A dual-functional laccase gene CsLAC17 from tea plant: Insights from VIGS and heterologous expression into lignin-mediated resistance and direct antifungal activity

A dual-functional laccase gene CsLAC17 from tea plant: Insights from VIGS and heterologous expression into lignin-mediated resistance and direct antifungal activity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A dual-functional laccase gene CsLAC17 from tea plant: Insights from VIGS and heterologous expression into lignin-mediated resistance and direct antifungal activity YuFeng Hu, Yichen Zhao This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9122877/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Tea ( Camellia sinensis (L.) O. Kuntze) is a globally significant beverage crop, yet its production is frequently compromised by various fungal diseases, therefore, enhancing its natural disease resistance is of great importance. Laccases (LACs) are key enzymes in plant defense responses. Here, we identified and functionally characterized a tea laccase gene, CsLAC17 , which has a full length of 1758 bp and encodes 585 amino acids. Silencing CsLAC17 in tea via virus-induced gene silencing (VIGS) significantly compromised resistance to anthracnose, accompanied by elevated MDA content. Its overexpression in tobacco increased lignin content and boosted resistance to Botrytis cinerea . Furthermore, the purified CsLAC17 protein, heterologously expressed in yeast, directly inhibited the growth of B. cinerea in vitro. These results indicate that CsLAC17 enhances plant systemic resistance by regulating lignin biosynthesis, and that its encoded protein also possesses direct antifungal activity. This finding not only provides new insights into tea disease resistance mechanisms but also offers a dual target for breeding disease-resistant cultivars and developing novel green fungicides. Camellia sinensis laccase Heterologous expression Pichia pastoris VIGS Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Key message This study first expresses CsLAC17 protein, functionally characterizing its dual role in enhancing disease resistance via lignin biosynthesis and direct antifungal activity, offering a target for breeding and green fungicides. 1 Introduction The tea plant ( Camellia sinensis (L.) O. Kuntze) is an evergreen woody species of the genus Camellia in the family Theaceae, native to Southwest China (Xia et al. 2017; Lu et al. 2021; Fang et al. 2021). Its young shoots and leaves are the primary raw materials for tea, the world’s most widely consumed non-alcoholic beverage (Wei et al. 2018; She et al. 2022). Consequently, the tea industry serves as a vital economic pillar for numerous producing nations and holds multifaceted significance in economic, medicinal, and cultural spheres (Xu et al. 2021; Pan et al. 2022). For numerous tea-producing countries, holding profound importance in economic, pharmaceutical, and cultural spheres. However, sustainable tea production is severely threatened by fungal and bacterial pathogens that cause devastating diseases such as tea anthracnose ( Colletotrichum spp.), blister blight ( Exobasidium vexans ), and brown blight ( Pestalotiopsis spp.). These diseases lead to drastic reductions in both yield and quality, resulting in substantial economic losses (Chen et al. 2025; Pandey et al. 2021; Samynathan et al. 2021). Currently, chemical fungicides are predominantly used in tea plantations to control tea plant diseases. This practice increases resistance and fungicides residues, leading to environmental pollution and compromising the safety of tea products (Yamada et al. 2016; Borah et al. 2022). Therefore, elucidating the mechanisms of disease resistance in tea plants and developing novel, eco-friendly control agents represent critical priorities for the sustainable advancement of the tea industry. Laccases, the largest subfamily of multicopper oxidases (MCOs), are widely distributed in plants, fungi, bacteria, and insects (Yu et al. 2021). Laccases possess three conserved catalytic domains (Cu-oxidase, Cu-oxidase_2, and Cu-oxidase_3) that bind four copper ions and exhibit broad substrate specificity (Arregui et al. 2019; Giardina et al. 2010). In nature, laccases play pivotal roles in organic matter cycling and ecosystem balance by mediating wood degradation, pigment formation, and antioxidant processes (Mayer and Staples 2002; Baldrian 2006). Their robust oxidative capacity also confers significant industrial application value, particularly in the textile, paper industry, and food processing sectors, where they are utilized for pollutant degradation, pulp bleaching, decolorization of dye wastewater, and product quality enhancement (Khatami et al. 2022; Bhardwaj et al. 2022). Plant laccases are localized in the cell walls of various higher plants. They were first identified in 1883 from the exudates of the Japanese lacquer tree Rhus vernicifera , and their presence in fungi was confirmed shortly thereafter (Yoshida 1883; Bertrand 1896). Laccases have been extensively reported to participate in lignin biosynthesis and enhance plant defense against stress, playing indispensable roles in plant growth and development. For instance, AtLAC4 and AtLAC17 are involved in the constitutive lignification of Arabidopsis thaliana stems. Simultaneous disruption of AtLAC4 , AtLAC11 , and AtLAC17 almost completely abolished lignin deposition, resulting in severe growth stagnation, reduced root diameter, indehiscent anthers, and impaired lignified vascular development (Berthet et al. 2011; Zhao et al. 2013). In Gossypium hirsutum , overexpression of GhLAC1 and GhLAC15 increased lignin accumulation, thereby enhancing cotton's resistance to the fungal pathogen Verticillium dahliae (Hu et al. 2018; Zhang et al. 2019). Overexpression of Phyllostachys edulis PeLAC10 in Arabidopsis elevated lignin content in transgenic plants, improving their drought tolerance and resistance to phenolic acids (Li et al. 2020b). Silencing of wheat TaLAC4 using VIGS increased susceptibility to Fusarium graminearum and reduced total lignin deposition, suggesting a potential role for TaLAC4 in defense-induced guaiacyl (G) lignin synthesis (Soni et al. 2020). Furthermore, PlLAC4 participates in lignin biosynthesis in Paeonia lactiflora , promoting dry lignin accumulation and secondary cell wall thickening (Tang et al. 2023). Transformation of tobacco with EuLAC1 from Eucommia ulmoides resulted in transgenic plants with higher lignin content and enhanced resistance to B. cinerea (Zhao et al. 2022). Collectively, these studies demonstrate that laccases play critical roles in plant growth, development, and stress responses primarily through the regulation of lignin biosynthesis. Laccase-mediated lignification is a crucial defense strategy for broad-spectrum disease resistance in plants. However, in the economically important crop tea plant, the disease resistance functions of the laccase gene family remain to be systematically elucidated. Previous studies have shown that the overexpression of CsLAC17 significantly increases lignin content and enhances resistance to gray blight in transgenic Arabidopsis (Yang et al. 2024). Additionally, the laccase gene CsLAC37 has been implicated in the tea plant’s response to fungal infection (Li et al. 2024). These findings suggest that laccase genes play crucial roles in tea plant disease resistance. Nevertheless, systematic studies on the protein expression and functional mechanisms of plant laccase proteins remain scarce. In this study, we focused on CsLAC17 , obtaining its purified protein through gene cloning and in vitro heterologous expression to explore the protein's resistance to anthracnose in tea plant. Furthermore, we systematically validated the biological function of CsLAC17 through heterologous expression in tobacco and gene silencing techniques in tea plant. These findings will contribute to a deeper understanding of the role of CsLAC17 protein in tea plant disease resistance, providing a scientific basis for developing laccase-based green pesticides, and ultimately promote the coordinated development of disease resistance and quality in tea plant. 2 Materials and Methods 2.1 Experimental materials The materials used in this experiment include two-year-old "Fudingdabai" tea plant cultivated at the College of Tea Science of Guizhou University. Additional experimental materials consist of Nicotiana benthamiana , Pichia pastoris strain GS115, Agrobacterium tumefaciens strain GV3101, Escherichia coli strain DH5α , viral vectors pTRV1 and pTRV2, as well as the plant expression vector pCAMBIA1300-35S-GUS, all provided by the Biotechnology Laboratory of Guizhou University. The pathogens utilized in this experiment include B. cinerea , a gray mold fungus from the genus Botrytis , and Glomerella cingulata , which is the causative agent of anthracnose. 2.2 Gene cloning Primers were designed based on the reference sequence of laccase gene from the tea plant transcriptome database. Using tea plant cDNA as a template, amplification was performed with Platinum® Taq DNA High Fidelity Polymerase (Invitrogen). The amplification primers were: Forward Primer (F): ATGGCTACTTATGTTCTTCTCT and Reverse Primer (R): ACATTTCGGAAGATCAGACG. The reaction system consisted of 25.0 µL 2× High Fidelity PCR Buffer, 1.0 µL dNTP mix (10 mM), 2.0 µL 50 mM MgSO 4 , 1.5 µL forward and reverse primers (10 µM), 5.0 µL cDNA, 1.0 µL Platinum® Taq High Fidelity (1 U/µL), and 13.0 µL PCR-grade water. The reaction program was as follows: pre-denaturation at 95 ℃ for 2 min, denaturation at 95 ℃ for 25 s, annealing at 55 ℃ for 25 s, extension at 68 ℃ for 90 s, for a total of 35 cycles, and finally extension at 68 ℃ for 5 min. 2.3 VIGS vector construction and tea plant infection This study employed Tobacco rattle virus (TRV) as a vector for Virus-Induced Gene Silencing (VIGS) to create a silencing vector targeting the CsLAC17 gene in tea plant. A 300 bp segment of CsLAC17 gene was chosen as the target sequence, and specific primers with homologous arms were designed for amplification using high-fidelity polymerase PCR. Concurrently, the pTRV2 empty vector underwent double digestion with EcoR I and Xho I. The purified PCR product was ligated to the linearized pTRV2 vector, which was then transformed into competent Escherichia coli cells. Following plasmid extraction and PCR verification, the recombinant plasmid was introduced into Agrobacterium tumefaciens strain GV3101 through electroporation. After colony PCR confirmation, the transformed bacteria were stored at -80°C for further use.The VIGS experiment was conducted according to the method described by Li et al. (2022). 2.4 Nicotiana benthamiana plant transformation and identification Design primers with homologous arms to clone laccase gene and perform double digestion with Sac I and Xba I on the pCAMBIA1300-35S-GUS vector. The digested laccase gene was ligated with the vector, and the recombinant vector, along with empty vector (EV), was transferred into Agrobacterium GV3101 using the freeze-thaw method. Colony PCR was then executed to verify positive clones. Co-cultivate the leaf discs with the Agrobacterium infection solution to generate transgenic tobacco explants. Further selection of resistant tobacco leaves was achieved through GUS histochemical staining. qRT-PCR was used to screen for transgenic lines with the highest expression levels of CsLAC17 , and tissue culture propagation of tobacco leaf segments was conducted to obtain plants with the same genetic background, ensuring the accuracy of subsequent experiments. 2.5 qRT-PCR analysis Leaves from the transgenic tobacco lines TP1 to TP11 were collected. To ensure sample quality and the accuracy of subsequent experiments, the leaf samples were rapidly frozen in liquid nitrogen and stored in a − 80 ℃ freezer. Total RNA was extracted and reverse transcribed into cDNA. According to the manufacturer's instructions, qRT-PCR was performed using the SYBR® Select Master Mix kit (Applied Biosystems Inc., Foster, CA, USA). The β-actin gene was used as the reference gene, and each sample included three biological replicates and three technical replicates. 2.6 Enzyme Activity Detection Infected tea plant and tobacco leaves were used to detect the activities of peroxidase (POD), catalase (CAT), superoxide dismutase (SOD), and malondialdehyde (MDA). The POD assay kit (BC0090), CAT assay kit (BC0200), SOD assay kit (BC5165), and MDA assay kit (BC0020) were used for the assays, following the manufacturer’s instructions (Beijing Solarbio Science & Technology Co., Ltd). In brief, 0.1 g sample was weighed and placed on ice, followed by the addition of 1 ml of extraction buffer for homogenization on ice. The mixture was then centrifuged at 8000 g for 10 min at 4°C, and the supernatant was collected for subsequent analysis. 2.7 Pathogen Inoculation and Treatment The preserved strains were inoculated onto PDA solid medium and incubated in the darkness at 28°C for 5–7 d. Appropriate leaves were selected, and their surfaces were disinfected with 75% ethanol and sterile distilled water. 5 mm thick colonies were then obtained from the edge of the fungal growth area and inoculated onto scratched leaf surface with the mycelium facing downward. Sterile PDA medium blocks and non-inoculated pathogens were used as control. Inoculated leaves were enclosed with plastic wrap and placed in a constant temperature incubator at 26°C for subsequent assessment of disease development at different leaf positions. The size of the leaf lesions was measured using Image J 1.53t software, with three biological replicates established for each treatment and at least 10 leaves inoculated per replicate. 2.8 Codon optimization DNA 2.0 software was used to optimize the codon of the tea plant CsLAC17 gene based on the Pichia pastoris codon usage preference table (Zhang et al. 2021). The optimized gene sequence was synthesized by GenScript Biology Company. 2.9 Vector construction and preparation of yeast engineering bacteria Restriction endonuclease sites EcoR I and Not I with protective bases were added to both ends of the tea plant CsLAC17 gene sequence, and the signal peptide encoding the first 33 amino acids at the N-terminus was removed. The vector pPIC9K and the gene were double-digested, and T4 DNA ligase was used for ligation. The resulting products were transformed into Escherichia coli . For detailed methodologies, refer to Liu et al. (2021b). Following the method of Pei et al. (2018), the constructed vector was digested with Sac I single enzyme, and the linear plasmid was transferred into competent yeast GS115 cells via electroporation. Single colonies grown on MD plate were subsequently inoculated onto MM plate and incubated at 30 ℃ for three days. Colonies that grow normally on MD plate but poor or no growth on MM plate are Muts type, while the remaining are Mut + type. After screening for Mut + recombinant Pichia pastoris , genomic DNA was extracted for PCR verification. 2.10 Induced expression of recombinant Pichia pastoris Ten positive clones were selected and inoculated in 10 mL YPD medium, and cultured at 28.5 ℃ with shaking at 220 rpm until reaching the logarithmic growth phase to prepare seed solution. A portion of 200 µL liquid was inoculated into 200 mL of BMGY medium, and continued to be cultured at the same condition until OD600 = 5. After centrifugation, the obtained cells were resuspended in 200 mL of BMMY medium and induced at 220 rpm and 28.5 ℃ in the presence of 1% methanol for 70 h. Protein expression was analyzed using the SDS-PAGE method, with results recorded by a Bio-Rad gel electrophoresis imager. 2.11 Fermentation condition optimization An appropriate volume of seed solution was inoculated into 100 mL of BMGY medium in a shake flask and incubated at 30 ℃ with shaking at 250 rpm for 20 h. After centrifugation, the cells were transferred to 100 mL of BMMY medium, adjusting the initial OD600 = 1. To induce laccase gene expression, Cu 2+ was added to achieve a final concentration of 0.5 mM, and methanol was added every 24 h to a final concentration of 1%. The culture was maintained at 250 rpm. To optimize fermentation conditions, various factors were considered, including induction time (0 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h), initial induction pH (2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6), induction temperature (20 ℃, 28 ℃, 30 ℃), and methanol concentration (0.25%, 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%). 2.12 Isolation and purification of CsLAC17 Protein To preserve protein activity, the protein was concentrated via ammonium sulfate precipitation. The target protein was precipitated by slowly adding ammonium sulfate solution to the supernatant to achieve 65% saturation. The mixture was sealed and incubated at 4 ℃ overnight for complete precipitation. The precipitate was then collected by centrifugation at 10,000 rpm for 10 min at 4 ℃ and redissolved in ddH₂O. To remove excess salt ions that could interfere with subsequent purification, the solution was dialyzed using a dialysis bag with a molecular weight cutoff of 3000 Mw, following the protocol described by Scheich et al. (2003). As the expression vector contained a histidine tag, the recombinant protein was further purified using a nickel-affinity column according to the method outlined by Sun (2020). 2.13 Laccase Enzyme Assay Laccase activity was determined using ABTS as the substrate. The reaction mixture contained 100 mM sodium acetate buffer (pH 4.0) and 0.5 mM ABTS. The reaction was initiated by adding 100 µL of appropriately diluted enzyme solution to 900 µL of the reaction mixture. The increase in absorbance at 420 nm was recorded every 30 s for 3 min using a spectrophotometer. One unit (U) of enzyme activity was defined as the amount of enzyme required to oxidize 1 µmol of ABTS per minute. The molar extinction coefficient of ABTS at 420 nm was taken as 36,000 M⁻¹ cm⁻¹. All assays were performed in triplicate. To evaluate the effect of metal ions on laccase activity, buffers containing different metal ions (Mg 2+ , K + , Fe 2+ , Cu 2+ , Zn 2+ , Fe 3+ ) at a concentration of 10 mmol/L were prepared. The tea plant laccase solution, prepared in citric acid buffer, was mixed with the respective metal ion buffers and incubated in a water bath at 40 ℃ for 30 min. A control group without the addition of any metal ions was included. All experiments were performed in triplicate. The enzyme activity of the control group was defined as 100%, and the activity of the experimental groups was expressed as a percentage relative to the control. 2.14 Statistical analysis All experiments were performed with at least three independent biological replicates. The value of each physiological index was expressed as the mean ± standard error (± SE). All data were verified by the Shapiro-Wilk test and the Levene test confirmed homogeneity of variance. Data that match normality and homogeneity of variance were compared between groups using one-way ANOVA (assuming test significance level α = 0.05) and were supplemented with the Tukey HSD test for post hoc multiple comparison corrections. 3 Results 3.1 Silencing of CsLAC17 compromises tea plant resistance to anthracnose In this study, we cloned the laccase gene CsLAC17 from the cDNA of tea plants, with a complete coding sequence (CDS) of 1758 bp, encoding 585 amino acids (Fig. S1). We employed Virus-Induced Gene Silencing (VIGS) to explore the role of laccase gene CsLAC17 in tea plant defense against anthracnose. The pTRV2- CsLAC17 vector was constructed using a specific fragment from a conserved region of the gene (Fig. S2). The VIGS system was first validated by the typical albino phenotype on new leaves (Fig. 1a), and the successful silencing of CsLAC17 was confirmed by qRT-PCR, which showed a significant reduction in its transcript levels compared to empty vector controls (Fig. 1b). Pathogenicity assays demonstrated that CsLAC17 -silenced plants were more susceptible to the pathogen, exhibiting significantly larger lesions than controls (Fig. 1c). To investigate the underlying physiological mechanism, we measured the activities of key protective enzymes. We found that the activities of SOD, POD, and CAT were significantly decreased, whereas MDA content, a marker of membrane damage, was significantly elevated in silenced plants (Fig. 1d). These findings indicate that CsLAC17 silencing weakens the plant’s reactive oxygen species (ROS) scavenging ability, thereby exacerbating pathogen-induced oxidative damage and ultimately compromising disease resistance. 3.2 CsLAC17 overexpression boosts tobacco resistance to B. cinerea To investigate the functional role of CsLAC17 in plant defense, we generated transgenic tobacco lines. Transgene integration was preliminarily confirmed by GUS staining (Fig. S3a), and the line with the highest expression level, designated tp-6, was identified by qPCR and propagated for subsequent analyses (Fig. S3b). Consistent with its elevated transcript level, the tp-6 line exhibited significantly higher laccase activity compared to wild-type (WT) and empty vector control (CK) plants, confirming the successful expression of functional CsLAC17 protein (Fig. 2a). We then evaluated the resistance of these plants to B. cinerea . Following inoculation, WT and CK plants developed typical water-soaked lesions that expanded rapidly over 1, 3, 5, and 7 dpi (Fig. 2b). In contrast, the TP displayed markedly milder disease symptoms and smaller lesion diameters at 7 dpi compared to controls (Fig. 2c). To elucidate the underlying mechanisms, we examined two key defense pathways. qPCR analysis showed that the transcript levels of key genes in the lignin biosynthesis pathway were significantly upregulated in TP plants compared to WT and CK (Fig. 2d). This finding corresponded with higher lignin content (Fig. 2f), suggesting that CsLAC17 may reinforce the cell wall by promoting lignin deposition. Simultaneously, the activities of protective enzymes (SOD, POD, CAT) were significantly elevated, while MDA content was notably reduced (Fig. 2e). This suggests that transgenic plants may suffer less membrane damage and lipid peroxidation induced by pathogens compared to control group. Collectively, these findings demonstrate that overexpressing CsLAC17 in tobacco confers enhanced resistance to B. cinerea through a dual mechanism: strengthening the physical barrier via lignin biosynthesis and enhancing biochemical defenses through the antioxidant system. 3.3 Codon optimization of CsLAC17 gene To accommodate the codon usage preferences of Pichia pastoris , we optimized the nucleotide sequence of the gene while maintaining the amino acid sequence unchanged. The specific optimization sites are indicated in the red regions of Fig. S4a, where an EcoR I restriction site was introduced at the N-terminus and a Not I restriction site was introduced at the C-terminus. The optimized sequence was synthesized by GenScript Biology Company. Subsequently, we constructed a recombinant plasmid of the target gene using the pPIC9K vector and transformed it into Escherichia coli . The successful construction of the recombinant vector was confirmed through double digestion analysis (Fig. S4b). 3.4 Induced expression of yeast The target protein was expressed using Pichia pastoris . The recombinant plasmid containing the target gene was successfully introduced into Pichia pastoris GS115 receptor cells, yielding a distinct PCR band of 1500–2000 bp, which confirmed the presence of multiple Mut + positive clones (Fig. S5). Following this, the engineered yeast cells were induced for protein expression, and the crude enzyme produced during fermentation was concentrated and dialyzed, followed by protein separation and purification using Ni-NTA. The purified protein was then analyzed by SDS-PAGE, which revealed a single band, indicating effective protein expression and confirming the successful preparation of the engineered yeast strain (Fig. 3). Moreover, the molecular weight of the expressed target protein was between 53–73 kDa, with an approximate value of 61 kDa, which aligns with the predicted molecular weight of 61 kDa. 3.5 Optimization of fermentation conditions of recombinant Pichia pastoris We determined the optimal conditions for expressing laccase in Pichia pastoris by optimizing induction time, initial pH, induction temperature, and methanol usage, while considering both the OD600-based growth profile and the enzyme activity (Fig. 4). Through systematic optimization of fermentation parameters, the optimal conditions for inducing laccase expression in Pichia pastoris were as follows: induction time of 96 h, initial pH of 4.5, induction temperature of 20°C, and methanol addition of 1.0%. The activity of the laccase expressed in Pichia pastoris under optimized conditions was determined by the ABTS assay. The enzyme showed a maximum activity of 2.58 U/mL, confirming the functionality of the purified protein . 3.6 Effect of metal ions on CsLAC17 activity and the antifungal effect of the enzyme preparation The effects of different metal ions on the activity of recombinant CsLAC17 were investigated. Cu 2+ significantly enhanced enzyme activity by 11.0%, whereas Fe 3+ , Zn 2+ and Fe 2+ strongly inhibited the enzymatic activity. Notably, Zn 2+ exhibited the most pronounced inhibitory effect, reducing the activity by 69.9%. Substantial inhibition was also observed with Fe 3+ and Fe 2+ (Fig. 5a). To evaluate the inhibitory effect of the laccase reagent prepared with copper sulfate, a filter paper inhibition assay was conducted. The treatments were as follows: (1) positive control treated with 1 mg/mL fluorescein solution; (2) 10 mg/mL laccase solution prepared with 0.01 M copper sulfate, which significantly inhibited B. cinerea growth; (3) 0.02 M copper sulfate solution alone, which showed no significant effect; and (4) 10 mg/mL laccase solution alone, which also had no significant effect on B. cinerea growth (Fig. 5b). These results demonstrate that the 10 mg/mL laccase reagent formulated with 0.01 M copper sulfate possesses inhibitory activity against B. cinerea . 4 Discussion As a vital economic crop, the healthy growth and disease resistance of the tea plant are crucial for the sustainable development of the tea industry. Lignin serves as a critical physical barrier against pathogens, and our previous research identified laccases—the key enzymes catalyzing lignin polymerization—as essential players in this defense mechanism (Zhao et al. 2022). Accordingly, we hypothesized that CsLAC17 also contributes to the plant’s defense responses. However, functional characterization in tea plants is challenging due to their perennial nature, self-incompatibility, and the lack of reliable stable transformation systems (Mukhopadhyay et al. 2016). Traditional breeding cycles are long, further limiting breeding efficiency. VIGS has emerged as a powerful tool for functional genomics in species recalcitrant to transformation, having been successfully applied in species such as Populus euphratica , Narcissus tazetta L., Jatropha curcas , and Malus crabapple (Li et al. 2022), yet it remains underutilized in tea. In this study, we successfully employed a VIGS system to dissect the function of CsLAC17 . Findings from both overexpression in tobacco and silencing in tea plants consistently demonstrate that CsLAC17 is essential for fortifying plant defenses by positively regulating lignin synthesis. Upon stress exposure, plants initiate a complex defense network, in which an early signal response centered on a reactive oxygen species (ROS) burst plays a crucial role (Tyagi et al. 2022). Since ROS are both key defense signals and cytotoxic, maintaining redox homeostasis between their production and scavenging is essential. This homeostasis is primarily regulated by antioxidant enzymes such as SOD, POD, and CAT. The extent of oxidative imbalance can be quantified by measuring MDA, a product of membrane lipid peroxidation (Mhamdi et al. 2018). In this study, we demonstrate that overexpression of CsLAC17 in transgenic tobacco significantly bolsters antioxidant capacity following pathogen infection. This was evidenced by markedly increased activities of SOD, POD, and CAT, coupled with reduced MDA content. Conversely, silencing CsLAC17 in tea plants compromised their defense, manifesting as diminished antioxidant enzyme activities and severe membrane lipid peroxidation. This phenomenon is not merely an isolated case in tea plants but points to a conserved function of the laccase gene family. For instance, silencing another tea plant laccase gene, CsLAC37 , using antisense oligonucleotide (AsODN) technology similarly resulted in increased H₂O₂ content and decreased POD activity, suggesting that laccases may play a conserved and important role in tea plant defense against anthracnose (Li et al. 2024). Overexpression of PeuLAC2 in poplar enhanced the antioxidant system’s activity, effectively protecting cell membranes from damage and improving the plant’s stress resistance (Niu et al. 2021). Similarly, tobacco plants overexpressing CsLAC18 exhibited significantly higher activities of CAT, SOD, and POD following cold stress compared to wild-type plants. Notably, the overexpression lines also exhibited higher laccase activity and lignin content, whereas silencing this gene in trifoliate orange ( Poncirus trifoliata ) via VIGS presented the opposite phenotype (Xu et al. 2022). Lignin deposition in the secondary cell wall provides a critical physical barrier against pathogen invasion (Boerjan et al. 2003). Its biosynthesis is a complex, multi-step process that begins with the phenylpropanoid pathway, generating three monolignols (H, G, and S) that are subsequently oxidatively polymerized by peroxidases and laccases to form the final lignin polymer (Vanholme et al. 2010; Zhao and Dixon 2011). In this study, we observed a significant upregulation of multiple key genes in the lignin biosynthesis pathway. The coordinated induction of these genes strongly suggests that the entire lignification process is activated as a defense response. The role of PAL and C4H in defense is well-established. For example, overexpression of CsPAL enhances resistance to blister blight infection (Nisha et al. 2018). In pear, PbC4H1 and PbC4H3 play key roles in lignin biosynthesis (Li et al. 2020a). Similarly, 4CL mediates the activation of various hydroxycinnamic acids, and suppression of Os4CL3 expression results in a significant reduction in lignin content, impaired plant growth, and other morphological changes (Gui et al. 2011). COMT and CCoAOMT are essential for methylation steps; in maize, ZmCCoAOMT2 is potentially involved in the biosynthesis of lignin and other phenylpropanoid metabolites (Yang et al. 2017). Overexpression of OsCOMT5 promotes lignin accumulation in rice and enhances tolerance to R. solani (Kaur et al. 2025). CCR and CAD catalyze the final stages of monolignol synthesis; overexpression of BnCCR2 in B. napus increases lignin content in the stem, thereby enhancing resistance to S. sclerotiorum (Liu et al. 2021a). Likewise, overexpression of TaCAD12 significantly enhances resistance to sharp eyespot in transgenic wheat lines (Rong et al. 2016). Taken together, the upregulation of this suite of genes in the present study indicates a comprehensive reinforcement of the lignin biosynthesis pathway, thereby fortifying the cell wall as a primary defense strategy against pathogen invasion. Plant laccases are ubiquitous polyphenol oxidases that contain four copper ions in their catalytic center. In this study, a laccase gene was cloned from tea plant and expressed in Pichia pastoris . Through the optimization of fermentation conditions to achieve high-level production of the recombinant protein, purified laccase was successfully obtained, which exhibited direct inhibitory activity against B. cinerea . As an integral component of the laccase active center, copper ions exert a significant influence on both the expression and activity of the enzyme. To further investigate the effects of inorganic ions on tea plant laccase activity, the enzyme solution was incubated with buffers containing various metal ions. The results indicated that Mg²⁺, Cu²⁺, and K⁺ activated the laccase, whereas Zn²⁺, Fe²⁺, and Fe³⁺ inhibited its activity. Consistent with these findings, previous research has shown that supplementing the culture medium of the white-rot fungus Pleurotus ostreatus with copper ions (0.5–5 mM) significantly enhances laccase activity and stability, with 1 mM Cu²⁺ resulting in an 8-fold increase in activity (Baldrian and Gabriel 2002). Similarly, Kang et al. (2024) demonstrated that 10 mM K⁺ improved the activity of Hericium erinaceus laccase, whereas 5 mM Fe²⁺ exhibited an inhibitory effect. Furthermore, Mg²⁺ and Cu²⁺ have been shown to promote the activity of DcLac1 and DcLac2 enzymes purified from carrot ( Daucus carota L.)(Ma et al. 2015). Notably, Mg²⁺ and K⁺ may maintain or enhance laccase activity by preserving the charge balance and the three-dimensional conformation of the protein structure. Conversely, Fe²⁺ and Fe³⁺ function as competitive inhibitors with similar modes of action; they can bind to the T1 site of laccase, thereby inhibiting enzyme activity by blocking substrate access to the T1 site or hindering electron transfer to the T1 active site (Umar et al, 2022). Collectively, our study positions CsLAC17 as a key node linking cell wall fortification and redox homeostasis in the plant defense network. For practical application, enhancing tea plant disease resistance relies on a combined approach: integrating traditional breeding with modern tools like Molecular Marker-Assisted Selection (MAS) and CRISPR/Cas9 gene editing for targeted cultivar improvement. 5 Conclusion In this study, we cloned CsLAC17 from Camellia sinensis and demonstrated, through complementary loss- and gain-of-function analyses, that CsLAC17 positively regulates antifungal resistance by coordinating cell-wall reinforcement and oxidative stress control. We further established a codon-optimized Pichia pastoris expression system to produce active CsLAC17 and characterize its regulation by metal ions. Notably, Cu²⁺ acted as a key activator, enabling the enzyme preparation to exhibit antifungal activity. This highlights a Cu-responsive laccase module with potential applications in resistance engineering and laccase-based biocontrol development. Collectively, these findings provide a promising target for breeding disease-resistant tea cultivars and developing eco-friendly control measures. Declarations Data availability All data presented in this research are available in the article and supplementary materials, further inquiries can be directed to the corresponding author. Funding This work was supported by the National Natural Science Foundation of China (Grant numbers [32160077]) and by the National Guiding Foundation for Local Science and Technology Development of China (Grant numbers [2023–009]). Competing Interests The authors have no relevant financial or non-financial interests to disclose. Author Contributions Yufeng Hu: Writing-original draft, Conceptualization, Methodology, Investigation, Formal analysis, Data curation. Yichen Zhao: Writing-review & editing, Writing-original draft, Conceptualization, Methodology, Project administration, Funding acquisition. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9122877","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":610768483,"identity":"4d28f550-0b60-40cb-b3f0-bdd4a427b38d","order_by":0,"name":"YuFeng Hu","email":"","orcid":"","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"YuFeng","middleName":"","lastName":"Hu","suffix":""},{"id":610768484,"identity":"1d9dde4a-e2bf-4d71-9955-b112b232e044","order_by":1,"name":"Yichen Zhao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxElEQVRIiWNgGAWjYHACNiC2gTB5SNCSRrqWwyRoMTh+/NmDHxXn7ebPSGB88LaNQd6coJYzCemGPWduJzfOSGA2nNvGYLizgZCWGwzHJHjbbiczSySwSfO2MSQYHCCohbFN8m/buWQ2iQT230RqYQYZfsCOB2gLM1FaJM+ksUnLnElOkOB52Cw555yE4QZCWviAISb5psLOXr49+eCHN2U28gRtUYAqSGxgYGwA0hIE1AOBfAOEtiesdBSMglEwCkYsAAAaEDy6E5JzGwAAAABJRU5ErkJggg==","orcid":"","institution":"Guizhou University","correspondingAuthor":true,"prefix":"","firstName":"Yichen","middleName":"","lastName":"Zhao","suffix":""}],"badges":[],"createdAt":"2026-03-14 13:25:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9122877/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9122877/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105381942,"identity":"59a8bab5-91dc-4841-8002-8485b1ca0f3d","added_by":"auto","created_at":"2026-03-25 11:29:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":770492,"visible":true,"origin":"","legend":"\u003cp\u003eImpact of silencing \u003cem\u003eCsLAC17\u003c/em\u003e on tea plant susceptibility to \u003cem\u003eGlomerella cingulata\u003c/em\u003e. (\u003cstrong\u003ea\u003c/strong\u003e) Phenotype of \u003cem\u003eCsLAC17\u003c/em\u003e-silenced tea plants. TRV:\u003cem\u003eCsPDS\u003c/em\u003e(Positive Control), WT (wild-type), TRV:EV (Negative Control), TRV:\u003cem\u003eCsLAC17. \u003c/em\u003e(b) Disease symptoms on WT, EV, and \u003cem\u003eCsLAC17\u003c/em\u003e-silenced tea plants at 7 dpi with \u003cem\u003eGlomerella cingulata\u003c/em\u003e. (c)Verification of \u003cem\u003eCsLAC17\u003c/em\u003e gene silencing efficiency by qRT-PCR. (d) Measurement of protective enzyme (SOD, POD, CAT) activities and MDA content in silenced, WT, and control plants. Note: Different lowercase letters represent significant differences (\u003cem\u003eP\u003c/em\u003e＜0.05)\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9122877/v1/792110f8ae9d442ceef849c2.png"},{"id":105381947,"identity":"a576abeb-5e33-4eb3-8219-8468a1025c4c","added_by":"auto","created_at":"2026-03-25 11:29:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2069845,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eCsLAC17\u003c/em\u003e overexpression confers enhanced resistance to gray mold in transgenic tobacco. (a) Measurement of laccase activity in transgenic tobacco. (b) Disease symptoms on WT, CK, and TP plants at 1, 3, 5, and 7 dpi with \u003cem\u003eBotrytis cinerea\u003c/em\u003e. (c) Lesion diameter on tobacco leaves at 7 dpi with \u003cem\u003eBotrytis cinerea\u003c/em\u003e. (d) qRT-PCR analysis of key genes in the lignin biosynthesis pathway. (e) Enzymatic activities of SOD, POD, and CAT, along with MDA content. (f) Lignin contents in leaves of wild-type and transgenic tobacco plants. Note: Different lowercase letters represent significant differences (\u003cem\u003eP\u003c/em\u003e＜0.05)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9122877/v1/7d16ec66853800e2e696d0f6.png"},{"id":105565387,"identity":"df5cdb16-3a20-4994-a99c-3e3c31ff5fe3","added_by":"auto","created_at":"2026-03-27 12:53:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":137477,"visible":true,"origin":"","legend":"\u003cp\u003eExpression and identification of \u003cem\u003eCsLAC17\u003c/em\u003e laccase protein. Note: M: Protein marker; 1: Purified protein bands\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9122877/v1/f0d8d9d4d372b5a39dbf8118.png"},{"id":105381944,"identity":"af08e6f5-0b6f-4ce7-8d8b-5d4f32c9c68d","added_by":"auto","created_at":"2026-03-25 11:29:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":355754,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of induction time (\u003cstrong\u003ea\u003c/strong\u003e), fermentation temperature (\u003cstrong\u003eb\u003c/strong\u003e), initial PH (\u003cstrong\u003ec\u003c/strong\u003e), and methanol addition (\u003cstrong\u003ed\u003c/strong\u003e) on the growth and enzyme production of \u003cem\u003epichia pastoris\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eNote: Different lowercase letters represent significant differences (\u003cem\u003eP\u003c/em\u003e＜0.05)\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9122877/v1/539d77af9df6b2a88b321b94.png"},{"id":105565980,"identity":"95d310a4-99a1-42bf-9fe9-6ca415855b33","added_by":"auto","created_at":"2026-03-27 12:54:56","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":286198,"visible":true,"origin":"","legend":"\u003cp\u003eResults of expanded culture of\u0026nbsp; \u003cem\u003eCsLAC17\u003c/em\u003e laccase produced by yeast engineering bacteria. (\u003cstrong\u003ea\u003c/strong\u003e) Effect of metal ions on the activity of tea plant laccase. (\u003cstrong\u003eb\u003c/strong\u003e) Study on antifungal effect of laccase by filter paper method Note: 1: 1 mg/ml fluconazole; 2: 0.01M CuSO\u003csub\u003e4\u003c/sub\u003e 10 mg/mL laccase; 3: 0.01M CuSO\u003csub\u003e4\u003c/sub\u003e; 4: 10mg/mL laccase; Bar: 1 cm\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9122877/v1/a6d98e3972b41973a58df5ea.png"},{"id":105570074,"identity":"56cf307b-b71f-42ba-bafd-9c36ddf89ccb","added_by":"auto","created_at":"2026-03-27 13:14:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5419643,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9122877/v1/0a30b3c3-2af0-4d2f-8b27-ca82e6eb910e.pdf"},{"id":105381945,"identity":"84294336-48b7-4779-aa1d-3507b7396ce4","added_by":"auto","created_at":"2026-03-25 11:29:09","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":836629,"visible":true,"origin":"","legend":"","description":"","filename":"1Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-9122877/v1/c82370fa30cb6c7b7faa0d8f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A dual-functional laccase gene CsLAC17 from tea plant: Insights from VIGS and heterologous expression into lignin-mediated resistance and direct antifungal activity","fulltext":[{"header":"Key message","content":"\u003cp\u003eThis study first expresses CsLAC17 protein, functionally characterizing its dual role in enhancing disease resistance via lignin biosynthesis and direct antifungal activity, offering a target for breeding and green fungicides.\u003c/p\u003e"},{"header":"1 Introduction","content":"\u003cp\u003eThe tea plant (\u003cem\u003eCamellia sinensis\u003c/em\u003e (L.) O. Kuntze) is an evergreen woody species of the genus Camellia in the family Theaceae, native to Southwest China (Xia et al. 2017; Lu et al. 2021; Fang et al. 2021). Its young shoots and leaves are the primary raw materials for tea, the world\u0026rsquo;s most widely consumed non-alcoholic beverage (Wei et al. 2018; She et al. 2022). Consequently, the tea industry serves as a vital economic pillar for numerous producing nations and holds multifaceted significance in economic, medicinal, and cultural spheres (Xu et al. 2021; Pan et al. 2022). For numerous tea-producing countries, holding profound importance in economic, pharmaceutical, and cultural spheres. However, sustainable tea production is severely threatened by fungal and bacterial pathogens that cause devastating diseases such as tea anthracnose (\u003cem\u003eColletotrichum\u003c/em\u003e spp.), blister blight (\u003cem\u003eExobasidium vexans\u003c/em\u003e), and brown blight (\u003cem\u003ePestalotiopsis\u003c/em\u003e spp.). These diseases lead to drastic reductions in both yield and quality, resulting in substantial economic losses (Chen et al. 2025; Pandey et al. 2021; Samynathan et al. 2021). Currently, chemical fungicides are predominantly used in tea plantations to control tea plant diseases. This practice increases resistance and fungicides residues, leading to environmental pollution and compromising the safety of tea products (Yamada et al. 2016; Borah et al. 2022). Therefore, elucidating the mechanisms of disease resistance in tea plants and developing novel, eco-friendly control agents represent critical priorities for the sustainable advancement of the tea industry.\u003c/p\u003e \u003cp\u003eLaccases, the largest subfamily of multicopper oxidases (MCOs), are widely distributed in plants, fungi, bacteria, and insects (Yu et al. 2021). Laccases possess three conserved catalytic domains (Cu-oxidase, Cu-oxidase_2, and Cu-oxidase_3) that bind four copper ions and exhibit broad substrate specificity (Arregui et al. 2019; Giardina et al. 2010). In nature, laccases play pivotal roles in organic matter cycling and ecosystem balance by mediating wood degradation, pigment formation, and antioxidant processes (Mayer and Staples 2002; Baldrian 2006). Their robust oxidative capacity also confers significant industrial application value, particularly in the textile, paper industry, and food processing sectors, where they are utilized for pollutant degradation, pulp bleaching, decolorization of dye wastewater, and product quality enhancement (Khatami et al. 2022; Bhardwaj et al. 2022). Plant laccases are localized in the cell walls of various higher plants. They were first identified in 1883 from the exudates of the Japanese lacquer tree \u003cem\u003eRhus vernicifera\u003c/em\u003e, and their presence in fungi was confirmed shortly thereafter (Yoshida 1883; Bertrand 1896). Laccases have been extensively reported to participate in lignin biosynthesis and enhance plant defense against stress, playing indispensable roles in plant growth and development. For instance, \u003cem\u003eAtLAC4\u003c/em\u003e and \u003cem\u003eAtLAC17\u003c/em\u003e are involved in the constitutive lignification of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e stems. Simultaneous disruption of \u003cem\u003eAtLAC4\u003c/em\u003e, \u003cem\u003eAtLAC11\u003c/em\u003e, and \u003cem\u003eAtLAC17\u003c/em\u003e almost completely abolished lignin deposition, resulting in severe growth stagnation, reduced root diameter, indehiscent anthers, and impaired lignified vascular development (Berthet et al. 2011; Zhao et al. 2013). In \u003cem\u003eGossypium hirsutum\u003c/em\u003e, overexpression of \u003cem\u003eGhLAC1\u003c/em\u003e and \u003cem\u003eGhLAC15\u003c/em\u003e increased lignin accumulation, thereby enhancing cotton's resistance to the fungal pathogen \u003cem\u003eVerticillium dahliae\u003c/em\u003e (Hu et al. 2018; Zhang et al. 2019). Overexpression of \u003cem\u003ePhyllostachys edulis PeLAC10\u003c/em\u003e in \u003cem\u003eArabidopsis\u003c/em\u003e elevated lignin content in transgenic plants, improving their drought tolerance and resistance to phenolic acids (Li et al. 2020b). Silencing of wheat \u003cem\u003eTaLAC4\u003c/em\u003e using VIGS increased susceptibility to \u003cem\u003eFusarium graminearum\u003c/em\u003e and reduced total lignin deposition, suggesting a potential role for \u003cem\u003eTaLAC4\u003c/em\u003e in defense-induced guaiacyl (G) lignin synthesis (Soni et al. 2020). Furthermore, \u003cem\u003ePlLAC4\u003c/em\u003e participates in lignin biosynthesis in \u003cem\u003ePaeonia lactiflora\u003c/em\u003e, promoting dry lignin accumulation and secondary cell wall thickening (Tang et al. 2023). Transformation of tobacco with \u003cem\u003eEuLAC1\u003c/em\u003e from \u003cem\u003eEucommia ulmoides\u003c/em\u003e resulted in transgenic plants with higher lignin content and enhanced resistance to \u003cem\u003eB. cinerea\u003c/em\u003e (Zhao et al. 2022). Collectively, these studies demonstrate that laccases play critical roles in plant growth, development, and stress responses primarily through the regulation of lignin biosynthesis.\u003c/p\u003e \u003cp\u003eLaccase-mediated lignification is a crucial defense strategy for broad-spectrum disease resistance in plants. However, in the economically important crop tea plant, the disease resistance functions of the laccase gene family remain to be systematically elucidated. Previous studies have shown that the overexpression of \u003cem\u003eCsLAC17\u003c/em\u003e significantly increases lignin content and enhances resistance to gray blight in transgenic \u003cem\u003eArabidopsis\u003c/em\u003e (Yang et al. 2024). Additionally, the laccase gene \u003cem\u003eCsLAC37\u003c/em\u003e has been implicated in the tea plant\u0026rsquo;s response to fungal infection (Li et al. 2024). These findings suggest that laccase genes play crucial roles in tea plant disease resistance. Nevertheless, systematic studies on the protein expression and functional mechanisms of plant laccase proteins remain scarce. In this study, we focused on \u003cem\u003eCsLAC17\u003c/em\u003e, obtaining its purified protein through gene cloning and in vitro heterologous expression to explore the protein's resistance to anthracnose in tea plant. Furthermore, we systematically validated the biological function of \u003cem\u003eCsLAC17\u003c/em\u003e through heterologous expression in tobacco and gene silencing techniques in tea plant. These findings will contribute to a deeper understanding of the role of CsLAC17 protein in tea plant disease resistance, providing a scientific basis for developing laccase-based green pesticides, and ultimately promote the coordinated development of disease resistance and quality in tea plant.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Experimental materials\u003c/h2\u003e \u003cp\u003eThe materials used in this experiment include two-year-old \"Fudingdabai\" tea plant cultivated at the College of Tea Science of Guizhou University. Additional experimental materials consist of \u003cem\u003eNicotiana benthamiana\u003c/em\u003e, \u003cem\u003ePichia pastoris\u003c/em\u003e strain GS115, \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain GV3101, \u003cem\u003eEscherichia coli\u003c/em\u003e strain \u003cem\u003eDH5α\u003c/em\u003e, viral vectors pTRV1 and pTRV2, as well as the plant expression vector pCAMBIA1300-35S-GUS, all provided by the Biotechnology Laboratory of Guizhou University. The pathogens utilized in this experiment include \u003cem\u003eB. cinerea\u003c/em\u003e, a gray mold fungus from the genus \u003cem\u003eBotrytis\u003c/em\u003e, and \u003cem\u003eGlomerella cingulata\u003c/em\u003e, which is the causative agent of anthracnose.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Gene cloning\u003c/h2\u003e \u003cp\u003ePrimers were designed based on the reference sequence of laccase gene from the tea plant transcriptome database. Using tea plant cDNA as a template, amplification was performed with Platinum\u0026reg; Taq DNA High Fidelity Polymerase (Invitrogen). The amplification primers were: Forward Primer (F): ATGGCTACTTATGTTCTTCTCT and Reverse Primer (R): ACATTTCGGAAGATCAGACG. The reaction system consisted of 25.0 \u0026micro;L 2\u0026times; High Fidelity PCR Buffer, 1.0 \u0026micro;L dNTP mix (10 mM), 2.0 \u0026micro;L 50 mM MgSO\u003csub\u003e4\u003c/sub\u003e, 1.5 \u0026micro;L forward and reverse primers (10 \u0026micro;M), 5.0 \u0026micro;L cDNA, 1.0 \u0026micro;L Platinum\u0026reg; Taq High Fidelity (1 U/\u0026micro;L), and 13.0 \u0026micro;L PCR-grade water. The reaction program was as follows: pre-denaturation at 95 ℃ for 2 min, denaturation at 95 ℃ for 25 s, annealing at 55 ℃ for 25 s, extension at 68 ℃ for 90 s, for a total of 35 cycles, and finally extension at 68 ℃ for 5 min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 VIGS vector construction and tea plant infection\u003c/h2\u003e \u003cp\u003eThis study employed Tobacco rattle virus (TRV) as a vector for Virus-Induced Gene Silencing (VIGS) to create a silencing vector targeting the \u003cem\u003eCsLAC17\u003c/em\u003e gene in tea plant. A 300 bp segment of \u003cem\u003eCsLAC17\u003c/em\u003e gene was chosen as the target sequence, and specific primers with homologous arms were designed for amplification using high-fidelity polymerase PCR. Concurrently, the pTRV2 empty vector underwent double digestion with \u003cem\u003eEcoR\u003c/em\u003e I and \u003cem\u003eXho\u003c/em\u003e I. The purified PCR product was ligated to the linearized pTRV2 vector, which was then transformed into competent \u003cem\u003eEscherichia coli\u003c/em\u003e cells. Following plasmid extraction and PCR verification, the recombinant plasmid was introduced into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain GV3101 through electroporation. After colony PCR confirmation, the transformed bacteria were stored at -80\u0026deg;C for further use.The VIGS experiment was conducted according to the method described by Li et al. (2022).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 \u003cem\u003eNicotiana benthamiana\u003c/em\u003e plant transformation and identification\u003c/h2\u003e \u003cp\u003eDesign primers with homologous arms to clone laccase gene and perform double digestion with \u003cem\u003eSac\u003c/em\u003e I and \u003cem\u003eXba\u003c/em\u003e I on the pCAMBIA1300-35S-GUS vector. The digested laccase gene was ligated with the vector, and the recombinant vector, along with empty vector (EV), was transferred into \u003cem\u003eAgrobacterium\u003c/em\u003e GV3101 using the freeze-thaw method. Colony PCR was then executed to verify positive clones. Co-cultivate the leaf discs with the \u003cem\u003eAgrobacterium\u003c/em\u003e infection solution to generate transgenic tobacco explants. Further selection of resistant tobacco leaves was achieved through GUS histochemical staining. qRT-PCR was used to screen for transgenic lines with the highest expression levels of \u003cem\u003eCsLAC17\u003c/em\u003e, and tissue culture propagation of tobacco leaf segments was conducted to obtain plants with the same genetic background, ensuring the accuracy of subsequent experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 qRT-PCR analysis\u003c/h2\u003e \u003cp\u003eLeaves from the transgenic tobacco lines TP1 to TP11 were collected. To ensure sample quality and the accuracy of subsequent experiments, the leaf samples were rapidly frozen in liquid nitrogen and stored in a\u0026thinsp;\u0026minus;\u0026thinsp;80 ℃ freezer. Total RNA was extracted and reverse transcribed into cDNA. According to the manufacturer's instructions, qRT-PCR was performed using the SYBR\u0026reg; Select Master Mix kit (Applied Biosystems Inc., Foster, CA, USA). The β-actin gene was used as the reference gene, and each sample included three biological replicates and three technical replicates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Enzyme Activity Detection\u003c/h2\u003e \u003cp\u003eInfected tea plant and tobacco leaves were used to detect the activities of peroxidase (POD), catalase (CAT), superoxide dismutase (SOD), and malondialdehyde (MDA). The POD assay kit (BC0090), CAT assay kit (BC0200), SOD assay kit (BC5165), and MDA assay kit (BC0020) were used for the assays, following the manufacturer\u0026rsquo;s instructions (Beijing Solarbio Science \u0026amp; Technology Co., Ltd). In brief, 0.1 g sample was weighed and placed on ice, followed by the addition of 1 ml of extraction buffer for homogenization on ice. The mixture was then centrifuged at 8000 g for 10 min at 4\u0026deg;C, and the supernatant was collected for subsequent analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Pathogen Inoculation and Treatment\u003c/h2\u003e \u003cp\u003eThe preserved strains were inoculated onto PDA solid medium and incubated in the darkness at 28\u0026deg;C for 5\u0026ndash;7 d. Appropriate leaves were selected, and their surfaces were disinfected with 75% ethanol and sterile distilled water. 5 mm thick colonies were then obtained from the edge of the fungal growth area and inoculated onto scratched leaf surface with the mycelium facing downward. Sterile PDA medium blocks and non-inoculated pathogens were used as control. Inoculated leaves were enclosed with plastic wrap and placed in a constant temperature incubator at 26\u0026deg;C for subsequent assessment of disease development at different leaf positions. The size of the leaf lesions was measured using Image J 1.53t software, with three biological replicates established for each treatment and at least 10 leaves inoculated per replicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Codon optimization\u003c/h2\u003e \u003cp\u003eDNA 2.0 software was used to optimize the codon of the tea plant \u003cem\u003eCsLAC17\u003c/em\u003e gene based on the \u003cem\u003ePichia pastoris\u003c/em\u003e codon usage preference table (Zhang et al. 2021). The optimized gene sequence was synthesized by GenScript Biology Company.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Vector construction and preparation of yeast engineering bacteria\u003c/h2\u003e \u003cp\u003eRestriction endonuclease sites \u003cem\u003eEcoR\u003c/em\u003e I and \u003cem\u003eNot\u003c/em\u003e I with protective bases were added to both ends of the tea plant \u003cem\u003eCsLAC17\u003c/em\u003e gene sequence, and the signal peptide encoding the first 33 amino acids at the N-terminus was removed. The vector pPIC9K and the gene were double-digested, and T4 DNA ligase was used for ligation. The resulting products were transformed into \u003cem\u003eEscherichia coli\u003c/em\u003e. For detailed methodologies, refer to Liu et al. (2021b). Following the method of Pei et al. (2018), the constructed vector was digested with \u003cem\u003eSac\u003c/em\u003e I single enzyme, and the linear plasmid was transferred into competent yeast GS115 cells via electroporation. Single colonies grown on MD plate were subsequently inoculated onto MM plate and incubated at 30 ℃ for three days. Colonies that grow normally on MD plate but poor or no growth on MM plate are Muts type, while the remaining are Mut\u003csup\u003e+\u003c/sup\u003e type. After screening for Mut\u003csup\u003e+\u003c/sup\u003e recombinant \u003cem\u003ePichia pastoris\u003c/em\u003e, genomic DNA was extracted for PCR verification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Induced expression of recombinant \u003cem\u003ePichia pastoris\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eTen positive clones were selected and inoculated in 10 mL YPD medium, and cultured at 28.5 ℃ with shaking at 220 rpm until reaching the logarithmic growth phase to prepare seed solution. A portion of 200 \u0026micro;L liquid was inoculated into 200 mL of BMGY medium, and continued to be cultured at the same condition until OD600\u0026thinsp;=\u0026thinsp;5. After centrifugation, the obtained cells were resuspended in 200 mL of BMMY medium and induced at 220 rpm and 28.5 ℃ in the presence of 1% methanol for 70 h. Protein expression was analyzed using the SDS-PAGE method, with results recorded by a Bio-Rad gel electrophoresis imager.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Fermentation condition optimization\u003c/h2\u003e \u003cp\u003eAn appropriate volume of seed solution was inoculated into 100 mL of BMGY medium in a shake flask and incubated at 30 ℃ with shaking at 250 rpm for 20 h. After centrifugation, the cells were transferred to 100 mL of BMMY medium, adjusting the initial OD600\u0026thinsp;=\u0026thinsp;1. To induce laccase gene expression, Cu\u003csup\u003e2+\u003c/sup\u003e was added to achieve a final concentration of 0.5 mM, and methanol was added every 24 h to a final concentration of 1%. The culture was maintained at 250 rpm. To optimize fermentation conditions, various factors were considered, including induction time (0 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h), initial induction pH (2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6), induction temperature (20 ℃, 28 ℃, 30 ℃), and methanol concentration (0.25%, 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Isolation and purification of CsLAC17 Protein\u003c/h2\u003e \u003cp\u003eTo preserve protein activity, the protein was concentrated via ammonium sulfate precipitation. The target protein was precipitated by slowly adding ammonium sulfate solution to the supernatant to achieve 65% saturation. The mixture was sealed and incubated at 4 ℃ overnight for complete precipitation. The precipitate was then collected by centrifugation at 10,000 rpm for 10 min at 4 ℃ and redissolved in ddH₂O. To remove excess salt ions that could interfere with subsequent purification, the solution was dialyzed using a dialysis bag with a molecular weight cutoff of 3000 Mw, following the protocol described by Scheich et al. (2003). As the expression vector contained a histidine tag, the recombinant protein was further purified using a nickel-affinity column according to the method outlined by Sun (2020).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13 Laccase Enzyme Assay\u003c/h2\u003e \u003cp\u003eLaccase activity was determined using ABTS as the substrate. The reaction mixture contained 100 mM sodium acetate buffer (pH 4.0) and 0.5 mM ABTS. The reaction was initiated by adding 100 \u0026micro;L of appropriately diluted enzyme solution to 900 \u0026micro;L of the reaction mixture. The increase in absorbance at 420 nm was recorded every 30 s for 3 min using a spectrophotometer. One unit (U) of enzyme activity was defined as the amount of enzyme required to oxidize 1 \u0026micro;mol of ABTS per minute. The molar extinction coefficient of ABTS at 420 nm was taken as 36,000 M⁻\u0026sup1; cm⁻\u0026sup1;. All assays were performed in triplicate.\u003c/p\u003e \u003cp\u003eTo evaluate the effect of metal ions on laccase activity, buffers containing different metal ions (Mg\u003csup\u003e2+\u003c/sup\u003e, K\u003csup\u003e+\u003c/sup\u003e, Fe\u003csup\u003e2+\u003c/sup\u003e, Cu\u003csup\u003e2+\u003c/sup\u003e, Zn\u003csup\u003e2+\u003c/sup\u003e, Fe\u003csup\u003e3+\u003c/sup\u003e) at a concentration of 10 mmol/L were prepared. The tea plant laccase solution, prepared in citric acid buffer, was mixed with the respective metal ion buffers and incubated in a water bath at 40 ℃ for 30 min. A control group without the addition of any metal ions was included. All experiments were performed in triplicate. The enzyme activity of the control group was defined as 100%, and the activity of the experimental groups was expressed as a percentage relative to the control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.14 Statistical analysis\u003c/h2\u003e \u003cp\u003eAll experiments were performed with at least three independent biological replicates. The value of each physiological index was expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (\u0026plusmn;\u0026thinsp;SE). All data were verified by the Shapiro-Wilk test and the Levene test confirmed homogeneity of variance. Data that match normality and homogeneity of variance were compared between groups using one-way ANOVA (assuming test significance level α\u0026thinsp;=\u0026thinsp;0.05) and were supplemented with the Tukey HSD test for post hoc multiple comparison corrections.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec18\"\u003e\n \u003ch2\u003e3.1 Silencing of \u003cem\u003eCsLAC17\u003c/em\u003e compromises tea plant resistance to anthracnose\u003c/h2\u003e\n \u003cp\u003eIn this study, we cloned the laccase gene \u003cem\u003eCsLAC17\u003c/em\u003e from the cDNA of tea plants, with a complete coding sequence (CDS) of 1758 bp, encoding 585 amino acids (Fig. S1). We employed Virus-Induced Gene Silencing (VIGS) to explore the role of laccase gene \u003cem\u003eCsLAC17\u003c/em\u003e in tea plant defense against anthracnose. The pTRV2-\u003cem\u003eCsLAC17\u003c/em\u003e vector was constructed using a specific fragment from a conserved region of the gene (Fig. S2). The VIGS system was first validated by the typical albino phenotype on new leaves (Fig. 1a), and the successful silencing of \u003cem\u003eCsLAC17\u003c/em\u003e was confirmed by qRT-PCR, which showed a significant reduction in its transcript levels compared to empty vector controls (Fig. 1b). Pathogenicity assays demonstrated that \u003cem\u003eCsLAC17\u003c/em\u003e-silenced plants were more susceptible to the pathogen, exhibiting significantly larger lesions than controls (Fig. 1c). To investigate the underlying physiological mechanism, we measured the activities of key protective enzymes. We found that the activities of SOD, POD, and CAT were significantly decreased, whereas MDA content, a marker of membrane damage, was significantly elevated in silenced plants (Fig. 1d). These findings indicate that \u003cem\u003eCsLAC17\u003c/em\u003e silencing weakens the plant’s reactive oxygen species (ROS) scavenging ability, thereby exacerbating pathogen-induced oxidative damage and ultimately compromising disease resistance.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\"\u003e\n \u003ch2\u003e3.2 CsLAC17 overexpression boosts tobacco resistance to \u003cem\u003eB. cinerea\u003c/em\u003e\u003c/h2\u003e\n \u003cp\u003eTo investigate the functional role of \u003cem\u003eCsLAC17\u003c/em\u003e in plant defense, we generated transgenic tobacco lines. Transgene integration was preliminarily confirmed by GUS staining (Fig. S3a), and the line with the highest expression level, designated tp-6, was identified by qPCR and propagated for subsequent analyses (Fig. S3b). Consistent with its elevated transcript level, the tp-6 line exhibited significantly higher laccase activity compared to wild-type (WT) and empty vector control (CK) plants, confirming the successful expression of functional CsLAC17 protein (Fig. 2a). We then evaluated the resistance of these plants to \u003cem\u003eB. cinerea\u003c/em\u003e. Following inoculation, WT and CK plants developed typical water-soaked lesions that expanded rapidly over 1, 3, 5, and 7 dpi (Fig. 2b). In contrast, the TP displayed markedly milder disease symptoms and smaller lesion diameters at 7 dpi compared to controls (Fig. 2c). To elucidate the underlying mechanisms, we examined two key defense pathways. qPCR analysis showed that the transcript levels of key genes in the lignin biosynthesis pathway were significantly upregulated in TP plants compared to WT and CK (Fig. 2d). This finding corresponded with higher lignin content (Fig. 2f), suggesting that \u003cem\u003eCsLAC17\u003c/em\u003e may reinforce the cell wall by promoting lignin deposition. Simultaneously, the activities of protective enzymes (SOD, POD, CAT) were significantly elevated, while MDA content was notably reduced (Fig. 2e). This suggests that transgenic plants may suffer less membrane damage and lipid peroxidation induced by pathogens compared to control group. Collectively, these findings demonstrate that overexpressing \u003cem\u003eCsLAC17\u003c/em\u003e in tobacco confers enhanced resistance to \u003cem\u003eB. cinerea\u003c/em\u003e through a dual mechanism: strengthening the physical barrier via lignin biosynthesis and enhancing biochemical defenses through the antioxidant system.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\"\u003e\n \u003ch2\u003e3.3 Codon optimization of \u003cem\u003eCsLAC17\u003c/em\u003e gene\u003c/h2\u003e\n \u003cp\u003eTo accommodate the codon usage preferences of \u003cem\u003ePichia pastoris\u003c/em\u003e, we optimized the nucleotide sequence of the gene while maintaining the amino acid sequence unchanged. The specific optimization sites are indicated in the red regions of Fig. S4a, where an \u003cem\u003eEcoR\u003c/em\u003e I restriction site was introduced at the N-terminus and a \u003cem\u003eNot\u003c/em\u003e I restriction site was introduced at the C-terminus. The optimized sequence was synthesized by GenScript Biology Company. Subsequently, we constructed a recombinant plasmid of the target gene using the pPIC9K vector and transformed it into \u003cem\u003eEscherichia coli\u003c/em\u003e. The successful construction of the recombinant vector was confirmed through double digestion analysis (Fig. S4b).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\"\u003e\n \u003ch2\u003e3.4 Induced expression of yeast\u003c/h2\u003e\n \u003cp\u003eThe target protein was expressed using \u003cem\u003ePichia pastoris\u003c/em\u003e. The recombinant plasmid containing the target gene was successfully introduced into \u003cem\u003ePichia pastoris\u003c/em\u003e GS115 receptor cells, yielding a distinct PCR band of 1500–2000 bp, which confirmed the presence of multiple Mut\u003csup\u003e+\u003c/sup\u003e positive clones (Fig. S5). Following this, the engineered yeast cells were induced for protein expression, and the crude enzyme produced during fermentation was concentrated and dialyzed, followed by protein separation and purification using Ni-NTA. The purified protein was then analyzed by SDS-PAGE, which revealed a single band, indicating effective protein expression and confirming the successful preparation of the engineered yeast strain (Fig. 3). Moreover, the molecular weight of the expressed target protein was between 53–73 kDa, with an approximate value of 61 kDa, which aligns with the predicted molecular weight of 61 kDa.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\"\u003e\n \u003ch2\u003e3.5 Optimization of fermentation conditions of recombinant \u003cem\u003ePichia pastoris\u003c/em\u003e\u003c/h2\u003e\n \u003cp\u003eWe determined the optimal conditions for expressing laccase in \u003cem\u003ePichia pastoris\u003c/em\u003e by optimizing induction time, initial pH, induction temperature, and methanol usage, while considering both the OD600-based growth profile and the enzyme activity (Fig. 4). Through systematic optimization of fermentation parameters, the optimal conditions for inducing laccase expression in \u003cem\u003ePichia pastoris\u003c/em\u003e were as follows: induction time of 96 h, initial pH of 4.5, induction temperature of 20°C, and methanol addition of 1.0%. The activity of the laccase expressed in \u003cem\u003ePichia pastoris\u003c/em\u003e under optimized conditions was determined by the ABTS assay. The enzyme showed a maximum activity of 2.58 U/mL, confirming the functionality of the purified protein .\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e3.6 Effect of metal ions on\u003c/strong\u003e \u003cstrong\u003eCsLAC17\u003c/strong\u003e \u003cstrong\u003eactivity and the antifungal effect of the enzyme preparation\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThe effects of different metal ions on the activity of recombinant CsLAC17 were investigated. Cu\u003csup\u003e2+\u003c/sup\u003e significantly enhanced enzyme activity by 11.0%, whereas Fe\u003csup\u003e3+\u003c/sup\u003e, Zn\u003csup\u003e2+\u003c/sup\u003e and Fe\u003csup\u003e2+\u003c/sup\u003e strongly inhibited the enzymatic activity. Notably, Zn\u003csup\u003e2+\u003c/sup\u003e exhibited the most pronounced inhibitory effect, reducing the activity by 69.9%. Substantial inhibition was also observed with Fe\u003csup\u003e3+\u003c/sup\u003e and Fe\u003csup\u003e2+\u003c/sup\u003e (Fig. 5a). To evaluate the inhibitory effect of the laccase reagent prepared with copper sulfate, a filter paper inhibition assay was conducted. The treatments were as follows: (1) positive control treated with 1 mg/mL fluorescein solution; (2) 10 mg/mL laccase solution prepared with 0.01 M copper sulfate, which significantly inhibited \u003cem\u003eB. cinerea\u003c/em\u003e growth; (3) 0.02 M copper sulfate solution alone, which showed no significant effect; and (4) 10 mg/mL laccase solution alone, which also had no significant effect on \u003cem\u003eB. cinerea\u003c/em\u003e growth (Fig. 5b). These results demonstrate that the 10 mg/mL laccase reagent formulated with 0.01 M copper sulfate possesses inhibitory activity against \u003cem\u003eB. cinerea\u003c/em\u003e.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eAs a vital economic crop, the healthy growth and disease resistance of the tea plant are crucial for the sustainable development of the tea industry. Lignin serves as a critical physical barrier against pathogens, and our previous research identified laccases\u0026mdash;the key enzymes catalyzing lignin polymerization\u0026mdash;as essential players in this defense mechanism (Zhao et al. 2022). Accordingly, we hypothesized that \u003cem\u003eCsLAC17\u003c/em\u003e also contributes to the plant\u0026rsquo;s defense responses. However, functional characterization in tea plants is challenging due to their perennial nature, self-incompatibility, and the lack of reliable stable transformation systems (Mukhopadhyay et al. 2016). Traditional breeding cycles are long, further limiting breeding efficiency. VIGS has emerged as a powerful tool for functional genomics in species recalcitrant to transformation, having been successfully applied in species such as \u003cem\u003ePopulus euphratica\u003c/em\u003e, \u003cem\u003eNarcissus tazetta\u003c/em\u003e L., \u003cem\u003eJatropha curcas\u003c/em\u003e, and \u003cem\u003eMalus crabapple\u003c/em\u003e (Li et al. 2022), yet it remains underutilized in tea. In this study, we successfully employed a VIGS system to dissect the function of \u003cem\u003eCsLAC17\u003c/em\u003e. Findings from both overexpression in tobacco and silencing in tea plants consistently demonstrate that \u003cem\u003eCsLAC17\u003c/em\u003e is essential for fortifying plant defenses by positively regulating lignin synthesis.\u003c/p\u003e \u003cp\u003eUpon stress exposure, plants initiate a complex defense network, in which an early signal response centered on a reactive oxygen species (ROS) burst plays a crucial role (Tyagi et al. 2022). Since ROS are both key defense signals and cytotoxic, maintaining redox homeostasis between their production and scavenging is essential. This homeostasis is primarily regulated by antioxidant enzymes such as SOD, POD, and CAT. The extent of oxidative imbalance can be quantified by measuring MDA, a product of membrane lipid peroxidation (Mhamdi et al. 2018). In this study, we demonstrate that overexpression of \u003cem\u003eCsLAC17\u003c/em\u003e in transgenic tobacco significantly bolsters antioxidant capacity following pathogen infection. This was evidenced by markedly increased activities of SOD, POD, and CAT, coupled with reduced MDA content. Conversely, silencing \u003cem\u003eCsLAC17\u003c/em\u003e in tea plants compromised their defense, manifesting as diminished antioxidant enzyme activities and severe membrane lipid peroxidation. This phenomenon is not merely an isolated case in tea plants but points to a conserved function of the laccase gene family. For instance, silencing another tea plant laccase gene, \u003cem\u003eCsLAC37\u003c/em\u003e, using antisense oligonucleotide (AsODN) technology similarly resulted in increased H₂O₂ content and decreased POD activity, suggesting that laccases may play a conserved and important role in tea plant defense against anthracnose (Li et al. 2024). Overexpression of \u003cem\u003ePeuLAC2\u003c/em\u003e in poplar enhanced the antioxidant system\u0026rsquo;s activity, effectively protecting cell membranes from damage and improving the plant\u0026rsquo;s stress resistance (Niu et al. 2021). Similarly, tobacco plants overexpressing \u003cem\u003eCsLAC18\u003c/em\u003e exhibited significantly higher activities of CAT, SOD, and POD following cold stress compared to wild-type plants. Notably, the overexpression lines also exhibited higher laccase activity and lignin content, whereas silencing this gene in trifoliate orange (\u003cem\u003ePoncirus trifoliata\u003c/em\u003e) via VIGS presented the opposite phenotype (Xu et al. 2022).\u003c/p\u003e \u003cp\u003eLignin deposition in the secondary cell wall provides a critical physical barrier against pathogen invasion (Boerjan et al. 2003). Its biosynthesis is a complex, multi-step process that begins with the phenylpropanoid pathway, generating three monolignols (H, G, and S) that are subsequently oxidatively polymerized by peroxidases and laccases to form the final lignin polymer (Vanholme et al. 2010; Zhao and Dixon 2011). In this study, we observed a significant upregulation of multiple key genes in the lignin biosynthesis pathway. The coordinated induction of these genes strongly suggests that the entire lignification process is activated as a defense response. The role of PAL and C4H in defense is well-established. For example, overexpression of \u003cem\u003eCsPAL\u003c/em\u003e enhances resistance to blister blight infection (Nisha et al. 2018). In pear, \u003cem\u003ePbC4H1\u003c/em\u003e and \u003cem\u003ePbC4H3\u003c/em\u003e play key roles in lignin biosynthesis (Li et al. 2020a). Similarly, 4CL mediates the activation of various hydroxycinnamic acids, and suppression of \u003cem\u003eOs4CL3\u003c/em\u003e expression results in a significant reduction in lignin content, impaired plant growth, and other morphological changes (Gui et al. 2011). COMT and CCoAOMT are essential for methylation steps; in maize, \u003cem\u003eZmCCoAOMT2\u003c/em\u003e is potentially involved in the biosynthesis of lignin and other phenylpropanoid metabolites (Yang et al. 2017). Overexpression of \u003cem\u003eOsCOMT5\u003c/em\u003e promotes lignin accumulation in rice and enhances tolerance to \u003cem\u003eR. solani\u003c/em\u003e (Kaur et al. 2025). CCR and CAD catalyze the final stages of monolignol synthesis; overexpression of \u003cem\u003eBnCCR2\u003c/em\u003e in \u003cem\u003eB. napus\u003c/em\u003e increases lignin content in the stem, thereby enhancing resistance to \u003cem\u003eS. sclerotiorum\u003c/em\u003e (Liu et al. 2021a). Likewise, overexpression of \u003cem\u003eTaCAD12\u003c/em\u003e significantly enhances resistance to sharp eyespot in transgenic wheat lines (Rong et al. 2016). Taken together, the upregulation of this suite of genes in the present study indicates a comprehensive reinforcement of the lignin biosynthesis pathway, thereby fortifying the cell wall as a primary defense strategy against pathogen invasion.\u003c/p\u003e \u003cp\u003ePlant laccases are ubiquitous polyphenol oxidases that contain four copper ions in their catalytic center. In this study, a laccase gene was cloned from tea plant and expressed in \u003cem\u003ePichia pastoris\u003c/em\u003e. Through the optimization of fermentation conditions to achieve high-level production of the recombinant protein, purified laccase was successfully obtained, which exhibited direct inhibitory activity against \u003cem\u003eB. cinerea\u003c/em\u003e. As an integral component of the laccase active center, copper ions exert a significant influence on both the expression and activity of the enzyme. To further investigate the effects of inorganic ions on tea plant laccase activity, the enzyme solution was incubated with buffers containing various metal ions. The results indicated that Mg\u0026sup2;⁺, Cu\u0026sup2;⁺, and K⁺ activated the laccase, whereas Zn\u0026sup2;⁺, Fe\u0026sup2;⁺, and Fe\u0026sup3;⁺ inhibited its activity. Consistent with these findings, previous research has shown that supplementing the culture medium of the white-rot fungus \u003cem\u003ePleurotus ostreatus\u003c/em\u003e with copper ions (0.5\u0026ndash;5 mM) significantly enhances laccase activity and stability, with 1 mM Cu\u0026sup2;⁺ resulting in an 8-fold increase in activity (Baldrian and Gabriel 2002). Similarly, Kang et al. (2024) demonstrated that 10 mM K⁺ improved the activity of \u003cem\u003eHericium erinaceus\u003c/em\u003e laccase, whereas 5 mM Fe\u0026sup2;⁺ exhibited an inhibitory effect. Furthermore, Mg\u0026sup2;⁺ and Cu\u0026sup2;⁺ have been shown to promote the activity of DcLac1 and DcLac2 enzymes purified from carrot (\u003cem\u003eDaucus carota\u003c/em\u003e L.)(Ma et al. 2015). Notably, Mg\u0026sup2;⁺ and K⁺ may maintain or enhance laccase activity by preserving the charge balance and the three-dimensional conformation of the protein structure. Conversely, Fe\u0026sup2;⁺ and Fe\u0026sup3;⁺ function as competitive inhibitors with similar modes of action; they can bind to the T1 site of laccase, thereby inhibiting enzyme activity by blocking substrate access to the T1 site or hindering electron transfer to the T1 active site (Umar et al, 2022).\u003c/p\u003e \u003cp\u003eCollectively, our study positions \u003cem\u003eCsLAC17\u003c/em\u003e as a key node linking cell wall fortification and redox homeostasis in the plant defense network. For practical application, enhancing tea plant disease resistance relies on a combined approach: integrating traditional breeding with modern tools like Molecular Marker-Assisted Selection (MAS) and CRISPR/Cas9 gene editing for targeted cultivar improvement.\u003c/p\u003e"},{"header":"5 Conclusion","content":"\u003cp\u003eIn this study, we cloned \u003cem\u003eCsLAC17\u003c/em\u003e from \u003cem\u003eCamellia sinensis\u003c/em\u003e and demonstrated, through complementary loss- and gain-of-function analyses, that \u003cem\u003eCsLAC17\u003c/em\u003e positively regulates antifungal resistance by coordinating cell-wall reinforcement and oxidative stress control. We further established a codon-optimized \u003cem\u003ePichia pastoris\u003c/em\u003e expression system to produce active CsLAC17 and characterize its regulation by metal ions. Notably, Cu\u0026sup2;⁺ acted as a key activator, enabling the enzyme preparation to exhibit antifungal activity. This highlights a Cu-responsive laccase module with potential applications in resistance engineering and laccase-based biocontrol development. Collectively, these findings provide a promising target for breeding disease-resistant tea cultivars and developing eco-friendly control measures.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data presented in this research are available in the article and supplementary materials, further inquiries can be directed to the corresponding author.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (Grant numbers [32160077]) and by the National Guiding Foundation for Local Science and Technology Development of China (Grant numbers [2023\u0026ndash;009]).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYufeng Hu:\u003c/strong\u003e Writing-original draft, Conceptualization, Methodology, Investigation, Formal analysis, Data curation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYichen Zhao:\u0026nbsp;\u003c/strong\u003eWriting-review \u0026amp; editing, Writing-original draft, Conceptualization, Methodology, Project administration, Funding acquisition.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eArregui L, Ayala M, G\u0026oacute;mez-Gil X, Guti\u0026eacute;rrez-Soto G, Hern\u0026aacute;ndez-Luna CE, Herrera de Los Santos M, Levin L, Rojo-Dom\u0026iacute;nguez A, Romero-Mart\u0026iacute;nez D, Saparrat MCN, Trujillo-Rold\u0026aacute;n MA, Valdez-Cruz NA (2019) Laccases: structure, function, and potential application in water bioremediation. 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Transgenic Res 31:215\u0026ndash;225. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11248-022-00297-8\u003c/span\u003e\u003cspan address=\"10.1007/s11248-022-00297-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-cell-tissue-and-organ-culture-pctoc","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcto","sideBox":"Learn more about [Plant Cell, Tissue and Organ Culture (PCTOC)](https://www.springer.com/journal/11240)","snPcode":"11240","submissionUrl":"https://submission.nature.com/new-submission/11240/3","title":"Plant Cell, Tissue and Organ Culture (PCTOC)","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Camellia sinensis, laccase, Heterologous expression, Pichia pastoris, VIGS","lastPublishedDoi":"10.21203/rs.3.rs-9122877/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9122877/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTea (\u003cem\u003eCamellia sinensis\u003c/em\u003e (L.) O. Kuntze) is a globally significant beverage crop, yet its production is frequently compromised by various fungal diseases, therefore, enhancing its natural disease resistance is of great importance. Laccases (LACs) are key enzymes in plant defense responses. Here, we identified and functionally characterized a tea laccase gene, \u003cem\u003eCsLAC17\u003c/em\u003e, which has a full length of 1758 bp and encodes 585 amino acids. Silencing \u003cem\u003eCsLAC17\u003c/em\u003e in tea via virus-induced gene silencing (VIGS) significantly compromised resistance to anthracnose, accompanied by elevated MDA content. Its overexpression in tobacco increased lignin content and boosted resistance to \u003cem\u003eBotrytis cinerea\u003c/em\u003e. Furthermore, the purified CsLAC17 protein, heterologously expressed in yeast, directly inhibited the growth of \u003cem\u003eB. cinerea\u003c/em\u003e in vitro. These results indicate that \u003cem\u003eCsLAC17\u003c/em\u003e enhances plant systemic resistance by regulating lignin biosynthesis, and that its encoded protein also possesses direct antifungal activity. This finding not only provides new insights into tea disease resistance mechanisms but also offers a dual target for breeding disease-resistant cultivars and developing novel green fungicides.\u003c/p\u003e","manuscriptTitle":"A dual-functional laccase gene CsLAC17 from tea plant: Insights from VIGS and heterologous expression into lignin-mediated resistance and direct antifungal activity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-25 11:29:04","doi":"10.21203/rs.3.rs-9122877/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-16T14:53:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"319926488404612514684180683157804655022","date":"2026-05-06T00:48:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"136096451039539764816102367624928226380","date":"2026-04-29T04:17:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-14T03:42:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"242358574394715810576842195888125714521","date":"2026-04-13T11:51:55+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-23T14:21:46+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-18T02:48:51+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-18T02:48:19+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell, Tissue and Organ Culture (PCTOC)","date":"2026-03-14T13:20:49+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-cell-tissue-and-organ-culture-pctoc","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcto","sideBox":"Learn more about [Plant Cell, Tissue and Organ Culture (PCTOC)](https://www.springer.com/journal/11240)","snPcode":"11240","submissionUrl":"https://submission.nature.com/new-submission/11240/3","title":"Plant Cell, Tissue and Organ Culture (PCTOC)","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"5f59f168-ac58-4761-9b0b-462a47bca4e3","owner":[],"postedDate":"March 25th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-16T14:53:55+00:00","index":20,"fulltext":""},{"type":"reviewerAgreed","content":"319926488404612514684180683157804655022","date":"2026-05-06T00:48:44+00:00","index":19,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-25T11:29:04+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-25 11:29:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9122877","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9122877","identity":"rs-9122877","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}