Clay nanosheet-mediated delivery of recombinant plasmids expressing artificial miRNAs via leaf spray to prevent infection by plant DNA viruses

Whitefly-transmitted begomoviruses are economically important plant pathogens that cause severe problems in many crop plants, such as tomato, papaya, cotton, and tobacco. Tomato yellow leaf curl virus (TYLCV) is a typical monopartite begomovirus that has been extensively studied, but methods that can efficiently control begomoviruses are still scarce. In this study, we combined artificial microRNA (amiRNA)-mediated silencing technology and clay nanosheet-mediated delivery by spraying and developed a method for efficiently preventing TYLCV infection in tomato plants. We designed three amiRNAs that target different regions of TYLCV to silence virus-produced transcripts. Three plant expression vectors expressing pre-amiRNAs were constructed, and recombinant plasmid DNAs (pDNAs) were loaded onto nontoxic and degradable layered double hydroxide (LDH) clay nanosheets. LDH nanosheets containing multiple pDNAs were sprayed onto plant leaves. We found that the designed amiRNAs were significantly accumulated in leaves 7 days after spraying, while the pDNAs were sustainably detected for 35 days after the spray, suggesting that the LDH nanosheets released pDNAs in a sustained manner, protected pDNAs from degradation and efficiently delivered pDNAs into plant cells. Importantly, when the LDH nanosheets coated with pDNAs were sprayed onto plants infected by TYLCV, both the disease severity and TYLCV viral concentration in sprayed plants were significantly decreased during the 35 days, while the levels of H2O2 were significantly increased in those plants. Taken together, these results indicate that LDH nanosheets loaded with pDNAs expressing amiRNAs can be a sustainable and promising tool for begomovirus control.


Introduction
Whitefly-transmitted begomoviruses are economically important plant pathogens that cause severe problems in many crop plants, such as tomato, papaya, cotton, and tobacco 1 . Tomato yellow leaf curl virus (TYLCV) is one of the most important viral pathogens of tomatoes. It belongs to the genus Begomovirus and is distributed worldwide, being the 3rd most important plant virus 1,2 . Begomoviruses have circular and single-stranded DNA molecules encapsulated in a twinned (quasi)-icosahedral virion 2 . They are classified into two groups based on their genome organization: monopartite, having a single genome component, and bipartite, having two genome components, DNA-A and DNA-B 3 . DNA-A and DNA-B are 2.7-2.8 kb in size, and each component has its own open reading frames (ORFs) oriented in a bidirectional fashion. Monopartite begomoviruses (or DNA-A of bipartite begomoviruses) have six ORFs, with two in the virion sense orientation (AV1/V1 and AV2/V2, encoding the coat protein (CP) and the V2/AV2 protein, respectively) and four in the complementary orientation (AC1/C1-AC4/C4, encoding the replication associated protein (Rep), or C1/AC1 protein; the transcriptional activator protein (TrAP), or C2/AC2 protein; the replication enhancer protein (REn), or C3/AC3 protein; and the C4/AC4 protein) 3 . TYLCV is a monopartite begomovirus.
Managing plant viruses is difficult because no chemicals are highly effective in suppressing viral infection in plants.
In recent years, interest in gene silencing mediated by artificial microRNAs (amiRNAs) has increased [4][5][6][7] . In this technology, an amiRNA is designed to target genes responsible for viral replication, transmission, or symptom development and interferes with the multiplication and spread of viruses in host plants 4 . amiRNAs are singlestranded (~21 nt long) and are designed by replacing a mature microRNA (miRNA) sequence with a duplex structure within pre-miRNAs 4 . Owing to certain attributes, such as their uniqueness, effectiveness and precision, gene silencing through amiRNAs is considered a second-generation technology of RNA interference (RNAi) 5 . It is easy to optimize single amiRNA sequences to target one or several mRNAs without disturbing the expression of other genes 6,7 .
Delivery of genetic materials and regeneration of transformed plants are very important for genetic transformation of plants, the latter being highly impacted by the liability and stability of the delivery method 8,9 . Methods such as electroporation, biolistics, agrobacterium-mediated delivery, and cationic delivery typically require the regeneration of genetically modified progeny plants. Furthermore, agrobacterium-mediated delivery introduces another pathogen into plants. These methods are not perfect and need improvement in applications [10][11][12][13] . Therefore, there is a need to develop more effective, simple, and economical methods.
To enhance the stability, translation efficiency, and cell targeting of a naked mRNA, nanomaterials have been used as effective transportation tools in the genetic engineering of plants [14][15][16] . Particularly, they have been applied for delivering amiRNAs into plants [17][18][19] . In this way, dsRNAs can be transferred into plant leaves 20 , roots 21,22 and flower buds 23 . Mitter et al. reported that a single spray of dsRNA loaded on a designed, nontoxic, and degradable layered double hydroxide (LDH) (BioClay) can protect plants against pepper mild mottle virus (PMMoV) and cucumber mosaic virus (CMV), which greatly prolongs the protection of plant leaves from 7 to 20 days 20 . dsRNA can enter plants and has a slow release of the RNAs carried with LDH delivery 20 . Recombinant DNA plasmids (pDNAs) are often used in plant genetic engineering, but the delivery of pDNA is an important element affecting the efficiency of plant transformation. The larger and sterically hindered pDNA brings new challenges for the loading and delivery of biomolecules with LDH. The aims of this study were to, for the first time, use LDH nanosheets as a delivery system for transporting recombinant pDNAs into plants and examine the effect of TYLCV suppression in plants.

amiRNA design and preparation of pDNA-LDH nanosheets
To improve amiRNA-mediated resistance in plants against TYLCV, three TYLCV-specific amiRNAs were designed based on the conserved region of the AV1 gene and a partial region of the AV2 gene. The designed amiRNAs were amiR-AV1/AV2-1, amiR-AV1-2, and amiR-AV1-3, which targeted three different regions of the AV1 gene (Table S1). AmiR-AV1/AV2-1 targeted the 5′ end of the AV1 transcript and the middle region of the AV2 transcript, as these two genes overlap each other with two different reading frames. AmiR-AV1-2 and amiR-AV1-3 targeted the middle and downstream regions of the AV1 transcript, respectively. No potential off-target effects of these amiRNAs were observed within the available tomato genome database. All three pre-amiRNAs (Table S3) were detected in the cloned plasmid pBI121 using PCR and sequencing (Fig. S1). The structures of the pre-miR159a backbone were predicted, including those for pre-amiRNA-1, pre-amiRNA-2, and pre-amiRNA-3 (Fig. S2).
To understand how LDH supported pDNA on a pDNA-LDH nanosheet, both LDH and pDNA-LDH were analyzed. LDH nanoparticles, which are made up of positively charged brucite-like layers, were synthesized by a microwave-assisted aqueous coprecipitation method. Energy-dispersive X-ray spectrometry (EDS) analysis gave an approximate chemical formula of Mg16.01Al6.44(OH) 12.78 (NO 3 )2.07(CO 3 )11.69•9.73H 2 O (Fig. S3). As measured by dynamic light scattering, the LDH nanoparticles possessed a positive zeta potential of +44.9 mV. The LDH nanoparticles exhibited a narrow size distribution with an average hydrodynamic diameter of 35.43 ± 0.07 nm (Zaverage particle size) and polydispersity index (PDI) of 0.24, which were much smaller than those of LDHs prepared by hydrothermal treatment (102.56 ± 1.01 nm, with a PDI of 0.16), suggesting that microwave-assisted treatment is a promising way to reduce the preparation time and maintain the necessary morphology.
X-ray diffraction and Fourier transform infrared (FTIR) spectrum analyses of the collected nanomaterials showed typical features of MgAl-LDH-type materials. The X-ray diffraction patterns of LDH samples (Fig. S4a) displayed a series of sharp peaks from basal diffractions at 10.7°, 22°, and 34°corresponding to planes (003), (006), and (009), indicating good crystallinity in the typical layered structure. The basal spacing (0.82 nm) was slightly larger than that reported in the literature, which indicated a greater chance of interlayer ion exchange. Based upon the full width at half maximum at the (003) plane and the Scherrer equation, the thickness of the pristine LDH nanoparticles was calculated to be 6.08 nm. FTIR spectra of LDH samples displayed characteristic absorption bands at 3486 and 1646 cm −1 , which were attributed to stretching vibrations of O-H and bending vibrations of hydroxyl groups of the interlayered water molecules (Fig.  S4b). The peak at 673 cm −1 was assigned to metal-oxygen-metal and metal-O-H stretching. Similar to previous observations, the absorption band at 1376 cm −1 was the asymmetric stretching vibration mode of C-O in carbonate, which overlapped the signal of the stretching vibration of NO 3 − . The morphological features of LDH samples were characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). SEM images showed that the LDH nanoparticles had lateral dimensions varying from 30 to 60 nm while maintaining a narrowly distinguished polygonal plateletlike nanosheet morphology (Fig. S4c). Most sheets were well formed in a hexagonal shape (Fig. S4d, e).

Stability of pDNAs on LDH nanosheets
For pDNA-LDH to provide prolonged pDNA-mediated protection against viruses, pDNA must not only remain on the leaf surface for a longer period than naked pDNA but also withstand field conditions, such as rain, sunlight, and nucleases. Atmospheric CO 2 and moisture can slowly breakdown LDH into biocompatible residue (Mg 2+ , Al (OH) 3 , and NO 3− , and so on), releasing the loaded biomolecules 24 . To evaluate the delivery and release of pDNA bonded on LDH nanosheets, the stability of pDNA-LDH was examined for delivering and releasing pDNA in acidic conditions. The pH increased from acidic to basic values within 60 min from the introduction of diluted nitric acid (pH 3.0) to LDH solution. The initial  Fig. 2b). At pH ≥ 6.0, most of the pDNA loaded on LDH nanosheets did not migrate on the gel during electrophoresis, as indicated by the florescence being retained in the well (Fig. 2b). The ability of the LDH nanosheets to protect loaded pDNA from potential environmental breakdown, such as enzymatic conditions, was investigated by exposure of pDNA and pDNA-LDH to DNase I. Gel electrophoresis showed that the treated pDNA was released from the LDH nanosheets and almost completely degraded ( Fig. 2c-a). The treated pDNA-LDH showed that some of the DNA was released from the LDH nanosheets and incompletely degraded ( Fig. 2c-b), while the other pDNA was released from the LDH nanosheets without degradation, which was indicated by the florescence being retained near the well (Fig. 2c-c). Accordingly, DNase I treatment degraded the naked pDNA to a greater extent than the pDNA bonded to LDH nanosheets, suggesting that the LDH nanosheets protected the pDNAs from DNase I degradation.

Delivery of pDNAs into plant cells and amiRNA expression
To examine the efficiency of foliar application of pDNA-LDH, YOYO-1 was used to label the nanosheets for visualization in plant cells. Once the pDNA/YOYO-1 complexes were formed, they were stable and exhibited a very high affinity between dye and the pDNA. The results indicated that cells of onion epidermis and Nicotiana benthamiana leaves treated with YOYO-pDNA-LDH showed an abundance of YOYO in the nucleus or membranes after 72 h post treatment (Fig. 3).
Systemic movement of YOYO florescence occurred from the outside to the inside of the plant cells in the YOYO-pDNA-LDH-treated plants. These observations suggested that pDNA can be taken up by plant cells with the help of LDH nanosheet transport and release. Moreover, at 35 days after spraying, the pBI121 vector was Fig. 2 Stability of pDNA on layered double hydroxide (LDH) nanosheets examined by acidic and enzymatic treatments. a pH value dynamics over 60 min from the introduction of diluted nitric acid (pH 3.0) to LDH solution. The pH increased from acidic to basic values within 60 min from the introduction of diluted nitric acid (pH 3.0) to LDH solution. The initial pH of 3.0 quickly changed in the first several minutes and stabilized at pH 7.12 after 1 h, indicating dissolution of LDH under acidic conditions. b pDNA-LDH was suspended in solutions at a range of initial pH values (2.0-12.0) and incubated for 24 h. pDNA release was complete in pH ≤ 5.0 solutions. At pH ≥ 6.0, most of the pDNA loaded on LDH nanosheets did not migrate on the gel during electrophoresis, as indicated by the florescence being retained in the well. M = 5 kb DNA ladder. c Treatment of pDNA and pDNA-LDH with DNase I. Gel electrophoresis showed that the treated pDNA was released from the LDH nanosheets and almost completely degraded (a). The treated pDNA-LDH showed that a part of the DNA was released from the LDH nanosheets and incompletely degraded (b). The other pDNA was released from the LDH nanosheets without degradation, which was indicated by the florescence being retained near the well (c). M = 5 kb DNA ladder. pDNA-LDH: pDNA loaded on LDH nanosheets, pDNA: plasmid DNA detected from nearby unsprayed new leaves, which indicated that the pDNA-LDH was delivered systemically from sprayed leaves to unsprayed new leaves in the treated N. benthamiana and Solanum lycopersicum plants (Fig. S5).
Expression of mixed mature amiRNAs was characterized by qRT-PCR using a Mir-X™ miRNA First-Strand Synthesis kit (TaKaRa Bio, USA) for diverse treatments of N. benthamiana and S. lycopersicum to investigate their role in suppressing TYLCV. In N. benthamiana treated with multiple pDNA-LDHs, amiRNA expression was upregulated to levels approximately seventy times that of the control group and greater than those of the other single pDNA-LDH treatments (Fig. 4a). In S. lycopersicum treated with multiple pDNA-LDHs, amiRNA expression was approximately one hundred and ninety times that of the control group (Fig. 4b).
Preventing TYLCV by foliar spray of pDNA-LDH Thirty minutes after foliar spraying, leaf cells and stomata of N. benthamiana and S. lycopersicum plants were not significantly different between pDNA-LDH, pDNA, and control treatments (Fig. S6). At 35 days post-TYLCV inoculation, the infectivity of TYLCV in N. benthamiana and S. lycopersicum plants harboring amiRNA was significantly suppressed compared to those of plants receiving the other treatments (Table S2). Disease incidence and severity were both lower in mixed pDNA-LDH-treated plants than in nontreated plants (Fig. 5a). All plants inoculated with TYLCV showed viral symptoms that spread from the top of the young leaves but the appearance times of symptoms in mixed pDNA-LDH-treated plants were significantly delayed (Fig. 5a). The symptom expression rate of mixed pDNA-LDH-treated S. lycopersicum plants was 41.7% (Table S2) over up to 35 days after inoculation, which was significantly lower than that for other treatments and closely related to the high expression of mixed mature amiRNAs (Fig. 4).
To determine the effect of mixed pDNA-LDH application on TYLCV prevention, the disease severity, accumulation of H 2 O 2 , and viral concentrations in TYLCV-infected N. benthamiana and S. lycopersicum were measured. H 2 O 2 significantly increased in the leaves of the plants treated with pDNA-LDH compared to other treated plants (Fig. 5b). These results suggest that mixed  (Fig. 6a, b).

Discussion
We have demonstrated that plasmids carrying amiRNAs were successfully delivered into plant cells with the help of LDH nanosheets and that the amiRNAs formed in the transformed plant cells were effective in suppressing the replication of TYLCV and symptom expression. The expression of amiRNA in S. lycopersicum leaves treated with multiple LDH-carrying pDNAs was significantly upregulated at 35 days post-TYLCV inoculation. In addition, by using multiple amiRNAs targeting different regions of the CP gene of TYLCV, the strategy reduced the risk of pathogens overcoming RNAi. LDH nanosheets were proven to be an effective carrier and delivery system for pDNA. More importantly, their slow release extended the period of effectiveness and assured long-lasting protection of plants against viral infection.
The stability of pDNA on LDH nanosheets was the key to effectively protecting and releasing pDNAs into different locations of plant cells. LDH nanosheets have been studied for carrying small RNAs and efficiently delivering them to mammalian cells in vitro 25 and for protecting and releasing large double-stranded RNAs into plant cells 20,26 . Genomic DNA fragments of~500-1000 bp 27 , PCR products of 800 bp 28  Solanum lycopersicum (b) after they were sprayed with pBI121-LDH (CK), pDNA1-LDH (amiRNA-AV1/AV2-1), pDNA2-LDH (amiRNA-AV1-2), pDNA3-LDH (amiRNA-AV1-3), and pDNA(1 + 2 + 3)-LDH (mixed with three pDNA-LDH), as determined by qRT-PCR assays. The bars for each column represent standard deviations. Values (means ± SEs) are the averages of three independent experiments, and significant differences (P ≤ 0.05) among treatments in each group are indicated by different letters intercalated in the interlayer galleries of LDHs by anion exchange or a direct self-assembly route and can then be delivered into animal cells. It seems that large and sterically hindered biomolecules such as pDNAs usually adopt a supercoiled structure in solution and do not fully intercalate in the interlayer galleries by means of anion exchange 30 . In this study, we also observed a small amount of incomplete intercalation of pDNA into LDH sheets, which should not impede cellular uptake and/or protection from degradation, which is in agreement with results from Ladewig 30 .
In delivering transgenic materials into plants, pDNA has some advantages over RNA 20 . High-quality pDNA is the key to successful genetic transformation in plants. pDNA can not only be propagated in Escherichia coli (E. coli) but also be produced in large quantities through fermentation. Methods for the rapid production of large amounts of pDNA include bioprocessing, boiling 31 , a modified boiling method 32 and alkaline lysis. Alkaline lysis is the most widely used method for extracting pDNAs from E. coli 33,34 . To achieve alkaline lysis at a large scale for the extraction of pDNAs from E. coli, Wright et al. used a simple stirred tank reactor system and increased the pDNA quantity very quickly by sequentially adding three precipitants, including a selective precipitation step with ammonium acetate 34 . This pDNA could be incorporated  between hydroxide layers by a simple ion-exchange reaction to form bio-LDH nanohybrids with gentle orbital agitation, which is the simplest method of nanocomplex formation 8,25,27,35 . It is a simple, effective, easy-tofollow method for the large-scale application of pDNAs, and it exhibits slow release and does not raise biosafety concerns.
In generating amiRNAs, the choice of target site was crucial in amiRNA-mediated virus resistance 36,37 . Fortunately, it is relatively easy to pick the ideal amiRNA sequences targeting mRNAs without disturbing the expression of other genes. This feature enhances the robustness of this technology 6 . The application of multiple amiRNAs targeting the AV1/AV2 gene of TYLCV resulted in a high level of viral suppression over a certain period of time in this study (Fig. 4). This is thematically sound and promising in practice. This result is supported by results from many other studies. These studies documented effects of viral diseases such as tomato leaf curl New Delhi virus 37,38 , TYLCV-Israel 39 , tomato leaf curl Java virus 40 , cotton leaf curl Burewala virus 41 , and bean golden mosaic virus 42 . Multiple resistance mediated by a single pre-amiRNA was often designed using the conserved sequence of virus genes to inhibit multiple virus strains simultaneously [43][44][45] . In addition, multiple pre-amiRNAs concatenated under the same promoter can express multiple amiRNAs and mediate multivirus resistance in plants 46,47 . These antiviral strategies mediated by amiRNA were simple and feasible in specific experimental operations. Aragao demonstrated the feasibility of genetically engineered Geminivirus-resistant plants 42 . They used RNAi constructs to silence viral genes. This resulted in high resistance to viral infection, with approximately 93% of the plants being symptom free. It is important that LDH-mediated amiRNA is delivered into plants before viral infection for successful resistance. LDH nanomaterials can protect and slow the release of pDNA on the leaves for a period of time. Most likely, nanoparticle delivery in the plant cells was accomplished via natural openings-e.g., stomata (microscopic pores in leaf surfaces), hydathodes (stomata-like openings that secrete water), or plasmodesmata, which was basically consistent with the results reported by many other researchers 8,20 .
In conclusion, LDH-based pDNA is a promising tool for enhancing plant resistance against TYLCV. This method is worthy of further investigation for thorough exploitation of host-induced gene silencing against geminiviruses in future work.
Natural pre-miRNA (ath-miR-159a) was used as a backbone to generate three artificial pre-miRNAs (pre-amiRNAs). The sequences of pre-miR159a were replaced by those of the amiRNAs, and the whole precursors with the amiRNA sequence were synthesized and cloned into pMD19-T (simple) via Xba I and Sac I enzyme cutting sites by Sangon Biotech (Shanghai, China). Subsequently, these sequences were cut from pMD19-T (simple) with Xba I and Sac I digestion enzymes and finally transferred into the binary vector pBI121 (also cut by Xba I and Sac I) via ligation by T4 DNA ligase. Expression of all the pre-amiRNAs was driven by the CaMV 35S promoter and terminated with the NOS terminator. The clones were confirmed by DNA sequencing. Thus, three pre-amiR-NAs, each containing amiR-AV1/AV2-1 (amir159-AV1/ AV2-1), amiR-AV1-2 (amir159-AV1-2), or amiR-AV1-3 (amir159-AV1-3), were cloned into the pBI121 vector and used in this study. Possible folding structures of the predicted transcripts from the pre-amiRNAs were determined using the mfold program (http://mfold.rna.albany. edu/?q=mfold/RNA-Folding-Form).
Preparation of pDNA-LDH nanosheets LDH nanoparticles were made up of positively charged brucite-like layers, which were synthesized by a microwave-assisted aqueous coprecipitation method 24,48 . The approximate chemical formula of LDH nanomaterials was determined by energy-dispersive X-ray spectrometry (EDX-720, Shimadzu, Japan). LDH nanoparticles in suspension were characterized for size distribution and zeta potential using a Nanosizer Nano ZS (Malvern Panalytical Ltd, Malvern, UK). Morphological images of the particles were captured by TEM using a JEM-1400Plus instrument (acceleration voltage of 120 kV, JEOL, Tokyo, Japan) and a ZEISS Merlin scanning electron microscope (SEM, Carl Zeiss SMT AG, Oberkochen, Germany). White pellet-like powder was yielded by freeze-drying a concentrated suspension. The powdery product was analyzed with FTIR spectroscopy using a Brucker TENSOR 27 instrument scanning from 4000 to 400 cm −1 and powder X-ray diffraction using a PANalytical X'pert3 Powder X-ray diffractometer (slit width DS = 1/4°, scanning rate: 5°min −1 , 2θ = 5°-80°, Co Kα radiation wavelength of 0.15418 nm) to verify the chemical composition and crystal structure of the nanoparticles.

Stability of pDNA on LDH nanosheets
To examine the stability of pDNA loaded on LDH nanosheets, acidic and enzymatic treatments were applied. pH changes were monitored by dissolving LDH nanosheets under strongly acidic conditions by adding 5 ml of 2 mM HNO 3 (pH 3.0) to 5 ml of 1 µg/µl LDH nanosheets. pH values were measured at 1, 3, 10, 20, 30, and 60 min after treatment. The release of pDNA from the pDNA-LDH complexes was determined at different pH values. Control-pDNA-LDH (1:10, 10 µl) was first precipitated by adding 30 µl of 1 M NaCl for 20 min. The precipitate was pelleted by centrifugation at 9000 × g for 30 min. Pellets were resuspended in 10 µl solutions (pH 2.0-12.0, adjusted with either HNO 3 or NaOH) and subjected to 1% agarose gel electrophoresis within 5 min of resuspension. Since LDH nanosheet-bound pDNA did not migrate, fluorescence could be observed in the well.

Uptake and transfection of pDNA in plant cells
qRT-PCR assays were performed to evaluate the expression levels of mixed mature miRNAs as previously described after 7 days. Total RNA and small RNA fractions of leaves of N. benthamiana and S. lycopersicum were extracted using a TRIzol (Life Technologies, USA)based method following the manufacturer's protocol. Expression of mixed miRNAs was quantified by qPCR using a Mir-X™ miRNA First-Strand Synthesis kit (TaKaRa Bio, USA), which is a complete, dual function system for performing first-strand cDNA synthesis and qPCR to precisely measure the level of miRNAs. The three upstream primers were miRNA-specific primers, which are described in Table S4. The downstream primers were the mRQ 3′ primer provided in the kit. The results were normalized to the U6 gene of N. benthamiana and S. lycopersicum (the primer sequences amplifying the U6 gene are shown in Table S4). Mixed samples were used in qRT-PCR analysis, and three biologically independent replicates were analyzed for each sample. Plants sprayed with pBI121-LDH were used as controls. Data were analyzed using the 2 −ΔΔCt method. Plants were kept at 25°C with 70% relative humidity and a 16 h/8 h photoperiod in an insect-free greenhouse, and symptom development was examined daily.

Detection of H2O2 in plants
TYLCV-inoculated leaves were subjected to 3,3-diaminobenzidine (DAB) staining 49 to investigate the accumulation of H 2 O 2 and its production location. Leaves of N. benthamiana and S. lycopersicum were excised from the plant and placed in 20 mL tubes, covered with DAB-HCl solution (1 mg/ml, pH 3.8) and incubated in a growth chamber for 8 h at 25°C. Once red-brown DAB solution moved up to the top of leaves along the veins, chlorophyll from whole leaves was removed by directly immersion into a fixing solution (anhydrous ethanol:acetic acid = 3:1) for 24 h. The stained leaves were processed with hydrated trichloroacetaldehyde three times and observed using an optical microscope.

Delivery of pDNA
To determine whether pDNAs were delivered into plant leaves, PCR was conducted on unsprayed new leaf samples of both N. benthamiana and S. lycopersicum. Total DNA of leaves was extracted using a plant genomic DNA extraction kit (TaKaRa, Dalian, China) following the manufacturer's protocol. The primers pBI121-F/pBI121-R (Table S4) were used to amplify the pBI121 vector.

Detection of TYLCV using qPCR
Expression of TYLCV was quantified by targeting leaves from mixed leaves collected from the top, middle, and bottom of plants by qPCR using SYBR Premix Ex Taq (TaKaRa) on an ABI Prism 7500 thermal cycler (Applied Biosystems, Waltham, USA) with the primer pairs Nb-GAPDH-F/Nb-GAPDH-R, TYLCV-YG-3/TYLCV-YG-4, and Tomato25s-Rrna-UNIV (+)/Tomato25s-Rrna-UNIV (−) ( Table S4). Triplicate PCRs were conducted for each DNA sample, and the assay consisted of three technical replicates. Data were analyzed using the 2 −ΔΔCt method.
Detection of TYLCV using ELISA analysis TYLCV was detected from mixed leaves collected from the top, middle and bottom of plants using antigen-coated plate (ACP)-ELISA according to standard methods. The antibody used in ACP-ELISA was reacted against the CP of TYLCV. The reactions were followed by measuring the optical density at 405 nm on a spectrum plate reader (Thermo Fisher Scientific, Waltham, USA). A sample was considered TYLCV positive when the absorbance values were two times higher than those of noninoculated plant samples.