NIR-assisted orchid virus therapy using urchin bimetallic nanomaterials in phalaenopsis

The synthesis route of urchin-like Au/Ag bimetallic nanomaterials was presented elsewhere [12]. In brief, the nanomaterials were fabricated by mixing 20 μl 10 mM chloroauric acid (HAuCl₄), 2 μl 10 mM aqueous silver nitrate (AgNO₃), and 1 ml milli-Q deionized water in a 2 ml centrifugal tube. Then, 4 μl of 100 mM ascorbic acid (AA) was quickly added to the mixture, and vigorously shaken for 20 s. The mixture instantly changed from a light yellowish solution into a bluish solution, implying the fabrication of urchin-like Au/Ag nanomaterials by reduction. After a 1 min reaction, 1 wt% aqueous O-carboxymethylchitosan or fluorescein isothiocyanate (FITC)-capped O-carboxymethylchitosan was chosen as surfactant and added into the solution for reacting one more day. The mixture was centrifuged at 9279 g three times and redispersed into deionized water for further investigation. The morphologies of urchin-like Au/Ag nanomaterials can be altered by tuning metal precursors.

Phalaenopsis 'Sogo Yukidian' was used in the analysis of near-infrared light (NIR)-assisted virus therapy. The plants were kept in a greenhouse with natural light and a controlled temperature of 27/22 °C (day/night). They were checked for the absence of two prevalent orchid viruses, CymMV and ORSV by detecting the coat protein through enzyme-linked immunoassay (ELISA) before experimentation.

The virus-free Phalaenopsis plants were inoculated by agroinfiltration of Agrobacterium tumefaciens containing CymMV or ORSV viral vector before the nanoparticle injection experiment. Agrobacterium was grown overnight at 28 °C and diluted to optical density at 600 nm (OD₆₀₀) = 0.5 with an infiltration buffer (0.5% d-glucose, 50 mM MES, 2 mM Na₃PO₄ċ12H₂O, and 100 μM acetosyringone) [13]. Successful systemic infection with the virus was confirmed by ELISA [14]. For leaf injection, nanoparticles were injected into the leaf surface with a 1 ml syringe in all treatments. After 1 day, the targeted leaf was irradiated using a 980 nm laser and collected 1, 3 and 7 days after treatment. The treated materials were frozen in liquid nitrogen and stored at 80 °C for future investigation.

Approximately 1 mm thick, hand-cut cross-sections of the samples were collected 48 and 72 h after application of FITC-labeled nanoparticles. Then, the cross-sections were observed under a microscope (Leica MZ FL-III). The images of the sections were taken with a Zeiss AxioCam MRc monochrome cooled-CCD camera. Transmission electron microscopic (TEM) and high resolution TEM (HRTEM) images were recorded on a Jeol JEM-2010 electron microscope operating at 200 kV

Total ribonucleic acid (RNA) was extracted from the dissected leaves by using a MaestroZol^TM RNA PLUS Extraction Reagent (Omics Biotechnology), and treated with RNase-free DNase (Stratagene) to remove residual DNA. For quantitative RT-PCR, the RNA template was mixed with KAPA SYBR Fast One-step qRT-PCR Kit (ABI Prism, KAPA Biosystem) on a Biometra RT-PCR system (TAIGEN Bioscience Corporation, Taiwan). The primers for CymMV coat protein gene were as follows: 5'-TACTACGCAAAAGTGGTGTGGAAT-3' as forward primer and 5'-AGAGCATAGAGAGTGTTGGTGGAG-3' as reverse primer. The ORSV coat protein gene was amplified using 5'-AATCAACCTTTGTACCAATTCTCTG-3' as forward primer and 5'-AGACTTGATTGTACGTACCAGTTCC-3' as reverse primer. The housekeeping gene actin (PACT4, AY134752) was amplified using 5'-GCTGGCATATATTGCTCTTGATTAT-3' as forward primer and 5'-ATCCAAACACTGTACTTCCTCTCTG-3' as reverse primer used for data normalization. The PCR reaction conditions were 42 °C for 5 min, then 95 °C for 5 min, and thermal cycling for 40 cycles (95 °C for 3 s and 60 °C for 30 s). The relative quantification was calculated according to the manufacturer's protocols.

The Phalaenopsis leaves (1 g) were homogenized by grinding in a mortar and pestle in 3 ml extraction buffer (100 mM Tris-HCl, pH 8.0, 10 mM KCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 30 mM sodium ascorbate, 0.7% 2-mercaptoethanol, 5 mM dithiothreitol and 2 mM phenylmethylsulfonyl fluoride); the total soluble proteins were then extracted as previously described [14]. The leaf homogenate contained approximately 1.0–1.2 mg of total soluble protein as determined by a Protein Assay Kit (Bio-Rad). The total soluble protein was used to coat the ELISA plate (PerkinElmer) at 10 μg per well in 50 μl of coating buffer (1.59 g l⁻¹ Na₂CO₃, 2.93 g l⁻¹ NaHCO₃, 0.2 g l⁻¹ NaN₃, pH 9.6), and the expression levels of coat protein were measured by ELISA. The ELISA plates were placed at 37 °C overnight, and then washed three times with phosphate buffered saline (PBS) containing 0.05% Tween-20 (Sigma) (PBST). After being washed with PBST, the wells were loaded with 1:1000 diluted rabbit anti-CymMV or anti-ORSV monoclonal antibody at 50 μl per well and incubated at 37 °C for 2 h. The wells were then washed three times with PBST, and loaded with 1:1000 diluted anti-rabbit Immunoglobulin G (IgG) and ABC Kit (Vector Laboratories Inc.). The wells were incubated at 37 °C for 2 h, followed by three washes with PBST. The plates were then developed with ρ-nitrophenylphosphate substrates and detected on an ELISA reader (Thermo) at 405 nm.

To fabricate urchin-like gold/silver bimetallic nanoparticles, the metal precursors HAuCl₄ and AgNO₃ were first mixed in aqueous solution at room temperature. An appropriate amount of aqueous AA solution was added into the mixed solution to fabricate diverse gold/silver bimetallic nanoparticles. Urchin-like, popcorn-like or irregularly spherical bimetallic nanoparticles were obtained in 1 min as varied by the mixed ratio of HAuCl₄/AgNO₃ (Au/Ag). In the reaction, the pH value was kept in an acidic condition (i.e. 3.0–3.4) for the growth of asymmetrical nanoparticles. The bio-friendly stabilizer, 1 wt% aqueous O-carboxymethylchitosan solution, was added into the solution.

Figure 1(A) shows the spiky urchin-like gold/silver bimetallic nanoparticles. As the proportion of gold ions increases, the thorns become fewer and larger. Figures 1(B) and (C) reveal that the morphologies of the nanoparticles become popcorn-like or irregular cores without apparent thorns when the metal precursor Au/Ag ratios increase to 25 or 100. We attribute the mechanism to the deposition of silver atoms on the surface of the pre-formed gold spherical nanostructures, which act as active sites for further growth of the thorns [12]. The calculation of the d-spacings in the HRTEM images shows that the nanoscale thorns grow along the [112] direction, which might be a consequence of a fast growth, i.e. controlled by kinetics, as being often observed in Si nanowires [15]. Figure 1(D) presents a typical single thorn. The crystal contains a large number of stacking-fault defects. The measured d-spacings of 0.235 and 0.201 nm are corresponding to the (111) and (200) planes of the face-centered cubic unit cell. Upon increasing the gold proportion, the urchin-like structure evolves into a popcorn-like structure with relatively smaller numbers and shorter thorns. Figure 1(E) shows an HRTEM image of a thorn from an urchin-like nanoparticle, showing a similar microstructure. Irregular bimetallic nanoparticles were obtained with an increase in Au/Ag ratio to 100. Figure 1(F) reveals that the branches grow to a broader form than those obtained in previous syntheses, and develop as quasi-spherical shapes instead of sharp-tip structures. Many domains can be observed and their crystal orientations are quite random. Moreover, the surface amorphous layers of the nanoparticles as seen in the HRTEM image in figure 1(F) show that the stabilizer molecules, O-carboxymethylchitosan, envelope the nanoparticles and maintain the morphologies or physical properties of the nanomaterials. Thus, the precursor ratio of Au/Ag significantly affects the morphology of the nanoparticles, which in turn changes the corresponding surface plasmon resonance.

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The morphology of metal nanoparticles strongly influences their corresponding extinction wavelength because of changes in surface plasmon resonance. In figure 2, the extinction spectra show that as the aspect ratio of the tips decreases, the surface plasmon resonance bands blue shift from the near infrared to the visible range. The urchin-like Au/Ag NPs, which were produced at an Au/Ag ratio of 10, provide a major 1000 nm near-infrared longitudinal band and a minor 700 nm red light transverse band because of the large aspect ratio of the tips. With the morphology of the nanoparticles changing from an urchin-like to popcorn-like or spherical morphology, the extinction also changes to major visible light because of the decreasing aspect ratio of the nanoparticles. According to previous studies, the Au/Ag NPs present a high light–heat transformation, providing strong local heat after specific surface plasmon band irradiation. The urchin-like Au/Ag NPs were selected as a candidate as photothermal reagents because of their strong near-infrared absorbance, which enables deeper penetration into tissues than that achieved with visible or ultraviolet light.

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Viral diseases affecting orchid flower quality is a critical dilemma in the global orchid industry. No effective solution has been developed for this problem. Although the application of nanotechnology to biology has mostly focused on animal and medical research, we demonstrate a nanomaterial that may be applied in plant science research and in the improvement of crop production. Accordingly, we developed a technical approach for the agricultural application of nanoparticles. In previous research, a mesoporous silica nanoparticle system was developed that can transport DNA and chemicals into tobacco protoplast and the intact leaves of young maize embryos [8]. Corredor et al [6] indicated that nanoparticles can penetrate living pumpkin plant tissue and migrate to different regions of the plant. Thus, further directed delivery of substances into plant cells is a feasible application. In the present study we also demonstrated that nanoparticles can penetrate Phalaenopsis leaf tissue and migrate to different regions of the plant (figure 3).

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To test nanoparticle penetration, labeling with FITC was carried out. Fluorescence microscopy analysis shows that without injection, protoplasts do not exhibit fluorescence in the FITC-specific filter set (figures 3(A), (D), (G)). By contrast, after 48 and 96 h, fluorescence occurs under FITC fluorescence. The fluorescence is expressed in the intercellular spaces of the tissue (figures 3(E) and (F)), indicating that FITC-labeled nanoparticles are present in the plant. The magnified image at the bottom of figures 3(G) and (I) show the same results. The penetration of nanomaterials into tissue can shorten the distance between viral tissue targeting and nanomaterials for enhancing therapeutic efficiency.

To investigate whether nanoparticles diminish virus density, we injected the nanoparticles into the infected Phalaenopsis leaves, and collected the leaves 1, 3 and 7 days after injection. RT-PCR was performed to confirm the expression of CymMV or ORSV coat protein gene. The results show that CymMV coat protein gene expression decreases immediately after injection with the nanoparticles. CymMV transcripts decrease in comparison with the control (injected with sterile distilled water), and messenger RNA (mRNA) expression slightly decreases with laser treatment (figure 4(A)). ORSV coat protein gene expression also shows a decreasing trend in the expression pattern, depending on the time of inoculation, and expression decreases in comparison with the control (injected with sterile distilled water) (figure 4(B)). The results reveal that laser is significantly related to decreased CymMV or ORSV gene expression and protein accumulation by enhancing the effect of nanoparticles. As treatment progressed, however, the transcript level of CymMV and ORSV transcripts gradually decreases. Consistent with this result, CymMV and ORSV coat proteins were also detected by ELISA. We found that in the plant injected with the nanoparticles, CymMV and ORSV coat protein accumulation levels are lower than that in the control. mRNA expression slightly decreases with laser treatment (figure 5).

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We found that CymMV and ORSV expression decreases two- to three-fold, and protein accumulation decreases one- to two-fold, suggesting that nanoparticle injection with laser treatment effectively reduces CymMV and ORSV coat protein gene expression and protein accumulation (figures 4 and 5). Chitosan, which was selected as nanoparticle stabilizer, may inhibit virus growth. Hirano [16] pointed out that chitinase isoforms are one group of pathogenesis-related proteins in plants. As a chitinase isoform, chitosan may be used to promote tissue growth and differentiation in tissue culture, as well as to induce plant defense. Moreover, virus reduction may be attributed to the ability of nanoparticles to convert absorbed near-infrared irradiation into heat (figure 6). Local temperature increases, thereby preventing virus differentiation and directly eliminating the virus. As presented in the results, photo-induced virus elimination efficiently enhances therapeutic behavior. In the future, nanomaterials may be conjugated with specific proteins to target CymMV and ORSV for a more highly efficient treatment system.

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