Seeds of tomato cv. Rio Grande plants were sown in hydroponic seed plugs (rockwool), germinated and grown under controlled greenhouse conditions (25 ± 2 °C, 16 h light/15 ± 2 °C, 8 h dark, and 60% relative humidity (RH). Two-week-old seedlings (two cotyledons) were transplanted into rockwool plugs (7.5 × 7.5 × 6.5 cm, Gordan Iberica, Spain). The experimental design consisted of three replicates of three plants per treatment. After two weeks, tomato leaves were sprayed with aqueous solutions of the peptides BP16, flg15, and BP358, an equimolar mixture of BP16 and flg15, at 125 µM, or with acybenzolar-S-methyl at 300 mg/L (Syngenta, Basel, Switzerland) until the run-off point. Water-sprayed plants were used as untreated controls. Twenty-four hours after product application, leaf samples were collected and processed to extract RNA for RT-qPCR assays. Plant material was ground to a fine powder in liquid nitrogen, adding 2 acid-washed glass beads (Sigma, 150 ± 600 μm) to the sample using the Tissuelyzer II system (Qiagen, Hilden, Germany). Total RNA was extracted from leaves using PureLink Plant RNA Reagent (Invitrogen, Life Technologies) according to the manufacturer’s manual. The RNA was solubilized in RNAse free water and was routinely subjected to DNAse treatment (Ambion® Turbo DNA-free™, Invitrogen Life Technologies, Carlsbad, CA, USA) to remove any contaminant DNA. In each step, the RNA was quantified using a Nanodrop N-2000 spectrophotometer, and its integrity was verified by denaturing agarose gel electrophoresis. First-strand of complementary DNA (cDNA) was generated from leave RNA using reverse transcriptase (High Capacity cDNA Reverse Transcription Kit, Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s manual.

To test gene defense induction in the treated tomato plants, a quantitative PCR (qPCR) assay was performed. qPCR was carried out in a fluorometric thermal cycler (qPCR Quant Studio 5, Applied Biosystems) by using a Mix SYBR®Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) as previously described [4]. Melting curve analysis was performed after each amplification to verify amplification specificity. A constitutive gene (actin) was used as a reference control, and the following genes implicated in plant defense response were analyzed: pathogenesis-related protein-1 (PR1), harpin (Harp), polyphenol oxidase (PPO), subtilisin-like protease (Sub1), blue copper binding-protein (BCB), osmotin (Osm2), acidic β-1,3 endoglucanase (GluA), lipoxygenase (LOX), protein inhibitor II (PinII), dehydrin (Tas14), and early-ripening tomato (ERT3). Specific oligonucleotides genes [4,55] were designed and used for their quantification. The primer concentration was 100 nM for all the genes except for the GluA, Harp, PR1, and actin genes which concentration was 300 nM. A calibration curve was prepared using decimal dilutions of recombinant plasmid DNA (target sequences were cloned into a vector pSpark (Canvax, Córdoba, Spain) in Escherichia coli DH5α cells). The efficiency for each standard curve was calculated to check that the efficiency within amplifications was similar.

Relative quantification of gene expression was done using the ΔΔCt method as previously described [4,52]. Ct values obtained for each repetition treatment were used to estimate the fold change value of the endogenous reference gene (actin) and the target plant defense genes. These results were used to calculate the ratios of the plant defense genes (relative to the actin gene and for all treatments analyzed, including the control plants). The statistical significance of the results for the selected peptides was determined using the REST2009 Software (Qiagen) [62].

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