The high-permeability cellulose nanocrystals carrier facilitates zinc utilization and enhances nsLTP2-mediated plant immunity

The high-permeability cellulose nanocrystals carrier facilitates zinc utilization and enhances nsLTP2-mediated plant immunity | 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 Article The high-permeability cellulose nanocrystals carrier facilitates zinc utilization and enhances nsLTP2-mediated plant immunity Xianchao Sun, Jing Wang, Shunyu Xiang, Xiaoyan Wang, Yang Shen, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5791872/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Zinc (Zn 2+ ) is an essential micronutrient that regulates plant growth, immunity, and antiviral defense mechanisms. However, its limited bioavailability often necessitates excessive application, resulting in inefficiencies in production and environmental stress. In response, we propose an environmentally friendly and sustainable approach to enhance the utilization of Zn 2+ . We developed CNC@PDA@Zn 2+ by embedding Zn 2+ into the polydopamine (PDA) coating of cellulose nanocrystals (CNCs). Leveraging the high cell permeability of CNCs, this material increased the transport capacity of Zn 2+ in plants and demonstrated the ability to inactivate viral particles in vitro . Moreover, CNC@PDA@Zn 2+ showed a superior induction of resistance while reducing Zn 2+ content, specifically by reprogramming the expression and localization of the resistance-related non-specific lipid transfer protein 2 (nsLTP2), which enhanced the salicylic acid (SA) signaling pathway in plants. Furthermore, the high conservation of nsLTP2 in flowering plants increases the potential application range of CNC@PDA@Zn 2+ . Importantly, CNC@PDA@Zn 2+ represents the most effective Zn 2+ -based antiviral nanomaterial to date, achieving its effects at the lowest reported Zn 2+ concentration. Overall, our results highlight that CNC@PDA@Zn 2+ can more effectively upregulate the conserved nsLTP2, thereby inducing viral defense responses via the SA pathway. This strategy not only improves the operation and utilization rate of Zn 2+ but also reduces its environmental residues, laying a theoretical foundation for the development of antivirus defense. Biological sciences/Plant sciences/Plant stress responses/Biotic Earth and environmental sciences/Environmental sciences/Environmental impact Zinc cellulose nanocrystal antivirus plant immunity TMV Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Plant viruses pose a significant threat to agriculture, causing substantial reductions in crop quality and yield upon infection 1 . The tobacco mosaic virus (TMV), recognized as one of the most detrimental plant viruses, has been identified to infect over 350 host plant species, including economically important crops such as tobacco, tomatoes, and peppers, leading to substantial economic losses 2 . Upon infection, TMV disrupts chloroplasts within plant cells, impedes photosynthesis, and results in mottled and discolored leaves, while also affecting cell division and causing leaf deformities 3 . Currently, there is no agent capable of fully eradicating TMV 4 . The prevention and management of viral diseases associated with TMV largely rely on the use of viral inhibitors, which face challenges such as limited storage stability, environmental pollution, excessive pesticide residues, and the development of pathogen resistance 5 . With advances in understanding plant immune mechanisms, there has been a shift towards utilizing plant immune inducers for disease prevention and control. These inducers trigger immune responses, including reactive oxygen species (ROS) accumulation, Ca 2+ signaling, callose deposition, salicylic acid (SA) production, and the expression of resistance-related genes 6, 7, 8, 9 , ultimately leading to systemic acquired resistance (SAR). During SAR, SA levels increase, and resistance genes such as pathogen-related protein (PR) genes are upregulated 10, 11, 12, 13 . The utilization of zinc (Zn²⁺) for disease control dates back to as early as 1939, and potentially even earlier 14, 15 . Zn 2+ , an essential micronutrient for plant growth, is integral to various biological processes, including metabolic pathways, enzymatic reactions, hormone metabolism, disease resistance, and stress tolerance. More recently, it has been recognized for its role in the regulation of nitrogen fixation efficiency within root nodule symbiosis 16 . As previously highlighted, plant immunity against pathogens is associated with SA accumulation in non-infected tissues, ROS burst, and the initiation of SA-dependent SAR. In this context, C9 dicarboxylic acids and azelaic acid (AzA) are known to participate in the long-distance signaling of SA 17 . It is significant that under Zn 2+ -rich and AzA conditions, the expression of PR1 is more readily upregulated compared to Zn 2+ -deficient conditions, indicating that adequate Zn 2+ levels can enhance the production of resistance-related metabolites, thereby influencing plant resistance levels. Prior research has shown that the lipid transfer protein-like protein encoded by AtAZI1 (Azelaic acid induced 1) is involved in transmitting SAR signals in response to AzA, thereby activating local or systemic nonspecific resistance to pathogens 18 , and this function of AtAZI1 is Zn 2+ -dependent 19 , establishing a link between plant nutritional immunity and micronutrient utilization. Moreover, the application of Zn 2+ fertilizers under drought conditions has been shown to increase antioxidant content (e.g., ascorbic acid, reduced glutathione, total flavonoids, total phenols) in wheat leaves, and foliar Zn 2+ spraying can enhance photosynthetic pigments, reduce lipid peroxidation of cell membranes, maintain cellular homeostasis, and mitigate drought-induced oxidative damage 20 . Thus, Zn 2+ emerges as a potent plant immune inducer and growth regulator 21, 22 . Despite the numerous studies highlighting Zn 2+ 's role in pathogen control, the application of Zn 2+ fertilizers still tends to be at relatively high concentrations 23 . Due to environmental complexities, the loss of active ingredients during the application of trace elements can reach 70%-90% 24 , which compromises the maximum absorption of Zn 2+ by plants 25 . Repeated applications not only increase economic costs but also impose environmental stress, leading to plant stress 26, 27 . In our previous study, we determined that 44 µg/mL of (CH 3 COO) 2 Zn (Zn 2+ content is 237.3 µM) exhibited antiviral properties while still allowing normal plant growth 28 . Furthermore, Zn 2+ toxicity symptoms have been reported to manifest at concentrations of 300 mg/kg leaf dry weight, although some crops have lower thresholds 26 . These findings underscore the high loss rates of Zn 2+ in practical applications and the potential environmental risks associated with its use. To address these challenges, we implemented various delivery strategies to mitigate Zn 2+ loss 29, 30 . However, more optimized delivery methods are still required to minimize Zn 2+ residues in the environment and enhance the efficiency of Zn 2+ application 31 . Due to the size effect and high permeability of nanomaterials, they can effectively enhance the stability of pharmaceutical ingredients, facilitate drug deposition on the application interfaces, and improve drug utilization 32 . Therefore, they are ideal for drug delivery in the development of plant disease prevention and control agents 33 . Rod-shaped nanomaterials, such as cellulose nanocrystals (CNCs), offer significant advantages as drug carriers for plant disease control due to their high surface area and aspect ratio 34, 35 . This feature facilitates cellular binding and uptake while minimizing cytotoxicity, and enhances deposition and retention on the surface of foliage 36, 37 . For instance, surface modification with polydopamine (PDA) enhances CNC’s affinity for cells. Additionally, CNCs can adsorb nanoparticles, such as silver, improving their bactericidal effect by increasing particle concentration on bacterial membranes 38 . Moreover, the properties of CNCs can be enhanced, such as sustaining control, via modification of the CNCs surface 39, 40 . Thus, it is reasonable to consider CNCs as pesticide carriers with high deposition efficiency, sustainable pesticide release, and effective antifungal and insecticidal capabilities 41 42 . Based on these analyses, we hypothesize that Zn 2+ functions as a signaling messenger during plant virus infection. Notably, the Zn 2+ concentration within plant cells is typically maintained at a steady state of 10–100 nM, while concentrations above 100 µM can induce cellular Zn 2+ toxicity. Our previous determination of the lowest antiviral concentration of (CH 3 COO) 2 Zn at 44 µg/mL, corresponding to 237.3 µM Zn 2+ , exceeds this threshold. However, increasing the concentration of (CH 3 COO) 2 Zn tenfold demonstrated enhanced resistance without inducing Zn 2+ toxicity 28 . This observation challenges the presumed upper limit of the capacity of Zn 2+ transporters in plants and underscores the substantial loss of (CH 3 COO) 2 Zn during resistance induction. Nonetheless, residual Zn 2+ , if not assimilated by the plant, poses environmental risks. Therefore, it is critical to reduce the applied concentration of Zn 2+ while enhancing its cellular uptake to optimize its role as a second messenger. To achieve this, we selected rod-shaped CNCs characterized by low cytotoxicity and high permeability for Zn 2+ modification. By ensuring minimal cytotoxicity, we increased the plant’s absorption and transport of Zn 2+ , thereby amplifying Zn 2+ -mediated SAR. This approach lays a theoretical foundation for the informed application of Zn 2+ in plant disease prevention and control. Results and Discussions Synthesis and characterization of CNC@PDA@Zn 2+ As mentioned above, although we systematically explored the minimum concentration of (CH 3 COO) 2 Zn that can induce plant disease defense, these concentrations still highly exceed the maximum Zn 2+ tolerance of plant cells. Moreover, as the (CH 3 COO) 2 Zn concentration increases, the resistance levels significantly improve while maintaining normal growth without any high Zn 2+ phenotypes. This phenomenon highlights the low efficiency of Zn 2+ absorption and the high environmental residue associated with the use of (CH 3 COO) 2 Zn. A recent study has shown that plants can enhance the utilization of metal ions in complex forms 43 . Therefore, we first synthesized CNC@PDA by exploiting the hydrogen bonding between CNC and PDA. This composite then served as a carrier for chelating Zn 2+ , leading to the creation of CNC@PDA@Zn 2+ (Fig. 1 a). To further confirm the chemical structure of CNC@PDA@Zn 2+ , we conducted verification by Transmission Electron Microscope (TEM), Fourier-transmitted infrared spectrometry (FTIR), Zeta potential, X-ray diffraction (XRD), and Thermogravimetric analysis (TGA). TEM showed that after grafting Zn 2+ on the surface of CNC, it displayed similar morphology (Fig. 1 b-c). In Fig. 1 d, the absorption peaks at 3359 cm − 1 , 2910 cm − 1 , and 1058 cm − 1 in CNC, CNC@PDA, PDA@Zn 2+ , and CNC@ PDA@Zn 2+ are assigned to -OH stretching, C–H vibration, and C–O stretching vibration respectively, which are the typical absorption peaks of CNC 44 . Additionally, the zeta potential shifted from − 31.8 ± 0.5 mV (CNC) to -29.7 ± 0.4mV (CNC@PDA) after coating PDA. Reduced zeta potential to -27.8 ± 0.4 mV (CNC@ PDA@ Zn 2+ ) after coordinating Zn 2+ on the CNC@PDA surface, indicating successful surface modification (Fig. 1 e). Additionally, TGA (Fig. 1 f) showed a considerable increase in the peak degradation temperature of CNC@PDA@Zn 2+ compared to CNC, suggesting that the modification improved the thermal stability of the composite. This indicates that the PDA coating enhances the thermal resistance of the CNC@PDA@Zn 2+ material. XRD analysis (Fig. 1 g) revealed that the main diffraction characteristics of CNC were still clearly present after modification, such as the diffraction peaks at 2θ angles around 14.7° (101), 16.6° (101̅), 22.7° (002), and 34.5° (040) 45 . These results showed that the modification didn’t disturb the crystalline integrity of the main cellulose structure. Besides, it indicated the decrease in crystallinity of nanoparticles is attributed to the introduction of a dense amorphous PDA diffusion layer on the CNC structure. Table S1 presents the results of the elemental analysis of zinc provided the anchoring of Zn 2+ onto CNCs by Inductively coupled plasma optical emission spectroscopy (ICP-OES). CNC@PDA@Zn 2+ demonstrates enhanced antiviral efficacy In our previous research, we observed that the antiviral activity of (CH 3 COO) 2 Zn exhibited a dose-dependent effect within a certain range 46 . However, even at the lowest concentration of (CH 3 COO) 2 Zn (44 µg/mL), the Zn 2+ concentration was approximately 237.3 µM, significantly exceeding the maximum Zn 2+ tolerance of plant cells 47 . To address this, we utilized the dimensional properties of CNCs to reduce the usage concentration of Zn 2+ while enhancing the overall antiviral efficacy of the material. We grafted Zn 2+ onto a crosslinked PDA coating on rod-shaped CNCs, resulting in CNC@PDA@Zn 2+ . Based on the ICP results ( Table S1 ), the Zn 2+ concentration of 1600 µg/mL CNC@PDA@Zn 2+ is approximately equivalent to the 44 µg/mL (CH3COO)2Zn acetate solution. Subsequently, detailed experiments were conducted using a concentration of 44 µg/mL (CH 3 COO) 2 Zn as a control. Various concentrations of CNC@PDA@Zn 2+ were sprayed daily for three days (10 mL per 10 plants each day). On the 4th day, GFP-tagged TMV was inoculated onto the sixth and seventh leaves of N. benthamiana via rub inoculation. GFP fluorescence intensity and changes were monitored under UV light to trace TMV infection (Fig. 2 a). Results showed that at 2 dpi, (CH 3 COO) 2 Zn treatment exhibited some antiviral activity, whereas CNC@PDA@Zn 2+ treatment significantly enhanced resistance, with higher concentrations correlating with increased resistance. Subsequent qPCR analysis of TMV-GFP nucleic acid accumulation confirmed these findings (Fig. 2 b). By the 4 dpi, the fluorescence intensity of inoculated leaves treated with (CH 3 COO) 2 Zn and water had approached the threshold, whereas CNC@PDA@Zn 2+ treatment resulted in slower infection progression (Fig. 2 c). qPCR showed that on this day, the TMV-GFP nucleic acid levels in (CH 3 COO) 2 Zn and water-treated leaves were similar, but significantly lower levels of TMV-GFP were observed in CNC@PDA@Zn 2+ -treated leaves. At 6 dpi, substantial TMV accumulation was evident in systemic leaves of water-treated plants, significantly reduced in (CH 3 COO) 2 Zn-treated leaves, and maintained at the lowest levels in CNC@PDA@Zn 2+ -treated leaves (Fig. 2 d). These results indicate that CNC@PDA@Zn 2+ effectively delays TMV-GFP infection. Notably, the antiviral function of CNC@PDA@Zn 2+ exhibited a dose-dependent response. At its maximum concentration (1600 µg/mL), the Zn 2+ content was approximately 237.3 µM (equivalent to the Zn 2+ content in 44 µg/mL (CH 3 COO) 2 Zn), and the material showed very strong antiviral activity, increasing efficacy more than threefold compared to the same Zn 2+ concentration of (CH 3 COO) 2 Zn. Even when diluted 16-fold (Zn 2+ content approximately 16.83 µM), CNC@PDA@Zn 2+ still exhibited excellent antiviral activity. Besides, this amount of Zn 2+ is much lower than that in the composite nanomaterials reported previously 29, 30 . These results demonstrate that embedding Zn 2+ in CNCs can enhance antiviral activity while reducing Zn 2+ concentration. The significant significance of this result is that we can manipulate the reduction of Zn 2+ content to induce stronger resistance, but significantly reduce the residual Zn 2+ in the environment. It is well-established that ZnO can promote the aggregation of viral particles in vitro , thereby inactivating the virus and ultimately inhibiting infection 48 . However, whether CNC@PDA@Zn 2+ possesses similar effects remains unclear. To investigate this, we first purified fresh TMV particles using differential centrifugation and then incubated them with 1600 µg/mL CNC@PDA@Zn 2+ . After 10 hours, we examined the morphological changes of TMV particles under a transmission electron microscopy (TEM) microscope. Surprisingly, the treated group exhibited a significant presence of fragmented viral particles with the average length of TMV particles being predominantly around 328.6 nm (Fig. 2 e, g), whereas in the CNC@PDA@ Zn 2+ treated group, the average length was reduced to 87.3 nm (Fig. 2 f, h). These findings indicate that CNC@PDA@Zn 2+ has an in vitro inactivation effect on TMV particles. To confirm whether this inactivation effect alters TMV infection in vivo , we inoculated N. benthamiana leaves with the treated suspensions. At 2 dpi, the water control group exhibited similar levels of viral infection, while the CNC@PDA@Zn 2+ treated group showed reduced accumulation of TMV-GFP in the inoculated leaves compared to the water control group (Fig. 2 i). By 4 dpi, the systemic leaves of the control groups showed a higher accumulation of TMV-GFP compared to the CNC@PDA@Zn 2+ treated group (Fig. 2 i). The qPCR and western blot results corroborated the fluorescence observations, showing a similar trend (Figs. 2 j-k). These results suggest that the composite material CNC@PDA@Zn 2+ demonstrates a strong inactivation effect on TMV-GFP particles. These results suggest that CNC@PDA@Zn 2+ can enhance antiviral effects by increasing in vitro passivation capacity while reducing Zn 2+ content. CNC@PDA@Zn enhanced Zn uptake and transport The rod-shaped CNC demonstrates low cytotoxicity and high permeability with a negatively charged surface, suggesting that the synthesized CNC@PDA@Zn 2+ may enhance the uptake of Zn 2+ by plant cells. To investigate this, CNC@PDA@Zn 2+ was sprayed onto the whole N. benthamiana leaves for three consecutive days, and its distribution within the plant was observed using TEM after the treatments. As expected, rod-shaped clusters were observed in the leaf tissues, indicating that CNC@PDA@Zn 2+ could enter plant cells and aggregate within the mesophyll cells (Fig. 3 a). The content of zinc also shows that Zn 2+ can be loaded and entered into plant (Fig. 3 b). More importantly, CNC@PDA@Zn 2+ was observed in chloroplasts (Fig. 3 a), demonstrating its ability to not only penetrate plant cells but also be actively transported to this key organelle. This localization suggests a targeted mechanism that may enhance its role in modulating chloroplast-associated immune responses and antiviral defenses. Besides, EDS analysis confirmed the presence of Zn 2+ signals ( Figure S1 a ), suggesting that Zn 2+ remained within the nano-carrier. In the stem tissues, dispersed CNC@PDA@Zn 2+ structures were also observed although it is difficult to pinpoint its exact location, with corresponding Zn 2+ signals detected by EDS ( Figure S1 b ), indicating that CNC@PDA@Zn 2+ could be transported from the leaves to the stem while still in its nano-carrier form (Fig. 3 a). However, no rod-shaped structures were observed in the roots (Fig. 3 a). This raises the possibility that Zn 2+ in the roots may exist in ionic form. To verify this hypothesis, we compared the Zn 2+ content in the roots, stems, and leaves after different treatments. The results showed an increased Zn 2+ content in the roots of the treated group, with higher overall Zn 2+ levels in CNC@PDA@Zn 2+ -treated plants compared to those treated with (CH 3 COO) 2 Zn (Fig. 3 d). Notably, when measuring the Zn 2+ content in various plant tissues following CNC@PDA@Zn 2+ treatment, an intriguing observation emerged. After foliar application of CNC@PDA@Zn 2+ at the highest tested concentration of 1600 µg/mL, the Zn 2+ content in roots, stems, and leaves remained relatively consistent and stable, ranging between 20–65 µg/g DW (Fig. 3 b-d). This level falls within the typical variability range of total Zn 2+ concentrations in plant tissues, suggesting that CNC@PDA@Zn 2+ maintains theoretical safety at this concentration. Furthermore, plant safety assessments confirmed that CNC@PDA@Zn 2+ is non-toxic to plants ( Figure S2 ). Compared with water-treated controls, CNC@PDA@Zn 2+ treatment significantly enhanced the growth of N. benthamiana , as evidenced by increased plant height and dry weight. These findings underscore CNC@PDA@Zn 2+ 's dual role in promoting plant growth while ensuring safety, even at higher application rates. Notably, rod-shaped structures were also observed in the leaves and stems following CNC-only treatment, suggesting that the transport of Zn 2+ to the stem is likely facilitated by CNC. This finding leads us to hypothesize that CNC@PDA@Zn 2+ utilizes the high permeability of CNC to transport Zn 2+ to the stem, where Zn 2+ is gradually released from CNC@PDA@Zn 2+ and subsequently transferred to the roots as free Zn 2+ . However, the exact mechanism by which Zn 2+ is efficiently regulated and transported to the roots remains unclear. To the best of our knowledge, Zn 2+ transport within plants primarily relies on four protein families: zinc-iron permease (ZIPs) family, responsible for transporting Zn 2+ from the external environment or organelles into the cytoplasm 49 ; metal tolerance proteins (MTPs) family, involved in Zn 2+ sequestration; heavy metal ATPase (HMAs) family, which pumps Zn 2+ into or out of organelles in an ATP-driven manner; and yellow stripe-like (YSLs) family, which assists in long-distance transport and distribution of Zn 2+ within the plant vascular system 50, 51 . To elucidate how CNC@PDA@Zn 2+ mediates long-distance Zn 2+ transport and efficient accumulation, we conducted a transcriptomic analysis of N. benthamiana leaves treated with CNC@PDA@Zn 2+ ( Figure S3 and Table S2 ). Compared to the water control, CNC@PDA@Zn 2+ induced differential expression of 6040 genes; when compared to (CH 3 COO) 2 Zn, this number increased to 7575 ( Figure S3a ). Among these differentially expressed genes which belong to ZIPs, MTPs, and HMAs family, 9 was related to Zn 2+ transport, with the YSLs family showing no significant changes ( Figure S3b ). Compared to (CH 3 COO) 2 Zn, these genes exhibited an upregulation trend, suggesting that CNC@PDA@Zn 2+ may stimulate the expression of Zn 2+ transporters in an unknown manner, thereby enhancing Zn 2+ transport capacity. It is important to note that the CNC@PDA@Zn 2+ components—dopamine and cellulose—lack inherent receptors in plants. However, cellulose is recognized by receptors such as wall-associated kinases (WAKs), leucine-rich repeat receptor-like kinases (LRR-RLKs), and catharanthus roseus RLK1-like kinases (CrRLK1L) 52, 53, 54 . The transcriptional levels of these receptors were also significantly upregulated following CNC@PDA@Zn 2+ treatment ( Figure S3c ), leading us to speculate that the upregulated Zn 2+ transport proteins might be finely regulated through the phosphorylation by these cellulose-recognizing receptors. Overall, although these transcriptomic analyses were limited to leaf tissues, they provide compelling evidence for the dual role of CNC@PDA@Zn 2+ in regulating Zn 2+ transport: facilitating efficient Zn 2+ transport to the stem via the high permeability of CNC and subsequently regulating Zn 2+ transport proteins through cellulose receptor-mediated phosphorylation mechanisms to enhance Zn 2+ translocation from stem to root. CNC@PDA@Zn 2+ alters nsLTP2 expression and localization. To investigate whether CNC@PDA@Zn 2+ contributes to and enhances broad-spectrum antiviral activity, we tested its inhibitory effects on the in vivo infection of various virus genera. The results revealed that while CNC@PDA@Zn 2+ exhibited antiviral activity against youcai mosaic virus (YoMV), with inhibitory effects surpassing those of (CH 3 COO) 2 Zn at 5 dpi, despite its efficacy was consistently lower than its inhibitory effect against TMV ( Figure S4 ). This disparity suggests that structural differences among these viruses may influence the direct inactivation capacity of CNC@PDA@Zn 2+ , highlighting its significant role in enhancing host resistance regulation induced by Zn 2+ . To the best of our knowledge, Zn 2+ 's known functions in plants include stabilizing enzyme structures or participating in catalytic reactions, affecting protein synthesis and gene expression, regulating chlorophyll synthesis, participating in energy metabolism, and enhancing plant disease resistance 55 . Specifically, in terms of enhancing disease resistance, Zn 2+ can reinforce the cell wall and increase the secretion of resistance proteins to inhibit pathogens in the apoplast, and it can also participate in hormone signaling pathways, stimulating the systemic acquired resistance (SAR) defense response 56 . To elucidate the role of CNC@PDA@Zn 2+ in enhancing antiviral activity, we conducted a detailed analysis of the obtained transcriptomic profiles. The results indicated that the differentially expressed genes enriched in KEGG pathways after CNC@PDA@Zn 2+ treatment were primarily associated with fundamental metabolic processes in plants, including spliceosome, purine metabolism, RNA transport, mRNA surveillance pathways, and ubiquitin-mediated proteolysis (Fig. 4 a). This suggests that CNC@PDA@Zn 2+ treatment alters various energy and metabolic processes within the host. Additionally, we observed that CNC@PDA@Zn 2+ induced the expression of many Zn 2+ -binding proteins, including zinc finger proteins, zinc metalloproteases, and superoxide dismutase (SOD) (Fig. 4 b) 56 . Interestingly, these proteins are directly or indirectly involved in plant defense mechanisms 56 . For instance, the SOD1 -encoded enzyme activity directly influences the host's reactive oxygen species (ROS) burst, thereby regulating cell wall formation. Recent works displayed that nanoparticles loaded zinc can affect the SOD enzyme activity 29, 57 . Notably, biochemical assays further confirmed that CNC@PDA@Zn 2+ enhances the SOD enzyme activity induced by zinc acetate within the host (Fig. 4 c), which is likely directly related to the increased resistance level observed. Nevertheless, the role of CNC@PDA@Zn 2+ in enhancing systemic acquired resistance (SAR) remains unclear. To investigate this further, we analyzed the top 100 differentially expressed genes (DEGs) with the most significant expression changes and categorized these genes based on their functions, among which nsLTP2 emerged as a particularly intriguing candidate, possibly serving as a target induced by CNC@PDA@Zn 2+ ( Figure S4 ). Following CNC@PDA@Zn 2+ treatment, nsLTP2 was highly expressed, with its expression level significantly exceeding that observed in the (CH 3 COO) 2 Zn treatment group (Figs. 4 d-e). Lipid transfer proteins (LTPs) are small, cysteine-rich proteins that primarily function in the transfer of various lipid molecules 58 . They play crucial roles in plant growth, defense against pathogens, and interactions with the environment 59 . It is particularly noteworthy that Zn 2+ -binding proteins possess highly conserved sequence motifs. In nsLTP2, we identified a C-Xn-C-Xn-CC-Xn-CXC-Xn-C-Xn-C motif, suggesting a potential Zn 2+ -binding capability. However, further research is needed to validate this binding affinity. Subsequently, we examined the tissue expression pattern of nsLTP2 in N. benthamiana and found that its expression was highest in the roots, followed by the leaves (Fig. 4 f), consistent with the expression pattern of NbLTP1 60 . Given the importance of subcellular localization for protein function, we used bioinformatics tools to predict that nsLTP2 contains a 24-residue N-terminal signal peptide and is primarily localized to the cell wall. To verify these predictions, we constructed nsLTP2-GFP fusion expression vectors driven by the cauliflower mosaic virus (CaMV) 35S promoter, as well as nsLTP2 △SP -GFP fusion constructs lacking the signal peptide (SP), and observed their localization in cells. Experimental results showed that nsLTP2-GFP was distributed in the cytoplasm and cell wall, while nsLTP2 △SP -GFP was found in the cytoplasm and chloroplasts. Interestingly, when CNC@PDA@Zn 2+ was applied to cells expressing these fusion proteins, their localization underwent significant changes. For nsLTP2-GFP, intracellular protein accumulation decreased and concentrated some in the chloroplast, with more protein accumulating extracellularly (Fig. 4 g). In contrast, nsLTP2 △SP -GFP became more concentrated in the chloroplasts, with the Pearson correlation curve of the GFP channel and chloroplast fluorescence channel completely overlapping (Fig. 4 h). Additionally, western blot analysis confirmed that CNC@PDA@Zn 2+ treatment not only altered nsLTP2/ nsLTP2 △SP localization but also increased nsLTP2 protein accumulation ( Figure S6 ). This result strengthens the correlation between CNC@PDA@Zn 2+ and nsLTP2. nsLTP2 as a positive regulator induces plant resistance To understand the functional significance of CNC@PDA@Zn 2+ -induced changes in nsLTP2 localization in plant resistance, we tested the antiviral function of nsLTP2. While the role of NbLTP1 in TMV infection is well characterized, we systematically explored the role of nsLTP2 during TMV infection. Stress expression analysis revealed that nsLTP2 expression significantly increased in inoculated leaves at 2 dpi with TMV-GFP, and its expression also markedly elevated in systemic leaves at 6 dpi ( Figure S7 ), suggesting a potential role for nsLTP2 in antiviral defense. Next, we utilized tobacco rattle virus (TRV)-mediated gene silencing to analyze nsLTP2’s role in TMV resistance. Following the agroinfiltration of N. benthamiana leaves with TRV1 + TRV2 (TRV:00) or TRV1 + TRV2:nsLTP2 (TRV:nsLTP2) for 12 days, RT-qPCR analysis revealed that nsLTP2 expression in systemic leaves was significantly lower in TRV:nsLTP2 plants compared to controls (Fig. 5 b). We then mechanically inoculated the 6th and 7th leaves with TMV-GFP and observed the movement of GFP under UV light at 2, 4, and 6 dpi to track TMV distribution. As shown in Fig. 5 a, GFP fluorescence signals were observed in the inoculated leaves at 2 dpi, and qPCR analysis demonstrated that TMV-GFP nucleic acid levels were significantly higher in silenced plants than in controls (Fig. 5 c). At 4 dpi, GFP signals appeared in the systemic leaves of silenced plants, whereas no GFP signals were detected in the systemic leaves of control plants; qPCR analysis of viral nucleic acids yielded similar results (Fig. 5 d). By 6 dpi, the GFP signal was more pronounced in silenced plants, and TMV-GFP accumulated significantly in their systemic leaves (Fig. 5 e). These results indicate that nsLTP2 silencing facilitates TMV-GFP infection. Subsequently, we compared the disease resistance conferred by nsLTP2 and nsLTP2 ΔSP . We fused nsLTP2 and nsLTP2 ΔSP with eGFP via a P2A linker, using eGFP:00 as a control. Following transient expression and TMV-GFP inoculation, we found that by 3 dpi, the control leaves contained numerous GFP fluorescence spots, while the treated leaves had significantly fewer spots, with the lowest number observed in the nsLTP2 ΔSP overexpression group (Fig. 5 f). qPCR results also showed that TMV-GFP nucleic acid levels were significantly lower in the nsLTP2 and nsLTP2 ΔSP overexpression groups compared to the control, with the nsLTP2 ΔSP group containing the least amount of TMV-GFP nucleic acids (Fig. 5 g). We then provided more compelling genetic evidence by stably overexpressing nsLTP2 ΔSP under the CaMV 35S promoter and analyzing its detailed antiviral phenotype. Consistent with the transient expression results, two independent overexpression lines exhibited remarkable antiviral activity, especially line #10 (Figs. 5 h-j). These findings suggest that chloroplast-localized nsLTP2 ΔSP confers stronger antiviral functions. As lipid transfer proteins, LTPs maintain the stability of organelle membranes by transporting lipids between various organelles 58 . For nsLTP2 containing the SP, CNC@PDA@Zn 2+ promotes its secretion to the extracellular space, potentially supporting the hypothesis that it stabilizes cell wall structures by transporting lipids 58 . However, we believe that its more crucial role lies in executing PR14 functions. LTP2 may have potential PR14-related functions, and the upregulation of this pathogenesis-related protein directly contributes to host resistance 58 . Nevertheless, nsLTP2 ΔSP demonstrates stronger resistance and accumulates in chloroplasts under CNC@PDA@Zn 2+ treatment. Chloroplasts are a primary battleground in plant-virus interactions, with chloroplast homeostasis directly influencing viral symptom development, and several chloroplast proteins exhibit antiviral activity 61, 62 . Notably, CNC@PDA@Zn 2+ seems to be able to enter chloroplasts and enhance the expression of various chloroplast-associated proteins ( Figure S8 ), strongly suggesting that CNC@PDA@Zn 2+ -induced nsLTP2 stabilizes chloroplast membrane structures by transporting lipids, leading to enhanced antiviral defense. Furthermore, we cannot entirely dismiss an important yet easily overlooked possibility: in roots lacking chloroplasts, nsLTP2 may function as a PR14 resistance protein when induced by CNC@PDA@Zn 2+ , while in chloroplast-rich leaves, nsLTP2 suppresses viral infection by maintaining chloroplast homeostasis. However, these hypotheses require further evidence for confirmation. Furthermore, Zn 2+ is known to facilitate cell wall fortification and is essential for chlorophyll synthesis 55, 56 , making it difficult not to associate these processes with the function of nsLTP2. We hypothesize that nsLTP2 may bind Zn 2+ and subsequently transport lipids to the cell wall and chloroplasts. This lipid-mediated process could involve the reversible transfer of Zn 2+ to the cell wall and chloroplasts, where it plays a crucial role. Notably, CNC@PDA@Zn 2+ appears to accelerate this process, possibly due to the increased intracellular Zn 2+ levels introduced by CNC@PDA@Zn 2+ or a chain reaction triggered by the CNC receptor hypothesis. However, more detailed genetic evidence is required to confirm these mechanisms. Regardless, these results support the role of CNC@PDA@Zn 2+ in enhancing resistance by inducing nsLTP2. The induction of nsLTP2 by CNC@PDA@Zn 2+ is dependent on the SA pathway for its disease response. The SAR in plants is intrinsically linked to SA, and Zn 2+ plays a crucial role in this process as well. Previous studies have already associated Zn 2+ with SAR 18, 19 , but whether Zn 2+ can promote SA accumulation by regulating the expression of nsLTP2 remains unclear. Notably, whether it functions as a PR14 protein or stabilizes chloroplast membrane structures, nsLTP2 appears to be closely linked to SA, as PR proteins exert their functions through the SA synthesized in chloroplasts. To explore this, we first treated N. benthamiana leaves with the SA analog MeSA and quantified the changes in nsLTP2 expression during this process. The results showed that nsLTP2 expression increased over time following MeSA treatment, indicating that nsLTP2 is an SA-inducible gene (Fig. 6 a). We then measured the SA content in two stable overexpression lines of nsLTP2 ΔSP and found that both lines exhibited increased SA levels (Fig. 6 b), suggesting a potential link between nsLTP2 and SA. Additionally, we searched the transcriptome database for SA-related genes induced by CNC@PDA@Zn 2+ and found that CNC@PDA@Zn 2+ enhanced the expression levels of several SA-related genes, including nsLTP2 (Fig. 6 c). We further quantified the expression of key genes involved in SA synthesis and response, such as NPR1 , pathogenesis-related gene 1 ( PR1 ), and pathogenesis-related gene 2 ( PR2 ). The results showed that both CNC@PDA@Zn 2+ and (CH 3 COO) 2 Zn treatments upregulated the expression of NPR1 , PR1 , and PR2 , with CNC@PDA@Zn 2+ promoting higher expression levels of NPR1 and PR1 (Fig. 6 d-f). Subsequently, we inoculated TMV-GFP after treating plants with CNC@PDA@Zn 2+ and (CH 3 COO) 2 Zn to examine the expression of SA-related genes under these complex conditions. The results demonstrated that under TMV-GFP infection, both (CH 3 COO) 2 Zn and CNC@PDA@Zn 2+ treatments induced high expression of PR2 (Fig. 6 f), while CNC@PDA@Zn 2+ also promoted NPR1 expression (Fig. 6 d). Furthermore, SA levels increased following (CH 3 COO) 2 Zn and CNC@PDA@Zn 2+ treatments (Fig. 6 g). Notably, after TMV-GFP inoculation, the SA content in the CNC@PDA@Zn 2+ -treated group was slightly higher than in the (CH 3 COO) 2 Zn-treated group (Fig. 6 g). These results suggest that the resistance induced by CNC@PDA@Zn 2+ in TMV-GFP-infected plants is more stable. To determine whether the resistance induced by CNC@PDA@Zn 2+ in N. benthamiana is entirely dependent on the SA pathway, we treated transgenic NahG plants, which express salicylate hydroxylase and are completely deficient in SA, with CNC@PDA@Zn 2+ and observed whether their antiviral phenotype changed. The results showed that at 2 dpi, NahG plants displayed stronger fluorescence signals in their leaves compared to the control, but this difference significantly diminished at 4 and 6 dpi (Fig. 6 h). RT-qPCR analysis indicated that the inhibitory effect of CNC@PDA@Zn 2+ on TMV-GFP infection was completely lost at 4 and 6 dpi (Fig. 6 i). These findings highlight the crucial role of CNC@PDA@Zn 2+ in inducing host systemic resistance, and this SAR resistance is largely dependent on SA signaling. Evolutionary conservation of nsLTP2 in flowering plants Finally, we conducted an in-depth analysis of the evolutionary relationships of nsLTP2 across the plant lineage, given its potential relevance to the functional scope of CNC@PDA@Zn 2+ . Notably, nsLTP2 originated in angiosperms, as it was first identified in Amborella trichopoda . A. trichopoda is the sole surviving representative of the basal sister lineage of all extant angiosperms, suggesting that nsLTP2 is one of the earliest genes to have emerged during the evolution of angiosperms. This highlights its critical role in the origin and early evolution of flowering plants. Subsequently, these nsLTP2 genes underwent duplication across different species, leading to a significant number of homologs in key crops such as Solanum lycopersicum (12 copies), Oryza sativa (12 copies), and Zea mays (7 copies) (Fig. 6 j). These findings suggest that CNC@PDA@Zn 2+ may broadly induce the expression of nsLTP2 across this vast phylogenetic group of angiosperms and that there are multiple nsLTP2 targets in some key crops. This further indicates the significant role of CNC@PDA@Zn 2+ in inducing resistance defenses throughout the plant kingdom. In summary, we developed a rode-shape nanomaterial, CNC@PDA@Zn 2+ , by embedding Zn 2+ into a PDA coating on CNCs. This innovative material demonstrated enhanced efficiency in controlling plant virus infection and facilitating trace element transport within plants, while significantly reducing Zn 2+ usage, positioning it as a key player in sustainable agricultural practices. Mechanistic analyses revealed that CNC@PDA@Zn 2+ induced a SA-mediated defense response by upregulating the secretion and expression of nsLTP2 across different organelles. Notably, this mechanism appears to be highly conserved among angiosperms. Our study presents a novel strategy for efficient Zn 2+ delivery in plants, strengthening plant immune responses and resistance to pathogens, thereby advancing the sustainable use of Zn 2+ as a plant disease control agent. Methods Plant materials and growth conditions Nicotiana benthamiana seeds were sterilized with 75% alcohol and inoculated in MS medium. After 1 week, they were transferred into soil. The growing condition was under 16 h day/8 h night cycle, 26°C, and 80% relative humidity. The plants were cultured to the six-leaves to eight-leaves stage for the next experiment, and fresh leaves were collected and frozen in liquid nitrogen for subsequent analysis. Synthesis of CNC@PDA@Zn The CNCs were prepared by hydrolyzing cotton linter with sulfuric acid according to our previous work 63 . The purified CNCs were used to prepare CNC@PDA@Zn 2+ . Dopamine hydrochloride (DA⋅HCl, 98%) and zinc chloride (ZnCl 2 ) were purchased from Shanghai Aladdin Biochemical Technology Co. Ltd. (Shanghai). Trimethylaminomethane (Tris) was purchased from Shanghai Yuanye Bio-Technology Co. Ltd. (Shanghai). Sodium hydroxide (NaOH, 96.0%) and hydrochloric acid (HCl, 36–38%) were purchased from Chongqing Chuandong Chemical Co. Ltd. (Chongqing). Characterization of CNC@PDA@Zn The FT-IR spectra of CNC, CNC@PDA, CNC@PDA@Zn 2+ , and PDA@Zn 2+ were measured by were characterized by a Nicolet 170SX FourierTransforman (Madison, WI, USA) with anhydrous KBr in the range of 4000–500 cm − 1 at attenuated total reflection cell by averaging 32 spectra with 4 cm − 1 . Zn 2+ content was measured by inductively coupled plasma atomic emission spectroscopy (ICP-AES) (iCAP7000, American). The morphology of CNC and CNC@PDA@Zn 2+ were measured by transmission electron microscope (TEM) (Talos F200X). The ζ-potential of CNC, CNC@PDA, and CNC@PDA@Zn 2+ was measured by a Zeasizer nano ZS (Malvern, UK). X-ray diffraction (XRD, D8 ADVANCE, Bruker) was employed to investigate the interaction of CNCs with PDA@Zn 2+ . Thermogravimetric analysis (TGA, STA 499 F5/F3 Jupiter, NETZSCH) was conducted to characterize the thermal stability and decomposition behavior of composites. Virus inoculation For TMV-GFP inoculation, the friction inoculation method was employed to eliminate potential experimental interference from Agrobacterium , which is commonly associated with the Agrobacterium infiltration method. Specifically, 0.1 g of fully TMV-GFP-infected N. benthamiana leaves were collected and ground into a fine powder using a mortar and pestle under liquid nitrogen. The resulting powder was dissolved in PBS buffer (pH = 7.4), and the suspension was adjusted to an OD 600 of 1. The prepared solution was then used to inoculate designated leaves via friction inoculation. Western blot For protein extraction, 0.1 g of leaf tissue was ground into a fine powder in a pre-chilled mortar with liquid nitrogen. Protein lysis buffer (0.1 M Tris-HCl, pH 7.4, 1% SDS, 0.02% β-mercaptoethanol) was added to the powder, and the mixture was incubated on ice for 30 min. The lysate was centrifuged at 5000 g for 15 minutes at 4°C, and the resulting supernatant was collected. SDS-protein loading buffer was added to the supernatant, and the mixture was boiled at 95°C for 5 min to denature the proteins. The prepared protein samples were subjected to SDS-PAGE using a Mini-PROTEAN® Tetra Cell system and subsequently transferred onto a PVDF membrane for western blot analysis. The membrane was probed with a mouse anti-GFP monoclonal antibody (ABclonal, AE012) as the primary antibody and HRP-conjugated goat anti-mouse IgG (H + L) (ABclonal, AS003) as the secondary antibody. RNA extraction and cDNA synthesis RNA was extracted using an Eastep® Super Total RNA Extraction Kit (Promega, LS1040, Beijing, China). The RNA sample was then reverse transcribed with a PrimeScript™ RT Reagent Kit (TaKaRa, RR037A, Shiga, Japan) in a 10 µL reaction. RNA-seq The N. benthamiana leaves of CNC@PDA@Zn 2+ treatments (PZ-1, PZ-2, and PZ-3), (CH 3 COO) 2 Zn treatments (Z-1, Z-2, and Z-3), CNC treatments (CNC-1, CNC-2 and CNC-3) and water treatments (CK-1, CK-2, CK-3) were collected and RNA was extracted using RNAiso Plus reagent (Takara, Japan). mRNA was isolated with oligo (dT) cellulose and broken into short fragments (200 nt) by adding a fragmentation buffer. First-strand cDNA was generated using random hexamer-primed reverse transcription, and second-strand cDNA was synthesized by DNA polymerase I and RNase H. After that, the synthesized cDNA fragments were purified and subjected to end pairing by adding a single “A” bases, and ligated with Illumina adapters. The ligation products were size-fractionated by agarose gel electrophoresis, and fragments were excised for PCR amplification. The amplified fragments were sequenced using Illumina HiSeqTM 2500 by Gene Denovo Co. (Guangzhou, China). De novo transcriptome assembly and differentially expressed gene (DEGs) analysis were described. In brief, the raw reads obtained by the sequencing platform were performed quality control (QC) and filtered to obtain high-quality clean reads. After that, clean reads were aligned to the reference genome (Niben.genome.v1.0.1). After performing the QC, a follow-up analysis was performed, followed by quantitative analysis and structural analysis. Quantitative analysis includes quantification of genes, exons, and transcripts. Subsequent differential expression analysis was based on quantitative analysis, functional enrichment analysis, and time series analysis. Structural analysis consists of alternative splicing analysis, gene structure optimization, new transcript analysis, and SNP/InDel analysis, gene fusion analysis. Real time quantitative PCR For real time quantitative PCR (qPCR), qTOWER2.0 real-time PCR (Analytikjena, Germany) and a QuantiNova™ SYBR Green PCR Kit (QIAGEN, Germany) were used to determine the relative expression levels of target genes. Three biological replicates were performed for each sample. ACTIN was selected as an internal control. Quantification of the relative changes in gene transcript levels was performed using the 2 −△△Ct method. All primers are shown in Table S3 . Virus particle purification and TEM observation To purify the virus particles, the first step is to inoculate TMV and collected 4 g leaves after 4 days. Add 0.2 mol/L PBS buffer (pH = 7.2) mixed with 1% β-mercaptoethanol (99 mL PBS + 1 mL β-mercaptoethanol) and ground it into a homogenate (1g tissue per 1mL buffer). The filter collects the supernatant as the crude extract. Under magnetic stirring, add 8% n-butanol (8 mL per 100 mL of solution), continue stirring for 15 minutes, and then centrifuge at 10000 rpm for 20 minutes. Collect the supernatant, add 4% NaCl and 4% PEG6000 (4 g each per 100 mL solution), stir for 1.5 hours, and centrifuge at 10,000 rpm for 15 minutes. Collect the precipitate, resuspend it in 0.01 mol/L PBS buffer (pH 7.2), and centrifuge at 8000 rpm for 5 minutes. Collect the precipitate, resuspend it in 0.01 mol/L PBS buffer (pH 7.2), and centrifuge at 8000 rpm for 5 minutes. The supernatant is the purified TMV extract, which is stored at 4°C. Mix the purified TMV particles with nanomaterials and ddH 2 O as the control. Allow all mixtures to interact in vitro at 25°C for 3 hours. Observe the TMV morphology under a JEOL JEM-2100 electron microscope. Eight fields of view were analyzed for each treatment, and representative TEM images were selected. The control group was processed using the same procedure. For the CNC nanomaterial transport in the plant, the leaf samples were sampled after spraying 3 days, stem samples were sampled at 4 days after spraying 3 days, and the root samples were sampled at 7 days after spraying 3 days. Vector construction For expression vector construction, two segments encoding nsLTP2 (Niben101Scf07951g02011) and nsLTP2 △SP (without the signal peptide) were designed with Xba I restriction sites and a 20-bp sequence overlapping with the region surrounding Xba I in the pART27-eGFP vector. These fragments were seamlessly cloned into the pART27-eGFP backbone, resulting in the binary expression vectors pART27-nsLTP2-eGFP and pART27-nsLTP2 △SP -eGFP. Similarly, the pART27-nsLTP2 △SP -7×Myc binary expression vector was constructed using the same seamless cloning approach. To construct the silencing vector, a 200-bp optimal silencing fragment for nsLTP2 was identified using the VIGS tool ( https://vigs.solgenomics.net/ ). This fragment was inserted into the pTRV2 vector at the Xba I and Xho I sites to generate the pTRV2-nsLTP2 vector. All primers are shown in Table S3 . N. benthamiana leaves infiltration and confocal observation Transient expression was performed as previously described, with minor modifications. Briefly, the constructs pART27-nsLTP1-GFP, pART27-nsLTP2 △SP -eGFP, pTRV1, pTRV2-nsLTP2, and P19 were transformed into Agrobacterium tumefaciens strain GV3101. The Agrobacterium cultures were activated with acetosyringone and prepared for infiltration. For expression vectors (pART27-based constructs), the cultures were adjusted to an OD 600 of 0.5. For silencing vectors (pTRV-based constructs), the cultures were adjusted to an OD 600 of 0.3 and co-infiltrated with P19 (OD 600 = 0.3) to enhance expression. The mixtures were infiltrated into the leaves of N. benthamiana using a syringe without a needle. For confocal observation, N. benthamiana leaves at the 6-leaf stage were infiltrated and leaf discs were isolated from the infiltrated leaves and visualized using a LSM780 confocal laser scanning microscope equipped with a 40*/1.2 water-immersion objective (Zeiss, Germany). GFP-derived fluorescence was excited a 488 nm and emission was captured with a 505- to 530 nm filter. SOD activity detection The enzyme activity was determined according to the previous report 64 . PBS was used to extract total protein from the treated N. benthamiana leaves, and enzyme activity kits were used to measure the activities SOD (at 560 nm) (Sinobest, YX-C-A500) according to the manuals, with three repetitions for each treatment. Detection of SA in leaves For SA quantification, 50 mg leaf tissue was finely ground in liquid nitrogen and extracted with 2 mL pre-cooled 80% methanol, then sealed with plastic wrap and cold-soaked at 4 ° C overnight. Centrifuge at 4 ° C, 5000 r/min for 10 min, take the supernatant and the residue continue to extract with 80% methanol. The supernatant was pooled by sonication two times, and the aqueous phase added 2mL petroleum ether was to decolorize 3 times, extract the aqueous phase with ethyl acetate 3 times, then blow it and add an acetic acid solution (pH = 3.5), purify through C18 column (Agilent C18,250*4.6mm; 5µm), elution with methanol, collect the eluate and dry before dissolving with mobile phase to a constant volume of 1mL, shake and mix the liquid then pass through 0.22 µm filter membrane and be tested. The quantification of SA was determined by HPLC (Agilent 1200), the equipment settings according to previous work 65 . Three independent replicates were performed with each experiment containing three biological repeats. Elemental analyses The concentration of Zn and Si in the tobacco plants (leaf, stem, and root) were assessed via inductively coupled plasma mass spectrometry (ICP-MS; ELAN DRC II, Perkin Elmer Inc.). Specifically, dried plant samples were fully digested in 6 mL of HNO 3 and hydrogen peroxide (5:1) solution at 150°C. Then, deionized water was added to the solution until the volume reached 50 mL. The supernatants were diluted with HNO 3 (1%) to 100 ppb. Finally, the filtered solution was used for elemental concentration analysis via ICP-MS. Phylogenetic analysis Homologous proteins of nbLTP2 were retrieved from the TAIR ( https://www.arabidopsis.org/ ) and Phytozome ( https://phytozome-next.jgi.doe.gov/ ) databases. The evolutionary relationships were analyzed using MEGA X. The phylogenetic tree was constructed with the following parameters: Jones-Taylor-Thornton (JTT) substitution model, Gamma Distributed (G) with four discrete Gamma categories, partial deletion with a 70% site coverage cutoff, and Subtree-Pruning-Regrafting (SPR) method using a BioNJ initial tree. A strong branch swap filter was applied to enhance the tree’s accuracy. Based on the phylogenetic analysis, proteins that fell outside the core tree were excluded. Subsequently, an evolutionary tree for the selected species was obtained from the Timetree database and visualized. Statistical analysis All experiments and data presented here involved three repeats. The data were presented as means and standard deviations. The statistical analysis was performed with SPSS software (version 22.0) using Student’s t -test (∗0.01 < p < 0.05, ∗∗0.001 < p < 0.01, ∗∗∗ p < 0.001) and One-way ANOVA test (LSD’s test, p < 005). Declarations Data and materials availability All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Acknowledgments We would like to thank Professor Yule Liu (Tsinghua University, Beijing, China) for his valuable comments on improving the experiment and providing the pSPDK661 (TMV-GFP) and pTRV2 vectors. This study was partly supported by the National Natural Science Foundation of China (31870147, X. Sun), the science and technology projects of Chongqing Company of China Tobacco Corporation (B20241NY1303 and B20241NY1310, X. Sun), and the China Scholarship Council (202306990064, X. Sun) Author contributions This study represents an interdisciplinary collaboration. Specifically, J. Wang, X. Wang, C. Liu, X. Zhu, and W. Liu conducted biological experiments under the guidance of S. Wang and X. Sun. The materials synthesis and characterization were performed by S. Xiang and Y. Shen under the supervision of X. Ma and J. Huang. All authors contributed to the writing and revision of the manuscript. Competing interests The authors declare no competing interests. References Jones RAC, Naidu RA. Global dimensions of plant virus diseases: Current status and future perspectives. Annual Review of Virology 6 , 387-409 (2019). Scholthof K-BG , et al. 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Pest Management Science 76 , 3636-3648 (2020). Cho K , et al. Quantification of jasmonic and salicylic acids in rice seedling leaves. Rice Protocols , 185-200 (2013). Additional Declarations There is NO Competing Interest. Supplementary Files Supplymentary.docx The high-permeability cellulose nanocrystals carrier facilitates zinc utilization and enhances nsLTP2-mediated plant immunity Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5791872","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":399726305,"identity":"4f60b5ed-a22e-48ab-9010-b8f12f309d39","order_by":0,"name":"Xianchao Sun","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA00lEQVRIiWNgGAWjYLCCBCA2YGBgfJBQUUOaFmaDB2eOkWATUAub5MMWZsIq5WfkHpN4UHNH3pz98LGKxAY2Bv727gT8ht/ISzZIOPbMcGdPWtqNxB0yDBJnzm7Ar0Uix/BBAtvhBIMDOWY3Es+wAUVy8WuRn5FjcCDhH1DL+TdmBYltzIS1MNwA2pLYBtRyI8eMgSgtBmfeGBsk9h023HDjWbJEwpljPAT9It+eYyb549theYPzyQc//qiokeNv7yXgMHTAQ5ryUTAKRsEoGAVYAQBWsEw3Bva1agAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-0062-4916","institution":"Southwest University","correspondingAuthor":true,"prefix":"","firstName":"Xianchao","middleName":"","lastName":"Sun","suffix":""},{"id":399726306,"identity":"602927a1-2f11-4e51-8e66-6a760503c323","order_by":1,"name":"Jing Wang","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Wang","suffix":""},{"id":399726307,"identity":"c03b858d-696d-4b18-ba09-9aba75232616","order_by":2,"name":"Shunyu Xiang","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Shunyu","middleName":"","lastName":"Xiang","suffix":""},{"id":399726308,"identity":"8c4c126e-ce51-42dd-b042-712e303d3a02","order_by":3,"name":"Xiaoyan Wang","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoyan","middleName":"","lastName":"Wang","suffix":""},{"id":399726309,"identity":"91810b59-c463-4f78-bf32-00ed2e7730d5","order_by":4,"name":"Yang Shen","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Shen","suffix":""},{"id":399726310,"identity":"613206a4-863e-4cae-8ab6-cc640eecdfec","order_by":5,"name":"Changyun Liu","email":"","orcid":"https://orcid.org/0000-0003-1683-5101","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Changyun","middleName":"","lastName":"Liu","suffix":""},{"id":399726311,"identity":"fedcd81e-0cb9-4d71-a264-33c3e65e15ee","order_by":6,"name":"Xin Zhu","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Zhu","suffix":""},{"id":399726312,"identity":"53730a43-2aa9-4df2-9915-6db8fb7b8fce","order_by":7,"name":"Weina Liu","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Weina","middleName":"","lastName":"Liu","suffix":""},{"id":399726313,"identity":"76a25eba-29de-4ede-8540-1d21937ce538","order_by":8,"name":"Shanzhi Wang","email":"","orcid":"","institution":"Department of Plant Pathology and the Ministry of Agriculture Key Laboratory of Pest Monitoring and Green Management, China Agricultural University, Beijing,","correspondingAuthor":false,"prefix":"","firstName":"Shanzhi","middleName":"","lastName":"Wang","suffix":""},{"id":399726314,"identity":"129349a0-f0c5-43d2-ac36-d640bcc87de2","order_by":9,"name":"Xiaozhou Ma","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Xiaozhou","middleName":"","lastName":"Ma","suffix":""},{"id":399726315,"identity":"5516fb96-34ad-4536-962f-105fe2d7858f","order_by":10,"name":"Jin Huang","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Jin","middleName":"","lastName":"Huang","suffix":""}],"badges":[],"createdAt":"2025-01-08 21:35:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5791872/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5791872/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":73465678,"identity":"14fc2880-ff4e-4eaa-9647-ed4f848d1be7","added_by":"auto","created_at":"2025-01-10 08:41:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":405376,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSynthesis and characterization of CNC@PDA@Zn\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e.\u003c/strong\u003e (a) synthesis method of CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e. (b) The TEM image of CNC. (c) The TEM image of CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e (d) The FTIR spectroscopy of CNC, CNC@PDA, Zn\u003csup\u003e2+\u003c/sup\u003e@PDA, and CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e. (e) ζ-potential of CNC, CNC@PDA, Zn\u003csup\u003e2+\u003c/sup\u003e@PDA and CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e. (f) TGA spectra of CNC, CNC@PDA, and CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e and (g) The X-ray diffraction patterns obtained for CNC, CNC@PDA, Zn\u003csup\u003e2+\u003c/sup\u003e@PDA, and CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-5791872/v1/03570ce37b4f45f9354018b7.png"},{"id":73465679,"identity":"602cf7b8-4298-482d-8f12-f34f56c4ec6f","added_by":"auto","created_at":"2025-01-10 08:41:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":867258,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of CNC@PDA@Zn\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2+ \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eon TMV-GFP infection \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro.\u003c/strong\u003e\u003c/em\u003e (a) antivirus activity of (CH3COO)\u003csub\u003e2\u003c/sub\u003eZn (44 μg/mL) and CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e (1600 μg/mL). CK as the control group, the concentration of Zn\u003csup\u003e2+\u003c/sup\u003e in 44 μg/mL (CH3COO)\u003csub\u003e2\u003c/sub\u003eZn was the same as the concentration of Zn\u003csup\u003e2+\u003c/sup\u003e loaded in 1600 μg/mL CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e. (b-d) TMV-GFP expression at 2 dpi, 4 dpi, and 6 dpi. The accumulation of TMV-GFP in inoculated leaves was tested at 2 dpi and 4 dpi, and the accumulation of TMV-GFP in systemic leaves was tested at 6 dpi. (e-f) TEM images of TMV particles treated with ddH\u003csub\u003e2\u003c/sub\u003eO and CNC@PDA@Zn\u003csup\u003e2\u003c/sup\u003e+. (g-h) Distribution histograms of the length for TMV particles treated with ddH\u003csub\u003e2\u003c/sub\u003eO and CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e. (i) Fluorescence accumulation of TMV-GFP at 2 and 4 days. (j) Quantitative virus accumulation at 2, 4, and 6 days by qPCR. (k) Western blot analysis of TMV-GFP protein content. Mean values displayed in each bar followed by different letters are significantly different according to Student’s \u003cem\u003et\u003c/em\u003e-test (∗\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, ∗∗\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ∗∗∗\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001) and LSD’s multiple range test (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). Vertical bars indicate standard errors (n = 5).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-5791872/v1/04b2efab064186983178e4f8.png"},{"id":73464562,"identity":"3545d437-7b1e-469f-8fbc-ee0e9cde15b4","added_by":"auto","created_at":"2025-01-10 08:33:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":570532,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranslocation of CNC@PDA@Zn\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eN. benthamiana\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003e(a) TEM images of translocation in leaves, stems, and roots. The red circles indicate the locations where nanomaterials are aggregated. (b-d) Contents of Zn\u003csup\u003e2+\u003c/sup\u003e in leaves, stems, and roots. Mean values displayed in each bar followed by different letters are significantly different according to LSD’s multiple range test (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). Vertical bars indicate standard errors (n = 3).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-5791872/v1/590c2fa6807f9b76c0f63831.png"},{"id":73465826,"identity":"3b5c6375-f5eb-4bbb-b1ff-c6cc7660fa69","added_by":"auto","created_at":"2025-01-10 08:49:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":581026,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCNC@PDA@Zn\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e alters nsLTP2 expression and localization.\u003c/strong\u003e (a) KEGG pathway enrichment of DEGs. (b) Heatmap representation of the expression of 100 DEGs between control and CNC@PDA @Zn\u003csup\u003e2+\u003c/sup\u003e. (c) SOD activity of (CH3COO)\u003csub\u003e2\u003c/sub\u003eZn and CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e. (d) FPKM of \u003cem\u003ensLTP2\u003c/em\u003e in transcriptome sequence analysis. (e) qPCR validation of \u003cem\u003ensLTP2\u003c/em\u003e. (f) Expression pattern of \u003cem\u003ensLTP2\u003c/em\u003e in different tissues. (g) Subcellular localization of nsLTP2. (h) Subcellular localization of nsLTP2\u003csup\u003eΔsp\u003c/sup\u003e. Mean values displayed in each bar followed by different letters are significantly different according to LSD’s multiple range test (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). Vertical bars indicate standard errors (n = 3).\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-5791872/v1/973580dad6ade7c4d7faae58.png"},{"id":73465681,"identity":"1c3910da-0174-40ac-b57f-34f1e8ebfefe","added_by":"auto","created_at":"2025-01-10 08:41:31","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":819656,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ensLTP2 as a positive regulator induce plant resistance. \u003c/strong\u003e(a) Symptom of TMV-GFP infection after silencing\u003cem\u003e nsLTP2\u003c/em\u003e for 12 days. (b) The expression of \u003cem\u003ensLTP2\u003c/em\u003e on silenced plants. (c)-(e) Accumulation of TMV-GFP at 2, 4, and 6 dpi were detected by RT-qPCR. TMV-GFP in the inoculated leaves was quantified at 2 dpi. 4, and 6 dpi were quantified the TMV-GFP in the systemic leaves. (f- h) Symptom maps of OE-nsLTP\u003csup\u003eΔsp\u003c/sup\u003e and WT plants inoculated with TMV-GFP; (i-j) TMV RNA levels in OE-nsLTP\u003csup\u003eΔsp\u003c/sup\u003e and WT plants were detected by qPCR at 3 and 5 dpi. Mean values displayed in each bar followed by different letters are significantly different according to Student’s \u003cem\u003et\u003c/em\u003e-test (∗\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, ∗∗\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ∗∗∗\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). Vertical bars indicate standard errors (n = 3)\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-5791872/v1/2e2d7deca586fa628d93632d.png"},{"id":73464573,"identity":"8b2b7f9e-2cc6-435a-b95b-89b5d8aeef3c","added_by":"auto","created_at":"2025-01-10 08:33:32","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":599007,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConserved nsLTP2 is involved in the SA-mediated resistance pathway.\u003c/strong\u003e (a) Relative expression of \u003cem\u003ensLTP2\u003c/em\u003e after being treated with MeSA. (b) SA content in two nsLTP overexpression lines. (c) DEGs related to SA. (d-f) Response of \u003cem\u003eNPR1\u003c/em\u003e, \u003cem\u003ePR1,\u003c/em\u003e and \u003cem\u003ePR2 \u003c/em\u003eafter spraying CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e 1 hours, spraying CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e 3 days, and spraying CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e 3 days then inoculate TMV-GFP. (g) SA content after CNC@PDA@Zn\u003csup\u003e2+ \u003c/sup\u003etreatment. (h) Accumulation of TMV-GFP after spraying CNC@PDA@Zn\u003csup\u003e2+ \u003c/sup\u003ein \u003cem\u003eNahG \u003c/em\u003eat 2 dpi (inoculated leaves) and 6 dpi (systemic leaves). (i) Related expression level of TMV-GFP in\u003cem\u003e NahG\u003c/em\u003e after treated with CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e. (j) Evolutionary relationships of nsLTP2. Mean values displayed in each bar followed by different letters are significantly different according to LSD’s multiple range test (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). Vertical bars indicate standard errors (n = 3).\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-5791872/v1/258e11b37e5fc77db592f4fa.png"},{"id":73465688,"identity":"41f27663-d0d9-4c7b-96ca-ab730e7a913b","added_by":"auto","created_at":"2025-01-10 08:41:33","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":310531,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic representation of CNC@PDA@Zn\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e-induced Zn\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e transport and its activity of induced plant immunity.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-5791872/v1/cd48680236037324fb0368c9.png"},{"id":73864540,"identity":"8be40bf7-ec08-49a7-ae31-3c1b42c97908","added_by":"auto","created_at":"2025-01-15 11:38:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6237659,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5791872/v1/2c6ef761-f107-4db1-bcef-c2b837bc98cc.pdf"},{"id":73464558,"identity":"770fd2c2-360c-44d5-a900-179122041f73","added_by":"auto","created_at":"2025-01-10 08:33:31","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3037143,"visible":true,"origin":"","legend":"The high-permeability cellulose nanocrystals carrier facilitates zinc utilization and enhances nsLTP2-mediated plant immunity","description":"","filename":"Supplymentary.docx","url":"https://assets-eu.researchsquare.com/files/rs-5791872/v1/547e33cbad7be99d2f23c0a0.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"The high-permeability cellulose nanocrystals carrier facilitates zinc utilization and enhances nsLTP2-mediated plant immunity","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePlant viruses pose a significant threat to agriculture, causing substantial reductions in crop quality and yield upon infection\u003csup\u003e1\u003c/sup\u003e. The tobacco mosaic virus (TMV), recognized as one of the most detrimental plant viruses, has been identified to infect over 350 host plant species, including economically important crops such as tobacco, tomatoes, and peppers, leading to substantial economic losses \u003csup\u003e2\u003c/sup\u003e. Upon infection, TMV disrupts chloroplasts within plant cells, impedes photosynthesis, and results in mottled and discolored leaves, while also affecting cell division and causing leaf deformities \u003csup\u003e3\u003c/sup\u003e. Currently, there is no agent capable of fully eradicating TMV \u003csup\u003e4\u003c/sup\u003e. The prevention and management of viral diseases associated with TMV largely rely on the use of viral inhibitors, which face challenges such as limited storage stability, environmental pollution, excessive pesticide residues, and the development of pathogen resistance \u003csup\u003e5\u003c/sup\u003e. With advances in understanding plant immune mechanisms, there has been a shift towards utilizing plant immune inducers for disease prevention and control. These inducers trigger immune responses, including reactive oxygen species (ROS) accumulation, Ca\u003csup\u003e2+\u003c/sup\u003e signaling, callose deposition, salicylic acid (SA) production, and the expression of resistance-related genes \u003csup\u003e6, 7, 8, 9\u003c/sup\u003e, ultimately leading to systemic acquired resistance (SAR). During SAR, SA levels increase, and resistance genes such as pathogen-related protein (PR) genes are upregulated \u003csup\u003e10, 11, 12, 13\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe utilization of zinc (Zn\u0026sup2;⁺) for disease control dates back to as early as 1939, and potentially even earlier \u003csup\u003e14, 15\u003c/sup\u003e. Zn\u003csup\u003e2+\u003c/sup\u003e, an essential micronutrient for plant growth, is integral to various biological processes, including metabolic pathways, enzymatic reactions, hormone metabolism, disease resistance, and stress tolerance. More recently, it has been recognized for its role in the regulation of nitrogen fixation efficiency within root nodule symbiosis \u003csup\u003e16\u003c/sup\u003e. As previously highlighted, plant immunity against pathogens is associated with SA accumulation in non-infected tissues, ROS burst, and the initiation of SA-dependent SAR. In this context, C9 dicarboxylic acids and azelaic acid (AzA) are known to participate in the long-distance signaling of SA \u003csup\u003e17\u003c/sup\u003e. It is significant that under Zn\u003csup\u003e2+\u003c/sup\u003e-rich and AzA conditions, the expression of \u003cem\u003ePR1\u003c/em\u003e is more readily upregulated compared to Zn\u003csup\u003e2+\u003c/sup\u003e-deficient conditions, indicating that adequate Zn\u003csup\u003e2+\u003c/sup\u003e levels can enhance the production of resistance-related metabolites, thereby influencing plant resistance levels. Prior research has shown that the lipid transfer protein-like protein encoded by \u003cem\u003eAtAZI1\u003c/em\u003e (Azelaic acid induced 1) is involved in transmitting SAR signals in response to AzA, thereby activating local or systemic nonspecific resistance to pathogens \u003csup\u003e18\u003c/sup\u003e, and this function of AtAZI1 is Zn\u003csup\u003e2+\u003c/sup\u003e-dependent \u003csup\u003e19\u003c/sup\u003e, establishing a link between plant nutritional immunity and micronutrient utilization. Moreover, the application of Zn\u003csup\u003e2+\u003c/sup\u003e fertilizers under drought conditions has been shown to increase antioxidant content (e.g., ascorbic acid, reduced glutathione, total flavonoids, total phenols) in wheat leaves, and foliar Zn\u003csup\u003e2+\u003c/sup\u003e spraying can enhance photosynthetic pigments, reduce lipid peroxidation of cell membranes, maintain cellular homeostasis, and mitigate drought-induced oxidative damage \u003csup\u003e20\u003c/sup\u003e. Thus, Zn\u003csup\u003e2+\u003c/sup\u003e emerges as a potent plant immune inducer and growth regulator \u003csup\u003e21, 22\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eDespite the numerous studies highlighting Zn\u003csup\u003e2+\u003c/sup\u003e's role in pathogen control, the application of Zn\u003csup\u003e2+\u003c/sup\u003e fertilizers still tends to be at relatively high concentrations \u003csup\u003e23\u003c/sup\u003e. Due to environmental complexities, the loss of active ingredients during the application of trace elements can reach 70%-90% \u003csup\u003e24\u003c/sup\u003e, which compromises the maximum absorption of Zn\u003csup\u003e2+\u003c/sup\u003e by plants \u003csup\u003e25\u003c/sup\u003e. Repeated applications not only increase economic costs but also impose environmental stress, leading to plant stress \u003csup\u003e26, 27\u003c/sup\u003e. In our previous study, we determined that 44 \u0026micro;g/mL of (CH\u003csub\u003e3\u003c/sub\u003eCOO)\u003csub\u003e2\u003c/sub\u003eZn (Zn\u003csup\u003e2+\u003c/sup\u003econtent is 237.3 \u0026micro;M) exhibited antiviral properties while still allowing normal plant growth \u003csup\u003e28\u003c/sup\u003e. Furthermore, Zn\u003csup\u003e2+\u003c/sup\u003e toxicity symptoms have been reported to manifest at concentrations of 300 mg/kg leaf dry weight, although some crops have lower thresholds \u003csup\u003e26\u003c/sup\u003e. These findings underscore the high loss rates of Zn\u003csup\u003e2+\u003c/sup\u003e in practical applications and the potential environmental risks associated with its use. To address these challenges, we implemented various delivery strategies to mitigate Zn\u003csup\u003e2+\u003c/sup\u003e loss \u003csup\u003e29, 30\u003c/sup\u003e. However, more optimized delivery methods are still required to minimize Zn\u003csup\u003e2+\u003c/sup\u003e residues in the environment and enhance the efficiency of Zn\u003csup\u003e2+\u003c/sup\u003e application \u003csup\u003e31\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eDue to the size effect and high permeability of nanomaterials, they can effectively enhance the stability of pharmaceutical ingredients, facilitate drug deposition on the application interfaces, and improve drug utilization\u003csup\u003e32\u003c/sup\u003e. Therefore, they are ideal for drug delivery in the development of plant disease prevention and control agents \u003csup\u003e33\u003c/sup\u003e. Rod-shaped nanomaterials, such as cellulose nanocrystals (CNCs), offer significant advantages as drug carriers for plant disease control due to their high surface area and aspect ratio\u003csup\u003e34, 35\u003c/sup\u003e. This feature facilitates cellular binding and uptake while minimizing cytotoxicity, and enhances deposition and retention on the surface of foliage \u003csup\u003e36, 37\u003c/sup\u003e. For instance, surface modification with polydopamine (PDA) enhances CNC\u0026rsquo;s affinity for cells. Additionally, CNCs can adsorb nanoparticles, such as silver, improving their bactericidal effect by increasing particle concentration on bacterial membranes\u003csup\u003e38\u003c/sup\u003e. Moreover, the properties of CNCs can be enhanced, such as sustaining control, via modification of the CNCs surface \u003csup\u003e39, 40\u003c/sup\u003e. Thus, it is reasonable to consider CNCs as pesticide carriers with high deposition efficiency, sustainable pesticide release, and effective antifungal and insecticidal capabilities\u003csup\u003e41 42\u003c/sup\u003e. Based on these analyses, we hypothesize that Zn\u003csup\u003e2+\u003c/sup\u003e functions as a signaling messenger during plant virus infection. Notably, the Zn\u003csup\u003e2+\u003c/sup\u003e concentration within plant cells is typically maintained at a steady state of 10\u0026ndash;100 nM, while concentrations above 100 \u0026micro;M can induce cellular Zn\u003csup\u003e2+\u003c/sup\u003e toxicity. Our previous determination of the lowest antiviral concentration of (CH\u003csub\u003e3\u003c/sub\u003eCOO)\u003csub\u003e2\u003c/sub\u003eZn at 44 \u0026micro;g/mL, corresponding to 237.3 \u0026micro;M Zn\u003csup\u003e2+\u003c/sup\u003e, exceeds this threshold. However, increasing the concentration of (CH\u003csub\u003e3\u003c/sub\u003eCOO)\u003csub\u003e2\u003c/sub\u003eZn tenfold demonstrated enhanced resistance without inducing Zn\u003csup\u003e2+\u003c/sup\u003e toxicity \u003csup\u003e28\u003c/sup\u003e. This observation challenges the presumed upper limit of the capacity of Zn\u003csup\u003e2+\u003c/sup\u003e transporters in plants and underscores the substantial loss of (CH\u003csub\u003e3\u003c/sub\u003eCOO)\u003csub\u003e2\u003c/sub\u003eZn during resistance induction. Nonetheless, residual Zn\u003csup\u003e2+\u003c/sup\u003e, if not assimilated by the plant, poses environmental risks. Therefore, it is critical to reduce the applied concentration of Zn\u003csup\u003e2+\u003c/sup\u003e while enhancing its cellular uptake to optimize its role as a second messenger. To achieve this, we selected rod-shaped CNCs characterized by low cytotoxicity and high permeability for Zn\u003csup\u003e2+\u003c/sup\u003e modification. By ensuring minimal cytotoxicity, we increased the plant\u0026rsquo;s absorption and transport of Zn\u003csup\u003e2+\u003c/sup\u003e, thereby amplifying Zn\u003csup\u003e2+\u003c/sup\u003e-mediated SAR. This approach lays a theoretical foundation for the informed application of Zn\u003csup\u003e2+\u003c/sup\u003e in plant disease prevention and control.\u003c/p\u003e"},{"header":"Results and Discussions","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e \u003ch2\u003eSynthesis and characterization of CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e\u003c/h2\u003e \u003cp\u003eAs mentioned above, although we systematically explored the minimum concentration of (CH\u003csub\u003e3\u003c/sub\u003eCOO)\u003csub\u003e2\u003c/sub\u003eZn that can induce plant disease defense, these concentrations still highly exceed the maximum Zn\u003csup\u003e2+\u003c/sup\u003e tolerance of plant cells. Moreover, as the (CH\u003csub\u003e3\u003c/sub\u003eCOO)\u003csub\u003e2\u003c/sub\u003eZn concentration increases, the resistance levels significantly improve while maintaining normal growth without any high Zn\u003csup\u003e2+\u003c/sup\u003e phenotypes. This phenomenon highlights the low efficiency of Zn\u003csup\u003e2+\u003c/sup\u003e absorption and the high environmental residue associated with the use of (CH\u003csub\u003e3\u003c/sub\u003eCOO)\u003csub\u003e2\u003c/sub\u003eZn. A recent study has shown that plants can enhance the utilization of metal ions in complex forms \u003csup\u003e43\u003c/sup\u003e. Therefore, we first synthesized CNC@PDA by exploiting the hydrogen bonding between CNC and PDA. This composite then served as a carrier for chelating Zn\u003csup\u003e2+\u003c/sup\u003e, leading to the creation of CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). To further confirm the chemical structure of CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e, we conducted verification by Transmission Electron Microscope (TEM), Fourier-transmitted infrared spectrometry (FTIR), Zeta potential, X-ray diffraction (XRD), and Thermogravimetric analysis (TGA). TEM showed that after grafting Zn\u003csup\u003e2+\u003c/sup\u003e on the surface of CNC, it displayed similar morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb-c). In Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, the absorption peaks at 3359 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 2910 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and 1058 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in CNC, CNC@PDA, PDA@Zn\u003csup\u003e2+\u003c/sup\u003e, and CNC@ PDA@Zn\u003csup\u003e2+\u003c/sup\u003e are assigned to -OH stretching, C\u0026ndash;H vibration, and C\u0026ndash;O stretching vibration respectively, which are the typical absorption peaks of CNC \u003csup\u003e44\u003c/sup\u003e. Additionally, the zeta potential shifted from \u0026minus;\u0026thinsp;31.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 mV (CNC) to -29.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4mV (CNC@PDA) after coating PDA. Reduced zeta potential to -27.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 mV (CNC@ PDA@ Zn\u003csup\u003e2+\u003c/sup\u003e) after coordinating Zn\u003csup\u003e2+\u003c/sup\u003e on the CNC@PDA surface, indicating successful surface modification (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). Additionally, TGA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef) showed a considerable increase in the peak degradation temperature of CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e compared to CNC, suggesting that the modification improved the thermal stability of the composite. This indicates that the PDA coating enhances the thermal resistance of the CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e material. XRD analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg) revealed that the main diffraction characteristics of CNC were still clearly present after modification, such as the diffraction peaks at 2θ angles around 14.7\u0026deg; (101), 16.6\u0026deg; (101̅), 22.7\u0026deg; (002), and 34.5\u0026deg; (040)\u003csup\u003e45\u003c/sup\u003e. These results showed that the modification didn\u0026rsquo;t disturb the crystalline integrity of the main cellulose structure. Besides, it indicated the decrease in crystallinity of nanoparticles is attributed to the introduction of a dense amorphous PDA diffusion layer on the CNC structure. \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e presents the results of the elemental analysis of zinc provided the anchoring of Zn\u003csup\u003e2+\u003c/sup\u003e onto CNCs by Inductively coupled plasma optical emission spectroscopy (ICP-OES).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e demonstrates enhanced antiviral efficacy\u003c/h2\u003e \u003cp\u003eIn our previous research, we observed that the antiviral activity of (CH\u003csub\u003e3\u003c/sub\u003eCOO)\u003csub\u003e2\u003c/sub\u003eZn exhibited a dose-dependent effect within a certain range \u003csup\u003e46\u003c/sup\u003e. However, even at the lowest concentration of (CH\u003csub\u003e3\u003c/sub\u003eCOO)\u003csub\u003e2\u003c/sub\u003eZn (44 \u0026micro;g/mL), the Zn\u003csup\u003e2+\u003c/sup\u003e concentration was approximately 237.3 \u0026micro;M, significantly exceeding the maximum Zn\u003csup\u003e2+\u003c/sup\u003e tolerance of plant cells \u003csup\u003e47\u003c/sup\u003e. To address this, we utilized the dimensional properties of CNCs to reduce the usage concentration of Zn\u003csup\u003e2+\u003c/sup\u003e while enhancing the overall antiviral efficacy of the material. We grafted Zn\u003csup\u003e2+\u003c/sup\u003e onto a crosslinked PDA coating on rod-shaped CNCs, resulting in CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e. Based on the ICP results (\u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e), the Zn\u003csup\u003e2+\u003c/sup\u003e concentration of 1600 \u0026micro;g/mL CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e is approximately equivalent to the 44 \u0026micro;g/mL (CH3COO)2Zn acetate solution. Subsequently, detailed experiments were conducted using a concentration of 44 \u0026micro;g/mL (CH\u003csub\u003e3\u003c/sub\u003eCOO)\u003csub\u003e2\u003c/sub\u003eZn as a control. Various concentrations of CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e were sprayed daily for three days (10 mL per 10 plants each day). On the 4th day, GFP-tagged TMV was inoculated onto the sixth and seventh leaves of \u003cem\u003eN. benthamiana\u003c/em\u003e via rub inoculation. GFP fluorescence intensity and changes were monitored under UV light to trace TMV infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Results showed that at 2 dpi, (CH\u003csub\u003e3\u003c/sub\u003eCOO)\u003csub\u003e2\u003c/sub\u003eZn treatment exhibited some antiviral activity, whereas CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e treatment significantly enhanced resistance, with higher concentrations correlating with increased resistance. Subsequent qPCR analysis of TMV-GFP nucleic acid accumulation confirmed these findings (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). By the 4 dpi, the fluorescence intensity of inoculated leaves treated with (CH\u003csub\u003e3\u003c/sub\u003eCOO)\u003csub\u003e2\u003c/sub\u003eZn and water had approached the threshold, whereas CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e treatment resulted in slower infection progression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). qPCR showed that on this day, the TMV-GFP nucleic acid levels in (CH\u003csub\u003e3\u003c/sub\u003eCOO)\u003csub\u003e2\u003c/sub\u003eZn and water-treated leaves were similar, but significantly lower levels of TMV-GFP were observed in CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e-treated leaves. At 6 dpi, substantial TMV accumulation was evident in systemic leaves of water-treated plants, significantly reduced in (CH\u003csub\u003e3\u003c/sub\u003eCOO)\u003csub\u003e2\u003c/sub\u003eZn-treated leaves, and maintained at the lowest levels in CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e-treated leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). These results indicate that CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e effectively delays TMV-GFP infection. Notably, the antiviral function of CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e exhibited a dose-dependent response. At its maximum concentration (1600 \u0026micro;g/mL), the Zn\u003csup\u003e2+\u003c/sup\u003e content was approximately 237.3 \u0026micro;M (equivalent to the Zn\u003csup\u003e2+\u003c/sup\u003e content in 44 \u0026micro;g/mL (CH\u003csub\u003e3\u003c/sub\u003eCOO)\u003csub\u003e2\u003c/sub\u003eZn), and the material showed very strong antiviral activity, increasing efficacy more than threefold compared to the same Zn\u003csup\u003e2+\u003c/sup\u003e concentration of (CH\u003csub\u003e3\u003c/sub\u003eCOO)\u003csub\u003e2\u003c/sub\u003eZn. Even when diluted 16-fold (Zn\u003csup\u003e2+\u003c/sup\u003e content approximately 16.83 \u0026micro;M), CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e still exhibited excellent antiviral activity. Besides, this amount of Zn\u003csup\u003e2+\u003c/sup\u003e is much lower than that in the composite nanomaterials reported previously \u003csup\u003e29, 30\u003c/sup\u003e. These results demonstrate that embedding Zn\u003csup\u003e2+\u003c/sup\u003e in CNCs can enhance antiviral activity while reducing Zn\u003csup\u003e2+\u003c/sup\u003e concentration. The significant significance of this result is that we can manipulate the reduction of Zn\u003csup\u003e2+\u003c/sup\u003e content to induce stronger resistance, but significantly reduce the residual Zn\u003csup\u003e2+\u003c/sup\u003e in the environment.\u003c/p\u003e \u003cp\u003eIt is well-established that ZnO can promote the aggregation of viral particles \u003cem\u003ein vitro\u003c/em\u003e, thereby inactivating the virus and ultimately inhibiting infection \u003csup\u003e48\u003c/sup\u003e. However, whether CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e possesses similar effects remains unclear. To investigate this, we first purified fresh TMV particles using differential centrifugation and then incubated them with 1600 \u0026micro;g/mL CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e. After 10 hours, we examined the morphological changes of TMV particles under a transmission electron microscopy (TEM) microscope. Surprisingly, the treated group exhibited a significant presence of fragmented viral particles with the average length of TMV particles being predominantly around 328.6 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee, g), whereas in the CNC@PDA@ Zn\u003csup\u003e2+\u003c/sup\u003e treated group, the average length was reduced to 87.3 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef, h). These findings indicate that CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e has an \u003cem\u003ein vitro\u003c/em\u003e inactivation effect on TMV particles. To confirm whether this inactivation effect alters TMV infection \u003cem\u003ein vivo\u003c/em\u003e, we inoculated \u003cem\u003eN. benthamiana\u003c/em\u003e leaves with the treated suspensions. At 2 dpi, the water control group exhibited similar levels of viral infection, while the CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e treated group showed reduced accumulation of TMV-GFP in the inoculated leaves compared to the water control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei). By 4 dpi, the systemic leaves of the control groups showed a higher accumulation of TMV-GFP compared to the CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei). The qPCR and western blot results corroborated the fluorescence observations, showing a similar trend (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ej-k). These results suggest that the composite material CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e demonstrates a strong inactivation effect on TMV-GFP particles. These results suggest that CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e can enhance antiviral effects by increasing \u003cem\u003ein vitro\u003c/em\u003e passivation capacity while reducing Zn\u003csup\u003e2+\u003c/sup\u003e content.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCNC@PDA@Zn enhanced Zn uptake and transport\u003c/h3\u003e\n\u003cp\u003eThe rod-shaped CNC demonstrates low cytotoxicity and high permeability with a negatively charged surface, suggesting that the synthesized CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e may enhance the uptake of Zn\u003csup\u003e2+\u003c/sup\u003e by plant cells. To investigate this, CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e was sprayed onto the whole \u003cem\u003eN. benthamiana\u003c/em\u003e leaves for three consecutive days, and its distribution within the plant was observed using TEM after the treatments. As expected, rod-shaped clusters were observed in the leaf tissues, indicating that CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e could enter plant cells and aggregate within the mesophyll cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). The content of zinc also shows that Zn\u003csup\u003e2+\u003c/sup\u003e can be loaded and entered into plant (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). More importantly, CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e was observed in chloroplasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003ea), demonstrating its ability to not only penetrate plant cells but also be actively transported to this key organelle. This localization suggests a targeted mechanism that may enhance its role in modulating chloroplast-associated immune responses and antiviral defenses. Besides, EDS analysis confirmed the presence of Zn\u003csup\u003e2+\u003c/sup\u003e signals (\u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea\u003c/b\u003e), suggesting that Zn\u003csup\u003e2+\u003c/sup\u003e remained within the nano-carrier. In the stem tissues, dispersed CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e structures were also observed although it is difficult to pinpoint its exact location, with corresponding Zn\u003csup\u003e2+\u003c/sup\u003e signals detected by EDS (\u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eb\u003c/b\u003e), indicating that CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e could be transported from the leaves to the stem while still in its nano-carrier form (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). However, no rod-shaped structures were observed in the roots (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). This raises the possibility that Zn\u003csup\u003e2+\u003c/sup\u003e in the roots may exist in ionic form. To verify this hypothesis, we compared the Zn\u003csup\u003e2+\u003c/sup\u003e content in the roots, stems, and leaves after different treatments. The results showed an increased Zn\u003csup\u003e2+\u003c/sup\u003e content in the roots of the treated group, with higher overall Zn\u003csup\u003e2+\u003c/sup\u003e levels in CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e-treated plants compared to those treated with (CH\u003csub\u003e3\u003c/sub\u003eCOO)\u003csub\u003e2\u003c/sub\u003eZn (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). Notably, when measuring the Zn\u003csup\u003e2+\u003c/sup\u003e content in various plant tissues following CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e treatment, an intriguing observation emerged. After foliar application of CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e at the highest tested concentration of 1600 \u0026micro;g/mL, the Zn\u003csup\u003e2+\u003c/sup\u003e content in roots, stems, and leaves remained relatively consistent and stable, ranging between 20\u0026ndash;65 \u0026micro;g/g DW (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eb-d). This level falls within the typical variability range of total Zn\u003csup\u003e2+\u003c/sup\u003e concentrations in plant tissues, suggesting that CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e maintains theoretical safety at this concentration. Furthermore, plant safety assessments confirmed that CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e is non-toxic to plants (\u003cb\u003eFigure S2\u003c/b\u003e). Compared with water-treated controls, CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e treatment significantly enhanced the growth of \u003cem\u003eN. benthamiana\u003c/em\u003e, as evidenced by increased plant height and dry weight. These findings underscore CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e's dual role in promoting plant growth while ensuring safety, even at higher application rates.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNotably, rod-shaped structures were also observed in the leaves and stems following CNC-only treatment, suggesting that the transport of Zn\u003csup\u003e2+\u003c/sup\u003e to the stem is likely facilitated by CNC. This finding leads us to hypothesize that CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e utilizes the high permeability of CNC to transport Zn\u003csup\u003e2+\u003c/sup\u003e to the stem, where Zn\u003csup\u003e2+\u003c/sup\u003e is gradually released from CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e and subsequently transferred to the roots as free Zn\u003csup\u003e2+\u003c/sup\u003e. However, the exact mechanism by which Zn\u003csup\u003e2+\u003c/sup\u003e is efficiently regulated and transported to the roots remains unclear. To the best of our knowledge, Zn\u003csup\u003e2+\u003c/sup\u003e transport within plants primarily relies on four protein families: zinc-iron permease (ZIPs) family, responsible for transporting Zn\u003csup\u003e2+\u003c/sup\u003e from the external environment or organelles into the cytoplasm \u003csup\u003e49\u003c/sup\u003e; metal tolerance proteins (MTPs) family, involved in Zn\u003csup\u003e2+\u003c/sup\u003e sequestration; heavy metal ATPase (HMAs) family, which pumps Zn\u003csup\u003e2+\u003c/sup\u003e into or out of organelles in an ATP-driven manner; and yellow stripe-like (YSLs) family, which assists in long-distance transport and distribution of Zn\u003csup\u003e2+\u003c/sup\u003e within the plant vascular system \u003csup\u003e50, 51\u003c/sup\u003e. To elucidate how CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e mediates long-distance Zn\u003csup\u003e2+\u003c/sup\u003e transport and efficient accumulation, we conducted a transcriptomic analysis of \u003cem\u003eN. benthamiana\u003c/em\u003e leaves treated with CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e (\u003cb\u003eFigure S3\u003c/b\u003e and \u003cb\u003eTable S2\u003c/b\u003e). Compared to the water control, CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e induced differential expression of 6040 genes; when compared to (CH\u003csub\u003e3\u003c/sub\u003eCOO)\u003csub\u003e2\u003c/sub\u003eZn, this number increased to 7575 (\u003cb\u003eFigure S3a\u003c/b\u003e). Among these differentially expressed genes which belong to ZIPs, MTPs, and HMAs family, 9 was related to Zn\u003csup\u003e2+\u003c/sup\u003e transport, with the YSLs family showing no significant changes (\u003cb\u003eFigure S3b\u003c/b\u003e). Compared to (CH\u003csub\u003e3\u003c/sub\u003eCOO)\u003csub\u003e2\u003c/sub\u003eZn, these genes exhibited an upregulation trend, suggesting that CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e may stimulate the expression of Zn\u003csup\u003e2+\u003c/sup\u003e transporters in an unknown manner, thereby enhancing Zn\u003csup\u003e2+\u003c/sup\u003e transport capacity. It is important to note that the CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e components\u0026mdash;dopamine and cellulose\u0026mdash;lack inherent receptors in plants. However, cellulose is recognized by receptors such as wall-associated kinases (WAKs), leucine-rich repeat receptor-like kinases (LRR-RLKs), and catharanthus roseus RLK1-like kinases (CrRLK1L) \u003csup\u003e52, 53, 54\u003c/sup\u003e. The transcriptional levels of these receptors were also significantly upregulated following CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e treatment (\u003cb\u003eFigure S3c\u003c/b\u003e), leading us to speculate that the upregulated Zn\u003csup\u003e2+\u003c/sup\u003e transport proteins might be finely regulated through the phosphorylation by these cellulose-recognizing receptors. Overall, although these transcriptomic analyses were limited to leaf tissues, they provide compelling evidence for the dual role of CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e in regulating Zn\u003csup\u003e2+\u003c/sup\u003e transport: facilitating efficient Zn\u003csup\u003e2+\u003c/sup\u003e transport to the stem via the high permeability of CNC and subsequently regulating Zn\u003csup\u003e2+\u003c/sup\u003e transport proteins through cellulose receptor-mediated phosphorylation mechanisms to enhance Zn\u003csup\u003e2+\u003c/sup\u003e translocation from stem to root.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCNC@PDA@Zn\u003c/b\u003e \u003csup\u003e \u003cb\u003e2+\u003c/b\u003e \u003c/sup\u003e \u003cb\u003ealters nsLTP2 expression and localization.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate whether CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e contributes to and enhances broad-spectrum antiviral activity, we tested its inhibitory effects on the \u003cem\u003ein vivo\u003c/em\u003e infection of various virus genera. The results revealed that while CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e exhibited antiviral activity against youcai mosaic virus (YoMV), with inhibitory effects surpassing those of (CH\u003csub\u003e3\u003c/sub\u003eCOO)\u003csub\u003e2\u003c/sub\u003eZn at 5 dpi, despite its efficacy was consistently lower than its inhibitory effect against TMV (\u003cb\u003eFigure S4\u003c/b\u003e). This disparity suggests that structural differences among these viruses may influence the direct inactivation capacity of CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e, highlighting its significant role in enhancing host resistance regulation induced by Zn\u003csup\u003e2+\u003c/sup\u003e. To the best of our knowledge, Zn\u003csup\u003e2+\u003c/sup\u003e's known functions in plants include stabilizing enzyme structures or participating in catalytic reactions, affecting protein synthesis and gene expression, regulating chlorophyll synthesis, participating in energy metabolism, and enhancing plant disease resistance \u003csup\u003e55\u003c/sup\u003e. Specifically, in terms of enhancing disease resistance, Zn\u003csup\u003e2+\u003c/sup\u003e can reinforce the cell wall and increase the secretion of resistance proteins to inhibit pathogens in the apoplast, and it can also participate in hormone signaling pathways, stimulating the systemic acquired resistance (SAR) defense response \u003csup\u003e56\u003c/sup\u003e. To elucidate the role of CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e in enhancing antiviral activity, we conducted a detailed analysis of the obtained transcriptomic profiles. The results indicated that the differentially expressed genes enriched in KEGG pathways after CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e treatment were primarily associated with fundamental metabolic processes in plants, including spliceosome, purine metabolism, RNA transport, mRNA surveillance pathways, and ubiquitin-mediated proteolysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). This suggests that CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e treatment alters various energy and metabolic processes within the host. Additionally, we observed that CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e induced the expression of many Zn\u003csup\u003e2+\u003c/sup\u003e-binding proteins, including zinc finger proteins, zinc metalloproteases, and superoxide dismutase (SOD) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eb)\u003csup\u003e56\u003c/sup\u003e. Interestingly, these proteins are directly or indirectly involved in plant defense mechanisms \u003csup\u003e56\u003c/sup\u003e. For instance, the \u003cem\u003eSOD1\u003c/em\u003e-encoded enzyme activity directly influences the host's reactive oxygen species (ROS) burst, thereby regulating cell wall formation. Recent works displayed that nanoparticles loaded zinc can affect the SOD enzyme activity\u003csup\u003e29, 57\u003c/sup\u003e. Notably, biochemical assays further confirmed that CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e enhances the SOD enzyme activity induced by zinc acetate within the host (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003ec), which is likely directly related to the increased resistance level observed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNevertheless, the role of CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e in enhancing systemic acquired resistance (SAR) remains unclear. To investigate this further, we analyzed the top 100 differentially expressed genes (DEGs) with the most significant expression changes and categorized these genes based on their functions, among which \u003cem\u003ensLTP2\u003c/em\u003e emerged as a particularly intriguing candidate, possibly serving as a target induced by CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e (\u003cb\u003eFigure S4\u003c/b\u003e). Following CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e treatment, \u003cem\u003ensLTP2\u003c/em\u003e was highly expressed, with its expression level significantly exceeding that observed in the (CH\u003csub\u003e3\u003c/sub\u003eCOO)\u003csub\u003e2\u003c/sub\u003eZn treatment group (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003ed-e). Lipid transfer proteins (LTPs) are small, cysteine-rich proteins that primarily function in the transfer of various lipid molecules \u003csup\u003e58\u003c/sup\u003e. They play crucial roles in plant growth, defense against pathogens, and interactions with the environment \u003csup\u003e59\u003c/sup\u003e. It is particularly noteworthy that Zn\u003csup\u003e2+\u003c/sup\u003e-binding proteins possess highly conserved sequence motifs. In nsLTP2, we identified a C-Xn-C-Xn-CC-Xn-CXC-Xn-C-Xn-C motif, suggesting a potential Zn\u003csup\u003e2+\u003c/sup\u003e-binding capability. However, further research is needed to validate this binding affinity. Subsequently, we examined the tissue expression pattern of \u003cem\u003ensLTP2\u003c/em\u003e in \u003cem\u003eN. benthamiana\u003c/em\u003e and found that its expression was highest in the roots, followed by the leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003ef), consistent with the expression pattern of \u003cem\u003eNbLTP1\u003c/em\u003e \u003csup\u003e60\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eGiven the importance of subcellular localization for protein function, we used bioinformatics tools to predict that nsLTP2 contains a 24-residue N-terminal signal peptide and is primarily localized to the cell wall. To verify these predictions, we constructed nsLTP2-GFP fusion expression vectors driven by the cauliflower mosaic virus (CaMV) 35S promoter, as well as nsLTP2\u003csup\u003e△SP\u003c/sup\u003e-GFP fusion constructs lacking the signal peptide (SP), and observed their localization in cells. Experimental results showed that nsLTP2-GFP was distributed in the cytoplasm and cell wall, while nsLTP2\u003csup\u003e△SP\u003c/sup\u003e-GFP was found in the cytoplasm and chloroplasts. Interestingly, when CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e was applied to cells expressing these fusion proteins, their localization underwent significant changes. For nsLTP2-GFP, intracellular protein accumulation decreased and concentrated some in the chloroplast, with more protein accumulating extracellularly (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eg). In contrast, nsLTP2\u003csup\u003e△SP\u003c/sup\u003e-GFP became more concentrated in the chloroplasts, with the Pearson correlation curve of the GFP channel and chloroplast fluorescence channel completely overlapping (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eh). Additionally, western blot analysis confirmed that CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e treatment not only altered nsLTP2/ nsLTP2\u003csup\u003e△SP\u003c/sup\u003e localization but also increased nsLTP2 protein accumulation (\u003cb\u003eFigure S6\u003c/b\u003e). This result strengthens the correlation between CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e and nsLTP2.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003ensLTP2 as a positive regulator induces plant resistance\u003c/h3\u003e\n\u003cp\u003eTo understand the functional significance of CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e-induced changes in nsLTP2 localization in plant resistance, we tested the antiviral function of nsLTP2. While the role of NbLTP1 in TMV infection is well characterized, we systematically explored the role of nsLTP2 during TMV infection. Stress expression analysis revealed that \u003cem\u003ensLTP2\u003c/em\u003e expression significantly increased in inoculated leaves at 2 dpi with TMV-GFP, and its expression also markedly elevated in systemic leaves at 6 dpi (\u003cb\u003eFigure S7\u003c/b\u003e), suggesting a potential role for nsLTP2 in antiviral defense. Next, we utilized tobacco rattle virus (TRV)-mediated gene silencing to analyze nsLTP2\u0026rsquo;s role in TMV resistance. Following the agroinfiltration of \u003cem\u003eN. benthamiana\u003c/em\u003e leaves with TRV1\u0026thinsp;+\u0026thinsp;TRV2 (TRV:00) or TRV1\u0026thinsp;+\u0026thinsp;TRV2:nsLTP2 (TRV:nsLTP2) for 12 days, RT-qPCR analysis revealed that \u003cem\u003ensLTP2\u003c/em\u003e expression in systemic leaves was significantly lower in TRV:nsLTP2 plants compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). We then mechanically inoculated the 6th and 7th leaves with TMV-GFP and observed the movement of GFP under UV light at 2, 4, and 6 dpi to track TMV distribution. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, GFP fluorescence signals were observed in the inoculated leaves at 2 dpi, and qPCR analysis demonstrated that TMV-GFP nucleic acid levels were significantly higher in silenced plants than in controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). At 4 dpi, GFP signals appeared in the systemic leaves of silenced plants, whereas no GFP signals were detected in the systemic leaves of control plants; qPCR analysis of viral nucleic acids yielded similar results (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). By 6 dpi, the GFP signal was more pronounced in silenced plants, and TMV-GFP accumulated significantly in their systemic leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). These results indicate that \u003cem\u003ensLTP2\u003c/em\u003e silencing facilitates TMV-GFP infection. Subsequently, we compared the disease resistance conferred by nsLTP2 and nsLTP2\u003csup\u003eΔSP\u003c/sup\u003e. We fused nsLTP2 and nsLTP2\u003csup\u003eΔSP\u003c/sup\u003e with eGFP via a P2A linker, using eGFP:00 as a control. Following transient expression and TMV-GFP inoculation, we found that by 3 dpi, the control leaves contained numerous GFP fluorescence spots, while the treated leaves had significantly fewer spots, with the lowest number observed in the nsLTP2\u003csup\u003eΔSP\u003c/sup\u003e overexpression group (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003ef). qPCR results also showed that TMV-GFP nucleic acid levels were significantly lower in the nsLTP2 and nsLTP2\u003csup\u003eΔSP\u003c/sup\u003e overexpression groups compared to the control, with the nsLTP2\u003csup\u003eΔSP\u003c/sup\u003e group containing the least amount of TMV-GFP nucleic acids (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003eg). We then provided more compelling genetic evidence by stably overexpressing nsLTP2\u003csup\u003eΔSP\u003c/sup\u003e under the CaMV 35S promoter and analyzing its detailed antiviral phenotype. Consistent with the transient expression results, two independent overexpression lines exhibited remarkable antiviral activity, especially line #10 (Figs.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003eh-j). These findings suggest that chloroplast-localized nsLTP2\u003csup\u003eΔSP\u003c/sup\u003e confers stronger antiviral functions. As lipid transfer proteins, LTPs maintain the stability of organelle membranes by transporting lipids between various organelles \u003csup\u003e58\u003c/sup\u003e. For nsLTP2 containing the SP, CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e promotes its secretion to the extracellular space, potentially supporting the hypothesis that it stabilizes cell wall structures by transporting lipids \u003csup\u003e58\u003c/sup\u003e. However, we believe that its more crucial role lies in executing PR14 functions. LTP2 may have potential PR14-related functions, and the upregulation of this pathogenesis-related protein directly contributes to host resistance \u003csup\u003e58\u003c/sup\u003e. Nevertheless, nsLTP2\u003csup\u003eΔSP\u003c/sup\u003e demonstrates stronger resistance and accumulates in chloroplasts under CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e treatment. Chloroplasts are a primary battleground in plant-virus interactions, with chloroplast homeostasis directly influencing viral symptom development, and several chloroplast proteins exhibit antiviral activity \u003csup\u003e61, 62\u003c/sup\u003e. Notably, CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e seems to be able to enter chloroplasts and enhance the expression of various chloroplast-associated proteins (\u003cb\u003eFigure S8\u003c/b\u003e), strongly suggesting that CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e-induced nsLTP2 stabilizes chloroplast membrane structures by transporting lipids, leading to enhanced antiviral defense. Furthermore, we cannot entirely dismiss an important yet easily overlooked possibility: in roots lacking chloroplasts, nsLTP2 may function as a PR14 resistance protein when induced by CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e, while in chloroplast-rich leaves, nsLTP2 suppresses viral infection by maintaining chloroplast homeostasis. However, these hypotheses require further evidence for confirmation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, Zn\u003csup\u003e2+\u003c/sup\u003e is known to facilitate cell wall fortification and is essential for chlorophyll synthesis \u003csup\u003e55, 56\u003c/sup\u003e, making it difficult not to associate these processes with the function of nsLTP2. We hypothesize that nsLTP2 may bind Zn\u003csup\u003e2+\u003c/sup\u003e and subsequently transport lipids to the cell wall and chloroplasts. This lipid-mediated process could involve the reversible transfer of Zn\u003csup\u003e2+\u003c/sup\u003e to the cell wall and chloroplasts, where it plays a crucial role. Notably, CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e appears to accelerate this process, possibly due to the increased intracellular Zn\u003csup\u003e2+\u003c/sup\u003e levels introduced by CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e or a chain reaction triggered by the CNC receptor hypothesis. However, more detailed genetic evidence is required to confirm these mechanisms. Regardless, these results support the role of CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e in enhancing resistance by inducing nsLTP2.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe induction of nsLTP2 by CNC@PDA@Zn\u003c/b\u003e \u003csup\u003e \u003cb\u003e2+\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eis dependent on the SA pathway for its disease response.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe SAR in plants is intrinsically linked to SA, and Zn\u003csup\u003e2+\u003c/sup\u003e plays a crucial role in this process as well. Previous studies have already associated Zn\u003csup\u003e2+\u003c/sup\u003e with SAR \u003csup\u003e18, 19\u003c/sup\u003e, but whether Zn\u003csup\u003e2+\u003c/sup\u003e can promote SA accumulation by regulating the expression of nsLTP2 remains unclear. Notably, whether it functions as a PR14 protein or stabilizes chloroplast membrane structures, nsLTP2 appears to be closely linked to SA, as PR proteins exert their functions through the SA synthesized in chloroplasts. To explore this, we first treated \u003cem\u003eN. benthamiana\u003c/em\u003e leaves with the SA analog MeSA and quantified the changes in \u003cem\u003ensLTP2\u003c/em\u003e expression during this process. The results showed that \u003cem\u003ensLTP2\u003c/em\u003e expression increased over time following MeSA treatment, indicating that \u003cem\u003ensLTP2\u003c/em\u003e is an SA-inducible gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). We then measured the SA content in two stable overexpression lines of nsLTP2\u003csup\u003eΔSP\u003c/sup\u003e and found that both lines exhibited increased SA levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e6\u003c/span\u003eb), suggesting a potential link between nsLTP2 and SA. Additionally, we searched the transcriptome database for SA-related genes induced by CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e and found that CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e enhanced the expression levels of several SA-related genes, including \u003cem\u003ensLTP2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). We further quantified the expression of key genes involved in SA synthesis and response, such as \u003cem\u003eNPR1\u003c/em\u003e, pathogenesis-related gene 1 (\u003cem\u003ePR1\u003c/em\u003e), and pathogenesis-related gene 2 (\u003cem\u003ePR2\u003c/em\u003e). The results showed that both CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e and (CH\u003csub\u003e3\u003c/sub\u003eCOO)\u003csub\u003e2\u003c/sub\u003eZn treatments upregulated the expression of \u003cem\u003eNPR1\u003c/em\u003e, \u003cem\u003ePR1\u003c/em\u003e, and \u003cem\u003ePR2\u003c/em\u003e, with CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e promoting higher expression levels of \u003cem\u003eNPR1\u003c/em\u003e and \u003cem\u003ePR1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e6\u003c/span\u003ed-f). Subsequently, we inoculated TMV-GFP after treating plants with CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e and (CH\u003csub\u003e3\u003c/sub\u003eCOO)\u003csub\u003e2\u003c/sub\u003eZn to examine the expression of SA-related genes under these complex conditions. The results demonstrated that under TMV-GFP infection, both (CH\u003csub\u003e3\u003c/sub\u003eCOO)\u003csub\u003e2\u003c/sub\u003eZn and CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e treatments induced high expression of \u003cem\u003ePR2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e6\u003c/span\u003ef), while CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e also promoted \u003cem\u003eNPR1\u003c/em\u003e expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). Furthermore, SA levels increased following (CH\u003csub\u003e3\u003c/sub\u003eCOO)\u003csub\u003e2\u003c/sub\u003eZn and CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e6\u003c/span\u003eg). Notably, after TMV-GFP inoculation, the SA content in the CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e-treated group was slightly higher than in the (CH\u003csub\u003e3\u003c/sub\u003eCOO)\u003csub\u003e2\u003c/sub\u003eZn-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e6\u003c/span\u003eg). These results suggest that the resistance induced by CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e in TMV-GFP-infected plants is more stable. To determine whether the resistance induced by CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e in \u003cem\u003eN. benthamiana\u003c/em\u003e is entirely dependent on the SA pathway, we treated transgenic \u003cem\u003eNahG\u003c/em\u003e plants, which express salicylate hydroxylase and are completely deficient in SA, with CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e and observed whether their antiviral phenotype changed. The results showed that at 2 dpi, \u003cem\u003eNahG\u003c/em\u003e plants displayed stronger fluorescence signals in their leaves compared to the control, but this difference significantly diminished at 4 and 6 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e6\u003c/span\u003eh). RT-qPCR analysis indicated that the inhibitory effect of CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e on TMV-GFP infection was completely lost at 4 and 6 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e6\u003c/span\u003ei). These findings highlight the crucial role of CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e in inducing host systemic resistance, and this SAR resistance is largely dependent on SA signaling.\u003c/p\u003e\n\u003ch3\u003eEvolutionary conservation of nsLTP2 in flowering plants\u003c/h3\u003e\n\u003cp\u003eFinally, we conducted an in-depth analysis of the evolutionary relationships of nsLTP2 across the plant lineage, given its potential relevance to the functional scope of CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e. Notably, nsLTP2 originated in angiosperms, as it was first identified in \u003cem\u003eAmborella trichopoda\u003c/em\u003e. \u003cem\u003eA. trichopoda\u003c/em\u003e is the sole surviving representative of the basal sister lineage of all extant angiosperms, suggesting that nsLTP2 is one of the earliest genes to have emerged during the evolution of angiosperms. This highlights its critical role in the origin and early evolution of flowering plants. Subsequently, these nsLTP2 genes underwent duplication across different species, leading to a significant number of homologs in key crops such as \u003cem\u003eSolanum lycopersicum\u003c/em\u003e (12 copies), \u003cem\u003eOryza sativa\u003c/em\u003e (12 copies), and \u003cem\u003eZea mays\u003c/em\u003e (7 copies) (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e6\u003c/span\u003ej). These findings suggest that CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e may broadly induce the expression of nsLTP2 across this vast phylogenetic group of angiosperms and that there are multiple nsLTP2 targets in some key crops. This further indicates the significant role of CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e in inducing resistance defenses throughout the plant kingdom.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn summary, we developed a rode-shape nanomaterial, CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e, by embedding Zn\u003csup\u003e2+\u003c/sup\u003e into a PDA coating on CNCs. This innovative material demonstrated enhanced efficiency in controlling plant virus infection and facilitating trace element transport within plants, while significantly reducing Zn\u003csup\u003e2+\u003c/sup\u003e usage, positioning it as a key player in sustainable agricultural practices. Mechanistic analyses revealed that CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e induced a SA-mediated defense response by upregulating the secretion and expression of nsLTP2 across different organelles. Notably, this mechanism appears to be highly conserved among angiosperms. Our study presents a novel strategy for efficient Zn\u003csup\u003e2+\u003c/sup\u003e delivery in plants, strengthening plant immune responses and resistance to pathogens, thereby advancing the sustainable use of Zn\u003csup\u003e2+\u003c/sup\u003e as a plant disease control agent.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials and growth conditions\u003c/h2\u003e \u003cp\u003e \u003cem\u003eNicotiana benthamiana\u003c/em\u003e seeds were sterilized with 75% alcohol and inoculated in MS medium. After 1 week, they were transferred into soil. The growing condition was under 16 h day/8 h night cycle, 26\u0026deg;C, and 80% relative humidity. The plants were cultured to the six-leaves to eight-leaves stage for the next experiment, and fresh leaves were collected and frozen in liquid nitrogen for subsequent analysis.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSynthesis of CNC@PDA@Zn\u003c/h3\u003e\n\u003cp\u003eThe CNCs were prepared by hydrolyzing cotton linter with sulfuric acid according to our previous work \u003csup\u003e63\u003c/sup\u003e. The purified CNCs were used to prepare CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e. Dopamine hydrochloride (DA\u0026sdot;HCl, 98%) and zinc chloride (ZnCl\u003csub\u003e2\u003c/sub\u003e) were purchased from Shanghai Aladdin Biochemical Technology Co. Ltd. (Shanghai). Trimethylaminomethane (Tris) was purchased from Shanghai Yuanye Bio-Technology Co. Ltd. (Shanghai). Sodium hydroxide (NaOH, 96.0%) and hydrochloric acid (HCl, 36\u0026ndash;38%) were purchased from Chongqing Chuandong Chemical Co. Ltd. (Chongqing).\u003c/p\u003e\n\u003ch3\u003eCharacterization of CNC@PDA@Zn\u003c/h3\u003e\n\u003cp\u003eThe FT-IR spectra of CNC, CNC@PDA, CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e, and PDA@Zn\u003csup\u003e2+\u003c/sup\u003e were measured by were characterized by a Nicolet 170SX FourierTransforman (Madison, WI, USA) with anhydrous KBr in the range of 4000\u0026ndash;500 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at attenuated total reflection cell by averaging 32 spectra with 4 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Zn\u003csup\u003e2+\u003c/sup\u003e content was measured by inductively coupled plasma atomic emission spectroscopy (ICP-AES) (iCAP7000, American). The morphology of CNC and CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e were measured by transmission electron microscope (TEM) (Talos F200X). The ζ-potential of CNC, CNC@PDA, and CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e was measured by a Zeasizer nano ZS (Malvern, UK). X-ray diffraction (XRD, D8 ADVANCE, Bruker) was employed to investigate the interaction of CNCs with PDA@Zn\u003csup\u003e2+\u003c/sup\u003e. Thermogravimetric analysis (TGA, STA 499 F5/F3 Jupiter, NETZSCH) was conducted to characterize the thermal stability and decomposition behavior of composites.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eVirus inoculation\u003c/h2\u003e \u003cp\u003eFor TMV-GFP inoculation, the friction inoculation method was employed to eliminate potential experimental interference from \u003cem\u003eAgrobacterium\u003c/em\u003e, which is commonly associated with the \u003cem\u003eAgrobacterium\u003c/em\u003e infiltration method. Specifically, 0.1 g of fully TMV-GFP-infected \u003cem\u003eN. benthamiana\u003c/em\u003e leaves were collected and ground into a fine powder using a mortar and pestle under liquid nitrogen. The resulting powder was dissolved in PBS buffer (pH\u0026thinsp;=\u0026thinsp;7.4), and the suspension was adjusted to an OD\u003csub\u003e600\u003c/sub\u003e of 1. The prepared solution was then used to inoculate designated leaves via friction inoculation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot\u003c/h2\u003e \u003cp\u003eFor protein extraction, 0.1 g of leaf tissue was ground into a fine powder in a pre-chilled mortar with liquid nitrogen. Protein lysis buffer (0.1 M Tris-HCl, pH 7.4, 1% SDS, 0.02% β-mercaptoethanol) was added to the powder, and the mixture was incubated on ice for 30 min. The lysate was centrifuged at 5000 \u003cem\u003eg\u003c/em\u003e for 15 minutes at 4\u0026deg;C, and the resulting supernatant was collected. SDS-protein loading buffer was added to the supernatant, and the mixture was boiled at 95\u0026deg;C for 5 min to denature the proteins. The prepared protein samples were subjected to SDS-PAGE using a Mini-PROTEAN\u0026reg; Tetra Cell system and subsequently transferred onto a PVDF membrane for western blot analysis. The membrane was probed with a mouse anti-GFP monoclonal antibody (ABclonal, AE012) as the primary antibody and HRP-conjugated goat anti-mouse IgG (H\u0026thinsp;+\u0026thinsp;L) (ABclonal, AS003) as the secondary antibody.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction and cDNA synthesis\u003c/h2\u003e \u003cp\u003eRNA was extracted using an Eastep\u0026reg; Super Total RNA Extraction Kit (Promega, LS1040, Beijing, China). The RNA sample was then reverse transcribed with a PrimeScript\u0026trade; RT Reagent Kit (TaKaRa, RR037A, Shiga, Japan) in a 10 \u0026micro;L reaction. \u003cb\u003eRNA-seq\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eN. benthamiana\u003c/em\u003e leaves of CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e treatments (PZ-1, PZ-2, and PZ-3), (CH\u003csub\u003e3\u003c/sub\u003eCOO)\u003csub\u003e2\u003c/sub\u003eZn treatments (Z-1, Z-2, and Z-3), CNC treatments (CNC-1, CNC-2 and CNC-3) and water treatments (CK-1, CK-2, CK-3) were collected and RNA was extracted using RNAiso Plus reagent (Takara, Japan). mRNA was isolated with oligo (dT) cellulose and broken into short fragments (200 nt) by adding a fragmentation buffer. First-strand cDNA was generated using random hexamer-primed reverse transcription, and second-strand cDNA was synthesized by DNA polymerase I and RNase H. After that, the synthesized cDNA fragments were purified and subjected to end pairing by adding a single \u0026ldquo;A\u0026rdquo; bases, and ligated with Illumina adapters. The ligation products were size-fractionated by agarose gel electrophoresis, and fragments were excised for PCR amplification. The amplified fragments were sequenced using Illumina HiSeqTM 2500 by Gene Denovo Co. (Guangzhou, China). De novo transcriptome assembly and differentially expressed gene (DEGs) analysis were described. In brief, the raw reads obtained by the sequencing platform were performed quality control (QC) and filtered to obtain high-quality clean reads. After that, clean reads were aligned to the reference genome (Niben.genome.v1.0.1). After performing the QC, a follow-up analysis was performed, followed by quantitative analysis and structural analysis. Quantitative analysis includes quantification of genes, exons, and transcripts. Subsequent differential expression analysis was based on quantitative analysis, functional enrichment analysis, and time series analysis. Structural analysis consists of alternative splicing analysis, gene structure optimization, new transcript analysis, and SNP/InDel analysis, gene fusion analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eReal time quantitative PCR\u003c/h2\u003e \u003cp\u003eFor real time quantitative PCR (qPCR), qTOWER2.0 real-time PCR (Analytikjena, Germany) and a QuantiNova\u0026trade; SYBR Green PCR Kit (QIAGEN, Germany) were used to determine the relative expression levels of target genes. Three biological replicates were performed for each sample. \u003cem\u003eACTIN\u003c/em\u003e was selected as an internal control. Quantification of the relative changes in gene transcript levels was performed using the 2\u003csup\u003e\u0026minus;△△Ct\u003c/sup\u003e method. All primers are shown in \u003cb\u003eTable S3\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eVirus particle purification and TEM observation\u003c/h2\u003e \u003cp\u003eTo purify the virus particles, the first step is to inoculate TMV and collected 4 g leaves after 4 days. Add 0.2 mol/L PBS buffer (pH\u0026thinsp;=\u0026thinsp;7.2) mixed with 1% β-mercaptoethanol (99 mL PBS\u0026thinsp;+\u0026thinsp;1 mL β-mercaptoethanol) and ground it into a homogenate (1g tissue per 1mL buffer). The filter collects the supernatant as the crude extract. Under magnetic stirring, add 8% n-butanol (8 mL per 100 mL of solution), continue stirring for 15 minutes, and then centrifuge at 10000 rpm for 20 minutes. Collect the supernatant, add 4% NaCl and 4% PEG6000 (4 g each per 100 mL solution), stir for 1.5 hours, and centrifuge at 10,000 rpm for 15 minutes. Collect the precipitate, resuspend it in 0.01 mol/L PBS buffer (pH 7.2), and centrifuge at 8000 rpm for 5 minutes. Collect the precipitate, resuspend it in 0.01 mol/L PBS buffer (pH 7.2), and centrifuge at 8000 rpm for 5 minutes. The supernatant is the purified TMV extract, which is stored at 4\u0026deg;C.\u003c/p\u003e \u003cp\u003eMix the purified TMV particles with nanomaterials and ddH\u003csub\u003e2\u003c/sub\u003eO as the control. Allow all mixtures to interact \u003cem\u003ein vitro\u003c/em\u003e at 25\u0026deg;C for 3 hours. Observe the TMV morphology under a JEOL JEM-2100 electron microscope. Eight fields of view were analyzed for each treatment, and representative TEM images were selected. The control group was processed using the same procedure.\u003c/p\u003e \u003cp\u003eFor the CNC nanomaterial transport in the plant, the leaf samples were sampled after spraying 3 days, stem samples were sampled at 4 days after spraying 3 days, and the root samples were sampled at 7 days after spraying 3 days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eVector construction\u003c/h2\u003e \u003cp\u003eFor expression vector construction, two segments encoding \u003cem\u003ensLTP2\u003c/em\u003e (Niben101Scf07951g02011) and \u003cem\u003ensLTP2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△SP\u003c/em\u003e\u003c/sup\u003e (without the signal peptide) were designed with \u003cem\u003eXba\u003c/em\u003eI restriction sites and a 20-bp sequence overlapping with the region surrounding \u003cem\u003eXba\u003c/em\u003eI in the pART27-eGFP vector. These fragments were seamlessly cloned into the pART27-eGFP backbone, resulting in the binary expression vectors pART27-nsLTP2-eGFP and pART27-nsLTP2\u003csup\u003e△SP\u003c/sup\u003e-eGFP. Similarly, the pART27-nsLTP2\u003csup\u003e△SP\u003c/sup\u003e-7\u0026times;Myc binary expression vector was constructed using the same seamless cloning approach. To construct the silencing vector, a 200-bp optimal silencing fragment for \u003cem\u003ensLTP2\u003c/em\u003e was identified using the VIGS tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://vigs.solgenomics.net/\u003c/span\u003e\u003cspan address=\"https://vigs.solgenomics.net/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). This fragment was inserted into the pTRV2 vector at the \u003cem\u003eXba\u003c/em\u003eI and \u003cem\u003eXho\u003c/em\u003eI sites to generate the pTRV2-nsLTP2 vector. All primers are shown in \u003cb\u003eTable S3\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eN. benthamiana\u003c/b\u003e \u003cb\u003eleaves infiltration and confocal observation\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTransient expression was performed as previously described, with minor modifications. Briefly, the constructs pART27-nsLTP1-GFP, pART27-nsLTP2\u003csup\u003e△SP\u003c/sup\u003e-eGFP, pTRV1, pTRV2-nsLTP2, and P19 were transformed into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain GV3101. The \u003cem\u003eAgrobacterium\u003c/em\u003e cultures were activated with acetosyringone and prepared for infiltration. For expression vectors (pART27-based constructs), the cultures were adjusted to an OD\u003csub\u003e600\u003c/sub\u003e of 0.5. For silencing vectors (pTRV-based constructs), the cultures were adjusted to an OD\u003csub\u003e600\u003c/sub\u003e of 0.3 and co-infiltrated with P19 (OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.3) to enhance expression. The mixtures were infiltrated into the leaves of \u003cem\u003eN. benthamiana\u003c/em\u003e using a syringe without a needle.\u003c/p\u003e \u003cp\u003eFor confocal observation, \u003cem\u003eN. benthamiana\u003c/em\u003e leaves at the 6-leaf stage were infiltrated and leaf discs were isolated from the infiltrated leaves and visualized using a LSM780 confocal laser scanning microscope equipped with a 40*/1.2 water-immersion objective (Zeiss, Germany). GFP-derived fluorescence was excited a 488 nm and emission was captured with a 505- to 530 nm filter.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eSOD activity detection\u003c/h2\u003e \u003cp\u003eThe enzyme activity was determined according to the previous report \u003csup\u003e64\u003c/sup\u003e. PBS was used to extract total protein from the treated \u003cem\u003eN. benthamiana\u003c/em\u003e leaves, and enzyme activity kits were used to measure the activities SOD (at 560 nm) (Sinobest, YX-C-A500) according to the manuals, with three repetitions for each treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eDetection of SA in leaves\u003c/h2\u003e \u003cp\u003eFor SA quantification, 50 mg leaf tissue was finely ground in liquid nitrogen and extracted with 2 mL pre-cooled 80% methanol, then sealed with plastic wrap and cold-soaked at 4 \u0026deg; C overnight. Centrifuge at 4 \u0026deg; C, 5000 r/min for 10 min, take the supernatant and the residue continue to extract with 80% methanol. The supernatant was pooled by sonication two times, and the aqueous phase added 2mL petroleum ether was to decolorize 3 times, extract the aqueous phase with ethyl acetate 3 times, then blow it and add an acetic acid solution (pH\u0026thinsp;=\u0026thinsp;3.5), purify through C18 column (Agilent C18,250*4.6mm; 5\u0026micro;m), elution with methanol, collect the eluate and dry before dissolving with mobile phase to a constant volume of 1mL, shake and mix the liquid then pass through 0.22 \u0026micro;m filter membrane and be tested. The quantification of SA was determined by HPLC (Agilent 1200), the equipment settings according to previous work\u003csup\u003e65\u003c/sup\u003e. Three independent replicates were performed with each experiment containing three biological repeats.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eElemental analyses\u003c/h2\u003e \u003cp\u003eThe concentration of Zn and Si in the tobacco plants (leaf, stem, and root) were assessed via inductively coupled plasma mass spectrometry (ICP-MS; ELAN DRC II, Perkin Elmer Inc.). Specifically, dried plant samples were fully digested in 6 mL of HNO\u003csub\u003e3\u003c/sub\u003e and hydrogen peroxide (5:1) solution at 150\u0026deg;C. Then, deionized water was added to the solution until the volume reached 50 mL. The supernatants were diluted with HNO\u003csub\u003e3\u003c/sub\u003e(1%) to 100 ppb. Finally, the filtered solution was used for elemental concentration analysis via ICP-MS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic analysis\u003c/h2\u003e \u003cp\u003eHomologous proteins of nbLTP2 were retrieved from the TAIR (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.arabidopsis.org/\u003c/span\u003e\u003cspan address=\"https://www.arabidopsis.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and Phytozome (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://phytozome-next.jgi.doe.gov/\u003c/span\u003e\u003cspan address=\"https://phytozome-next.jgi.doe.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) databases. The evolutionary relationships were analyzed using MEGA X. The phylogenetic tree was constructed with the following parameters: Jones-Taylor-Thornton (JTT) substitution model, Gamma Distributed (G) with four discrete Gamma categories, partial deletion with a 70% site coverage cutoff, and Subtree-Pruning-Regrafting (SPR) method using a BioNJ initial tree. A strong branch swap filter was applied to enhance the tree\u0026rsquo;s accuracy. Based on the phylogenetic analysis, proteins that fell outside the core tree were excluded. Subsequently, an evolutionary tree for the selected species was obtained from the Timetree database and visualized.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll experiments and data presented here involved three repeats. The data were presented as means and standard deviations. The statistical analysis was performed with SPSS software (version 22.0) using Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test (\u0026lowast;0.01\u0026thinsp;\u0026lt;\u0026thinsp;\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u0026lowast;\u0026lowast;0.001\u0026thinsp;\u0026lt;\u0026thinsp;\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, \u0026lowast;\u0026lowast;\u0026lowast;\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and One-way ANOVA test (LSD\u0026rsquo;s test, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;005).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData and materials availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Professor Yule Liu (Tsinghua University, Beijing, China) for his valuable comments on improving the experiment and providing the pSPDK661 (TMV-GFP) and pTRV2 vectors.\u0026nbsp;This study was partly supported by the National Natural Science Foundation of China (31870147, X. Sun), the science and technology projects of Chongqing Company of China Tobacco Corporation (B20241NY1303 and B20241NY1310, X. Sun), and the China Scholarship Council (202306990064, X. Sun)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study represents an interdisciplinary collaboration. Specifically, J. Wang, X. Wang, C. Liu, X. Zhu, and W. Liu conducted biological experiments under the guidance of S. Wang and X. Sun. The materials synthesis and characterization were performed by S. Xiang and Y. Shen under the supervision of X. Ma and J. Huang. All authors contributed to the writing and revision of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJones RAC, Naidu RA. 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However, its limited bioavailability often necessitates excessive application, resulting in inefficiencies in production and environmental stress. In response, we propose an environmentally friendly and sustainable approach to enhance the utilization of Zn\u003csup\u003e2+\u003c/sup\u003e. We developed CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e by embedding Zn\u003csup\u003e2+\u003c/sup\u003e into the polydopamine (PDA) coating of cellulose nanocrystals (CNCs). Leveraging the high cell permeability of CNCs, this material increased the transport capacity of Zn\u003csup\u003e2+\u003c/sup\u003e in plants and demonstrated the ability to inactivate viral particles \u003cem\u003ein vitro\u003c/em\u003e. Moreover, CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e showed a superior induction of resistance while reducing Zn\u003csup\u003e2+\u003c/sup\u003e content, specifically by reprogramming the expression and localization of the resistance-related non-specific lipid transfer protein 2 (nsLTP2), which enhanced the salicylic acid (SA) signaling pathway in plants. Furthermore, the high conservation of nsLTP2 in flowering plants increases the potential application range of CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e. Importantly, CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e represents the most effective Zn\u003csup\u003e2+\u003c/sup\u003e-based antiviral nanomaterial to date, achieving its effects at the lowest reported Zn\u003csup\u003e2+\u003c/sup\u003e concentration. Overall, our results highlight that CNC@PDA@Zn\u003csup\u003e2+\u003c/sup\u003e can more effectively upregulate the conserved nsLTP2, thereby inducing viral defense responses via the SA pathway. This strategy not only improves the operation and utilization rate of Zn\u003csup\u003e2+\u003c/sup\u003e but also reduces its environmental residues, laying a theoretical foundation for the development of antivirus defense.\u003c/p\u003e","manuscriptTitle":"The high-permeability cellulose nanocrystals carrier facilitates zinc utilization and enhances nsLTP2-mediated plant immunity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-10 08:33:26","doi":"10.21203/rs.3.rs-5791872/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c6aa5228-a23a-4a3d-9811-9985829f6f7b","owner":[],"postedDate":"January 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":42592791,"name":"Biological sciences/Plant sciences/Plant stress responses/Biotic"},{"id":42592792,"name":"Earth and environmental sciences/Environmental sciences/Environmental impact"}],"tags":[],"updatedAt":"2025-01-15T11:30:40+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-10 08:33:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5791872","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5791872","identity":"rs-5791872","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}