1.1 Plant material, bacterial strains, and pathogen cultures
Annual P. frutescens seeds were sown in an outdoor experimental plot of the College of Life Sciences, Chongqing Normal University, Chongqing, China (29°36′50″ N, 106°18′2″ E) in April, and different tissues at flowering stage (roots, stems, leaves, and flowers) and mature seeds were collected in July, snap-frozen in liquid nitrogen and stored in -80°C refrigerator until use. B. napus (Zhongshuang No. 11, WT) seeds were sterilized with 75% ethanol for 30–60 s, then 0.1% mercuric chloride for 10 min, and finally washed 4–6 times with sterile double distilled water (ddH2O). The sterilized B. napus seeds were germinated on 1/2 Murashige and Skoog (MS) medium (Coolaber, Beijing) and cultured at 25°C for 4–6 d in the dark condition to obtain asepsis seedling.
The competent Escherichia coli DH5α strain and Agrobacterium tumefaciens GV3101 strain were purchased from WEIDI (China) and used. To obtain vigorously growing pathogens, the pathogenic fungi (A. brassicae and B. cinerea) were inoculated in potato dextrose agar (PDA) medium, respectively, and then incubated at 26/26 ℃ (Light/Dark), 0 h/24 h (Light/Dark) conditions for 7 d.
1.2 Cloning and sequence characterization of PfPDF genes
Cloning primers were designed based on the sequences annotated as PfPDF genes in transcriptome database of P. frutescens [21], and then PfPDF sequences were cloned by PCR amplification using P. frutescens seed genomic DNA (gDNA) and cDNA as templates, respectively (Table S1). Each reaction comprised 1.5 µl template (10 ng), 12 µl Premix Taq (TaKaRa, Japan), 1.5 µl of the primers pairs (10 µM), and 10 µl ddH2O. PCR was performed as follows: 94 ℃ for 5 min, followed by 30 cycles of 94 ℃ for 30 s, 57 ℃ for 30 s, 72 ℃ for 60 s, and 72 ℃ for 10 min. PCR products were detected by 1% (w/v) agarose gel electrophoresis and collected using the SanPrep Column DNA Gel Extraction Kit (Sangon, China). The recombinant intermediate vectors pMD19T-PfPDF were constructed using the pMD19-T Vector Cloning Kit (TaKaRa, Japan) according to the manufacturer's instructions. Primer synthesis and sequencing were performed by ThermoFisher (China) and Sangon (China), respectively.
The intron-exon structures of the PfPDF genes were visualized by the GSDS 2.0 server (http://gsds.gao-lab.org/). The ProtoParam (https://web.expasy.org/protparam/) was used to analyze the basic characteristics of PfPDF, including the number of amino acids (NA), molecular weight (MW), theoretical isoelectric point (pI), grand average of hydropathicity (GRAVY) and instability index (II). The amino acids translation of PfPDF genes was performed using Vector NTI software. DiANNA server (http://clavius.bc.edu/~clotelab/DiANNA/) was used to predict the disulfide bond topology [22]. Analysis of transmembrane helices and signal peptides was performed using the TMHMM-2.0 and SignalP-6.0 resources in the DTU.dk service (https://www.dtu.dk/), respectively. The secondary structure and 3D-model of PfPDF proteins were executed by Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2) [23].
1.3 Analysis of protein-protein interaction (PPI) network
PPI network provides an intuitive and rapid understanding of gene function, and is also important for regulatory relationships between family proteins. Based on this, we constructed a PPI network of PfPDF proteins using the information of Arabidopsis homologs in the String database (v11.5, https://cn.string-db.org/), with the following parameters: all PfPDF protein sequences and the organisms being Arabidopsis.
1.4 Phylogenetic analysis
The Arabidopsis AtPDF protein sequences were downloaded from TAIR database (https://www.arabidopsis.org/). Multiple sequence alignment of AtPDF and PfPDF proteins using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) and visualized by Jalview. The evolutionary relationship among AtPDF and PfPDF was elucidated by constructing an unrooted phylogenetic tree by the Neighbor-Joining (N-J) method in MEGA X [24]. Various technical parameters (e.g., 1000 bootstrap replicates, Pairwise deletion and the JTT + G evolutionary model) were used to construct, and visualized with the iTOL website (https://itol.embl.de). Detailed accession numbers of AtPDF sequences were available in Table S2.
1.5 PfPDF expression pattern analysis
The real-time quantitative polymerase chain reaction (RT-qPCR) was performed for analysis the expression pattern of PfPDF genes. Total RNA was extracted from different tissues (roots, stems and leaves, and flowers at flowering stage) and mature seeds of P. frutescens using the RNAprep pure plant kit (Tiangen, China), and then reverse transcribed to cDNA using PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa, Beijing). Efficient RT-qPCR primers were designed based on the coding sequences (CDS). The reaction system was 10 µL SYBR Green super mix (Bio-Rad, USA), 1 µL primer pairs, 4.0 µL cDNA, and 5.0 µL Nuclease-free H2O. RT-qPCR reaction conditions were performed as follows: 95 ℃ for 60 s, followed by 40 cycles of 95 ℃ for 10 s、60 ℃ for 30 s. All RT-qPCR reactions were performed on a CFX96 Touch Real-Time PCR system (Bio-Rad, USA) and Pf18S as an internal reference gene. The 2−△△CT method was used to calculate the relative gene expression.
1.6 Construction of plant expression vector
The pCAMBIA1303 vector was double digested using Nco I and Spe Ⅰ, and purified PfPDF2 fragment was ligated to the digested linearized pCAMBIA1303 vector using In-Fusion HD Cloning Kit (TaKaRa, Japan) in accordance with the manufacturer's instructions. The recombinant vector was transferred into DH5α, and the positive strain was picked for sequencing. Finally, plasmid was extracted using TIANprep Rapid Mini Plasmid Kit (Tiangen, China) and transformed into GV3101 using the freeze-thaw method.
1.7 Production of transgenic plants
The cotyledons of B. napus were co-incubated with A. tumefaciens infestation solution for 5 min, and then cultured on 1/2 MS medium in dark conditions. After 2 d, the cotyledons were transferred to the callus induction medium (MS contain 6-BA 1 mg/L, 2,4-D 1 mg/L, Kan 50 mg/L, and Cef 500 mg/L) and cultured for 21–28 d. Subsequently, transferred into bud induction medium (MS contain 6-BA 3 mg/L, Cef 500 mg/L, ZT 2 mg/L, and Kan 20 mg/L). Transfer to stem induction medium (MS contain 6-BA 0.05 mg/L, Cef 250 mg/L, and Kan 20 mg/L) after it has grown young shoots. Finally, they were transferred to root induction medium (MS contain IBA 1 mg/L and Cef 50 mg/L) and cultured for 7 d. The well-grown regenerated seedlings were transplanted into plastic boxes with nutrient soil and coarse soil (1:2, v:v) after acclimatizing and grown at 25 ℃ with a photoperiod of 16/8 h (Light/Dark) until mature seeds were harvested.
The binary vector pCAMBIA1303, which carried a gene encoding hygromycin phosphotransferase as plant selection marker and β-glucosidase (GUS) as a reporter, and driven by the CaMV 35S promoter. The gDNA were extracted from different lines of T1 generation transgenic B. napus and molecular identification using primers targeting the T-DNA region of pCAMBIA1303 (Table S1), gDNA of WT was used as a negative control. PCR was performed as follows: 94℃ for 5min, followed by 35 cycles of 94°C for 30 s, 57°C for 30 s, 72°C for 1 min, and 72◦C for 10 min. The leaves of transgenic B. napus plants were stained and identified using the GUS stain Kit (Coolaber, China). Finally, total RNA was extracted from young leaves of transgenic and WT B. napus, and the expression level of PfPDF2 was analyzed by RT-qPCR. The T1 generation seeds were screened by 1/2 MS medium (20 mg/L hygromycin B), and transplanted to soil. The above procedure was repeated, the T4 generation transgenic B. napus without trait segregation was considered as a homozygous line for further studies.
1.8 Resistance of B. napus expressing PfPDF2 to fungal pathogens
In vitro leaf antifungal bioactivity was measured on T4 generation transgenic B. napus (A1, A2) and WT (50 days after sown). A 0.5 cm2 wound was created on the adaxial surface of B. napus leaf through a scalpel, and a PDA block with actively growing pathogens (0.5 cm2) was placed on the proximal surface of the wound. Subsequently, co-cultured in an artificial incubator: 24 ℃/20 ℃ (Light/Dark), 0 h/24 h (Light/Dark), 80% RH. Observed the disease symptoms and degree of damage after 7 DPI (days post inoculation). The farthest distance from the inoculation center to the lesion was recorded as lesion radius. The experiment was repeated four times.
1.9 RNA-Seq and KEGG analysis
Total RNA was extracted from leaves of 1-month-old WT and A2 plants, and cDNA libraries were prepared by the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, England) in accordance with the supplier’s instructions, and subsequently sequenced by Illumina. Low quality bases (Qphred < = 20), adapters and lower quality sequences were removed from the raw sequence reads. The number of reads mapped to each gene was then calculated by featureCounts (1.5.0-p3) and the fragments per kilobase of transcript per million mapped reads (FPKM) mapped to that gene was calculated. Finally, KEGG analysis of the differentially expressed genes (DEGs) were performed by clusterProfile [25].
1.10 Agronomic trait analysis
To determine whether the over-expression of PfPDF2 function might affect agronomic traits, WT, A1 and A2 plants were grown in plastic boxes with the same nutrient soil under natural short-day (NSD, < 14-h light/day) conditions. Ten plants of each line were randomly selected before plant harvest to measure plant height and number of branches. The number of seeds per pod, 1000-grain weight, total yield and the number of pods per 20 cm were measured after maturity. The total protein content of A1, A2, and WT B. napus was determined by using a Protein Content (SP) Kit (Grace, China).
To determine whether the introduction of PfPDF2 would have an effect on the production of B. napus seed oil, we also extracted the total oil from mature seeds to analyze oil content and fatty acid (FA) composition. Fistly, mature seeds were dried at 50°C for 48 h. The total oil of WT, A1 and A2 were extracted using the Soxhlet extraction method with petroleum ether (30–60°C) as solvent. The FA composition of the seed oil was analyzed by gas chromatograph (GC) according to Zhou et al [26].
1.11 Statistical analysis
The experimental results were statistically analyzed using Student’s t-test in IBM SPSS Statistics. * and ** indicates significant difference at P < 0.05, P < 0.01, respectively. Graphs were drawn by using GraphPad Prism v8.0.