template DNA for the synthesis of dsRNA specific to the gene encoding G-protein β-subunit (GPB1), which was used as a target in the RNAi experiment, was produced by polymerase chain reaction (PCR) using cDNA and GPB1 specific primers with the T7 promoter sequence tagged at their 5′ end (Table S1, Supplementary material). The 50 μL PCR reaction con-  Table S2, Supplementary material) was synthesized using MEGAscrip T7 Transcription Kit (Thermo Scientific, Waltham, MA, USA) following the manufacturer's instructions. The dsRNA was eluted in nuclease-free water and stored at −20 °C.
A similar procedure was followed to produce dsRNA specific for the gene encoding green fluorescent protein (GFP) with a size of 546 bp, which was used as a control with the following differences. The DNA template for the dsRNA synthesis was produced by PCR using DNA extracted from Eschericha coli harboring a GFP-containing plasmid and GFP specific primers with a T7 promoter sequence tagged at their 5′ end (Table S1, Supplementary material).

Quantitative RT-PCR
For quantification of the GPB1 expression, P. infestans sporangia were treated with GPB1-dsRNA with or without the addition of CQDs and with GFP-dsRNA as a control. A mixture of GPB1-dsRNA and CQDs was made by mixing 12.57 μL GPB1-dsRNA (70 ng/μL) with 0.44 μL CQDs (0.02 mg/mL) and 8.99 μL nuclease free water in a total volume of 22 μL, vortexed vigorously and incubated at room temperature for 30 min. The experiment was performed in 384-well High Content Screening Microplates with Glass Bottom (Corning) in a total volume of 20 μL of which 10 μL sporangia suspension, 5 μL potato dextrose broth and 5 μL of either treatments: GPB1-dsRNA/CQDs mixture; GPB1-dsRNA (in final concentration of 10 ng/μL) or GFP-dsRNA (in a final concentration of 30 ng/μL). After 72 h of incubation, the liquid containing the dsRNA was piped out and the cultures were washed two times by adding 20 μL of sterile distilled water in each well. Total RNA was extracted from washed sporangia and developing hyphae using an RNAqueous-Micro Kit (Ambion, Waltham, MA, USA), including DNase treatment, following the manufacturer's instructions. Copy DNA (cDNA) was synthesized using a SYBR Green Fast Advanced Cells-to-Ct Kit (Invitrogen, Waltham, MA, USA). The quantitative PCR (qPCR) was performed on a 7300 real-time PCR system (Applied Biosystems, Waltham, MA, USA) using SG qPCR Master Mix (EURx, Gdansk, Poland) with the addition of uracil-N-glycosylase (UNG). Amplification was as follows: 1 min at 50 °C, 10 min at 95 °C, 40 cycles of 15 s at 95 °C, 15 s at 55 °C, 45 s at 72 °C finalized by melt curve analysis using instrument default settings. The primers used for GPB1 quantification were PiGPB1-qRT-F and PiGPB1-qRT-R [33]. The reference gene was P. infestans actin, amplified by primers pi-actin-F and pi-actin-R [34]. Three biological and two technical replicates per treatment were used, and the relative expression was calculated using the 2 -ΔΔCT method. The experiment was repeated twice. Data were analyzed with ANOVA followed by Tukey's Honest Significant Difference test for multiple comparison.

Electrophoretic mobility shift assay
To determine the interaction between dsRNA and the CQDs, 200 ng of GPB1-dsRNA were mixed with different amounts of CQDs in a total volume of 10 μL and a final CQDs concentration of 10, 20, 40, 80 and 160 μg/mL. In the same manner high-charge cationic polymer polyethylenimine (PEI) was mixed with dsRNA to test the complexation ability in the following nitrogen-to-phosphate (N/P) ratios: 2, 6, 10, 15 and 30. The reactions were incubated at room temperature for 30 min and mixed by vortexing two times. The amount of 5 μL were mixed with 1 μL 6X loading buffer, loaded in 2% agarose gel and run for 15 min at 150 V in 0.5 TBE buffer. For staining the gels were submerged in a 3X GelRed solution for 30 min, followed by 10 min washing in distilled water. Visualization was performed using an EC3 Imaging System (UVP, Cambridge, UK).

HaCaT cell culture
Immortalized human keratinocytes (HaCaT cells) were used to study the cytotoxicity effect of CQDs. The cells were obtained from the cell culture collection of the in vitro laboratory for evaluation of biological activity and toxicity, BioPharm, Research and Development and Innovation Consortium (Sofia Tech Park, Sofa, Bulgaria). The cell cultures were grown in DMEM medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin. The cultures were kept in a humidified incubator HEPA Class 100, IncuSafe MCO-15 (Panasonic, Amsterdam, The Netherlands) at 37 °C under 5% CO 2 in the air. For routine passages, the cells were detached using a mixture of 0.05% trypsin and 0.02% EDTA and were passaged 2 times per week (1:2 to 1:3 split). The experiments were performed during the exponential phase of cell growth.
Before conducting the experiment, cells were trypsinized with 0.25% trypsin-EDTA solution (Sigma) for 5-7 min, counted and diluted with DMEM medium without added serum. Cells (1 × 10 4 cells) were plated in each well of a 96-well plate (Biologix, Hallbergmoos, Germany). Twenty-four hours after the cells adhered to the plate, the cells were treated with a CQDs solution.

MTT assay
The cells were incubated in medium with CQDs, and were added in concentrations ranging from 1 to 160 µg/mL. The cultures were kept in a humidified incubator HEPA Class 100, IncuSafe MCO-15 (Panasonic) at 37 °C under 5% CO 2 in the air for 24 and 48 h. The MTT test was performed according to Mosmann [35]. Briefly, the cells were incubated for 3 h with MTT solution (0.5 mg MTT in 10 mL DMEM) at 37 °C under 5% CO 2 . The formed blue MTT formazan was extracted with a mixture of absolute ethanol and DMSO (1:1, v/v). The quantitative analysis was performed by absorbance measurements in an automated microplate reader (BioTek Cytation 3 Hybrid Multi-Mode Reader; Agilent Technologies, Santa Clara, CA, USA) at 540/620 nm. Data were exported and processed in Excel and GraphPad Prism 5.0, and the treatments were compared with ANOVA followed by Newman Keuls and Dunnett post-hoc test.

Double staining with acridine orange (AO) and propidium iodide (PI)
The ability of CQDs to induce cytopathological changes was assessed using double staining with acridine orange (AO) and propidium iodide (PI) according to a standard procedure [36]. The cells were seeded 3 × 10 5 cells per well, and 24 h after the cells adhered, they were treated with CQDs. The cells were grown on cover slips in 6-well plates in the presence of CQDs concentrations from 20 to 160 µg/mL. Non-treated cells were used as control. After 48 h of incubation, the cover slips were removed and washed with PBS for 2 min. Equal volumes of fluorescent dyes containing AO (10 µg/mL in PBS) and PI (10 µg/mL in distilled water) were added to the cells. Fresh stained cells were placed on a glass slide and examined under fluorescence microscope BioTek Cytation 3 Hybrid Multi-Mode Reader (Agilent Technologies) within 30 min before the fluorescent color started to fade.

Effect of CQDs on P. infestans
The effect of CQDs on the mycelium of P. infestans was assessed by measuring the radial growth on solid medium after 9 days of incubation. Significant difference between the control treatment and some of the tested CQDs concentrations was found (ANOVA, p < 0.001). The mycelial growth in the treatment containing 20 µg/mL CQDs was reduced by 87%, while the concentration of 40 µg/mL CQDs completely inhibited the P. infestans growth ( Figure 1A). There was no significant difference in the radial growth of the mycelium between the other three CQDs concentrations (2.5, 5 and 10 µg/mL) and the control treatment, however the visual assessment of the cultures revealed an effect to the mycelium density in a dose response manner ( Figure 1B).
In liquid medium, P. infestans sporangia develop by growing hyphae (direct germination) or by releasing zoospores that subsequently encyst (indirect germination). The effect of CQDs to sporangia germination was quantified by measuring the optical density of the medium. No difference between the treatments was evident after the first 24 h (Figure 2). After 48 h, however, a significant difference between the highest CQDs concentration (160 µg/mL) and the water control was found (Tukey's HSD, p = 0.02), and after 72 h, the sporangia development was reduced by 61, 63 and 75% in the treatments containing 40, 80 and 160 µg/mL CQDs, respectively. The microscopic observations of sporangia indicated less germination and restricted hyphal length in correlation with the CQDs concentrations ( Figure 2B)

Effect of CQDs to three other plant pathogenic fungi
After determining the inhibitory activity of CQDs against P. infestans, the effect to other plant pathogenic fungi was investigated. The optical density of the medium was used as an estimate for the spores germination and growth in the presence of CQDs of three species, namely B. cinerea, A. alternata and F. oxysporum. After 24 h of incubation, the absorbance of the media containing the highest CQDs concentrations was significantly lower compared to the control treatments in all the three species (Figure 3). The strongest effect was detected in B. cinerea with about 90% reduction in the absorbance at CQDs concentrations of 80 and 160 µg/mL ( Figure 3A). The absorbance of the medium with spores from A. alternata and F. oxysporum was reduced by 36 and 56%, respectively, in the presence of 160 µg/mL CQDs ( Figure 3B, C).
The inhibitory effect of the CQDs weakened on the second day of incubation in all three fungal species.
After 48 h, the growth of B. cinerea and F. oxysporum exposed to 160 µg/mL CQDs was reduced by 48 and 17%, respectively, while no significant difference in the growth between all the treatments in A. alternata was detected.

Effect of CQDs on RNAi efficiency in P. infestans
The effect of CQDs to the RNAi efficiency in P. infestans was studied by placing sporangia in liquid medium supplemented with a mixture of dsRNA specific for the P. infestans GPB1 gene and CQDs. A sub-inhibitory concentration of CQDs of 10 µg/mL was used and the expression of GPB1 in growing hyphae was measured after 72 h of incubation. As a negative control, dsRNA specific for the GFP gene was used. No difference between the relative expression of GPB1 in P. infestans treated with GPB1-dsRNA alone and treated with GFP-dsRNA was found (p = 0.8267, Tukey's HSD). However, the level of GPB1 transcripts was reduced by about 50% in P. infestans treated with GPB1-dsRNA/ CQDs mixture in comparison with both the GPB1-dsRNA alone (p = 0.001, Tukey's HSD) and the negative control GFP-dsRNA (p = 0.004, Tukey's HSD) (Figure 4).
An electrophoretic mobility shift assay was used to study the interaction between the dsRNA and CQDs in the mixture. The mobility of 100 ng GBP1-dsRNA was compared to 100 ng of GBP1-dsRNA mixed with CQDs in concentration of 10, 20, 40, 80 and 160 µg/mL ( Figure 5A). The migration speed or the band intensity did not differ between the treatments demonstrating lack of interaction between the components. In contrast, when GBP1-dsRNA was mixed with the cationic polymer PEI in different N/P ratios, the dsRNA migration in the gel was highly affected ( Figure 5B). Figure 1. inhibition of the mycelial growth by cqDs in P. infestans. influence of cqDs to the colony diameter of 9-day-old cultures of P. infestans (a). error bars represent the standard deviation of the means from three replicates. Different letters represent significant differences between treatments at p < 0.001 (anoVa followed by tukey hSD test). images of the P. infestans mycelial growth and density on V8 agar plates with different cqDs concentrations (B).

Effect of CQDs on the survival and proliferation of human epidermal keratinocytes
To evaluate the cytotoxicity of the CQDs, normal human keratinocytes were exposed to increasing concentrations, ranging from 1 to 160 µg/mL, and treatment intervals of 24 and 48 h. The cells viability was determined using MTT test. There was no cytotoxic effect at both time intervals based on the percentage of cell survival even at the highest CQDs concentration ( Figure 6A). Notably, an increase in the cells proliferation in some of the treatments was detected after the first 24 h; however, the differences were no longer present after 48 h of incubation.
CQDs concentrations of 20, 40, 80 and 160 µg/mL were used for cell morphology examination by double staining with acridine orange and propidium iodide ( Figure 6B-F). The human keratinocytes treated with CQDs were of a normal shape and size in all the treatments. The cell nuclei were with clearly defined borders and regular oval/round shape with several nucleoli. No vacuoles were observed. Individual cells were in the mitosis stage.

Discussion
There are reports about the antimicrobial activity of the CQDs, but most of them concern bacteria, and very few mention fungi or fungal-like organisms [16,17,19,[37][38][39]. To our knowledge, there is only one study which describes the inhibitory activity of CQDs against an oomycete organism [19], and such has never been investigated in P. infestans. Therefore, in this study, we investigated the effect of different CQDs concentrations on the development of this important plant pathogen. . effect of cqDs on the Rnai efficiency in P. infestans. Relative expression of GPB1 in growing hyphae upon 72 h of incubation after treatment with GFP-dsRna, GPB1-dsRna mixed with cqDs and GPB1-dsRna alone. a significant difference was found between the GPB1 transcripts level in the hyphae growing in the presence of GPB1-dsRna/cqDs mixture compared to both GPB1-dsRna alone and the control GFP-dsRna (F = 11.63, df = 15, p = 0.00089). three biological and two technical replicates per treatment were used and the whole experiment was repeated twice. error bars represent the standard error of the means. Different letters represent significant differences between treatments at p < 0.01 (anoVa followed by tukey's hSD test). The growth of P. infestans mycelium was completely inhibited in the presence of 40 µg/mL CQDs, which is a relatively low concentration, compared for example with 300 µg/mL CQDs, causing the same effect, in another two fungal species, namely Rhizoctonia solani and Pyricularia grisea [18]. Notably, in liquid medium, no effect on sporangia development was detected during the first 24 h. This is probably due to the initially slower development, due to the time needed for sporangia germination. However, the inhibition was much stronger on the second day, and after 72 h it reached up to 75% growth reduction at 160 µg/mL CQDs.
Since P. infestans is not a fungal, but an oomycete species, an intriguing question was if the CQDs would inhibit the growth of other plant pathogenic fungi. Therefore, spores of B. cinerea, A. alternata and F. oxysporum were treated with the same range of CQDs concentrations, and the growth was determined by measuring the optical density of the medium. In contrast with P. infestans, the inhibitory activity was the most significant after the first 24 h, and weakened with time. The effect differed among species: after 48 h the inhibition was moderate in B. cinerea (with 48% growth reduction), low in F. oxysporum (with 17% growth reduction) and absent in A. alternata.
The significant effect of the CQDs on P. infestans together with the species-specific response observed in the other fungal species indicate their broad-spectrum antifungal activity, to which, however, the different species exhibit different sensitivity. Such selective antimicrobial activity was described in silver nanoparticles against different species of bacteria and fungi [40,41]. The distinct response of P. infestans to the CQDs exposure can be explained with the significant differences in physiology and biochemistry between the oomycete and fungal organisms [42].
The mechanisms behind the CQDs activity against fungi are unknown and there are very few studies addressing this question in bacteria. So far the role of the surface functionalization for the antibacterial activity has been proven, and the involvement of light activated accumulation of reactive oxygen species (ROS) and cell wall damage were demonstrated [43,44]. Whether the same mechanisms are involved in the activity against fungi; however, needs further investigation.
Following the assessment of the CQDs antifungal activity, we evaluated the ability of CQDs to increase the RNAi efficiency in P. infestans. The RNAi induced by exogenous dsRNA in plant pathogenic fungi and fungal-like organisms is a promising plant protection alternative; however, its efficiency is strongly dependent on the ability of the species to take up the silencing molecules. It has been demonstrated that the dsRNA uptake by P. infestans is limited and differs among structures, for example, it was observed in some hyphae and in about 5-10% of the sporangia and zoospores [33]. We found that the growth of P. infestans decreases with the increase in the CQDs concentration and we estimated the range of this effect. Hence, a sub-inhibitory dose of 10 µg/mL CQDs was selected and used to treat sporangia in combination with gene specific dsRNA. As a target, we used the gene coding the GPB1 protein. The GPB1 is important for the vegetative growth and the sporangia formation, making it a suitable target to suppress P. infestans development via RNAi [33,45,46]. In another study, GPB1 was efficiently silenced by the introduction of additional copies into the genome, which led to changes in the mycelium morphology and reduced the sporangia production [47]. Gene silencing of GPB1 also restricted the disease progress in transgenic plants demonstrating the highest efficiency among three target genes [45].
To expose the P. infestans sporangia to GPB1-dsRNA and to assess the effect of the CQDs to the RNAi efficiency we used low volume, in vitro culture method, followed by RNA extraction and quantification of the transcripts level by qRT-PCR. The expression level of the GPB1 did not differ between the sporangia treated with naked GPB1-dsRNA and the control GFP-dsRNA, indicating that in vitro treatment with dsRNA alone do not trigger gene silencing in P. infestans.
The efficiency of the RNAi caused by the environmental dsRNA in P. infestans was studied recently by two groups, which, however, report contradictory results [33,48]. Qiao et al. [33] came to a conclusion that the traditional spray-induced gene silencing (SIGS) approach is not effective against P. infestans, since the treatment with naked dsRNA targeting several genes failed to reduce their expression levels and had no effect on the disease severity [33]. On the contrary, Kalyandurg et al. [48] stated that SIGS have a potential to be used for potato blight control, as they obtained the opposite outcomes in a similar experimental setup, also using some of the same target genes (e.g. GPB1 and the haustorium membrane protein PiHMP1). This inconsistency was attributed to methodological differences, such as the application method of the dsRNA on leaves and the inoculum concentration [48]. Although we used different methodology, our results are in better agreement with Qiao et al. [33]. In another recent study, an efficient RNAi in P. infestans was achieved in combination with nanoclay, also confirming the low ability of the naked dsRNA to cause gene silencing [46]. Furthermore, we found that CQDs increase the efficiently of RNAi in P. infestans. A significant reduction (about 50%) in the GPB1 transcripts was found when sporangia were treated with GPB1-dsRNA mixed with CQDs compared to the naked GPB1-dsRNA and to the control GFP-dsRNA. To explain this result, we studied the interaction between the dsRNA and CQDs, using electrophoretic mobility shift assay. The ability of the dsRNA to form a complex was demonstrated with the positively charged PEI; however, no binding between the dsRNA and CQDs was found. An explanation for this lack of interaction would be that both dsRNA and CQDs have the same negative charge. This result demonstrated that the CQDs effect to the gene silencing is not a consequence of the dsRNA/CQDs complexation. A possible explanation is related to the ability of the CQDs to increase cell membrane permeability, which has been observed in human cells [49]. Such an increase would allow the entry of dsRNA in the P. infestans structures and subsequently initiating RNAi, which, however, needs further confirmation.
Any potential use of CQDs would require safety assessment on a case-by-case basis keeping in mind their intended application [50]. Here we evaluated the toxicity of the CQDs to human skin cells and found no increased toxicity, as the cell viability was above 90% in all the treatments. After 48 h even at the highest dose of 160 µg/mL CQDs, the viability of the cells was similar to the control treatment. In several studies, the same types of human cells (HaCaT) are exposed to CQDs reported comparable results. No loss of viability in cells treated with CQDs synthesized from cabbage was measured in the dose range we tested; however, low cytotoxicity occurred at concentrations above 500 μg/mL [51]. The viability of HaCaT cells was 85% after treatment with 200 μg/mL N-doped CQDs [52]. Other studies reported the results from CQDs cytotoxicity assessment using different types of cells (human, mammalian, mouse, etc.) across different concentrations [53]. In general, higher CQDs quantities can have certain cytotoxicity, the magnitude of which varies according to the precursors and surface functionalization [15].
In much rarer occasions, an increase in the cell proliferation due to CQDs treatment has been reported, for example, in standard mouse fibroblasts by negatively charged CQDs, which was accompanied with the accumulation of ROS [15]. We found an increase in cell proliferation, up to 19%, after the first 24 h in some of the CQDs treatments. The cell morphology did not show any alternations and, after 48 h, the cell viability was similar across all treatments.
The potential applications of different types of nanoparticles in agriculture have received growing attention recently, involving the development of nanopesticides, nanofertilizers, plant growth promoters and nanocarriers [54]. Here we determined the inhibitory activity of CQDs against several plant pathogens, as well as their ability to enhance RNAi in P. infestans. The low toxicity toward human cells was also demonstrated. Altogether, the described CQDs characteristics can be useful in the development of new plant protection strategies. However, there is still lack of understanding about the fate of the carbon nanomaterials in the environment, and many questions related to their safety still need to be addressed [55]. The inhibition of fungal and fungal-like plant pathogens by CQDs is intriguing and their possible application as nanopesticides deserves investigating. On the other hand, this rather broad activity implies the need to study their effect on the microbial communities in soils.

Data availability statement
The data that support the findings of this study are available on reasonable request from the corresponding author.