The synthetic DNA sequence encoding PAP (GenBank accession no. M34840.1) was cloned as a fusion to the Fc fragment of the human IgM μ chain (GenBank accession No. X57086.1). The PAP sequence was modified by the addition of an N-terminal extension encoding a signal peptide (MATQRRANPSSLHLITVFSLLAAVVSAEVD; Lu et al., 2012). The gene encoding PAP-IgM Fc was cloned under the control of the enhanced cauliflower mosaic virus (CaMV) 35S promoter and the tobacco etch virus 5’-leader sequence (TEV; Figure 1A). The PAP-IgM Fc expression cassette was subcloned into the HindIII and EcoRI restriction enzyme sites of the binary plant expression vector pBI121 to generate pBI PAP-IgM Fc. Then, the vector was transformed into competent Escherichia coli DH5α cells for amplification.

The recombinant pBI PAP-IgM Fc vector was transferred into Agrobacterium tumefaciens strain LBA4404 by electroporation. Then, transgenic tobacco (Nicotiana tabacum) plants were generated by Agrobacterium-mediated transformation (Ko et al., 2003; Lu et al., 2012; So et al., 2013). Transgenic plant lines were selected on Murashige and Skoog (MS) medium [30 g⋅L^-1 of Sucrose, 6 g⋅L^-1 of Phyto agar, and 4.8 g⋅L^-1 of MS B5 vitamin (Duchefa Biochemie, Haarlem, Netherlands)] containing 100 mg⋅L^-1 kanamycin and 250 mg⋅L^-1 cefotaxime. Transgenic plants were grown in a chamber at constant temperature (23°C) and light intensity of 50 μmol⋅m^-2⋅s^-1 under a long-day photoperiod (16:8 h light-dark cycle). Among the transgenic plants with high protein expression, three PAP-IgM Fc expressing transgenic lines (T502, T506, and T509) were randomly selected for in vitro tissue subculture.

Transgenic plants expressing PAP-IgM Fc were grown in vitro as the first generation tissue culture (SG₁) in a Magenta GA-7 vessel (Sigma–Aldrich, St. Louis, MO, USA) for 4 weeks. Plant stems were divided into three sections [top (T), middle (M), and base (BA)]. T, M, and BA plantlet stem pieces were transplanted into new media and grown as the second-generation tissue subculture (SG₂) for 2 weeks. This tissue subculture was repeated to generate the third generation SG₃ (Figure 1A).

Leaf tissue samples were harvested from the top portion of transgenic and non-transgenic plantlets. Genomic DNA from fresh leaf tissue was isolated using a DNA extraction kit (RBC Bioscience, Seoul, South Korea) according to the manufacturer’s protocol. The extracted genomic DNA was analyzed by polymerase chain reaction (PCR) to confirm the presence of the recombinant PAP-IgM Fc gene using the following primer pairs: forward primer 5′-GCC CTC GTT TTC AAG AAC TTG-3′ and reverse primer 5′-CGG GAT CCT CAG TAG CAG GTG CCA GCT GTG-3′. The PCR was performed with 30 cycles of 94°C for 20 s, 62°C for 20 s, and 72°C for 120 s. Genomic DNA isolated from non-transgenic plant leaves was used as the negative control, and the pBI PAP-IgM Fc gene was used as a positive control. The expected size of PAP-IgM Fc PCR product was 1,685 bp. PCR analysis was performed for more than three times.

Leaf tissue samples were used from each top portion of transgenic plantlets. Total RNA was isolated from the leaves of transgenic plant samples (SG₁, SG₂, and SG₃) using an Rneasy plant mini kit (Quagen, Valencia, CA) according to the manufacturer’s recommendations, and the isolated RNA samples were stored at -80°C. Genomic DNA removal and cDNA synthesis were performed using the Quantitect reverse transcription kit (Quagen, Valencia, CA) according to the manufacturer’s protocol. Each RNA sample was used as a template for RT-PCR and RT-qPCR analyses. RT-PCR and RT-qPCR reactions were performed using the Maxime PCR premix kit (Intron Biotechnology, Seoul, South Korea) and Rotor-Gene SYBR Green PCR kit (Quagen, Valencia, CA), respectively. PAP-IgM Fc primers were as follows: 5′-CTC ATG CTA CCT GGT TGC AG-3′; forward, 5′-GGT GGG ACG AAG ACG CTC A-3′. Quantitative RT-PCR was analyzed using real-time PCR machine (Rotor-Gene Q, Quagen) with the following cycling parameters: 5 min at 95°C, 5 s at 95°C, 10 s at 60°C, and 40 cycles of 5 s at 95°C and 10 s at 60°C. The elongation factor 1-α (EF-1α) gene was used as a housekeeping gene. RT PCR and RT-qPCR analyses were performed for 3 times.

The PAP-IgM Fc protein expression level in transgenic lines T502, T506, and T509 was investigated using immunoblot analysis. Leaf samples (100 mg) were harvested from the plantlet top and homogenized in 300 μL of 1 × PBS (137 mM NaCl, 10 mM Na₂HPO₄, 2.7 mM KCl, and 2 mM KH₂PO₄) to extract total soluble proteins. PAP-IgM Fc leaf extracts were boiled with 5 × protein loading buffer (1 M Tris-HCl, 50% glycerol, 10% SDS, 5% 2-mercaptoethanol, and 0.1% bromophenol blue) for 10 min and cooled for 2 min. Total soluble proteins were separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane (Millipore, Billerica, MA, USA). Membranes were incubated with 5% skim milk (Sigma, St. Louis, MO, USA) in 1 × PBS buffer at 4°C overnight. Blots were incubated with a goat anti-human IgM (μ chain) antibody conjugated to horseradish peroxidase (Abcam Inc., Cambridge, MA, USA) diluted 1:5,000 in 1 × PBS solution for 2 h at room temperature (Figure 2A). After three 10-min washes, proteins were detected with the SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA) and visualized by exposing the membrane to X-ray film (Fuji, Tokyo, Japan). Leaf tissue extract from non-transgenic plants was used as a negative control. Detected protein bands were digitized as an electronic image, and band intensity was measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Immunoblot analysis was performed for more than three times.

Expression of PAP-IgM Fc in plant leaf tissue was analyzed using a chemiluminescence immunoassay (CLIA). Leaf samples from PAP-IgM Fc transgenic plants were harvested from the plantlet top (T of SG₂ and T-T’, M-T’, and BA-T’ of SG₃; Figure 1A) and homogenized in 300 μL of 1 × PBS (137 mM NaCl, 10 mM Na₂HPO₄, 2.7 mM KCl, and 2 mM KH₂PO₄) for total soluble protein extraction. All samples were diluted 1:100 in 1 × PBS solution. A cuvette containing the anti-PAP coated beads (Siemens Healthcare Diagnostics Inc., Llanberis, UK) was incubated with PAP-IgM Fc and an anti-PAP antibody conjugated to alkaline phosphatase for 30 min at 37°C (Figure 2F). After the antibodies were removed and the beads were washed in deionized distilled water as a centrifugal washing buffer, the PAP-IgM Fc proteins were detected using the chemiluminescent substrate. The samples were analyzed on an IMMULITE 2000 xpi (Siemens Healthcare Diagnostic Inc., Flanders, NJ, USA). CLIA was performed for more than three times.

All values are shown as the mean ± SD. Recombinant PAP-IgM Fc protein expression in transgenic plant leaves was compared by using the unpaired t-test, and p values less than 0.01 (^∗∗∗) or 0.05 (^∗∗) were considered statistically significant. Statistical significance was assessed using Excel (Microsoft Office Excel 2013; Microsoft Corporation, Redmond, WA, USA).

Transgenic tobacco plant lines were obtained by Agrobacterium-mediated transformation with plant expression vectors carrying PAP-IgM Fc. Genomic DNA extracted from three transgenic lines (T502, T506, and T509) was analyzed by PCR to confirm insertion of the PAP-IgM Fc transgene. The PAP-IgM Fc gene was detected in the top leaves of each subculture generation (SG₁, SG₂, and SG₃) obtained from the T, M, and BA stems harvested from tobacco transgenic plants (SG₁). The size of the PAP-IgM Fc PCR product in the genome of the transgenic plants was 1,685 bp (Figure 1B). No PAP-IgM Fc PCR product was detected in non-transgenic plants.

In real-time PCR and quantitative real-time PCR, the relative transcription level was observed in the top portions of all subculture generations (SG₁, SG₂, and SG₃) leaf tissues (Supplementary Figure S1). The transgene was not detected in non-transgenic plant (Figure 1C). RT-PCR product of the expected size of PAP-IgM Fc was detected in all samples of transgenic plants (T502, T506, and T509; Figure 1C). In RT-qPCR, the PAP-IgM Fc transcriptional levels were consistently higher than 0.9 level in SG₁, SG₂, and SG₃ leaf section (T, M, and BA; Supplementary Figure S1). Non-transgenic plant showed no PAP-IgM Fc signal.

PAP-IgM Fc protein expression in leaf samples from transgenic plants was analyzed by western blotting. All transgenic plant lines harboring the PAP-IgM Fc transgene showed a protein band of approximately 83 kDa (Figure 2B). Recombinant PAP-IgM Fc fusion proteins were expressed in all subculture generations (SG₁, SG_2, and SG₃) obtained from the T, M, and BA stems of SG₁ transgenic plants (Figure 2B). No PAP-IgM Fc protein band was detected in non-transgenic plants, which were used as a negative control (Figure 2B).

Quantitative western blot analysis was performed to determine the expression level of the recombinant PAP-IgM Fc fusion protein in the top leaves of SG₁, SG₂, and SG₃ (Figure 2B). The intensity of the PAP-IgM Fc fusion protein bands in the top leaf samples was quantified using Image J software. Although the PAP-IgM Fc recombinant protein expression level differed slightly among the SG₁, SG₂, and SG₃ samples, all leaf samples showed stable expression of the recombinant protein (Figures 2C–E).

A CLIA was performed to quantify the expression of recombinant PAP-IgM Fc protein in the transgenic leaves of SG₂ and SG₃ plants. In T502, PAP-IgM Fc expression levels in each leaf position were slightly different. However, the mean values at each position in SG₃ were not significantly different (Figure 2G). In T506, the PAP-IgM Fc expression levels were significantly different at each leaf position, and the mean values at each position in SG₃ were significantly different (Figure 2H). In T509, the PAP-IgM Fc expression levels in SG₂, T-T’ of SG₃, and M-T- of SG₃ differed. However, the mean values at all positions were not significantly different (Figure 2I). Overall, the expression of the recombinant PAP-IgM Fc fusion protein was maintained without loss in all in vitro SG samples (Figures 2G–I).