Rational design of ROS scavenging and fluorescent gold nanoparticles to deliver siRNA to improve plant resistance to Pseudomonas syringae

doi:10.21203/rs.3.rs-3852889/v1

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Research Article

Rational design of ROS scavenging and fluorescent gold nanoparticles to deliver siRNA to improve plant resistance to Pseudomonas syringae

https://doi.org/10.21203/rs.3.rs-3852889/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 29 Jul, 2024

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Bacterial diseases are one of the common issues to result in crop loss world-widely, and the increasing usage of chemical pesticides has caused the occurrence of resistance in pathogenic bacteria and environmental pollution problems. Nanomaterials mediated gene silencing is started to display powerful efficiency and environmental friendliness for improving plant disease resistance. However, the internalization of nanomaterials and the physiological mechanism behind nano-improved plant disease resistance are still rarely understood. Herein, we engineered the polyethyleneimine (PEI) functionalized gold nanoparticles (PEI-AuNPs) with fluorescent property and ROS scavenging activity to act as siRNA delivery platform. Besides the loading, protection and delivering of nucleic acid molecules in plant mature leaf cells by PEI-AuNPs, its fluorescent property further enables the traceability of the distribution of the loaded nucleic acid molecules in cells. In addition, PEI-AuNPs delivery system successfully mediated defense regulated gene silencing, thus improving plant disease resistance by reducing bacterial number, balancing ROS content, enhancing antioxidant enzyme activity, and improving chlorophyll fluorescence performance. Our results showed the benefits of AuNP-based RNAi technology in improving plant disease resistance and the potential of plant nanobiotechnology to protect agricultural production.

Fluorescent gold nanoparticles

RNA delivering

Gene silencing

ROS scavenging ability

Disease resistance

1. Polyethyleneimine functionalized gold nanoparticles (PEI-AuNPs) with fluorescent properties and reactive oxygen species (ROS) scavenging ability are synthesized rationally;

2. PEI-AuNPs with fluorescent properties act as the indicator to evaluate the nuclear acid internalization efficiency;

3. PEI-AuNPs platform successfully mediate AtWRKY1 gene silencing and Pst DC3000 resistance by scavenging ROS, increasing antioxidant enzyme activity, and improving photosynthesis.

Disease is an obvious problem that affects plant growth and has a major impact on global food production. Approximately 10% of the world’s food production is lost each year causing by various diseases, such as viruses, bacteria, fungi, and nematodes [1, 2]. Among these, bacterial plant diseases pose a particular challenge for plant pathologists [3, 4]. Currently, several methods are employed to control bacterial diseases in plants. These methods include the use of plant varieties that are resistant or less susceptible to diseases, interventions involving chemical and/or biological controls, and cultivation practices aimed at reducing the presence of disease-causing agents [3, 5]. Chemical control, in particular, is widely utilized in agricultural production. However, it is important to note that the excessive use of chemicals can lead to the development of antibiotic resistance in pathogens and environmental pollution [6].

RNA interference (RNAi) control is a widely recognized method for disease control. This technology involves the delivery of small RNA molecules, such as double-stranded RNA (dsRNA) or small interfering RNA (siRNA), directly into the plant cells. By efficiently and specifically degrading their complementary mRNA, the RNAi method silences the relative expression level of the targeted gene [7]. Extensive research has demonstrated that genetically generating RNAi transgenic lines targeting negative defense regulated genes is applicable to increase plants’ disease resistance and enhance crop growth [8–10]. However, the process of genetic modification is time-consuming.

Recently, the use of exogenous RNA inducers offers several advantages over genetic modification. [11] Firstly, it provides a quicker and more efficient method for introducing RNAi into plants, bypassing the time-consuming process of generating transgenic lines. Secondly, the exogenous application of RNA inducers allows for precise control over the targeted genes. Thirdly, by designing specific RNA molecules that target disease-related genes, researchers can tailor the treatment to the specific pathogen or disease of interest, which minimizes off-target effects and reduces the potential for unintended consequences. However, the traditional methods of exogenous application of dsRNA/siRNA, like spraying, infiltration, injection, and root/seed soaking [12], or transient methods mediated by agrobacterium, plant virus vector-mediated, and gene gun-mediated approaches suffer similar problems: 1) the efficacy of dsRNA/siRNA internalization in the plant is limited; and 2) RNA has short half-life time and species limitations. Therefore, it is urgent to explore a new gene silencing carrier that helps RNA to get into cells efficiently and extends their duration time in cells.

A promising approach for controlling plant diseases is the use of nano-technology. Nanomaterials can be used as nanocarriers to deliver DNA, RNA, and protein into plants for plant genetic engineering [13, 14]. Single-walled carbon nanotubes have been proven to deliver functional genetic material into chloroplasts and nuclei [15, 16]. Based on this idea, cell-penetrating peptide (CPP)-based nanocarriers [17], layered double hydroxide (LDH) clay nanosheets [18], polymer functionalized graphene oxide nanoparticles (GONs) [19], functionalized carbon dots [20], polyethyleneimine-coated gold nanoclusters (AuNCs) [21], and gold nanoparticles (AuNPs) [22] have been successfully used to deliver siRNA into intact plants and result in gene silencing. These methods probably extend the duration time of RNA and improve the translocation rate of RNA. However, our knowledge about how nanomaterials can be internalized into plant cells and the physiological mechanisms underlying nano-improved plant bacterial disease resistance via mediating gene silencing is still rare. In addition, without targeting genes, polyethyleneimine-coated MXene quantum dots (PEI-MQDs) with reactive oxygen species (ROS) scavenging activity directly enhanced cotton Verticillium. dahlia resistance by reducing the ROS content at the infection site [23]. Therefore, combining ROS scavenging nanomaterials with an RNAi approach to control plant diseases is probably the next generation of nano-technology.

In this study, we optimized gold nanoparticles to enable them with auto-fluorescence and ROS scavenging activity to efficiently deliver siRNA into plant cells and to evaluate the efficacy of rational designed PEI-AuNPs in gene silencing and thus plant disease resistance.

2.1 Chemicals

Polyethyleneimine (PEI, 10 kD and 25 kD), 30% ammonium hydroxide solution, and tetrachloroauric (III) acid (HAuCl₄·3H₂O) were purchased from Sigma-Aldrich. Cy3-ssDNA, siRNA, and primer were obtained from Tsingke. Ultrapure water (18.25 MΩ cm^− 1) provided by the Millipore system was utilized throughout this study.

2.2 Synthesis and purification of PEI-modified gold nanoparticles

The synthesis of PEI-modified gold nanoparticles (PEI-AuNPs) followed the previous method with modification. In Brief, 5 mL solution of 3.2% 10 kD or 25 kD branched PEI was added dropwise to a 20 mL glass bottle containing 5 mL of 0.28% chloroauric acid solution. The mixture was stirred at 1000 rpm continuously for 5 minutes at room temperature (25°C). Then, the mixture was added dropwise to a 50 mL conical flask containing 15 mL of 60% ammonia solution. The reaction was carried out at 30°C for 24 hours with a rotation speed of 500 rpm to obtain PEI-AuNPs. The reaction solution was collected when its color was no longer changed. The solution was filtered with a 0.2 µm filter membrane. Thereafter, a 30 kD dialysis bag was used to dialyze the solution for 24 h to remove the unreacted PEI. The solution of PEI-AuNPs was stored in a refrigerator at 4°C without light. The concentration of PEI-AuNPs was calculated by drying the PEI-AuNPs solution in an oven at 70°C.

2.3 Plant material and growth conditions

Arabidopsis thaliana (Col-0) was used in this study, and the Arabidopsis seeds were directly sown in standard soil mix under the following growth conditions: temperature was 23 ± 2°C, humidity was 60%, light intensity was 200 µmol m^− 2 s^− 1 PAR, photoperiod was 14 h/10 h (light/dark). Arabidopsis plants were grown in a growth chamber (Boante company, Wuhan, China). 4-week-old Arabidopsis seedlings were used.

2.4 Strain culture

Pseudomonas syringae pv. tomato (Pst) DC3000 pathogen is provided by Prof. Tao Chen from Huazhong Agriculture University. Pst DC3000 pathogen was cultured in LB liquid medium containing rifampicin. The medium was incubated at 28°C until the cell concentration reached OD₆₀₀ = 1. After centrifugation, the Pst DC3000 bacterial was resuspended in 10 mM sterile MgCl₂ buffer to reach OD₆₀₀ = 0.001.

2.5 Fluorescence imaging of Arabidopsis leaf cells

To prove that PEI-AuNPs could deliver the nucleic acid molecules, Cy3-ssDNA was synthesized by the company. 30 µL of 20 mM Cy3-ssDNA was mixed 70 µL of 0.9 g/L PEI-AuNPs to get PEI-AuNPs-Cy3-ssDNA solution which was diluted 15 times with leaf infiltration buffer (10 mM MgCl₂ and 10 mM MES), and infiltrated 4-week-old Arabidopsis leaves with a 1 mL needle-free syringe. After 3 h of incubation, the fluorescence imagings of mesophyll cells of leaves were observed by laser scanning confocal microscopy (LASM SP8). Briefly, to prepare the sections, a 5 mm diameter disc was removed from the treated leaves using a hole taker, and then placed with the leaves side up on top of a glass slide. One drop of perfluoronaphthylamine (PFD) was applied to the leaves, and the leaves were covered with a cover slip to ensure that there were no air bubbles. The sections were observed under a Leica laser scanning confocal microscope. LASM settings were as follows: excitation light was 405 nm and 552 nm for dual channels, strength was 20%, PMT1 was 450–490 nm, PMT2 was 560–600 nm, PMT3 was 700–790 nm.

2.6 Gene silencing by PEI-AuNPs-siAtWRKY1

For gene silencing, 20 mM AtWRKY1-targeted siRNA (siAtWRKY1) was loaded on 0.9 g/L PEI-AuNPs at a PEI-AuNPs: siAtWRKY1 = 3: 7 volume ratio (v: v) and diluted 15 times with leaf infiltration buffer, followed by mixing 30 µL siAtWRKY1, 70 µL PEI-AuNPs and diluting the mixture with 1400 µL leaf infiltration buffer. Then PEI-AuNPs-siAtWRKY1 solution was infiltrated into the 4-week-old Arabidopsis leaves with a 1 mL needle-free syringe. Three control solutions were established as follow: 30 µL of siAtWRKY1 was mixed with 70 µL of free RNA water, then diluted 15 times with leaf infiltration buffer, 70 µL of PEI-AuNPs was loaded on 30 µL free-RNA water, then diluted 15 times with leaf infiltration buffer, and 100 µL of free RNA water was diluted 15 times with leaf infiltration buffer. After the post-infiltration for 0, 1, 2, and 3 days, total RNA was extracted from the infiltrated leaves using a Plant RNA Extraction Kit (RN38, Aidlai, Beijing). And cDNA synthesized for RT-qPCR was performed using TRUEscript First Strand cDNA Synthesis (PC5402, Aidlai, Beijing). Primers designed by NCBI to amplify the AtWRKY1 sequence as shows in (Table S1). The qRT-PCR was performed using the BioRad 298 CFX Connect Real-Time PCR System (Bio-Rad, California, USA). Four replicates were analyzed for each treatment with relative gene expression based on 2^−ΔΔCt.

2.7 Pathogenic bacteria infection, detection, and statistics analysis

Arabidopsis leaves were treated with buffer, PEI-AuNPs, PEI-AuNPs-siAtWRKY1 and siAtWRKY1. Two days later, leaves were injected with Pst DC3000 bacteria diluted using 10 mM MgCl₂ OD₆₀₀ = 0.001).

The amount of bacterial was analyzed based on previous publications with brief modifications. To quantify Pst DC3000 bacterial communities in Arabidopsis leaves, leaves were surface-sterilized with 75% ethanol for 1 min and washed twice in sterile water. After air-dried, leaves were imaged and homogenated in 1 mL sterile 10 mM MgCl₂ buffer. Serially diluted in 1 mL (up to 10^− 4 for the PEI-AuNPs-siAtWRKY1 group and up to 10^− 6 for the control, PEI-AuNPs, and siAtWRKY1 group) and plated on LB plates containing rifampicin. Under experimental conditions in this study, plates were incubated in dark conditions in a 28°C incubator. Almost all Pst DC3000 bacteria grew to microcolonies large enough in LB plates within 2–3 days for efficient counting. The number of colonies calculated from the dilution factor was used to infer the total number of bacteria in the leaves. Leaf area was performed by Image J software. Calculate the amount of bacterial growth using the following formula:

Colony Count/Leaf area (CFU/cm²) = Log₁₀[Colony Count * 10^(Dilution Factor + 1)/Leaf Area].

For statistical analysis, the logarithm of bacterial growth could reduce the error and visually show the difference in colony count between different treatment groups.

2.8 DAB staining, NBT staining, ROS determination, and enzyme activity assay

Pst DC3000 can significantly induce the accumulation and bursting of reactive oxygen species in Arabidopsis leaves after infection [24]. DAB (3,3’-diaminobenzidine) and NBT (nitro blue tetrazolium) histochemical staining were determined following the protocol of Kumar et al. [25] with some modifications. In brief, 0.05 g DAB and 0.1 g NBT were dissolved in 45 mL of distilled water (pH adjusted to 3.8 with 0.1 M HCl) and 50 mL sodium phosphate buffer (50 mM, pH = 7.5), respectively. Arabidopsis leaves were immersed in staining solution and rotated at 50 rpm overnight in the dark at the room temperature (25°C). After removal of the staining solution, the leaves were washed with deionized water. The chlorophyll of leaves was removed by using a boiling mixture of ethanol and glycerol (9: 1, v: v). and the photographs were taken with a Cannon 90D camera. The DAB intensity was calculated using Image J software. The determination of H₂O₂ content was performed with an assay kit from Nanjing Jiancheng Biotechnology Co., Ltd. Total protein content was determined by using Coomassie brilliant blue method and measured at 488 nm using a UV-vis spectrophotometer. The superoxide dismutase (SOD) activity was spectrophotometrically determined at 560 nm using the nitro-blue tetrazolium method [26]. The peroxidase (POD) activity was spectrophotometrically assessed by monitoring the formation of guaiacol at 470 nm [27]. The activity of catalase (CAT) was determined according to standard protocols as described by Rezazad Bari et al [28].

2.9 Evaluation of Arabidopsis leaves growth status

The growth status of different treatments was evaluated by leaf size, chlorophyll content index (CCI), chlorophyll content, chlorophyll fluorescence imaging. The leaves were photographed and their size was analyzed using Image J software. CCI was acquired by using a SPAD chlorophyll meter, and each leaf was measured at least six times to obtain an average value. The measurement of chlorophyll content was performed following an earlier publication. Leaf samples (after 3 days of infection) were soaked in a mixture solution containing ethanol and acetone (1: 1, v: v) and rotated at 50 rpm for 24 h in the dark to measure chlorophyll content. To collect the supernatant, the mixed solution was centrifuged at 2000 rpm for 10 min. The absorbance of supernatant was measured at 644 nm and 662 nm by using a UV-vis spectrophotometer. The chlorophyll content was calculated using the following equations:

Chlorophyll a content = (9.784 * A₆₆₂ − 0.99 * A₆₄₄) / leaf quality

Chlorophyll b content = (21.426 * A₆₄₄ − 4.65 * A₆₆₂) / leaf quality

Total chlorophyll content = chlorophyll a content + chlorophyll b content

Where A₆₄₄ and A₆₆₂ were the absorbance values measured at 644 nm and 622 nm, respectively.

2.10 Analysis of chlorophyll fluorescence imaging

Chlorophyll fluorescence imaging of Arabidopsis leaves was performed using the Mini-Imaging-PAM (Walz, Germany), and chlorophyll fluorescence parameters were taken from a previous publication [29]. After 30 min of dark adaptation, the response of the plants was completely open. The operating parameters of the unit were described below: the detection light intensity was 0.5 µmol m^− 2 s^− 1, the actinic light intensity was 200 µmol m^− 2 s^− 1, the saturation pulse light intensity was 2500 µmol m^− 2 s^− 1, the pulse light time was 0.8 s, and the time interval was 20 s. In the absence of actinic light, the initial fluorescence (Fo) and maximum fluorescence in the dark were measured with the saturating pulse light. And the kinetics curve of chlorophyll fluorescence was determined. The steady state was obtained by stimulating normal photosynthesis with actinic light for 5 minutes and was measured.

Fv’/ Fm’ =(Fm’- Fo’)/Fm’

Y(II) = (Fm’-Fs)/Fm’

NPQ = (Fm- Fm’)/Fm

qP = (Fm’-Fs)/(Fm’-Fo’)

qN = (Fm- Fm’)/(Fm-Fo’)

Where the maximum fluorescence yield (Fm’) was obtained after 0.8 s of saturated pulse light irradiation. When photochemical light was turned off and far-red light was turned on, initial fluorescence under light (Fo’).

2.11 Statistical analysis

A difference analysis was performed by a one-way of variance (ANOVA) using SPSS 26.0 (IBM, Inc., Armonk, NY, USA). The graphs were constructed in Excel 2016 (Microsoft, USA) and Origin 2021 (OriginLab, Northampton, MA, USA). Tukey’s t-test was used to evaluate the separation of means, and significant differences were detected at p < 0.05.

3.1 Preparation and characterization of PEI-AuNPs

To synthesize PEI-AuNPs, two sizes of PEI (10 kD and 25 kD) were mixed up with HAuCl₄·3H₂O with the additional ammonium hydroxide. PEI ligands provide a high density of positively charged amino functional groups to improve stability and dispersion in gold nanoparticle synthesis [30, 31]. The pH of the gold precursor solution has a strong influence on the final morphology of gold nanoparticles, and alkaline conditions favour the formation of uniformly sized Au nanoparticles [32]. Ammonium hydroxide enhances the reduction reaction and provides an alkaline environment for the synthesis of PEI-AuNPs, resulting in the fluorescence of PEI-AuNPs. Therefore, we obtained two types of uniformly dispersed spherical PEI-AuNPs (10 kD and 25 kD) (Fig. 1a). The original color of 10 kD PEI-AuNPs was magenta, whereas 25 kD PEI-AuNPs displayed darker magenta color (Fig. S1a). Next, the characteristics of PEI-AuNPs (10 kD and 25 kD) were measured by UV-vis absorption spectra, transmission electron microscopy (TEM), dynamic light scattering (DLS), and zeta potentials (Fig. 1, Fig. S1b and Fig. S2). Interestingly, the two types of PEI-AuNPs displayed almost the similar UV absorption pattern (Fig. S1b), by which the absorbance peaks of 10 kD PEI-AuNPs were at 346 nm and 551 nm and that of 25 kD PEI-AuNPs were at 353 nm and 523 nm. Both PEI-AuNPs were uniformly sized and dispersed, while the diameter of 25 kD PEI-AuNPs was significantly larger than that of 10 kD PEI-AuNPs (8.08 ± 0.24 nm vs 7.33 ± 0.25 nm) (Fig. 1b and Fig S2a-S2b). Although both PEI-AuNPs were positively charged and the hydrodynamic diameters of two types of PEI-AuNPs were not statistically different (Fig. S2c), the zeta potential value of 25 kD PEI-AuNPs was 49.87% higher than that of 10 kD PEI-AuNPs (Fig. S2d) with the value 15.53 ± 2.63 mV and 28.86 ± 3.55 mV for 10 kD and 25 kD PEI-AuNPs, respectively.

Considering PEI ligands that contain positively charged amine groups bind negatively charged nucleic acid through electrostatic adsorption and the greatest positive zeta potential of PEI-functionalized gold nanoclusters allows the highest loading capacity of nucleic acid carriers [21, 33, 34], 25 kD PEI-AuNPs (represented by PEI-AuNPs) was used for siRNA loading and downstream internalization experiments. Before siRNA loading, the core structure of the PEI-AuNPs was observed by the high-resolution transmission electron microscopy (HRTEM) image, which showed that PEI-AuNPs had a lattice spacing of 0.23 nm (Fig. S3). The X-ray diffraction (XRD) pattern of the PEI-AuNPs was also recorded in the range of 10–80 °. The lattice plane (111) was dominant and had a much lower ratio between the intensities of the diffraction peaks above the lattice planes (200), (220), and (311) (Fig. S4). Next, PEI-AuNPs were infiltrated into Arabidopsis leaves to monitor their biocompatibility. After five days of continuous observation, plants infected with PEI-AuNPs grew as healthy as the control groups (Fig. S5), suggesting that PEI-AuNPs exhibit an extremely low level of toxicity and are relatively safe for plants.

To verify whether PEI-AuNPs and nucleic acid molecules, such as siRNA, could form a complex and examine their optimal binding ratio, we assessed the loading efficiency of siRNA by PEI-AuNPs. The binding of siRNA on PEI-AuNPs did not affect the morphology, dispersion, and homogeneity of PEI-AuNPs. The average diameter of PEI-AuNPs-siRNA (8.97 ± 0.24 nm) was marginally statistically larger than that of PEI-AuNPs (Fig. 1c) and the hydrodynamic diameter of PEI-AuNPs-siRNA (12.06 ± 1.18 nm) was 4 times larger than that of PEI-AuNPs (61.17 ± 8.68 nm) (Fig. 1d), indicating loading of RNA was successful and the formation of a supramolecular complex [21]. Consistently, siRNA loading on PEI-AuNPs decreased the zeta potential of PEI-AuNPs-siRNA, but PEI-AuNPs-siRNA remained positively charged (Fig. 1e). However, siRNA loading had no influence on the optical properties of PEI-AuNPs, as reflected by the fluorescence spectra and UV-vis spectra (Fig. 1f, S6). Moreover, agarose gel electrophoresis showed that, in contrast to siRNA alone, PEI-AuNPs and siRNA were able to form a complex so that siRNA did not move in the positive direction of the electrophoresis tank (Fig. 1g). The binding ratio between PEI-AuNPs and siRNA was further measured, by which the ratio of 3: 7 (v: v) allowed the maximal loading of siRNA and was therefore used for subsequent experiments. Overall, though the fluorescence spectra, zeta potential, and TEM images of PEI-AuNPs before and after siRNA loading, siRNA could tightly bind to PEI-AuNPs without altering the morphology, fluorescence property, and colloidal stability of PEI-AuNPs.

3.2 Cy3-ssDNA-loaded PEI-AuNPs internalized into mature plant cells

To evaluate the efficiency of nucleic acid molecules internalization into the mature plant cells through PEI-AuNPs, Cy3 labeled ssDNA oligos were first loaded onto PEI-AuNPs to detect their penetration into epidermal and mesophyll cells. Numerous studies have been conducted on nanomaterials carrying nucleic acids, primarily examining their entry into epidermal cells [35], except for their penetration into mesophyll cells, the crucial functional units in plants. Specifically, 10 mM of Cy3-ssDNA was bound to PEI-AuNPs by a typic 7: 3 (v: v) ratio. After infiltration into the Arabidopsis leaves, the infected plants were placed in the dark for further incubation for about 3 h to avoid the fluorescence quenching of either PEI-AuNPs or Cy3-ssDNA [36, 37]. Next, the confocal microscopy observation showed that both free Cy3-ssDNA and PEI-AuNPs trapped Cy3-ssDNA (AuNPs-Cy3-ssDNA) were localized at leaf epidermal cells, whereas the fluorescence of AuNPs-Cy3-ssDNA was uniformly distributed across the entire leaf surface rather than those free Cy3-ssDNA (Fig. 2a). This is consistent with the fact that PEI-AuNPs enter into tobacco leaves [21, 38]. In addition to the epidermal cells, the fluorescence signal was observed in the mesophyll cells as well, where the Cy3 signal was found to be closely associated with chlorophyll (Fig. S7). This finding suggests that PEI-AuNPs may facilitate the entry of nucleic acids into the functional cells of plants.

In addition, the interactions between nanoparticles, Cy3-ssDNA, and plant cells were further explored utilizing the fluorescence properties of PEI-AuNPs. Indeed, PEI-AuNPs exhibit enhanced penetration capability, therefore allowing PEI-AuNPs-Cy3-ssDNA to easily enter mesophyll cells compared with the control group of Cy3-ssDNA (Fig. 2b). This finding further supports the notion that PEI-AuNPs significantly enhance the efficiency of nucleic acid delivery into plants. Next, the amount of PEI-AuNPs present in Arabidopsis leaves was quantified and a calibration curve was established. By measuring the fluorescence intensity of various concentrations of PEI-AuNPs in Arabidopsis leaf extracts, a linear equation y = 95914x + 4355.9 with an R² value of 0.9978 was established (Fig. 2c). The accuracy of this equation was also assessed with the fact that the actual internalization of PEI-AuNPs per leaf was 0.02 g, while the theoretical internalization per leaf was calculated to be 0.0324 g. Thus, this results in an internalization efficiency of 61.73% for the Arabidopsis leaves.

3.3 PEI-AuNPs-siAtWRKY1 silenced AtWRKY1 to improve plant disease resistance

Since nuclear acids easily entered plant cells with the help of PEI-AuNPs, we next sought to evaluate whether these molecules could be functional by loading AtWRKY1 siRNA. The AtWRKY1 gene has been discovered to have a negative impact on plant resistance to Pst DC3000. AtWRKY1 loss-of-function mutants have impeded the pathogen's propagation, suggesting targeting the suppression of the AtWRKY1 gene appears to be an effective strategy for enhancing plant disease resistance. To dissect the relationship between gene silencing and plant pathogen resistance mediated by PEI-AuNPs, AtWRKY1 siRNA was loaded on PEI-AuNPs by the similar binding ratio as before. Next, Arabidopsis leaves were treated with control buffer, PEI-AuNPs, PEI-AuNPs-siAtWRKY1, and siAtWRKY1, then sampled for 0, 1, 2, and 3 days after growth, respectively, and the total RNA was extracted from the leaves to determine the gene expression of AtWRKY1. The results indicated that there was no significant change in AtWRKY1 expression at 0 and 1 day after treatments (Fig. 3a and 3b). However, after 2 days of treatments, the PEI-AuNPs-siAtWRKY1 group showed a significant reduction in AtWRKY1 expression compared to the control groups (Fig. 3c). The siAtWRKY1 treatment led to a slight decrease, but it was not statistically significant (Fig. 3c). The maximum efficiency of AtWRKY1 silencing was observed on the third day, resulting in an impressive 80% decrease in AtWRKY1 gene expression. In contrast, siRNA alone displayed no effect at all (Fig. 3d).

The maximum efficiency of AtWRKY1 silencing implies that this phase is optimal for testing plant resistance against Pst DC3000. To investigate the impact of silenced AtWRKY1 on disease susceptibility in Arabidopsis, we conducted an experiment where Arabidopsis leaves were infected with Pst DC3000. Prior to the infection, the leaves were pre-treated for three days with a control buffer, PEI-AuNPs, PEI-AuNPs-siAtWRKY1, and siAtWRKY1. Subsequently, the infected area was inoculated with Pst DC3000 with an OD₆₀₀ of 0.001. After three days, the morphological, physiological, and disease susceptibility of the infected leaves were evaluated. Our results showed that Arabidopsis leaves treated with PEI-AuNPs-siAtWRKY1 exhibited minimal disease area (Fig. 4a) and unaffected leave growth (Fig. 4b), with a significantly increased content of total chlorophyll (Fig. 4c). The chlorophyll index was monitored for three consecutive days following Pst DC3000 infection. It was observed that the chlorophyll index continued to decrease as the infection time prolonged (Fig. S8), while treatment with PEI-AuNPs-siAtWRKY1 significantly helped maintain the chlorophyll index (Fig. S8). Furthermore, the number of colonies in infested leaves was quantified by extracting Pst DC3000. Initially, the extract solution was incubated at LB solid medium containing rifampicin to count the number of colonies right after Pst DC3000 infestation. No difference was observed between the treatments, ruling out the bacteria's effect on disease resistance (Fig. 4d and S9a). The colonies were confirmed to be Pst DC3000 using specific 16 s primers (Fig. S9b). Interestingly, after three days of infection, the log₁₀ of bacterial growth number was significantly lower in the PEI-AuNPs-siAtWRKY1 treatment (Fig. 4e), as observed in the serial dilution (Fig. 4f). Collectively, these results indicate that pre-treatment with PEI-AuNPs-siAtWRKY1 enhances plant resistance to Pst DC3000.

3.5 PEI-AuNPs-siAtWRKY1 leads to a reduction of H₂O₂, changes of antioxidant enzyme activity system

Pathogen infections like Pst DC3000 cause a burst of reactive oxygen species (ROS) in plants, resulting in severe growth damage of infected leaves [39]. Extensive studies have shown that delivering ROS-scavenging nanomaterials to plants are able to alleviate abiotic stress [40–42], such as salt and heat stress. These materials are designed as nanozymes possessing ROS removing enzymatic activity. PEI-AuNPs were confirmed to contain ability to scavenge H₂O₂ in vitro with a scavenging rate of 24%, and siAtWRKY1 did not affect the scavenging ability of PEI-AuNPs (Fig. S2). To further investigate whether PEI-AuNPs delivery system contributes plant defense response by regulating ROS system, the level of hydrogen peroxide (H₂O₂), superoxide anion (˙O₂⁻) and ROS scavenging system by antioxidant enzyme were measured. H₂O₂ level was visualized by 3,3'-diaminobenzidine (DAB) staining. DAB is a stain that reacts with H₂O₂ in the presence of certain proteins, such as peroxidases, producing a dark brown precipitate. It is thus used to detect the presence and distribution of H₂O₂ in plant cells. Interestingly, only treatment with PEI-AuNPs-siAtWRKY1, but not PEI-AuNPs, resulted in a decrease in DAB staining (Fig. 5a and S10) and total content of H₂O₂ (Fig. 5b). However, the measurement of superoxide anion (˙O₂⁻) by nitro blue tetrazolium (NBT) straining and the content analysis revealed no significant difference among all treatments (Fig. 5c and 5d). These results suggest that the impact of PEI-AuNPs-siAtWRKY1 primarily lies in its effect on the H₂O₂ level, and PEI-AuNPs mediated AtWRKY1 gene silencing leads to reduced H₂O₂.

Besides, ROS scavenging system by antioxidant enzyme activity is also important for plants survival [43]. In our study, we measured the activities of SOD, POD, and CAT enzymes, and found that only CAT activity (Fig. 5e) increased by PEI-AuNPs-siAtWRKY1 treatment, while SOD (Fig. 5f) and POD activities (Fig. 5g) decreased. Consistently, the total protein content was significantly higher by PEI-AuNPs-siAtWRKY1 treatment (Fig. 5h). These findings suggest that PEI-AuNPs-siAtWRKY1 probably specifically targets the CAT enzyme system.

3.5 AtWRKY1 gene silencing improves plant photosynthetic efficiency

Pst DC3000 infections frequently lead to leaf necrosis and hinder plant growth by affecting the production of photosynthetic assimilates [1]. However, the Arabidopsis leaves treated with PEI-AuNPs-siAtWRKY1 exhibited notably increased total protein content (Fig. 5h), which was likely due to the enhanced photosynthetic assimilates.

Chlorophyll fluorescence imaging is a powerful tool for discriminating photosynthetic function in leaves [44]. To assess the impact on photosynthesis, several chlorophyll fluorescence indices, such as fluorescence origin (Fo), fluorescence maximum (Fm), photochemical efficiency of PSⅡin the light (Y(II)), Nonphotochemical quenching (NPQ), photochemical quenching coefficient (qP), Non-photochemical quenching coefficient (qN), and (electron transport rate) ETR were monitored by imaging and quantification in vivo (Fig. 6a). Fo and Fm represent the minimum and maximum fluorescence intensities of plants after a sufficiently long period of dark acclimation, respectively, and PEI-AuNPs-siAtWRKY1 significantly increased the Fo and Fm of the plant (Fig. 6b and 6c). Y(II) is the actual quantum efficiency of PSII in plants under the action of light [45]. NPQ dissipates the excess light energy absorbed as heat and is an important photoprotective process [46]. PEI-AuNPs-siAtWRKY1 improved Y(II) and NPQ and the differences were statistically significant (Fig. 6d and 6e). qP indicates the proportion of open reaction centers in all PSII reaction centers [47, 48]. qN correlates with non-photochemical burst energy dissipation [49]. PEI-AuNPs-siAtWRKY1 significantly increased qP and qN (Fig. 6f and 6g), suggesting that it can enhance the ability of light energy captured by PSII in plants to be used for photochemical reactions and dissipated for non-photochemical reactions. PEI-AuNPs-siAtWRKY1 was able to increase the level of ETR (Fig. S11), which reflects the actual electron transfer rate [50]. Taken together, The PEI-AuNPs-siAtWRKY1 treated Arabidopsis leaves had better chlorophyll fluorescence compared to other groups (Fig. 6, Fig. S11), indicating AtWRKY1 gene silencing by PEI-AuNPs-siAtWRKY1 could enhance disease resistance by promoting photosynthetic performance.

4.1 Enabling fluorescent property to PEI-AuNPs delivery system is important for its traceability in plants

Numerous extensive studies have consistently shown that multifunctional AuNPs exhibit high biocompatibility, stability, and do not adversely affect cell viability [51, 52]. AuNPs are usually used to deliver nucleic acid molecules based on their diverse properties [22, 38, 51]. These investigations have laid a strong foundation for employing AuNPs as an effective vehicle for nucleic acid delivery. Here, we engineered AuNPs with 10 kD and 25 kD PEI, and imparted them with auto-fluorescence property. Previous studies have reported fluorescence enhancement of gold nanoparticles assisted by specific surface ligands [53]. Compared with previous AuNPs [22, 51], besides the enhanced auto-fluorescence property, the modified PEI-AuNPs had reduced particle size, but improved homogeneity and dispersibility.

The delivery of nucleic acid molecules and their destination after delivery is a key aspect in nano-improved plant disease resistance via mediating gene silencing [54]. Numerous studies have demonstrated that nanoparticles possess the ability to traverse the cell wall and internalize into the plant cell membrane, facilitating the delivery of molecules to the plant nucleus or chloroplast [16, 22]. Nevertheless, Zhang et al. [38] concluded that the transport of biomolecules into plant cells can be independent of the cellular internalization of nanoparticles, and nanoparticle carriers do not need to be internalized into the cell to achieve effective molecular delivery. Notably, we also noticed that nanomaterials and nucleic acid molecules were electrostatically adsorbed. Nucleic acid molecules may dislodge when the nanocarriers are translocated into the cells. By enhancing the reduction reaction, our synthesized PEI-AuNPs possess an inherent fluorescent property, which sets them apart from conventional nanomaterials, modified with organic dye fluorescent molecules [22]. The utilization of photoluminescent nanomaterials offers several advantages, including enhanced optical stability and the ability to easily manipulate their morphology and size. In this study, observing by Cy3-labelled ssDNA attached PEI-AuNPs, Cy3-labelled ssDNA and PEI-AuNPs were visualized simultaneously with confocal microscopy, which allows the evaluation of delivery efficiency of nucleic acid and PEI-AuNPs more accurate. Therefore, fluorescent PEI-AuNPs may enhance living cell observation and expands their potential applications in the field of biology.

4.2 PEI-AuNPs-siAtWRKY1 mediates siRNA delivery and induces efficient gene silencing in plants

Viral vector-mediated VIGS technology and PEG-transformed protoplast regeneration are commonly used methods for transient gene silencing. However, their application is limited to certain species due to host-range limitations [55]. A promising alternative is nanomaterial-mediated gene silencing, a non-viral vector biotechnology that overcomes these constraints. It can internalize into plant cells and deliver siRNA to mature plant tissues without external assistance [56, 57].

Several nanomaterial delivery platforms have been developed to efficiently silence genes in plants, although they may have varying levels of efficacy. For example, GONs demonstrated a 97.2% silencing efficiency in plant tissues after 24 hours, but this effect returned to normal levels after 5 days [19]. Au materials have also been shown to deliver siRNA with different silencing rates. PEI-AuNCs, for instance, achieved a silencing efficiency of 76.5 ± 5.9% when delivering GFP siRNA [21] [24], while AuNPs mediated NPR1 siRNA silencing up to 80% [22]. PEI-modified AuNPs proved to be highly in delivering nucleic acids for gene silencing in plants. By optimizing the ratio of the of PEI-AuNPs-siRNA complex, we were able to significantly enhance the adsorption of siRNA onto PEI-AuNPs and successfully deliver siAtWRKY1 to silence AtWRKY1 in leaf cells. This resulted in a remarkable 70.6 ± 1.5% silencing efficiency, with no observed toxicity. It is important to note that different nanomaterials exhibit varying levels of efficiency in achieving gene silencing in plants, likely due to differences in concentration, properties, and interaction processes with plants. Therefore, further research is warranted to explore how the silencing efficiency of nanomaterials can be improved through the scientific design of these materials.

Although many nano-delivery platforms have been developed and proven to have RNA silencing effects, relatively few studies have established the correlation between silencing effect and biological phenotype. In our study, we established the relationship among PEI-AuNPs-siAtWRKY1, AtWRKY1 gene silencing and disease resistance in Arabidopsis. Consistent with traditional studies, which have shown that loss-of-function of AtWRKY1 mutants improve plant disease resistance [58], we found that AtWRKY1 gene silencing by EI-AuNPs-siAtWRKY1 can achieve the same effect. Therefore, our study paves the way for the application of the PEI-AuNPs siRNA system in the field.

4.3 Use of ROS scavenging and fluorescent PEI-AuNPs to deliver siRNA could be an efficient way to improve plant disease resistance

Reactive oxygen species (ROS) play a crucial role in plant signaling, allowing them to respond to both abiotic and biotic stress. However, an excessive accumulation of ROS can be detrimental, leading to the oxidation of membrane lipids, proteins, and genetic material [43]. Pst DC3000 leads to a large accumulation of reactive oxygen species during the infestation [59]. To combat this, inorganic nanoparticles with enzyme-like properties have been developed to mimic the activity of natural peroxidases and help plants scavenge ROS, thus enhancing their resistance to oxidative stress [40]. However, previous studies on nanomaterial delivery of nucleic acid molecules for gene silencing in plants have overlooked the potential of these nanomaterials to also function as ROS scavengers due to their enzyme-mimetic nature.

In this study, the ability of PEI-AuNPs to scavenge ROS in vitro was demonstrated. Although PEI-AuNPs alone did not exhibit any H₂O₂ reducing activity, the introduction of PEI-AuNPs-siAtWRKY1 successfully silenced AtWRKY1 and helped maintain ROS homeostasis. Specifically, it effectively reduced H₂O₂ level without affecting the˙O₂⁻ level (Fig. 5). Moreover, PEI-AuNPs-siAtWRKY1 not only aid in enhancing CAT enzyme activities for hydrogen peroxide scavenging, but they also exhibit a potential drawback of decreasing the activities of SOD and POD enzymes. This reduction could be attributed to the excessive scavenging of reactive oxygen species (ROS) by PEI-AuNPs or the silencing of the AtWRKY1 gene expression. Nevertheless, this dual effect is consistent with the improved plant health and a decrease in ROS-scavenging enzyme synthesis [26].

Chloroplasts play a crucial role in plant defense against Pst DC3000, which cause significant damage to plant leaves by depleting chlorophyll a and b [60]. This depletion results in the development of pronounced chlorosis symptoms, characterized by a rapid and widespread yellowing of the affected leaves [60, 61]. Our study demonstrated that the use of PEI-AuNPs-siAtWRKY1 effectively suppressed the expression of AtWRKY1, resulting in elevated chlorophyll levels and enhanced photosynthetic performance. This improvement in photosynthesis holds great significance for crop production as it directly correlates with increased crop yield. Consequently, the gene silencing capabilities of PEI-AuNPs offer promising prospects for enhancing crop production efficiency by targeting negatively regulated genes.

This study evaluated the loading efficiency of fluorescent PEI-AuNPs with siRNA, the internalization efficiency of PEI-AuNPs into plant cells, and the co-localization between PEI-AuNPs and nucleic acid molecules during delivery. PEI-AuNPs-siAtWRKY1 effectively induced AtWRKY1 gene silencing, with a silencing efficiency of up to 71%. Additionally, the results of bacterial resistance assay, ROS content assay, enzyme activity, and chlorophyll fluorescence assay showed that the PEI-AuNPs-siAtWRKY1 treated group exhibited enhanced disease resistance in Arabidopsis. Hence, PEI-AuNPs offer a promising solution for delivering siRNA to plants, facilitating efficient gene silencing. It further indicates that biocompatible PEI-AuNPs with fluorescence hold immense potential as carriers for enhancing plant disease resistance, which presents a novel strategy for breeding with improved disease resistance.

Supplementary Information

The supplementary materials are available at the online version.

Acknowledgements

We thank Mr. Jianbo Cao and Ms. Limin He for their help in TEM imaging at Public Laboratory of Electron Microscopy, Huazhong Agricultural University.

Authors’ contributions

Jie Qi: Data curation, Formal analysis, Validation, Writing-original draft. Yanhui Li: Data curation, Methodology, Writing-original draft. Xue Yao: Methodology. Guangjing Li: Methodology. Wenying Xu: Formal analysis. Zhouli Xie: Writing-original draft. Jiangjiang Gu: Writing-original draft. Zhaohu Li: Supervision, Conceptualization, Funding acquisition, Formal analysis, Writing-review & editing. Honghong Wu: Supervision, Conceptualization, Funding acquisition, Formal analysis, Writing-review & editing.

Funding

This work was supported by the National Key Research and Development Program of China (2022YFD2300205), the National Natural Science Foundation of China (No. 32120103008, 32071971), the HZAU-AGIS Cooperation Fund (SZYJY2021008), Fundamental Research Funds for the Central Universities (2662023ZKPY002), and the Hubei Agricultural Science and Technology Innovation Center Program (2021-620-000-001-032).

Data availability

All data generated or analyzed during this study are included in this published article.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing financial interests.

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You are reading this latest preprint version

Rational design of ROS scavenging and fluorescent gold nanoparticles to deliver siRNA to improve plant resistance to Pseudomonas syringae

Status:

Journal Publication

Version 1

Abstract

Figures

Highlights

1. Introduction

2. Materials and methods

2.1 Chemicals

2.2 Synthesis and purification of PEI-modified gold nanoparticles

2.3 Plant material and growth conditions

2.4 Strain culture

2.5 Fluorescence imaging of Arabidopsis leaf cells

2.6 Gene silencing by PEI-AuNPs-siAtWRKY1

2.7 Pathogenic bacteria infection, detection, and statistics analysis

2.8 DAB staining, NBT staining, ROS determination, and enzyme activity assay

2.9 Evaluation of Arabidopsis leaves growth status

2.10 Analysis of chlorophyll fluorescence imaging

2.11 Statistical analysis

3. Results

3.1 Preparation and characterization of PEI-AuNPs

3.2 Cy3-ssDNA-loaded PEI-AuNPs internalized into mature plant cells

3.3 PEI-AuNPs-siAtWRKY1 silenced AtWRKY1 to improve plant disease resistance

3.5 PEI-AuNPs-siAtWRKY1 leads to a reduction of H₂O₂, changes of antioxidant enzyme activity system

3.5 AtWRKY1 gene silencing improves plant photosynthetic efficiency

4. Discussion

4.1 Enabling fluorescent property to PEI-AuNPs delivery system is important for its traceability in plants

4.2 PEI-AuNPs-siAtWRKY1 mediates siRNA delivery and induces efficient gene silencing in plants

5. Conclusions

Declarations

References

Supplementary Tables

Additional Declarations

Status:

Journal Publication

Version 1

Rational design of ROS scavenging and fluorescent gold nanoparticles to deliver siRNA to improve plant resistance to Pseudomonas syringae

Status:

Journal Publication

Version 1

Abstract

Figures

Highlights

1. Introduction

2. Materials and methods

2.1 Chemicals

2.2 Synthesis and purification of PEI-modified gold nanoparticles

2.3 Plant material and growth conditions

2.4 Strain culture

2.5 Fluorescence imaging of Arabidopsis leaf cells

2.6 Gene silencing by PEI-AuNPs-siAtWRKY1

2.7 Pathogenic bacteria infection, detection, and statistics analysis

2.8 DAB staining, NBT staining, ROS determination, and enzyme activity assay

2.9 Evaluation of Arabidopsis leaves growth status

2.10 Analysis of chlorophyll fluorescence imaging

2.11 Statistical analysis

3. Results

3.1 Preparation and characterization of PEI-AuNPs

3.2 Cy3-ssDNA-loaded PEI-AuNPs internalized into mature plant cells

3.3 PEI-AuNPs-siAtWRKY1 silenced AtWRKY1 to improve plant disease resistance

3.5 PEI-AuNPs-siAtWRKY1 leads to a reduction of H2O2, changes of antioxidant enzyme activity system

3.5 AtWRKY1 gene silencing improves plant photosynthetic efficiency

4. Discussion

4.1 Enabling fluorescent property to PEI-AuNPs delivery system is important for its traceability in plants

4.2 PEI-AuNPs-siAtWRKY1 mediates siRNA delivery and induces efficient gene silencing in plants

5. Conclusions

Declarations

References

Supplementary Tables

Additional Declarations

Status:

Journal Publication

Version 1

3.5 PEI-AuNPs-siAtWRKY1 leads to a reduction of H₂O₂, changes of antioxidant enzyme activity system