Deciphering molecular events behind Systemin-induced resistance against Botrytis cinerea in tomato plants.

Plant defense peptides are paramount endogenous danger signals secreted after a challenge intensifying the plant immune response. The peptidic hormone Systemin (Sys) was shown to participate in resistance in several plant-pathosystems, although the mechanisms behind Sys-IR when exogenously applied remain elusive. We performed proteomic, metabolomic and enzymatic studies to decipher the Sys-induced changes in tomato plants either in the absence or the presence of Botrytis cinerea infection. Sys-treatments triggered direct proteomic rearrangement mostly involved in carbon metabolism and photosynthesis. However, the final induction of defense proteins required concurrent challenge, triggering priming of pathogen-targeted proteins. Conversely, at the metabolomic level, Sys-treated plants showed an alternative behaviour following a general priming profile. Out of the primed metabolites, the flavonoids rutin and isorhamnetin and two alkaloids correlated with the proteins 4-coumarate-CoA-ligase and chalcone-flavanone-isomerase triggered by Sys treatment. In addition, the proteomic and enzymatic analyses revealed that Sys conditioned the primary metabolism towards the production of available sugars that could be fuelling the priming of callose deposition in Sys-treated plants, furthermore PR1 appeared as as key element in Sys-induced resistance. Collectively, the direct induction of proteins and priming of specific secondary metabolites in Sys-treated plants indicated that posttranslational protein regulation is an additional component of priming against necrotrophic fungi.

The simultaneous perception of self- and non-self-danger signals potentiates plant innate immunity responses.

MAIN CONCLUSION: The simultaneous perception of endogenous and exogenous danger signals potentiates PAMP-triggered immunity in tomato and other downstream defence responses depending on the origin of the signal. Abstract Plant cells perceive a pathogen invasion by recognising endogenous or exogenous extracellular signals such as Damage-Associated Molecular Patterns (DAMPs) or Pathogen-Associated Molecular Patterns (PAMPs). In particular, DAMPs are intracellular molecules or cell wall fragments passive or actively released to the apoplast, whose extracellular recognition by intact cells triggers specific immune signalling, the so-called DAMP-triggered immunity. The extracellular recognition of DAMPs and PAMPs leads to a very similar intracellular signalling, and this similarity has generated a biological need to know why plants perceive molecules with such different origins and with overlapped innate immunity responses. Here, we report that the simultaneous perception of DAMPs and a PAMP strengthens early and late plant defence responses. To this aim, we studied classical PTI responses such as the generation of ROS and MAPK phosphorylation, but we also monitored the biosynthesis of phytocytokines and performed a non-targeted metabolomic analysis. We demonstrate that co-application of the bacterial peptide flagellin with the DAMPs cyclic AMP or cellobiose amplifies PAMP-triggered immunity responses. Both co-applications enhanced the synthesis of phytocytokines, but only simultaneous treatments with cAMP strengthened the flagellin-dependent metabolomic responses. In addition, cAMP and cellobiose treatments induced resistance against the hemibiotrophic bacteria Pseudomonas syringae pv. tomato DC3000. Overall, these results indicate that the complex mixture of DAMPs and PAMPs carries specific information that potentiates plant defence responses. However, downstream responses seem more specific depending on the composition of the mixture.

Phosphorus availability drives mycorrhiza induced resistance in tomato.

Arbuscular mycorrhizal (AM) symbiosis can provide multiple benefits to the host plant, including improved nutrition and protection against biotic stress. Mycorrhiza induced resistance (MIR) against pathogens and insect herbivores has been reported in different plant systems, but nutrient availability may influence the outcome of the interaction. Phosphorus (P) is a key nutrient for plants and insects, but also a regulatory factor for AM establishment and functioning. However, little is known about how AM symbiosis and P interact to regulate plant resistance to pests. Here, using the tomato-Funneliformis mosseae mycorrhizal system, we analyzed the effect of moderate differences in P fertilization on plant and pest performance, and on MIR against biotic stressors including the fungal pathogen Botrytis cinerea and the insect herbivore Spodoperta exigua. P fertilization impacted plant nutritional value, plant defenses, disease development and caterpillar survival, but these effects were modulated by the mycorrhizal status of the plant. Enhanced resistance of F. mosseae-inoculated plants against B. cinerea and S. exigua depended on P availability, as no protection was observed under the most P-limiting conditions. MIR was not directly explained by changes in the plant nutritional status nor to basal differences in defense-related phytohormones. Analysis of early plant defense responses to the damage associated molecules oligogalacturonides showed primed transcriptional activation of plant defenses occurring at intermediate P levels, but not under severe P limitation. The results show that P influences mycorrhizal priming of plant defenses and the resulting induced-resistance is dependent on P availability, and suggest that mycorrhiza fine-tunes the plant growth vs defense prioritization depending on P availability. Our results highlight how MIR is context dependent, thus unravel molecular mechanism based on plant defence in will contribute to improve the efficacy of mycorrhizal inoculants in crop protection.

Extracellular DNA as an elicitor of broad-spectrum resistance in Arabidopsis thaliana.

Like in mammals, the plant immune system has evolved to perceive damage. Damaged-associated molecular patterns (DAMPs) are endogenous signals generated in wounded or infected tissue after pathogen or insect attack. Although extracellular DNA (eDNA) is a DAMP signal that induces immune responses, plant responses after eDNA perception remain largely unknown. Here, we report that signaling defenses but not direct defense responses are induced after eDNA applications enhancing broad-range plant protection. A screening of defense signaling and hormone biosynthesis marker genes revealed that OXI1, CML37 and MPK3 are relevant eDNA-Induced Resistance markers (eDNA-IR). Additionally, we observed that eDNA from several Arabidopsis ecotypes and other phylogenetically distant plants such as citrus, bean and, more surprisingly, a monocotyledonous plant such as maize upregulates eDNA-IR marker genes. Using 3,3'-Diaminobenzidine (DAB) and aniline blue staining methods, we observed that H2O2 but not callose was strongly accumulated following self-eDNA treatments. Finally, eDNA resulted in effective induced resistance in Arabidopsis against the pathogens Hyaloperonospora arabidopsidis, Pseudomonas syringae, and Botrytis cinerea and against aphid infestation, reducing the number of nymphs and moving forms. Hence, the unspecificity of DNA origin and the wide range of insects to which eDNA can protect opens many questions about the mechanisms behind eDNA-IR.

Expression of a Fungal Lectin in Arabidopsis Enhances Plant Growth and Resistance Toward Microbial Pathogens and a Plant-Parasitic Nematode.

Coprinopsis cinerea lectin 2 (CCL2) is a fucoside-binding lectin from the basidiomycete C. cinerea that is toxic to the bacterivorous nematode Caenorhabditis elegans as well as animal-parasitic and fungivorous nematodes. We expressed CCL2 in Arabidopsis to assess its protective potential toward plant-parasitic nematodes. Our results demonstrate that expression of CCL2 enhances host resistance against the cyst nematode Heterodera schachtii. Surprisingly, CCL2-expressing plants were also more resistant to fungal pathogens including Botrytis cinerea, and the phytopathogenic bacterium Pseudomonas syringae. In addition, CCL2 expression positively affected plant growth indicating that CCL2 has the potential to improve two important agricultural parameters namely biomass production and general disease resistance. The mechanism of the CCL2-mediated enhancement of plant disease resistance depended on fucoside-binding by CCL2 as transgenic plants expressing a mutant version of CCL2 (Y92A), compromised in fucoside-binding, exhibited wild type (WT) disease susceptibility. The protective effect of CCL2 did not seem to be direct as the lectin showed no growth-inhibition toward B. cinerea in in vitro assays. We detected, however, a significantly enhanced transcriptional induction of plant defense genes in CCL2- but not CCL2-Y92A-expressing lines in response to infection with B. cinerea compared to WT plants. This study demonstrates a potential of fungal defense lectins in plant protection beyond their use as toxins.

Roots drive oligogalacturonide-induced systemic immunity in tomato.

Oligogalacturonides (OGs) are fragments of pectin released from the plant cell wall during insect or pathogen attack. They can be perceived by the plant as damage signals, triggering local and systemic defence responses. Here, we analyse the dynamics of local and systemic responses to OG perception in tomato roots or shoots, exploring their impact across the plant and their relevance in pathogen resistance. Targeted and untargeted metabolomics and gene expression analysis in plants treated with purified OGs revealed that local responses were transient, while distal responses were stronger and more sustained. Remarkably, changes were more conspicuous in roots, even upon foliar application of the OGs. The treatments differentially activated the synthesis of defence-related hormones and secondary metabolites including flavonoids, alkaloids and lignans, some of them exclusively synthetized in roots. Finally, the biological relevance of the systemic defence responses activated upon OG perception was confirmed, as the treatment induced systemic resistance to Botrytis cinerea. Overall, this study shows the differential regulation of tomato defences upon OGs perception in roots and shoots and reveals the key role of roots in the coordination of the plant responses to damage sensing.

Arabidopsis Plants Sense Non-self Peptides to Promote Resistance Against Plectosphaerella cucumerina.

Peptides are important regulators that participate in the modulation of almost every physiological event in plants, including defense. Recently, many of these peptides have been described as defense elicitors, termed phytocytokines, that are released upon pest or pathogen attack, triggering an amplification of plant defenses. However, little is known about peptides sensing and inducing resistance activities in heterologous plants. In the present study, exogenous peptides from solanaceous species, Systemins and HypSys, are sensed and induce resistance to the necrotrophic fungus Plectosphaerella cucumerina in the taxonomically distant species Arabidopsis thaliana. Surprisingly, other peptides from closer taxonomic clades have very little or no effect on plant protection. In vitro bioassays showed that the studied peptides do not have direct antifungal activities, suggesting that they protect the plant through the promotion of the plant immune system. Interestingly, tomato Systemin was able to induce resistance at very low concentrations (0.1 and 1 nM) and displays a maximum threshold being ineffective above at higher concentrations. Here, we show evidence of the possible involvement of the JA-signaling pathway in the Systemin-Induced Resistance (Sys-IR) in Arabidopsis. Additionally, Systemin treated plants display enhanced BAK1 and BIK1 gene expression following infection as well as increased production of ROS after PAMP treatment suggesting that Systemin sensitizes Arabidopsis perception to pathogens and PAMPs.

Exogenous strigolactones impact metabolic profiles and phosphate starvation signalling in roots.

Strigolactones (SLs) are important ex-planta signalling molecules in the rhizosphere, promoting the association with beneficial microorganisms, but also affecting plant interactions with harmful organisms. They are also plant hormones in-planta, acting as modulators of plant responses under nutrient-deficient conditions, mainly phosphate (Pi) starvation. In the present work, we investigate the potential role of SLs as regulators of early Pi starvation signalling in plants. A short-term pulse of the synthetic SL analogue 2'-epi-GR24 promoted SL accumulation and the expression of Pi starvation markers in tomato and wheat under Pi deprivation. 2'-epi-GR24 application also increased SL production and the expression of Pi starvation markers under normal Pi conditions, being its effect dependent on the endogenous SL levels. Remarkably, 2'-epi-GR24 also impacted the root metabolic profile under these conditions, promoting the levels of metabolites associated to plant responses to Pi limitation, thus partially mimicking the pattern observed under Pi deprivation. The results suggest an endogenous role for SLs as Pi starvation signals. In agreement with this idea, SL-deficient plants were less sensitive to this stress. Based on the results, we propose that SLs may act as early modulators of plant responses to P starvation.

1-Methyltryptophan Modifies Apoplast Content in Tomato Plants Improving Resistance Against Pseudomonas syringae.

Plants can produce numerous natural products, many of which have been shown to confer protection against microbial attack. In this way, we identified 1-methyltryptophan (1-MT), a natural compound produced by tomato plants in response to Pseudomonas syringae attack, whose application by soil drench provided protection against this pathogen. In the present work, we have studied the mechanisms underlying this protection. The results demonstrated that 1-MT can be considered a new activator of plant defense responses that acts by inhibiting the stomatal opening produced by coronatine (COR) and could thereby, prevent bacteria entering the mesophyll. Besides, 1-MT acts by blocking the jasmonic acid (JA) pathway that, could avoid manipulation of the salicylic acid (SA) pathway by the bacterium, and thus hinder its growth. Although the concentration of 1-MT reached in the plant did not show antimicrobial effects, we cannot rule out a role for 1-MT acting alone because it affects the expression of the fliC gene that is involved in synthesis of the flagellum. These changes would result in reduced bacterium motility and, therefore, infective capacity. The results highlight the effect of a tryptophan derivative on induced resistance in plants.

Starch degradation, abscisic acid and vesicular trafficking are important elements in callose priming by indole-3-carboxylic acid in response to Plectosphaerella cucumerina infection.

A fast callose accumulation has been shown to mediate defence priming in certain plant-pathogen interactions, but the events upstream of callose assembly following chemical priming are poorly understood, mainly because those steps comprise sugar transfer to the infection site. ß-Amino butyric acid (BABA)-induced resistance in Arabidopsis against Plectosphaerella cucumerina is known to be mediated by callose priming. Indole-3-carboxylic acid (ICOOH, also known as I3CA) mediates BABA-induced resistance in Arabidopsis against P. cucumerina. This indolic compound is found in a common fingerprint of primed metabolites following treatments with various priming stimuli. In the present study, we show that I3CA induces resistance in Arabidopsis against P. cucumerina and primes enhancement of callose accumulation. I3CA treatment increased abscisic acid (ABA) levels before infection with P. cucumerina. An intact ABA synthesis pathway is needed to activate a starch amylase (BAM1) to trigger augmented callose deposition against P. cucumerina during I3CA-IR. To verify the relevance of the BAM1 amylase in I3CA-IR, knockdown mutants and overexpressors of the BAM1 gene were tested. The mutant bam1 was impaired to express I3CA-IR, but complemented 35S::BAM1-YFP lines in the background of bam1 restored an intact I3CA-IR and callose priming. Therefore, a more active starch metabolism is a committed step for I3CA-IR, inducing callose priming in adult plants. Additionally, I3CA treatments induced expression of the ubiquitin ligase ATL31 and syntaxin SYP131, suggesting that vesicular trafficking is relevant for callose priming. As a final element in the callose priming, an intact Powdery Mildew resistant4 (PMR4) gene is also essential to fully express I3CA-IR.

Accurate and easy method for systemin quantification and examining metabolic changes under different endogenous levels.

BACKGROUND: Systemin has been extensively studied since it was discovered and is described as a peptidic hormone in tomato plants and other Solanaceae. Jasmonic acid and systemin are proposed to act through a positive feed-back loop with jasmonic acid, playing synergistic roles in response to both wounding and insect attack. Despite its biological relevance, most studies regarding the function of systemin in defence have been studied via PROSYSTEMIN (PROSYS) gene expression, which encodes the propeptide prosystemin that is later cleaved to systemin (SYS). Interestingly, hardly any studies have been based on quantification of the peptide. RESULTS: In this study, a simple and accurate method for systemin quantification was developed to understand its impact on plant metabolism. The basal levels of systemin were found to be extremely low. To study the role of endogenous systemin on plant metabolism, systemin was quantified in a transgenic line overexpressing the PROSYS gene (PS+) and in a silenced antisense line (PS-). We evaluated the relevance of systemin in plant metabolism by analysing the metabolomic profiles of both lines compared to wildtype plants through untargeted metabolomic profiling. Compounds within the lignan biosynthesis and tyrosine metabolism pathways strongly accumulated in PS+ compared to wild-type plants and to plants from the PS- line. The exogenous treatments with SYS enhanced accumulation of lignans, which confirms the role of SYS in cell wall reinforcement. Unexpectedly, PS+ plants displayed wild-type levels of jasmonic acid (JA) but elevated accumulation of 12-oxo-phytodienoic acid (OPDA), suggesting that PS+ should not be used as an over-accumulator of JA in experimental setups. CONCLUSIONS: A simple method, requiring notably little sample manipulation to quantify the peptide SYS, is described. Previous studies were based on genetic changes. In our study, SYS accumulated at extremely low levels in wild-type tomato leaves, showed slightly higher levels in the PROSYSTEMIN-overexpressing plants and was absent in the silenced lines. These small changes have a significant impact on plant metabolism. SA and OPDA, but not JA, were higher in the PROSYS-overexpressing plants.

The sterol-binding activity of PATHOGENESIS-RELATED PROTEIN 1 reveals the mode of action of an antimicrobial protein.

Pathogenesis-related proteins played a pioneering role 50 years ago in the discovery of plant innate immunity as a set of proteins that accumulated upon pathogen challenge. The most abundant of these proteins, PATHOGENESIS-RELATED 1 (PR-1) encodes a small antimicrobial protein that has become, as a marker of plant immune signaling, one of the most referred to plant proteins. The biochemical activity and mode of action of PR-1 proteins has remained elusive, however. Here, we provide genetic and biochemical evidence for the capacity of PR-1 proteins to bind sterols, and demonstrate that the inhibitory effect on pathogen growth is caused by the sequestration of sterol from pathogens. In support of our findings, sterol-auxotroph pathogens such as the oomycete Phytophthora are particularly sensitive to PR-1, whereas sterol-prototroph fungal pathogens become highly sensitive only when sterol biosynthesis is compromised. Our results are in line with previous findings showing that plants with enhanced PR-1 expression are particularly well protected against oomycete pathogens.

The Nitrogen Availability Interferes with Mycorrhiza-Induced Resistance against Botrytis cinerea in Tomato.

Mycorrhizal plants are generally quite efficient in coping with environmental challenges. It has been shown that the symbiosis with arbuscular mycorrhizal fungi (AMF) can confer resistance against root and foliar pathogens, although the molecular mechanisms underlying such mycorrhiza-induced resistance (MIR) are poorly understood. Tomato plants colonized with the AMF Rhizophagus irregularis display enhanced resistance against the necrotrophic foliar pathogen Botrytis cinerea. Leaves from arbuscular mycorrhizal (AM) plants develop smaller necrotic lesions, mirrored also by a reduced levels of fungal biomass. A plethora of metabolic changes takes place in AMF colonized plants upon infection. Certain changes located in the oxylipin pathway indicate that several intermediaries are over-accumulated in the AM upon infection. AM plants react by accumulating higher levels of the vitamins folic acid and riboflavin, indolic derivatives and phenolic compounds such as ferulic acid and chlorogenic acid. Transcriptional analysis support the key role played by the LOX pathway in the shoots associated with MIR against B. cinerea. Interestingly, plants that have suffered a short period of nitrogen starvation appear to react by reprogramming their metabolic and genetic responses by prioritizing abiotic stress tolerance. Consequently, plants subjected to a transient nitrogen depletion become more susceptible to B. cinerea. Under these experimental conditions, MIR is severely affected although still functional. Many metabolic and transcriptional responses which are accumulated or activated by MIR such NRT2 transcript induction and OPDA and most Trp and indolic derivatives accumulation during MIR were repressed or reduced when tomato plants were depleted of N for 48 h prior infection. These results highlight the beneficial roles of AMF in crop protection by promoting induced resistance not only under optimal nutritional conditions but also buffering the susceptibility triggered by transient N depletion.

Systemic resistance in citrus to Tetranychus urticae induced by conspecifics is transmitted by grafting and mediated by mobile amino acids.

Recent research suggests that systemic signalling and communication between roots and leaves plays an important role in plant defence against herbivores. In the present study, we show that the oviposition of the two-spotted spider mite Tetranychus urticae in the systemic leaves of citrus rootstock Citrus aurantium (sour orange) was reduced by 50% when a lower leaf was previously infested with conspecifics. Metabolomic and gene expression analysis of the root efflux revealed a strong accumulation of glutamic acid (Glu) that triggered the expression of the citrus putative glutamate receptor (GRL) in the shoots. Additionally, uninfested sour orange systemic leaves showed increased expression of glutamate receptors and higher amounts of jasmonic acid (JA) and 12-oxo-phytodienoic acid in plants that were previously infested. Glu perception in the shoots induced the JA pathway, which primed LOX-2 gene expression when citrus plants were exposed to a second infestation. The spider mite-susceptible citrus rootstock Cleopatra mandarin (C. unshiu) also expressed systemic resistance, although the resistance was less effective than the resistance in sour orange. Surprisingly, the mobile signal in Cleopatra mandarin was not Glu, which suggests a strong genotype-dependency for systemic signalling in citrus. When the cultivar Clemenules (C. clementina) was grafted onto sour orange, there was a reduction in symptomatic leaves and T. urticae populations compared to the same cultivar grafted onto Cleopatra mandarin. Thus, systemic resistance is transmitted from the roots to the shoots in citrus and is dependent on rootstock resistance.

Metabolic transition in mycorrhizal tomato roots.

Beneficial plant-microorganism interactions are widespread in nature. Among them, the symbiosis between plant roots and arbuscular mycorrhizal fungi (AMF) is of major importance, commonly improving host nutrition and tolerance against environmental and biotic challenges. Metabolic changes were observed in a well-established symbiosis between tomato and two common AMF: Rhizophagus irregularis and Funneliformis mosseae. Principal component analysis of metabolites, determined by non-targeted liquid chromatography-mass spectrometry, showed a strong metabolic rearrangement in mycorrhizal roots. There was generally a negative impact of mycorrhizal symbiosis on amino acid content, mainly on those involved in the biosynthesis of phenylpropanoids. On the other hand, many intermediaries in amino acid and sugar metabolism and the oxylipin pathway were among the compounds accumulating more in mycorrhizal roots. The metabolic reprogramming also affected other pathways in the secondary metabolism, mainly phenyl alcohols (lignins and lignans) and vitamins. The results showed that source metabolites of these pathways decreased in mycorrhizal roots, whilst the products derived from α-linolenic and amino acids presented higher concentrations in AMF-colonized roots. Mycorrhization therefore increased the flux into those pathways. Venn-diagram analysis showed that there are many induced signals shared by both mycorrhizal interactions, pointing to general mycorrhiza-associated changes in the tomato metabolome. Moreover, fungus-specific fingerprints were also found, suggesting that specific molecular alterations may underlie the reported functional diversity of the symbiosis. Since most positively regulated pathways were related to stress response mechanisms, their potential contribution to improved host stress tolerance is discussed.

The 'prime-ome': towards a holistic approach to priming.

Plants can be primed to respond faster and more strongly to stress and multiple pathways, specific for the encountered challenge, are involved in priming. This adaptability of priming makes it difficult to pinpoint an exact mechanism: the same phenotypic observation might be the consequence of unrelated underlying events. Recently, details of the molecular aspects of establishing a primed state and its transfer to offspring have come to light. Advances in techniques for detection and quantification of elements spanning the fields of transcriptomics, proteomics, and metabolomics, together with adequate bioinformatics tools, will soon allow us to take a holistic approach to plant defence. This review highlights the state of the art of new strategies to study defence priming in plants and provides perspectives towards 'prime-omics'.

Tetranychus urticae-triggered responses promote genotype-dependent conspecific repellence or attractiveness in citrus.

The citrus rootstocks sour orange and Cleopatra mandarin display differential resistance against Tetranychus urticae. Sour orange plants support reduced oviposition, growth rates and damage compared with Cleopatra mandarin plants. Jasmonic acid signalling and flavonoid accumulation have been revealed as key mechanisms for the enhanced resistance of sour orange plants. In this study, we observed that the release of T. urticae herbivore-induced plant volatiles (HIPVs) from sour orange plants has a marked repellent effect on conspecific mites associated with the production of the terpenes α-ocimene, α-farnesene, pinene and d-limonene, and the green leaf volatile 4-hydroxy-4-methyl-2-pentanone. By contrast, T. urticae HIPVs from Cleopatra mandarin plants promote conspecific mite attraction associated with an increase in (2-butoxyethoxy) ethanol, benzaldehyde and methyl salicylate levels. HIPVs released from sour orange plants following T. urticae infestation induce resistance in Cleopatra mandarin plants, thereby reducing oviposition rates and stimulating the oxylipin biosynthetic gene lipoxygenase2 (LOX2). Cleopatra HIPVs do not affect the response to T. urticae of these rootstocks. We conclude that sour orange plants promote herbivore-induced resistance in Cleopatra mandarin plants and, despite the weak basal resistance of these rootstocks, herbivore resistance can be induced through the combination of HIPVs, such as α-ocimene and d-limonene.

Preparing to fight back: generation and storage of priming compounds.

Immune-stimulated plants are able to respond more rapidly and adequately to various biotic stresses allowing them to efficiently combat an infection. During the priming phase, plant are stimulated in absence of a challenge, and can accumulate and store conjugates or precursors of molecules as well as other compounds that play a role in defense. These molecules can be released during the defensive phase following stress. These metabolites can also participate in the first stages of the stress perception. Here, we report the metabolic changes occuring in primed plants during the priming phase. ß-aminobutyric acid (BABA) causes a boost of the primary metabolism through the tricarboxylic acids (TCA) such as citrate, fumarate, (S)-malate and 2-oxoglutarate, and the potentiation of phenylpropanoid biosynthesis and the octodecanoic pathway. On the contrary, Pseudomonas syringae pv tomato (PstAvrRpt2) represses the same pathways. Both systems used to prime plants share some common signals like the changes in the synthesis of amino acids and the production of SA and its glycosides, as well as IAA. Interestingly, a product of the purine catabolism, xanthosine, was found to accumulate following both BABA- and PstAvrRpt2-treatement. The compounds that are strongly affected in this stage are called priming compounds, since their effect on the metabolism of the plant is to induce the production of primed compounds that will help to combat the stress. At the same time, additional identified metabolites suggest the possible defense pathways that plants are using to get ready for the battle.

Disruption of the ammonium transporter AMT1.1 alters basal defenses generating resistance against Pseudomonas syringae and Plectosphaerella cucumerina.

Disruption of the high-affinity nitrate transporter NRT2.1 activates the priming defense against Pseudomonas syringae, resulting in enhanced resistance. In this study, it is demonstrated that the high-affinity ammonium transporter AMT1.1 is a negative regulator of Arabidopsis defense responses. The T-DNA knockout mutant amt1.1 displays enhanced resistance against Plectosphaerella cucumerina and reduced susceptibility to P. syringae. The impairment of AMT1.1 induces significant metabolic changes in the absence of challenge, suggesting that amt1.1 retains constitutive defense responses. Interestingly, amt1.1 combats pathogens differently depending on the lifestyle of the pathogen. In addition, N starvation enhances the susceptibility of wild type plants and the mutant amt1.1 to P. syringae whereas it has no effect on P. cucumerina resistance. The metabolic changes of amt1.1 against P. syringae are subtler and are restricted to the phenylpropanoid pathway, which correlates with its reduced susceptibility. By contrast, the amt1.1 mutant responds by activating higher levels of camalexin and callose against P. cucumerina. In addition, amt1.1 shows altered levels of aliphatic and indolic glucosinolates and other Trp-related compounds following infection by the necrotroph. These observations indicate that AMT1.1 may play additional roles that affect N uptake and plant immune responses.

The plasticity of priming phenomenon activates not only common metabolomic fingerprint but also specific responses against P. cucumerina.

Previously we described that different priming stimuli trigger common metabolomic responses against P. cucumerina. Furthermore we showed that several primed metabolites were present following independent priming inducers such as natural constitutive priming promoted by gene mutations and chemical priming induced by the ß-aminobutyric acid (BABA). Despite we found a common metabolomic fingerprint, in the present research we focus our attention in specific metabolites that are primed differentially by a mutation in the NRT2.1 gene (lin1 mutant) and BABA treatments against P. cucumerina. Around eight hundred compounds were overaccumulated in the resistant mutant lin1 and in BABA treated plants upon infection. Among them 404 and 412 were specific of each priming condition while 103 compounds were shared by both. Flavonoids and lignans were specifically accumulated in lin1 in response to the fungal attack, while tyrosine, purine metabolism, and aromatic carbon degradation compounds were only accumulated in BABA primed plants upon infection. However, most metabolites differentially accumulated by the two priming conditions belonged to the same metabolic pathways, suggesting that different priming stimuli, upon a given biotic stress, may stimulate similar pathways but activate specific differences depending on the priming stimulus.