Boosting lactoferrin yields in Nicotiana tabacum via HC-Pro co-agroinfiltration

Abstract

Introduction

Plant-based recombinant protein production provides a safe and cost-effective method for producing functional medicinal proteins. However, transgene silencing, a natural defense mechanism in plants, often reduces protein production by causing transgenes to behave differently than expected. To overcome this issue, some viruses encode a Helper-Component Proteinase (HC-Pro) that can suppress RNA silencing.

Objectives

This study evaluated the potential of a Potato Virus A (PVA)-derived HC-Pro suppressor to enhance the expression of recombinant Camelus dromedarius lactoferrin (CdLF) in Nicotiana tabacum.

Methods

Constructs for expressing CdLF were generated using the pGWB17-PVA-HC-Pro and pART27 vectors [1]. A cDNA library from dromedary camel mammary glands was used to amplify the CdLF gene using specific primers and clone it into a pTZ57R vector (Thermo Fisher Scientific, Catalog No. K1213). This was then transformed into Escherichia coli (DH5α). N. tabacum plants were transformed using Agrobacterium tumefaciens. PCR confirmed the presence of the CdLF gene, and expression levels were measured by qRT-PCR. A cold extraction buffer was used for protein extraction, followed by ELISA to quantify lactoferrin levels.

Results and conclusion

Co-expression of the HC-Pro suppressor effectively increased recombinant CdLF production in transgenic plants. Transcriptional increases of 5.7-fold in wild-type agroinfiltrated plants and 3.6-fold in T1 plants, compared to controls, resulted in a 3.4-fold and 1.8-fold rise in recombinant CdLF protein production, respectively. Functional analysis also confirmed that the recombinant CdLF protein had significant antimicrobial properties. Overall, this study demonstrated that the HC-Pro suppressor, derived from the PVA virus, significantly enhances CdLF yield and has the potential to improve plant-based recombinant protein production.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-026-08465-2.

Introduction

Lactoferrin, first discovered in 1939 during research into the role of iron in milk [2], is a versatile protein that, due to its iron-binding capabilities, was initially named lactotransferrin and later renamed lactoferrin [3]. This protein, a key member of the transferrin family, is recognized for its vital role in iron transport and absorption, as well as its antimicrobial, anti-inflammatory, and immunomodulatory properties [4, 5]. Lactoferrin consists of two structural lobes, the N-lobe and the C-lobe, each featuring an iron-binding site. It is found in mammalian biological fluids, including milk, saliva, tears, and nasal secretions [6].

Lactoferrin’s iron-binding capacity is not exclusive to iron. However, it extends to a diverse range of other biological molecules, including heparin, glycosaminoglycans, and lipopolysaccharides [7]. This diverse functionality contributes to lactoferrin’s broad range of functions. Although lactoferrin is a highly conserved protein performing essential functions across mammalian species, subtle variations between species lead to distinct properties and potential health benefits. These differences define the various types of lactoferrin, which are primarily categorized based on their source. Major types include bovine and human lactoferrin derived from cow and human milk, respectively. Other species include caprine, ovine, and camel lactoferrin. Camelus dromedarius lactoferrin (CdLF), a complex protein comprising 689 amino acids, 17 disulfide bonds, and 4 potential glycosylation sites, offers distinct advantages for various health applications. Its ability to cross-react with anti-human lactoferrin antibodies enhances its absorption and bioactivity [8], since antibodies generated against human lactoferrin can bind to camel lactoferrin. This structural similarity highlights CdLF as a potential substitute for human lactoferrin in therapeutic applications. Its strong antimicrobial and antiviral activities [9], together with unique glycosylation that improves stability, iron-binding [10], and solubility, support its role as a cytotoxic and anti-pathogenic agent [9, 11].

The lactoferrin market, valued at USD 245 million in 2021, is expected to reach USD 505 million by 2030 [12]. Limited abundance in milk and high extraction costs have driven interest in heterologous expression systems, including microbes and animal cells [13]. Plants are promising cost-effective hosts for recombinant production [14], though variability in transgene expression remains a challenge.

In higher plants, RNA silencing (a natural antiviral defense mechanism), along with other factors, including transgene copy number and insertion site, contributes to variations in transgene expression [15, 16]. The paper “Isolation and expression of antimicrobial Camel lactoferrin (cLf) gene in tobacco” by Sohrabi et al. [17] successfully demonstrated the expression of the camel lactoferrin gene in tobacco plants aiming for the production of this valuable protein, which indicates that although many efforts have been made to improve lactoferrin production in plants, researchers are still working to solve the problem of low gene expression [17]. The Helper Component Proteinase (HC-Pro) is a crucial and well-conserved multifunctional protein that plays diverse roles in multiple processes, such as potyvirus replication, long- and cell-distance movement [18], aphid-mediated virus transmission [19], symptom development [20], viral synergism [21], inhibition of endonuclease activity [22], modulation of the protease activity of the 20 S proteasome [23], and inhibition of RNA silencing-based antiviral protection in plants [24]. Furthermore, HC-Pro contributes to enhancing the yield of viral particles, interacts with host factors [25], modulates various cellular processes, including altering gene expression profiles, suppressing the activity of miRNAs, affecting the regulation of transcription factors, and influencing the expression of energy production and stress-related genes in plants [26].

Our study builds on previous work producing CdLF in tobacco (N. tabacum) [17], where low expression levels (0.1–0.3% of total soluble protein) and variability in transgene expression limited production [27]. To address this, we employed the HC-Pro viral silencing suppressor, which blocks post-transcriptional gene silencing (PTGS) in transgenic plants. Agroinfiltration of tobacco leaves was performed using two plasmids: one carrying the CdLF gene and the other carrying HC-Pro. T1 generation plants constitutively expressing CdLF were also agroinfiltrated with HC-Pro to further boost expression. This approach increased CdLF levels without stable HC-Pro integration, avoiding potential phenotypic alterations [28]. Other viral silencing suppressors have been similarly used to enhance recombinant protein production in plants, highlighting the broader applicability of this strategy.

Materials and methods

Hc-Pro and pART27:CdLF constructs generation

The pGWB17-PVA-HC-Pro construct, provided by the Virology Research Center of Shiraz University [29], was used to express the open reading frame (ORF) of the Potato Virus A (PVA) HC-Pro gene. The cDNA library, prepared from the mammary gland of dromedary camels, was used as a template for amplification of the lactoferrin (CdLF) gene. The CdLF gene was amplified using a forward primer with an XhoI restriction site (5′-ATACTCGAGCGCCTCAGGACCCCAGACAT-3′) and a reverse primer with a HindIII restriction site (5′-GAAAGCTTGGGAGAGGAAGGTGCAGCACA-3′). The primers were designed based on conserved regions of the following CdLF gene sequences (accession numbers): M81327, L19981, M83202, AJ010930, D88510, X78902, EU523857, AJ311874, and AJ131674 [17]. These primers flank the entire coding sequence of CdLF, encompassing 2150 bp. The amplified CdLF fragment was initially cloned into the pTZ57R cloning vector using the SmaI restriction site and transformed into Escherichia coli (DH5α) by electroporation.

The pART27 binary vector was modified to create the recombinant expression vector, pART27:CdLF. This was achieved by inserting the amplified CdLF gene fragment into the multiple cloning site (MCS) region of the pART27 vector using the XhoI and HindIII restriction sites. The pART27:CdLF vector places the CdLF gene under the control of the 2X CaMV 35 S promoter. It includes a neomycin phosphotransferase (nptII) gene as a selectable marker. Both the pGWB17 PVA: HC-Pro and pART27:CdLF plasmids were separately introduced into Agrobacterium tumefaciens C58 via electroporation. Recombinant Agrobacterium colonies were screened using Luria-Bertani (LB) selective medium containing kanamycin (50 mg/l) and confirmed by colony PCR using CdLF-specific primers.

Transformation and verification of the CdLF gene in tobacco plants

N. tabacum cv. Turkish seeds were kindly provided by the Seed and Plant Certification & Registration Research Institute (SPCRI). The seeds were germinated in a greenhouse and cultivated under controlled conditions, with a 16-hour light/8-hour dark cycle and a temperature range of 22 °C to 28 °C. Leaf discs were excised from one-month-old tobacco plants, wounded using sterile tweezers, and immersed in liquid Luria-Bertani (LB) medium containing A. tumefaciens C58 cells harboring the pART27:CdLF binary vector. After a 10-minute incubation period, the explants were transferred to solid, full-strength Murashige and Skoog (MS) medium [30] enriched with 4.4 g/L MS basal salts with vitamins (Sigma-Aldrich, catalog number M5524), 30 g/L sucrose, and 8 g/L agar. The pH of the medium was adjusted to 5.8 before autoclaving.

The explants were then placed on the medium and incubated in the dark at 25°C for two days. Subsequently, the explants were transferred to a fresh hormone-free MS medium containing the same composition, but with the addition of 30 mg/L meropenem to inhibit bacterial growth and 100 mg/L kanamycin for the selection of transformed plant cells. After ten days on selective medium, surviving shoots were transferred to sterile soil for further growth under controlled greenhouse conditions. Genomic DNA was extracted from leaf tissue of regenerated plants using the CTAB method [31]. The presence of the CdLF gene in the putative transgenic plants was confirmed by polymerase chain reaction (PCR). PCR reactions were performed with an initial denaturation step at 94°C for 1 minute, followed by 35 cycles of denaturation at 94°C for 1 minute, annealing at 56°C for 1 minute, and extension at 72°C for 1 minute. A final extension step at 72°C for 10 minutes was also performed. Specific primers for the CdLF gene, designed for amplification of a 330 bp region (forward: 5’-GCTGCTGTGCCTCAATGG-3’ and reverse: 5’-GTGTGCTACCTTATCAATCCG-3’), were used for the PCR. These primers, also referred to as CdLF_Short, were later used for the quantitative real-time PCR (qRT-PCR) analysis.

Transient expression in N. tabacum and T1 CdLF transgenic plants

To investigate the effect of HC-Pro on CdLF expression in wild-type N. tabacum and in the T1 generation of CdLF-expressing transgenic plants, A. tumefaciens cultures harboring either the pGWB17-PVA-HC-Pro construct or the pART27:CdLF vector were prepared. Both Agrobacterium cultures were grown separately in Luria-Bertani (LB) medium overnight at 28 °C with shaking at 200 rpm. Following overnight growth, the bacterial cultures were centrifuged at 4000 × g for 10 min, and the resulting bacterial pellets were resuspended in an infiltration buffer containing 10 mM 2-(N-morpholino)ethanesulfonic acid (MES) and 10 mM MgSO4, adjusted to a pH of 5.8.

Agroinfiltration of wild-type N. tabacum Plants: Six-week-old N. tabacum plants, at the six-leaf stage, were selected for transient expression assays. Equal volumes of the A. tumefaciens cultures containing pGWB17-PVA-HC-Pro and pART27:CdLF were mixed to achieve a final optical density at 600 nm (OD600) of 0.6. This A. tumefaciens mixture was then infiltrated into the leaves of the plants using a needleless syringe, ensuring even distribution and minimal tissue damage.

Agroinfiltration of T1 Generation Plants: For T1 generation plants stably expressing CdLF, only the A. tumefaciens culture containing the pGWB17-PVA-HC-Pro construct was used. This culture was adjusted to an OD600 of 0.6, and half of a selected leaf of each plant at the six-leaf stage was infiltrated using a needleless syringe. The other half of the leaf was not infiltrated and served as a control, allowing for localized analysis of HC-Pro’s impact on gene expression.

Following agroinfiltration, the plants were transferred to a greenhouse under controlled environmental conditions that promote growth and development. The leaf samples were collected at 3-, 5-, and 9-days post-infiltration (dpi) to assess temporal changes in gene expression. The collected leaf samples were immediately frozen in liquid nitrogen and stored at -80 °C to preserve RNA integrity for subsequent analyses.

Camel lactoferrin expression measurements (Quantitative RT- PCR analysis)

To quantify CdLF gene expression and assess the impact of HC-Pro co-expression, quantitative real-time PCR (qRT-PCR) was performed. Total RNA was extracted from leaf tissue using a commercial kit (Denazist, Iran). RNA quality and quantity were assessed by agarose gel electrophoresis and a NanoDrop ND-1000 spectrophotometer. Extracted RNA was treated with DNase I to eliminate any genomic DNA contamination, followed by reverse transcription into cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). This kit includes M-MuLV reverse transcriptase, an RNase inhibitor, and a dNTP mix. cDNA synthesis was verified by standard PCR using primers specific to the elongation factor (EF) gene [32].

QRT-PCR reactions were performed using a Bioer Real-Time PCR System. Three biological replicates and two technical replicates were analyzed for each sample. Cycle threshold (CT) values were calculated as the average of technical replicates. They were used to determine relative gene expression using the 2^-ΔΔCT method [33]. EF served as the reference gene for normalization [32]. Amplification was carried out in a LineGeneK thermal cycler (Bioer, China) under the following conditions: 10 minutes at 94°C, followed by 40 cycles of 15 seconds at 94°C, 15 seconds at 55.4°C, and 20 seconds at 72°C. Specific primers, distinct from those used for the cDNA amplification, were designed for qRT-PCR. Forward-CdLF_Short and reverse-CdLF_Short for CdLF, were used for CdLF, resulting in a 330 bp amplicon, while 5’-GCCACACCTCGCACATTG-3’ (forward) and 5’-GCCAGCATCACCATTCTTG-3’ (reverse) were used for EF, resulting in a 150 bp amplicon.

The samples analyzed by qRT-PCR were: leaf tissue from N. tabacum plants transformed with the pART27:CdLF vector, leaf tissue from plants transformed with the empty pART27 vector (negative control); leaf tissue from agroinfiltrated plants treated with pART27:CdLF alone, or treated with both pART27:CdLF and pGWB17-PVA-HC-Pro vectors; and leaf tissue from both non-agroinfiltrated and pGWB17-PVA-HC-Pro agroinfiltrated T1 generation plants stably expressing CdLF.

Protein extraction and ELISA test

To quantify recombinant CdLF expression in transgenic tobacco plants, protein extraction and direct ELISA were performed. Freshly harvested leaf tissue (200 mg) was ground to a fine powder in liquid nitrogen. The resulting homogenate was incubated for 2 h at 4 °C with an equal volume (v/v) of cold extraction buffer, consisting of 50 mM potassium phosphate, pH 7.0. Following incubation, the homogenate was centrifuged at 12,000 x g for 20 min at 4 °C, and the supernatant containing the total soluble protein was collected. The total protein concentration was then determined using the Bradford assay [34].

To ensure consistency in treatment, Agrobacterium infiltration was performed using a standardized concentration of 1 × 10⁹ cells/mL. Recombinant CdLF expression was compared between two groups: Group 1 consisted of plants co-infiltrated with pART27:CdLF and pGWB17-PVA-HC-Pro constructs. At the same time, Group 2 included plants infiltrated only with the pART27:CdLF construct.

For the T1 generation transgenic plants constitutively expressing the CdLF gene, recombinant CdLF concentration was compared between the two halves of a single leaf. One half of the leaf was infiltrated with the pGWB17-PVA-HC-Pro construct, while the other half remained un-infiltrated as a control. For ELISA, 60 µg of total extracted protein from each sample was added to wells of a 96-well microplate pre-coated with the same extraction buffer. Plates were incubated for 2 h at 4 °C, followed by three washes with PBS containing 0.05% Tween-20 (PBST). A primary lactoferrin antibody (MyBio-Source, USA) was applied at a 1:1000 dilution and incubated for 1 h at room temperature. After three additional washes with PBST, an HRP-conjugated secondary antibody at 1:5000 dilution was added and incubated for 1 h at room temperature. The wells were washed three times with PBST, and a TMB chromogenic substrate was added. The reaction was stopped with sulfuric acid, and absorbance was measured at 450 nm using a microplate reader. Negative controls included wells with extraction buffer only and protein from non-transformed plants. At the same time, recombinant lactoferrin served as a positive control. All samples were assayed in triplicate, and results were normalized to total protein content.

Antimicrobial activity assay

The antimicrobial activity of recombinant CdLF protein was evaluated against a panel of microbial strains, including Gram-positive bacteria Staphylococcus aureus (PTCC 1112, ATCC 6538) and Bacillus subtilis (PTCC 1023, ATCC 6633), Gram-negative bacterium Escherichia coli (PTCC 1330, ATCC 8739), and the fungal pathogen Candida albicans (PTCC 5027, ATCC 10231). All strains were sourced from the Iranian Research Organization for Science and Technology (IROST). For each microbial strain, cultures were grown in liquid Luria-Bertani (LB) medium at 37 °C with shaking at 200 rpm until reaching mid-log phase, standardized to an optical density (OD600) of 0.4. Subsequently, 1 mL aliquots of these cultures were transferred into sterile microcentrifuge tubes.

Protein samples were prepared by extracting total soluble proteins from freshly harvested transgenic N. tabacum leaves using 50 mM potassium phosphate buffer (pH 7.0), followed by centrifugation at 12,000 × g for 20 min at 4 °C to clear debris. Protein concentrations were quantified using the Bradford assay, and 50 µg of total soluble protein was added to each microbial culture. The test mixtures were incubated at 37 °C with shaking at 200 rpm. Antimicrobial efficacy was assessed using Colony Forming Unit (CFU) assays performed after incubation. CFU assays involved preparing serial dilutions of each mixture in sterile saline and plating aliquots on nutrient agar. Plates were incubated under strain-specific conditions before the colonies were enumerated.

Appropriate controls were included throughout the assays: protein extracts from non-transformed plants and protein extraction buffer served as negative controls, whereas commercially obtained lactoferrin acted as a positive control, ensuring assay validity and specificity. Biological replicates corresponded to protein samples extracted from individual plants. Each experiment was conducted in triplicate, and results are presented as mean values ± standard deviation (SD). Comparisons of microbial growth were made between samples treated with protein extracts from transgenic plants and those treated with non-transgenic extracts, enabling a precise evaluation of CdLF’s antimicrobial potency.

Statistical Analysis

Statistical analyses were performed using Minitab 16 software (Minitab Inc., State College, PA, USA). To determine significant differences among experimental groups, a one-way analysis of variance (ANOVA) was conducted. A significance threshold of p ≤ 0.05 was used for all analyses. Where the ANOVA indicated a statistically significant difference, Tukey’s post-hoc test was used to identify significant differences between individual group means. Data were visualized using GraphPad Prism version 8.0.1 (GraphPad Software Inc., San Diego, CA, USA). The data are presented as the mean ± standard error (SE) from three independent biological replicates to ensure the robustness and reliability of the analyses.

Results

Transgenic plants and molecular analysis

To confirm the successful construction of the recombinant strain, colony PCR was first performed on A. tumefaciens C58 using primers specific to the CdLF gene. A clear 2150 bp product was amplified, verifying that the pART27:CdLF expression plasmid had been correctly introduced into Agrobacterium. Following plant transformation, a total of 40 regenerated shoots were obtained on kanamycin-selective medium, from which 34 healthy shoots were selected for further study. After transfer to soil, 21 plants survived and developed normally. Genomic PCR analysis of these 21 plants confirmed the presence of the CdLF gene in all samples.

In contrast, no amplification was detected in non-transformed control plants (Fig. 1- uncropped, unedited images attached in supplementary file). To further evaluate gene expression, RNA was extracted from these 21 plants, and cDNA was synthesized for PCR analysis. 17 plants produced a specific 330 bp amplification product, indicating transcription of the CdLF gene, while 4 plants did not show detectable transcript levels. These 17 plants were thus confirmed as true transgenic lines. Among them, 6 representative plants were subjected to detailed molecular characterization, and 5 plants with high-quality RNA were ultimately selected for quantitative real-time PCR to assess CdLF expression levels.

Fig. 1.

PCR analysis of CdLF gene integration and expression. A Electrophoresis of PCR products using genomic DNA as template. A 2150 bp band corresponding to the entire CdLF coding sequence is observed in the positive control (lane 4) and in transgenic plant samples (lanes 5–10), with each transgenic plant sample representing an independent T1 transgenic line, but not in the negative control (lane 1) or in non-transformed plants (lane 3). A 1 kb DNA ladder (bands at 250, 500, 750, 1000, 1500, 2000 bp) is shown in lane 2. B Electrophoresis of PCR products using cDNA as a template. A 330 bp band confirming CdLF gene expression is observed in transgenic plant samples (lanes 2–6, each representing an independent T1 transgenic line) and in the positive control (lane 9, plasmid DNA containing CdLF), but not in the negative control (lane 7) or in the non-transformed plant (lane 8). A 100 bp DNA ladder (bands at 100, 200, 300, 400, 500 bp) is shown in lane 1. Lane 10, Positive Control

Quantitative RT-PCR

To determine CdLF expression levels, quantitative real-time PCR (qRT-PCR) was performed on transgenic and non-transformed tobacco plants. Initial analysis of CdLF transcript accumulation in T0 transgenic lines revealed five lines with significant differences in gene expression. The third transgenic line showed the highest CdLF expression, closely followed by the second line, with both exhibiting statistically comparable expression levels (Fig. 2A).

Fig. 2.

Quantitative RT-PCR analysis of CdLF expression. A CdLF mRNA expression levels in T0 transgenic tobacco plants. Individual transgenic lines are labeled 1t, 2t, 3t, 4t, and 5t. B Enhancement of CdLF expression in T1 generation transgenic plants by HC-Pro viral suppressor, measured five days after infiltration with pGWB17-PVA-HC-Pro. The x-axis represents different T1 lines, and the y-axis indicates the increased expression achieved by viral suppression. C Enhancement of CdLF expression in wild-type agroinfiltrated plants by HC-Pro viral suppressor. The x-axis represents different agroinfiltrated plant samples, and the y-axis shows the increase in transgene expression caused by the viral suppressor of RNA silencing HC-Pro. D Time-course analysis of CdLF expression in wild-type agroinfiltrated N. tabacum plants. Six-week-old plants were infiltrated with A. tumefaciens, and leaf samples were collected at 3, 5, and 9 days post-infiltration (dpi)

To assess the impact of HC-Pro co-agroinfiltration, the expression of the CdLF gene was analyzed in agroinfiltrated plants. This revealed no significant increase in CdLF transcript levels at 3 days post-infiltration (dpi). However, by 5 dpi, CdLF expression reached its peak with a 5.2-fold increase relative to baseline levels (Fig. 2D). While expression declined slightly by 9 dpi, it remained elevated (4.7-fold increase compared to baseline). Based on these results, all subsequent analyses were standardized to 5 dpi.

HC-Pro co-expression positively impacted CdLF production in wild-type agroinfiltrated plants. Specifically, plants co-expressing pGWB17-PVA-HC-Pro and pART27:CdLF constructs showed a 1.5- to 5.7-fold increase in CdLF transcript accumulation compared to those expressing pART27:CdLF alone (Fig. 2C). In the T1 generation, seeds were selected from transgenic plants with the highest CdLF expression levels. Analysis of T1 plants 5 dpi after infiltration with pGWB17-PVA-HC-Pro showed a 3.6-fold increase in CdLF transcript levels compared to T1 plants without HC-Pro infiltration (Fig. 2B).

Protein production quantification and antimicrobial activity Analysis

Recombinant CdLF protein concentrations were quantitatively measured using ELISA. Co-agroinfiltration of wild-type plants with both pART27:CdLF and pGWB17-PVA-HC-Pro constructs resulted in a 3.4-fold increase in CdLF production compared to plants agroinfiltrated solely with pART27:CdLF (Fig. 3A). In T1 generation transgenic plants, the CdLF protein concentration in the half-leaf infiltrated with the pGWB17-PVA-HC-Pro construct was 1.8-fold higher compared to the non-infiltrated half-leaf (Fig. 3B). Following the use of the HC-Pro silencing suppressor, lactoferrin yields are comparable to, or even exceed, those reported in other plant-based expression systems. Specifically, transient expression in N. benthamiana has typically yielded approximately 27–40 µg/g fresh weight of lactoferrin, with performance dependent on the specific expression vectors and targeting strategies employed [6, 35]. In comparison, stable transformants of tobacco expressing camel lactoferrin previously showed yields of 0.1–0.3% of total soluble protein, which generally translates to lower microgram quantities per gram of tissue [17]. This demonstrates that HC-Pro effectively enhances protein accumulation, supporting its utility for the scalable and efficient production of recombinant proteins in plants.

Fig. 3.

Recombinant CdLF protein levels in leaf extracts, as measured by ELISA using a lactoferrin antibody. Leaf samples were collected at 5 days post-infiltration (dpi). Values represent the mean ± standard error of the mean (SEM) from three independent experiments. A Recombinant CdLF levels in agroinfiltrated wild-type N. tabacum plants: plants infiltrated with pART27:CdLF alone (S1); plants co-infiltrated with pART27:CdLF and pGWB17-PVA-HC-Pro (S2); and plants infiltrated with buffer only (negative control; S3). B Recombinant CdLF levels in T1 generation transgenic plants: non-infiltrated T1 plants (S1), T1 plants infiltrated with pGWB17-PVA-HC-Pro (S2), and non-transgenic N. tabacum plants infiltrated with buffer only (negative control; S3). Different letters denote statistically significant differences in CdLF protein levels (p < 0.05)

The inhibitory effects of recombinant CdLF on microbial growth were assessed using total protein extracts from transgenic tobacco leaves. These extracts were tested against the gram-positive bacteria S. aureus PTCC 1112, B. subtilis PTCC 1023, the gram-negative bacteria E. coli PTCC 1330, and the fungus C. albicans PTCC 5027. The total protein extracts from transgenic tobacco plants exhibited significant antimicrobial activity, while extracts from non-transgenic plants showed no significant effect on microbial growth (Fig. 4). These results indicate that the recombinant CdLF protein, as expressed in the total protein extracts, can inhibit the growth of both bacterial and fungal pathogens.

Fig. 4.

The antimicrobial activity of total protein extracts (50 µg) from transgenic N. tabacum plants expressing recombinant CdLF against S. aureus, E. coli, B. subtilis, and C. albicans. Data are presented as the mean ± standard error of the mean (SEM) of three independent experiments. Statistically significant differences (p < 0.05), as determined by a one-way ANOVA with Tukey’s post-hoc test for multiple comparisons, are indicated by asterisks (*) when comparing the transgenic plant protein extracts to those from non-transgenic plants for each microorganism

Using 50 µg of total protein extracts from transgenic plants, growth rates were significantly reduced by 43.8% for S. aureus, 53.33% for E. coli, 35.2% for B. subtilis, and 59.45% for C. albicans, relative to the 100% growth observed with protein extracts from non-transgenic plants. These findings reveal varying levels of susceptibility to recombinant CdLF among the different microorganisms, as demonstrated by the differing degrees of colony growth inhibition. In contrast, protein extracts from non-transgenic plants showed no inhibitory effect on microbial growth.

Discussion

Lactoferrin (LF) is a multifunctional iron-binding glycoprotein with well-established antimicrobial, anti-inflammatory, anticancer, and immunomodulatory properties, highlighting its therapeutic potential [4, 36]. Plant molecular farming offers a promising system for recombinant protein production, providing safety, scalability, and eukaryotic post-translational modifications. However, it is often limited by low yields due to post-transcriptional gene silencing (PTGS), a natural plant defense mechanism against foreign nucleic acids [14, 37]. Viral silencing suppressors, such as HC-Pro from Potato Virus A (PVA), have emerged as effective tools to mitigate PTGS and enhance transgene expression [38].

In this study, co-expression of PVA HC-Pro in N. tabacum significantly increased CdLF mRNA levels by approximately 5.7-fold in wild-type agroinfiltration and 3.6-fold in T1 transgenic lines. Protein accumulation increased correspondingly by 3.4-fold and 1.8-fold, reflecting HC-Pro’s ability to modulate RNA silencing and stress response pathways [28]. This enhancement surpasses earlier reports of camel lactoferrin expression in tobacco, where yields were generally below 0.3% of total soluble protein [17], and is comparable to or exceeds transient expression of human lactoferrin in N. benthamiana [35].

However, in T1 transgenic plants, CdLF protein production was only increased by 1.8-fold despite a 3.6-fold increase in gene expression, suggesting the influence of other factors such as inherited small interfering RNAs (siRNAs) or post-transcriptional regulation [39, 40].

Other viral suppressors, including p19 from Tomato bushy stunt virus, CMV 2b, and PO, have also been shown to improve recombinant protein yields across various plant species and target proteins [15, 41]. HC-Pro was selected for this study due to its documented efficiency in tobacco systems [28]. The lower fold increase in protein compared to mRNA in T1 lines suggests that additional factors, such as inherited siRNAs, translational regulation, or protein turnover, affect ultimate protein accumulation [39, 40]. This indicates that combining HC-Pro with complementary strategies may further enhance yields. HC-Pro expression can also impact plant physiology, including growth dynamics and photosynthetic efficiency, emphasizing the need to balance suppressor activity with plant health to optimize production [28]. Despite these considerations, our findings demonstrate that HC-Pro can substantially improve CdLF production in plants, supporting the potential of plant-derived lactoferrin as a scalable and cost-effective platform for recombinant protein production.

In conclusion, this study provides valuable insights into leveraging viral silencing suppressors to enhance the yield of recombinant therapeutic proteins in plants. Future work will focus on comparative evaluation of different suppressors, optimization of expression conditions, and elucidation of molecular mechanisms governing post-transcriptional regulation and protein stability in transgenic plants to maximize both productivity and functional quality of lactoferrin for pharmaceutical applications [15]. [42]

Conclusion

This study demonstrates the potential of the Potato Virus A HC-Pro gene as an effective suppressor of transgene silencing, significantly enhancing the yield of recombinant proteins in plants while maintaining their native functionality. The observed enhancement by HC-Pro appears to be mediated through several potential cellular mechanisms, including the induction of stress response pathways, modification of the protein folding environment in the endoplasmic reticulum, and inhibition of RNA silencing. These findings offer significant implications for plant-based protein production and suggest a promising approach for developing a cost-effective and safe production method. Further investigation is still needed to fully explore the potential of the PVA HC-Pro suppressor in enhancing gene expression and optimizing its application in various plant-based production systems.

Abbreviations

ANOVA

Analysis of variance

CaMV

Cauliflower mosaic virus

cDNA

Complementary DNA

CdLF

Camelus dromedarius lactoferrin

CFU

Colony forming unit

CT

Cycle threshold

DNA

Deoxyribonucleic acid

DNase I

Deoxyribonuclease I

dpi

Days post-infiltration

EF

Elongation factor

ELISA

Enzyme-linked immunosorbent assay

ER

Endoplasmic reticulum

HC-Pro

Helper component proteinase

HRP

Horseradish peroxidase

LB

Luria–Bertani

LF

Lactoferrin

MCS

Multiple cloning site

MES

2-(N-morpholino)ethanesulfonic acid

MgSO₄

Magnesium sulfate

miRNA

MicroRNA

MS

Murashige and Skoog

nptII

Neomycin phosphotransferase II

OD600

Optical density at 600 nm

ORF

Open reading frame

PBS

Phosphate-buffered saline

PBST

Phosphate-buffered saline with Tween-20

PCR

Polymerase chain reaction

PTGS

Post-transcriptional gene silencing

PVA

Potato virus A

qRT-PCR

Quantitative real-time polymerase chain reaction

RNA

Ribonucleic acid

SD

Standard deviation

SE

Standard error

SEM

Standard error of the mean

siRNA

Small interfering RNA

TMB

3,3′,5,5′-Tetramethylbenzidine

Authors’ contributions

M.L., F.A. and A.N. wrote the main manuscript text. Y.-H.L., B.S.-K., and M.S. contributed to data analysis and interpretation. F.A. and B.F.-N. assisted in literature review and experimental design. J.A.P., W.J.L., and A.S. prepared figures and tables. P.K. and R.M.G. supervised the project and contributed to manuscript revision. All authors reviewed and approved the final manuscript.

Funding

Open access funding provided by Parul University.

Data availability

The dataset(s) supporting the conclusions of this article are included within the article (and its additional file(s)).

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.