*2.3. DNA Isolation*

Genomic DNA was isolated using DNeasy Plant Mini Kit (QIAGEN, Hilden, Germany) following the manufacturer's protocol. The DNA integrity was examined by gel electrophoresis on 1.0% (w/v) agarose and the DNA amount was checked by spectrophotometry method (Spectrophotometer Implen NanoPhotometer Pearl, München, Germany) at 260 nm.

### *2.4. Determination of Methylation Patterns of Genes*

The methylation pattern of DNA was determined using the combination of restricted cleavage of the gene sequence at methylation sites (CCGG islands) using the restriction enzymes *MspI* and *HpaII* (New England Biolabs, Ipswich, Massachusetts, USA) and real-time PCR using primers flanking the methylation islands (cleavage sites). The difference between these two isoschizomers is that *HpaII* can cleave unmethylated CCGG, but *MspI* can cleave unmethylated CCGG as well as the sequence when the internal C residue is methylated CCmGG. Both of these enzymes do not cleave the sequence CCGG when the external and internal C is methylated (CmCmGG).

The reaction (in final volume 50 μL) of restriction cleavage by *HpaII* (250 units) and *MspI* (250 units) was run at 37 ◦C overnight on the template DNA (0.75μg), which later served as a template for real-time PCR reactions. Restrictions enzymes were inactivated by incubation at 80 ◦C for 20 min. The program used for real-time PCR was 95 ◦C for 10 min, and 40 cycles of denaturation for 10 s at 95 ◦C, annealing for 20 s at 57 ◦C, and extension for 25 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 37.5 ng DNA (final concentration 1.5 ng/μL).

The obtained results of digested and undigested DNA were obtained as x-fold of the relative quantification to *actin* (the reference gene). The pattern of gene methylation sites was calculated according to the equations: CCGG (non-methylated cytosines) = total DNA – DNA left after digest by *HpaII*; CCmGG (internal methylated cytosine) = DNA left after digest by *HpaII* - DNA left after digest by *MspI*; CmCmGG (external and internal methylated cytosine) = DNA left after digest by *MspI*. The changes in the different methylated cytosine in DNA (CCGG, CCmGG, and CmCmGG) were presented as x-fold relatively to the control.

Seven CCGG sites were analyzed (three in the promoter (P) and three in the exon (E) and one in the intron (I)) in the β*-1,3-glucanase 1* gene, twelve sites (eleven in the exon and one in the intron) in the β*-1,3-glucanase 2* gene, and eight sites in the exon in the *chitinase* gene. The β*-1,3-glucanase 1* gene sites were characterized by the following positions starting from ATG in DNA: P1 -1204-1201; P2 -1147-1144; P3 -344-341; E1 + 440-443; E2 + 1479-1482; E3 + 1763-1766; I1 + 2341-2344; the β*-1,3-glucanase 2* gene: E1 +50-53; E2 +432-435; E3 +690-693; E4 +883-886; E5 +891-894; E6 +1009-1012; E7 +1024-1027; E8 +1034-1037; E9 +1097-1100; E10 +1178-1181; E11 +1421-1424; I1 +1874-1877; and the *chitinase* gene: E1 +118-121; E2 +138-141; E3 +163 – 167; E4 +227-230; E5 +401-404; E6 +467-470; E7 +957-960; E8 +1105-1108. A schematic diagram of the position of all putative sites and the relative position of the primers that were used to perform qPCR to estimate methylation levels is presented in Figure S2.

### *2.5. Determination of Total DNA Methylation*

The global level of DNA methylation of flax infected by *Fusarium* strains was determined colorimetrically by measuring the level of 5-methylcytosine using the dedicated MethylFlash Methylated DNA Quantification Kit (EpiGentek Group Inc., Farmingdale, NY, USA) according to the manufacturer's instructions.
