DOTAP-functionalized DNA nanocages enable efficient miR168d trafficking to antagonize PVY infection by modulating HSP90-5 homeostasis

DOTAP-functionalized DNA nanocages enable efficient miR168d trafficking to antagonize PVY infection by modulating HSP90-5 homeostasis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article DOTAP-functionalized DNA nanocages enable efficient miR168d trafficking to antagonize PVY infection by modulating HSP90-5 homeostasis yanwei gong, Junying zhang, liu yang, Xinyi Zhao, lingdie wang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7756582/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Background Plant viruses cause severe agricultural losses. Conventional pesticides have issues such as residues and resistance, while the delivery efficiency of functional microRNAs in RNA interference strategies is low. This study aimed to evaluate the inhibitory effect of miR168d on Potato virus Y in Nicotiana benthamiana and construct a high-efficiency nanodelivery system. Results MicroRNA168d significantly reduces the replication and spread of Potato virus Y by targeting and inhibiting Heat Shock Protein 90-5. For the nanocomplex with a tetrahedral DNA nanostructure as the carrier and the cationic lipid 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) as the coating—characterization showed that it enhances the vascular transport efficiency, nuclease resistance, and cellular permeability of miR168d. Additionally, this nanocomplex exhibits low toxicity and good biocompatibility toward tobacco suspension cells. After foliar application, the nanocomplex group showed higher accumulation of miR168d in the leaves and stems of N.benthamiana compared with that in the control group. Specifically, the accumulation of mRNA and protein of the PVY coat protein in the nanocomplex group decreased by 64.3% and the corresponding percentage (consistent with the reduction at the protein level), respectively. As a result, the disease resistance of the plants was significantly improved . Conclusion This study reveals the antiviral mechanism of the miR168d-HSP90-5 regulatory module, provides a green non-transgenic nanoscale strategy, and is of great significance for agricultural antiviral breeding and sustainable agriculture. miRNA Antiviral immunity Nanocarrier Heat shock protein Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Among major plant pathogens that threaten global food security, viruses account for approximately half of the plant infections, resulting in estimated annual economic losses of over US $ 30 billion [ 1 ]. After fungal pathogens, these infections are the second most major cause of reduced worldwide agricultural output [ 2 ]. Viral disease management is complicated and a constant concern because of the significant diversity of plant viruses [ 3 ]. These diseases can alter the structure of plant communities and cause genetic erosion, which might lead to significant output losses or even crop failure [ 4 ]. It has been observed that vectors are crucially involved in the dissemination of plant viruses. For example, upon an outbreak, the aphid-transmitted Potato Virus Y (PVY) can rapidly spread throughout tobacco fields, making containment extremely challenging. A danger to global agriculture, PVY is a systemic infectious pathogen that causes necrotic disease. It can infect > 170 plant species from 34 genera, including those in the Solanaceae, Chenopodiaceae, and Fabaceae families. Furthermore, crops like potatoes, tomatoes, and tobacco are especially vulnerable [ 5 , 6 ]. Moreover, PVY is among the top ten most damaging plant viruses worldwide and has an extensive geographical distribution [ 7 , 8 ]. A number of variables, such as host cultivar, vector activity, and virus strain, affect the incidence and severity of PVY. Based on how they interact with genes that confer resistance to potatoes, PVY strains are categorized into various types, including C, O, N, Z, and E [ 9 – 11 ]. While strain N causes veinal necrosis, strains C and O have been found to cause mosaic and vein-clearing symptoms in tobacco. In common tobacco, recombinant strains arising from genetic recombination between O and N strains and carrying the N-type HC-Pro protein, such as N:O, NTN, N-Wi, NTN-NW, and NA-NTN, frequently induce severe veinal necrosis [ 12 , 13 ]. As green plant protection has progressed, more focus has been placed on the environmental contamination and safety hazards brought on by excessive use of chemical pesticides. The "3R" problems, Residue, Resistance, and Resurgence, pose major risks to ecosystems and human health due to the misuse of these pesticides. In agriculture, biological control refers to the management of pests and diseases by using natural or genetically modified organisms and their byproducts. Currently, 3 main methods are employed in biological control tactics against PVY: genetic engineering, biological agents, and biopesticides. Furthermore, PVY management has progressed because of the discovery and use of RNA interference (RNAi) [ 14 ]. RNAi has been widely acknowledged as a key mechanism of post-transcriptional gene silencing since its discovery in the early 21st century. RNAi synthesizes non-coding small RNAs (sRNA; about 20–30 nt long) or their encoded products to modulate gene expression. RNAi functions as a crucial molecular immune system in plants, especially during viral infection, and is essential for the early antiviral responses [ 15 , 16 ]. The RNAi process is primarily mediated by sRNAs, which, based on their origin and functional mechanisms, can be categorized into three classes: microRNAs (miRNAs), small interfering RNAs (siRNAs), and PIWI-interacting RNAs (piRNAs) [ 17 ]. Although these sRNAs have different biogenesis pathways, they all require Argonaute (AGO) proteins to produce effector complexes, which are the fundamental components of RNAi [ 18 ]. These complexes mediate sequence-specific gene silencing by recognizing target molecules via base complementarity [ 19 ]. In miRNA and siRNA pathways, this complex is called the RNA-induced silencing complex (RISC) and binds target mRNAs via a guide strand to induce translational repression or mRNA breakage, which in turn silences the gene [ 20 , 21 ]. Previous literature suggests that miRNAs modulate various biological processes by regulating the expression of downstream target genes [ 22 ]. In plants, miRNAs have near-perfect complementarity to their target genes, allowing significantly accurate target prediction. Compared to animals, plant miRNAs can inhibit translation to lower protein levels rather than reducing the expression of target mRNA, indicating a more complex regulatory mechanism. Translational repression and mRNA cleavage are the two major mechanisms by which plant miRNAs mediate gene silencing. AGO proteins' endonuclease activity is required for mRNA cleavage. When the mature miRNA is assembled into the RISC complex, it directs selective recognition and binding to the target mRNA, which is followed by phosphodiester bond cleavage at the location corresponding to nucleotides 10–11 of the miRNA. The fragments that result are then degraded by exonucleases [ 23 ]. HSP90 is a class of heat shock proteins that are extensively found in both eukaryotic and prokaryotic organisms. Furthermore, it acts as a molecular chaperone that is essential for cellular development, differentiation, apoptosis, and stress responses [ 24 , 25 ]. Moreover, different plant species have different numbers of HSP90 isoforms; for example, Arabidopsis thaliana has seven members that are found in the cytoplasm, mitochondria, chloroplasts, and endoplasmic reticulum [ 26 , 27 ]. Several studies have indicated that HSP90 modulates the replication and pathogenesis of various plant viruses. It promotes the replication of Bamboo mosaic virus (BaMV) by recognizing the 3′-UTR of the viral RNA [ 28 ] and facilitates the replication of Red clover necrotic mosaic virus (RCNMV) [ 29 ]. Similarly, tomato yellow leaf curl virus (TYLCV) infection is mediated by HSP90 and SGT1, and its deactivation may accumulate ubiquitinated proteins or cause cell death [ 30 ]. Therefore, to develop new methods for managing viral illnesses, this study selected HSP90 as the target of miRNA interference because of its importance as a vital host component that is less vulnerable to inhibition by direct pathogen targeting and plays a critical role in the viral life cycle. Because of its tunable physicochemical features, nanotechnology has attracted a lot of interest in the materials and biological sciences. However, establishing nanomaterials with specific size and shape remains challenging [ 31 , 32 ]. DNA-based nanostructures, such as tetrahedral DNA nanoparticles (TDNs), are developed using Watson–Crick base pairing. They provide programmable architecture, significant biocompatibility, and low toxicity, thus making them ideal platforms for delivering RNAi-based therapeutics [ 33 ]. TDNs are self-assembled by annealing four single-stranded DNAs, and are structurally characterized via non-denaturing gel electrophoresis, atomic force microscopy (AFM), transmission electron microscopy (TEM), and dynamic light scattering (DLS) for size, zeta potential, and dispersion stability [ 34 ]. In plants, their small size allows for effective cellular absorption. TDNs can load miRNA through complementary pairing for antiviral applications by adding a DNA linker at the 5′ end. Additional protein or enzyme functionalization improves stability and expands their use in targeted distribution and plant protection. Recently, various nanocarriers, such as liposomes, polymers, silica, and carbon-based materials, have been developed [ 35 ] and showed significant success in animal systems [ 36 ]. Furthermore, several nanoplatforms capable of crossing plant tissue barriers have been engineered to deliver nucleic acids, peptides, and phytohormones [ 37 ]. For example, liposomes can transport proteins and stimulate immunological responses in Arabidopsis [ 38 ]; functionalize carbon nanotubes to promote gene silencing [ 39 ]. Moreover, gold nanoclusters deliver siRNA to induce phenotypic changes [ 40 ], and mesoporous silica nanoparticles transport siRNA with substantial efficiency, achieving up to 98% silencing rates [ 41 ]. These nanoparticles provide a potential non-transgenic crop protection method, decrease enzymatic degradation, and improve nucleic acid stability. Since their development in the 1960s [ 42 ], liposomes (self-assembled phospholipid-cholesterol vesicles) have been employed as essential drug delivery vehicles because of their biocompatibility, biodegradability, and simplicity of surface modification [ 43 ]. They are frequently utilized to improve patient compliance and therapeutic efficacy via various administration methods, including parenteral, pulmonary, oral, and topical delivery [ 44 ]. Liposomes improve efficacy while decreasing toxicity by protecting encapsulated drugs from degradation, prolonging half-life, facilitating regulated release, and enabling targeted distribution via passive or active processes. Cationic liposomes are useful for gene therapy applications, producing stable complexes that resist nuclease degradation and enhance transport efficiency through electrostatic interactions with negatively charged macromolecules, such as DNA and RNA. Nanotechnology breakthroughs have substantially increased the range of available medication delivery methods, providing improved sustainability, biocompatibility, and multifunctional benefits. This study employed the inherent characteristics of liposomes to create a stable and highly effective miRNA delivery system with self-assembled TDNs as the main structural component of a liposomal carrier. This hybrid method overcomes species-specific barriers, decreases the cytotoxic effects associated with traditional virus-induced gene silencing, and significantly increases the stability and transport efficiency of miRNA. This investigation proposed a novel, sustainable, safe, and green approach to developing next-generation antiviral nanomedicines for plants. Experimental methods Synthesis of tetrahedral DNA nanoparticles and formation of its nucleic acid/lipid complex (TDN-miR168d@DOTAP) TDNs (approximately 8nm in size) were prepared according to a previous research procedure with slight modifications [ 45 ]. First, 1.21 g of Tris base, 5.08g of MgCl2·6H2O and 45mL of sterile enzyme-free water were mixed, the pH value of the solution was adjusted to 8.0 with HCl, and the mixed solution was filtered through a 0.22µm filter membrane to obtain 10×TM buffer. Then, 1µL of each the A (100µM), B (100µM), C (100µM) and D chains (100µM) were combined (Sequences are in Table S1 , materials were synthesized by Qingdao Weilai Biotechnology Co., LTD, and 10µL of 10× TM buffer and 86µL of sterile enzyme-free water were added to make a 100µL system. The mixed liquid was heated to 95℃for 10min in a PCR instrument (Eppendorf, Hamburg, Germany) and then cooled to 4℃ for 30min to form a TDN with the miR168d linker sequence. To evaluate the subcellular localization of nanostructures, we used single-stranded A with Cy3 labeling to assemble the TDN.The TDN can be stored at 4℃ for 1 month. miR168d duplexes were prepared according to previous research procedures with slight modifications [ 24 ]. To synthesize double-stranded miR168d with a linker sequence, 1µL of each of the two corre sponding fully complementary RNA oligonucleotides miR168d (100µM) and antisense-miR168d (100µM) were mixed (sequences are in Table S1 , materials were synthesized by Qingdao Weilai Biotechnology Co., LTD, and 10µL of 10×PBS and 88µL of sterile enzyme-free water were added to make a 100µL system. The mixed liquid was heated to 95℃ for 5min in a PCR instrument and then kept at 25℃ for 30 min. One hundred microliters of each TDN (1µM) and miR168d (1µM) that had been synthesized in the above steps were mixed with 800µL of 1× PBS, and the mixture was heated at 37℃ for 30 min to bind the TDN to miR168d at a final concentration of 100nM. The concentration can be adjusted according to experimental needs. To assess the internalization of the nanostructures, we used Cy3-labeled single-stranded A to assemble TDNs. This method ensures that the final concentration of the Cy3 dye in TDN is equal to that in TDN-miR168d so we can evaluate both the internalization of the nano structures based on the Cy3 fluorescence that is colocalized with the cytoplasm and the internalization efficiency of the nanostructures. The synthesized TDN-miR168d was taken and dispersed in ultrapure water. An appropriate amount of DOTAP was added into DMSO and dispersed by ultrasonic treatment with a high-intensity ultrasonic processor for 1h. The two were mixed at a volume ratio of 1:1 and incubated at 4℃ for 2h to prepare the antiviral nanomedicine TDN-miR168d@DOTAP. Characterization TDN-miR168d and TDN-miR168d@DOTAP were subjected to gel mobility shift assays in a 3.0% (w/v) agarose gel matrix. The successful assembly of TDN-miR168d was confirmed according to the migration rate, and the extent of DOTAP coating on TDN-miR168d was determined from the bright ness of the band. A nanoparticle size and zeta potential analyzer (OPP TRONIY-919SZ, Beijing, China) was used to measure the hydrated particle size and surface charge of various the complexes by DLS. The structure and morphology of the prepared nanoparticles were deter mined by using the JEOL JEM-F200 (Japan), TESCAN MIRA LMS (Czech Republic) and the Bruker Dimension (Germany). The Brunauer-Emmett-Teller (BET) method (ASAP 2010, Micromeritics, USA) and the Barrett-Joyner-Halenda (BJH) method were used to calculate the pore size distribution and surface area, respectively. Plant materials and viral strains Host and virus source: N.benthamiana cultured in a climate chamber at 25°C with a 16 h light/8 h dark cycle; Potato Virus Y (PVY) and the infectious clone PVY-GFP, which were propagated on N. tabacum var. Samsun NN (Sansheng NN) and preserved by the Virus Research Group of Tobacco Research Institute, Chinese Academy of Agricultural Sciences (CAAS). TRV-Induced Silencing of miR168d Agrobacteria containing correctly sequenced plasmids (pTRV1, pTRV2, pTRV2-STTM-miR168d, and pTRV2-PDS) were cultured in a shaker at 28°C for 24 hours. The bacterial cells were collected by centrifugation and resuspended in an infiltration buffer. The resuspended pTRV1 solution was mixed at a 1:1 ratio (OD600 = 0.5) with three different resuspended solutions separately: pTRV2 (negative control), pTRV2-STTM-miR168 (treatment group), and pTRV2-PDS (positive control). Using a sterile syringe (without the needle), the mixed solutions were gently infiltrated into the abaxial surface of leaves of Nicotiana benthamiana at the 4–5 leaf stage. Approximately 10 days after infiltration, when albino phenotypes appeared on the top leaves of N. benthamianain filtrated with pTRV2-PDS, PVY-GFP was inoculated onto the two largest fully expanded leaves in the middle of the tobacco plants. At 1-7dpi, the inoculated leaves were sampled. The expression level of the viral coat protein (CP) was detected using quantitative real-time PCR (RT-qPCR) and Western blot analysis. Transient overexpression of miR168d The resuspended agrobacterial solution containing the correctly sequenced p35S:miR168d plasmid (OD600 = 0.5) was incubated at room temperature in the dark for 3 hours, then infiltrated into the two largest fully expanded leaves in the upper-middle part of Nicotiana benthamiana plants. After 24 hours of cultivation, PVY-GFP was inoculated onto the infiltrated leaves. From 1 to 7 days post-inoculation (dpi), the infiltrated leaves were sampled to detect the expression level of the viral coat protein (CP). Prediction and Functional Analysis of the Target Gene (HSP90-5) of miR168d in N.benthamiana The potential target genes of miR168d in N.benthamiana were predicted using the "psRobot_tar" functional module on the web interface of the PsRobot plant small RNA analysis system ( http://omicslab.genetics.ac.cn/psRobot/ ). Based on the transcriptome sequencing results of N.benthamiana infected with PVY, a heatmap was generated to analyze the expression levels of these potential target genes after viral infection, and quantitative real-time PCR (RT-qPCR) was performed for verification. Bioinformatics analysis was used to examine the binding sites between miR168d and its target genes. Additionally, target gene silencing and overexpression experiments were conducted to detect their effects on PVY infection. Western blotting (WB) To detect the presence of viral proteins, total plant proteins were extracted using protein extraction kit (ABclonal, Wuhan, China) and subjected to WB using virus-specifc antibodies against PVY and GFP (Agdia, Elkhart, IN, USA), respectively. In addition, β-actin was used as the internal reference and detected using β-actin antibodies (ABclonal, Wuhan, China). Real-time fuorescence relative quantitative PCR (RT-qPCR) Total RNAs were extracted and used as the template to synthesize cDNA samples following the program of the reverse transcription kit (Transgen, Beijing, China). The relative mRNA expression levels of target genes were quantitatively detected following the instructions of the real-time fuorescence quantitative PCR kit (Vazyme, Nanjing, China). Te primer sets PVY-F/PVY-R, β-ActinQF/β-ActinQR (S1) were used to amplify PVY CPand β-actin, respectively. The 2 −ΔΔCt method was employed to calculate the relative expression levels of these genes. All experiments were repeated three times with three biological replicates. Data are expressed as mean ± standard deviation of at least three independent experiments. Statistical analyses were performed using SPSS (v21, IBM, Armonk, NY, USA) with Duncan’s multiple range test analysis of variance (ANOVA) and independent sample t-test. Statistical significance was set at p < 0.05. Results miR168d negatively regulates PVY infection in N. benthamiana The rapid development of high-throughput technologies and the increasing data on plant genomes and transcriptomes have improved our understanding of the roles of miRNA in plant growth and stress responses, including antiviral defense. A high-throughput sequencing analysis of roots, stems, and leaves miRNA of PVY-infected N. benthamiana identified 1,677 known miRNAs, of which 60 were significantly differentially expressed. Furthermore, a regulatory network linking these miRNAs to antiviral responses was generated by integrating transcriptome co-analysis, GO and KEGG enrichment, and target prediction [ 45 ]. To investigate the role of miR168d in PVY-infected plants, a 35S promoter-driven construct was employed for transient overexpression of miR168d (35S:miR168d). When infected with PVY, 35S:miR168d plants indicated strong resistance compared to empty vector (35S:00) controls. Moreover, the newly developed leaves had no curling symptoms, and systemic virus movement was significantly delayed (Fig. 1 A). RT-qPCR analysis showed substantially decreased PVY RNA accumulation (Fig. 1 B). Western blotting validated the decrease in viral coat protein (CP) levels (Fig. 1 C), suggesting that miR168d overexpression improves antiviral immunity. For loss-of-function analysis, miR168d was silenced using a short tandem target mimic (STTM) construct (TRV:miR168d). The TRV:PDS-positive control indicated photobleaching was predicted (Supplementary Fig. 1A), whereas the TRV:miR168d plants had a 71.3% reduction in miR168d levels ( p < 0.001 ; Supplementary Fig. 1B). After PVY-GFP treatment, TRV:miR168d plants showed stronger GFP fluorescence relative to controls (Fig. 1 D). RT-qPCR (Fig. 1 E) and western blot (Fig. 1 F) revealed that both PVY’s CP transcript and protein levels were significantly elevated in silenced plants, confirming increased viral susceptibility. It was found that miR168d acts as a negative regulator of PVY infection and its overexpression can effectively restrict viral replication and spread, providing a new molecular target for miRNA-mediated antiviral defense in plants. miR168d targets HSP90-5 to inhibit PVY infection To elucidate the response mechanism of N. benthamiana miR168d during PVY infection, the expression dynamics of miR168d in different tissues were evaluated after infection. Differential miRNA expressions were identified via sRNA sequencing and were validated by RT-qPCR. The data revealed that miR168d expression was significantly reduced during PVY infection, suggesting its potential role as a regulatory factor in host innate immunity (Fig. 2 A) [ 45 ]. Furthermore, transcriptomic and RT-qPCR heatmap analyses showed that the HSP90-5 transcript was substantially upregulated in PVY-infected leaves (Figs. 2 B–C). Moreover, Bioinformatics prediction and subsequent target validation identified a specific binding site between miR168d and the 3′UTR of HSP90-5 (Fig. 2 D). miR168d overexpression substantially inhibited HSP90-5 expression (Fig. 2 E), confirming that HSP90-5 is a direct target of miR168d, consistent with the previous reports indicating that heat shock proteins, including HSP90, promote infection and replication of various plant viruses [ 12 ] To functionally characterize the miR168d–HSP90-5 regulatory axis, the gain- and loss-of-function assays were carried out to evaluate its impact on PVY infection. Transient HSP90-5 overexpression in N.benthamiana , followed by PVY-GFP inoculation, significantly increased GFP fluorescence under UV light (Fig. 2 F). Furthermore, RT-qPCR and Western blot analyses validated the upregulated levels of viral CP mRNA and protein (Figs. 2 G– 2 H). Whereas HSP90-5 silencing inhibited viral CP accumulation and reduced fluorescence intensity (Fig. 2 I– 2 K). These findings revealed that miR168d post-transcriptionally suppresses HSP90-5 expression, which inhibits PVY accumulation, thus indicating a novel miRNA–target module crucial for plant antiviral defense. Cationic lipid-coated DNA nanostructure for efficient miRNA delivery in plants This study addressed the problem of low gene delivery efficiency due to the plant cell wall barrier and nuclease degradation, improving effective delivery of the resistance gene miR168d. Using a TDN as the framework and the cationic lipid DOTAP (1,2-dioleoyl-3-trimethylammonium propane) as the coating, a unique nano-delivery system was developed that significantly improved the antiviral gene miR168d's transmembrane permeability and vascular long-distance transport capability. Furthermore, 3% agarose gel electrophoresis indicated that the four single-stranded DNAs (A, B, C, D) were of approximately equal length (Fig. 3 A). The effective assembly of TDN-miR168d was confirmed by the synthesized TDN's band size, which was 190 bp and increased to about 210 bp following conjugation with miR168d. Hydrodynamic size measurements revealed that TDN-miR168d and TDN-miR168d@DOTAP had diameters of 23.6 nm and 165.1 nm, respectively (Fig. 3 B). Furthermore, zeta potential analysis demonstrated that TDN-miR168d had a negative charge of -8 mV, whereas DOTAP had a positive charge of + 55 mV. The completely encapsulated TDN-miR168d@DOTAP complex had a positive zeta potential of + 35.6 mV. After pretreatment, the TEM showed that uncoated TDN-miR168d had lost its tetrahedral integrity (Supplementary Fig. 3). DOTAP's encapsulation of TDN-miR168d was confirmed using infrared spectroscopy. The spectrum of TDN-miR168d showed distinctive peaks at 1309.35 cm − 1 and 1436.10 cm − 1 (molecular skeleton vibrations), 2912.06 cm − 1 and 2994.86 cm − 1 (C–H stretching vibrations of alkyl chains), and 1041.53 cm − 1 (ascribed to P–O or C–O vibrations in nucleic acids). In the composite spectrum, the 1041.53 cm⁻¹ peak disappeared because of electrostatic interactions between phosphate groups and DOTAP’s cationic amine groups. The absence of alkyl peaks and the appearance of a new peak at 1652.31 cm − 1 , along with a reorganization of molecular skeleton vibrations, confirmed that TDN-miR168d was successfully encapsulated by DOTAP instead of physically mixing (Fig. 3 C). This study also performed AFM and TEM analyses, which revealed good dispersion without significant aggregation. AFM indicated significant differences in the morphology and height between TDN-miR168d and TDN-miR168d@DOTAP, highlighting structural changes post-encapsulation. Moreover, TEM images of TDN-miR168d@DOTAP revealed significantly enlarged particle sizes and localized protrusions, which might be due to the formation of an electrostatic complex between cationic DOTAP and TDN-miR168d, leading to a thicker composite structure (Figs. 3 D– 3 E). These morphological alterations further support successful encapsulation. To prepare the Cy3A-TDN-miR168d complex for real-time intracellular tracking, the A strand was substituted with a Cy3-labeled A-Cy3 strand. After 24 hours of leaf infiltration, confocal microscope imaging revealed a particular Cy3 fluorescence enrichment in the cytoplasm of N. benthamiana cells (Fig. 3 F), demonstrating that TDN, with its virus-like size and efficient endocytosis, enables transmembrane delivery of miR168d to the target site. This finding highlights TDN's advantage in tissue penetration as a gene carrier and lays the groundwork for future functional research on antivirals. Cationic lipid-modified nanocarrier enhances antiviral miRNA delivery and efficacy in plants This study employed the cationic lipid DOTAP to generate a charge-adapted nanocomplex, TDN-miR168d@DOTAP, to systematically improve the transmembrane permeability and long-distance vascular transport capacity of the antiviral gene miR168d. This was performed to improve the efficiency of the gene delivery system, which was limited by the plant cell wall barrier and nuclease degradation. Furthermore, cytotoxicity analyses were conducted on TDN-miR168d and TDN-miR168d@DOTAP to assess their biosafety and constituents. Neither substance substantially impacted the typical growth and development of tobacco suspension cells (By2) (Fig. 4 A). Moreover, in both treatments, plants had intact cellular morphology with cell survival above 95%, suggesting that the TDN-miR168d@DOTAP nano-delivery system has low cytotoxicity, good biocompatibility, and no effect on typical plant growth. TDN-miR168d (bare carrier) and TDN-miR168d@DOTAP (lipid-modified) were sprayed foliarly on healthy N. benthamiana plants to evaluate the enhanced effect of DOTAP modification on miR168d delivery. After 3 to 7 days, miR168d expression was observed in the stems (the systemic transport terminus) and leaves (the local delivery site). RT-qPCR revealed that from day 5 to 7, the TDN-miR168d@DOTAP group's miR168d expression in leaf tissues was consistently and significantly higher than that of the bare carrier group ( p < 0.01 , Fig. 4 B). This suggests that DOTAP-mediated membrane affinity successfully passes through the cell wall and epidermal wax barriers, improving the efficiency of local delivery. In stem tissues, no significant differences were observed in miR168d levels between the two groups within the first 3 days ( p > 0.05 ). However, by day 5, the TDN-miR168d@DOTAP group indicated increased expression ( p < 0.001 , Fig. 4 C). This delayed increase suggests that DOTAP provides prolonged systemic transport capacity via nuclease resistance and vascular-targeted penetration in addition to improving local delivery. In particular, during apoplastic and symplastic transport, the lipid coating protects the TDN structure from phosphoesterases and plant RNAses. Furthermore, the nanocomplex's small size and surface charge alteration allow for intercellular transport through plasmodesmata and phloem sieve tubes, which ultimately permits the genetic payload to be distributed systemically. To validate the antiviral efficacy of the TDN-miR168d@DOTAP nanocomplex, its ability to confer resistance against PVY infection was evaluated. N. benthamiana plants with 6 to 8 leaves were rub-inoculated with PVY. After 24 hours, the infected leaves were completely sprayed with TDN-miR168d or TDN-miR168d@DOTAP; the control group received an equivalent amount of water. On the 7th day after therapy, samples were collected for qRT-qPCR analysis, which revealed that compared to the control, PVY CP mRNA levels decreased by 49.4% in the TDN-miR168d group and by 64.3% in the TDN-miR168d@DOTAP group (Figs. 4 D– 4 E). These findings were validated by Western blot analysis (Fig. 4 F). These results suggest that the antiviral gene was successfully delivered via the TDN-miR168d@DOTAP nanocomplex, which also provides substantial resistance against PVY, thus providing a new and effective way of delivering antiviral genes in plants. Discussion This study elucidated a novel antiviral mechanism in N.benthamia mediated by the miR168d-HSP90-5 regulatory module, and also established an efficient nanocarrier system, TDN-miR168d@DOTAP, for targeted miRNA delivery. The findings not only deepen the understanding of host-virus interactions but also demonstrate the potential of nanotechnology in non-transgenic crop protection strategies. MicroRNAs have emerged as key regulators of plant immune responses, fine-tuning gene expression during pathogen challenges. Previous studies have reported differential expression of certain miRNAs during viral infections, showing that they participate in defense responses by targeting either viral genomes or host factors linked to infection [ 46 , 47 ]. For instance, miR168 regulates AGO1 expression to modulate RNAi efficiency, while miR160 influences auxin signaling to affect susceptibility [ 48 ]. The present study identified miR168d as a negative regulator of PVY infection, extending the functional repertoire of miRNAs in antiviral defense. Notably, unlike canonical RNAi approaches that target viral genes directly, miR168d operates through a host-centered mechanism by suppression of HSP90-5, a chaperone protein critical for viral replication. This strategy may reduce the selective pressure on mutants involved in viral escape, a common drawback of pathogen-targeted RNAi [ 49 ]. The present results align with reports that HSP90 facilitates the replication of various viruses, including bamboo mosaic virus and tomato yellow leaf curl virus [ 50 ]. However, in contrast to earlier studies that focused on genetic or chemical inhibition of HSP90, we demonstrate that miRNA-mediated post-transcriptional regulation offers a tunable and specific means of control. Furthermore, the observed downregulation of miR168d during PVY infection suggests the presence of a viral counter-defense strategy, possibly leading to modulation of HSP90-5 levels and the promotion of replication. These dynamic miRNA-target interactions highlight the co-evolutionary arms race between plants and viruses [ 51 ]. The present study thus contributes to a growing body of evidence that miRNAs represent central players in plant immunity, and their manipulation offers a promising approach for the engineering of resistant crops. The effective delivery of nucleic acids within plants remains a major challenge due to plant cell walls, enzymatic degradation, and limited vascular mobility. While nanoparticle-based delivery has been widely explored in mammalian systems [ 52 ], its application in plants is still emerging. Recent studies have used clay nanosheets, carbon nanotubes, and silica nanoparticles for the delivery of siRNAs and DNA constructs [ 53 , 54 ]. However, many of these systems are associated with low efficiency, cytotoxic effects, or limited tissue penetration. Our designed nanocarrier, TDN-miR168d@DOTAP, integrates the structural programmability of DNA nanostructures with the membrane affinity of cationic lipids. TDNs offer excellent biocompatibility, enable precise functionalization, and are easy to synthesize [ 55 ], while DOTAP enhances cellular uptake and nuclease resistance through electrostatic interactions with negatively charged membranes. The resulting nanocomplex achieved significantly higher accumulation of miR168d in both local and systemic tissues compared to the non-lipidated TDN, underscoring the importance of surface engineering in overcoming plant-specific barriers. The cytoplasmic delivery of Cy3-labeled TDNs was confirmed using confocal microscopy, demonstrating their ability to cross cell walls and membranes, possibly via endocytosis or plasmodesmata-mediated transport. This represents a significant advance over conventional methods, such as the use of agrofiltration or viral vectors, which are often limited by host range, insert size, or regulatory constraints [ 56 , 57 ]. The present system provides a non-transgenic, scalable, and environmentally friendly alternative for gene delivery in plants. From a practical perspective, this study offers a promising strategy for the control of infection by PVY and potentially other plant viruses. The foliar application of TDN-miR168d@DOTAP significantly reduced accumulation of the PVY coat protein, demonstrating its efficacy under controlled conditions. This approach aligns with the growing demand for sustainable alternatives to chemical pesticides responsible for both environmental contamination and the development of resistance [ 57 – 59 ]. Theoretically, the findings indicate the value of targeting host factors, instead of viral components, for broad-spectrum resistance. The strong conservation of HSP90 in plants and its ability to facilitate the replication of multiple viruses suggest that miR168d-based strategies could apply to a range of pathosystems. Furthermore, the modularity of the TDN platform would enable adaptation for the delivery of other miRNAs or siRNAs, making it a versatile tool for both research and application. Additionally, this study bridges molecular biology and nanotechnology, illustrating the effectiveness of interdisciplinary approaches in addressing longstanding challenges in agriculture. The integration of molecular mechanisms (miRNA-target interactions) with engineered solutions (nanocarriers) provides a holistic framework for the development of next-generation techniques for plant protection. Despite these advances, several challenges should be addressed before translational application. 1. Target Specificity and Potential Off-Target Effects: While silencing HSP90-5 was effective in the suppression of PVY, HSP90 proteins are involved in a variety of essential cellular processes, including stress responses, hormone signaling, and protein folding [ 58 ]. Constitutive downregulation may therefore impair plant growth and development. To improve specificity, future studies could focus on the identification of downstream effectors of HSP90-5 that are specifically hijacked by viruses, using proteomic or interactome analyses, leading to the development of tissue-specific or infection-inducible promoters to drive miRNA expression. Another possibility is the use of CRISPR-based methods to engineer HSP90-5 variants that retain physiological functions while resisting viral exploitation. 2. Environmental Stability and Field Applicability: It is possible that the described nanocomplex is susceptible to degradation under field conditions (e.g., UV radiation, rainfall, temperature fluctuations). DOTAP, in particular, is prone to oxidation under strong light. The following strategies could be used to enhance its resilience: encapsulation in light-responsive polymers that release payloads under specific wavelengths; the use of natural lipids (e.g., soybean lecithin or phytosterols) to improve biodegradability and reduce cost; surface functionalization with protective ligands (e.g., cellulose-binding domains or chitosan) to enhance adhesion and retention on leaf surfaces. 3. Scalability and Regulatory Considerations: Large-scale synthesis of TDNs is both technically challenging and costly. Future work should explore the use of automated assembly platforms for high-throughput production, the use of alternative nanomaterials (e.g., biodegradable polymers or peptide-based carriers) that provide similar functionality with easier scalability, and thorough biosafety assessments to ensure non-toxicity to non-target organisms and compliance with regulatory standards for nano-agricultural products. 4. Resistance Management: Viral evolution may lead to escape mutants, as commonly seen in resistance strategies. Continuous monitoring of the conservation of the target site and miRNA efficacy is essential. It is proposed that a virus surveillance network be established to track mutations in real-time, as well as the design of multi-target miRNA systems or the combination of miRNA delivery with other RNAi constructs to reduce the risk of escape. The integration of nanomaterial application with other management practices (e.g., crop rotation or biological controls) would also assist sustainable disease management. In summary, this study provides a comprehensive molecular and technological framework for the application of miRNA-mediated antiviral defense in plants. The study not only identified miR168d as a key regulator of PVY infection, mediated by suppression of HSP90-5, but also developed an efficient nanodelivery system that enhanced miRNA stability, uptake, and systemic mobility. These findings advance our fundamental understanding of plant-virus interactions and provide a translatable strategy for sustainable agriculture. Future efforts should focus on refining target specificity, improving environmental stability, and enabling scalable production. The integration of tools from nanotechnology, synthetic biology, and precision agriculture has significant potential for addressing the challenges associated with crop disease and contributing to global food security. Conclusion Nanodelivery technology serves as a pivotal solution for enhancing miRNA delivery efficiency, offering considerable potential to mitigate environmental safety concerns and pesticide resistance. In this study, we successfully developed a novel and efficient miRNA-based nanoplatform that specifically targets the viral auxiliary factor HSP90-5, functioning as a key antiviral regulator in N.benthamiana. The system, designated TDN-miR168d@DOTAP, integrates a tetrahedral DNA nanocage (TDN) with a cationic lipid coating. This formulation significantly improved miR168d stability and systemic transport, leading to a substantial reduction in Potato virus Y (PVY) accumulation and enhanced plant resistance. Further investigations revealed that the delivery system effectively downregulates HSP90-5 expression and confers significant protective effects against PVY infection. Importantly, in vivo assessments confirmed that the system caused no adverse effects on plant growth and development, inducing only minimal plant cell necrosis, thereby demonstrating its favorable biosafety profile. Collectively, these findings highlight the promising application prospects of the biocompatible and environmentally benign TDN-miR168d@DOTAP system for targeted inhibition of plant gene expression and sustainable management of plant viral diseases. Declarations Authors' contributions Yanwei Gong: conceptualization, data curation, investigation, methodology, formal analysis, writing-original draft, and writing-review and editing. Junying Zhang: conceptualization, investigation. Liu Yang: supervision, funding acquisition and validation. Xinyi Zhao: formal analysis and data curation. Lingdie Wang: review and editing. Dong An: formal analysis and data curation. Lianqiang Jiang: investigation, methodology and writing-review and editing. Yubing Jiao: conceptualization, methodology, formal analysis, data curation and editing. Lili Shen: conceptualization, investigation, methodology and writing-review and editing. Funding This work was supported by Science and Technology Project of Liangshan Prefecture Company, Sichuan Provincial Tobacco Company (SCYC202311) Availability of data and material no datasets were generated or analysed during the current study. 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07:02:51","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":114083,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7756582/v1/e7b98ea0c8b154a6d2273488.png"},{"id":94988830,"identity":"2402f167-0086-4961-887e-4a3fda19d2d1","added_by":"auto","created_at":"2025-11-03 07:11:05","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":114711,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7756582/v1/ce7aed015df3c5519a8e64be.png"},{"id":94930193,"identity":"1d9b9025-7830-454a-8e43-048118a134bc","added_by":"auto","created_at":"2025-11-01 18:40:26","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":87325,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7756582/v1/ba44ffc5481a820fe282b723.png"},{"id":94988400,"identity":"200d7853-53d1-4db3-8d74-8f39dd44ed18","added_by":"auto","created_at":"2025-11-03 07:08:53","extension":"xml","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":150937,"visible":true,"origin":"","legend":"","description":"","filename":"aff064ffabca4db2bf060f815de9d1401structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7756582/v1/c40adc629024f04b00348de9.xml"},{"id":94930206,"identity":"d39171e9-042f-4cfc-ba41-82047b4944cd","added_by":"auto","created_at":"2025-11-01 18:40:26","extension":"html","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":165322,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7756582/v1/27b412d64785910d41039497.html"},{"id":94930181,"identity":"4efc3408-6802-42e6-b474-ca3f42aed406","added_by":"auto","created_at":"2025-11-01 18:40:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":351521,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of viral infection properties based on transient infiltration of miR168d. (A) Phenotype of \u003cem\u003eN. benthamiana\u003c/em\u003e after miR168d overexpression. (B) PVY-CP content of the miR168d overexpressing plant was detected by RT-qPCR. (C) and Western blotting. (D) Phenotype of \u003cem\u003eN. benthamiana\u003c/em\u003e after STTM silencing of miR168d. (E) PVY expression after STTM silencing was assessed by RT-qPCR and (F) Western blotting. Data are indicated as means ± SD; n = 3; *\u003cem\u003ep \u0026lt; 0.05\u003c/em\u003e; **\u003cem\u003ep \u0026lt; 0.01\u003c/em\u003e; ***\u003cem\u003ep \u0026lt; 0.001\u003c/em\u003e, t test.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7756582/v1/93af2fc1d6f67f544ecfb53f.png"},{"id":94930182,"identity":"b66be466-20e9-4c54-945b-f6c485bca544","added_by":"auto","created_at":"2025-11-01 18:40:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":327579,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional analysis of HSP90-5, which regulates PVY infection \u003cem\u003evia \u003c/em\u003emiR168d. (A) RT-qPCR analysis of differentially expressed miRNA168 based on sRNA sequencing; (B) Heat map analysis based on transcriptome sequencing identified differentially expressed genes in PVY-infected plants. (C) RT-qPCR results of HSP90-5 expression in PVY-infected plants. (D) Prediction of the binding sites of miR168d and HSP90-5. (E) Target genes were screened by transiently overexpressing miR168d. (F) Phenotype analysis of \u003cem\u003eN. benthamiana\u003c/em\u003e after HSP90-5 overexpression. (G) PVY expression in the HSP90-5 overexpressing plant was assessed\u003cem\u003e via\u003c/em\u003e RT-qPCR and (H) Western blotting. (I) Phenotype of \u003cem\u003eN. benthamiana\u003c/em\u003e after VIGS silencing of HSP90. (J) PVY expression after VIGS silencing was assessed using RT-qPCR and (K) Western blotting. Data are depicted as means ± SD; n = 3; *\u003cem\u003ep \u0026lt; 0.05\u003c/em\u003e; **\u003cem\u003ep \u0026lt; 0.01\u003c/em\u003e; ***\u003cem\u003ep \u0026lt; 0.001\u003c/em\u003e, t-test.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7756582/v1/baa7fbadb40d1963331965bb.png"},{"id":94930189,"identity":"aa2cacc1-0e23-402a-bbe5-09ba3fe5bfdd","added_by":"auto","created_at":"2025-11-01 18:40:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":322260,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization diagram of TDN-miR168d. (A) TDN-miR168d agarose gel electrophoresis. (B) Particle sizes of TDN-miR168d and TDN-miR168d@DOTAP. (C) FTIR spectra. (D) AFM imaging of TDN-miR168d and TDN-miR168d@DOTAP. Scale bar = 400 nm. (E) TEM imaging. Scale bar = 100 nm. (F) The internal localization of Cy3-TDN-miR168d was detected by confocal laser microscopy. Scale bar = 75 mm.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7756582/v1/d545be8573192a8b38f2b1b4.png"},{"id":94930203,"identity":"34cc9644-e694-4b52-b0f0-2214a67e70ea","added_by":"auto","created_at":"2025-11-01 18:40:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":336385,"visible":true,"origin":"","legend":"\u003cp\u003eTDN-miR168d@DOTAP nano-delivery and antiviral assessment. Cell viability assays of By2 cell suspensions treated with TDN-miR168d and TDN-miR168d@DOTAP for 48 hours. Scale bar = 100 mm. (B) miR168d content in leaves and stems at different time points after foliar spraying with TDN-miR168d and TDN-miR168d@DOTAP. (D) Phenotype of \u003cem\u003eN. benthamiana\u003c/em\u003e after TDN-miR168d and TDN-miR168d@DOTAP overexpression. Scale bar = 2 cm. (E) PVY expression after 3 different treatments was quantified by RT-qPCR and (F) Western blot analyses. Data are provided as means ± SD; n = 3; *\u003cem\u003ep \u0026lt; 0.05\u003c/em\u003e; **\u003cem\u003ep \u0026lt; 0.01\u003c/em\u003e; ***\u003cem\u003ep \u0026lt; 0.001\u003c/em\u003e, t-test.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7756582/v1/2ceac0ed1f2d7dcb1b04f698.png"},{"id":95312034,"identity":"94b3d0c5-a3f3-4e42-a733-d116321f8a82","added_by":"auto","created_at":"2025-11-06 15:45:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2313944,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7756582/v1/457f7e5c-de53-4e19-8f5b-dec9713d3468.pdf"},{"id":94930199,"identity":"58d01afc-cb88-4433-a0be-a88dd093949f","added_by":"auto","created_at":"2025-11-01 18:40:26","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":9310420,"visible":true,"origin":"","legend":"","description":"","filename":"S1.tif","url":"https://assets-eu.researchsquare.com/files/rs-7756582/v1/f2aedfe84edb90661e06914d.tif"},{"id":94930184,"identity":"5e6b4f34-568a-4db5-a05d-65cf20aeff81","added_by":"auto","created_at":"2025-11-01 18:40:25","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":11050,"visible":true,"origin":"","legend":"","description":"","filename":"S1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7756582/v1/9ca7481b3fb858cd9d664c88.xlsx"},{"id":94930190,"identity":"63382281-520d-4839-88cf-462b7136358d","added_by":"auto","created_at":"2025-11-01 18:40:25","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":147494,"visible":true,"origin":"","legend":"","description":"","filename":"GRAPHICALABSTRACT.png","url":"https://assets-eu.researchsquare.com/files/rs-7756582/v1/5b7489747eed35c4682fb1fb.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"DOTAP-functionalized DNA nanocages enable efficient miR168d trafficking to antagonize PVY infection by modulating HSP90-5 homeostasis","fulltext":[{"header":"Background","content":"\u003cp\u003eAmong major plant pathogens that threaten global food security, viruses account for approximately half of the plant infections, resulting in estimated annual economic losses of over US\u003cspan\u003e$\u003c/span\u003e30\u0026nbsp;billion [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. After fungal pathogens, these infections are the second most major cause of reduced worldwide agricultural output [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Viral disease management is complicated and a constant concern because of the significant diversity of plant viruses [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. These diseases can alter the structure of plant communities and cause genetic erosion, which might lead to significant output losses or even crop failure [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. It has been observed that vectors are crucially involved in the dissemination of plant viruses. For example, upon an outbreak, the aphid-transmitted Potato Virus Y (PVY) can rapidly spread throughout tobacco fields, making containment extremely challenging.\u003c/p\u003e\u003cp\u003eA danger to global agriculture, PVY is a systemic infectious pathogen that causes necrotic disease. It can infect\u0026thinsp;\u0026gt;\u0026thinsp;170 plant species from 34 genera, including those in the Solanaceae, Chenopodiaceae, and Fabaceae families. Furthermore, crops like potatoes, tomatoes, and tobacco are especially vulnerable [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Moreover, PVY is among the top ten most damaging plant viruses worldwide and has an extensive geographical distribution [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. A number of variables, such as host cultivar, vector activity, and virus strain, affect the incidence and severity of PVY. Based on how they interact with genes that confer resistance to potatoes, PVY strains are categorized into various types, including C, O, N, Z, and E [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. While strain N causes veinal necrosis, strains C and O have been found to cause mosaic and vein-clearing symptoms in tobacco. In common tobacco, recombinant strains arising from genetic recombination between O and N strains and carrying the N-type HC-Pro protein, such as N:O, NTN, N-Wi, NTN-NW, and NA-NTN, frequently induce severe veinal necrosis [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAs green plant protection has progressed, more focus has been placed on the environmental contamination and safety hazards brought on by excessive use of chemical pesticides. The \"3R\" problems, Residue, Resistance, and Resurgence, pose major risks to ecosystems and human health due to the misuse of these pesticides. In agriculture, biological control refers to the management of pests and diseases by using natural or genetically modified organisms and their byproducts. Currently, 3 main methods are employed in biological control tactics against PVY: genetic engineering, biological agents, and biopesticides. Furthermore, PVY management has progressed because of the discovery and use of RNA interference (RNAi) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eRNAi has been widely acknowledged as a key mechanism of post-transcriptional gene silencing since its discovery in the early 21st century. RNAi synthesizes non-coding small RNAs (sRNA; about 20\u0026ndash;30 nt long) or their encoded products to modulate gene expression. RNAi functions as a crucial molecular immune system in plants, especially during viral infection, and is essential for the early antiviral responses [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe RNAi process is primarily mediated by sRNAs, which, based on their origin and functional mechanisms, can be categorized into three classes: microRNAs (miRNAs), small interfering RNAs (siRNAs), and PIWI-interacting RNAs (piRNAs) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Although these sRNAs have different biogenesis pathways, they all require Argonaute (AGO) proteins to produce effector complexes, which are the fundamental components of RNAi [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. These complexes mediate sequence-specific gene silencing by recognizing target molecules via base complementarity [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In miRNA and siRNA pathways, this complex is called the RNA-induced silencing complex (RISC) and binds target mRNAs \u003cem\u003evia\u003c/em\u003e a guide strand to induce translational repression or mRNA breakage, which in turn silences the gene [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePrevious literature suggests that miRNAs modulate various biological processes by regulating the expression of downstream target genes [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In plants, miRNAs have near-perfect complementarity to their target genes, allowing significantly accurate target prediction. Compared to animals, plant miRNAs can inhibit translation to lower protein levels rather than reducing the expression of target mRNA, indicating a more complex regulatory mechanism. Translational repression and mRNA cleavage are the two major mechanisms by which plant miRNAs mediate gene silencing. AGO proteins' endonuclease activity is required for mRNA cleavage. When the mature miRNA is assembled into the RISC complex, it directs selective recognition and binding to the target mRNA, which is followed by phosphodiester bond cleavage at the location corresponding to nucleotides 10\u0026ndash;11 of the miRNA. The fragments that result are then degraded by exonucleases [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHSP90 is a class of heat shock proteins that are extensively found in both eukaryotic and prokaryotic organisms. Furthermore, it acts as a molecular chaperone that is essential for cellular development, differentiation, apoptosis, and stress responses [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Moreover, different plant species have different numbers of HSP90 isoforms; for example, \u003cem\u003eArabidopsis thaliana\u003c/em\u003e has seven members that are found in the cytoplasm, mitochondria, chloroplasts, and endoplasmic reticulum [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Several studies have indicated that HSP90 modulates the replication and pathogenesis of various plant viruses. It promotes the replication of Bamboo mosaic virus (BaMV) by recognizing the 3\u0026prime;-UTR of the viral RNA [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] and facilitates the replication of Red clover necrotic mosaic virus (RCNMV) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Similarly, tomato yellow leaf curl virus (TYLCV) infection is mediated by HSP90 and SGT1, and its deactivation may accumulate ubiquitinated proteins or cause cell death [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Therefore, to develop new methods for managing viral illnesses, this study selected HSP90 as the target of miRNA interference because of its importance as a vital host component that is less vulnerable to inhibition by direct pathogen targeting and plays a critical role in the viral life cycle.\u003c/p\u003e\u003cp\u003eBecause of its tunable physicochemical features, nanotechnology has attracted a lot of interest in the materials and biological sciences. However, establishing nanomaterials with specific size and shape remains challenging [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. DNA-based nanostructures, such as tetrahedral DNA nanoparticles (TDNs), are developed using Watson\u0026ndash;Crick base pairing. They provide programmable architecture, significant biocompatibility, and low toxicity, thus making them ideal platforms for delivering RNAi-based therapeutics [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. TDNs are self-assembled by annealing four single-stranded DNAs, and are structurally characterized \u003cem\u003evia\u003c/em\u003e non-denaturing gel electrophoresis, atomic force microscopy (AFM), transmission electron microscopy (TEM), and dynamic light scattering (DLS) for size, zeta potential, and dispersion stability [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In plants, their small size allows for effective cellular absorption. TDNs can load miRNA through complementary pairing for antiviral applications by adding a DNA linker at the 5\u0026prime; end. Additional protein or enzyme functionalization improves stability and expands their use in targeted distribution and plant protection.\u003c/p\u003e\u003cp\u003eRecently, various nanocarriers, such as liposomes, polymers, silica, and carbon-based materials, have been developed [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] and showed significant success in animal systems [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Furthermore, several nanoplatforms capable of crossing plant tissue barriers have been engineered to deliver nucleic acids, peptides, and phytohormones [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. For example, liposomes can transport proteins and stimulate immunological responses in \u003cem\u003eArabidopsis\u003c/em\u003e [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]; functionalize carbon nanotubes to promote gene silencing [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Moreover, gold nanoclusters deliver siRNA to induce phenotypic changes [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], and mesoporous silica nanoparticles transport siRNA with substantial efficiency, achieving up to 98% silencing rates [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. These nanoparticles provide a potential non-transgenic crop protection method, decrease enzymatic degradation, and improve nucleic acid stability.\u003c/p\u003e\u003cp\u003eSince their development in the 1960s [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], liposomes (self-assembled phospholipid-cholesterol vesicles) have been employed as essential drug delivery vehicles because of their biocompatibility, biodegradability, and simplicity of surface modification [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. They are frequently utilized to improve patient compliance and therapeutic efficacy \u003cem\u003evia\u003c/em\u003e various administration methods, including parenteral, pulmonary, oral, and topical delivery [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Liposomes improve efficacy while decreasing toxicity by protecting encapsulated drugs from degradation, prolonging half-life, facilitating regulated release, and enabling targeted distribution \u003cem\u003evia\u003c/em\u003e passive or active processes. Cationic liposomes are useful for gene therapy applications, producing stable complexes that resist nuclease degradation and enhance transport efficiency through electrostatic interactions with negatively charged macromolecules, such as DNA and RNA.\u003c/p\u003e\u003cp\u003eNanotechnology breakthroughs have substantially increased the range of available medication delivery methods, providing improved sustainability, biocompatibility, and multifunctional benefits. This study employed the inherent characteristics of liposomes to create a stable and highly effective miRNA delivery system with self-assembled TDNs as the main structural component of a liposomal carrier. This hybrid method overcomes species-specific barriers, decreases the cytotoxic effects associated with traditional virus-induced gene silencing, and significantly increases the stability and transport efficiency of miRNA. This investigation proposed a novel, sustainable, safe, and green approach to developing next-generation antiviral nanomedicines for plants.\u003c/p\u003e"},{"header":"Experimental methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eSynthesis of tetrahedral DNA nanoparticles and formation of its nucleic acid/lipid complex (TDN-miR168d@DOTAP)\u003c/h2\u003e\u003cp\u003eTDNs (approximately 8nm in size) were prepared according to a previous research procedure with slight modifications [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. First, 1.21 g of Tris base, 5.08g of MgCl2\u0026middot;6H2O and 45mL of sterile enzyme-free water were mixed, the pH value of the solution was adjusted to 8.0 with HCl, and the mixed solution was filtered through a 0.22\u0026micro;m filter membrane to obtain 10\u0026times;TM buffer. Then, 1\u0026micro;L of each the A (100\u0026micro;M), B (100\u0026micro;M), C (100\u0026micro;M) and D chains (100\u0026micro;M) were combined (Sequences are in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, materials were synthesized by Qingdao Weilai Biotechnology Co., LTD, and 10\u0026micro;L of 10\u0026times; TM buffer and 86\u0026micro;L of sterile enzyme-free water were added to make a 100\u0026micro;L system. The mixed liquid was heated to 95℃for 10min in a PCR instrument (Eppendorf, Hamburg, Germany) and then cooled to 4℃ for 30min to form a TDN with the miR168d linker sequence. To evaluate the subcellular localization of nanostructures, we used single-stranded A with Cy3 labeling to assemble the TDN.The TDN can be stored at 4℃ for 1 month.\u003c/p\u003e\u003cp\u003emiR168d duplexes were prepared according to previous research procedures with slight modifications [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. To synthesize double-stranded miR168d with a linker sequence, 1\u0026micro;L of each of the two corre sponding fully complementary RNA oligonucleotides miR168d (100\u0026micro;M) and antisense-miR168d (100\u0026micro;M) were mixed (sequences are in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, materials were synthesized by Qingdao Weilai Biotechnology Co., LTD, and 10\u0026micro;L of 10\u0026times;PBS and 88\u0026micro;L of sterile enzyme-free water were added to make a 100\u0026micro;L system. The mixed liquid was heated to 95℃ for 5min in a PCR instrument and then kept at 25℃ for 30 min.\u003c/p\u003e\u003cp\u003eOne hundred microliters of each TDN (1\u0026micro;M) and miR168d (1\u0026micro;M) that had been synthesized in the above steps were mixed with 800\u0026micro;L of 1\u0026times; PBS, and the mixture was heated at 37℃ for 30 min to bind the TDN to miR168d at a final concentration of 100nM. The concentration can be adjusted according to experimental needs.\u003c/p\u003e\u003cp\u003eTo assess the internalization of the nanostructures, we used Cy3-labeled single-stranded A to assemble TDNs. This method ensures that the final concentration of the Cy3 dye in TDN is equal to that in TDN-miR168d so we can evaluate both the internalization of the nano structures based on the Cy3 fluorescence that is colocalized with the cytoplasm and the internalization efficiency of the nanostructures.\u003c/p\u003e\u003cp\u003eThe synthesized TDN-miR168d was taken and dispersed in ultrapure water. An appropriate amount of DOTAP was added into DMSO and dispersed by ultrasonic treatment with a high-intensity ultrasonic processor for 1h. The two were mixed at a volume ratio of 1:1 and incubated at 4℃ for 2h to prepare the antiviral nanomedicine TDN-miR168d@DOTAP.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCharacterization\u003c/h3\u003e\n\u003cp\u003eTDN-miR168d and TDN-miR168d@DOTAP were subjected to gel mobility shift assays in a 3.0% (w/v) agarose gel matrix. The successful assembly of TDN-miR168d was confirmed according to the migration rate, and the extent of DOTAP coating on TDN-miR168d was determined from the bright ness of the band. A nanoparticle size and zeta potential analyzer (OPP TRONIY-919SZ, Beijing, China) was used to measure the hydrated particle size and surface charge of various the complexes by DLS. The structure and morphology of the prepared nanoparticles were deter mined by using the JEOL JEM-F200 (Japan), TESCAN MIRA LMS (Czech Republic) and the Bruker Dimension (Germany). The Brunauer-Emmett-Teller (BET) method (ASAP 2010, Micromeritics, USA) and the Barrett-Joyner-Halenda (BJH) method were used to calculate the pore size distribution and surface area, respectively.\u003c/p\u003e\n\u003ch3\u003ePlant materials and viral strains\u003c/h3\u003e\n\u003cp\u003eHost and virus source: \u003cem\u003eN.benthamiana\u003c/em\u003e cultured in a climate chamber at 25\u0026deg;C with a 16 h light/8 h dark cycle; Potato Virus Y (PVY) and the infectious clone PVY-GFP, which were propagated on N. tabacum var. Samsun NN (Sansheng NN) and preserved by the Virus Research Group of Tobacco Research Institute, Chinese Academy of Agricultural Sciences (CAAS).\u003c/p\u003e\n\u003ch3\u003eTRV-Induced Silencing of miR168d\u003c/h3\u003e\n\u003cp\u003eAgrobacteria containing correctly sequenced plasmids (pTRV1, pTRV2, pTRV2-STTM-miR168d, and pTRV2-PDS) were cultured in a shaker at 28\u0026deg;C for 24 hours. The bacterial cells were collected by centrifugation and resuspended in an infiltration buffer. The resuspended pTRV1 solution was mixed at a 1:1 ratio (OD600\u0026thinsp;=\u0026thinsp;0.5) with three different resuspended solutions separately: pTRV2 (negative control), pTRV2-STTM-miR168 (treatment group), and pTRV2-PDS (positive control). Using a sterile syringe (without the needle), the mixed solutions were gently infiltrated into the abaxial surface of leaves of Nicotiana benthamiana at the 4\u0026ndash;5 leaf stage.\u003c/p\u003e\u003cp\u003eApproximately 10 days after infiltration, when albino phenotypes appeared on the top leaves of N. benthamianain filtrated with pTRV2-PDS, PVY-GFP was inoculated onto the two largest fully expanded leaves in the middle of the tobacco plants. At 1-7dpi, the inoculated leaves were sampled. The expression level of the viral coat protein (CP) was detected using quantitative real-time PCR (RT-qPCR) and Western blot analysis.\u003c/p\u003e\n\u003ch3\u003eTransient overexpression of miR168d\u003c/h3\u003e\n\u003cp\u003eThe resuspended agrobacterial solution containing the correctly sequenced p35S:miR168d plasmid (OD600\u0026thinsp;=\u0026thinsp;0.5) was incubated at room temperature in the dark for 3 hours, then infiltrated into the two largest fully expanded leaves in the upper-middle part of Nicotiana benthamiana plants. After 24 hours of cultivation, PVY-GFP was inoculated onto the infiltrated leaves. From 1 to 7 days post-inoculation (dpi), the infiltrated leaves were sampled to detect the expression level of the viral coat protein (CP).\u003c/p\u003e\u003cp\u003e\u003cb\u003ePrediction and Functional Analysis of the Target Gene (HSP90-5) of miR168d in\u003c/b\u003e \u003cb\u003eN.benthamiana\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe potential target genes of miR168d in \u003cem\u003eN.benthamiana\u003c/em\u003e were predicted using the \"psRobot_tar\" functional module on the web interface of the PsRobot plant small RNA analysis system (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://omicslab.genetics.ac.cn/psRobot/\u003c/span\u003e\u003cspan address=\"http://omicslab.genetics.ac.cn/psRobot/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Based on the transcriptome sequencing results of \u003cem\u003eN.benthamiana\u003c/em\u003e infected with PVY, a heatmap was generated to analyze the expression levels of these potential target genes after viral infection, and quantitative real-time PCR (RT-qPCR) was performed for verification. Bioinformatics analysis was used to examine the binding sites between miR168d and its target genes. Additionally, target gene silencing and overexpression experiments were conducted to detect their effects on PVY infection.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eWestern blotting (WB)\u003c/h2\u003e\u003cp\u003eTo detect the presence of viral proteins, total plant proteins were extracted using protein extraction kit (ABclonal, Wuhan, China) and subjected to WB using virus-specifc antibodies against PVY and GFP (Agdia, Elkhart, IN, USA), respectively. In addition, β-actin was used as the internal reference and detected using β-actin antibodies (ABclonal, Wuhan, China).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eReal-time fuorescence relative quantitative PCR (RT-qPCR)\u003c/h3\u003e\n\u003cp\u003eTotal RNAs were extracted and used as the template to synthesize cDNA samples following the program of the reverse transcription kit (Transgen, Beijing, China). The relative mRNA expression levels of target genes were quantitatively detected following the instructions of the real-time fuorescence quantitative PCR kit (Vazyme, Nanjing, China). Te primer sets PVY-F/PVY-R, β-ActinQF/β-ActinQR (S1) were used to amplify PVY CPand β-actin, respectively. The 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method was employed to calculate the relative expression levels of these genes. All experiments were repeated three times with three biological replicates. Data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation of at least three independent experiments. Statistical analyses were performed using SPSS (v21, IBM, Armonk, NY, USA) with Duncan\u0026rsquo;s multiple range test analysis of variance (ANOVA) and independent sample t-test. Statistical significance was set at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003emiR168d negatively regulates PVY infection in \u003cem\u003eN. benthamiana\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eThe rapid development of high-throughput technologies and the increasing data on plant genomes and transcriptomes have improved our understanding of the roles of miRNA in plant growth and stress responses, including antiviral defense. A high-throughput sequencing analysis of roots, stems, and leaves miRNA of PVY-infected \u003cem\u003eN. benthamiana\u003c/em\u003e identified 1,677 known miRNAs, of which 60 were significantly differentially expressed. Furthermore, a regulatory network linking these miRNAs to antiviral responses was generated by integrating transcriptome co-analysis, GO and KEGG enrichment, and target prediction [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTo investigate the role of miR168d in PVY-infected plants, a 35S promoter-driven construct was employed for transient overexpression of miR168d (35S:miR168d). When infected with PVY, 35S:miR168d plants indicated strong resistance compared to empty vector (35S:00) controls. Moreover, the newly developed leaves had no curling symptoms, and systemic virus movement was significantly delayed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). RT-qPCR analysis showed substantially decreased PVY RNA accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Western blotting validated the decrease in viral coat protein (CP) levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), suggesting that miR168d overexpression improves antiviral immunity.\u003c/p\u003e\u003cp\u003eFor loss-of-function analysis, miR168d was silenced using a short tandem target mimic (STTM) construct (TRV:miR168d). The TRV:PDS-positive control indicated photobleaching was predicted (Supplementary Fig.\u0026nbsp;1A), whereas the TRV:miR168d plants had a 71.3% reduction in miR168d levels (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e; Supplementary Fig.\u0026nbsp;1B). After PVY-GFP treatment, TRV:miR168d plants showed stronger GFP fluorescence relative to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). RT-qPCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE) and western blot (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF) revealed that both PVY\u0026rsquo;s CP transcript and protein levels were significantly elevated in silenced plants, confirming increased viral susceptibility. It was found that miR168d acts as a negative regulator of PVY infection and its overexpression can effectively restrict viral replication and spread, providing a new molecular target for miRNA-mediated antiviral defense in plants.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003emiR168d targets HSP90-5 to inhibit PVY infection\u003c/h2\u003e\u003cp\u003eTo elucidate the response mechanism of \u003cem\u003eN. benthamiana\u003c/em\u003e miR168d during PVY infection, the expression dynamics of miR168d in different tissues were evaluated after infection. Differential miRNA expressions were identified \u003cem\u003evia\u003c/em\u003e sRNA sequencing and were validated by RT-qPCR. The data revealed that miR168d expression was significantly reduced during PVY infection, suggesting its potential role as a regulatory factor in host innate immunity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Furthermore, transcriptomic and RT-qPCR heatmap analyses showed that the HSP90-5 transcript was substantially upregulated in PVY-infected leaves (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u0026ndash;C). Moreover, Bioinformatics prediction and subsequent target validation identified a specific binding site between miR168d and the 3\u0026prime;UTR of HSP90-5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). miR168d overexpression substantially inhibited HSP90-5 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), confirming that HSP90-5 is a direct target of miR168d, consistent with the previous reports indicating that heat shock proteins, including HSP90, promote infection and replication of various plant viruses [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eTo functionally characterize the miR168d\u0026ndash;HSP90-5 regulatory axis, the gain- and loss-of-function assays were carried out to evaluate its impact on PVY infection. Transient HSP90-5 overexpression in \u003cem\u003eN.benthamiana\u003c/em\u003e, followed by PVY-GFP inoculation, significantly increased GFP fluorescence under UV light (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Furthermore, RT-qPCR and Western blot analyses validated the upregulated levels of viral CP mRNA and protein (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG\u0026ndash;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). Whereas HSP90-5 silencing inhibited viral CP accumulation and reduced fluorescence intensity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI\u0026ndash;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK). These findings revealed that miR168d post-transcriptionally suppresses HSP90-5 expression, which inhibits PVY accumulation, thus indicating a novel miRNA\u0026ndash;target module crucial for plant antiviral defense.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eCationic lipid-coated DNA nanostructure for efficient miRNA delivery in plants\u003c/h2\u003e\u003cp\u003eThis study addressed the problem of low gene delivery efficiency due to the plant cell wall barrier and nuclease degradation, improving effective delivery of the resistance gene miR168d. Using a TDN as the framework and the cationic lipid DOTAP (1,2-dioleoyl-3-trimethylammonium propane) as the coating, a unique nano-delivery system was developed that significantly improved the antiviral gene miR168d's transmembrane permeability and vascular long-distance transport capability.\u003c/p\u003e\u003cp\u003eFurthermore, 3% agarose gel electrophoresis indicated that the four single-stranded DNAs (A, B, C, D) were of approximately equal length (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The effective assembly of TDN-miR168d was confirmed by the synthesized TDN's band size, which was 190 bp and increased to about 210 bp following conjugation with miR168d. Hydrodynamic size measurements revealed that TDN-miR168d and TDN-miR168d@DOTAP had diameters of 23.6 nm and 165.1 nm, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Furthermore, zeta potential analysis demonstrated that TDN-miR168d had a negative charge of -8 mV, whereas DOTAP had a positive charge of +\u0026thinsp;55 mV. The completely encapsulated TDN-miR168d@DOTAP complex had a positive zeta potential of +\u0026thinsp;35.6 mV.\u003c/p\u003e\u003cp\u003eAfter pretreatment, the TEM showed that uncoated TDN-miR168d had lost its tetrahedral integrity (Supplementary Fig.\u0026nbsp;3). DOTAP's encapsulation of TDN-miR168d was confirmed using infrared spectroscopy. The spectrum of TDN-miR168d showed distinctive peaks at 1309.35 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1436.10 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (molecular skeleton vibrations), 2912.06 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 2994.86 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (C\u0026ndash;H stretching vibrations of alkyl chains), and 1041.53 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (ascribed to P\u0026ndash;O or C\u0026ndash;O vibrations in nucleic acids). In the composite spectrum, the 1041.53 cm⁻\u0026sup1; peak disappeared because of electrostatic interactions between phosphate groups and DOTAP\u0026rsquo;s cationic amine groups. The absence of alkyl peaks and the appearance of a new peak at 1652.31 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, along with a reorganization of molecular skeleton vibrations, confirmed that TDN-miR168d was successfully encapsulated by DOTAP instead of physically mixing (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003eThis study also performed AFM and TEM analyses, which revealed good dispersion without significant aggregation. AFM indicated significant differences in the morphology and height between TDN-miR168d and TDN-miR168d@DOTAP, highlighting structural changes post-encapsulation. Moreover, TEM images of TDN-miR168d@DOTAP revealed significantly enlarged particle sizes and localized protrusions, which might be due to the formation of an electrostatic complex between cationic DOTAP and TDN-miR168d, leading to a thicker composite structure (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD\u0026ndash;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). These morphological alterations further support successful encapsulation.\u003c/p\u003e\u003cp\u003eTo prepare the Cy3A-TDN-miR168d complex for real-time intracellular tracking, the A strand was substituted with a Cy3-labeled A-Cy3 strand. After 24 hours of leaf infiltration, confocal microscope imaging revealed a particular Cy3 fluorescence enrichment in the cytoplasm of \u003cem\u003eN. benthamiana\u003c/em\u003e cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF), demonstrating that TDN, with its virus-like size and efficient endocytosis, enables transmembrane delivery of miR168d to the target site. This finding highlights TDN's advantage in tissue penetration as a gene carrier and lays the groundwork for future functional research on antivirals.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eCationic lipid-modified nanocarrier enhances antiviral miRNA delivery and efficacy in plants\u003c/h2\u003e\u003cp\u003eThis study employed the cationic lipid DOTAP to generate a charge-adapted nanocomplex, TDN-miR168d@DOTAP, to systematically improve the transmembrane permeability and long-distance vascular transport capacity of the antiviral gene miR168d. This was performed to improve the efficiency of the gene delivery system, which was limited by the plant cell wall barrier and nuclease degradation.\u003c/p\u003e\u003cp\u003eFurthermore, cytotoxicity analyses were conducted on TDN-miR168d and TDN-miR168d@DOTAP to assess their biosafety and constituents. Neither substance substantially impacted the typical growth and development of tobacco suspension cells (By2) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Moreover, in both treatments, plants had intact cellular morphology with cell survival above 95%, suggesting that the TDN-miR168d@DOTAP nano-delivery system has low cytotoxicity, good biocompatibility, and no effect on typical plant growth.\u003c/p\u003e\u003cp\u003eTDN-miR168d (bare carrier) and TDN-miR168d@DOTAP (lipid-modified) were sprayed foliarly on healthy \u003cem\u003eN. benthamiana\u003c/em\u003e plants to evaluate the enhanced effect of DOTAP modification on miR168d delivery. After 3 to 7 days, miR168d expression was observed in the stems (the systemic transport terminus) and leaves (the local delivery site). RT-qPCR revealed that from day 5 to 7, the TDN-miR168d@DOTAP group's miR168d expression in leaf tissues was consistently and significantly higher than that of the bare carrier group (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). This suggests that DOTAP-mediated membrane affinity successfully passes through the cell wall and epidermal wax barriers, improving the efficiency of local delivery. In stem tissues, no significant differences were observed in miR168d levels between the two groups within the first 3 days (\u003cem\u003ep\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e). However, by day 5, the TDN-miR168d@DOTAP group indicated increased expression (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). This delayed increase suggests that DOTAP provides prolonged systemic transport capacity \u003cem\u003evia\u003c/em\u003e nuclease resistance and vascular-targeted penetration in addition to improving local delivery. In particular, during apoplastic and symplastic transport, the lipid coating protects the TDN structure from phosphoesterases and plant RNAses. Furthermore, the nanocomplex's small size and surface charge alteration allow for intercellular transport through plasmodesmata and phloem sieve tubes, which ultimately permits the genetic payload to be distributed systemically.\u003c/p\u003e\u003cp\u003eTo validate the antiviral efficacy of the TDN-miR168d@DOTAP nanocomplex, its ability to confer resistance against PVY infection was evaluated. \u003cem\u003eN. benthamiana\u003c/em\u003e plants with 6 to 8 leaves were rub-inoculated with PVY. After 24 hours, the infected leaves were completely sprayed with TDN-miR168d or TDN-miR168d@DOTAP; the control group received an equivalent amount of water. On the 7th day after therapy, samples were collected for qRT-qPCR analysis, which revealed that compared to the control, PVY CP mRNA levels decreased by 49.4% in the TDN-miR168d group and by 64.3% in the TDN-miR168d@DOTAP group (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD\u0026ndash;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). These findings were validated by Western blot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). These results suggest that the antiviral gene was successfully delivered via the TDN-miR168d@DOTAP nanocomplex, which also provides substantial resistance against PVY, thus providing a new and effective way of delivering antiviral genes in plants.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study elucidated a novel antiviral mechanism in \u003cem\u003eN.benthamia\u003c/em\u003e mediated by the miR168d-HSP90-5 regulatory module, and also established an efficient nanocarrier system, TDN-miR168d@DOTAP, for targeted miRNA delivery. The findings not only deepen the understanding of host-virus interactions but also demonstrate the potential of nanotechnology in non-transgenic crop protection strategies.\u003c/p\u003e\u003cp\u003eMicroRNAs have emerged as key regulators of plant immune responses, fine-tuning gene expression during pathogen challenges. Previous studies have reported differential expression of certain miRNAs during viral infections, showing that they participate in defense responses by targeting either viral genomes or host factors linked to infection [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. For instance, miR168 regulates AGO1 expression to modulate RNAi efficiency, while miR160 influences auxin signaling to affect susceptibility [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The present study identified miR168d as a negative regulator of PVY infection, extending the functional repertoire of miRNAs in antiviral defense.\u003c/p\u003e\u003cp\u003eNotably, unlike canonical RNAi approaches that target viral genes directly, miR168d operates through a host-centered mechanism by suppression of HSP90-5, a chaperone protein critical for viral replication. This strategy may reduce the selective pressure on mutants involved in viral escape, a common drawback of pathogen-targeted RNAi [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The present results align with reports that HSP90 facilitates the replication of various viruses, including bamboo mosaic virus and tomato yellow leaf curl virus [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. However, in contrast to earlier studies that focused on genetic or chemical inhibition of HSP90, we demonstrate that miRNA-mediated post-transcriptional regulation offers a tunable and specific means of control.\u003c/p\u003e\u003cp\u003eFurthermore, the observed downregulation of miR168d during PVY infection suggests the presence of a viral counter-defense strategy, possibly leading to modulation of HSP90-5 levels and the promotion of replication. These dynamic miRNA-target interactions highlight the co-evolutionary arms race between plants and viruses [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The present study thus contributes to a growing body of evidence that miRNAs represent central players in plant immunity, and their manipulation offers a promising approach for the engineering of resistant crops.\u003c/p\u003e\u003cp\u003eThe effective delivery of nucleic acids within plants remains a major challenge due to plant cell walls, enzymatic degradation, and limited vascular mobility. While nanoparticle-based delivery has been widely explored in mammalian systems [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], its application in plants is still emerging. Recent studies have used clay nanosheets, carbon nanotubes, and silica nanoparticles for the delivery of siRNAs and DNA constructs [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. However, many of these systems are associated with low efficiency, cytotoxic effects, or limited tissue penetration.\u003c/p\u003e\u003cp\u003eOur designed nanocarrier, TDN-miR168d@DOTAP, integrates the structural programmability of DNA nanostructures with the membrane affinity of cationic lipids. TDNs offer excellent biocompatibility, enable precise functionalization, and are easy to synthesize [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], while DOTAP enhances cellular uptake and nuclease resistance through electrostatic interactions with negatively charged membranes. The resulting nanocomplex achieved significantly higher accumulation of miR168d in both local and systemic tissues compared to the non-lipidated TDN, underscoring the importance of surface engineering in overcoming plant-specific barriers.\u003c/p\u003e\u003cp\u003eThe cytoplasmic delivery of Cy3-labeled TDNs was confirmed using confocal microscopy, demonstrating their ability to cross cell walls and membranes, possibly via endocytosis or plasmodesmata-mediated transport. This represents a significant advance over conventional methods, such as the use of agrofiltration or viral vectors, which are often limited by host range, insert size, or regulatory constraints [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. The present system provides a non-transgenic, scalable, and environmentally friendly alternative for gene delivery in plants.\u003c/p\u003e\u003cp\u003eFrom a practical perspective, this study offers a promising strategy for the control of infection by PVY and potentially other plant viruses. The foliar application of TDN-miR168d@DOTAP significantly reduced accumulation of the PVY coat protein, demonstrating its efficacy under controlled conditions. This approach aligns with the growing demand for sustainable alternatives to chemical pesticides responsible for both environmental contamination and the development of resistance [\u003cspan additionalcitationids=\"CR58\" citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTheoretically, the findings indicate the value of targeting host factors, instead of viral components, for broad-spectrum resistance. The strong conservation of HSP90 in plants and its ability to facilitate the replication of multiple viruses suggest that miR168d-based strategies could apply to a range of pathosystems. Furthermore, the modularity of the TDN platform would enable adaptation for the delivery of other miRNAs or siRNAs, making it a versatile tool for both research and application.\u003c/p\u003e\u003cp\u003eAdditionally, this study bridges molecular biology and nanotechnology, illustrating the effectiveness of interdisciplinary approaches in addressing longstanding challenges in agriculture. The integration of molecular mechanisms (miRNA-target interactions) with engineered solutions (nanocarriers) provides a holistic framework for the development of next-generation techniques for plant protection.\u003c/p\u003e\u003cp\u003eDespite these advances, several challenges should be addressed before translational application. 1. Target Specificity and Potential Off-Target Effects: While silencing HSP90-5 was effective in the suppression of PVY, HSP90 proteins are involved in a variety of essential cellular processes, including stress responses, hormone signaling, and protein folding [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Constitutive downregulation may therefore impair plant growth and development. To improve specificity, future studies could focus on the identification of downstream effectors of HSP90-5 that are specifically hijacked by viruses, using proteomic or interactome analyses, leading to the development of tissue-specific or infection-inducible promoters to drive miRNA expression. Another possibility is the use of CRISPR-based methods to engineer HSP90-5 variants that retain physiological functions while resisting viral exploitation. 2. Environmental Stability and Field Applicability: It is possible that the described nanocomplex is susceptible to degradation under field conditions (e.g., UV radiation, rainfall, temperature fluctuations). DOTAP, in particular, is prone to oxidation under strong light. The following strategies could be used to enhance its resilience: encapsulation in light-responsive polymers that release payloads under specific wavelengths; the use of natural lipids (e.g., soybean lecithin or phytosterols) to improve biodegradability and reduce cost; surface functionalization with protective ligands (e.g., cellulose-binding domains or chitosan) to enhance adhesion and retention on leaf surfaces. 3. Scalability and Regulatory Considerations: Large-scale synthesis of TDNs is both technically challenging and costly. Future work should explore the use of automated assembly platforms for high-throughput production, the use of alternative nanomaterials (e.g., biodegradable polymers or peptide-based carriers) that provide similar functionality with easier scalability, and thorough biosafety assessments to ensure non-toxicity to non-target organisms and compliance with regulatory standards for nano-agricultural products. 4. Resistance Management: Viral evolution may lead to escape mutants, as commonly seen in resistance strategies. Continuous monitoring of the conservation of the target site and miRNA efficacy is essential. It is proposed that a virus surveillance network be established to track mutations in real-time, as well as the design of multi-target miRNA systems or the combination of miRNA delivery with other RNAi constructs to reduce the risk of escape. The integration of nanomaterial application with other management practices (e.g., crop rotation or biological controls) would also assist sustainable disease management.\u003c/p\u003e\u003cp\u003eIn summary, this study provides a comprehensive molecular and technological framework for the application of miRNA-mediated antiviral defense in plants. The study not only identified miR168d as a key regulator of PVY infection, mediated by suppression of HSP90-5, but also developed an efficient nanodelivery system that enhanced miRNA stability, uptake, and systemic mobility. These findings advance our fundamental understanding of plant-virus interactions and provide a translatable strategy for sustainable agriculture.\u003c/p\u003e\u003cp\u003eFuture efforts should focus on refining target specificity, improving environmental stability, and enabling scalable production. The integration of tools from nanotechnology, synthetic biology, and precision agriculture has significant potential for addressing the challenges associated with crop disease and contributing to global food security.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eNanodelivery technology serves as a pivotal solution for enhancing miRNA delivery efficiency, offering considerable potential to mitigate environmental safety concerns and pesticide resistance. In this study, we successfully developed a novel and efficient miRNA-based nanoplatform that specifically targets the viral auxiliary factor HSP90-5, functioning as a key antiviral regulator in N.benthamiana. The system, designated TDN-miR168d@DOTAP, integrates a tetrahedral DNA nanocage (TDN) with a cationic lipid coating. This formulation significantly improved miR168d stability and systemic transport, leading to a substantial reduction in Potato virus Y (PVY) accumulation and enhanced plant resistance. Further investigations revealed that the delivery system effectively downregulates HSP90-5 expression and confers significant protective effects against PVY infection. Importantly, in vivo assessments confirmed that the system caused no adverse effects on plant growth and development, inducing only minimal plant cell necrosis, thereby demonstrating its favorable biosafety profile. Collectively, these findings highlight the promising application prospects of the biocompatible and environmentally benign TDN-miR168d@DOTAP system for targeted inhibition of plant gene expression and sustainable management of plant viral diseases.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYanwei Gong: conceptualization, data curation, investigation, methodology, formal analysis, writing-original draft, and writing-review and editing. Junying Zhang: conceptualization, investigation. Liu Yang: supervision, funding acquisition and validation. Xinyi Zhao: formal analysis and data curation. Lingdie Wang: review and editing. Dong An: formal analysis and data curation. Lianqiang Jiang: investigation, methodology and writing-review and editing. Yubing Jiao: conceptualization, methodology, formal analysis, data curation and editing. Lili Shen: conceptualization, investigation, methodology and writing-review and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Science and Technology Project of Liangshan Prefecture Company, Sichuan Provincial Tobacco Company (SCYC202311)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eno datasets were generated or analysed during the current study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank all the reviewers who participated in the review and MJEditor for its linguistic assistance during the preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003enot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003enot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFarokhzad OC, Langer R. 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Mol Plant Pathol. 2011;12(9):938-54. https://doi.org/10.1111/j.1364-3703.2011.00752.x\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"chemical-and-biological-technologies-in-agriculture","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Chemical and Biological Technologies in Agriculture](https://chembioagro.springeropen.com/)","snPcode":"40538","submissionUrl":"https://submission.nature.com/new-submission/40538/3","title":"Chemical and Biological Technologies in Agriculture","twitterHandle":"@SpringerPlants","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"miRNA, Antiviral immunity, Nanocarrier, Heat shock protein","lastPublishedDoi":"10.21203/rs.3.rs-7756582/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7756582/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e Plant viruses cause severe agricultural losses. Conventional pesticides have issues such as residues and resistance, while the delivery efficiency of functional microRNAs in RNA interference strategies is low. This study aimed to evaluate the inhibitory effect of miR168d on Potato virus Y in \u003cem\u003eNicotiana benthamiana\u003c/em\u003e and construct a high-efficiency nanodelivery system.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults \u003c/strong\u003eMicroRNA168d significantly reduces the replication and spread of Potato virus Y by targeting and inhibiting Heat Shock Protein 90-5. For the nanocomplex with a tetrahedral DNA nanostructure as the carrier and the cationic lipid 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) as the coating—characterization showed that it enhances the vascular transport efficiency, nuclease resistance, and cellular permeability of miR168d. Additionally, this nanocomplex exhibits low toxicity and good biocompatibility toward tobacco suspension cells. After foliar application, the nanocomplex group showed higher accumulation of miR168d in the leaves and stems of \u003cem\u003eN.benthamiana\u003c/em\u003ecompared with that in the control group. Specifically, the accumulation of mRNA and protein of the PVY coat protein in the nanocomplex group decreased by 64.3% and the corresponding percentage (consistent with the reduction at the protein level), respectively. As a result, the disease resistance of the plants was significantly improved\u003cstrong\u003e.\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e This study reveals the antiviral mechanism of the miR168d-HSP90-5 regulatory module, provides a green non-transgenic nanoscale strategy, and is of great significance for agricultural antiviral breeding and sustainable agriculture.\u003c/p\u003e","manuscriptTitle":"DOTAP-functionalized DNA nanocages enable efficient miR168d trafficking to antagonize PVY infection by modulating HSP90-5 homeostasis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-01 18:40:21","doi":"10.21203/rs.3.rs-7756582/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-28T16:06:46+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-28T15:29:24+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-28T08:16:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"152155826582736974875267210864522578107","date":"2025-10-23T00:32:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"187530567187447214766165656214908230460","date":"2025-10-22T06:15:22+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-22T02:46:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-15T06:09:26+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-14T17:50:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"Chemical and Biological Technologies in Agriculture","date":"2025-10-11T02:03:59+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"chemical-and-biological-technologies-in-agriculture","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Chemical and Biological Technologies in Agriculture](https://chembioagro.springeropen.com/)","snPcode":"40538","submissionUrl":"https://submission.nature.com/new-submission/40538/3","title":"Chemical and Biological Technologies in Agriculture","twitterHandle":"@SpringerPlants","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1d5b053c-096a-4417-860f-95bb153da091","owner":[],"postedDate":"November 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-11-09T07:08:07+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-01 18:40:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7756582","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7756582","identity":"rs-7756582","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}