Polyglutamate-loaded chitosan nanogels reprogram plant metabolism for increased growth and viral resistance

Domain: biology
Published: 2026-03-27
Editorial outcome: minor_revision

Abstract
Asparagine synthetase B (AS-B) is essential for nitrogen metabolism, but its broader physiological functions remain poorly understood. Here we show that the evolutionarily conserved Nicotiana benthamiana NbAS-B confers expression-dependent antiviral resistance and promotes plant growth. Multi-omics analyses indicate that NbAS-B-mediated antiviral immunity relies on glutamate-induced activation of Ca²⁺ signaling through the receptor GLR3.3, whereas its growth-promoting effect results from photosynthetic reprogramming. Building on these insights, we develop polyglutamate-loaded chitosan nanogels (PGANPs) to artificially manipulate this pathway. These nanogels efficiently enter plant tissues and enable sustained in situ release of glutamate, thereby mimicking and amplifying NbAS-B signaling outputs. PGANPs provide long-lasting systemic antiviral immunity while concurrently enhancing plant growth, without incurring metabolic costs. Our work identifies NbAS-B as a dual-function regulator linking metabolic status to immune activation and establishes PGANPs as an eco-friendly, controllable, and durable nanobiotechnology for managing viral diseases in crops.

Introduction
Tobacco mosaic virus (TMV) was the first plant virus to be discovered, isolated and visualized under an electron microscope 1, marking a significant milestone in virology. TMV poses a considerable challenge due to its broad host range and the difficulty of its management. Under natural conditions, TMV inflicts damage to on key Solanaceae crops such as tomatoes, potatoes and peppers, as well as a variety of other vegetables and numerous weeds 2. This has led to increased attention on tobacco mosaic disease, driving greater awareness and efforts toward effective prevention and control strategies. Current TMV management primarily follows a “prevention-first, comprehensive management”. However, post-infection treatments are largely reliant on chemical agents, raising concerns over escalating drug resistance and environmental pollution. Consequently, there is an urgent need for the development of novel antiviral agents to enhance TMV control strategi es 3.

Plant virus diseases have consistently been regarded as the cancer of plants, with no effective drug therapy available. And the most efficacious approach currently is to undertake disease resistance breeding by employing the plant resistance strategy on the basis of comprehending the mechanism of plant disease resistance. The salicylic acid (SAT) signaling pathway serves as a crucial signaling pathway for the antiviral activity of plants. The infection of plant viruses can cause the accumulation of SA expression in plants, triggering a series of antiviral immune responses 4, 5. Previous research conducted in our laboratory has discovered that coat protein-interacting protein (IP-L), a negative regulator of TMV infection, is capable of regulating virus infection by affecting photosynthesis 6. And further investigation reveals that IP-L can interact with and stabilize asparagine synthetase B (NbAS-B) by inhibiting the ubiquitin-26S proteasome pathway, thereby synergistically affecting the biosynthesis of asparagine which inhibits TMV infection by activating the SA signaling pathway7. These studies have confirmed that NbAS-B, functioning as an asparagine synthetase, catalyzes the biosynthesis of asparagine and glutamate.

Asparagine synthetase (AS) is a catalytic enzyme that primarily utilizes energy derived from ATP hydrolysis and employs glutamine as a nitrogen source to catalyze the conversion of aspartate to asparagine, concomitantly producing glutamate. This process involves NH₃ as a signaling molecule in regulation8, 9, 10. AS is distributed in animals, plants, and microorganisms, with its isoform AS-B being mainly present in plants11, 12. Current research on AS has primarily focused on nitrogen transport during seed germination and growth stages13, 14, 15, 16, as well as nitrogen remobilization during plant senescence17, 18, 19.

Glutamate (Glu), a prevalent amino acid in plants, plays a pivotal role in amino acid metabolism, coordinating essential physiological metabolic functions such as ammonia assimilation or disdimilation and amino acid transmination in plants. It serves as a framework for the synthesis of other stress-resistant amino acids in plants 20. Glu not only serves as a crucial metabolite and nutrient, but also functions as a signaling molecule in the physiological metabolism of plants. It gets involved in various growth and development processes such as seed germination, root growth, pollen tube elongation and photosynthesis, as well as pathogen resistance and non-biological stress response (such as salt, cold, heat and drought)21, 22, 23, 24, 25. Additionally, recent research has revealed that glutamic acid plays a role in the synthesis of SA: iso-branched acid and glutamic acid form an unstable iso-branched acid-glutamic acid adduct (ISC-9-Glu), which can spontaneously decompose or be catalyzed by EPS1 to produce SA 26, 27. This finding suggests a potential avenue for further investigation into the disease resistance function of asparagine synthetase.

Plant glutamate receptor-like proteins (GLRs) constitute a family of ligand-gated ion channels structurally related to mammalian ionotropic glutamate receptors (iGluRs)28. These receptors function predominantly as Ca²⁺-permeable cation channels and are integral to the perception of internal and external cues, enabling the coordination of developmental programs and stress adaptation28, 29. Glu acts as an important plant signaling molecule; its binding to GLRs initiates a downstream signaling cascade in which the induction of salicylic acid (SA) biosynthesis serves as a central event in establishing disease resistance30. SA, a major defense hormone, potentiates immune outputs through the transcriptional activation of pathogenesis-related (PR) genes31. Thus, GLRs form a key signaling bridge that translates external stress perception into SA-dependent immunity via a sequential process involving Glu perception, Ca²⁺ signature generation, and hormonal reprogramming32.

Functional evidence supports this model: plants harboring gain-of-function GLR3.3 alleles display amplified systemic Ca²⁺ waves in response to Glu33. Ligand-activated GLR3.3 mediates prolonged Ca²⁺ influx, leading to the activation of calcium-dependent protein kinases CPK5/6. These CPKs subsequently phosphorylate the promoter of isochorismate synthase 1 (ICS1), a rate-limiting enzyme in SA biosynthesis, elevating its transcriptional activity more than threefold and increasing leaf SA accumulation by 2.5-4-fold. In a distinct regulatory mechanism, GLR2.9a/b modulates nuclear Ca²⁺ concentrations to alter the nuclear localization of the transcription factor WRKY40, thereby synergistically enhancing the expression of ICS1 and PR134.

In this study, we constructed a multi-omics network to elucidate the potential mechanisms by which NbAS-B regulates nitrogen allocation, finely tunes plant growth, and affects SA signaling, thereby contributing to plant disease resistance. By integrating the chemical properties of Glu, a downstream product of NbAS-B, we developed targeted PGANP nanomaterials to modulate this pathway. These nanomaterials have the potential to simultaneously regulate plant growth and balance disease resistance, while also reducing the pollution and resistance issues caused by chemical agents in agricultural production, providing a theoretical foundation for the scientific prevention and control of plant viral diseases.

Results
NbAS-B enhances plant resistance to viruses while promotes plant growth

In our previous study, virus-induced gene silencing (VIGS) of NbAS-B promotes TMV-GFP infection, whereas its transient overexpression suppresses viral infection. To further investigate the physiological significance, we generated NbAS-B loss-of-function mutants using CRISPR/Cas9. Two independent homozygous lines harbored deletion mutations at the gRNA1 target site, resulting in two distinct mutation types—a missense mutation and a frameshift mutation—which disrupted the conserved PAM domain (Supplementary Figure 1). Upon TMV-GFP inoculation, both nbas-b lines displayed markedly accelerated viral spread: by 5 dpi, GFP fluorescence nearly saturated the second systemic leaf, whereas wild-type (WT) plants showed substantially lower infection levels (Figure 1a). qPCR analysis confirmed more than a twofold increase in viral RNA accumulation in both mutant lines (Figure 1c). To complement these findings, we established NbAS-B-overexpressing (NbAS-B-OE) transgenic lines driven by the CaMV 35S promoter. Unexpectedly, initial screening revealed that the antiviral phenotype was expression-dependent: higher NbAS-B abundance correlated with stronger inhibition of TMV-GFP (Figure 1a). We therefore selected multiple independent overexpression lines (OE#1, OE#5, OE#4, OE#27) exhibiting a gradient of NbAS-B transcript and protein levels (Supplementary Figure 1). Consistently, TMV-GFP inoculation showed a proportional reduction in viral spread in plants with higher NbAS-B expression (Supplementary Figure 2), and qPCR analysis confirmed a corresponding decrease in systemic viral RNA accumulation at 5 dpi (Supplementary Figure 2). Correlation analysis demonstrated a significant negative relationship between NbAS-B expression and TMV-GFP abundance (Supplementary Figure 2). To determine whether this regulatory role extends to other RNA viruses, we challenged both nbas-b#1 and NbAS-B-OE27 plants with PVX-GFP, TuMV-GFP, and TRV-GFP. NbAS-B knockout consistently enhanced infection by all three viruses, whereas its overexpression markedly suppressed their accumulation (Supplementary Figure 2). Collectively, these results identify NbAS-B as a dose-dependent positive regulator of broad-spectrum antiviral immunity in N. benthamiana.

Previous studies have suggested that AS-B also promotes plant growth. To assess the effect of NbAS-B overexpression and knockout on plant development, we analyzed growth phenotypes at multiple stages—seedling, vegetative, and mature phases. Seedling growth assays on MS medium using physiologically uniform seeds revealed a significant increase in root length in NbAS-B-overexpressing lines, which positively correlated with NbAS-B expression levels (Figure 1b, Supplementary Figure 3). Further analysis during vegetative and mature stages showed that overexpression plants exhibited enhanced growth compared to WT, as indicated by increased plant height and leaf width (Figure 1d–f). This growth-promoting effect was particularly evident in fresh and dry weight measurements at the mature stage (Figure 1g–h), confirming that NbAS-B overexpression modulates plant growth. Conversely, nbas-b knockout lines exhibited significantly suppressed growth across developmental stages compared to controls. Collectively, these results indicate that NbAS-B plays a pivotal role in promoting plant growth throughout various developmental phases, likely via enhanced nitrogen allocation and reutilization.

Integrated transcriptome and metabolome reveal NbAS-B-mediated resistance and growth balance

To elucidate the potential regulatory pathways underlying the NbAS-B–mediated balance between disease resistance and growth, we first performed transcriptome sequencing of NbAS-B–overexpressing and knockout lines. Reference transcriptome analysis was conducted using two overexpressing lines (NbAS-B-OE#5 and NbAS-B-OE#27) and two knockout lines (nbas-b#1 and nbas-b#2). Compared with the wild type, 146 genes were up-regulated and 276 genes were down-regulated in NbAS-B-OE#5, whereas 366 genes were up-regulated and 406 genes were down-regulated in NbAS-B-OE#27 (Figure. 2a-b), indicating that higher NbAS-B expression levels were associated with a greater number of differentially expressed genes (DEGs). Notably, only 201 DEGs (23.51%) overlapped between the two overexpressing lines, and the number of DEGs in NbAS-B-OE#27 was 28.53% higher than that in NbAS-B-OE#5 (Figure. 2c). This suggests that approximately one-quarter of the DEGs were induced by NbAS-B overexpression and that higher expression levels triggered more extensive changes in gene expression. These findings imply that differential expression of NbAS-B across tissues or under stress conditions may enable distinct transcriptional responses to environmental stimuli, such as the up-regulation of NbAS-B observed upon TMV infection. In contrast, We identified a 5-bp deletion in nbas-b#1, causing a frameshift and a predicted complete loss of function, and a 3-bp deletion in nbas-b#2, expected to lead to a partial loss of function via a missense mutation (Supplementary Figure 1). RNA-seq profiling revealed starkly contrasting global expression changes: nbas-b#2 significantly altered only 26 transcripts, whereas nbas-b#1 dysregulated 518 genes (Figure. 2h-j). Subsequent KEGG pathway analysis revealed that NbAS-B overexpression primarily regulated pathways related to photosynthesis, starch and sucrose metabolism, and carbon fixation in photosynthetic organisms. Additionally, pathways associated with amino acid metabolism and plant defense responses were significantly enriched. It is noteworthy that the induction of these pathways exhibited a dose-dependent manner—higher NbAS-B expression levels correlated with increased numbers of DEGs involved (Figure. 2d-e). In contrast, nbasb knockout lines were mainly enriched in pathways such as plant–pathogen interaction, plant hormone signal transduction, and amino acid metabolism (Figure. 2h-i). Given the dual role of NbAS-B in promoting growth and enhancing resistance, we found that growth-related DEGs were predominantly enriched in photosynthesis and starch/polysaccharide metabolism pathways (Figure. 2d), whereas defense-related DEGs were mainly concentrated in Ca2+ signaling and MAPK signaling pathways (Figure. 2d-e). The extent of alterations in these pathways also positively correlated with NbAS-B expression levels (Figure. 2f-g). To validate these findings, qPCR analysis confirmed that genes associated with the Ca2+ pathway (CNGC family), photosynthetic electron transport chain (Pet family), jasmonic acid signaling (JAZ family), and abscisic acid signaling (SnRK2 family) were significantly up-regulated in transgenic plants (Supplementary Figure 4). KEGG pathway enrichment analysis of differentially expressed genes in nbas-b#1 revealed that its molecular phenotype is characterized by systemic defense activation and metabolic reprogramming (Figure. 2k). Significantly enriched pathways included "Plant-pathogen interaction" and "Phenylpropanoid biosynthesis". Concurrently, GO term analysis demonstrated specific enrichment for cell wall-associated biological processes, with the most prominent terms being "xyloglucan metabolic process", "cell wall organization", and "cell wall polysaccharide metabolic process" (Figure. 2l). These results strongly suggest that the most direct and primary consequence of NbAS-B loss of function is the disruption of cell wall synthesis and assembly. By integrating transcriptomic profiles from NbAS-B overexpression and CRISPR/Cas9 plants, we established a coherent regulatory framework underlying NbAS-B function. The DEGs were predominantly enriched in pathways associated with “Plant-pathogen interaction”，“MAPK signaling” and “Phenylpropanoid biosynthesis”, This indicates extensive reprogramming of metabolic and signaling pathways. Notably, calcium ion-associated genes constituted a major component of this regulatory network, as evidenced by substantial alterations in numerous calcium-related genes (Figure. 2m). The widespread remodeling of calcium-responsive transcription strongly suggests that NbAS-B may function as a key upstream regulator in calcium signaling–mediated immune activation.These results provide new insights into the transcriptional regulatory mechanisms by which NbAS-B coordinates growth and disease resistance.

As mentioned above, NbAS-B overexpression significantly enriched DEGs related to amino acid metabolism pathways. To further investigate whether NbAS-B–induced balance between disease resistance and growth depends on amino acid metabolism, we performed targeted amino acid metabolomics analysis. Interestingly, the two overexpressing lines (NbAS-B-OE#5 and NbAS-B-OE#27) exhibited high correlation and collectively showed differential changes in 56 amino acid–related metabolites. Among these, 50 metabolites were significantly decreased and 6 were significantly increased (Figure. 2n), indicating that differences in NbAS-B expression levels did not alter the overall pattern of amino acid metabolite reprogramming induced by its overexpression. Classification of these differential metabolites revealed that the vast majority were organic acids and derivatives (84.31%), followed by organoheterocyclic compounds (7.84%) (Figure. 2o). It is particularly noteworthy that the organoheterocyclic compounds were enriched with multiple nitrogen-rich amino acids, which may play key roles in promoting the redistribution of nitrogen within the plant. Subsequent variable importance in projection (VIP) analysis identified glutamine, serine, alanine, and glutamate as critical contributors to this metabolic shift (Figure. 2p). Heatmap analysis showed that glutamate levels were significantly increased in NbAS-B transgenic plants, while its catalytic substrate glutamine was correspondingly decreased, suggesting a forward shift in the reaction (Figure. 2q-s). Especially noteworthy is that glutamine and glutamate serve as the direct catalytic precursor and product of NbAS-B, respectively. This finding not only confirms the central role of this enzyme in the metabolic process but also highlights the importance of glutamate in the NbAS-B–mediated regulatory network.

NbAS-B-enhanced growth promotion and resistance induction partly depend on the increase of glutamate

NbAS-B primarily participates in the synthesis of asparagine and glutamate in plants, with NbAS-B overexpressing plants exhibiting increased levels of these two metabolites. We hypothesize that asparagine and glutamate contribute to the promotion of growth, particularly glutamate, which plays a role in enhancing virus resistance through NbAS-B. To test this hypothesis, we first conducted antiviral experiments on N. benthamiana plants by externally applying Glu and Asn (Supplementary Figure 5), and then treated the plants with a glutamate inhibitor 4,5-Difluoro-2-methoxy-N-(4-phenylthiazol-2-yl) benzamide (DFMTI) and an asparagine inhibitor (L-asparaginase) to assess the inhibition efficiency (Figure. 3a, Supplementary Figure 6) before being inoculated with TMV-GFP. Following L-asparaginase treatment, there was no significant difference in viral content between wild-type and transgenic plants (Figure. 3b-c). However, plants treated with DFMTI exhibited significantly higher viral loads compared to those treated with L-asparaginase (Figure. 3b-c), underscoring the crucial role of glutamate in antiviral defense. Given that NbAS-B overexpression did not alter the profile of differential amino acid changes, we selected NbAS-B-OE#27 for further analysis. DFMTI treatment reduced the antiviral activity conferred by NbAS-B overexpression, whereas L-asparaginase treatment did not (Figure 3b). Notably, DFMTI treatment could not fully offset the enhanced resistance conferred by NbAS-B overexpression, indicating that the antiviral activity was partially dependent on increased glutamate levels (Figure 3c). Further validation of glutamate's antiviral role was conducted through external application of glutamate. We found that glutamate application significantly enhanced N. benthamiana's resistance to TMV infection, with resistance closely correlated to glutamate concentration. The strongest inhibition, at 75%, was observed at a glutamate concentration of 0.26 mg/mL (Figure. 3d-e). These results suggest that NbAS-B enhances resistance, in part, by mediating glutamate synthesis.

Glutamate is known to activate plant glutamate receptors (GLRs) and subsequently trigger Ca2+-dependent defense signaling 35 26, 27. Given that NbAS-B overexpression elevates glutamate levels and has previously been linked to SA-mediated immunity, we hypothesized that NbAS-B–induced glutamate accumulation contributes directly to antiviral resistance. Consistent with this model, NbAS-B-OE#27 plants exhibited markedly higher transcript levels of the SA-responsive marker genes PR1 and ICS1 compared with WT plants (Figure. 3f-g). Similarly, the content changes of Glu and SA in transgenic plants and wild-type plants under virus-inoculated and non-inoculated conditions were detected, the results showed that the Glu content in NbAS-B-OE#27 and NbAS-B-OE#5 plants was higher than that in WT; after inoculation with TMV-GFP, the levels of Glu and SA increased significantly (Figure. 3h-i). Additionally, in the SA-deficient mutant NahG, exogenous application of 0.26 mg/mL glutamate completely failed to induce TMV-GFP resistance (Figure. 3j-k), further confirming that glutamate-induced resistance is entirely dependent on the SA signaling pathway. Together, these data indicate that NbAS-B–derived glutamate activates a Ca2+-dependent immune pathway—likely through GLR-mediated signaling—which subsequently engages the SA pathway to establish effective antiviral defense.

NbAS-B promotes plant growth by enhancing organic matter accumulation and plant photosynthesis

As previously established, NbAS-B-OE exhibits an expression-dependent growth-promoting effect, with transcriptome sequencing highlighting the critical role of the photosynthesis pathway in this expression dependence. It is reasonable to infer that the growth enhancement induced by NbAS-B is due to reprogramming of the photosynthesis pathway, driven by its differential expression pattern. To explore this hypothesis, We evaluated the photosynthetic parameters of the two overexpression lines and the knockout mutant lines. Chlorophyll content was the first parameter examined. While no significant differences were observed at the seedling stage, chlorophyll levels, particularly chlorophyll b, were markedly higher in NbAS-B-OE plants compared to the control at the flowering and mature stages. This induction was expression-dependent, with NbAS-B-OE#27 exhibiting significantly higher chlorophyll content than NbAS-B-OE#5 at the flowering stage (Figure. 4a-c). In addition to chlorophyll levels, other key photosynthetic parameters, including net photosynthetic rate, stomatal conductance, intercellular CO2 concentration, and transpiration rate, were significantly elevated in NbAS-B-OE plants during both the growth and mature stages compared to WT plants. Most of these parameters also demonstrated expression dependence, with higher NbAS-B expression correlating with stronger photosynthetic performance (Figure. 4d-g). These results suggest that the growth-promoting effect of NbAS-B overexpression may be attributed to its ability to induce reprogramming of the photosynthesis pathway, enhancing photosynthetic efficiency and ultimately supporting increased plant growth (Figure. 4h-i). In the nbas-b mutants, we similarly detected chlorophyll content and photosynthesis-related parameters. The knockout lines exhibited significantly lower chlorophyll levels and markedly reduced photosynthetic parameters compared to the control group, further confirming NbAS-B's involvement in the plant photosynthetic process (Supplementary Figure 7).

In summary, we speculate that the reprogramming of the photosynthetic pathway induced by NbAS-B may also be partially attributed to the glutamate it synthesizes, as glutamate metabolism occurs within chloroplasts and is linked to both the structure and function of these organelles. Furthermore, the glutamate metabolic pathway is closely associated with the development of photosynthetic organs, leaf functionality, and the dynamic nitrogen and carbon metabolism in chloroplasts. It is worth noting that we suspect the dual role of NbAS-B in promoting growth and enhancing resistance is evolutionarily conserved. The AS-B gene was identified in the unicellular eukaryotic green alga Chlamydomonas reinhardtii, followed by its appearance in the semi-terrestrial alga Klebsormidium nitens and the terrestrial moss Marchantia polymorpha. This gene has since been retained in basal angiosperms Amborella trichopoda as well as dicots and monocots, suggesting that AS-B's functions in promoting growth and increasing resistance are highly conserved across plant lineages (Supplementary Figure 8).

Polyglutamate (PGA) showed enhanced anti-TMV effect compared to glutamate

As mentioned previously, the dual growth-promoting and resistance-enhancing effects of NbAS-B are likely conserved, and both functions are linked to the glutamate it produces. A key question, however, is how this discovery can be leveraged to support sustainable agricultural development. With this goal in mind, we explored the potential of glutamate as a tool for crop protection. Our earlier findings established the importance of glutamate concentration in TMV resistance. Polyglutamic acid (PGA), which has excellent biocompatibility and biodegradability, is widely used in the food industry, medicine, and biomaterials. We therefore investigated whether PGA could offer similar, or enhanced, resistance to TMV-GFP infection compared to glutamate. N. benthamiana plants were treated with varying concentrations of glutamate and PGA for three days, followed by inoculation with TMV-GFP via mechanical friction. At 7 dpi, systemic leaf infection was assessed under UV light. The results demonstrated a significantly enhanced inhibitory effect on TMV-GFP infection in plants treated with different concentrations of glutamate compared to the positive control (CHI). Consistent with previous findings, the most pronounced effect was observed in plants treated with 0.26 mg/mL glutamate (Supplementary Figure 9). Notably, at the same concentration, PGA exhibited an even stronger inhibitory effect, likely due to the higher number of glutamate monomers present in the PGA polymer. These observations were further supported by qPCR analysis (Supplementary Figure 9), which confirmed the enhanced effect of polyglutamic acid in suppressing TMV infection. These results suggest that PGA could serve as an effective agent for improving plant resistance, offering potential applications in sustainable agriculture.

Synthesized PGANPs have a stronger ability to induce N. benthamiana resistance to TMV

Despite the promising antiviral efficacy of PGA, its polyanionic properties significantly limit its ability to enter plant cells, restricting its broader potential in enhancing antiviral activity. To address this limitation, we aimed to develop a more efficient delivery system by selecting a cationic carrier for PGA. This approach would improve cell permeability and ensure environmental compatibility and sustainability. CS, a biopolymer with strong cationic properties, was selected to encapsulate PGA, taking advantage of their electrostatic interactions (Figure. 5a). We synthesized six different concentrations of chitosan-loaded PGA nanocomposites (PGANPs) to enhance cellular uptake.

Transmission electron microscopy (TEM) analysis of the PGANPs revealed spherical particles with smooth surfaces across all concentrations, though particle size varied (Figure. 5h, Supplementary Figure 10). The measured sizes were 56.44 ± 2.12 nm, 72.08 ± 2.35 nm, 63.64 ± 2.25 nm, 59.26 ± 2.08 nm, 47.44 ± 2.16 nm, and 30.83 ± 2.42 nm, respectively (Figure. 5b), which fall within the nanoscale range. As expected, PGA's polyanionic nature allowed it to electrostatically interact with the positively charged CS. This interaction significantly influenced the Zeta potential of the PGANPs, with values of +41.07 ± 0.10 mV, +29.32 ± 0.20 mV, +19.65 ± 0.10 mV, and +5.32 ± 0.15 mV for different concentrations (Figure. 5c), indicating a proportional increase in negative charge with higher PGA concentrations. This trend suggests enhanced binding between PGA and CS as more positive charges on CS were neutralized36. To confirm the successful interaction between PGA and CS, Fourier-transform infrared (FTIR) analysis was performed37. The presence of an amide bond (υ-NH-CO) at 1645 cm-1 (Figure. 5d) confirmed that the interaction between PGA and CS occurred, forming a stable compound38. X-ray diffraction (XRD) analysis further supported these findings, showing a distinct peak for CS at 20°, consistent with its crystalline structure. However, both PGA and PGANPs lacked significant diffraction peaks, indicating the formation of a bonded compound with an ionic crystal structure (Figure. 5g). The presence of a NaCl impurity peak in PGANPs was attributed to insufficient washing during preparation, which nonetheless confirmed the successful loading of PGA 39.

X-ray photoelectron spectroscopy (XPS) analysis (Figure. 5e) and organic element analysis (Supplementary Table 2) estimated a 65% loading efficiency for PGA across different PGANP concentrations, highlighting their significant induction effect even at low concentrations40. Thermogravimetric analysis (TGA) further revealed variations in thermal stability between PGANP concentrations, with degradation occurring at 200-300°C due to water evaporation from the material. Residual degradation at 800°C decreased with increasing PGA loading, suggesting that PGANPs possess inherent thermal stability (Figure. 5f).

To investigate whether PGANPs have a stronger ability to induce plant resistance to TMV, we applied 10 mL of the PGANPs to the experimental plants for three consecutive days. TMV-GFP was then inoculated on the plants via friction. Systemic leaves were collected on the 7 dpi, and TMV content was assessed. The results revealed that plants treated with 44 μg/mL of PGANPs exhibited a significantly reduced green fluorescence signal under UV light compared to those treated with CHI or PGA alone (Figure. 6a). qPCR analysis of TMV-GFP expression levels in the systemic leaves confirmed that plants treated with 44 μg/mL PGANPs had significantly lower viral expression levels than the control groups treated with PGA or CHI (Figure. 6b). To further validate these observations, we conducted repeated experiments with the most effective concentrations of PGANP and PGA. Consistently, 44 μg/mL PGANP demonstrated the strongest antiviral effect. These results indicate that PGANP significantly enhances the resistance of tobacco plants to TMV-GFP, outperforming both PGA and Glu treatments. This suggests that PGANP’s higher efficacy may be attributed to its enhanced ability to penetrate plant cells and activate related resistance pathways. For subsequent experiments, we selected PGANPs at a concentration of 44 μg/mL and a particle size of 47.44 ± 2.157 nm. The results also suggest that the combined inhibitory effect of PGA and CS is not merely additive. This may be explained by the improved cellular uptake of nanomaterialized PGA, resulting in a stronger induction effect. Given that glutamate-induced resistance is concentration-dependent, the higher intracellular levels of PGA may have contributed to the enhanced antiviral activity observed in PGANP-treated plants.

NbGLR3.3 is required for PGANP-induced antiviral signaling

As indicated above, PGANP likely exhibits a resistance-inducing effect analogous to that of Glu. Further investigation revealed that PGANP treatment elevates endogenous Glu levels, thereby promoting salicylic acid (SA) accumulation and ultimately enhancing plant-induced resistance (Figure. 6c-d). Previous studies have shown that Glu activates SA signaling through binding to glutamate receptors (GLRs), a process that triggers calcium influx and, via calmodulin, initiates downstream SA signaling41. Notably, GLR3.3 plays a critical role in this calcium signal transduction42, 43. However, whether exogenous application of PGANP activates calcium flux to induce antiviral immunity remains unclear, and it is also unknown if PGANP enhances SA signaling responses by strengthening its binding to GLRs. To address this, we generated knockout mutants of the glutamate receptor gene nbglr3.3. In these mutant lines, exogenous application of PGANP failed to confer resistance against TMV (Figure. 6e-f). Moreover, the expression levels of SA signaling-related defense genes NPR1 and PR1 were significantly downregulated in the nbglr3.3 mutant (Figure. 6g-i). These results demonstrate that the enhanced TMV resistance conferred by PGANP is dependent on GLR3.3. We therefore conclude that PGANP modulates the interaction between glutamate and the GLR3.3 receptor, thereby influencing SA levels and ultimately activating the plant immune defense response. Additionally, we examined the effects of PGANP on the expression of genes associated with the abscisic acid (ABA) and ethylene (ET) pathways (Supplementary Figure 11), but no significant correlations with these pathways were observed. Collectively, these findings indicate that PGANP enhances systemic resistance by modulating the binding capacity to downstream GLR receptors, leading to increased SA accumulation-a phenomenon further validated in the salicylic acid-deficient mutant NahG (Supplementary Figure 12).

PGANP Nanoparticles Enhance Plant Disease Resistance against TMV through Stomatal Uptake and Glutamate-Mediated Induction

To facilitate cellular uptake, PGANP was designed to carry a positive charge, enhancing its entry into plant cells. To visualize its behavior, the fluorescent probe 7-amino-4-methylcoumarin (AMC) was covalently coupled to glutamate, PGA, and PGANP via amidation, generating Glu-AMC, PGA-AMC, and PGANP-AMC (Supplementary Figure 13). These compounds were applied exogenously to plants for three consecutive days, and fluorescence was monitored using confocal laser scanning microscopy (excitation: 350 nm; emission: 430-450 nm). PGANP-AMC exhibited the strongest fluorescence at the stomata, followed by PGA-AMC, whereas Glu-AMC fluorescence was minimal (Figure 7a). Similarly, protoplasts isolated one day after treatment displayed markedly higher intracellular fluorescence in the PGANP group, indicating superior cellular uptake compared with PGA (Figure 7b). qPCR analysis revealed that PGANP treatment significantly upregulated the expression of defense-related genes NbPR1 and NbNPR1 within 24 hours, whereas glutamate treatment triggered a more rapid transcriptional response within 0–6 hours (Figure 7c-d). Consistently, endogenous glutamate levels in PGANP-treated plants was significantly elevated within 24 hours (Figure 7e), suggesting that PGANP-mediated antiviral effects are glutamate-dependent. Supporting this, in vitro incubation of PGANP with total plant protein resulted in a time-dependent increase in glutamate, becoming significant after 12 hours (Figure 7f), confirming that plant proteases can degrade PGANP into bioactive glutamate. Collectively, these results indicate that PGANP nanoparticles primarily enter plant cells via stomata, are enzymatically converted to glutamate, and subsequently activate systemic defense responses against TMV.

PGANPs ensure high foliar persistence while supporting plant health

Plant virus infections are often unpredictable, and ensuring a high, steady-state presence of PGANPs on plant leaves is crucial for sustained protection against potential viral invasions. CS, known for its ability to maintain sustained release kinetics, was combined with PGA to synthesize PGANPs, with the goal of providing long-lasting antiviral effects (Figure. 8a). To verify whether these nanocomposites possess sustained release properties, we loaded the fluorescent group 7-amino-4-methylcoumarin onto the PGA surface via amidation and measured the fluorescence at excitation and emission wavelengths of 351 nm and 430 nm, respectively. We generated a standard curve using four concentrations (1, 0.5, 0.25, and 0.125 mg/mL), obtaining the equation Y = 688.8X + 4.609 (R² = 0.9993). This curve allowed us to calculate the cumulative release of PGA from PGANPs over time. The results demonstrated a gradual increase in cumulative fluorescence from 0 to 72 hours, with the release not reaching 100% even at 72 hours (Figure. 8c), indicating a sustained release over time. To further assess the biological relevance of this sustained release, we analyzed the expression of one key resistance-related genes PR1 in N. benthamiana leaves treated with PGANPs. The expression of PR1 in the PGANP-treated group showed an initial increase, peaking at 24 hours, followed by a gradual decline, although the expression at 72 hours remained significantly higher than in other treatments. This expression pattern closely mirrored the release kinetics of PGA from PGANPs, underscoring a direct correlation between PGA release and PR1 activation. In comparison, PGA treatment alone induced PR1 expression until 48 hours, after which it declined, while CS sustained the induction of PR1 but at a lower level than PGANPs. (Supplementary Figure 14). These findings indicate that PGANPs offer a sustained release mechanism that can continuously activate defense pathways, presenting significant potential for broad agricultural applications, especially in the management of unpredictable viral infections.

Although PGANPs demonstrate strong persistence on the leaf surface, environmental challenges such as rain pose a significant threat to their long-term retention. To address this issue, the adhesion ability of PGANPs on leaves was evaluated by simulating rainwater scouring. A watering can, positioned 0.2 meters above the plants, delivered water at a rate of 100 mL/min to mimic the effects of rainfall. The retention of PGANPs on the leaf surface was analyzed after 1, 3, and 5 wash cycles with tap water. The results revealed that PGANPs exhibited significantly higher retention on the leaf surface compared to PGA alone (Figure. 8b-d). This enhanced retention can be attributed to the properties of CS, an environmentally friendly material with abundant positive charges on its surface. The electrostatic interactions between the positively charged CS and the negatively charged PGA create strong hydrogen bonds between carboxyl (-COOH) and hydroxyl (-OH) groups on the leaf surface. These interactions not only facilitate greater entry of nanomaterials into plant cells but also contribute to improved adhesion to the leaf surface, ensuring longer-lasting effects under environmental stressors like rain.

The adhesion and retention properties of PGANPs provide a promising approach to enhance the efficacy of pesticide applications. By improving the leaf surface adherence of PGANPs, it is possible to reduce the frequency and amount of pesticide use, thus promoting a more efficient and sustainable agricultural practice. This novel strategy aligns with the goal of reducing pesticide usage while maximizing effectiveness, offering a potential solution to environmental and economic challenges in crop protection.

The notable persistence of PGANPs on the leaf surface raises concerns about potential residue accumulation, necessitating a closer examination of its safety. Despite its classification as environmentally friendly and sustainable, its prolonged presence on plant surfaces warrants an investigation into its broader effects. To address this, we assessed the impact of PGANP application on the growth and development of N. benthamiana plants. The results indicated that PGANPs not only preserved the growth-promoting effects observed from the elevated glutamate content induced by NbAS-B overexpression but also exhibited a general growth-enhancing effect on the N. benthamiana plants themselves (Figure. 8e-j). Furthermore, we evaluated the disease resistance and growth performance of plants inoculated with TMV-GFP over an 8-21dpi post-inoculation period. The results demonstrated that the induced resistance effect persisted for two to three weeks, with no significant difference in viral infection ultimately observed compared to the control group (Supplementary Figure 15).This growth-promoting action is likely linked to nitrogen redistribution and the reprogramming of photosynthetic pathways20, 44. Previous studies have shown that nitrogen metabolism and photosynthesis are closely associated with glutamate production, both of which play critical roles in plant growth and development. Therefore, the application of PGANPs seems to benefit plants by boosting both resistance and growth, potentially through mechanisms involving the dynamic regulation of key metabolic processes. In conclusion, PGANPs stand out as a highly efficient and environmentally friendly resistance inducer with dual benefits: they not only enhance the plant's innate resistance mechanisms but also promote overall growth and development. This dual functionality positions PGANPs as a promising tool for sustainable agricultural practices, with the potential to improve crop resilience and productivity while minimizing environmental impact. Based on the findings of this study, we propose a model in which NbAS-B–derived Glu exerts dual functions in plant physiology. Under normal conditions, Glu enhances photosynthetic efficiency, thereby promoting plant growth and development. During viral infection, NbAS-B elevates Glu levels in an expression-dependent manner, which activates Ca2+ signaling via the glutamate receptor NbGLR3.3, ultimately triggering systemic activation of the SA defense pathway. To translate these insights into practical applications, we developed a nanotechnology-based strategy in which positively charged PGANPs facilitate efficient delivery of Glu into plant cells. This approach directly engages NbGLR3.3-dependent Ca2+ signaling and SA-mediated defense, simultaneously enhancing antiviral immunity and supporting plant growth (Figure. 8k).

Methods
Materials

Nicotiana benthamiana was cultured in a greenhouse at 25℃ with 80% humidity (10 h of light and 14 h of darkness). The plants cultivated to 4-6 leaf stage would be used for subsequent experiments. N,N-diisopropylethylamine (DIPEA) and trifluoroacetic acid (TFA) were obtained from Thermo Scientific. Boc-Glu(OH)-OtBu was purchased from Bachem. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide×HCl (EDC×HCl) was obtained from Alfa Aesar. N-hydroxybenzotriazole (HOBt) was purchased from Polypeptide group. 7-Amino-4-methylcoumarine and HCl 4N in 1,4-dioxane were bought from Sigma Aldrich. Sodium carbonate and magnesium sulphate were obtained from Fisher Scientific and citric acid from VWR Chemicals. Dichloromethane (DCM), cyclohexane, ethyl acetate (AcOEt), diethyl ether, and acetonitrile (ACN) were purchased from Carlo Erba.Polyglutamic acid (>99%), chitosan(CS) (200-500 mPas), Span-80, Tween-80, CaCl (>99%) , 4,5-Difluoro-2-methoxy-N-(4-phenylthiazol-2-yl)benzamide（DFMTI）, L-asparaginase and 7-amino-4 methylcoumarin (>98%) were purchased from AdNamas-beta (Shanghai, China). Paraffin oil (AR) was purchased from General-Reagent and chlor oinconazide was provided by Shandong Chambroad HoldingGroup Co., Ltd. Cellulase R-10L and Macerozyme R-10 purchased from Yakult Pharmaceutical Industry Co., Ltd. TMV-GFP (pSPDK 661) was a gift from Yule Liu (Tsinghua University, Beijing, China)45. Asparagine synthetase reagent kit was purchased from Suzhou Grace Biotechnolgy Co., Ltd. Eastep Super Total RNA extraction kit was purchased from Promega Biotechnology (Beijing, China) and Prime Script RT reverse transcription kit was purchased from Titan Technology (Shanghai, China). The primers used in the experiment were synthesized by Sangon (Shanghai, China). QuantinovaTM SYBR green PCR kit was purchased from Shuguang Biotechnology (Chongqing, China). The CFXTouch real-time PCR instrument (Bio Rad, USA) was used for fluorescence quantitative detection of the samples. The transmission electron microscopy (TEM) was measured with a Tecnai G2 F20 (FEI, USA) at 100 kV. The scanning electron microscope (SEM) used for bacterial observation was SU8010 (Hitachi, Japan) at 20 kV. The thermogravimetric analysis of the nanogels was measured by Netzsch STA 449 F5/F3 Jupiter (Netzsch, Germany), and the zeta potential was measured by Malvern Zetasizer Nano ZS90 (Malvern, UK). XRD was measured by D8 Advance (BrukerAXS Germany). GFP antibodies: Primary antibody (Mouse anti-GFP) was incubated overnight at 4℃, followed by incubation with the secondary antibody (Goat anti-Mouse) for 1 h at room temperature. Both antibodies were purchased from CooLab.

Transgenic plants generation

For the overexpression construct, the full-length coding sequence of NbAS-B was cloned into the PCMV-C-Myc vector. Transgenic Nicotiana benthamiana seeds were generated by Chongqing Baoguang Biotechnology Co., Ltd. Expression levels of NbAS-B in the overexpression transgenic plants were analyzed by RT–qPCR and Western blot. For CRISPR–Cas9-targeted knockout mutants of nbas-b and nbglr3.3（The nbglr3.3 mutant used in this study was generously supplied by Dr. Feng Zhu at Yangzhou University.）, genomic fragments covering the target sites were amplified and sequenced in T₀ plants. The edited T₀ plants were subsequently self-pollinated to produce T₁ seeds，and T₂ seeds were then propagated for subsequent phenotypic analysis and related experiments.

Virus inoculation

Five wild-type plants, five NbAS-B transgenic plants (#5 and #27), and five gene-edited plants (nbas-b#1 and nbas-b#2 mutants) were mechanically inoculated with TMV-GFP. The fluorescence distribution of the inoculated leaves and new leaves was observed under a portable ultraviolet lamp on the third and fifth days after inoculation. The expression levels of the virus were analyzed by real-time PCR and western-blot for induced resistance to the virus. The experiment was repeated at least three times.

RNA extraction and qPCR

RNA was extracted with Trizol method46. The specific method was as follows. 0.1 g of tobacco leaves treated with different materials were collected to grind in liquid nitrogen. Then 1 mL of RNA Trizol and 200 μL of trichloromethane were added to mix well, and the resulted mixture was centrifuged at 12,000 rpm/min for 15 min. After being placed at -20℃ for 4 h, the mixture was further centrifuged at 12000 rpm/min for 5 min, and finally dissolved with 50 μL of RNA-free water. The obtained RNA was reverse transcribed into cDNA with qPCR reaction followed. The qPCR reaction was performed using a CFX Touch real-time PCR instrument and a Quantinova™ SYBR green PCR kit, in which Actin was used as the internal reference gene. The expression levels of gene transcripts were quantified by the 2-ΔΔCT method47. The entire primer sequences used in this experiment were presented in Supplementary Table 1. Data results were quantified using Origin 2021 software and the related gene expression profiles were plotted.

Protein extraction and western blotting

Protein extraction was performed by a protein extraction lit. 0.4 g of tobacco leaves was fully crushed with a sterile pestle in liquid nitrogen. Then 200 μL of protein extraction solution was added to mix thoroughly. The obtained mixture was placed on ice for 30 min and then centrifuged at 12,000 rpm/min at 4°C for 15 min. After centrifugation, 120 μL of the supernatant was removed with 30 μL of 5× protein loading buffer added, which was boiled in boiling water for 5-10 min and placed on ice for another 5 min. Protein samples were tested by polyacrylamide gel electrophoresis (PAGE). The expression levels of the virus under different treatment conditions were analyzed by examining the expression of TMV-GFP viral protein by western blotting experiment.

Determination of Agronomic Traits

The plant height, root length, and leaf width of wild type tobacco plants and transgenic NbAS-B plants were measured at different growth stages including seedling, flourishing, and maturity stages to analyze the impact of NbAS-B on plant growth and development. The experiment was repeated three times for each group.

Measurement of Photosynthesis

Photosynthetic parameters were measured using a portable photosynthesis system on five WT and five transgenic plants, respectively. To minimize diurnal fluctuations, all measurements were conducted between 9:00 and 11:00 AM. Key parameters, including the net photosynthetic rate (Pn), intercellular CO₂ concentration (Ci), and transpiration rate (E), were recorded once stable readings were obtained from the instrument. A minimum of three replicates were performed for each dataset.

Determination of Reducing Sugar and Crude Protein Contents

The effects of NbAS-B on photosynthesis were assessed through the content of chlorophyll, reducing sugar and crude protein. Four wild type tobacco plants and four transgenic tobacco plants at different growth stages were used in this experiment, and their photosynthesis was measured through a portable photosynthesizer. The crude protein content was determined using the Kjeldahl nitrogen determination method 48, while reducing sugar levels were determined using a micromethod. Each group underwent three repetitions of the experiment.

Transcriptome analysis

RNAiso Plus kit (Takara, Japan) was employed for RNA extraction from OENbAS-B transgenic plants and WT plants. Magnetic beads with Oligo (dT) were paired with polyA for A-T base pairing to isolate mRNA from total RNA for transcriptome analysis. The mRNA was divided into fragments about 300bp by fragmentation buffer. A strand of cDNA was synthesized by random primers using the fragmented mRNA as templates under the action of reverse transcriptase, and then double synthesis was performed to form a stable double-strand structure. Then, the End Repair Mix was added to make it a flat end, and an A base was appended to the 3’ end so as to connect the Y-shaped junction. Purification and fragment sorting were performed on the adapter-linked products, and the sorted products were amplified by PCR to obtain the final library, which would be sequenced on the Illumina NovaSeq 6000 platform. RSEM (http://deweylab.github.io/RSEM) was utilized for expression level analysis, while DESeq2 (https://www.edocumentation.org/packages/DEGseq/versions/1.16.0) was employed for expression difference analysis. The default screening criteria for significantly differentially expressed genes were as follows: FDR < 0.05 and |log2FC|≥1. A gene meeting both of these conditions simultaneously was classified as a differentially expressed gene (DEG). KOBAS (http://kobas.cbi.pku.eud.cn/home.do) was utilized for KEGG PATHWAY enrichment analysis, and the results were adjusted for multiple testing using the Benjamini-Hochberg method to control false discovery rate (FDR). The Corrected P-Value was set at 0.05, and the KEGG pathway that met this criterion was defined as the KEGG pathway that was significantly enriched in differentially expressed genes.

Targeted metabolome analysis

A certain quality of NbAS-B transgenic plants and wild-type plants was weighed, lyophilized, and ground in 2 mL tubes containing 5 mm tungsten beads. Metabolites were extracted using a precooled mixture of methanol, acetonitrile, and double-distilled water (ddH O), followed by ultrasonic shaking in an ice bath for one hour. After storage at -20°C and centrifugation, the supernatants were concentrated under vacuum. For metabolomic analysis, the processed samples were subjected to an ultra-performance liquid chromatography-electrospray ionization-quadrupole-Orbitrap mass spectrometry (UPLC-ESI-Q-Orbitrap-MS) system. Positive ion scanning was used for the acquisition of mass spectrometry signals, and specific columns and mobile phases were employed for sample processing. Raw mass spectrometry data was processed for peak alignment, retention time correction, and peak area extraction. Metabolites were identified by matching accurate mass and MS/MS data against established databases and a self-built metabolite standard library, with a focus on features exhibiting significant measurement values across samples.

Treatment with Glu and PGA and Virus Inoculation

The tobacco plants from 4-6 leaf stage with the same growth trend were used as experimental materials. Glutamate solutions with different concentrations (0.13, 0.26, 0.52, 1 mmol/L) were prepared, with solvent water as the negative control and CHI as the positive control. 10 mL of glutamate solutions with different concentrations were sprayed on the surface of the tobacco leaves. After three days of continuous spraying, the leaves were inoculated with TMV-GFP through friction. The fluorescence distribution of the inoculated leaves and new leaves was observed under a portable ultraviolet lamp on the third and fifth days after inoculation. The expression levels of the virus were analyzed by real-time PCR to select the best glutamate concentration for induced resistance to the virus. The experiment was repeated at least three times.

Based on the analysis of the inductive effect and disease resistance of glutamate, the inductive effect of PGA was further explored. The detection method of PGA induced resistance was the same as that of glutamate, but the analysis of viral expression levels should be analyzed by westernblot besides PCR. And the experiment was also repeated at least three times.

Synthesis and characterization of PGANP

The synthesis of PGANP with different concentrations mainly referred to the method of Wang with appropriate improvements49. In detail,

1 g of CS was dissolved in 50 mL of 3% glacial acetic acid, and was stirred evenly to obtain a solution (step A). Span-80 (1.5 mL) and Tween-80 (0.45 mL) were dropped into liquid paraffin (150 mL), and stirred using a magnetic stirrer (800 rpm/min) for more than 30 min (step B). The solution obtained in step A was dropped into the solution of B, and then the mixed solution was stirred in a magnetic stirrer for 1 h to obtain a nanoemulsion. PGA (10, 22.5, 35, 52.5, 70, 100 mg) was dissolved into 5 mL of water, and then was added to the above nanomulsion. The obtained solution would be stirred using a magnetic stirrer for more than 1 h. Finally, 25 mL of the saturated NaCl solution was added. After further stirring for 20 min, the mixed solution was centrifuged in a refrigerated centrifuge (7, 500 rpm/min) for 20 min, and then washed with ddH O three times. The precipitate was collected, and used for subsequent experiments after freeze-drying. PGANP synthesized by the above method were characterized by transmission electron microscope (TEM), dynamic light scattering (DLS), Zeta potential analyzer, and infrared spectroscopy. The total content of PGA was determined by thermogravimetric analysis and differential scanning calorimetry (TGA-DSC) to analyze the characteristics and properties of PGANP.

Synthesis of protected Glu-AMC

To a solution of Boc-Glu(OH)-O OtBu (361 mg, 1.19 mmol, 1 eq) in DCM (20 mL) at 0°C, DIPEA (829 µL, 4.76 mmol, 4 eq), EDC×HCl (456 mg, 2.38 mmol, 2eq) and HOSBt (322 mg, 2.38 mmol, 2 eq) were added. After 15 min, a solution of 7-amino-4-methylcoumarine (250 mg, 1.43 mmol, 1.2 eq) in DCM (5 mL) was added to the mixture. The solution was stirred at room temperature overnight, and the reaction was monitored by thin-layer chromatography using cyclohexane/diethyl ether 6:4 as eluent, and HPLC. As the reaction was not complete, fresh EDC×HCl (456 mg, 2.38 mmol, 2 eq) and HOBt (322 mg, 2.38 mmol, 2 eq) were added and the mixture was heated at reflux (50 °C) for 7 h and stirred at room temperature overnight. After two days, fresh EDC×HCl (456 mg, 2.38 mmol, 2 eq) and HOBt (322 mg, 2.38 mmol, 2 eq) were again added and the mixture was heated at reflux (50 °C) for 6 h and stirred at room temperature overnight. The reaction was quenched with water (15 mL) and the aqueous phase was extracted with DCM. The organic phase was washed three times with a solution of 10% of sodium bicarbonate, three times with water, three times with a solution of 10 % of citric acid, three times with water and was dried over magnesium sulphate. The solvent was evaporated under vacuum and the product was purified by silica gel chromatography starting with a pure cyclohexane solution and continuing with cyclohexane/AcOEt at 60:40 and with cyclohexane/AcOEt at 50:50 and finishing with 100% AcOEt. Two pure fractions (70 mg) were obtained using the mixture of cyclohexane/AcOEt 50:50 and 100% AcOEt. The contaminated fractions were further purified by preparative HPLC using a gradient from 20% ACN + 0.08% TFA to 80% ACN + 0.08% TFA for 60 min The combination of the HPLC pure fractions afforded protected Glu-AMC (59 mg). Total yield: 23.5 %.

Synthesis of deprotected Glu-AMC

Protected Glu-AMC (40 mg 0.087 mmol) was dissolved in 4 mL of HCl 4N in 1,4-dioxane and the reaction mixture was stirred overnight at room temperature. Fresh HCl 4N in 1,4-dioxane (2 mL) was added again and the reaction was stirred 3 h. The solvent was removed under vacuum and diethyl ether was added to precipitate the deprotected Glu-AMC (19.5 mg). Yield: 75%.

Inductive effects of PGANP

PGANP with different concentrations were prepared by the method described above. 10 mL of the obtained nanomaterials were taken to spray on the surface of tobacco plants until the droplets dropped, and five plants were repeated for each treatment. After three days of continuous spraying, the plants were inoculated with TMV-GFP through friction. 0.1 g of the inoculated leaves and 0.1 g of new leaves were collected on the third and seventh days respectively after inoculation. Meanwhile, the viral infection, and the size and distribution of the diseased spots were observed under a portable ultraviolet lamp. The optimal concentration of PGA for resistance was selected by quantitative analysis of viral content after RNA was extracted from the collected leaf tissue. Each experiment was repeated at least three times.

Effects of PGANP on related phytohormone signaling

Similarly, 10 mL of PGANP with different concentrations were sprayed on the surface of the tobacco plants. After three days of continuous spraying, the materials were sampled separately. The expression of genes involved in SA, ET, and ABA synthesis was determined by quantitative real-time PCR after the extracted RNA was reverse transcribed into cDNA. Based on the quantitative results, the content of related hormones with differential gene expression was determined by high performance liquid chromatography. The signaling pathways mainly affected by PGA and the effects of PGA on the synthesis of related hormones were further analyzed. Each experiment was repeated at least three times.

Determination of Glu and Asn Contents

10 mL of PGANP (22 μg/mL) was sprayed on the unilateral leaves of the tobacco plants. The Glu and Asn content in the lateral leaves was analyzed by high performance liquid chromatography. And on this basis, the effect of PGANP on glutamate content in plants was further analyzed to determine whether PGA improved its induced resistance by enhancing Glu synthesis.

Isolation of Tobacco Mesophyll Protoplasts:

(1) Treat fresh tobacco leaves by rinsing thoroughly with distilled water and gently drying the surface moisture. Remove the leaf epidermis and cut the tissue into approximately 2 mm fine strips. Transfer 1 g of the strips into a Petri dish containing 20 mL of enzyme digestion solution (comprising 2.0% Cellulase R-10 and 0.5% Macerozyme R-10), ensuring complete submersion of the leaf strips.
(2) Incubate the Petri dish on a shaker at 23-28℃ with gentle agitation (40-60 rpm) for 3-6 h to allow digestion.
(3) Filter the digestion mixture through a 100 μm nylon mesh into a 50 mL round-bottom centrifuge tube to remove undigested tissue and large debris.
(4) Centrifuge the filtered solution at 100-150 g for 2–5 min at room temperature to pellet the protoplasts.
(5) Carefully remove the supernatant. Gently layer the resuspended protoplasts over a cushion of 3 mL 20% sucrose solution (prepared in W5 buffer: 154 mM NaCl, 125 mM CaCl₂, 5 mM KCl, 2 mM MES, pH 5.7) in a centrifuge tube.
(6) Centrifuge at 100-150 g for 5-10 min. Collect the supernatant, which contains the purified protoplasts.

Sustained-release capacity of PGANP on leaves

Firstly, the fluorescent molecule 7-amino-4-methylcoumarin (C H NO ) was loaded onto the surface of PGA through amidation reaction. The specific process of the synthesis was as follows: PGA was mixed with 7-amino-4-methylcoumarin at a 1:1 ratio, and then 2-aminoimidazole hemisulfate was added to perform the overnight reaction, which would be dialyzed and freeze-dried the next day. As a common fluorescent signal substance, 7-amino-4-methylcoumarin exhibits blue fluorescence at the excitation wavelength of 351 nm and the emission wavelength of 430 nm. Therefore, the residual PGA content on the surface of plant leaves could be analyzed by detecting the intensity of blue fluorescence through a fluorescence spectrophotometer. Then, 2 mL of PGA@C H NO nanomaterials with different concentrations (1, 0.5, 0.25, 0.125 mg/mL) were taken to measure their fluorescence values at the excitation wavelength of 351 nm and the the emission wavelength of 430 nm under a fluorescence spectrophotometer. The experiment of each concentration value was repeated three times, and the obtained results were made into a standard curve. Finally, the PGA@ C H NO composite was dissolved in distilled water at a concentration of 1 mg/mL. The supernatants obtained at 6 h, 12 h, 24 h, 48 h and 72 h were centrifuged at 5,000 rpm/min for 5 min. The supernatant at 0 h was used as the control. The fluorescence values of these supernatants were measured under a fluorescence spectrophotometer. Each result was measured three times so as to analyze the sustained-release capacity of PGA@ C H NO composite on plant leaves.

Adhesion ability of PGANP on leaves

10 mL of PGA@ C H NO nanomaterials at a concentration of 1 mg/mL was sprayed on the surface of tobacco plants, and PGA was used as the control. After the sprayed solution was fully absorbed, the plant surfaces were washed with 100 mL distilled water. And the samples were taken after washing 1, 3 and 5 times respectively. The absorption values of the samples at the excitation wavelength of 351 nm and the emission wavelength of 430 nm were measured by a fluorescence spectrophotometer, so as to analyze the adhesion ability of PGA nanocomposites on leaves. Each experiment was repeated three times.

PGANP safety test

The dried seeds were transferred to petri dishes containing wet filter paper treated with ddH O, PGANP (1 mg/mL) and CS (1 mg/mL). 200 seeds were placed in each dish and cultured in an artificial growth chamber at 25 ± 2℃ with a relative humidity of 85 % for 14 h. The germination rate of seeds was observed for 5 days. Each treatment was repeated for 3 times.

Statistical analysis

All of the above experiments were performed three times independently, and the mean and standard errors of the experimental values were calculated. The data were presented as mean ± standard errors. Statistical and correlation analyses were performed using a one-way analysis of variance with Duncan’s test (SPSS 17.0, Chicago, IL, USA). P <0.05 was considered as statistical significance.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this study, the authors discovered that the evolutionarily conserved NbAS-B induces both antiviral and growth-promoting responses in Nicotiana benthamiana, in a manner dependent on expression intensity. They demonstrated that these effects are mediated through a glutamate-dependent enhancement of salicylic acid (SA) signaling and a reprogramming of the photosynthesis pathway. However, it is somewhat confusing that they developed nanogels (PGANPs) by loading PGA onto chitosan via electrostatic interactions to regulate the glutamate-dependent salicylic acid (SA) signaling. In general, the research content of this manuscript is too broad and lacks depth, which is not suitable for publication in Nature Communications. The following is what I think the authors need to further verify and improve.\n1. The authors suggested that the induction effects of NaAS-B dependent on expression intensity. However, in fact, the authors only verified two expression intensities, namely, NbAS-B-OE#5 and NbAS-B-OE#27, which can only reflect the differences between the two transgenic lines, and cannot prove that the expression intensity of NaAS-B is dependent. If this conclusion is to be proved, it is necessary to add another transgenic line with different expression intensity.\n2. In line 176, regarding the KEGG pathway analysis in Fig. 2, the pathway enrichment of the two different expression levels is the highest for fatty acid biosynthesis. However, the analysis in the article does not mention this pathway.\nIn lines 196-214, targeted metabolomic profiling focused on amino acids was performed, and it was concluded that the reaction catalyzed by NbAS-B in NbAS-B transgenic plants proceeded in the forward direction. After NbAS-B overexpression, the reaction catalyzed by NbAS-B proceeded positively, which was originally an expected result, and this result could be directly verified by analyzing the expression levels of precursors and products catalyzed by NbAS-B (Fig. 2l-m). Therefore, this metabolomics did not reveal anything interesting.\n3. In this manuscript, the explanation of the disease resistance mechanism of NbAS-B is mostly at the level of phenotype and physiological indicators, and there is a lack of in-depth analysis at the molecular level. For example, the disease resistance mechanism of glutamate was basically concluded through literature analysis, and the author did not carry out innovative experiments to study the molecular targets of the disease resistance mechanism.\n5. The proposal of PGA nanogel is too far-fetched and has little correlation with previous studies. PGA and glutamate are completely different substances, and their influence mechanisms on plants are also completely different, and there are many substances with similar structure of glutamate, which cannot be simply tied together. On the part of PGA nanogel, there is an obvious sense of splicing and data stacking, which reduces the research depth of the manuscript. In addition, the relative molecular weight of PGA is not indicated, and it is well known that different molecular weights of PGA have different effects on plant growth.\n6. Many of the statements in the manuscript lack clear evidence.\nLine 407 “These results indicate that ... outperforming both PGA and Glu treatments. Please provide relevant data description.\nLine 409 “This suggests that ... activate related resistance pathways.\" Please demonstrate that PGANP can penetrate plant cells.\nLine 437 “However, no significant correlations with these pathways were found.” However, in Figure S8, there are significant differences between Nb-ERF017 and Nb-ERN1 after PGANP treatment.", "reviewer_2": "Qiao et al. suggested a pivotal role of Nicotiana benthamiana (Nb) asparagine synthetase NbAS-B in enhancement of growth and induction of antiviral resistance. Furthermore, the authors showed a promising measure to induce both the antiviral and growth-promoting responses by application of chitosan-loaded PGA nanocomposites (PGANPs). I feel that data in this study could bring a novelty to open a path for sustainable control of plant viral disease control. However, there are several points to make clear or to further discuss.\nI would like to give some comments as below.\nResults and discussion\nTransgenic overexpression of NbAS-B enhances plant resistance to viruses and growth promotion\nLines 124-132\nThe NbAS-B overexpressing transgenic NB lines #5 and #27 both enhanced antiviral responses against TMV infection and promoted plant growth. However, it would be better to show data on host responses against other plant pathogenic virus infections to further validate the dual role of NbAS-B in antiviral response and plant growth.\nIn the section of introduction, the authors have referred to recent studies on the roles of asparagine synthetase (AS) in the transport and distribution of nitrogen throughout plant development. However, the molecular mechanism associated with salicylic acid (SA) synthesis, which could be regulated by NbAS-B, remains unknown. Glutamate (Glu) has also an important role in amino acid metabolism involved in ammonia assimilation or dissimilation and amino acid transamination in plants, involving in various growth and development processes such as seed germination, root growth, pollen tube elongation and photosynthesis, as well as pathogen resistance and non-biological stress response in addition to the synthesis of SA. It would be possible to explain involvement of NbAS-B in induction of antiviral response and plant growth using NbAS-B-OE#5 and NbAS-B-OE#27. However, NbAS-B-silencing lines are necessary for complement of the results from NbAS-B-OE#5 and NbAS-B-OE#27.\nNbAS-B-enhanced growth promotion and resistance induction partly depend on the increase of glutamate\nLines 266-270\nUnlike NbAS-B-OE#5, the content of SA in the non-inoculated plants showed no significance between WT and NbAS-B-OE#27. On the other hand, SA accumulation level in the TMV-inoculated NbAS-B-OE#27 was significantly higher than that in WT (Fig. 3i). How do you explain the differences in SA levels between NbAS-B-OE#27 and NbAS-B-OE#5 under non-inoculated and inoculated conditions?\nSynthesized PGANPs have a stronger ability to induced N. benthamiana resistance to TMV\nThe authors considered negative effect of polyanionic properties of PGA on entering plant cells, resulting restriction of broader potential in its enhancing antiviral activity. The authors, therefore, developed and examined PGANPs to enhance ability of PGA to induce plant resistance. Interestingly, gene expression of ICS1, but not PAL2, was strongly induced without any significant correlations with the abscisic acid- and ethylene-mediated pathways, and a substantial increase in SA level was measured in the plants treated with PGANPs. By additional assay using the SA-deficient mutant NahG Nb plants, the authors concluded that glutamate-induced resistance entirely depends on the SA signaling pathway. Did the authors see a significant upregulation of ICS1 expression in the transcriptome analysis using NbAS-B-OE#27 and NbAS-B-OE#5 in comparison with WT Nb plants?\nPGANPs ensure high foliar persistence while supporting plant health\nInduction of host resistance by PGANPs was evaluated at 3 and 7 days post-treatment in this assay. Do the authors think that the time course for this assay was enough to evaluate long-lasting dual effect of PGANPs on antiviral response and plant growth? Did the authors observe any intensification of TMV-induced symptoms after withdrawal of effect of PGANPs in WT Nb plants?", "reviewer_3": "Tobacco is an important commercial crop, while tobacco mosaic virus (TMV) is a major biotic stress factor that affects tobacco industry. Improving tobacco growth, quality, and resistance to TMV is an importantly real problem in tobacco industry. Based on this problem, this manuscript found that Nicotiana benthamiana asparagine synthetase-B (NbAS-B) could induce both antiviral and growth-promoting responses in an expression intensity-dependent manner in Nicotiana benthamiana by the crosstalk of glutamate (Glu) and salicylic acid (SA) signaling pathways. Meanwhile, the developed nanogels (PGANPs) also showed an antiviral and growth-promoting effect on tobacco plants by regulating Glu-dependent SA signaling. It is important to understand the possible mechanisms by which NbAS-B and PGANPs alone improve antiviral and growth-promoting response in tobacco plants, which is a promising approach to enhance growth and induce resistance against viral infection. Also, the experiment design is scientific and reasonable, and the research content and methods have major innovation. However, I think the following comments should be considered.\n(1) ABSTRACT: The research GAP and what is NbAS-B, might be Nicotiana benthamiana asparagine synthetase-B, should be clearly stated in the ABSTRACT.\n(2) Key words: What is Transcriptional metabolome?\n(3) INTRODUCTION: Lines 86-87, the authors stated the research gap “However, the precise mechanism by which NbAS-B regulates SA synthesis remains unresolved”, but the manuscript has not completely answered this question according to figure 7 (w), which should be further revised.\n(4) RESULTS and DISCUSSION: (a) Figure 1h, i, and j, longth and stem longth are difficult to understand. (b) Similarly, Figure 1k and l, y-axis might be fresh/dry weight (g/plant), not (g). (c) Also, according to the catalytic mechanism of NbAS-B, whose products, besides Glu, should include Asn, which might take part in tobacco growth and resistance to TMV, but Asn content in figure 2 i and m was not seen (note, figure legend was described).Also, ammonia (NH3) as a signaling molecule, the conversion of Gln and Asp into Glu and Asn under the catalysis of AS is involved in the change in mmonia (NH3) signaling, which should be mentioned. (d) For tobacco plants, Figure 4, the content (not concentration) of chlorophyll a, b, and total chlorophyll should be mg/g fresh weight (FW) or  g/ g FW, not  g/  L; correspondingly, figure 4 h and i, as well as figure 6f, might be 100 g FW and g FW, respectively. (e) Figures 3, 6, and 7, what is DFMTI or CS, whose full names is not seen in the whole manuscript. Also, in figure 3, L-asparaginase, as an asparagine inhibitor, is a protein, how it enters into the plant cells should be stated in the text.\n(5) METHODS: Many subtitles in the METHODS have not described methods but results, which should be carefully revised. For examples, “Induction effect of transgenic plants expressing asparagine synthetase B on TMV-GFP”, “Asparagine synthetase B enhances plant growth and development”, “Antiviral effects of glutamate and PGA”, “Inductive effects of PGANP”, and so on.\n(6) WRITING: The language should be further concise. For example, after the full names and abbreviations of some terms were represented in the first position, the abbreviations only were used subsequently, including them in figures, such as AS, SA, Glu, Gln, Asp, Asn, and so on. Also, the seeds in line 154; abscisic acid biosynthesis (SnRK2 genes) in line 189, which should be abscisic acid signaling; Asp, Gln, Glu, and Asn in line 230, Asp and Asn should be deleted according to figure 2 l and m, and so on."}, "author_response": "1. Response to the Reviewer #1\nComment 1. The authors suggested that the induction effects of NaAS-B dependent on expression intensity. However, in fact, the authors only verified two expression intensities, namely, NbAS-B-OE#5 and NbAS-B-OE#27, which can only reflect the differences between the two transgenic lines, and cannot prove that the expression intensity of NbAS-B is dependent. If this conclusion is to be proved, it is necessary to add another transgenic line with different expression intensity.\nResponse: We thank this reviewer very much for his/her helpful comment. To address this comment stating that the experimental design involving two transgenic plant lines was insufficient to demonstrate a dose-dependent effect of NbAS-B expression, we have supplemented the data with an additional transgenic line. The results after inoculationn with TMV-GFP show that as the expression level of NbAS-B increases from low to high, the induced resistance against TMV is progressively enhanced. Therefore, we can conclude that the inductive effect of NbAS-B is dependent on its expression strength.\nThe following sentences(line 150) and figureS2 were added in the revised manuscript.\nFig. S2 (a) Symptoms of TMV-GFP infection in transgenic NbAS-B plants with varying expression levels. (b)Viral accumulation in systemic leaves at 5 days post-inoculation with TMV-GFP as determined by qPCR. (c) Symptom phenotypes of PVX-GFP, TRV-GFP, TUMV-GFP infections in WT, NbAS-B-overexpressing and nbas-b knockout plants. (d-f) Viral accumulation in systemic leaves after infection with different viruses as determined by qPCR.\nComment 2. In line 176, regarding the KEGG pathway analysis in Fig. 2, the pathway enrichment of the two different expression levels is the highest for fatty acid biosynthesis. However, the analysis in the article does not mention this pathway. In lines 196-214, targeted metabolomic profiling focused on amino acids was performed, and it was concluded that the reaction catalyzed by NbAS-B in NbAS-B transgenic plants proceeded in the forward direction. After NbAS-B overexpression, the reaction catalyzed by NbAS-B proceeded positively, which was originally an expected result, and this result could be directly verified by analyzing the expression levels of precursors and products catalyzed by NbAS-B (Fig. 2l-m). Therefore, this metabolomics did not reveal anything interesting.\nResponse: We thank this reviewer for his/her suggestions and constructive comments. Regarding the comment on the fatty acid biosynthesis pathway, we acknowledge that our initial analysis overlooked this aspect. Our further investigation reveals that the fatty acid biosynthesis pathway primarily participates in the plastidial fatty acid synthesis pathway in plants. This process provides essential precursors for the synthesis of membrane lipids, as well as secondary metabolites such as epidermal waxes and cuticle. Given its significant impact on plant growth and development, we confirm that this pathway is indeed involved in the NbAS-B-mediated nitrogen synthesis process, thereby promoting plant growth. Accordingly, we have addressed this point in the revised manuscript. On the other hand, this reviewer noted that the metabolomics results did not yield particularly interesting conclusions. Indeed, our primary goal with the metabolomics analysis was to address two key aspects: first, we aimed to use targeted metabolomics to demonstrate that the upregulation of NbAS-B promotes the accumulation of Asn and Glu in plants — a finding that could also be validated through direct analysis or product quantification. Second, we sought to examine the expression levels of other amino acids to explore the potential synergy between growth and disease resistance. However, the metabolomics data showed significant increases only in specific amino acids, notably Asn and Glu, while no marked differences were observed in other growth-related amino acids. Nevertheless, both Glu and Asn, as key nitrogen transport molecules, do contribute to plant growth and development processes.\nComment 3. In this manuscript, the explanation of the disease resistance mechanism of NbAS-B is mostly at the level of phenotype and physiological indicators, and there is a lack of in-depth analysis at the molecular level. For example, the disease resistance mechanism of glutamate was basically concluded through literature analysis, and the author did not carry out innovative experiments to study the molecular targets of the disease resistance mechanism.\nResponse: We thank this reviewer for the suggestion to strengthen the molecular underpinnings of our study. We agree that investigating the direct molecular targets is key to enhance the mechanistic analysis. In response, we generated NbAS-B loss-of-function mutants and performed the transcriptomic analysis. This revealed that NbAS-B not only influences plant growth and development but is also significantly involved in plant defense pathways against pathogens, as evidenced by strong enrichment in both overexpression and knockout datasets. We further demonstrated that NbAS-B exerts this function primarily by modulating glutamate (Glu) levels to influence the downstream salicylic acid (SA) signaling pathway. To investigate whether the nano-material PGANP can activate this pathway, we synthesized coumarin-labeled PGA and confirmed its entry into plant cells via stomata. We then monitored its effect on Glu synthesis over time. The results showed that PGA indeed elevates intracellular Glu levels and induces the expression of defense genes NPR1 and PR1. We propose that this Glu-inducing effect of PGANP results from its degradation by plant proteases. To test this, we incubated PGANP with total plant protein extracts and measured Glu levels in the mixture in the period of 0–12 h. A significant increase in Glu was observed at 8 h post-incubation. These findings explain that the antiviral activity of PGANP is largely mediated by Glu released upon its degradation. The nano-formulation of PGA serves primarily to enhance the cellular uptake efficiency (Figure 2). Furthermore, transcriptome analysis of overexpression and knockout lines highlighted differentially expressed genes clustered in plant-pathogen interaction, MAPK signaling, and phytohormone pathways, with notable alterations in calcium signaling. Given prior reports that Glu binding to glutamate receptors (GLRs) can trigger Ca2+ influx and subsequently activate SA signaling by affecting PR1 and ICS1 expression, we generated a nbglr3.3 knockout mutant. Exogenous application of PGANP to this mutant abolished the induced resistance against TMV that was observed in wild-type plants. This result confirms that PGANP-enhanced TMV resistance depends on Glu-GLR interaction, which amplifies calcium signaling and thereby potentiates SA-mediated defense (Figure 3). In summary, we have elucidated the functional link between PGANP and Glu in planta and, using genetic approaches, demonstrated that Glu derived from PGANP degradation primarily activates SA signaling through binding to GLR3.3, leading to upregulation of PR1 and ICS1 expression.\nThe following sentences (line528-549)and figures were added in the revised manuscript:\nFigure 2. PGANP enters plant cells via stomata and participates in the induction of systemic resistance.(a) Fluorescence distribution observed by confocal microscopy in N. benthamiana leaves treated with Glu-AMC, PGA-AMC, or PGANP-AMC.(b) Intracellular fluorescence distribution in protoplasts extracted from leaves one day after treatment with different compounds.(c–d) Expression levels of resistance-related genes NbNPR1and NbPR1following exogenous application of different materials.(e) Endogenous glutamate (Glu) content in plants from 6-24h post-treatment.(f) Changes in Glu levels following co-incubation of plant total protein with PGANP in vitro.\nFigure 3. PGANP enhances the induced resistance to TMV-GFP by responding to SA signals (a) Viral infection of heart-leaves after being sprayed with different concentrations of PGANP. (b) Virus expression detected by qPCR. (c) The content of Glu after spraying PGANP. (d) Effect of PGANP on the content of SA. (e) Disease symptoms in nbglr3.3mutant plants after PGANP treatment and TMV-GFP infection. (f) Viral accumulation measured by qPCR in PGANP-treated nbglr3. 3mutant. (g–i) Expression levels of SA signaling-related genes (NbICS1, NbNPR1, NbPR1, NbPR2) in nbglr3.3 mutant analyzed by qPCR.\nComment 4. The authors did a comparative transcriptomic comparison of two transgenic lines that overexpress NbAS-B. From a rigorous design perspective, the transcriptomic comparison should also be made for those that silence NbAS-B.\nResponse: We thank this reviewer for his/her suggestion. We agree that comparing both gain- and loss-of-function genotypes is essential for robust conclusions. Accordingly, our study included RNA-seq analysis of two NbAS-B-overexpressing (OE) lines and two CRISPR/Cas9 knockout (KO) lines (nbas-b#1 and nbas-b#2). The KO data provided critical complementary evidence:the complete KO mutant nbas-b#1 exhibited a strong transcriptomic shift, with 518 dysregulated genes versus only 26 in the partial KO nbas-b#2 (Fig. 4h-j), confirming the dose-dependent regulatory impact of NbAS-B.KEGG analysis of nbas-b#1 showed significant enrichment in \"Plant-pathogen interaction\" and \"Phenylpropanoid biosynthesis\" pathways (Fig. 4k), indicating that loss of NbAS-B activates systemic defense and metabolic reprogramming.GO analysis specifically highlighted enrichment in cell wall-related processes such as \"xyloglucan metabolic process\" (Fig. 4l).Thus, our experimental design already incorporated the suggested loss-of-function transcriptomic comparison. The bidirectional evidence from OE and KO lines has been essential in establishing a coherent framework for NbAS-B function in balancing growth and defense, as detailed in the Results.\nThe following sentences(line188) and figure2 were added in the revised manuscript:\nFigure 4 Transcriptome targeted metabolomics analysis of NbAS-B-OE, nbas-b and WT. (a-b) Analysis of DEGs with a volcano plot. Up-regulated, down-regulated and no differentially expressed genes are indicated by red, blue and grey dots, respectively. (c) Differential gene Venn diagram of NbAS-B-OE and WT. (d-e) KEGG enrichment analysis of NbAS-B-OE and WT. (f-g) Heatmaps of defense, plant growth and defense genes. Highly and lowly expressed transcripts are labeled in red and blue, respectively. (h-i) Volcano plot analysis of differentially expressed genes between nbas-b and WT, where up-regulated, down-regulated, and non-differentially expressed genes are represented in red, green, and blue, respectively. (j) Differential gene Venn diagram of nbas-b and WT. (k) KEGG enrichment analysis of nbas-b#1 and WT. (l) GO enrichment analysis of nbas-b#2 and WT. (m) Enrichment of calcium signaling-related genes in the transcriptomic data of NbAS-B-OE, nbas-b, and WT plants. (n) Statistics on the differential expression of amino acids between WT and NbAS-B-OE, with red representing the number of upregulated genes and blue representing the number of downregulated genes. (o) HMDB map of differential metabolites, where each pie chart in the figure represents different HMDB classifications with various colors, and the area of each segment indicates the relative proportion of metabolites in that classification. (p) Metabolite VIP bubble chart: comparison of amino acid expression levels between OENbAS-B and WT. (q) Heatmap of targeted amino acid metabolism expression in WT and NbAS-B-OE. (r-s) Validation of the expression levels of key amino acids Gln and Glu in the NbAS-B pathway based on metabolome data.\nComment 5. The proposal of PGA nanogel is too far-fetched and has little correlation with previous studies. PGA and glutamate are completely different substances, and their influence mechanisms on plants are also completely different, and there are many substances with similar structure of glutamate, which cannot be simply tied together. On the part of PGA nanogel, there is an obvious sense of splicing and data stacking, which reduces the research depth of the manuscript. In addition, the relative molecular weight of PGA is not indicated, and it is well known that different molecular weights of PGA have different effects on plant growth.\nResponse: We thank this reviewer for his/her comment, Regarding the point that polyglutamic acid (PGA) and glutamic acid (Glu) are distinct compounds, we subsequently applied PGANP exogenously to plants and observed an increase in glutamate levels. Since PGA is a high-molecular-weight polymer, we hypothesized that it might be degraded by proteases. To test this, we extracted total plant protein and co-incubated it with PGANP in the presence of protease inhibitors. The mixture was sampled at different time points to measure Glu content. As shown in Figure 4f, Glu levels increased significantly by 8 h and showed a more pronounced increase by 12 h, supporting that PGA is likely degraded by certain plant proteasomes to release Glu monomers. For reference, the PGA used in our experiments had a molecular mass of 700,000 Da. Regarding the uptake of nanomaterial, in addition to PGA-AMC and PGANP-AMC tested in the original paper, we synthesized the new control fluorescently-labeled glutamic acid (Glu-AMC). We applied these three molecules to Nicotiana benthamiana leaves. Leaf samples were collected at 12 h to observe fluorescence distribution, and protoplasts were prepared at 24 h to examine subcellular localization. As shown in Figure 4a-e, PGANP notably accumulated in stomata and subsequently entered plant cells. Consistent with this, both the expression levels of NPR1 and PR1 and the Glu content were significantly higher in PGANP-treated plants compared to controls, confirming that PGANP enhances Glu levels and thereby promotes plant immunity.\nFigure 5 PGANP enters plant cells via stomata and participates in the induction of systemic resistance.(a) Fluorescence distribution observed by confocal microscopy in N. benthamianaleaves treated with Glu-AMC, PGA-AMC, or PGANP-AMC.(b) Intracellular fluorescence distribution in protoplasts extracted from leaves one day after treatment with different compounds.(c–d) Expression levels of resistance-related genes NbNPR1 and NbPR1 following exogenous application of different materials.(e) Endogenous glutamate (Glu) content in plants from 6-24h post-treatment.(f) Changes in Glu levels following co-incubation of plant total protein with PGANP in vitro.\nComment 6. Many of the statements in the manuscript lack clear evidence.\na、Line 407 “These results indicate that ... outperforming both PGA and Glu treatments. Please provide relevant data description.\nb、Line 409 “This suggests that ... activate related resistance pathways.\" Please demonstrate that PGANP can penetrate plant cells.\nc、Line 437 “However, no significant correlations with these pathways were found.” However, in Figure S8, there are significant differences between Nb-ERF017 and Nb-ERN1 after PGANP treatment.\nResponse: We thank the reviewer for his/her helpful comments. Point a. As shown in Figure S9, we screened the induced resistance effects of PGA and Glu at different concentrations against TMV-GFP. The results demonstrated that PGA (0.26) exhibited significantly stronger induced resistance compared to other Glu treatment groups. Therefore, we further evaluated the resistance effect of PGANP (Fig 6) using PGA (0.26) as the control, and found that PGANP showed superior induced resistance to PGA (0.26). Based on these findings, we conclude that \"these results indicate that PGANP significantly enhances the resistance of tobacco plants to TMV-GFP, outperforming both PGA and Glu.\"\nFigure S9 (a) Foliar application of different concentrations of Glu and PGA followed by inoculation with TMV-GFP. (b) Virus expression levels in plants treated with PGA detected by qPCR.\nFigure. 6 PGANP enhances the induced resistance to TMV-GFP by responding to SA signals (a) Viral infection of heart-leaves after being sprayed with different concentrations of PGANP. (b) Virus expression detected by qPCR. (c) The content of Glutamic acid after spraying PGANP. (d-e) The expression of NbICS1 and NbPAL2 under PGANP treatment. (f) Effect of PGANP on the content of Salicylic acid.\nPoint b、As shown in Figure 4, fluorescence tracking of PGANP-AMC in tobacco revealed that the labeled molecules entered plant cells via stomatal uptake, as confirmed by protoplast assays.\nPoint c、We evaluated the activation of the relevant signaling pathways by measuring the expression levels of key genes in the ethylene (ET) and abscisic acid (ABA) signaling pathways. The results showed a significant increase in the expression of the transcription factor genes Nb-ERF017 and Nb-ERN1, which function downstream of the ET signaling pathway, indicating successful activation of the ET pathway. It is worth noting that Nb-ERF017 acts as a responsive factor in ET signaling rather than being involved in ethylene biosynthesis. On the other hand, no significant change was observed in the expression of Nb-ABF, a key transcription factor gene in the ABA signaling pathway, suggesting that the ABA pathway was likely not activated under these conditions. In summary, our data demonstrate that the treatment specifically activated the ET signaling pathway but had no significant effect on the ABA signaling pathway. Consequently, our initial characterization was imprecise and has been amended in the text.\n1. Response to the Reviewer #2\nComment 1.Transgenic overexpression of NbAS-B enhances plant resistance to viruses and growth promotion Lines 124-132 The NbAS-B overexpressing transgenic NB lines #5 and #27 both enhanced antiviral responses against TMV infection and promoted plant growth. However, it would be better to show data on host responses against other plant pathogenic virus infections to further validate the dual role of NbAS-B in antiviral response and plant growth.\nResponse: We thank this reviewer for his/her comment. Following the suggestion, we constructed a knockout mutant of nbas-b. Upon inoculation with TMV-GFP, we observed a significant increase in viral accumulation compared to WT plants. Additionally, assessment of agronomic traits indicated that the growth of nbas-b mutants was also inhibited relative to WT (Figure. 1). Furthermore, we inoculated these lines with other viruses (TRV-GFP, TuMV-GFP, PVX-GFP), and the experimental results demonstrated that NbAS-B also exhibits notable induced resistance against these viruses(Figure.S2).\nFigure. 1 NbAS-B enhances the induced resistance of plants to TMV-GFP and promotes plant growth. (a) Symptom charts of NbAS-B-OE#5, NbAS-B-OE#27, nbas-b#1, nbas-b#2 and WT after 5 days of TMV-GFP inoculation. (b) Growth phenotype images of NbAS-B-OE#5, NbAS-B-OE#27, nbas-b#1, nbas-b#2 and WT. (c) Virus expression level detected by qPCR. (d-f) Stem length, leaf width and leaf length of NbAS-B-OE#5, NbAS-B-OE#27, nbas-b#1, nbas-b#2 and WT measured at seedling, growth and maturity stages. (g-h) Fresh weight and dry weight of plants at maturity stages.\nFig. S2 (a) Symptoms of TMV-GFP infection in transgenic NbAS-B plants with varying expression levels. (b)Viral accumulation in systemic leaves at 5 days post-inoculation with TMV-GFP as determined by qPCR. (c) Symptom phenotypes of PVX-GFP, TRV-GFP, TUMV-GFP, and TMV-GFP infections in WT, NbAS-B-overexpressing and nbas-b knockout plants. (d-g) Viral accumulation in systemic leaves after infection with different viruses as determined by qPCR.\nComment 2. In the section of introduction, the authors have referred to recent studies on the roles of asparagine synthetase (AS) in the transport and distribution of nitrogen throughout plant development. However, the molecular mechanism associated with salicylic acid (SA) synthesis, which could be regulated by NbAS-B, remains unknown. Glutamate (Glu) has also an important role in amino acid metabolism involved in ammonia assimilation or dissimilation and amino acid transamination in plants, involving in various growth and development processes such as seed germination, root growth, pollen tube elongation and photosynthesis, as well as pathogen resistance and non-biological stress response in addition to the synthesis of SA. It would be possible to explain involvement of NbAS-B in induction of antiviral response and plant growth using NbAS-B-OE#5 and NbAS-B-OE#27. However, NbAS-B-silencing lines are necessary for complement of the results from NbAS-B-OE#5 and NbAS-B-OE#27.\nResponse: We thank this reviewer for his/her comment. Following this suggestion, we constructed knockout mutants of nbas-b. Upon inoculation with TMV-GFP, we observed a significant increase in viral accumulation compared to WT plants. Additionally, assessment of agronomic traits indicated that the growth of nbas-b mutants was also inhibited relative to WT (Figure 1).\nFigure. 1 NbAS-B enhances the induced resistance of plants to TMV-GFP and promotes plant growth. (a) Symptom charts of NbAS-B-OE#5, NbAS-B-OE#27, nbas-b#1, nbas-b#2 and WT after 5 days of TMV-GFP inoculation. (b) Growth phenotype images of NbAS-B-OE#5, NbAS-B-OE#27, nbas-b#1, nbas-b#2 and WT. (c) Virus expression level detected by qPCR. (d-f) Stem length, leaf width and leaf length of NbAS-B-OE#5, NbAS-B-OE#27, nbas-b#1, nbas-b#2 and WT measured at seedling, growth and maturity stages. (g-h) Fresh weight and dry weight of plants at maturity stages.\nComment 3.Lines 266-270\nUnlike NbAS-B-OE#5, the content of SA in the non-inoculated plants showed no significance between WT and NbAS-B-OE#27. On the other hand, SA accumulation level in the TMV-inoculated NbAS-B-OE#27 was significantly higher than that in WT (Fig. 3i). How do you explain the differences in SA levels between NbAS-B-OE#27 and NbAS-B-OE#5 under non-inoculated and inoculated conditions?\nResponse: We thank this reviewer for his/her suggestions and constructive comments. The observed variation likely stems from T-DNA position effects. In NbAS-B-OE#5, the insertion may have created a pre-activated state through subtle perturbation of endogenous genes, resulting in constitutively elevated basal SA levels. In contrast, the transgene in NbAS-B-OE#27 appears integrated at a neutral site, where NbAS-B overexpression alone does not significantly activate SA biosynthesis without pathogen challenge. About the differences following TMV inoculation: both overexpression lines exhibited enhanced SA accumulation upon TMV infection. NbAS-B-OE#27 showed particularly strong induction, suggesting effective amplification of immune signaling through the Glu-Ca²⁺ pathway. While NbAS-B-OE#5 also responded to infection, its already elevated basal SA levels may have resulted in less pronounced relative induction or altered response kinetics compared to NbAS-B-OE#27.\nComment 4.The authors considered negative effect of polyanionic properties of PGA on entering plant cells, resulting restriction of broader potential in its enhancing antiviral activity. The authors, therefore, developed and examined PGANPs to enhance ability of PGA to induce plant resistance. Interestingly, gene expression of ICS1, but not PAL2, was strongly induced without any significant correlations with the abscisic acid- and ethylene-mediated pathways, and a substantial increase in SA level was measured in the plants treated with PGANPs. By additional assay using the SA-deficient mutant NahG Nb plants, the authors concluded that glutamate-induced resistance entirely depends on the SA signaling pathway. Did the authors see a significant upregulation of ICS1 expression in the transcriptome analysis using NbAS-B-OE#27 and NbAS-B-OE#5 in comparison with WT Nb plants?\nResponse: We thank this reviewer for his/her comment. While our transcriptomic data did not reveal significant differential expression of the ICS1 gene, the analysis of both overexpression and knockout lines showed significant enrichment of the calcium signaling pathway. Notably, it has been reported that glutamate (Glu) can bind to glutamate receptor-like (GLR) proteins, leading to calcium influx and subsequent upregulation of ICS1 and PR1 expression, thereby activating the SA signaling pathway. Furthermore, through genetic experiments using the nbglr3.3 mutant, we confirmed that the disease resistance conferred by Glu, released from PGANP degradation, depends on GLR-mediated calcium signaling. Together, these findings elucidate the antiviral mechanism mediated by NbAS-B.\nFigure. 2 Transcriptome targeted metabolomics analysis of NbAS-B-OE, nbas-b and WT. (a-b) Analysis of DEGs with a volcano plot. Up-regulated, down-regulated and no differentially expressed genes are indicated by red, blue and grey dots, respectively. (c) Differential gene Venn diagram of NbAS-B-OE and WT. (d-e) KEGG enrichment analysis of NbAS-B-OE and WT. (f-g) Heatmaps of defense, plant growth and defense genes. Highly and lowly expressed transcripts are labeled in red and blue, respectively. (h-i) Volcano plot analysis of differentially expressed genes between nbas-b and WT, where up-regulated, down-regulated, and non-differentially expressed genes are represented in red, green, and blue, respectively. (j) Differential gene Venn diagram of nbas-b and WT. (k) KEGG enrichment analysis of nbas-b#1 and WT. (l) GO enrichment analysis of nbas-b#2 and WT. (m) Enrichment of calcium signaling-related genes in the transcriptomic data of NbAS-B-OE, nbas-b, and WT plants. (n) Statistics on the differential expression of amino acids between WT and NbAS-B-OE, with red representing the number of upregulated genes and blue representing the number of downregulated genes. (o)The HMDB map of differential metabolites, where each pie chart in the figure represents different HMDB classifications with various colors, and the area of each segment indicates the relative proportion of metabolites in that classification. (p) Metabolite VIP bubble chart: Comparison of amino acid expression levels between OENbAS-B and WT. (q) Heatmap of targeted amino acid metabolism expression in WT and NbAS-B-OE. (r-s) Validation of the expression levels of key amino acids Gln, Glu in the NbAS-B pathway based on metabolome data.\nFig. 6 PGANP enhances the induced resistance to TMV-GFP by responding to SA signals (a) Viral infection of heart-leaves after being sprayed with different concentrations of PGANP. (b) Virus expression detected by qPCR. (c) The content of Glu after spraying PGANP. (d) Effect of PGANP on the content of SA. (e)Disease symptoms in nbglr3.3mutant plants after PGANP treatment and TMV-GFP infection. (f)Viral accumulation measured by qPCR in PGANP-treated nbglr3.3mutant. (g–i) Expression levels of SA signaling-related genes (NbICS1, NbNPR1, NbPR1, NbPR2) in nbglr3.3mutant analyzed by qPCR.\nComment 5. Induction of host resistance by PGANPs was evaluated at 3 and 7 days post-treatment in this assay. Do the authors think that the time course for this assay was enough to evaluate long-lasting dual effect of PGANPs on antiviral response and plant growth? Did the authors observe any intensification of TMV-induced symptoms after withdrawal of effect of PGANPs in WT Nb plants?\nResponse: We thank this reviewer for his/her suggestions and constructive comments. We further monitored the induced resistance efficacy and the impact on plant growth and development in tobacco plants treated with PGANP from 8 to 21 days post-inoculation (dpi) with TMV-GFP. As shown in Figure S14, the induced resistance effect of PGANP remained evident at 14 dpi, but diminished by 21 dpi. Notably, PGANP-treated plants exhibited more vigorous growth compared to the control group. While WT plants developed noticeable leaf necrosis symptoms in the later stages, PGANP-treated plants maintained relatively healthy foliage.\nFigure. S14 Phenotypic evaluation of PGANP-mediated resistance against TMV-GFP in Nicotiana benthamiana.(a) Symptom development in plants pre-treated with PGANP or PGA followed by TMV-GFP challenge, photographed at 8, 14, and 21 days post-inoculation (dpi).(b) Plant growth and developmental phenotypes under the same treatment conditions at 8, 14, and 21 dpi.(c) qPCR analysis of viral accumulation in leaves at 8, 14, and 21 dpi.\n1. Response to the Reviewer #3\nComment 1. Abstract: The research GAP and what is NbAS-B, might be Nicotiana benthamiana asparagine synthetase-B, should be clearly stated in the Abstract.\nResponse: We thank this reviewer for the suggestion. We clearly stated in the Abstract that NbAS-B refers to the N. benthamiana asparagine synthetase gene B.\nComment 2. Key words: What is Transcriptional metabolome?\nResponse: We thank this reviewer for the question. Transcriptional metabolome refers to the transcriptome and metabolome, and it has been corrected to \"Transcriptome and metabolome.\"\nComment 3. Introduction: Lines 86-87, the authors stated the research gap “However, the precise mechanism by which NbAS-B regulates SA synthesis remains unresolved”, but the manuscript has not completely answered this question according to figure 7 (w), which should be further revised.\nResponse: We thank this reviewer for his/her comment. Regarding the mode NbAS-B affects SA signaling, our study found that NbAS-B primarily influences the synthesis of downstream Glu. It has been reported that Glu can affect Ca²⁺ flow, thereby responding to the SA signaling pathway. This study primarily reveals that PGANP can be degraded by plant proteases to produce Glu, which subsequently triggers plant immune responses. Additionally, Glu promotes plant growth by influencing nitrogen transport (Figure. 8k).\nComment 2. Results and Discussion: (a) Figure 1h, i, and j, longth and stem longth are difficult to understand. (b) Similarly, Figure 1k and l, y-axis might be fresh/dry weight (g/plant), not (g). (c) Also, according to the catalytic mechanism of NbAS-B, whose products, besides Glu, should include Asn, which might take part in tobacco growth and resistance to TMV, but Asn content in figure 2 i and m was not seen (note, figure legend was described).Also, ammonia (NH3) as a signaling molecule, the conversion of Gln and Asp into Glu and Asn under the catalysis of AS is involved in the change in mmonia (NH3) signaling, which should be mentioned. (d) For tobacco plants, Figure 4, the content (not concentration) of chlorophyll a, b, and total chlorophyll should be mg/g fresh weight (FW) or g/ g FW, not g/ L; correspondingly, figure 4 h and i, as well as figure 6f, might be 100 g FW and g FW, respectively. (e) Figures 3, 6, and 7, what is DFMTI or CS, whose full names is not seen in the whole manuscript. Also, in figure 3, L-asparaginase, as an asparagine inhibitor, is a protein, how it enters into the plant cells should be stated in the text.\nResponse: We thank this reviewer for his/her comment.\n(a) We have changed \"longth\" to \"length\" in Fig. 1.\n(b) The unit for fresh/dry weight has been corrected from \"(g)\" to \"(g/plant)\".\n(c) Regarding the detection of asparagine and aspartic acid content, we observed an increase in asparagine levels (Fig. S5). As for the disease resistance effect of asparagine, exogenous application of the inhibitor L-asparaginase revealed that it does not play a dominant role in NbAS-B-induced resistance against TMV; instead, Glu has a more significant effect (Fig. 3b). Furthermore, the main text highlights that ammonia (NH₃), as a signaling molecule, is involved in the catalytic process of asparagine synthetase (AS), where glutamine (Gln) and aspartic acid (Asp) are converted to glutamate (Glu) and asparagine (Asn), reflecting changes in ammonia signaling.\n(d) The units for chlorophyll a, b, and total chlorophyll in Figure 4 have been updated to \"μg/g FW\", while the units in Figures 4h and 4i have been corrected to \"(100g FW)\".\n(e) We have provided the full names of DFMTI (4,5-difluoro-2-methoxy-N-(4-phenylthiazol-2-yl)benzamide) and CS (chitosan) in the experimental materials. L-asparaginase, an inhibitor of asparagine, is a protein that may be absorbed by plants through stomatal pathways.\nComment 5. Methods: Many subtitles in the METHODS have not described methods but results, which should be carefully revised. For examples, “Induction effect of transgenic plants expressing asparagine synthetase B on TMV-GFP”, “Asparagine synthetase B enhances plant growth and development”, “Antiviral effects of glutamate and PGA”, “Inductive effects of PGANP”, and so on.\nResponse: We thank this reviewer for his/her suggestions. We have carefully revised the subheadings throughout the Experimental Methods section, changing previously conclusive statements to descriptive language about the experimental methods.\nComment 6. Writing: The language should be further concise. For example, after the full names and abbreviations of some terms were represented in the first position, the abbreviations only were used subsequently, including them in figures, such as AS, SA, Glu, Gln, Asp, Asn, and so on. Also, the seeds in line 154; abscisic acid biosynthesis (SnRK2 genes) in line 189, which should be abscisic acid signaling; Asp, Gln, Glu, and Asn in line 230, Asp and Asn should be deleted according to figure 2 l and m, and so on.\nResponse: We thank this reviewer for his/her suggestions. We have carefully reviewed the formatting throughout the entire paper. For terms appearing for the first time with their full names and abbreviations, subsequent mentions have been corrected to use the abbreviations only. Additionally, \"abscisic acid biosynthesis\" has been revised to \"abscisic acid signaling,\" and references to \"Asp and Asn,\" which were not discussed, were deleted."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have provided satisfactory responses to my comments. In the revised manuscript, they have supplemented sufficient experimental data to address the previously raised concerns, and the results are convincing. I deem that the revised manuscript has met the standards of Nature Communications and recommend its acceptance.", "reviewer_2": "In the revised manuscript, NbAS-B has been proven to induce resistance against TRV-GFP, TuMV-GFP, and PVX-GFP infections in Nicotiana benthamiana (Nb), indicating induction of antiviral resistance encompassing wide range of plant virus species. The authors additionally constructed knockout mutants of nbas-b-as-b to show significant increase in TMV level in comparison with WT Nb plants. The authors also extended monitoring of the induced resistance and plant growth of Nb after treatment with PGANP, resulting in persistence of the PGANP-induced resistance at 14 days post inoculation (dpi) and vigorous growth of the plants at 21 dpi.\nOverall, the results from additional experiments in the revised manuscript meet requirements suggested in my comments."}, "author_response": ""}]

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