Flax (Linum usitatissimum) is a valuable source of oil and fibers used in many industrial products. Its cultivation is restricted by environmental stress factors, but the biggest crop losses worldwide are caused by Fusarium infection, with Fusarium oxysporum f.sp. lini (Fol) being the most common. The Fusarium genus has a broad host range, although individual isolates usually only cause disease in a narrow range of plant species. The fungus is soil-borne and infection takes place mainly through the roots, although the hypocotyl may also be infected.
Fusarium oxysporum generally causes symptoms such as wilting, chlorosis, necrosis, premature leaf drop, and browning of the vascular system. Vascular wilt can account for up to 20% of the annual losses in flax cultivation. The fungus infects healthy plants by penetrating the root tips, root wounds, or lateral roots. It advances intracellularly into the xylem by producing microconidia, which germinate and thus block the vascular vessels, preventing water and nutrient translocation. This leads to epinasty and yellowing of the lower leaves, progressive wilting, and eventually death. After the plant dies, the fungus invades all of the tissues, sporulates, and infects neighboring plants (Kroes et al. 1998; Michielse and Rep 2009).
Most isolates of F. oxysporum do not produce mycotoxins, although there are reports that the fungi are able to synthesize moniliformin, beauvericin, diacetoxyscirpenol, T-2 toxin, HT-2 toxin, zearalenone, fumonisin and fusaric acid (Li et al. 2013). Mycotoxins can inhibit eukaryotic protein synthesis and facilitate the spread of the fungus because they inhibit plant defense mechanisms (Wagacha and Muthomi 2007). However, plants can produce compounds capable of interfering with mycotoxin production and they can degrade or detoxify trichothecenes by glycosylation (Karlovsky 2011; Shi et al. 2017).
The initial response of a plant to pathogen infection involves the production of reactive oxygen species (ROS). These can act directly against phytopathogen attack by killing the microorganism or limiting pathogen penetration of plant tissues by stiffening the cell wall. ROS achieve this by facilitating peroxidase reactions that catalyze intra- and inter-molecular cross-links between structural components of the cell wall and lead to lignin polymerization. In addition, ROS can act as signal molecules that activate plant cell defense mechanisms (De Gara et al. 2003). The reaction to the pathogen also involves production of pathogenesis-related (PR) proteins, including glucanases, chitinases and peroxidases, production of pectinase inhibitors, and changes in the cell wall, in particular due to pectin methylesterification and lignification, which create a physical barrier to fungal growth. ROS secretion must be strictly controlled due to the potentially harmful side effects, so plants also secrete antioxidative enzymes (superoxide dismutase, ascorbate peroxidase) and compounds (glutathione, secondary metabolites) (Wojtasik et al. 2011).
These secondary metabolites with antioxidative properties can be divided into two groups. Those produced via the phenylpropanoid pathway (phenolic acids, flavonoids, lignin and lignans) have been proven to participate in plant defense against pathogen invasion (Kostyn et al. 2012; Boba et al. 2016). The second group consists of isoprenoids, also called terpenoids. This large and most diverse class of compounds has a number of functions, including signaling and defense against pathogens.
The classification of terpenoids is based on the number of 5-carbon isoprene units, which constitute the molecule scaffold. Carotenoids, cytokinins, ABA, gibberellic acids, brassinosteroids, the phytol side chain of chlorophyll, and many more compounds, derive from terpenoid precursors. They serve a range of biological functions, acting as plant growth regulators, pigments, volatile attractants, and toxic deterrents in plant-animal interactions.
Two main pathways are employed in isoprene production: the mevalonate (MVA) pathway, which occurs in the cytosol of all types of organisms (Sonawane et al. 2016), and the non-mevalonate (MEP) pathway, which takes place in plastids (Lushchak and Semchuk 2012; Finkelstein 2013; Nisar et al. 2015) (Fig. 1). It is understood that the MVA pathway generates units for synthesis of 15- and 30-carbon terpenoids, such as squalene, sterols, stanols and brassinosteroids, while the MEP pathway leads to the synthesis of carotenoids (including xanthophylls), ABA, and gibberellins, and provides units for the synthesis of tocopherols and chlorophylls.
Very little is known about the genes controlling the terpenoid synthesis pathway in flax. Only a few gene sequences had been presented in GenBank before genome sequencing projects were undertaken. Even now, a large amount of the data remains unannotated. On the other hand, the manipulation of terpenoid components in flax proved to be successful in increasing flax resistance to Fusarium infection (Boba et al. 2011). To understand the functioning of the terpenoid pathway in flax and provide the tools for genetic manipulation, we cloned the key genes of the flax terpenoid pathway and investigated their expression in response to Fusarium attack. Gene expression analysis results were correlated with the key compounds: carotenoids, tocopherols, sterols and ABA. The levels of callose and callose synthase gene expression, known to be regulated by ABA, and participating in plant defense responses, were assayed. To the best of our knowledge, this is the first report of a comprehensive study on the terpenoid pathway response in flax after F. oxysporum infection.
A particularly important role during a pathogen infection in plants is played by phytohormones and more specifically the interplay between their signaling. Salicylic acid (SA) is known to reduce plant susceptibility to pathogen infection, while jasmonic acid (JA), ethylene, ABA and auxin are involved in a more complex system of action, often exhibiting contradictory effects. All these hormones are part of a larger signaling network that integrates environmental inputs and provides a powerful system against microbial manipulations (Di et al. 2016). The inter-phytohormonal interactions are only partially understood, and especially the role of ABA in plant resistance to pathogens is controversial. According to current knowledge ABA influences some responses of plant pathogen resistance (stomatal closure, red-ox homeostasis), though its effect may vary with a number of variables, such as type of tissue, age of the plant, pathogen type, and stage of the infection (Maksimov 2009).
Under pathogenic F. oxysporum infection, the disease development is controlled by phytohormones, though the constant and correct mechanism of their actions cannot be defined, mainly due to the variable influence of different F. oxysporum strains. Thus, the ability of a particular strain to adapt to a particular host plant, which could explain the narrow host range observed in the F. oxysporum species, might be closely connected with the ability to manipulate the phytohormone signaling network. This hypothesis, although not proven yet, is strongly supported by some results on mutants with impaired hormone signaling, in which despite undisturbed colonization by the fungus, disease symptoms were significantly reduced (Thatcher et al. 2009; Cole et al. 2014). The current understanding is that the role of ABA in defense cannot be generalized as it appears to have a pathogen- and context-dependent role. Divergent ABA functions have been reported amongst the different necrotrophic species, including between B. cinerea, S. sclerotiorum, and A. brassicicola despite their common virulence and pathogenicity strategies. For example, in a study of tomato seedling–Alternaria solani interaction, ABA-treated plants showed improved resistance against infection (Song et al. 2011). Also, flax plants with increased ABA levels that were previously generated in our laboratory were more resistant to pathogen infection (Boba et al. 2018). However, the impaired ABA biosynthesis in Arabidopsis thaliana resulted in enhanced resistance to Plectosphaerella cucumerina (Sanchez-Vallet et al. 2012).
The first effect of ABA is closure of stomata to hinder further pathogen penetration. A group of ABA receptors (RCAR/PYR/PYL) was discovered recently which activate a cascade of responses after binding to the hormone (Cao et al. 2011; Lim and Lee 2015). Production of free radicals by NADPH oxidases and elevation of cellular calcium level are among the effects regulated by ABA (Mittler and Blumwald 2015). Transcriptome analysis of tomato after exogenous ABA application showed changes in the expression of many genes involved in the plant’s response to stress and infection. Among them, genes responsible for production and quenching of free radical and PR genes, including those connected with signal transduction through JA, were found (Wang et al. 2013). This is not surprising as about 15% of gene promoters possess ABA-responsive elements in A. thaliana (though they are not equally common in plant genomes of different species) (Gomez-Porras et al. 2007). A growing body of literature indicates that ABA is an indispensable element of the JA based response in some plant–pathogen interactions (Adie et al. 2007; García-Andrade et al. 2011).
Overall, ABA appears as a key regulator of defense against necrotrophs with both negative and positive contributions. Clarification of the nature of ABA function is further confounded by its interactions with other resistance pathways and the potential trade-offs resulting from the occurrence of abiotic stresses during infection (Ton et al. 2009).