2.6. Validation of DEGs by RT-qPCR

To confirm the transcriptome data obtained by RNA-seq analysis, 27 DEGs were selected (Log₂(FC) > |1.4| and good repeatability among the three biological replicates) and their expression level was validated by RT-qPCR ( Supplementary Table 2 ). The UBQ gene, coding for the Ubiquitin-conjugating enzyme, was used in this study as the endogenous gene for data normalization. This endogenous gene was previously selected according to the method described by Silver et al. (2006) ( Supplementary Figure 1 ).

Standard curves for DEGs and the endogenous gene were obtained using decimal dilutions of extracted recombinant plasmid DNA (target sequences were cloned into a vector pSpark^® in Escherichia coli DH5α cells) corresponding to copy numbers ranging between 10² and 10⁷. Ct values in each dilution were measured in triplicate and a negative non-template control was included in each run. Real-time PCR reactions included 10 μL SYBR^® Green PCR Master Mix (Applied Biosystems), 6 μL RNase-free water, 1 μL of each forward and reverse primer ( Supplementary Table 2 ) at the corresponding concentration, and 2 μL DNA in a final volume of 20 μL. The optimal primer concentration (100, 300 or 600 nM) was previously defined. The thermal cycling conditions were as follows: 10 min at 95˚C for initial denaturation; 40 cycles of 15 s at 95˚C, and 1 min at 60˚C; and a final melting curve program of 60 to 95˚C with a heating rate of 0.5˚C s^-1. Ct values were plotted against the logarithm of their initial template copy numbers and each standard curve was generated by a linear regression of the plotted points. The efficiency of each standard curve was calculated using the formula E = (10^(-1/a) -1) x100, where “a” is the slope of the curve.

For RT-qPCR, total RNA was extracted from leaf samples of treated plants using Spectrum™ Plant Total RNA Kit (Sigma-Aldrich) as explained above. First-strand of cDNA was synthetized from RNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer’s instructions. The absence of chromosomal DNA contamination was confirmed by minus-reverse transcriptase control in qPCR. Quantitative PCR was carried out in a QuantStudio™ 5 Real-Time PCR System (Applied Biosystems) to assess the transcriptional level of 27 DEGs. All the information of the selected genes and primers designed by Primer-BLAST tool from the Nacional Centre for Biotechnology Information (NCBI) are shown in Supplementary Table 2 . Optimized qPCR reactions and the thermal cycling conditions were described above. Each qPCR assay included duplicates of each cDNA sample, no-template and RNA controls to check for contamination. Ct values from three biological replicates were averaged, and UBQ gene was used for data normalization.

The comparative critical threshold (ΔΔCt) method was used to assess the relative quantification of gene expression. Similar amplification efficiencies of all gene primer pairs were checked ( Supplementary Table 3 ) making the ΔΔCt method appropriate to calculate the Fold-Change (FC). The ΔCt of the NTC leaf samples was used as the calibrating condition to calculate the FC. Genes were considered to be up- or downregulated if their FC were at least two-fold (FC = 2¹ or 2⁻¹) higher or less than the calibrator condition (Livak and Schmittgen, 2001) and showed statistically significant differences with the NTC.