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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.

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., This research was funded by the European Union’s Horizon 2020 Research and Innovation program, grant 765290; by the Spanish Government grants RTI2018-094350-B-C31 and RED2018-02407-T; and CSIC intramural project 201440E046. We acknowledge support by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI)

There is increased interest in research on yeasts as potential phytopathogen biocontrol agents due to increasing restrictions in the use of chemical pesticides. Yeast strains from a range of genera and species have been reported to inhibit postharvest decay in different fruits. However, the mechanisms behind these yeast biocontrol capacities have not been completely deciphered because they are complex and act synergistically. In this study, we performed a thorough untargeted analysis of the exometabolome generated in a co-culture of the fungal plant pathogen Botrytis cinerea with four antagonistic yeast strains: Pichia fermentans (two strains), Issatchenkia terricola and Wickerhamomyces anomalus. As a result, general and strain-specific antifungal mechanisms and molecules were identified. The P. fermentans strains secreted the highest number of differential metabolites to the extracellular medium when co-cultured with B. cinerea. In vitro antagonistic and in vivo pathogen protection assays were performed with the selected metabolites. Among a plethora of 46 differentially secreted metabolites related to yeast-fungus competitive interaction, the phenylpropanoid trans-cinnamic acid and the alkaloid indole-3-carboxaldehyde were identified as the best antagonistic metabolites against gray mold infection under in vivo protection assays. Both metabolites caused damage to the fungal membrane and increased ROS generation in spores of B. cinerea. In addition, enhanced yeast secretion to the extracellular medium of oxylipins, dipeptides, alkaloids or antibiotics deserve to be further investigated as signaling or antagonistic molecules. This study opens the door to future investigations of roles of these molecules in yeast metabolism and application of this knowledge for biotechnological purposes. This work was financed by the Departamento de Desarrollo Económico y Empresarial from the Gobierno de Navarra (Spain): grants 0011-1365-2021-000079 and 0011-1411-2019-000009. Open Access funding provided by Universidad Pública de Navarra.

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 towards 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 disease susceptibility. The protective effect of CCL2 did not seem to be direct as the lectin showed no growth-inhibition towards 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 wild type plants. This study demonstrates a potential of fungal defense lectins in plant protection beyond their use as toxins.

International audience; Plant metabolomics is a set of fast-moving, analytical and chemometric tools and methods for plant functional genomics, phenotyping and systems biology. This multidisciplinary “omics” science can deliver qualitative and quantitative data that provide a detailed description of biochemical systems that are influenced by environmental changes, such as plant–pathogen interactions. This chapter examines key insights and recent outputs in the field of phytopathological metabolomics with a specific focus on the perturbations of primary compounds involved in central metabolism and infection-related metabolites including phytohormones. Priming of plant immune responses is also considered through the angle of metabolomics. Furthermore, the chapter highlights the recurring challenges in the distinction of host and pathogen metabolomes and the hurdles of metabolome annotation. The chapter concludes with perspectives indicating key avenues in ongoing efforts to decode metabolic landscapes of plants under biotic stress conditions.

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., Oligogalacturonide perception in tomato roots or shoots triggers differential transient local responses, but also a myriad of systemic responses at the metabolic, transcriptomic and enzymatic level leading to pathogen resistance. The results highlight the key role of roots in the regulation of these systemic responses.

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 c1loser 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 BAK1 gene expression following infection as well as increased production of ROS after PAMP treatment suggesting that Systemin sensitizes Arabidopsis perception to pathogens and PAMPs., This work was funded by the Plan de Promoción de la Investigación Universitat Jaume I UJI-B2016-43; the Spanish Ministry MICIU; RTI2018-094350-B-C33; the grant to JP-F 19I045 from the Plan Propio de Investigación Universitat Jaume I and PRIMA-INTOMED.

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.

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., Consejo Superior de Investigaciones Científicas, Grant/Award Number: 201640I040; European Regional Development Fund, Grant/Award Numbers: AGL2017‐88‐083‐R, RTI2018‐094350‐B‐C31; Generalitat Valenciana, Grant/Award Number: CDEIGENT/2018/015; Ministerio de Ciencia, Innovación y Universidades, Grant/Award Numbers: AGL2017‐88‐083‐R, RTI2018‐094350‐B‐C31, FJCI‐2015‐23575; National Network of Carotenoids, Grant/Award Number: BIO2017‐90877‐REDT

Indole-3-carboxylic acid (I3CA) is an indolic compound that induces resistance in Arabidopsis adult plants against the necrotrophic fungus Plectosphaerella cucumerina through primed callose accumulation. In this study, we confirm the relevance of ATL31 and SYP121 genes involved in vesicular trafficking in I3CA priming of defenses and we discard camalexin as a mediator of I3CA-induced resistance (IR) in adult plants. In addition, we observed that an intact I3CA biosynthetic pathway is necessary for I3CA-IR functionality.

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., This work was supported by the Spanish Ministry of Science and Innovation (AGL2013-49023-C03-02-R) and by Pla de Promoció de la Investigació of Universitat Jaume I (P1 1B2013-75, UJI-A2016-09, and UJI-B2017-30), Research Staff Training grant of AG-H (PREDOC/2016/27) of Universitat Jaume I.

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., This work was financially supported by the Plan de Promoción de la Investigación de la Universitat Jaume I P1- 1B2015-33, the Spanish Ministry MINECO AGL2015-64990-C2-2-R and the Generalitat Valenciana project AICO/2016/029.

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. β-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., The authors thank the Serveis Centrals de la Universitat Jaume I and particularly Cristian Barrera for their technical support. This work was financially supported by the Plan de Promoción de la Investigación de la Universitat Jaume I UJI‐B2016‐43, MINECO AGL2015‐64990‐C2‐2 and the Generalitat Valenciana AICO/2016/029.

Summary Pathogenesis-related proteins played a pioneering role fifty 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. However, the biochemical activity and mode of action of PR-1 proteins has remained elusive. 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 sterol sequestration 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. This article is protected by copyright. All rights reserved.

Being sessile organisms, plants must respond to various challenges in the environment. The priming process consists of three clear stages. The first stage includes all the cellular changes in the absence of the challenge so-called pre-challenge priming stage. These changes are expected to be rather subtle, affecting the preparation of the plant to properly manage subsequent responses to pathogens with no major fitness costs. Most of the research that has been conducted at this stage has been dedicated to the study of changes in gene expression and protein phosphorylation. However, the metabolic changes that occur during the pre-challenge priming stage are poorly understood. The second stage affects the early to late stages of the defence response, which occurs after the interaction with a pathogen has been established. Most studies involving priming are dedicated to the molecular events that take place during this stage. Most studies have shown that defence priming is strongly hormonally regulated; however, there is also evidence of the involvement of phenolic derivative compounds and many other secondary metabolites, leading to stronger and faster plant responses. The third priming phase ranges from long lasting defence priming to trans-generational acquired resistance. Long-term metabolic transitions, that occur in the offspring of primed plants, remain to be elucidated. Here we review existing information in the literature that relates to the metabolic changes that occur during all three defence priming stages and highlight the metabolic transitions that are associated with the stimulation of priming and the characteristics of the pathogens whenever possible.

1 BACKGROUND Life history parameters of the phytophagous spider mite Tetranychus urticae in citrus depend on the rootstock where the cultivar is grafted. To unveil the mechanisms responsible for this effect, the authors have carried out comparative experiments of T. urticae performance on two citrus rootstocks, the highly T. urticae-sensitive Cleopatra mandarin and the more tolerant sour orange. 2 RESULTS Sour orange showed reduced leaf damage symptoms, supported lower mite populations and reduced oviposition rates compared with Cleopatra mandarin. Hormonal, metabolomic and gene expression analyses of the main defence pathways suggest a relevant role of the oxylipin and the flavonoid pathways in the response against T. urticae. Sour orange showed an increased activity of the JA pathway, which was hardly active in the most susceptible rootstock. Moreover, treatments with the LOX inhibitor Phenidone abolished the enhanced tolerance of sour orange. Therefore, oxylipin-dependent defence seems to be rootstock dependent. The metabolomic analysis showed the importance of the flavonoid pathway, which is implicated in the interaction between plants and their environment. 3 CONCLUSION The findings suggest that sour-orange enhanced tolerance to spider mites can be sustained by a combination of pre-existing and induced responses depending on high levels of flavonoids and a fast and effective activation of the oxylipin pathway. © 2013 Society of Chemical Industry

Highlight Systemic resistance in citrus against Tetranychus urticae is rootstock dependent and is transmitted by grafting. Roots release glutamic acid after infestation, inducing GRL expression and promoting resistance in the canopy., 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.

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., This study was financially supported by grants AGL2012-39923 from the Spanish MINECO and P10-AGR-6516 from Junta de Andalucía. J. Rivero is supported by Ph.D. fellowship BES-2013-062638

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. This work was partially funded by the Spanish Plan Nacional I+D (AGL2011-30538-C03-01 and AGL2012-39923-C02-02). The authors are grateful to Cristian Vicent (Servicio de Instrumentación Científica–Universitat Jaume I) for technical assistance. We also thank Ivo Feussner (University of Goettingen, Germany) for providing Mar-Vis software and training to J.G.

Disruption of the high-affinity nitrate transporter NRT2.1 activates the priming defence 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 defence 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 defence 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.

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 β-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.

Life history parameters of the phytophagous spider mite Tetranychus urticae in citrus depend on the rootstock where the cultivar is grafted. To unveil the mechanisms responsible for this effect, the authors have carried out comparative experiments of T. urticae performance on two citrus rootstocks, the highly T. urticae-sensitive Cleopatra mandarin and the more tolerant sour orange.Sour orange showed reduced leaf damage symptoms, supported lower mite populations and reduced oviposition rates compared with Cleopatra mandarin. Hormonal, metabolomic and gene expression analyses of the main defence pathways suggest a relevant role of the oxylipin and the flavonoid pathways in the response against T. urticae. Sour orange showed an increased activity of the JA pathway, which was hardly active in the most susceptible rootstock. Moreover, treatments with the LOX inhibitor Phenidone abolished the enhanced tolerance of sour orange. Therefore, oxylipin-dependent defence seems to be rootstock dependent. The metabolomic analysis showed the importance of the flavonoid pathway, which is implicated in the interaction between plants and their environment.The findings suggest that sour-orange enhanced tolerance to spider mites can be sustained by a combination of pre-existing and induced responses depending on high levels of flavonoids and a fast and effective activation of the oxylipin pathway. © 2013 Society of Chemical Industry.

Summary Priming is a physiological state for protection of plants against a broad range of pathogens, and is achieved through stimulation of the plant immune system. Various stimuli, such as beneficial microbes and chemical induction, activate defense priming. In the present study, we demonstrate that impairment of the high-affinity nitrate transporter 2.1 (encoded by NRT2.1) enables Arabidopsis to respond more quickly and strongly to Plectosphaerella cucumerina attack, leading to enhanced resistance. The Arabidopsis thaliana mutant lin1 (affected in NRT2.1) is a priming mutant that displays constitutive resistance to this necrotroph, with no associated developmental or growth costs. Chemically induced priming by β–aminobutyric acid treatment, the constitutive priming mutant ocp3 and the constitutive priming present in the lin1 mutant result in a common metabolic profile within the same plant–pathogen interactions. The defense priming significantly affects sugar metabolism, cell-wall remodeling and shikimic acid derivatives levels, and results in specific changes in the amino acid profile and three specific branches of Trp metabolism, particularly accumulation of indole acetic acid, indole-3–carboxaldehyde and camalexin, but not the indolic glucosinolates. Metabolomic analysis facilitated identification of three metabolites in the priming fingerprint: galacturonic acid, indole-3–carboxylic acid and hypoxanthine. Treatment of plants with the latter two metabolites by soil drenching induced resistance against P. cucumerina, demonstrating that these compounds are key components of defense priming against this necrotrophic fungus. Here we demonstrate that indole-3–carboxylic acid induces resistance by promoting papillae deposition and H2O2 production, and that this is independent of PR1, VSP2 and PDF1.2 priming.

Plant resistance against the necrotrophic pathogen Plectosphaerella cucumerina is mediated by a combination of several hormonal-controlled signalling pathways. The priming agent β-aminobutyric acid (BABA) is able to induce effective resistance against this pathogen by stimulating callose-rich cell wall depositions. In the present research it is demonstrated that BABA-Induced Resistance (BABA-IR) against P. cucumerina in Arabidopsis has additional components such as the induction of defences mediated by indolic derivatives. Chromatographic approach for the detection and characterization of metabolites enhanced by BABA compared with water-treated plants only when the challenge is present has been developed. The metabolites matching this criteria are considered to be primed by BABA. The analytic procedure is based on the combination of liquid chromatography (LC) with a triple quadrupole (TQD) detector in a precursor ion scanning mode. Using this analytical system a signal in negative electro-spray mode of 160 m/z is primed by BABA in infected plants. A subsequent exact mass analysis in a quadrupole time-of-flight mass spectrometer demonstrated that this ion was the indole-derivative metabolite indole-3-carboxylic acid (I3CA). The identity of indole-3-carboxilic acid was definitively confirmed by comparing its retention time and fragmentation spectra with a commercial standard. Quantification of I3CA in primed plants showed that this indolic metabolite is specifically primed by BABA upon P. cucumerina infection, while other indolic compounds such as IAA and camalexin are not. Taking together these observations with the known role of callose in priming against this pathogen, suggests that priming is not a single mechanism but rather a multicomponent defence.

© 2015 Elsevier Ltd. 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'.