**3. Results**

### *3.1. DNA Methylation Patterns of* β*-1,3-Glucanase and Chitinase Genes*

Due to the lack of flax genes' annotation in a database, in the first step we had to extract whole gene sequences for two isoforms of β*-1,3-glucanase* and *chitinase* genes on the basis of known gene fragments to find whole DNA sequences. The obtained sequences were analyzed in silico in order to identify the potential methylation sites. We found 7 CCGG sites in the β*-1,3-glucanase 1* gene (three in the promoter, three in exons, and one in the intron), 12 CCGG sites in the β*-1,3-glucanase 2* gene (eleven in exons and one in the intron), and 8 CCGG sites in exons in the *chitinase* gene. The analysis of methylation profiles of PR genes included all potential CCGG sites. Here we present only data for those sites where differences in a methylation pattern were observed between treatments. The differentiating CCGG sites for the β-1,3-glucanase 1 gene (3 sites) and chitinase gene (3 sites) are shown as x-fold of different methylation of CCGG (CCGG, CCmGG, and CmCmGG) in Figure 1 and as the DNA methylation pattern in Figure S3. We found that the methylation in all analyzed CCGG sequences for the β*-1,3-glucanase 2* gene did not change.

**Figure 1.** Changes in DNA methylation pattern of β*-glucanase 1*and *chitinase* in flax seedlings treated with non-pathogenic or pathogenic strains of *Fusarium oxysporum* and after sensitizing effects of the

non-pathogenic strain treated with pathogenic Foln3 at 6, 12, 24, 36, and 48 h after inoculation presented as x-fold change in relation to control. The analysis of the third position in the exon, the first in the intron, and the third in the promoter of β*-1,3-glucanase 1* and the first, seventh, and eighth positions in the exon of *chitinase* was performed by the digestion of genomic DNA by the restriction enzymes *HpaII* and *MspI* and then the real-time PCR reaction. The data represent the mean from three biological repetitions. Asterisks mark statistically significant differences (*p* < 0.05) between the treated samples and their own controls.

The DNA methylation pattern of the β*-1,3-glucanase 1* gene in flax incubated with the non-pathogenic strain of *Fusarium oxysporum* was characterized by changes in the level of methylation of internal and/or external cytosine in three CCGG sequences (Figure 1). Analyzing the third CCGG place in the promoter of the β*-1,3-glucanase 1* gene, it was observed that the level of non-methylated cytosines (CCGG) significantly increased over 2.7-fold at the 6th, 12th, 24th, and 48th hour of incubation and decreased to 12% at the 36th hour. The levels of internal methylated cytosine (CCmGG) and internal and external methylated cytosines (CmCmGG) did not change significantly in flax plants incubated with the non-pathogenic strain of *F. oxysporum*. Other changes were shown for the third place in the exon, where a significant increase in non-methylated cytosines (CCGG) was observed at the 12th and 48th hour after incubation, a reduced level to 50% at the 24th hour, and a lack of non-methylated cytosines at the 36th hour. It was followed by a decrease at the 12th hour in the CmCmGG and 1.4-fold and 1.7-fold increases at the 36th and 48th hour, respectively. It was interesting that for CCGG islands located in the intron, the level of CCmGG and CmCmGG remained unchanged, but the level of non-methylated cytosines increased over 1.9-fold at the 12th and 48th hour of incubation and decreased to 64% at the 24th and 7% at the 36th hour.

The analysis of eight CCGG places in the *chitinase* gene revealed that only three of them were characterized by changes in the DNA methylation pattern in flax incubated with the non-pathogenic strain of *Fusarium oxysporum* (Figure 1). In the first CCGG site in the exon, the level of non-methylated cytosines decreased to 56% and 68% at the 12th and 24th hour of incubation with the non-pathogenic strain. An increase in non-methylated cytosines in the CCGG site was noted in the seventh exon (10-fold increase at the 12th hour and more than 6-fold increase at the 48th hour of incubation). It was followed by a decrease in the CCGG to 43% and 8% at the 6th and 24th hour, respectively. In addition, in the eighth site in the exon, the largest increase of the non-methylated cytosines in the CCGG was observed at the 24th and 36th hour after incubation together with the reduced level of the CCGG to 42% at the 6th hour.

In flax plants incubated with the pathogenic strain of *F. oxysporum* the profile status of methylation of cytosines in CCGG sites of the β*-1,3-glucanase 1* gene differed from that determined for flax incubated with the non-pathogenic strain, but the same differentiating CCGG sites remained. We observed a reduced level of non-methylated cytosines at the 6th, 24th, 36th, and 48th hour (a reduction to 20% and to 42% at the 6th and 48th hour and a total reduction at the 24th and 36th hour) and its significantly higher level at the 12th hour in the third CCGG place in the promoter. In the third place in the exon a reduced level of non-methylated cytosines in CCGG was observed at the 6th, 12th, 24th, and 48th hour of incubation (a reduction to 43% at the 6th and a total reduction at the 12th, 24th, and 48th hour) and a significant increase at the 36th hour. In the intron, the decrease in non-methylated cytosines in the CCGG site to 21% and 14% at the 24th and 36th hour after incubation, respectively, was subsequently replaced by an increase (2.9-fold) in non-methylated cytosines at the 48th hour.

The first, seventh, and eighth CCGG sites in the exons in the *chitinase* gene are di fferentiating sites in flax inoculated with pathogen. A thorough analysis of these CCGG sites in the *chitinase* gene revealed about a two-fold increase in non-methylated cytosines at the 6th, 12th, and 48th hour of incubation in the first CCGG site as well as the largest increase in non-methylated cytosines at the 12th and 48th hour in the seventh site. In the eighth CCGG site, we noted the largest increase in the non-methylated cytosines at the 12th hour of incubation and also a significant but smaller increase at the 48th hour of incubation. Additionally, the lower level of the non-methylated cytosines (a reduction to about 50%) at the 6th and the 36th hour after incubation was observed. The increase in the non-methylated cytosines at the 12th hour was correlated with the decrease in the internal methylated cytosine at this time.

The methylation profile of the β*-1,3-glucanase 1* gene in flax seedlings incubated with the pathogenic strain after previous sensitization by Fo47 was determined based on an analysis of the third place in the promoter, the first place in the intron, and the third place in the exon (Figure 1). The third CCGG site in the promoter was characterized by a total reduced level of non-methylated cytosines at the 12th, 24th, and 36th hour and a significant increase in non-methylated cytosines at the 6th and 48th hour.

Moreover, the methylation profile of the *chitinase* gene was determined by analyzing the methylation status of eight CCGG sites in exons, of which three sites were altered (the first, seventh, and eighth). The level of non-methylated cytosines remained unchanged in relation to the sensitized plants in all time points investigated. However, we observed a reduced level (about 50%) of internal and external methylated cytosines (CmCmGG) at the 12th and 36th hour. The seventh CCGG site in the exon showed a considerable increase in the non-methylated cytosines at all the time-points but the 12th, where the increase was smaller. Constant, reduced levels (about 50%) of internal and external methylated cytosines (CmCmGG) were observed. The eighth site in the exon was characterized by a reduced level of non-methylated cytosines at the 6th, 24th, and 36th hour.

### *3.2. Level of Total DNA Methylation*

After determining the changes in DNA methylation pattern of β*-glucanase 1* and *chitinase* in flax seedlings incubated with non-pathogenic and pathogenic strains of *F. oxysporum* and in flax infected by *Foln3* after previous treatment with the non-pathogenic strain we examined the total level of DNA methylation, and the results are shown in Figure 2.

**Figure 2.** Total DNA methylation in flax seedlings treated with non-pathogenic (Fo47) or pathogenic strains of *Fusarium oxysporum* (Foln3) (panel **A**) and after sensitizing effects of the non-pathogenic strain treated with pathogenic Foln3 (+ Foln3) (panel **B**) at 6, 12, 24, 36, and 48 h after inoculation. The data represent the mean from three biological repetitions. Asterisks mark statistically significant di fferences (*p* < 0.05) between the treated samples and their own controls.

The level of total DNA methylation increased by about 1.5-fold at the 6th, 36th, and 48th hour of flax seedlings' incubation with the non-pathogen, while at the other analyzed time points only a slight decrease (approximately 20%) was observed when compared to control plants. In the case of the flax plants incubated with the pathogenic strain, a 1.6-fold increase in total DNA methylation at the 36th hour and a reduction to 62% and 43% at the 12th and 48th hour of incubation with the pathogen, respectively, were noted. However, in the flax sensitized with Fo47 the total level of DNA methylation increased 1.8-fold at the 12th hour of incubation, and was reduced to 70%, 40%, and 74% at the 6th, 24th, and 36th hour, respectively.

### *3.3. Expression Levels of PR Genes*

The expression levels of PR genes, two isoforms of β*-1,3-glucanase* and one of *chitinase,* were determined in flax seedlings treated with non-pathogenic and pathogenic strains of *Fusarium oxysporum* as well as in flax seedlings sensitized by the non-pathogenic strain Fo47 and then incubated with the pathogenic strain Foln3. In order to present the precise kinetics of changes, the analyses were performed after 6, 12, 24, 36, and 48 h of flax incubation with appropriate *Fusarium* strains and the obtained results are presented in Figure 3.

**Figure 3.** Expression level of β*-glucanase 1,* β*-glucanase 2,* and *chitinase* in flax seedlings treated with non-pathogenic (Fo47) or pathogenic strains of *Fusarium oxysporum* (Foln3) (panel **A**) and after sensitizing e ffects of the non-pathogenic strain treated with pathogenic Foln3 (+ Foln3) (panel **B**) at 6, 12, 24, 36, and 48 h after inoculation. The data were obtained from real-time RT-PCR analysis. *Actin* was used as a reference gene and the transcript levels were normalized to the control plant. The data represent the mean ± standard deviations from three biological repetitions. Asterisks mark statistically significant di fferences (*p* < 0.05) between the treated samples and their own controls.

In flax seedlings incubated with the non-pathogenic strain of *Fusarium oxysporum,* the analysis of the expression level of β*-1,3-glucanase 1* gene revealed a two-fold increase at the 12th hour of incubation. An increase in the transcript level was also noted at subsequent time points (the 24th and 36th hour), but it was lower compared to the earlier time (the 12th hour). In the case of flax incubated with the pathogenic strain, the expression level of the β*-1,3-glucanase 1* gene increased continuously with the incubation time (from a 2.6-fold increase at the 12th hour to 11-fold at the 48th hour). However, in the sensitized flax, the transcript level of the β*-1,3-glucanase 1* gene increased during incubation from 3.6-fold at the 6th hour to 10.3-fold at the 24th hour and then decreased to the initial level (after 12 h priming) and then finally a 3.4-fold increase at the 48th hour was observed.

In flax plants incubated with the non-pathogenic strain, the second isoform of the β*-1,3-glucanase* gene was characterized by a 1.85-fold increase in expression level after 24 h of incubation and a decrease to 65%, 20%, and 12% at the 6th, 36th, and 48th hour, respectively. Furthermore, the analysis of this gene in flax seedlings incubated with the pathogenic strain revealed the smallest changes in the expression level as compared to other PR genes. However, in comparison with control plants the mRNA level of this gene increased 1.7-fold, 2.3-fold, 1.6-fold, and 2-fold at the 12th, 24th, 36th, and 48th hour of incubation with pathogenic *F. oxysporum*, respectively. In contrast, the incubation of flax seedlings with the pathogen after previous sensitization by the non-pathogenic strain had a slight e ffect on the mRNA level of the β*-1,3-glucanase 2* gene. A significant increase in the expression of this gene (about 2-fold) occurred at the 6th, 12th, and 24th hour of incubation with the pathogenic strain and 1.6-fold increase at 48th hour was observed.

For the *chitinase* gene, an increase in the mRNA levels at each time point after incubation with the non-pathogenic strain was observed, with the largest increase at the 12th and 24th hour of incubation (6.4-fold and 5.8-fold increase, respectively). In flax seedlings incubated with the pathogenic strain, the transcript level of *chitinase* increased from 2.6-fold at the 12th hour to 4.9-fold at the 36th hour and then decreased, but still remained higher compared to the control (2.5-fold increase). However, flax treatment with pathogenic Foln3, after earlier sensitization with non-pathogenic Fo47, initially caused a significant 8.5-fold increase in *chitinase* gene expression at the 6th hour, 27.7-fold increase at the 12th hour, and a 20-fold increase at the 24th hour. Then, it decreased to 8-fold of the control at the 36th hour, and finally a large, 25.8-fold increase in the expression of *chitinase* at the 48th hour was observed.

### *3.4. Expression Levels of Genes Involved in DNA Methylation*

Due to the changes in methylation profiles of PR genes and the need to explain such changes, the next stage of the study was to analyze the expression of genes involved in the DNA modifications: Methylation (*chromomethylase 1, CMT1* and *chromomethylase 3*, *CMT3*) and demethylation (*repressor of silencing 1*, *ROS1* and *demeter*, *DME*). Additionally, *argonaute 1* (*AGO1*), *argonaute 4* (*AGO4*), and *RNA dependent RNA polymerase 2* (*RDR2*) genes involved in mechanisms of DNA modification were assessed. Changes in expression levels of genes analyzed in flax seedlings incubated with non-pathogenic and pathogenic strains of *F. oxysporum* and in flax incubated with Foln3 after having a sensitizing e ffect by Fo47 are shown in Figure 4.

In flax incubated with the non-pathogenic *F. oxysporum*, expression levels of *CMT1* and *CMT3* genes were decreased: A reduction to 40% at the 6th hour of incubation. Additionally, a lower expression level of the *CMT1* gene was maintained until the 36th hour. In contrast, the expression level of the *CMT3* gene was increased at the 12th hour of incubation (up to a level equal to the control), but it again decreased by 79%, 67%, and 55% at the 24th, 36th, and 48th hour, respectively. The *DME* gene exhibited a slight (1.25- to 1.5-fold) increase in the transcript level over time of the flax incubation with the non-pathogenic strain of *Fusarium*, and finally, at the 48th hour, the expression level of this gene increased more than two-fold. An analysis of a second gene involved in DNA demethylation (*ROS1*) revealed a decrease in the expression level of this gene to 63% at the 36th hour and a 2.3-fold increase at the 48th hour compared to the control. Furthermore, the incubation of flax with the non-pathogenic strain of *Fusarium* affected the expression of genes involved in other mechanisms that modify the DNA. Expression of *AGO4* and *RDR2* genes increased (1.8-fold and 1.7-fold, respectively) at the 48th hour of incubation, while at earlier time points it did not change at all. The *AGO1* gene was characterized by greater variability, wherein the level of expression was increased 2.9-fold, 2.1-fold, and 4.7-fold at the 24th, 36th, and 48th hour, respectively.

**Figure 4.** *Cont*.

**Figure 4.** Expression levels of genes involved in DNA methylation (*chromomethylase 1*, *chromomethylase 3*, *demeter*, *repressor of silencing 1*, *argonaute 1*, *argonaute 4*, *RNA-dependent RNA polymerase 2*) in flax seedlings treated with non-pathogenic (Fo47) or pathogenic strains of *Fusarium oxysporum* (Foln3) (panel **A**) and after sensitizing effects of the non-pathogenic strain treated with pathogenic Foln3 (+ Foln3) (panel **B**) at 6, 12, 24, 36, and 48 h after inoculation. The data were obtained from real-time RT-PCR analysis. *Actin* was used as a reference gene and the transcript levels were normalized to the control plant. The data represent the mean ± standard deviations from three biological repetitions. Asterisks mark statistically significant differences (*p* < 0.05) between the treated samples and their own controls.

After incubation of flax plants with the pathogenic *F. oxysporum*, we observed a decrease in the expression of genes involved in the process of DNA methylation. The transcript level of the *CMT1* gene was reduced to approximately 50% for all analyzed times, while the transcript level of the *CMT3* gene was reduced to 50% at the 6th hour, then it returned to the control level at the 12th hour and 24th hour, and then decreased to 30% at the 36th and 48th hour. The DNA demethylation genes responded differently in flax incubated with the pathogen. The expression level of the *ROS1* gene was about half of the control at the majority of time points, with the lowest value (a reduction to 20%) at the 36th hour. The level of expression of the *DME* gene significantly increased by 3.6-fold at the 24th hour, but was also slightly reduced to 65% at the 6th hour. As in the case of the treatment of flax seedlings with the non-pathogen, flax incubation with the pathogen induced similar changes in the expression of *AGO4* and *RDR2* genes and various changes of the *AGO1* gene. The transcript levels of *AGO4* genes were reduced to 50–75% during incubation with pathogen. Only 12 and 48 h after incubation did the mRNA level of the *AGO4* gene remain unchanged. The level of mRNA of the *RDR2* gene was reduced to 51% and 58% at the 6th and 48th hour, respectively. Similarly to the non-pathogenic strain of *Fusarium*, the pathogenic strain caused a 2.3-fold increase in expression of the *AGO1* gene at the 12th hour and a two-fold increase after 48 h of incubation.

In flax incubated with pathogenic Foln3 after prior sensitization by non-pathogenic Fo47 the expression levels of *CMT1* and *CMT3* genes di ffered significantly from each other, which was not observed in flax after incubation either with pathogenic or with non-pathogenic *F. oxysporum*. The expression of the *CMT1* gene was reduced below 80% in all analyzed time points. The second gene, *CMT3*, showed a decrease in the level of mRNA (to about 40% at the 6th, to 70% at the 12th, to 50% at the 24th, and to 30% at the 48th hour). Among genes participating in DNA demethylation, only the transcript level of the *DME* gene changed, whereas the mRNA level of *ROS1* remained unchanged. The expression level of *DME* declined to 70% at the 6th hour; then showed a 1.7-fold, 1.8-fold and 2.7-fold increase at the 12th, 24th and 36th hour. The mRNA level of the *RDR2* gene was reduced to 40% at the 6th hour, but starting from the 12th hour a return to the state before incubation was observed. A decrease in the expression was also noted for the *AGO4* gene (to 60% and 70% at the 6th and 48th hour, respectively). The *AGO1* gene showed another pattern of expression, wherein initially at the 6th hour of incubation there was a 40% decrease in expression level, then a 1.6-fold and a 2.8-fold increase at the 24th and 36th hour, and a drop back to 90% compared to the control.

### *3.5. Phenotypic Changes in Flax after Sensitization through Non-Pathogenic Strain of F. Oxysporum*

The additional step in evaluation of the sensitizing action of the *Fusarium oxysporum* non-pathogenic strain on flax was based on the phenotypic analysis of plants from in vitro cultures, which were first incubated for two days with the non-pathogenic strain of *Fusarium oxysporum* (for comparison, plants were also incubated without fungi (control plants)) and then through the subsequent days with the pathogenic strain (as previously, appropriate controls were prepared). The first pictures of plants were taken after four and six days (Figure S4).

It is visible at 4 days after inoculation that the prior plant incubation with the non-pathogenic strain reinforced the resistance to pathogen infection compared to plants which had not been pre-incubated with the non-pathogenic strain. These di fferences increased at later time points. It seems that the selected two-day period of sensitization was optimal for the plants. Plants that have been incubated for the first two days and a further four and six days on medium with the pathogen looked the worst.

The phenotypic analysis of flax after the sensitizing action caused by the non-pathogenic strain of *F. oxysporum* and then after incubation with pathogenic *F. oxysporum* confirmed our supposition that non-pathogenic strains could enhance the flax plants' resistance to the pathogen infection.
