Plant materials and S. lycopersici inoculation
Tomato plants of the resistant cultivar Motelle (MO) were provided by the Chinese Academy of Agricultural Sciences. Seedlings of the transgenic line Micro-Tom (MT) and Nicotiana benthamiana were obtained from our laboratory. Tomato and tobacco plants were subsequently grown in a greenhouse at 25-28°C and 60% relative humidity under a 14 h/10 h light/dark photoperiod.
S. lycopersici was isolated from tomato plants and plated on potato dextrose agar (PDA) in Petri dishes at 25-28°C for 10 days under a 12 h/12 h photoperiod. Afterward, 4-week-old tomato seedlings of MO, Moneymaker and MT were inoculated with a conidial suspension (1×104 conidia mL-1), while control plants were sprayed with sterilized water. The plants were maintained in a greenhouse (25-28°C) under a relative humidity of >80%. The disease indexes were evaluated post inoculation, and leaves were harvested at 0 and 3 days post inoculation (dpi) for further analysis.
Gene cloning and bioinformatic analysis
The 5’- and 3’-ends of cDNA sequences were cloned by homologous recombination via PCR Cloning Kit. Specific primers used for the target sequence were designed via Primer 6.0 software, and the target gene SlERF01 was cloned via PCR implemented in accordance with the following reaction protocol: 94°C for 3 min; 35 cycles of 94°C for 30 s, 60°C for 45 s, and 72°C for 30 s kb-1; and 72°C for 10 min. A part-CAM-SLERF01 vector was constructed for the identification of positive clones. All the primers used in the study are shown in Table S1.
The SlERF01 sequence was examined by checking the NCBI Conserved Domain Database (CDD) (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi), and the identified sequences were analyzed via DNAMAN 5.0 (Data S2). A phylogenetic tree of the AP2/ERF family proteins of tomato was subsequently constructed by MEGA 5.2.
Subcellular localization
The full-length SLERF01 open reading frame (ORF) without the termination codon was amplified via PCR in conjunction with a high-fidelity polymerase together with the primers GFP-SLERF01-F and GFP-SLERF01-R. A pCAM35::SlERF01-GFP fusion construct was prepared by inserting the PCR products into a pCAM35::GFP vector between its KpnI and XbaI sites. The pCAM35::GFP (control) and pCAM35::SLERF01-GFP vectors were subsequently transformed into Agrobacterium tumefaciens GV3101. Single clones were selected and then cultured in Luria-Bertani (LB) liquid media containing corresponding antibiotics. The transformed Agrobacterium cells were concentrated by centrifugation, after which they were harvested, diluted to an OD600 of 0.4, and injected into N. benthamiana leaves via a syringe. Two days after agroinfiltration, the green fluorescent proteins (GFPs) were imaged by a laser scanning confocal microscope (FV10-ASW, Olympus).
Transformation of tomato
The full-length coding DNA sequence (CDS) of SlERF01 was amplified via PCR and cloned into a part-CAM vector harboring XhoI and XbaI sites. A pCAM-SLERF01 overexpression vector was constructed, and the pCAM-SlERF01 recombinant plasmid and the pCAM plasmid were transferred into A. tumefaciens strain GV3101 (BioVector NTCC Inc., Beijing, China). The pCAM-SlERF01 (overexpression) and pCAM (empty) vectors were transferred into the susceptible cultivar MT via a tomato genetic transformation technique [25]. Ten-day-old tomato seedlings were used as explants and precultured for 2 days on MR (Murashige and Skoog (MS) media supplemented with 0.2 mg l-1 zeatin and 1.0 mg l-1 indoleacetic acid (IAA), pH 5.8) media.
A single colony of A. tumefaciens was selected from LB liquid media that was supplemented with corresponding antibiotics. Bacterial cells were then collected, after which tomato cotyledons were immersed in the bacterial suspension for 3-5 min and cocultivated for 2 days. Infected cotyledons were transferred to suitable media and allowed to grow for 2 weeks, and the explants were subcultured every 3 weeks. After acclimatization, plantlets with well-developed roots were transplanted into soil.
Two different A. tumefaciens strains were used for virus-induced gene silencing (VIGS). One carried TRV1, which encoded viral proteins needed for replication and movement, while the other, TRV2, harbored the coat protein and sequence used for VIGS [26]. The target sequence of SlERF01 was amplified via PCR with specific primers. After digestion with EcoRI and BamHI, the TRV vector was ligated to the PCR product. TRV::SlERF01, TRV::00 and TRV::PDS vectors were constructed and propagated in LB media that containing 50 mg mL-1 kanamycin. The recombinant plasmids were then transferred into A. tumefaciens strain GV3101, after which the transformed cells were cultured in induction media (10 mM 2-(N-morpholino)ethanesulfonic acid (MES), 10 mM MgCl2, 2.50 μg mL-1 kanamycin, 100 μg mL-1 rifampicin and 200 µM acetosyringone) to an OD600 of 0.3. Lst, TRV1 and TRV2 were mixed together at a volumetric ratio of 1:1 and incubated for 3 h; MO plants at the 3-4-leaf stage were then infiltrated with each mixture via a 1 mL syringe containing approximately 0.5-1 mL of the Agrobacterium cell culture solution. The treated plants were sampled at indicated time points for further analysis, and 3 biological replicates were included in the test.
Real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis and determination of physiological indexes
Expression analysis of the overexpression and VIGS plants was performed via qRT-PCR. Total RNA was extracted from tomato leaves by TRIzol reagent [27]. cDNA was synthesized by a reverse transcription kit (TaKaRa) according to the manufacturer’s instructions. The qRT-PCR system consisted of 10 μL of 2× TransStart Top Green qPCR SuperMix (TransGen, China), 0.5 μL of forward/reverse primers, and 2 μL of cDNA template, and ddH2O was added to bring the total volume to 20 μL. The qRT-PCR program was as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 5 s, 62°C for 15 s and 72°C for 30 s. The 2–∆∆CT method [28] was subsequently used to analyze the qRT-PCR data, with EF1α serving as a reference gene [29]. The qRT-PCR primers used are listed in Table S1.
For exogenous hormone treatment, 0.2 mM SA and 0.4 mM JA solutions were sprayed onto tomato plants (the control plants were sprayed with water) at different time points (SA: 0, 12, 24, 48 and 72 h; JA: 0, 24, 48, 72 and 96 h). The levels of the endogenous SA and JA hormones were measured via high-performance liquid chromatography (HPLC). SA and JA were extracted from the leaves according to a modified method described by Llugany et al. [30], after which their concentrations were measured by an AB SCIEX QTRAP 5500 instrument (USA) according to the manufacturer’s instructions. Samples were collected from three individual plants for analyses of the SA content, JA content and gene expression. Data from three independent experiments were statistically analyzed according to Student’s t-tests, and P < 0.05 was considered statistically significant.
Microscopy observations
Trypan blue staining [31], 3,3-diaminobenzidine (DAB) staining, toluidine blue (TB) staining and aniline blue (AB) staining were used to observe the progression of S. lycopersici infection and the production of H2O2, lignin and callose in SlERF01-overexpressing and SlERF01-VIGS plants. The leaves were collected at 0 and 3 days after inoculation.
Cell death was observed by the use of TB staining, with destaining in Farmer’s solution (95% ethanol:chloroform:acetic acid at a volumetric ratio of 6:3:1) for 3 h and boiling in 0.1% trypan blue solution at 65°C for 2 h, followed by transfer to a saturated chloral hydrate solution for 4 h. The leaves were ultimately observed under a light microscope.
The production of H2O2 was detected via DAB staining [32]. Infected tomato leaves were incubated in 0.1% DAB solution at room temperature in the dark for 12 h and then boiled in a 96% ethanol solution for 10 min. The leaves were ultimately observed under a light microscope. Lignin was observed by the use of the TB staining method [33]. The infected tomato leaves were placed in formaldehyde:acetic acid:ethanol (FAA) solution for 24 h and then stained with a 0.05% TB solution. The leaves were subsequently observed under a light microscope. Callose was detected by the use of the AB staining method [34]. The infected tomato leaves were placed in FAA solution, cleared with 100% ethanol solution and then stained with 0.07 M K2HPO4 in a 0.01% AB solution for 24 h. The leaves were ultimately observed under a fluorescence microscope. Leaf samples were collected from three individual plants for analyses of the HR, H2O2 production, and lignin and callose accumulation.