CaAMP1 synthesis and vector construction
The nucleotide sequence of C. annuumCaAMP1 (GenBank ID: AAT35532.1) was synthesized with added Xba I and Sac I recognition sites at the 5′ and 3′ ends, respectively (Sangon Biotech, Shanghai, China). The modified CaAMP1 gene was inserted into a pCambia3300 vector containing a modified CaMV 35S promoter [48] (GenBank: GI3319906) to facilitate its constitutive expression in soybean. The gene sequence was amplified using the CaAMP1-F/R primer pair, with a final primer concentration of 0.4 μM, under the following conditions: 94°C for 5 min; followed by 35 cycles of 94°C for 30 s, 59°C for 30 s, and 72°C for 30 s; and final extension at 72°C for 7 min. All primers used in this study are listed in Table S1. The purified fragment was then subcloned into a pCambia3300 plasmid containing a phosphinothricin acetyl transferase (bar) resistance gene, encoding PAT, as a plant selection marker driven by a modified CaMV 35S promoter [48] (GenBank: GI3319906). The constructed pCambia3300-CaMV 35S-CaAMP1 plasmid was subsequently transformed into competent A. tumefaciens strain EHA101 cells, by the freeze-thaw method [49, 50].
Regeneration and screening of transgenic plants
Agrobacterium-mediated transformation was used for regenerating transgenic soybean, with the soybean cultivar Williams 82 as the recipient, which was provided by Prof. Fudi Xie of Shenyang Agricultural University, China (ID: WDD00587, Chinese Crop Germplasm Information System, http://www.cgris.net), following the method described in Yang et al. (2018) and Zhang et al. (2014) [51, 52]. The regenerated PAT-tolerant plants were screened using LibertyLink® strip test (cat #AS 013 LS; EnviroLogix Inc., Portland, ME, USA) and PCR. Herbicide-tolerant T1–T3 transgenic lines were identified by spraying the leaves with 500 mg∙L-1 glufosinate (EnviroLogix Inc., Portland, Maine, USA) on complete expansion of the first trifoliate leaves, and then analyzed by PCR using the CaAMP1-F1/R1 and Bar-F/R primer pairs (Table S1) until homozygous transgenic plants were obtained. DNA was extracted from the leaves of transgenic and wild-type soybean, using a simple homogenization and ethanol precipitation method, for PCR analysis [53]. PCR was performed with a final primer concentration of 0.2 μM, with the following conditions: 94°C for 5 min; followed by 35 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 30 s; and final extension at 72°C for 7 min.
To confirm the integration of T-DNA in transgenic soybean, T2 transgenic plants were selected for genomic DNA extraction, using a modified high salt cetyl-trimethyl ammonium bromide method [54]. DIG High Prime DNA Labeling and Detection Starter Kit I (No. 11745832910; Roche Applied Science, Indianapolis, IN, USA) was used for Southern blot analysis, according to the manufacturer’s instructions. Approximately 30 μg of the genomic DNA from transgenic soybean and control plants was digested completely with EcoR I (New England Biolabs Inc., Beverly, Massachusetts). The digested DNA was then transferred onto positively charged nylon membranes (GE Amersham, RPN303B, USA). Hybridization was carried out at 42°C for 12–16 h, using CaAMP1 labeled with digoxigenin-(DIG)11-dUTP as a probe. The washing conditions and signal detection were as described in Yang et al. (2018) [51].
Expression analysis in transgenic soybean
Total RNA and proteins were extracted for expression analysis. Total RNA was extracted from 2-week-old leaves of T3 transgenic plants (8096, 8101, 8111, 8130, 8197, 8253) using a EasyPure PlantRNA Kit (TransGen Biotech, Beijing, China), and DNase I was used to eliminate the contaminant genomic DNA. cDNA was then synthesized using the ThermoScript RT-PCR system (Invitrogen, USA), and RT-PCR was performed using CaAMP-RF/RR primers (Table S1). GmACT (GeneBank ID: NM 001289231), amplified using the primers 5′-CACCGGAGTTTTCACCGATA-3′ and 5′-AGGAATGATGTTAA-3′, was used as the control.
Crude proteins were extracted from ~100 mg fresh leaves of the control and T3 transgenic soybean lines (8096, 8101, 8111, 8130, 8197, 8253), separated on a 12% (w/v) SDS-PAGE gel, and then transferred electrophoretically onto a PVDF membrane (AmershamTM HybondTM, GE Healthcare, USA) [41]. After blocking with 3% dried skimmed milk diluted in PBST (1× PBS, 0.1% Tween-20), the membrane was blotted with a rabbit polyclonal antibody (1:500 dilution) raised against recombinant CaAMP1 protein (GenScript Co., Ltd. Nanjing, China) and horseradish peroxidase (HRP)-labeled goat-anti-rabbit IgG (1:5,000 dilution; Abcam, UK) at 25°C for 4 h. The bands observed following western blotting were visualized using the BiodlightTM Western Chemiluminescent HRP substrate (Bioworld Technology, Inc., St. Louis, MN, USA) after extensive washing.
Evaluation of PRR tolerance under greenhouse conditions
To evaluate the tolerance of transgenic soybean to P. sojae race 1, the T2−T4 generations of transgenic lines 8096, 8101, 8111, 8130, 8197, and 8253 were infected with P. sojae race 1, following the method described by Schmitthenner et al. (1994) [55]. Isolation and cultivation of the inoculum were performed as described by Akamatsu et al. (2010) and Du et al. (2018) [56, 28]. Transgenic soybean, wild-type Williams 82, and the PRR-susceptible cultivar Jiunong 21 (ID:ZDD22796), which were provided by the Soybean Research Institute of Jilin Academy of Agricultural Sciences, were grown in a greenhouse, and the hypocotyls of 15-day-old seedlings were inoculated with macerated mycelia of P. sojae race 1. The plants were then maintained in a humid environment for 15–24 h, before being transferred to the greenhouse for symptom development, at 25°C under an 18-h light/6-h dark photoperiod [28]. After 5 to 10 days of inoculation, plant infection data were collected and survival rates were calculated [57]. All experiments were performed with three replicates of 20 inoculated plants each replicate.
Differences in the survival rates of the control and transgenic lines were quantitatively assessed by t-test at a significance level of P = 0.05 or 0.01, using Microsoft Analysis Tool.
Quantitative RT-PCR analysis of disease-responsive genes
Leaves were collected from T3 transgenic lines (8096, 8101, 8111, 8130, 8197, 8253) and wild-type Williams 82 plants, 0, 1, 2, 4, 8, 12, and 24 h after inoculation with P. sojae race 1 mycelia, for quantitative PCR. Total RNA extraction and cDNA synthesis were performed as described in previous sections. The relative expression levels of 12 genes involved in different stress response pathways, including PR1 (AF136636), PR2 (M37753), PR3 (AF202731), PR5 (BU765509), PR12 (BU964598), PAL (X52953), PPO (EF158428), AOS (DQ288260), SGT1 (NM_001249656), GmNPR1-1 (FJ418594), GmNPR1-2 (FJ418596), and GmRAR1 (FJ222386), were analyzed by qRT-PCR, with GmACT (U60500) as the internal control. Amplification was performed in a final reaction volume of 20 µL, with ~80 ng cDNA and 0.4 µL each of forward and reverse primers (Table S1), using a SYBR Green-based One-Step qRT-PCR kit (TransGen Biotech, China). The conditions for the qRT reaction were as follows: 50°C for 2 min; 95°C for 10 min; and 45 cycles of 95°C for 2 min, 62°C for 30 s, and 72°C for 30 s. The relative expression level of each gene was determined using the 2-ΔΔCt method [58]. To improve the accuracy of the data, three biological and three technical replicates were performed for each experiment.
Agronomic traits of transgenic lines
Nine agronomic traits of T3 transgenic lines and wild-type soybean were assessed, including maturity period, leaf shape, plant height, flower color, hilum color, branch number, node number, podding height, and 100-seed weight, and t-test was used for quantitative analysis.