*4.11. Gene Expression*

According to the manufacturer's protocol, total mRNA was isolated from 0.5 g tomato plant root of control and all treatments using the Plant RNA Kit (Sigma-Aldrich, St. Louis, MO, USA). The purified RNA was quantitated using SPECTROstar Nano (BMG LABTECH, Ortenberg, Germany). For each sample, 10 μg total RNA was treated with DNAse RNAsefree (Fermentas, Waltham, MA, USA), 5 μg of which was reverse transcribed in a reaction mixture consisting of oligo dT primer (10 pmL/μL), 2.5 μL 5X buffer, 2.5 μL MgCl2, 2.5 μL 2.5 mM dNTPs, 4 μL from oligo (dT), 0.2 μL (5 Unit/μL) reverse transcriptase (Promega, Walldorf, Germany), and 2.5 μL RNA. RT-PCR amplification was performed in a thermal cycler PCR, programmed at 42 ◦C for 1 h and 72 ◦C for 20 min. Quantitative real-time PCR was carried out on 1 μL 1:10 diluted cDNA templates by triplicate using the realtime analysis (Rotor-Gene 6000, QIAGEN GmbH, Hilden, Germany) system. The primer sequences used in qRT-PCR are given in Table 4. Primers of three PRs (PR3, PR12) genes, three WRKY transcriptional factors (WRKY1, WRKY4, WRKY33, and WRKY53 TFs genes), and housekeeping gene (reference gene) were used for gene expression analysis using SYBR® Green-based method. The reaction mixture consists of 1 μL of template, 10 μL of SYBR Green Master Mix, 2 μL of reverse primer, 2 μL of forwarding primer, and sterile distilled water for a total reaction volume of 20 μL. PCR assays were performed using the following conditions: 950 ◦C for 15 min followed by 40 cycles of 950 ◦C for 30s and 600 ◦C for 30 s. The CT of each sample was used to calculate ΔCT values (target gene CT subtracted from β-Actin gene CT [86]). The relative gene expression was determined using the 2-ΔΔCt method [87].


**Table 4.** Sequences of primers used in qRT-PCR analysis.
