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Plants have evolved a plethora of defense strategies to persist in a hostile environment that abounds with pathogens. Besides constitutive structural and biochemical defenses, plants possess sophisticated defense machinery that is put to use against those few pathogens that succeed in overcoming the preformed barriers. The plant possesses an arsenal of these inducible defenses, including the reactive oxygen species (ROS) burst, hypersensitive (HR) cell death, cell wall fortifications and de novo synthesis of various antimicrobial compounds, which ultimately deprive a (hemi)biotrophic pathogen of the plant's nutritional resources (Pieterse et al., 2009). However, the efficacy of such ROS/HR-mediated defense strategies against necrotrophic pathogens, which favor host cell death, remains questionable (Robert-Seilaniantz et al., 2007).
Botrytis cinerea has recently been nominated as the most scientifically and economically important necrotrophic fungal plant pathogen (Dean et al., 2012). As a true necrotroph, induction of cell death in the host tissue is known as a crucial mode of pathogenicity (Van Baarlen et al., 2004; Govrin et al., 2006). To accomplish this, B. cinerea is armed with an array of virulence factors, such as phytotoxic metabolites, oxidative burst-evoking enzymes and oxalic acid (Govrin & Levine, 2000; Van Kan, 2006). On the other hand, resistance to necrotrophic pathogens such as B. cinerea depends, to some extent, on the ability of the host to control the balance between cell death and survival (Mengiste, 2012). It has been shown that expression of a senescence-associated gene of Arabidopsis (SAG12) is induced by B. cinerea infection, while transgenic nonsenescing tomatoes exhibit some degree of resistance to the pathogen (Swartzberg et al., 2008). A model has been proposed for Arabidopsis nonhost resistance to B. cinerea, according to which presence of a cellular zone around the primary infection site with a slower rate of cell death can restrict pathogen colonization (Van Baarlen et al., 2007).
We have previously demonstrated that abscisic acid (ABA) deficiency in the sitiens mutant of tomato results in a strong resistance response to B. cinerea (Audenaert et al., 2002). Additionally, it was recently shown that cuticle permeability and specific cell wall structure in the mutant lead to rapid defensive responses, including an early and localized production of hydrogen peroxide (H2O2) and HR followed by cell wall fortifications in the epidermis, which ultimately impede further spreading of the pathogen (Asselbergh et al., 2007; Curvers et al., 2010). Microarray analysis revealed that, in addition to groups of genes coding for pathogenesis-related (PR) proteins and enzymes related to cell wall structure being up-regulated, expression of a cluster of genes involved in primary amino acid metabolism significantly increased in the resistant mutant at 8 h postinoculation (hpi; Asselbergh et al., 2007). The most notable of the latter group of genes were peroxisomal/glyoxysomal aspartate transaminase (AAT), the lysine catabolic gene ketoglutarate reductase/saccharopin dehydrogenase (LKR/SDH) and the γ-aminobutyric acid (GABA) biosynthetic gene glutamate decarboxylase (GAD). However, the cause and the putative role of these amino acid-associated transcriptional alterations in the resistance mechanism of sitiens against B. cinerea remained unclear.
Although there are dozens of studies corroborating the link between primary metabolism and plant defense responses (reviewed by Bolton, 2009), molecular knowledge on the role of primary amino acid metabolism in plant resistance mechanisms against pathogens is still fragmentary. We have recently reviewed the role of glutamate metabolism in plant–pathogen interactions, highlighting the importance of infection-triggered alterations in central amino acid metabolism in molding a resistance/susceptibility response in the host (Seifi et al., 2013). A causal link has been proposed between the disruption in glutamate/glutamine homeostasis and induction of cellular redox imbalances in leaf cells of Arabidopsis, leading to ROS-mediated cell death and resistance against the biotrophic fungus Erysiphe cichoracearum and the hemibiotrophic fungus Colletotrichum higginsianum (Liu et al., 2010). Conversely, drastic depletion in glutamate storage in distal, noninvaded regions of sunflower was postulated as a defensive strategy against B. cinerea. It was hypothesized that, through this mechanism, glutamate-derived N-rich amino acids are supplied to the infected area, delaying the necrotroph-induced senescence (Dulermo et al., 2009). In Arabidopsis, LKR/SDH is significantly up-regulated by abiotic stresses, but also by B. cinerea infection (Genevestigator; Zimmermann et al., 2005). It is suggested that stress-induced lysine catabolism serves as a generator of glutamate, the key precursor for important stress-related metabolites such as arginine, proline and GABA (Galili et al., 2001). Similarly, the observed increased susceptibility to B. cinerea in cytosolic aspartate transaminase (AAT1) overexpressing lines of Arabidopsis was suggested to be caused through the repression of glutamate-consuming pathways to maintain normal glutamate concentrations. In this way, less glutamate would be converted to key defense-related metabolites such as proline or GABA (Brauc et al., 2011).
γ-Aminobutyric acid is known to accumulate in plant tissues in response to various abiotic and biotic stresses, suggesting assorted functions for the molecule, ranging from involvement in central carbon : nitrogen (C : N) metabolism to functioning as a signaling molecule during plant–microbe interactions (Kinnersley & Turano, 2000; Roberts, 2007; Fait et al., 2008). Making up a considerable fraction of total free amino acid content in some plants (Roberts, 2007), GABA might be an attractive nutritional target, particularly for intercellularly growing pathogens. For instance, infection of tomato by the biotrophic fungus Cladosporium fulvum was shown to induce high concentrations of GABA in the apoplast, providing the pathogen with a rich nitrogen source (Solomon & Oliver, 2002). GABA also appears to be a nutrient source for Pseudomonas syringae pv. tomato in Arabidopsis. However, conditions promoting high concentrations of GABA can increase plant resistance by repressing the expression of the hrp genes in the bacterium (Park et al., 2010). The involvement of GABA in plant–stress interactions has been also attributed to the metabolic functions of the GABA shunt, a cytosolic-mitochondrial pathway with an important role in the plant central C : N metabolism, connecting amino acid metabolism to the tricarboxylic acid (TCA) cycle (Fait et al., 2008). The GABA shunt consists of three key enzymes: GAD, GABA transaminase (GABAT) and succinic-semialdehyde dehydrogenase (SSADH). Specifically using glutamate as the main precursor for GABA biosynthesis, GAD activity catalyzes the first step of the GABA shunt in the cytosol, while the other two steps of the shunt, GABAT and SSADH, occur in mitochondria (Shelp et al., 2012). Although there are a number of studies demonstrating involvement of the GABA shunt in response to abiotic stresses (Bouché et al., 2003; Ludewig et al., 2008), information on the molecular mechanism underpinning the role of this pathway in plant defense mechanisms against pathogens is still scarce (Wu et al., 2006).
This study aimed to unravel the role of the GABA shunt in the resistance response of the sitiens mutant to B. cinerea. In addition, we investigated how alterations in GABA metabolism may be linked to the HR-mediated epidermal arrest of the pathogen seen in the mutant. The efficacy of the ROS-fueled defense of sitiens against a ROS-favoring pathogen, such as B. cinerea, appears to be vitally dependent on a secondary anti-cell-death defense mechanism, activated in the area surrounding the invaded cells via concurrent overactivation of the GABA shunt and the cytosolic glutamine synthetase. Our sitiens model has revealed that maintaining cell viability via alterations in central C : N metabolisms is crucial in the resistance response to a necrotrophic pathogen.
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In this study, the sitiens–B. cinerea pathosystem was used as a model to understand the mechanisms needed to effectively resist a necrotrophic pathogen. The focus was mainly directed toward the role of GABA metabolism in the sitiens defense response, inspired by previous studies (Asselbergh et al., 2007; Brauc et al., 2011). The results indicate that a timely overactivation of the GS/GOGAT cycle and the GABA shunt maintains cell viability, slowing down senescence in the site of primary invasion. Induction of such a cell death-alleviating mechanism in the cells surrounding the epidermal sites penetrated by B. cinerea may also explain how the HR-mediated defense response observed in sitiens (Asselbergh et al., 2007) can effectively suppress a necrotrophic pathogen.
In angiosperms, there are two distinct GS isoforms, cytosolic (GS1) and chloroplastic (GS2), which feed ammonium in the GS/GOGAT cycle. GS1 is commonly believed to be involved in ammonium reassimilation during natural and stress-induced senescence, as the GS1 transcript is known as a putative senescence-associated gene (SAG)/marker (Pageau et al., 2006). The chloroplastic isoform (GS2), though, plays a crucial role in the assimilation of ammonium obtained from nitrate reduction and photorespiration (Perez-Garcia et al., 1998; Bernard & Habash, 2009). Coinduction of SAGs and defense-related genes has previously been observed in many cases, highlighting the necessity for efficient protection against opportunistic pathogens during the critical process of senescence (Quirino et al., 2000). For instance, overlapping expression of GS1 and PAL has been reported in a number of studies on plant tissues undergoing some form of senescence, such as lignin depositing sclerenchyma cells in naturally maturing leaf blades of rice (Sakurai et al., 2001), and senescing leaves of Phaseolus vulgaris in response to Colletotrichum lindemuthianum (Tavernier et al., 2007). Accordingly, the concurrent high levels of GS1/PAL7 expression and GS/PAL activities observed in inoculated sitiens suggest a metabolic link between the phenylpropanoid pathway and the GS/GOGAT cycle, presumably through reassimilation of PAL-derived ammonium. However, according to the results presented here, the rapid up-regulation of GS1 in sitiens seems not to be associated with senescence, as senescence is delayed in the mutant, particularly during the early stages of the interaction. Under the pathogen challenge, and considering the down-regulation of GS2, the cytosolic isoform might play a compensatory role to ensure the critical functionality of the GS-GOGAT cycle. Therefore, it could be hypothesized that overactivation of the GS-GOGAT cycle is not merely involved in scavenging the unwanted byproduct of a defensive pathway, but, more importantly, might be an influential part of an activated anti-cell death mechanism in sitiens. This was further confirmed when either VIGS of GS1 or inhibition of GS by MSO promoted cell death and abolished the capacity of sitiens to contain the pathogen during the later stages of the interaction (Fig. 2a). However, a late up-regulation of GS1 was observed in wild-type plants, which may be linked to the putative senescence-related role of the gene in agreement with the strong senescence occurring in these plants (Fig. 5c).
Besides functioning as the basic step in forming different amino acids, glutamate generation via the GS-GOGAT cycle serves as a key source for various compounds with possible roles in plant–pathogen interaction, such as GABA (Galili et al., 2001; Brauc et al., 2011). Cytosolic decarboxylation of glutamate by GAD is the main route for GABA biosynthesis (Baum et al., 1996), as well as a controlling point in the GABA shunt (Busch & Fromm, 1999). It is also known that this route has a regulatory role in central C : N metabolism by providing a major link between amino acid metabolism and the TCA cycle (Fait et al., 2008). The genes encoding GABA-shunt enzymes exhibited in sitiens an early up-regulation in response to the pathogen, concomitant to the PAL7 and GS1 expression pattern, suggesting a concerted interplay between defense response and primary metabolism in the mutant. Further evidence of this interplay is provided by the UPLC analysis, showing an increase in GABA concentrations in the resistant sitiens mutant at 8 hpi, followed by a considerable consumption of the amino acid. Since absolute GABA concentrations are higher in wild-type plants than in the sitiens mutant, it seems that, rather than the concentration of GABA per se, temporal activation of the GABA shunt is important in defense.
Under oxidative stress conditions, the TCA enzymes aconitase, succinyl-CoA ligase and the NADH-generating α-ketoglutarate dehydrogenase are deactivated, resulting in accelerated cell death in the tissue undergoing stress (Sweetlove et al., 2002). Although involvement of the GABA shunt in maintaining redox equilibrium during plant responses to abiotic stresses or natural senescence has gained some attention (Ansari et al., 2005; Fait et al., 2005; Shelp et al., 2012), the literature relating to the cell death-alleviating role of the GABA shunt in plant responses to pathogen-induced oxidative stress is scarce. According to the data presented here, overactivation of the GABA shunt in sitiens plays a vital role in resistance to B. cinerea. Likewise, exogenous application of GABA to the wild-type could effectively restrict the pathogen progress by forming an HR-like ring around the spreading lesion (Fig. 3b), mimicking the mesophyllic ring observed as the secondary wave of defense in sitiens (Fig. 2a). Taken together, it could be hypothesized that timely activation of the GABA shunt in sitiens might restrict the extent of cell death caused by the H2O2-mediated defense response to B. cinerea, particularly in cells in the vicinity of the pathogen penetration sites. The redox regulating effect of the GABA shunt may be explained by generation of NADH via SSADH and/or by ensuring the functionality of the TCA cycle, under oxidative stress, through bypassing the ROS-sensitive enzymes of the cycle. This hypothesis is in agreement with the report of Wu et al. (2006), suggesting that the GABA shunt may play a role in restricting cell death in both the incompatible and compatible interaction of rice with the blast fungus Magnaporte oryzae. Likewise, we have recently proposed a model describing how overactivation of metabolic pathways that can maintain the functionality of the GS/GOGAT and TCA cycle in invaded cells may function as anti-cell death defense strategy, termed as ‘endurance’, resulting in resistance against necrotrophic pathogens (Seifi et al., 2013).
Defense responses in plants are known to be highly energy-demanding processes (Heil & Bostock, 2002; Berger et al., 2007), heavily draining the TCA cycle-generated energy and intermediates (Kinnersley & Turano, 2000; Bolton, 2009). The TCA cycle plays a crucial anabolic role in supporting the costly defense-related metabolic pathways, such as stress-induced phenylpropanoid metabolism, which may consume up to 20% of the total photosynthetic carbon in the plant (Dennis & Blakeley, 1995). This huge demand highlights the necessity of anaplerotic reactions (‘filling-up reactions’; Kornberg, 1965) to replenish the cycle and ensure its constant functionality during such circumstances (Kinnersley & Turano, 2000). Under pathogen invasion pressure, the GABA shunt might also operate as an important anaplerotic route to the TCA cycle, providing carbon skeleton in the form of succinate (Michaeli et al., 2011). Accordingly, the race-nonspecific Lr34-mediated resistance response against the wheat leaf rust pathogen Puccinia triticina has been shown to be considerably energy-intensive, entailing concurrent up-regulation of the TCA cycle and the GABA shunt (Bolton et al., 2008). The results presented here may also support this hypothesis, suggesting concurrent overactivation of the energy-intensive phenylpropanoid pathway and the GABA shunt during the early stages of the sitiens–B. cinerea interaction. In this context, and regarding the observed delayed senescence in the mutant, the early and moderate transcriptional up-regulation of GDH1 in sitiens might be associated with the PAL-GS1 pattern and the deaminating activity of the enzyme, forming 2-oxoglutarate from glutamate, functioning as another anaplerotic entry point into the TCA cycle. However, the late and substantial increase of GDH in the wild-type is seemingly associated with the aminating activity of the enzyme, exhausting the TCA cycle and facilitating cell death, as suggested previously (Pageau et al., 2006).
It seems that the lower concentrations of ABA in sitiens lead to a metabolic state in which the process of senescence is slowed down (Fig. 5c, Sit-Mock). Accordingly, ABA was shown to play a major role in the regulation of leaf senescence in Lilium leaves (Arrom & Munné-Bosch, 2012). Interestingly, in the inoculated sitiens, cell viability and photosynthesis are retained within the pathogen-infested area (beneath the inoculation droplet) until at least 21 dpi, while still containing viable B. cinerea mycelia. The term ‘green bionissia’ was first defined by Walters et al. (2008) as a ‘localized green area of host tissue, in which both host and pathogen cells are alive, surrounded by yellow, senescent host tissue’. This is typically seen in plants invaded by obligate biotrophs, such as rusts and mildews, benefiting the pathogen by prolonging availability of a living nutrient source. Here, however, we report that ABA deficiency in the sitiens mutant of tomato results in a rare type of green bionissia, encircled within a mesophyllic HR-like ring, functioning as a resistance mechanism to retard the necrotrophic pathogen-induced senescence in the invaded area. In wild-type tomato plants, none of the anti-senescence defense mechanisms discussed here were observed at early time points, suggesting that they are suppressed by basal ABA concentrations in the susceptible interaction. It should be noted that exogenous application of ABA suppresses all defense responses in sitiens (Asselbergh et al., 2007; Curvers et al., 2010). However, how exactly ABA may negatively control such defense mechanisms still remains unanswered.
In summary, through our model sitiens–B. cinerea pathosystem, molecular features of a biphasic resistance mechanism in response to a true necrotrophic invasion strategy were eventually unraveled. We propose that, in addition to the epidermal HR-mediated defense response (Asselbergh et al., 2007), timely reconfiguration of the host central C : N metabolism plays a vital role in shaping a secondary line of defense response in the resistant mutant (Fig. 8). Initially, the ‘fast pathogen arrest’ phase, consisting of a rapid epidermal ROS accumulation followed by cell wall fortifications, in which the phenylpropanoid pathway plays a vital role, arrests the penetrating pathogen in the epidermis. Concurrently, a ‘maintenance’ phase of basic metabolism begins presumably in the mesophyll cells surrounding the infection site, tightly controlling the extent of the epidermal HR-mediated defense response, and slowing down the infection-induced senescence. The ammonium produced by PAL activity is reassimilated by the cytosolic GS, reutilizing the byproduct of the primary phase to reduce the damage to the GS/GOGAT cycle in the absence of the chloroplastic isoform. Subsequently, the GS-GOGAT-generated carbon skeleton is supplied into the TCA cycle via the overactivated GABA shunt, replenishing the redox-regulating and anabolically critical Krebs cycle under the constant pressure of infectious mycelia of B. cinerea. Ultimately, activation of such a survival mechanism in the vicinity of the penetration site culminates in forming a rarely seen antinecrotrophic type of ‘green bionissia’ in sitiens, enabling an HR-mediated defense strategy to be ultimately effective against an HR-favoring necrotrophic pathogen.
Figure 8. The multifaceted resistance mechanism in tomato (Solanum lycopersicum) sitiens against Botrytis cinerea. The model depicts the interplay between two spatially and functionally different defensive phases, working synergistically to suppress the pathogen progress: the epidermal hypersensitive response (HR)-mediated response (fast pathogen arrest phase, red asterisks), and the mesophyllic GS1/GABA-shunt-mediated anti-cell death mechanism (maintenance phase, green asterisks). The immunologically stained micrograph of an infected sitiens leaf section has been adopted from our previous study (Curvers et al., 2010), showing two HR-undergone epidermal cells in response to the pathogen penetration (red asterisks). Enzymes are indicated in gray rectangles. PAL, phenylalanine ammonia lyase; GS1, cytosolic glutamine synthetase; GAD, glutamate decarboxylase; GABA, γ-aminobutyric acid; GABA-T, GABA-transaminase; SSA, succinate semialdehyde; SSADH, SSA dehydrogenase; GDH, glutamate dehydrogenase; TCA, tricarboxylic acid.
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