Plant materials
One-year-old P. notoginseng plants were collected in Wenshan city, Qiubei county, Yunnan province, China and then cultivated under shade in a greenhouse at Yunnan Agricultural University. Sterile wild-type (WT) tobacco (N. tabacum) seedlings were grown in a light incubator and then used for genetic transformations.
Fungal materials
Four pathogens, including C. destructans, F. solani, and F. oxysporum, were isolated from diseased P. notoginseng plants with root rot symptoms and then characterized in terms of their morphological and molecular features. The fungal materials (C. destructans, F. solani, and F. oxysporum) were preserved on slants at 4°C. The mycelia on the slants were used to inoculate potato dextrose agar (PDA) medium and carrot agar medium in plates, which were then incubated at 28°C until the mycelial diameter was approximately 3 cm. A mycelial plug was transferred to fresh medium in a new plate and then incubated at 28°C. This step was repeated three times.
Chemical treatments and fungal inoculation
The roots of healthy 1-year-old P. notoginseng plants were wounded with scissors and then inoculated with C. destructans. Specifically, the wounded roots were immersed in a C. destructans culture for 30 min. For the chemical treatments, the wounded roots were immersed in 200 µM SA, 100 µM MeJA, 1 mM ethephon (ETH), 1 mM gibberellic acid (GA3), 1 mM abscisic acid (ABA), and 1 mM hydrogen peroxide (H2O2) solutions for 30 min. The roots treated with sterile water were used as the control for the fungal inoculation and chemical treatments. The P. notoginseng roots were collected at 12, 24, 48, and 72 h after the fungal inoculation and chemical treatments. All samples were immediately frozen in liquid nitrogen and stored at -80°C prior to the subsequent analyses.
Identification of the P. notoginseng PR4 gene
The Pfam number for the PR4 gene is PF00967 (Chen et al., 2020). The P. notoginseng reference genome (Yang et al., 2021) was searched using PF00967 and the ‘Find Best Homology’ module of TBtools to identify PnPR4. The PnPR4 coding sequence (CDS) was obtained by analyzing the genomic sequence and then used to design the following primers for the PCR amplification of PnPR4 cDNA: PnPR4-F (5′-ATGCAAGTTGTACACAT-3′) and PnPR4-R (5′-TCAGTCTCCGCAGTTCAC-3′). The amplified product was inserted into the pMD18-T vector and sequenced. The accuracy of the sequence was confirmed on the basis of an alignment with the PnPR4 CDS. The molecular weight and isoelectric point of PnPR4 were predicted using the ExPASy Molecular Biology Server (www.ExPASy.ProtParamtool.html). The TMHMM 2.0 program (DTU Health Tech) was used to predict the presence of a signal peptide and a transmembrane region in PnPR4. The similarity and evolutionary relationships between PnPR4 and other plant PR4 proteins were analyzed using MEGA.
Quantitative real-time PCR
For the quantitative real-time PCR (qRT-PCR) analysis, PnPR4-specific primers were designed using the Primer5 software and PnACT2 (GenBank accession number KF815706.2) was selected as the internal reference gene (Liu et al., 2012). Total RNA was extracted from healthy and uniformly growing (i.e., height) 1-year-old P. notoginseng plants. The P. notoginseng root tips were wounded using scissors and then treated with C. destructans, SA, GA3, ABA, ETH, MeJA, or H2O2. The P. notoginseng root samples were immediately frozen using liquid nitrogen and stored at -80°C. The RNA was reverse transcribed to synthesize cDNA using the PrimeScript™ RT Reagent Kit with gDNA Eraser (Perfect Real Time) (Takara Bio, China). The cDNA and the SYBR qPCR master mix and enzyme (Vazyme Biotech, China) were used for the qRT-PCR analysis, which was completed on the QuantStudio™ Real-time PCR system, with four replicates per gene. The QuantStudio™ Design & Analysis software was used to analyze the generated data. Relative gene expression levels were calculated using the 2−ΔΔCt method.
Subcellular localization analysis
The PnPR4-GFP fusion gene was constructed to determine the subcellular localization of PnPR4. The PnPR4 open reading frame (ORF) was inserted into the pCAMBIA1301-GFP vector. Agrobacterium tumefaciens cells were transformed with the pCAMBIA1301-PnPR4-GFP recombinant plasmid. The positive clones carrying the recombinant plasmid were identified by PCR and then injected into the lower epidermis of the leaves from 4-week-old tobacco plants. After 3–4 days, the subcellular localization of PnPR4 was examined using the UltraVIEW VoX spinning disc confocal microscope (Perkin Elmer, USA).
PnPR4 recombinant protein production and assessment of its antifungal activity
The PnPR4 ORF without the fragment encoding the N-terminal signal peptide was inserted into the pET-32a vector. Escherichia coli BL21 cells were transformed with the pET-32a-PnPR4 recombinant plasmid. The transformed cells were treated with 0.5 mM isopropyl β-D-thiogalactopyranoside (IPTG) to induce the production of the recombinant protein, which was subsequently purified using a Ni-NTA affinity chromatography column (Sangon Biotech, China).
The antifungal activity of the recombinant protein was evaluated using three fungi (C. destructans, F. oxysporum, and F. solani), which were cultured on PDA medium in plates at 28°C until the colony diameter was approximately 2 cm. Five sterile filter paper discs (0.6 mm diameter) were placed around the colonies, after which 20 µL solutions consisting of 10, 15, or 20 µg recombinant protein were added. Sterile water and the elution buffer (20 µL) were used as the blank controls. All plates were incubated at 28°C for 3–5 days and then photographed using the PowerShot G7X Mark III camera (Canon, Japan). To determine the inhibitory effects of the recombinant protein on fungal growth, the growth-inhibition area was calculated using the Photoshop software.
Generation and screening of transgenic tobacco plants
The PnPR4 ORF was obtained by digesting the pGEM-T-PnPR4 plasmid with SacI and BamHI. The PnPR4 ORF was then inserted into the pC1300s plant expression vector digested with the same two restriction enzymes. Competent E. coli DH5α cells were transformed with the pC1300s-PnPR4 recombinant plasmid and then the positive clones were confirmed by PCR. The recombinant plasmid was then inserted into A. tumefaciens EHA105 cells using a CaCl2 freeze-thaw method. A leaf disc transformation method was used to infect tobacco with the transformed A. tumefaciens EHA105 cells. Transgenic tobacco plants were obtained by tissue culture and by screening for antibiotic resistance. Genomic DNA was extracted from the leaves of the transgenic tobacco plants using the CTAB method to verify the plants were successfully transformed (McCormick et al., 1986). The T0 generation transgenic tobacco plants verified by PCR were grown in a greenhouse to produce the T2 generation.
Analysis of the transgenic tobacco plants in terms of PnPR4 expression and disease resistance
The PnPR4 transcription levels in the T2 tobacco lines were analyzed by qRT-PCR. The WT tobacco plants were used as the negative control, whereas the transgenic tobacco line with the highest Ct value was used as the positive control.
To functionally characterize PnPR4, the transgenic tobacco lines in which PnPR4 was highly expressed were examined regarding their resistance to a C. destructans infection, with WT plants serving as the control. Fully expanded tobacco leaves with a uniform size were wounded and then inoculated with a 20 µL C. destructans spore suspension (106 spores/mL). The inoculated leaves were placed on sterile water-soaked filter paper in a foam box, which was sealed with plastic wrap and maintained at 25°C in a light incubator for 1 week. The inoculated leaves were collected and the lesions due to the C. destructans infection were examined.