*2.6. Gene Expression Analysis*

The level of transcript for PR genes (β*-1,3-glucanase 1*, β*-1,3-glucanase 2*, *chitinase*), genes involved in chromatin modification (*chromomethylase 1*, *chromomethylase 3*, *repressor of silencing 1*, *demeter*, *argonaute 1*, *argonaute 4*, *RNA-dependent RNA polymerase 2*), and *actin* gene was measured using the quantitative PCR technique.

The total RNA was isolated from plant tissues by treatment with Trizol reagen<sup>t</sup> (Life Technologies, Carlsbad, California, USA) according to the manufacturer's manual. Its quality was verified by gel electrophoresis on 1.5% (w/v) agarose containing 15% (v/v) formaldehyde and quantitated spectrophotometrically (Spectrophotometer Implen NanoPhotometer Pearl, München, Germany) at λ = 260 nm. In order to remove the residual genomic DNA, the samples were subjected to digestion with DNase I (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Then, purified RNA served as a template for reverse transcription PCR to obtain cDNA. The reaction was designed using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, California, USA).

The quantitative PCR reactions were run using a SYBR dye-based set of reagents (DyNAmo HS SYBR Green qPCR Kit, Thermo Fisher Scientific, Waltham, Massachusetts, USA) on an Applied Biosystems StepOnePlus Real-Time PCR System (Life Technologies, Carlsbad, California, USA). Primers were designed to selectively amplify only plant sequences (not fungal, see Table S1) using LightCycler Probe Design Software 2 (Roche, Basel, Switzerland). Their specificity at annealing temperature was verified by analyzing the melting curves of the obtained reaction products. The reaction setup was designed according to the kit manufacturer's protocol. The program used for real-time PCR was 95 ◦C for 10 min, and 40 cycles of denaturation for 15 s at 95 ◦C, annealing for 20 s at 57 ◦C, and extension for 30 s at 72 ◦C, and reaction mixtures contain (in final volume 25 μL): Master Mix 2x, forward and reverse primers in final concentration 0.5 μM and 125 ng cDNA (final concentration 5 ng/μL) All the reactions were run in triplicate. Changes in gene expression levels are presented as x-fold of relative quantities (RQ) standardized for *actin* (the reference gene) in relation to the control, non-treated plants.
