**1. Introduction**

Currently, the greatest losses in flax crops are caused by fungal infections. Fungal diseases are the cause of about 20% of losses of flax cultivation. They result in the reduction of crop yield, both the seed and fiber and deterioration of their quality, as well as feed and food obtained from them [1]. Among the genus *Fusarium*, saprophytic *Fusarium oxysporum* is the major flax pathogen. The strains of *Fusarium oxysporum* can be divided depending on their virulence into pathogenic and non-pathogenic strains. The first group infects various plant species causing wilt or root rot and finally leads to death of the plant. By contrast, the latter do not invade the vascular system of plants and do not kill the host. Moreover, non-pathogenic strains of *F. oxysporum* colonizing plants without symptoms of fusariosis can protect the host from infection by pathogenic strains [2,3]. Therefore, some selected, non-pathogenic

strains are perceived as potential biological control agents because their resistance-inducing activity correlates with an increase in the activity of pathogenesis-related (PR) proteins: β-1,3-glucanase and chitinase [4,5].

PR proteins play a very important role in the plant response to pathogenic infections. They are locally accumulated in infection sites and surrounding tissues, as well as in uninfected tissues, providing resistance to plants against subsequent infection [6]. β-1,3-glucanase is responsible for the degradation of the fungal cell wall by the hydrolysis of β-1,3-glycosyl bonds in β-1,3-glucan, thereby releasing the fungal cell wall fragments (oligosaccharides), which act as elicitors (damage-associated molecular patterns, DAMPs) in a defensive reaction stimulating the production of other PR proteins and antifungal low molecular weight components (phytoalexins) [7]. Chitinases catalyze the cleavage of bonds between C1 and C4, two residues of N-acetyl-D-glucosamine in chitin, inhibiting the growth of fungal hyphae during the invasion of the intercellular space, and releasing fungal elicitors (chitooligosaccharides or chitin oligomers) that induce a plant's defense response by biosynthesis of Chitinases and other PR proteins [8].

There is a significant increase in the expression of both β*-1,3-glucanase* and *chitinase* genes during the infection, as confirmed in many plants, e.g., tomato, tobacco, soybeans, wheat, and peas [9,10]. At present, genetic engineering tools are frequently and routinely used to improve the plant defense system and thus plants' resistance against pathogens. The results of such an approach are genetically modified organisms (GMOs). One possibility to strengthen the plant defense system making it pathogen-resistant is upregulation or overexpression of pathogen-related genes or genes encoding enzymes involved in the synthesis of secondary metabolites [11–13].

A similar e ffect, intense synthesis of pathogen-related proteins, was observed in various plant species after plant treatment with a non-pathogenic strain of *F. oxysporum* when compared to the control plants [3,14]. This was followed by an increase in the resistance against pathogenic strains of *F. oxysporum* in cucumber and peas [3]. In contrast, a study conducted by Aime and co-workers revealed that colonization of tomato by a non-pathogenic strain resulted in a decreased mRNA level of β*-1,3-glucanases* and *chitinases* genes. Furthermore, the expression of this group of genes was observed only when tomato plants subsequently underwent infection with a pathogenic strain of *F. oxysporum*. Such response, called priming, implies the protective role of non-pathogenic strains by accelerating and strengthening the plant defense mechanisms only upon contact with the pathogen [15].

After exposure to non-pathogenic fungal strains, the plants develop acquired immunity to pathogenic infections. Moreover, in this way the immune memory associated with the process of preparing cells (priming) may be transferred to the next generation. Priming results from the accumulation in the cell of inactive mitogen-activated protein kinases (MPK3, MPK6), and nonexpressor of pathogenesis-related genes 1 (NPR1) (both mRNA and protein) that are activated after infection [16]. Priming also a ffects chromatin modifications: Mostly methylation and acetylation of histones and DNA, associated with activation of genes involved in plant resistance. A local pathogenic infection can change the status of methylation and acetylation of histones and in particular can a ffect the promoter sequences of genes in systemic tissues. Such epigenetic changes provide plants with a long-term immune memory inherited from generation to generation [17].

Among eukaryotes, the plant genome bears the highest levels of DNA methylation, with up to 50% in some species. DNA methylation and modification of associated chromatin proteins determines the epigenetic status of the genome. Depending on the sequence context of the cytosine residue to be methylated, we can define CG, CNG, or CNN (C = cytosine; G = guanine; N = nucleotide other than G) specific methylation types. Two types of maintenance methyltransferase exist in plants: DNA methyltransferase (MET1), which predominantly methylates CG sites, and plant-specific chromomethylase 3 (CMT3) that methylates CNG (N = A, C, or T) sites. The third type methylates CNN and is controlled by domains of rearranged methyltransferase 1 (DRM2), a homologue of mammalian de novo DNA methyltransferase DNMT3 [18]. De novo DNA methylation is conducted by the pathway that includes specific transcripts that are copied into dsRNA by RNA-dependent RNA polymerase 2 (RDR2). This siRNA associates with argonaute 4 (AGO4) in a complex that mediates DNA methylation [19].

Our preliminary data indicate that single base pair mapping (bisulfite-seq) of methylated cytosine in flax seedlings reveals that over 8% of CG, 4% of CNG, and 7.3% of CNN is methylated. For comparison, the methylation state of maize genome (mainly composed of repetitive elements) ranges around 75% and 62% of CG and CNG sequence contexts, respectively [20]. Genome-wide mapping of methylated cytosine in *Arabidopsis thaliana* (*A. thaliana*) revealed levels of 24% CG, 6.7% CNG, and 1.7% CNN methylation [21].

Many literature reports have described changes in DNA at the genome-wide level caused by environmental stress, which can be inherited [22]. Pathogenic infection and abiotic stress can lead to two contrasting effects at the level of methylation in plants: Hypermethylation at the genome-wide level and hypomethylation of resistance-related genes, which can correlate with regulation of the expression of the defense-related genes in plants [23].

Based on the current state of knowledge about the mechanisms of epigenetic inheritance, it is anticipated that changes in the flax epigenome introduced by infection with a non-pathogenic *Fusarium oxysporum* strain would be directional and transmitted to the offspring. The aim of the study is to accurately understand and compare the mechanisms of infection of pathogenic and non-pathogenic strains as well as the sensitizing effect of the non-pathogenic strain, and in particular to examine the DNA methylation not only throughout the genome but also in specific genes. PR proteins are essential to plant resistance to biotic stress, which makes them the main focus of this research. The findings of our research can help to improve crop resistance in the future.
