The Evaluation of the Significance of Cell Wall Polymers in Flax Response to Fusariosis

Having been priced for centuries as a source of valuable raw materials, flax (Linum usitatissimum) thanks to its multipurpose applications, nowadays, is perceived as a non-waste plant. Residues after pressing the oil (the seedcake) and extracting the fiber (the shives) are both unappreciated materials containing important compounds which have antioxidant, antibacterial, antifungal and anticancer properties. The factors that limit flax growth and development are connected with the climate conditions, however, the pathogen infections, especially with fungi from genus Fusarium are responsible for the biggest losses in flax crop yield. The most dangerous flax pathogens are: Fusarium oxysporum whose infection leads to Fusarium-derived leaves wilting, and Fusarium culmorum which causes Fusarium rot base of the shoots. Both types of Fusarium reduce the crop yield and the quality of raw materials derived from flax. The cell wall is the first, mechanical barrier against pathogen infection. The pathogens’ attack starts when they secrete enzymes degrading the host’s cell wall. At the first stage, by decomposing pectin with pectinases, the cell wall structure loosens and other cell wall components are exposed to degradation by fungal celullases and hemicellulases. As a result of polygalacturonases, belonging tofungal pectinases, action oligogalactouronides (OG) are released. OGs are short pectin fragments, which acting as the elicitors, activate the plant defense mechanisms. Through the signal pathways OGs induce the expression of genes involved in the pathogenesis and other genes related to the metabolic and systemic response. The main aim of the thesis was to evaluate the role of the cell wall polymers in the flax plant response to the infection with Fusarium oxysporum and Fusarium culmorum. For this purpose, the flax seedlings were incubated with fungal strains from genus Fusarium, and theplant tissue was collected duringthe progress of the infection (in 6th, 12nd, 24th, 36th and 48th hour after the infection beginning). The next step was to investigate changes in the expression of genes involved in the cell wall polymers metabolism using real time PCR technique. These analyzes have to be preceded by the identification and verification of the exact, flax mRNA sequences of tested genes. Due to the lack in the database of the flax mRNA sequence of the majority of investigated genes as well as the lack of flax genome the cDNA library was used to isolate particular genes using degenerate primers. These primers were designed for the most homologous mRNA fragments from other plants. Successfully, a few dozens of partial sequences of PR genes and genes involved inthe cell wall polymer metabolismwere identified, and later they were confirmed after the publication of the flax genome sequence. Particular attention was paid to pecitn, constituting the first target of pathogenic enzymes.Other polymers like cellulose, hemicellulose and lignin were also investigated to fully understand the defense mechanism of plants. Additionally, polyamines were taken into consideration, in particular those linked to the cell wall, as it is known from the literature that they are involved in the plant response to the biotic stress. In order to accurately determine the molecular background of successive stages of plant-pathogen interactions in flax and to correlate them with the changes in cell wall polymers, genes related to pathogenesis (β-1,3-glucanase and chitinase) were examined.