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

Journal of Nanobiotechnology volume 22, Article number: 446 (2024) Cite this article

Abstract

Bacterial diseases are one of the most common issues that result in crop loss worldwide, and the increasing usage of chemical pesticides has caused the occurrence of resistance in pathogenic bacteria and environmental pollution problems. Nanomaterial mediated gene silencing is starting to display powerful efficiency and environmental friendliness for improving plant disease resistance. However, the internalization of nanomaterials and the physiological mechanisms behind nano-improved plant disease resistance are still rarely understood. We engineered the polyethyleneimine (PEI) functionalized gold nanoparticles (PEI-AuNPs) with fluorescent properties and ROS scavenging activity to act as siRNA delivery platforms. Besides the loading, protection, and delivery 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. Additionally, the PEI-AuNPs-based RNAi delivery system successfully mediated the silencing of defense-regulated gene AtWRKY1. Compared to control plants, the silenced plants performed better resistance to Pseudomonas syringae, showing a reduced bacterial number, decreased ROS content, increased antioxidant enzyme activities, and improved chlorophyll fluorescence performance. Our results showed the advantages of AuNP-based RNAi technology in improving plant disease resistance, as well as the potential of plant nanobiotechnology to protect agricultural production.

Highlights

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

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

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

Introduction

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 due to 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].

Controlling RNA interference (RNAi) 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. The RNAi method silences the relative expression level of the targeted gene by efficiently and specifically degrading its complementary mRNA [7]. Extensive research has demonstrated that genetically generating RNAi transgenic lines targeting negative defense-regulated genes are applicable to increase plants’ disease resistance and enhance crop growth [8,9,10]. However, the process of genetic modification is time-consuming.

Recently, the use of exogenous RNA inducers has offered 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. Third, researchers can tailor the treatment to the specific pathogen or disease of interest by designing specific RNA molecules that target disease-related genes. This 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 a short half-life and species limitations. Therefore, it is urgent to explore a new gene silencing carrier that helps RNA get into cells efficiently and extends its duration in cells.

Nanotechnology is a promising approach for controlling plant diseases. Nanomaterials can be used as nanocarriers to deliver DNA, RNA, and protein into plants for plant genetic engineering [13]. Single-walled carbon nanotubes have been proven to deliver functional genetic material into chloroplasts and nuclei [14, 15]. Based on this idea, cell-penetrating peptide (CPP)-based nanocarriers [16], layered double hydroxide (LDH) clay nanosheets [17], polymer functionalized graphene oxide nanoparticles (GONs) [18], functionalized carbon dots [19], polyethyleneimine-coated gold nanoclusters (AuNCs) [20], and gold nanoparticles (AuNPs) [21] have been successfully used to deliver siRNA into intact plants and result in gene silencing. These methods probably extend the RNA’s duration and improve its translocation rate. 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 [22]. Therefore, combining ROS scavenging nanomaterials with an RNAi approach to control plant diseases is probably the next generation of nanotechnology.

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 rationally designed PEI-AuNPs in gene silencing and thus plant disease resistance.

Materials and methods

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.

Synthesis and purification of PEI-modified gold nanoparticles

The synthesis of PEI-modified gold nanoparticles (PEI-AuNPs) followed the previous method with modifications. In brief, a 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 1,000 rpm continuously for 5 min at room temperature (25 °C). Then, the mixture was added dropwise to a 50 mL conical flask containing 15 mL of a 60% ammonia solution. The reaction was carried out at 30 °C for 24 h with a rotation speed of 500 rpm to obtain PEI-AuNPs. The reaction solution was collected when its color 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.

Plant material and growth conditions

Arabidopsis thaliana (Col-0) was used in this study, and the Arabidopsis seeds were directly sown in a standard soil mix under the following growth conditions: The temperature was 23 ± 2 °C, the humidity was 60%, the light intensity was 200 µmol m− 2 s− 1 PAR, and the 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.

Strain culture

The Pseudomonas syringae pv. tomato (Pst) DC3000 pathogen is provided by Prof. Tao Chen from Huazhong Agriculture University. The 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 bacteria were resuspended in 10 mM sterile MgCl2 buffer to reach OD600 = 0.001.

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 with 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 imaging of mesophyll cells in leaves was 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.

Assessment of PEI-AuNPs absorption in leaves

The efficiency of PEI-AuNPs in Arabidopsis leaves was determined using the method described in Avellan et al. paper [23]. PEI-AuNPs were infiltrated into Arabidopsis leaves and were incubated for 3 h at darkness. Subsequently, the leaves were washed with a 10− 3 M KCl solution for 1 min. Then, the leaves were sealed in the envelope bags and drying it for 72 h at 105 °C. The analysis of Au content in the dried leaves was measured by ICP-MS (Aglient 7800, USA). Moreover, the dissolution of PEI-AuNPs was assessed by an in vitro experiment. Briefly, Arabidopsis leaves mixed with PBS buffer (0.05 M, pH = 7.8) were ground by a grinding instrument (65 Hz, 300s, Tissuelyser-32 L, Shanghai Jingxin Industrial Development Co., Ltd.). The final leaf extract solution was incubated with 0.06 g/L PEI-AuNPs on a shaker (200 rpm) for 120 h. 2 mL mixture solution was added into a dialysis bag (MWCO = 30,000). The dialysis bag was then put into a falcon tube containing 50 mL of ddH2O for 120 h. Then, the collected eluent was filtered through a 220 nm pore size syringe filter (Merck Millipore Ltd.). The Au content was measured by ICP-MS (Aglient 7800, USA).

The stability and dislodging assay of PEI-AuNPs-nucleic acids complex

To evaluate the stability of PEI-AuNPs and nucleic acids (Cy3-ssDNA) complex, the Cy3 fluorescence under different pH conditions was measured. In brief, PEI-AuNPs and Cy3-ssDNA were mixed at a ratio of 3:7 (v: v), and the mixture solution was diluted by 15 times with different pH solutions (pH 5, 6, 7, and 8). Subsequently, the diluted solutions were transferred to a dialysis bag (MWCO = 30,000). Then, the dialysis bag was immersed in a 50 mL centrifuge tube filled with 40 mL ddH2O for dialysis. The Cy3 fluorescence intensity of the dialysate was measured using a fluorescence spectrophotometer at 0 h, 2 h, and 8 h respectively. Similarly, to evaluate the dislodging ability of PEI-AuNPs and nucleic acid complex, PEI-AuNPs-Cy3-ssDNA complex was mixed with a 100 nM H2O2 solution, and then transfer it to a dialysis bag (MWCO = 30,000). The dialysis bag was immersed into a 50 mL centrifuge tube filled with 40 mL of ddH2O for dialysis. The Cy3 fluorescence intensity of dialysate was measured at 0, 2, and 8 h, respectively.

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 the 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 follows: 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 period 0, 1, 2, and 3 days, total RNA was extracted from the infiltrated leaves using a Plant RNA Extraction Kit (RN38, Aidlai, Beijing). The cDNA synthesized for RT-qPCR was performed using TRUEscript First Strand cDNA Synthesis (PC5402, Aidlai, Beijing). Primers were designed by NCBI to amplify the AtWRKY1 sequence, as shown 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. Moreover, the Western blot test was conducted as described elsewhere.

Pathogenic bacterial infection, detection, and statistical 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 number of bacteria 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 being air-dried, leaves were imaged and homogenated in 1 mL of sterile 10 mM MgCl2 buffer. Serially diluted in 1 mL (up to 10− 4 for the PEI-AuNPs-siAtWRKY1 group and up to the 10− 5 for the control, PEI-AuNPs, and siAtWRKY1 groups) 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. The 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.

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 a staining solution and rotated at 50 rpm overnight in the dark at room temperature (25 °C). After the removal of the staining solution, the leaves were washed with deionized water. The chlorophyll of the 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 the 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].

Evaluation of Arabidopsis leaf growth status

The growth status of different treatments was evaluated by leaf size, chlorophyll content index (CCI), chlorophyll content, and 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 the 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.

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 a saturating pulse light. The kinetic curve of chlorophyll fluorescence was determined. The steady state was obtained by stimulating normal photosynthesis with actinic light for 5 min 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’).

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’).

Statistical analysis

A difference analysis was performed by a one-way analysis 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. ** represents for p < 0.01. *** represents p < 0.001.

Results

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]. Due to the high charge density inherent in both PEI and nucleic acid molecules, PEI amines sustain polymer stability through robust electrostatic interactions, predominantly with the phosphate groups of nucleic acids, while occasionally engaging with groove atoms within the nucleic acid structure [32, 33]. 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 [34]. Ammonium hydroxide enhances the reduction reaction and provides an alkaline environment for the synthesis of PEI-AuNPs, resulting in their fluorescence. 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 a 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), in which the absorbance peaks of 10 kD PEI-AuNPs were at 346 nm and 551 nm and those 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 values of 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 [20, 35, 36], 25 kD PEI-AuNPs (represented by PEI-AuNPs) were 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 °C. The diffraction peaks above the lattice planes (200), (220), and (311) had a much lower ratio between the intensities than those above the lattice plane (111), which was the dominant plane (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. Furthermore, the dissolution assay showed that the detected content of dissolved gold content from the eluent after dialysis of the mixture of PEI-AuNPs and leaf extracts (3 h of incubation) is 49.62 ± 0.57 ng, which only accounts for 4.14% of the used PEI-AuNPs (1.2 µg) in the mixture, showing the good stability of PEI-AuNPs in leaf extracts (Fig. S6).

We assessed the loading efficiency of siRNA by PEI-AuNPs to verify whether PEI-AuNPs and nucleic acid molecules, such as siRNA, could form a complex and examine their optimal binding ratio. The binding of siRNA on PEI-AuNPs did not affect the morphology, dispersion, or 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 the loading of RNA was successful and the formation of a supramolecular complex [20]. The zeta potential of PEI-AuNPs (28.86 ± 3.55 mV) was consistently dropped when siRNA was loaded on them, but PEI-AuNPs-siRNA (13.57 ± 2.88 mV) 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, S7). Besides that, agarose gel electrophoresis showed that PEI-AuNPs and siRNA were able to form a complex, unlike siRNA alone. So, 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, and the ratio of 3: 7 (v: v) allowed the maximal loading of siRNA and was therefore used for subsequent experiments. The results of Cy3-ssDNA release by PEI-AuNPs-Cy3-ssDNA under different pH conditions indicated that significant amounts of free Cy3-ssDNA were released into the external fluid of the dialysis bag after 2 h. However, the Cy3-ssDNA adsorbed by PEI-AuNPs remained undetected in the external fluid of the dialysis bag across different pH environments. This observation suggests that PEI-AuNPs-nucleic acid complex exhibited good stability, at least under different pH (Fig. S6). Overall, through 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, or colloidal stability of PEI-AuNPs.

Fig. 1
figure 1

The synthesis and characterization of PEI-AuNPs and PEI-AuNPs-siRNA. (a) Schematic illustration of the synthesis procedure to obtain PEI-AuNPs and PEI-AuNPs-siRNA; (b-c) TEM images of 25 kD PEI-AuNPs (b) and 25 kD PEI-AuNPs-siRNA; (d) The hydrodynamic diameters of AuNPs and AuNPs-siRNA (n = 4 samples per group); (e) Zeta potentials of PEI-AuNPs and PEI-AuNPs-siRNA (n = 4 samples per group); (f) Fluorescence emission spectra of PEI-AuNPs and PEI-AuNPs-siRNA under 405 nm excitation; (g) Gel electrophoresis images of different ratios of PEI-AuNPs and siRNA. **p < 0.01. The data presented were the mean ± SE

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

To evaluate the uptake efficiency of nucleic acid molecules into 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 [37], 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 [38, 39]. 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 that of free Cy3-ssDNA (Fig. 2a). This is consistent with the fact that PEI-AuNPs enter tobacco leaves [20, 40]. 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. S9). This finding suggests that PEI-AuNPs may facilitate the entry of nucleic acids into the functional cells of plants.

Fig. 2
figure 2

Internalization of Cy3-ssDNA-PEI-AuNPs into Arabidopsis leaf cells. (a-b) Confocal images of Cy3-ssDNA, PEI-AuNPs and PEI-AuNPs-Cy3-ssDNA in plant epidermal cells (a) and mesophyll cells (b) 3 h post infiltration; (c) A calibration curve was established by measuring the fluorescence intensity of various concentrations of PEI-AuNPs in Arabidopsis leaf

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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, allowing PEI-AuNPs-Cy3-ssDNA to enter mesophyll cells more easily than 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 by 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, the Arabidopsis leaves have an uptake efficiency of 61.73%. In addition, the results of the ICP-MS analysis assay showed that the Au content in PEI-AuNPs treated Arabidopsis leaves was 69.98 ± 0.73 µg/g, accounting for 58.3% of the total Au content in applied PEI-AuNPs (Fig. S10).

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 that 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 with a 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 days after treatments (Fig. 3a and b). 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). This is further confirmed by the Western blot test, showing that after 3 days treatment, the PEI-AuNPs-siAtWRKY1 group showed the lowest band intensity compared with control, PEI-AuNPs, and siAtWRKY1 groups (Fig. S11). Overall, the above results clearly showed that compared with no gene silencing in treatment group with single used siAtWRKY1, use of AuNPs to deliver siAtWRKY1 helped to silence the targeted AtWRKY1 gene.

Fig. 3
figure 3

Arabidopsis leaf extracts were used for qRT-PCR analysis to quantify the fold changes of AtWRKY1 mRNA expression at 0 (a), 1 (b), 2 (c), and 3 (d) days after infiltration with buffer control, PEI-AuNPs, PEI-AuNPs-siAtWRKY1, and siAtWRKY1. (a-d) n = 4 samples per group. The data presented were the mean ± SE

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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 susceptibilities 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 leaf growth (Fig. 4b), with a significantly increased content of total chlorophyll (Fig. 4c). The chlorophyll index was monitored for three consecutive days following the Pst DC3000 infection. It was observed that the chlorophyll index continued to decrease as the infection time prolonged, while treatment with PEI-AuNPs-siAtWRKY1 significantly helped maintain the chlorophyll index (Fig. S12). Furthermore, the number of colonies in infested leaves was quantified by extracting Pst DC3000. Initially, the extract solution was incubated in 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 S13a). The colonies were confirmed to be Pst DC3000 using specific 16 s primers (Fig. S13b). 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). It should be noted that no statistically significant difference of the colony counts was observed between the cohort subjected to PEI-AuNPs treatment group and the control group, indicating that the use of PEI-AuNPs does not directly reduce bacterial numbers. Collectively, these results indicate that pre-treatment with PEI-AuNPs-siAtWRKY1 enhances plant resistance to Pst DC3000.

Fig. 4
figure 4

PEI-AuNPs-siAtWRKY1 silenced AtWRKY1 to improve plant disease resistance. Phenotype (a), leaf area (b) and chlorophyll content of leaves from buffer control, PEI-AuNPs, PEI-AuNPs-siAtWRKY1, and siAtWRKY1 groups after 3 days of Pst DC3000 infection; (d-e) Statistically quantify the number of colonies of Pst DC3000 in buffer control, PEI-AuNPs, PEI-AuNPs-siAtWRKY1, and siAtWRKY1 groups at day 0 (d) and day 3 (e) of infection, respectively; (f) Images of Pst DC3000 cultured in LB solid medium containing rifampicin resistance at day 3 of infection. (b-e) n = 6 samples per group. The data presented were the mean ± SE

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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 to infected leaves [41]. Extensive studies have shown that delivering ROS-scavenging nanomaterials to plants are able to alleviate abiotic stress [42,43,44], such as salt and heat stress. These materials are designed as nanozymes possessing ROS removing enzymatic activity. PEI-AuNPs were confirmed to contain the ability to scavenge H2O2in vitro with a scavenging rate of 24%, and siAtWRKY1 did not affect the scavenging ability of PEI-AuNPs (Table S2). To further investigate whether the PEI-AuNPs delivery system contributes to plant defense responses by regulating the ROS system, the levels of hydrogen peroxide (H2O2), superoxide anion (˙O2−) and ROS scavenging systems by antioxidant enzymes 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 S13) and total H2O2 content (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 d). 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, the ROS scavenging system’s antioxidant enzyme activity is also important for plant survival [45]. 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.

Fig. 5
figure 5

AtWRKY1 silencing led to reduced H2O2 levels and systemic changes in antioxidant enzyme activity. (a-d) DAB staining (a), H2O2 content (b), NBT staining (c), and ·O2− content (d) of leaves from buffer control, PEI-AuNPs, PEI-AuNPs-siAtWRKY1, and siAtWRKY1 groups after 3 days of Pst DC3000 infection; enzyme activities of SOD (e), POD (f), CAT (g) in leaves of buffer control, AuNPs, AuNPs-siAtWRKY1 and siAtWRKY1 groups after 3 days of Pst DC3000 infection; (h) total protein content of buffer control, PEI-AuNPs, PEI-AuNPs-siAtWRKY1 and siAtWRKY1 groups. (a-h) n = 6 samples per group. The data presented were the mean ± SE

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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 assimilation.

Chlorophyll fluorescence imaging is a powerful tool for discriminating photosynthetic function in leaves [46]. To assess the impact on photosynthesis, several chlorophyll fluorescence indices, such as fluorescence origin (Fo), fluorescence maximum (Fm), photochemical efficiency of PSII in the light (Y(II)), nonphotochemical quenching (NPQ), photochemical quenching coefficient (qP), nonphotochemical 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 c). Y(II) is the actual quantum efficiency of PSII in plants under the action of light [47]. NPQ dissipates the excess light energy absorbed as heat and is an important photoprotective process [48]. PEI-AuNPs-siAtWRKY1 improved Y(II) and NPQ and the differences were statistically significant (Fig. 6d and e). qP indicates the proportion of open reaction centers in all PSII reaction centers [49, 50]. qN correlates with non-photochemical burst energy dissipation [51]. PEI-AuNPs-siAtWRKY1 significantly increased qP and qN (Fig. 6f and g), 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. S15), which reflects the actual electron transfer rate [52]. Taken together, the PEI-AuNPs-siAtWRKY1 treated Arabidopsis leaves had better chlorophyll fluorescence compared to other groups (Fig. 6, S14), indicating AtWRKY1 gene silencing by PEI-AuNPs-siAtWRKY1 could enhance disease resistance by promoting photosynthetic performance.

Fig. 6
figure 6

Chlorophyll fluorescence parameters of Arabidopsis leaves in buffer control, PEI-AuNPs, PEI-AuNPs-siAtWRKY1, and siAtWRKY1 groups. (a) Chlorophyll fluorescence images of Arabidopsis leaves; (b-g) Fo (b), Fm (c), Y(ll) (d), NPQ (e), qP (f), qN (g) chlorophyll fluorescence parameters for PEI-AuNPs, PEI-AuNPs-siAtWRKY1 and siAtWRKY1 groups. (b-g) n = 6 samples per group. The data presented were the mean ± SE

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Discussion

Enabling fluorescent properties 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 [53]. AuNPs are usually used to deliver nucleic acid molecules based on their diverse properties [21, 40, 53]. PEI-based nanocomposites possessing distinctive features, including high affinity, thermal stability, and mechanical strength, were synthesized. Positively charged PEI served as both reducing and stabilizing agents in the synthesis of PEI-AuNPs, effectively preventing agglomeration and ensuring colloidal stability at ambient conditions [54, 55]. The formation of gold nanoparticles was attributed to the direct redox reaction between amine groups and AuCl4−, wherein the amine groups in PEI captured and reduced AuCl4−, leading to the nucleation and gradual formation of gold nanoparticles [56]. 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 properties. Previous studies have reported fluorescence enhancement of gold nanoparticles assisted by specific surface ligands [57]. Compared with previous AuNPs [21, 53], 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 of nano-improved plant disease resistance via mediating gene silencing [58]. 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 [15, 21]. Nevertheless, Zhang et al. [40] 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 [21]. 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 Cy3-labelled ssDNA attached to PEI-AuNPs, Cy3-labelled ssDNA and PEI-AuNPs were visualized simultaneously with confocal microscopy, which makes the evaluation of the delivery efficiency of nucleic acid and PEI-AuNPs more accurate. Therefore, fluorescent PEI-AuNPs may enhance living cell observation and expand their potential applications in the field of biology.

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. Many studies have used RNAi techniques to achieve plant resistance to Pseudomonas syringae [59,60,61]. For example, a weakened miR472-RDR6 silencing pathway in Arabidopsis, necessary for the repression of NB-LRRs, enhanced plant defense against Pseudomonas syringae by facilitating the detection of the avirulence effector AvrPphB [62]. Canto-Pastor et al. reported that the miR482/2118 family functions to inhibit NB-LRRs in tomato plants. The introduction of a short tandem target mimic RNA to sequester miR482/2118 improves resistance to Pseudomonas syringae [63]. However, their application is limited to certain species due to host-range limitations [64]. 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 [65, 66].

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 h, but this effect returned to normal levels after 5 days [18]. Au materials have also been shown to deliver siRNA with different silencing rates. In recent years, PEI-AuNPs has emerged as a method for siRNA delivery in plants. Li et al. [21] and Zhang et al. [53] utilized AuNPs to deliver siRNANPR1 and microRNA (AmiRNA), respectively, for silencing the NPR1 and ATG6 genes in Arabidopsis thaliana. AuNPs deliver nucleic acid molecules that help in functional studies of specific genes. In addition, the FAM fluorescent sequence to the 5′ end of the siRNA to track AuNPs-siRNA in plant cells. PEI-AuNCs, for instance, achieved a silencing efficiency of 76.5 ± 5.9% when delivering GFP siRNA [20, 24], while AuNPs mediated NPR1 siRNA silencing up to 80% [21]. PEI-modified AuNPs proved to be highly effective at delivering nucleic acids for gene silencing in plants. By optimizing the ratio of the 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 the silencing effect and biological phenotype. In our study, we established the relationship between 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 improves plant disease resistance [67], we found that AtWRKY1 gene silencing by EI-AuNPs-siAtWRKY1 can achieve the same effect. Therefore, our study paves the way for the field application of the PEI-AuNPs siRNA system.

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 [45]. During the infestation, Pst DC3000 leads to a large accumulation of reactive oxygen species [68]. Meanwhile, it is commonly known that the over-accumulated ROS could be utilized to trigger the release of cargos from nano-delivery system. Similarly, in this study, ROS could be an elicitor to trigger the dislodging of siWRKY1 from the PEI-AuNPs-siWRKY1 complex. Indeed, the detection of Cy3 fluorescence in the eluent solution after the dialysis of the PEI-AuNPs-Cy3-ssDNA complex suggests that ROS can trigger the dislodging of siRNA from the PEI-AuNPs-siRNA complex (Fig. S16). Moreover, to combat the issue of over-accumulated ROS induced oxidative damage, 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 [42]. 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 the 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 AtWRKY1 gene expression. Nevertheless, this dual effect is consistent with improved plant health and a decrease in ROS-scavenging enzyme synthesis [26]. Moreover, previous studies showed that Arabidopsis plants with knockdown of AtWRKY1 had significantly lower reactive oxygen species content after inoculation compared to controls [67, 69]. Also, it is known that the use of ROS scavenging nanomaterials can help plants to maintain ROS homeostasis under stress [22, 44, 70, 71]. Thus, we argue that both enzyme-mimetic nature and silencing of AtWRKY1 via siRNA could contribute to the maintenance of ROS homeostasis in PEI-AuNPs-siWRKY1 treated plants under Pst DC3000.

Chloroplasts play a crucial role in plant defense against Pst DC3000, which causes significant damage to plant leaves by depleting chlorophyll a and b [72]. This depletion results in the development of pronounced chlorosis symptoms, characterized by a rapid and widespread yellowing of the affected leaves [72, 73]. Our study demonstrated that the use of PEI-AuNPs-siAtWRKY1 effectively suppressed AtWRKY1 expression and improved the resistance to Pst DC3000, thus exhibiting elevated chlorophyll levels and enhanced photosynthetic performance than control plants. 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.

Conclusions

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 the bacteria 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.

Data availability

No datasets were generated or analysed during the current study.

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Acknowledgements

We thank Mr. Jianbo Cao and Ms. Limin He for their help in TEM imaging at the Public Laboratory of Electron Microscopy, Huazhong Agricultural University. We also thank Mr. Ashadu Nyande from Huazhong Agricultural University for reading and polishing the manuscript’s English. The Pst DC3000 was donated by Prof. Tao Chen at Huazhong Agricultural University.

Funding

This work was supported by the National Key Research and Development Program of China (2023YFD19101700-3), the National Natural Science Foundation of China (No. 32120103008, 32071971), the HZAU-AGIS Cooperation Fund (SZYJY2021008), Fundamental Research Funds for the Central Universities (2662023ZKPY002), the Key Research and Development Projects of Henan Province (231111113000) and the Hubei Agricultural Science and Technology Innovation Center Program (2021-620-000-001-032).

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  1. Jie Qi and Yanhui Li are contributed equally to this work.

Authors and Affiliations

  1. National Key Laboratory for Germplasm Innovation & Utilization of Horticultural Crops, The Center of Crop Nanobiotechnology, College of Plant Science & Technology, Huazhong Agricultural University, Wuhan, 430070, China

    Jie Qi, Yanhui Li, Xue Yao, Guangjing Li, Wenying Xu, Lingling Chen, Zhouli Xie, Jiangjiang Gu, Honghong Wu & Zhaohu Li

  2. Hubei Hongshan Laboratory, Wuhan, 430070, China

    Jie Qi, Yanhui Li, Xue Yao, Guangjing Li, Wenying Xu, Lingling Chen, Zhouli Xie, Honghong Wu & Zhaohu Li

  3. College of Chemistry, Huazhong Agricultural University, Wuhan, 430070, China

    Jiangjiang Gu

  4. Shenzhen Institute of Nutrition and Health, Huazhong Agricultural University, Shenzhen, 511464, China

    Jiangjiang Gu & Honghong Wu

  5. Shenzhen Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Genome Analysis Laboratory of the Ministry of Agriculture, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, 511464, China

    Jiangjiang Gu & Honghong Wu

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  1. Jie Qi
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Contributions

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

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Correspondence to Honghong Wu.

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Qi, J., Li, Y., Yao, X. et al. Rational design of ROS scavenging and fluorescent gold nanoparticles to deliver siRNA to improve plant resistance to Pseudomonas syringae. J Nanobiotechnol 22, 446 (2024). https://doi.org/10.1186/s12951-024-02733-9

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Journal of Nanobiotechnology

ISSN: 1477-3155

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