Beneficial microbe-plant associations are common in nature, and their benefits to plant health and their potential application in agriculture are under extensive scrutiny (Campos-Soriano and San Segundo, 2011; Smith and Smith, 2011). Among these associations, arbuscular mycorrhizal (AM) symbiosis is one of the most widespread mutualistic associations worldwide. AM symbioses are established between soil-borne fungi from the phylum Glomeromycota, known as arbuscular mycorrhizal fungi (AMF), and the roots of more than 80% of plant species. These fungi are ubiquitous obligate biotrophs that colonize plant roots to obtain plant carbohydrates for the formation, maintenance, and function of mycorrhizal structures and to complete their life cycle (Bago et al., 2000). In return, the AMF improve plant acquisition of water and mineral nutrients, as well as plants' ability to overcome biotic and abiotic stresses (Gianinazzi et al., 2010; Jung et al., 2012; Cameron et al., 2013; Pieterse et al., 2014; Selosse et al., 2014).

Multiple studies show that mycorrhizal plants are more resistant not only to root attackers but also to foliar pathogens and herbivorous insects (Campos-Soriano et al., 2011; Jung et al., 2012; Song et al., 2013, 2015). Although no antimicrobial compounds released by AMF have yet been described, it is known that AM plants can successfully resist root pathogens as well as certain foliar diseases. Among the mechanisms operating in the enhanced resistance, increased plant nutrition, competition for nutrients and colonization sites, and induction of defense mechanisms have been reported (Jung et al., 2012). In addition to these changes in the host plant, beneficial effects have been associated with changes in the microrhizosphere (Cameron et al., 2013). In fact, most reports suggest an induced resistance mechanism rather than increased tolerance or fungal competition between the AMF and the pathogen since AM plants are able to reduce the extent of the pathogen infection (Cordier et al., 1998; Campos-Soriano et al., 2011; Song et al., 2013; Hayek et al., 2014). This protection is known as mycorrhiza-induced resistance (MIR) (Pozo and Azcón-Aguilar, 2007; Jung et al., 2012) and in many respects resembles to rhizobacteria-induced systemic resistance (ISR) (Pieterse et al., 2014). The expression of defense genes in many of those studies was only reported during infection, hence following a defense priming pattern (Balmer et al., 2015), although direct activation of defenses has also been reported in mycorrhizal plants. For example, colonization of rice roots by AMF triggers signals that provide resistance against the blast fungus Magnaporthe orizae (Campos-Soriano et al., 2011). The mycorrhizal rice plants display resistance against M. orizae through a two-step mechanism: direct induction of defense genes in the absence of the pathogen and triggering defense priming once the infection is established. In this respect, it has been proposed that MIR acts through defense priming by pre-conditioning plant tissues for efficient defense activation (Pozo and Azcón-Aguilar, 2007; Jung et al., 2012). AMF can promote defense priming such that there are no fitness costs for the plant in terms of growth and yield depending on the developmental stages of the partners and the environmental conditions (e.g., nutrient availability and light intensity). In fact, there are reports describing the benefits in terms of fruit quality and plant fitness achieved mainly through improved uptake of phosphorous and nitrogen, although there are also reports showing a reduction in growth (Gianinazzi et al., 2010; Smith and Smith, 2011).

MIR has been associated with the metabolic and genetic rearrangement that occurs as a consequence of the AMF colonization of roots that affects plant primary and secondary metabolism (Rivero et al., 2015). The establishment and functioning of the symbiosis requires a high degree of coordination between both partners, and bidirectional (plant and fungal) control assures a fair trade of resources between the symbionts (Smith and Smith, 2011). Indeed, precise regulation of host hormone levels has been proposed as a central mechanism in the regulation of the interaction and could modulate plant defenses (Fernández et al., 2014; Gutjahr, 2014; Pozo et al., 2015). Several metabolic pathways have been shown to change following AMF root colonization, particularly phenol alcohols and oxylipin derivatives in tomato (Rivero et al., 2015). Seemingly, AMF colonization affects root gene expression and, remarkably, gene expression in shoots as well (Liu et al., 2007; Fiorilli et al., 2009; López-Ráez et al., 2010); metabolic changes in leaves have also been reported in mycorrhizal plants (Schweiger et al., 2014).

Although, significant progress has been made in recent years, the mechanisms by which AMF modulate plants' immune system are far from being understood. MIR may be a side effect of plants' mechanisms for regulating the level of fungal colonization in roots, which implies important changes in root hormone homeostasis (Pozo and Azcón-Aguilar, 2007; Pozo et al., 2015). During the first stages of colonization, mycorrhizal plants display a type of biotrophic pathogen response that involves regulation of SA responses (Güimil et al., 2005; Paszkowski, 2006; Jung et al., 2012). As mycorrhization progresses, there is a switch to an increase in JA-dependent responses. In fact, Rivero et al. (2015) demonstrated that most metabolites derived from α-linoleic acid, a precursor of JA, are induced in AMF-colonized roots.

Mycorrhizal associations and their benefits for plant health are affected by environmental conditions (Pozo et al., 2015). In particular, nutrient availability can have a strong impact on symbiosis and plant defenses (Pastor et al., 2014a,b). Despite the clear advantages acquired by mycorrhizal plants in the uptake of P, few studies have described the effect on N uptake and metabolism (Ames et al., 1983; Hawkins et al., 2000; Reynolds et al., 2005). Several ammonium (López-Pedrosa et al., 2006; Pérez-Tienda et al., 2011, 2012) and amino acid (Cappellazzo et al., 2008) transporters have been characterized in Rhizophagus irregularis. The sensing of nitrogen in the root environment has been a popular topic since it was demonstrated that nitrate transporters deliver signals to the shoot, regulating the C/N imbalance in the plant when there is an alteration of nitrogen in the root (Léran et al., 2015). In Arabidopsis, these signals contribute to plants' tendencies regarding growth or resistance to stress that is cross-regulated by the ABA repressor ABI2 and the nitrogen transporter NRT1.1. These dual functions of transport and signaling demonstrated by the membrane proteins were first described in animals, and the term transceptor was applied to them (Gojon et al., 2011).

The effect of nitrogen and its metabolism in plant immune defenses was extensively investigated by Fagard et al. (2014). A reduction in the N supply produces a decrease in chitinase, chitosanase and peroxidase activities, reducing Arabidopsis defenses (Dietrich et al., 2004). However, it is also known that high N fertilization may result in an increase in N-rich compounds, contributing to the activation of pathogenic virulence. In addition, the nature of the nitrogen supply is also a relevant factor in understanding how N affects immune responses. A continuous ammonium supply in tomato plants triggers Pseudomonas syringae resistance through systemic acclimation, activating basal defenses prior to infection (Fernández-Crespo et al., 2015). In fact, the high affinity ammonium transporter 2.3 (AMT2.3) is related to the suppression of premature arbuscule degeneration (PAD) in Medicago truncatula during the dynamic establishment and degeneration of the arbuscule process in root cells (Breuillin-Sessoms et al., 2015). Thus, it is clear that not only are the levels or sources of N important for AM establishment and the plant immune system, but the regulation and activity of nitrate and ammonium transporters are as well.

Four recent discoveries may suggest a relevant link between MIR and nitrate transporters. (1) The uptake of nitrate is a major issue in AM symbiosis (Fellbaum et al., 2012). (2) Changes in root architecture are a direct effect of AMF in the plant (García-Garrido and Ocampo, 2002). (3) In Arabidopsis, it was shown that nitrate transporters regulate the root architecture, particularly the NRT2.1 gene that encodes a high-affinity transport system (Little et al., 2005). Finally, (4) the Atnrt2.1 and lin1 mutants, which are impaired in NRT2.1, display constitutive defense priming (Camañes et al., 2012; Gamir et al., 2014) because NRT2.1 acts as a repressor of plant immune responses against Plectosphaerella cucumerina and P. syringae.

In the present research, we aim to determine the effectiveness of MIR against Botrytis cinerea in tomato and the mechanisms behind it. We also aim to determine whether nitrogen sensing in the root environment and the consequent activation of nitrate transporters have an impact on the functionality of MIR. Here, we show that tomato plants colonized with R. irregularis display enhanced resistance against the aerial pathogen B. cinerea. AM plants show an enhanced horizontal defense that involves the potentiation of different defense pathways participating in basal resistance. Reasonably, transient nitrogen depletion induces NRT gene expression, which stimulates susceptibility. Despite this susceptibility, there are specific signals that still remain induced upon MIR. Those permanent changes may explain why AM plants are still more resistant against the pathogen even under low N conditions.

Mycorrhiza-induced resistance has been shown to be functional against different biotic stressors such as pathogens and insects (Jung et al., 2012; Cameron et al., 2013; Smith and Smith, 2015). AMF confers enhanced resistance not only against root infections but also against leaf pathogens. Reports on MIR suggest that this induced resistance occurs through a defense priming mechanism (Campos-Soriano et al., 2012; Song et al., 2015). In tomato, symbiosis with the AMF Funneliformis mosseae has been shown to reduce early blight promoting oxylipin-related gene expression and primed induction of glucanase, chitinase, PAL, and LOX enzymatic activities upon Alternaria solani infection (Song et al., 2015). F. mosseae has also been shown to protect tomato against B. cinerea; although, no molecular mechanisms have been proposed for such protection (Fiorilli et al., 2011).

In the present study, we provide evidence of the ability of the widely distributed AMF R. irregularis to protect tomato plants against the foliar necrotrophic pathogen B. cinerea. Furthermore, we also found that this protection occurs through defense priming. As indicated by the PCA of the metabolic profiles obtained by untargeted analysis, however, a certain effect in the leaf metabolome is associated with mycorrhization in the absence of challenge. The changes were particularly pronounced when plants were under pathogen attack or perceiving nutritional depletion.

Indeed, quantification of the fungal biomass showed that MIR is still functional under nutritional stress, although to a lesser extent than in plants under normal N conditions. As expected, tomato plants reorganize with transcriptional and metabolic profiles upon infection. First, the JA-related genes LOXD and PROSYS are significantly induced during MIR. Systemin was shown to be an activator of the oxylipin pathway (Farrokhi et al., 2008), acting through a positive feedback loop with JA biosynthesis (Ryan and Pearce, 1998). In agreement with this enhanced activation of JA biosynthetic genes, levels of JA and its precursor OPDA are higher in infected mycorrhizal plants. In contrast, the finding that leaves from mycorrhizal infected plants show a significant reduction in SA levels can be explained by the negative interference of the elevated levels of JA (Flors et al., 2008). Remarkably, it has been shown that in tomato, enhanced SA levels benefits B. cinerea infection by antagonizing the effective JA-dependent defenses (El Oirdi et al., 2011). Our data suggest that this manipulation by the pathogen is somehow avoided in mycorrhizal plants. As part of the complex defense priming triggered by AMF, the indolic compounds may play a relevant role. Trp, Indole carboxadehyde, N-acetyl-5 metoxytriptamine and 5-hydroxy-3-acetic acid were over-accumulated in infected AM tomato compared with their accumulation levels in non-mycorrhizal plants. Notably, Met, Tyr, Gln, and Glu were also induced in BBAM-infected plants. Vitamins such as riboflavin and pyridoxal phosphate (PLP) have been also related to defense priming in Arabidopsis against B. cinerea and other pathogens (Zhang et al., 2009, 2014, 2015). Remarkably, the N required for PLP biosynthesis appears to be supplied by the hydrolysis of GLn to Glu through the salvage biosynthetic pathway of vitamin B6 (Zhang et al., 2014); thus, elevated Glu and PLP levels may be related. Glu participates in signal transduction from systemic tissues upon wounding (Christmann and Grill, 2013). Whether Glu is translocated systemically from the roots to the infected shoots still remains to be determined, although recently Rivero et al. (2015) showed that Glu is strongly accumulated in AM roots; however, it is noteworthy that Glu has also been associated with other defense priming inducers in Arabidopsis, such as BABA or infection with avirulent bacteria (Pastor et al., 2014a). In addition, one of the most representative pathways among the hits in the over-accumulated signals of infected AM plants was the phenolic acid derivatives pathway. Again, this pathway was induced in R. irregularis colonized tomato roots, showing enhanced accumulation of the lignans precursors and shikimate derivatives (Rivero et al., 2015). The potential contribution of the metabolite changes in the roots to the observed alterations in shoots deserves further investigation.

As a partial conclusion, it is worth noting that all these metabolic and genetic changes are likely to contribute to the resistant phenotype of AM tomato plants because most of them have been previously linked to plant resistance against pathogens.

Surprisingly, most of these signals are repressed when the plant senses nutritional stress. In Arabidopsis, N starvation on one side of the root system leads to an up-regulation of nitrate uptake on the other side of the root system accompanied by an up-regulation of the nitrogen transporter NRT2.1. (Gansel et al., 2001). The closest sequence homologies to the Arabidopsis NRT2.1 are tomato NRT2.4 with 70% homology and NRT2.3 with 69% homology, both encoding functional transporters (Fu et al., 2015). Remarkably, both genes are expressed in leaves upon N starvation, suggesting additional functions in N uptake in the roots.

Plants subjected to 48 h of N depletion induce NRT2.1 gene expression, as described previously for other NRT family members in Arabidopsis (Camañes et al., 2012; Kiba et al., 2012). There is increasing evidence that N supply modulates not only primary plant responses such as root architecture and C/N metabolism (Lejay et al., 1999; Remans et al., 2006; Feng et al., 2011) but also plant immune responses (Camañes et al., 2012; Fagard et al., 2014; Pastor et al., 2014b; Fernández-Crespo et al., 2015). In the present study, we showed that roots' perception of nitrogen starvation enhanced B. cinerea susceptibility and reduced but did not impair MIR. The experimental conditions we implemented prevented N scarcity in the plant because we only exposed adult tomato plants to a depletion period of 48 h, which is not sufficiently long to alter levels of total N in the leaves. Despite this observation, the transient depletion has a strong effect on the plant metabolome as it is observed in the unsupervised PCA analysis. This finding demonstrates that the sensing of N depletion induces a metabolic rearrangement preparing the plant for likely N starvation. Previous studies have shown that lower N regime levels and transient N starvation increase the susceptibility of Arabidopsis to A. alternata and P. cucumerina (Camañes et al., 2012; Pastor et al., 2014b). Furthermore, Vega et al. (2015) showed that tomato cv Micro-Tom is more susceptible to B. cinerea under low nitrogen fertilization regimes (<4 mM KNO₃). This reduction of N has a strong effect on primary and secondary metabolism gene transcripts and appears to be directly linked to oxidative stress. These studies propose a reorganization of plants' metabolism to address N starvation, which interferes with the plant immune system, transforming the plant into a more susceptible host for the pathogens. In our study, we showed that although MIR effectiveness is severely affected by transient N depletion, it is still functional, allowing for a reduction in pathogen proliferation in the plant tissues. This finding demonstrates the relevance of using AMF in field crops, which may preserve a better status of plants' immune system during nutritional fluctuations.

The strong effect of N depletion, likely mediated by NRT2 transcript induction, was the abolishment of the accumulation of OPDA, most Trp and indolic derivatives, riboflavin, folic acid and amino acids upon infection compared with levels measured in BBAM plants not subjected to N starvation prior to infection. These compounds have been previously shown to participate in defense against P. cucumerina, as is the case for Trp derivatives (Bednarek et al., 2009; Gamir et al., 2014), against P. syringae in the case of riboflavin (Zhang et al., 2009) and against B. cinerea in the case of Glu (Hatmi et al., 2015). Our observations indicate that all members of the NRT2 family are induced upon N depletion without infection. It is likely that in tomato these members function as transceptors, as demonstrated in Arabidopsis for NRT2 family members (Gojon et al., 2011; Camañes et al., 2012). In Arabidopsis, the de-repression of NRT2.1 triggers susceptibility by repressing defenses against P. syringae and P. cucumerina (Camañes et al., 2012; Gamir et al., 2014). Notably, lin1 Arabidopsis mutants, blocked in NRT2.1, express constitutive defense priming and are more resistant to necrotroph pathogens. Our results show that NRT2.1 and NRT2.2 are induced in mycorrhizal plants but that NRT2.3 is repressed. Considering that NRT2.3 shows high homology with Arabidopsis NRT2.1, it is likely that AMF modulates its induction upon B. cinerea infection, and subsequently, the plant becomes more resistant. Further research is required to understand how NRT2 members regulate plant immune responses in tomato. However, the fact that under N depletion all three members become strongly induced in infected plants and MIR is less effective confirms the hypothesis that NRT2.3 may function as a sensor of nutritional stress repressing immune defense responses in the plant. Clearly, NRT2.3 induction and the repression of all the metabolic signals described above upon transient N deprivation simply block portion of the primed defenses induced in mycorrhizal plants. Indeed, there is a significant part of the phytometabolome that still remains strongly induced, and therefore, these compounds are enhanced in a N-independent manner. The indole-3-carboxaldehyde is of particular interest. Although its role in defense responses is not currently known, its oxidized form, indole-3-carboxylic acid, was recently described as a signal found in a common fingerprint in several priming stimuli (Gamir et al., 2014). There are many hits in the phenolic acid pathway and derivatives accumulated in AM-N-infected plants. Previous reports describe the antifungal and defensive function of many of these compounds, particularly the flavonoid quercetin and chlorogenic acid (Jia et al., 2010). The protective roles of malic acid and vitamin B6, mainly on its vitamer piridoxal phosphate, are also well known (Zhang et al., 2014). Both compounds have been described to participate in induced defense and priming mechanisms (Pastor et al., 2014a; Zhang et al., 2014 respectively). How sedohetulose-7-phosphate, creatine, glucose1,6-diphosphate, adenosine or tropine, and other partially identified compounds with similar accumulation patterns mediate MIR either in normally fertilized plants (BBAMinf) or under N depletion (BBAM-Ninf) remains unknown at the moment.

In conclusion, we have shown that R. irregularis colonization of tomato roots results in an increased resistance of the shoots to B. cinerea, likely through defense priming involving the induction of a set of secondary metabolites, including the oxyilipins JA and OPDA; Trp derivatives; the amino acids Tyr, Met, Gln, and Glu; some indoles; and phenolic acids and their derivatives. All of these metabolites appear to participate as an integral part of a complex tuning mechanism of the immune system in mycorrhizal plants. However, plants that sense potential N starvation (transient N depletion in our experimental system) reorganize their metabolism to prepare for a nutritional battle antagonizing defense responses against biotic stress. Interestingly, this defense repression observed in non-mycorrhizal plants is partially antagonized in R. irregularis colonized plants that maintain an active part of the N-independent phytometabolome changes related to resistance, mounting a less effective but still functional MIR against B. cinerea.

PS: Genetic análisis, mycorrhization expériments, hormonal análisis. PT: mycorrhization assays, fungal inoculation. JG: metabolomic analysis. MP: Mycorrhization % determination, microscopy. GC: nitrogen content analysis. MC: NRT gene expression. VF: data analysis and processing, bioinformatic analysis of the metabolimic data, figure elaboration, manuscript writing, coordination.