**3.3. Effector protein recognition**

**3.2. Signaling and defense response**

ment of local resistance in the infected region.

Recognition of pathogen elicitors that are released at the site of infection is rapidly followed by changes in ion flow and the production of reactive oxygen species. These events activate signaling cascades, which lead to the activation of the transcription factors involved in the activation of defense genes [66]. These responses are known to be regulated through complex signaling pathways involving various phytohormones. The FOL-activated signaling network integrates signals shared and mediated by synergistic or antagonistic interactions between salicylic acid (SA), jasmonic acid (JA), ethylene (ET), abscisic acid (ABA), and ROS [67, 68]. The SA plays an essential role in plant defense signaling since the recognition of FOL-derived components allows the accumulation of this phytohormone, with the subsequent establish-

86 Fusarium - Plant Diseases, Pathogen Diversity, Genetic Diversity, Resistance and Molecular Markers

In the same way as the systemic resistance of the whole plant [69], the biosynthesis of SA is regulated by the Arabidopsis defense-related gene (SID2) [70]. This pathway requires the high-affinity protein SABP2, responsible for the conversion of methyl salicylic acid to SA [71], as well as the nonexpresser of pathogenesis-related (PR) genes positive regulator (NPR1) [72], which is regulated by the transcription factors of the TGA and WRKY family [73, 74]. On the other hand, the function of Ca as a second messenger has been characterized in numerous signaling pathways of plants, transporting a wide range of environmental and developmental stimuli to the physiological response [75]. An example is its participation in the regulation of SA levels through the interaction of a Ca/calmodulin with the transcription factor–enhanced disease susceptibility 1 (EDS1), through the activation of the Ca channels for the influx and subsequent mobilization of the intracellular Ca stores [76]. The increase of Ca in the cytoplasm is the first step in the signaling pathway of PAMP-triggered immunity (PTI). This elevation may occur in response to the perception of PAMPs, interactions of the R gene due to

phosphorylation events, G protein signaling, and/or cyclic nucleotide increase [77].

death, processes that inhibit the invasion of the pathogen through isolation [82].

Activation of MAPKs is critical in components of basal defense pathways as well as in more specific interactions involving R-gene–mediated resistance. The oxidative burst activates an MAPK cascade that induces the downstream defensive mechanisms regulated by SA, ET, JA,

The SA is crucial to induce the production of superoxide anion and hydrogen peroxide, by the activation of apoplastic peroxidase, and subsequently NADPH oxidase of the plasma membrane [78], which are connected to each other through the activation of Ca channels, as it has been pointed out that the increase of the cytoplasmic Ca coincides with the concomitant increase of ROS, or by the phosphorylation of proteins [79]. ROS are known for their direct antimicrobial role against pathogens as well as their relation to the activation of second messengers related to the expression of genes related to the production of response proteins [80], such as the peroxidases of class III, which are important due to their involvement in the reinforcement of the cell wall in the site of interaction with the pathogen, through catalysis of the reticulation of cell wall components including glycoproteins, lignin, and suberin [81]. Also, the oxidative burst is associated with the hypersensitivity response or programmed cell

Once the first line of defense is activated through recognition of PAMPs, FOL employs mechanisms that allow it to suppress such activated responses. During the infection process, it secretes small proteins rich in cysteine (effector or virulence proteins). The function of these proteins is to promote infection and colonization in the host plant, by disrupting various cellular processes such as signal transduction or modifying the proteins in the host plant [98].

The set of these effectors determines the specificity of the host, as well as the ability of the pathogen to manipulate the host immunity [99, 100]. In FOL, these effectors are designated as proteins secreted in the xylem (SIX) and six genes have been reported to encode them [101]. These genes related to pathogenicity are located on a small chromosome within the FOL genome [102].

Recognition of effectors occurs through receptors known as R proteins, which contain at the amino terminus a predicted conserved central domain that functions as a nucleotide-binding site (NBS) and a variable number of leucine-rich repeats (LRR) in the extreme C-terminal [103]. It is considered that this LRR domain of proteins could contribute to the recognition of various ligands derived from pathogens, while the amino-terminal domain determines the specificity of signaling. These receptors are also referred to as NBS-LRR [104–106]. The way in which R proteins activate the signal transduction pathway leading to plant defense is not yet fully understood, but recognition of pathogens is thought to trigger nucleotide-dependent conformational changes that may induce oligomerization, thus providing a scaffold for the activation of downstream signaling components [107].

The perception of the effectors triggers the second branch of the plant defense system or ETI. This is based on the gene-for-gene hypothesis, where resistance to disease is thought to be conferred by R genes, or immunity (I) genes in tomato, but requires the coincidence of avirulence genes (Avr) from FOL [12]. This is why FOL is divided into physiological races based on the ability of the individual strains to overcome the tomato-specific immunity genes (**Figure 3**). Therefore, compatible or incompatible interactions are controlled by three avirulence genes (Avr 1–3) in FOL and the corresponding resistance genes (I-I3) in tomato [108].

The SIX4 protein has been identified as a virulence effector designated as Avr1 gene, and the mature protein is made up of 184 amino acids and contains 6 cysteines in its structure. Its expression was initially reported for race FOL 1 and is required to activate resistance mediated by gene I and I-1. In addition, it is related to the suppression of disease resistance linked to the I-2 gene and the I-3 gene for tomato [109]. The SIX3 protein corresponds to the Avr2 gene, which is made up of 144 amino acids and contains 2 repeated cysteines. It is required for the development of disease symptoms, as demonstrated by its deletion, resulting in a reduction in virulence. This protein can be recognized by the I-2 resistance gene [11].

Expression of the Avr3 gene encoding the SIX1 protein is essential for virulence in tomato. Its structure consists of 189 amino acids and contains 8 repeated cysteines. Its recognition is necessary to activate the resistance through the I-3 gene. A relation between the expression of this gene and Avr1, for the evasion of resistance activated by I-3, has been suggested. The Avr3 gene requires the presence of live plant cells, and its secretion is performed immediately after the penetration of the root cortex [110–112]. Another important effector protein in the pathogenicity of FOL is SIX6. It is made up of 199 amino acids and contains 7 cysteines. It is expressed in early and late stages of infection and its expression suppresses the cellular death triggered by the I-2 protein and requires the presence of live host cells [113]. Close homologs have been found in other special forms for the SIX6 and SIX7 effectors, suggesting that these genes may have a more general role in pathogenicity [114].

parasitic growth, which produces an extensive growth in the plant and leads to the symptoms

**Figure 3.** Classification of the physiological races of FOL according to the interaction of Avr genes of FOL and immunity

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One of the strategies of FOL to overcome the tomato defense response is the participation of chitinases, through the synergistic action of two proteases that are required for the complete separation of the binding domain of class I and class IV chitinase of tomato, allowing full

Detoxification of α-tomatine by the action of the enzyme tomatinase in FOL has also been identified. It is encoded by the TOM1 gene, which plays an essential role for the successful infection of the fungus [117]. The role of fusaric acid, which plays an important role in fungal pathogenicity,

of the disease [115].

genes in tomato.

virulence of FOL [116].

**3.4. Overcoming defense response**

The expression of Avr genes in FOL is regulated by the transcription factor SIX Gene Expression 1 (SGE1). Although its expression is not required for the vegetative growth of the fungus, it is essential for the pathogenicity of FOL, by playing an important role during A Molecular Vision of the Interaction of Tomato Plants and *Fusarium oxysporum* f. sp. *lycopersici* http://dx.doi.org/10.5772/intechopen.72127 89

**Figure 3.** Classification of the physiological races of FOL according to the interaction of Avr genes of FOL and immunity genes in tomato.

parasitic growth, which produces an extensive growth in the plant and leads to the symptoms of the disease [115].

#### **3.4. Overcoming defense response**

These genes related to pathogenicity are located on a small chromosome within the FOL

88 Fusarium - Plant Diseases, Pathogen Diversity, Genetic Diversity, Resistance and Molecular Markers

Recognition of effectors occurs through receptors known as R proteins, which contain at the amino terminus a predicted conserved central domain that functions as a nucleotide-binding site (NBS) and a variable number of leucine-rich repeats (LRR) in the extreme C-terminal [103]. It is considered that this LRR domain of proteins could contribute to the recognition of various ligands derived from pathogens, while the amino-terminal domain determines the specificity of signaling. These receptors are also referred to as NBS-LRR [104–106]. The way in which R proteins activate the signal transduction pathway leading to plant defense is not yet fully understood, but recognition of pathogens is thought to trigger nucleotide-dependent conformational changes that may induce oligomerization, thus providing a scaffold for the

The perception of the effectors triggers the second branch of the plant defense system or ETI. This is based on the gene-for-gene hypothesis, where resistance to disease is thought to be conferred by R genes, or immunity (I) genes in tomato, but requires the coincidence of avirulence genes (Avr) from FOL [12]. This is why FOL is divided into physiological races based on the ability of the individual strains to overcome the tomato-specific immunity genes (**Figure 3**). Therefore, compatible or incompatible interactions are controlled by three avirulence genes (Avr 1–3) in FOL and the corresponding resistance genes (I-I3) in tomato [108].

The SIX4 protein has been identified as a virulence effector designated as Avr1 gene, and the mature protein is made up of 184 amino acids and contains 6 cysteines in its structure. Its expression was initially reported for race FOL 1 and is required to activate resistance mediated by gene I and I-1. In addition, it is related to the suppression of disease resistance linked to the I-2 gene and the I-3 gene for tomato [109]. The SIX3 protein corresponds to the Avr2 gene, which is made up of 144 amino acids and contains 2 repeated cysteines. It is required for the development of disease symptoms, as demonstrated by its deletion, resulting in a reduc-

Expression of the Avr3 gene encoding the SIX1 protein is essential for virulence in tomato. Its structure consists of 189 amino acids and contains 8 repeated cysteines. Its recognition is necessary to activate the resistance through the I-3 gene. A relation between the expression of this gene and Avr1, for the evasion of resistance activated by I-3, has been suggested. The Avr3 gene requires the presence of live plant cells, and its secretion is performed immediately after the penetration of the root cortex [110–112]. Another important effector protein in the pathogenicity of FOL is SIX6. It is made up of 199 amino acids and contains 7 cysteines. It is expressed in early and late stages of infection and its expression suppresses the cellular death triggered by the I-2 protein and requires the presence of live host cells [113]. Close homologs have been found in other special forms for the SIX6 and SIX7 effectors, suggesting that these

The expression of Avr genes in FOL is regulated by the transcription factor SIX Gene Expression 1 (SGE1). Although its expression is not required for the vegetative growth of the fungus, it is essential for the pathogenicity of FOL, by playing an important role during

tion in virulence. This protein can be recognized by the I-2 resistance gene [11].

genes may have a more general role in pathogenicity [114].

activation of downstream signaling components [107].

genome [102].

One of the strategies of FOL to overcome the tomato defense response is the participation of chitinases, through the synergistic action of two proteases that are required for the complete separation of the binding domain of class I and class IV chitinase of tomato, allowing full virulence of FOL [116].

Detoxification of α-tomatine by the action of the enzyme tomatinase in FOL has also been identified. It is encoded by the TOM1 gene, which plays an essential role for the successful infection of the fungus [117]. The role of fusaric acid, which plays an important role in fungal pathogenicity, has also been described by decreasing the cell viability of the plant. It is directly related to programmed cell death through damage to photosynthetic machinery, increase in protease activity, ROS production, low regulation of antioxidant enzyme activities, and positive regulation in lipid peroxidation, as well as the disorder of the nitrogen metabolism resulting in the collapse of the cell [118–120]. Its biosynthesis requires expression of the FUB1 and FUB4 genes [121], and L-aspartate is suggested as the precursor amino acid in the biosynthetic pathway [122].

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